Upregulation of CaMKIIβ and Nogo-C mRNA in schizophrenia and the prevalence of CAA insert in the 3’UTR of the Nogo gene.
by
Gabriela Novak
A thesis submitted in conformity with the requirements for the degree of Philosophy
Graduate Department of Pharmacology University of Toronto
© Copyright by Gabriela Novak 2008
ii
UPREGULATION OF CAMKIIβ AND NOGO-C MRNA IN SCHIZOPHRENIA AND THE PREVALENCE OF CAA INSERT IN THE 3’UTR OF THE NOGO GENE.
Gabriela Novak
Doctor of philosophy, 2008
Graduate Department of Pharmacology University of Toronto
ABSTRACT
Schizophrenia may result from altered gene expression leading to abnormal neurodevelopment.
In a search for genes with altered expression in schizophrenia, cDNA library subtractive hybridization
experiments using post-mortem human frontal cerebral cortices from schizophrenia individuals and
neurological controls were performed.
I found the mRNA of two neurodevelopmentally important genes, Nogo (RTN4) and
calcium/calmodulin-dependent protein kinase II beta (CaMKIIβ), to be overexpressed in post-mortem
frontal cortex tissues from patients who suffered with schizophrenia. I used the quantitative real-time
polymerase chain reaction method to determined the mRNA levels of these genes in tissues from age-
and sex-matched individuals.
Nogo is a myelin-associated protein which inhibits the outgrowth of neurites and nerve
terminals. The gene produces three splice variants, Nogo-A, B and C. I found Nogo-C mRNA to be
overexpressed by 26% in schizophrenia. I also found a 17% reduction of Nogo-B mRNA in samples
from individuals who had been diagnosed with severe depression. Furthermore, I showed that there is a
direct correlation between the expression of both Nogo-A and -C and the presence of a CAA insert in
the 3’UTR of the Nogo gene.
CaMKII is a kinase localized at the postsynaptic density. The holoenzyme is primarily composed
of the subunits α and β, encoded by two separate genes. It influences the expression of many
neuroreceptors, in particular receptors of the glutamatergic pathway. CaMKII also mediates neural
maturation during puberty, a time of onset of schizophrenia. The expression of CaMKIIα was elevated
29% in frontal cortex tissues of patients who suffered from depression. The expression of CaMKIIβ was
elevated 27% in tissues of schizophrenia patients and 36% in tissues of patients diagnosed with
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depression. Upregulation of CaMKIIβ was associated with the presence of the CAA insert in at least one
copy of the Nogo gene in a group containing both healthy subjects and patients with mental illness,
possibly linking the CaMKII and Nogo pathways. The values for the expression of Nogo, CaMKIIα and
CaMKIIβ were normalized to β-glucuronidase expression to minimize the effects of mRNA
degradation. These results confirm that upregulation of Nogo-C and CaMKIIβ is likely associated with
schizophrenia.
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ACKNOWLEDGEMENTS
During my Ph.D. studies, I was fortunate to work with a team that has, in their friendship,
assistance and support, far surpassed my expectations. I am deeply grateful to Dr. Philip Seeman for his
guidance, advice, patience and support, which made these past years an incredible learning experience
both in science and in life. It is rare to meet a person with such a profound influence on one's life. I
thank Dr. Teresa Tallerico for her excellent insight in science, for her friendship and for sharing her
invaluable life and parenting experience. The lab would not be the same without Elaine Jack, her
friendship and professional assistance, who also deserves my thanks. I am also grateful to Francoise Ko,
Ph.D., for her friendship and camaraderie. She was a source of contagious optimism, energy and
motivation. The lab would, of course, not be complete without Dr. Guan's contribution and friendly
smiles. I thank my parents and my brother for their support and love. I am grateful to my daughter
Jannine and my husband for their love, their patience, and their understanding, as well as to our
youngest daughters Elizabeth and Kira, who have come into our lives in the past few years, for enriching
our lives with their love.
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TABLE OF CONTENTS
Page
Title page i
Abstract ii
Acknowledgements iv
Table of contents v
List of figures ix
List of tables x
List of publications xi
Abbreviations xii
INTRODUCTION 1
1. SCHIZOPHRENIA 1
1.1. Pathophysiology 2
1.2. Search for genes dysregulated in schizophrenia. 4
2. CaMKII 5
2.1. Activation of CaMKII 6
2.2. Fine tuning of CaMKII activity 8
2.3. Subunit composition and holoenzyme characteristics 9
2.4. CaMKII splicing and expression 10
2.5. Developmental expression 12
2.6. Synaptic plasticity and memory formation 14
2.7. Sensitization 16
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2.8. Involvement of the D2 receptors 18
3. NOGO 18
3.1. The expression of Nogo 22
3.2. Nogo-A in neurodevelopment 23
3.3. The Gi protein – PKC / IP3 pathways 24
3.4. Other pathways involved in Nogo signaling 25
4. DEPRESSION 27
RESULTS 28
Study No. 1: Schizophrenia and Nogo: elevated mRNA in cortex, and high
prevalence of a homozygous CAA insert. 28
1. Abstract for study No. 1 28
2. Introduction for study No. 1 28
3. Materials and Methods for study No. 1 30
3.1. Post-mortem tissues. 30
3.2. Blood samples. 30
3.3. PCR-select cDNA subtractive hybridization. 31
3.4. Quantitative RT-PCR. 32
3.5. Genomic DNA extraction. 33
3.6. Amplification and sequencing of the Nogo DNA template. 33
4. Results of study No. 1 33
vii
Study No. 2: Increased Expression of Calcium/calmodulin-dependent protein
kinase IIβ in Frontal Cortex in Schizophrenia and Depression. 39
1. Abstract for study No. 2 39
2. Introduction for study No. 2 39
3. Materials and Methods for study No. 2 40
3.1. Extraction of total RNA from tissues 41
3.2. First-strand cDNA synthesis 43
3.3. Real-time Quantitative Polymerase Chain Reaction 43
3.4. Determination of mRNA degradation. 44
4. Results of study No. 2 45
Study No. 3: Nogo A, B and C Expression in Schizophrenia, Depression
and Bipolar Frontal Cortex, and correlation of Nogo expression with CAA/TATC
polymorphism in 3’UTR. 51
1. Abstract for study No. 3 51
2. Introduction for study No. 3 51
3. Materials and Methods for study No. 3 52
3.1. Extraction of total RNA from tissues 52
3.2. First-strand cDNA synthesis 53
3.3. Real-time Quantitative Polymerase Chain Reaction 53
3.4. Determination of mRNA degradation. 55
3.5. Detection of a CAA / TATC insert 55
3.6. Statistical methods. 55
4. Results of study No. 3 56
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Supplemental Results
1. Effects of antipsychotics on Nogo expression. 64
2. The mRNA levels of CaMKIIα and CaMKIIβ and their correlation with
the presence of CAA insert in the 3’UTR of the Nogo gene. 65
DISCUSSION 68
Discussion for study No. 1 68
Discussion for study No. 2 69
Discussion for study No. 3 71
General Discussion 74
1. CaMKII plays an important role in age of onset of schizophrenia 74
2. Dysregulation of the CaMKII pathway can explain key pathophysiological
signs of schizophrenia. 75
3. Environmental factors 77
4. Depression 78
5. Nogo and its role in schizophrenia 79
6. The link between CaMKIIβ expression and the CAA insert in the Nogo gene. 81
REFERENCES 83
APPENDICES 110
ix
LIST OF FIGURES
Figure 1. Risk of developing schizophrenia. 2
Figure 2. Functional domains of CaMKIIα. 7
Figure 3. Tissue specificity of variable domain utilization. 13
Figure 4. The RTN4/Nogo gene. 20
Figure 5. Functional domains of Nogo/RTN4. 22
Figure 6. Nogo cDNA sequence. 35
Figure 7. CaMKIIα in schizophrenia and depression. 46
Figure 8. CaMKIIβ in schizophrenia and depression. 47
Figure 9. Effect of mRNA degradation 50
Figure 10. Standardization eliminates effects of degradation. 57
Figure 11. Nogo A in schizophrenia. 60
Figure 12. Nogo B in depression 61
Figure 13. Nogo C in schizophrenia. 62
Figure 14. Nogo A, B and C levels versus the presence of CAA insert 63
Figure 15. Nogo mRNA levels in antipsychotic treated rats 64
Figure 16. CaMKIIα mRNA levels and their correlation with the presence
of the CAA insert in 3’UTR of the Nogo gene. 66
Figure 17. CaMKIIβ mRNA levels and their correlation with the presence
of the CAA insert in 3’UTR of the Nogo gene. 67
x
LIST OF TABLES
Table 1. List of templates identified through DNA subtraction. 4
Table 2. Alternative names and chromosomal location of the
reticulon family members. 19
Table 3. Quantitative RT-PCR of Nogo gene expression and clinical
summaries of samples 36
Table 4. Frequency of CAA insert in schizophrenia and controls. 37
Table 5. Clinical data and findings on post-mortem frontal cerebral cortex tissues 42
Table 6. Levels of CaMKIIα and CaMKIIβ transcripts 48
Table 7. Levels and percent change 58
Table 8. Clinical summaries for postmortem brain tissues 59
Table 9. Nogo A, B, C, CaMKIIα and CaMKIIβ levels in patients
treated with antipsychotics. 65
xi
LIST OF PUBLICATIONS
1. Novak, G., Seeman, P., Tallerico, T., 2000. Schizophrenia: elevated mRNA for calcium-calmodulin-dependent protein kinase II beta in frontal cortex. Brain Res Mol Brain Res. 82, 95-100
2. Tallerico, T., Novak, G., Liu, I.S., Ulpian, C., Seeman, P., 2001. Schizophrenia: elevated mRNA for dopamine D2(Longer) receptors in frontal cortex. Brain Res Mol Brain Res. 87, 160-165.
3. Novak, G., Kim, D., Seeman, P., Tallerico, T., 2002. Schizophrenia and Nogo: elevated mRNA in cortex, and high prevalence of a homozygous CAA insert. Brain Res Mol Brain Res. 107, 183-189
4. Novak, G., Seeman, P., Tallerico, T., 2006. Increased expression of calcium/calmodulin-dependent protein kinase II beta in frontal cortex in schizophrenia and depression. Synapse. 59, 61-68.
5. Novak, G., Tallerico, T., 2006. Nogo A, B and C expression in schizophrenia, depression and bipolar frontal cortex, and correlation of Nogo expression with CAA/TATC polymorphism in 3'-UTR. Brain Res. 1120, 161-171.
6. Greenstein, R., Novak, G., Seeman, P., 2007. Amphetamine sensitization elevates CaMKIIβ mRNA. Synapse. 61, 827-834.
7. Novak, G. and Seeman, P., 2008. Hyperactive mice show elevated D2High receptors, a model for schizophrenia: calcium/calmodulin-dependent kinase II alpha knockouts (In manuscript)
xii
ABBREVIATIONS
Aβ β-amyloid peptide
AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate
APP amyloid precursor protein
ATF1 activating transcription factor 1
ATP adenosine5'-triphosphate
BACE1 β-secretase
BDNF brain-derived neurotrophic factor
bp Base pair
CaM calmodulin
CaMKII Ca2+/calmodulin-dependent protein kinase II (CAMK2)
CaMKIIN Ca2+/calmodulin-dependent protein kinase II inhibitor
cAMP cyclic AMP (adenosine monophosphate)
CaMRE CaM kinase responsive element
CBP CREB binding protein
cDNA deoxyribonucleic acid complementary to mRNA
CDK5 cyclin-dependent protein kinase 5
CNS central nervous system
CPEB cytoplasmic polyadenylation element binding protein
CRE cAMP response element, cis-regulatory elements
CREB cAMP response element-binding protein
DA dopamine
DAG diacylglycerol
DAT dopamine transporter
ddNTP dideoxynucleoside 5'-triphosphate
DNA Deoxyribonucleic acid
dNTP Deoxynucleoside-5'-triphosphate
DPFC dorsolateral prefrontal cortex
DP1 Deleted in Polyposis Locus Protein 1 (Yop1p)
D2R dopamine 2 receptor
xiii
ds double stranded
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
EF-2 kinase elongation factor-2 kinase, also CaMKIII
EGFR epidermal growth factor receptor
EtOH Ethanol
GABA gamma-aminobutyric acid
GABA-R gamma-aminobutyric acid receptor
GDPs giant depolarizing potentials
GluR1 glutamate receptor subunit 1
GPDH Glyceraldehyde-3-phosphate dehydrogenase
LINGO-1 leucine rich repeat and Ig domain containing 1, NgR1 ligand
LRR leucine rich repeat
LTP Long term potentiation
LTD Long term depression
MAG myelin associated glycoprotein
MAPK Mitogen activated protein kinase
MAP2 microtubule associated protein 2
MRI magnetic resonance imaging
mRNA Messenger ribonucleic acid
NF-AT nuclear factor of activated T cells
NF-M Neurofilament - M (medium size)
NGF nerve growth factor
NgR Nogo receptor (RTN4-R)
NMDA N-methyl-D-aspartate
NMDA-R N-methyl-D-aspartate receptor
NR2A-D N-methyl-D-aspartate receptor (NMDA) subunits 2A through D
NR2B N-methyl-D-aspartate receptor (NMDA) subunit 2B
Nt Amino terminus of a protein
OMgp Oligodendrocyte-myelin glycoprotein
PCP Phencyclidine
xiv
PCR Polymerase chain reaction
PEST protein tyrosine phosphatase
PET positron emission tomography
PFC prefrontal cortex
PKA cAMP-dependent protein kinase A
PKC protein kinase C
PLC phospholipase C
PP1 Protein phosphatase-1
PP2A protein phosphatase 2A
PSD postsynaptic density
PSD-95 postsynaptic density protein of 95kD
Ras regulatory GTP hydrolase
RCE retinoblastoma control element
RHD reticulon homology domain
RNA ribonucleic acid
RNase Ribonuclease
RT reverse transcription
SAP mammalian synapse-associated-protein
SPECT Single photon emission computed tomography
SynGAP Synaptic Ras-GTPase-activating protein
TAE tris acetate buffer (4 mM) containing 2 mM EDTA (pH 8.0)
TBE Tris buffer containing 45 mM Tris-borate, 2mM EDTA (pH 8.0)
TE Buffer containing 10mM Tris (pH 8.0), 1 mM EDTA (pH 8.0)
TH tyrosine hydroxylase
TROY TNFRSF19, a transmembrane receptor, member of the tumor necrosis
factor receptor (TNFR) superfamily
uORF upstream of open reading frame
UV ultraviolet
VTA ventral tegmental area
5-HT 5-hydroxytryptamine, serotonin
1
INTRODUCTION
1. SCHIZOPHRENIA
Schizophrenia is a devastating illness, affecting approximately 1% of the population. At the
present time, there is no consensus as to the causes of the disease, although a number of risk factors have
been identified. I have identified two genes that are upregulated in human schizophrenia frontal cortex,
Ca2+/calmodulin-dependent protein kinase II (CaMKII) and a reticulon Nogo/RTN4. My PhD work is an
attempt to explain the contribution of the dysregulation of these genes to the etiology of schizophrenia.
Schizophrenia has a strong genetic component (Figure 1). This follows from the observation that
disease concordance is 50% for monozygotic twins, while the prevalence of the disease in the general
population is only 1%. (Cardno et al., 1999). In other words, approximately 50% of individuals carrying
schizophrenia genes will develop the disease. One may argue that, in twins, the shared in utero
environment plays a determining role in predisposition to schizophrenia but this is refuted by the fact
that dizygotic twins, who share 50% of their genetic material and enjoy the same in utero environment,
only show 10-14% concordance. Furthermore, offspring of both the affected and unaffected
monozygotic twin carry equal risk of developing schizophrenia (17%) (Gottesman and Bertelsen, 1989;
Kringlen and Cramer, 1989). Compared to other complex genetic disorders, schizophrenia has one of the
highest heritabilities. Its heritability (85%) is similar to that of type I diabetes (72%–88%) and greater
than that of breast cancer (30%), coronary heart disease in males (57%), and type II diabetes (26%)
(Kirov et al., 2005).
The analysis of the inheritance pattern of schizophrenia in families suggests the involvement of
several genes, acting in combination, each lending a small contribution to the disease (Cardno and
Gottesman, 2000; Gogos and Gerber, 2006; Risch, 1990). Many genes have been implicated so far,
namely alterations in the levels of parvalbumin, reelin and BDNF (Knable et al., 2004). Other genes
linked to schizophrenia include apolipoprotein L1 (APLOL1) and APOL2 (Mimmack et al., 2002),
DISC1 (Ekelund et al., 2004), RTN4R (Sinibaldi et al., 2004), RGS4 (Mirnics et al., 2001b) and many
others.
Studies also show schizophrenia to be genetically related to other psychoses, such as bipolar
disorder (Shih et al., 2004) and depression (Siris, 2001), but sharing only some of the risk factors
(Cardno and Gottesman, 2000).
2
1.1. Pathophysiology
The first evidence of gross neuropathology in schizophrenia was the demonstration of enlarged
lateral ventricles in approximately one third of those diagnosed with schizophrenia (Johnstone, 1989).
MRI studies subsequently showed enlarged lateral and third ventricle in twins discordant for
schizophrenia and smaller anterior hippocampi in the affected twins (Suddath et al., 1990b; Zipursky et
al., 1992). Further studies have implicated the prefrontal cortex (PFC) as a major locus of dysfunction in
Figure 1. Risk of developing schizophrenia. Risk of developing schizophrenia, depending on a relationship to a family member with schizophrenia. (Gottesman, 1991; Owen, 2005).
schizophrenia (Andreasen et al., 1997; Weinberger et al., 1986). PFC is a brain region critically
involved in the control of cognition, reasoning, perception, and emotion (Goldman-Rakic, 1995),
cognitive tasks impaired by schizophrenia (Goda and Davis, 2003). At the cellular level, a number of
consistent structural abnormalities of the PFC have been reported. These include the maldistribution of
interstitial neurons (Akbarian et al., 1996). Other structural and metabolic abnormalities have also been
reported (Benes, 2000; Moore et al., 1999; Weinberger and Berman, 1996). There are consistent
decreases in nonpyramidal, largely GABAergic (inhibitory), cells (Benes, 2000), alterations in
50%
40%
30%
20%
10%
twins twins
50% 50% 50% 25%
proportion of genes shared
Uncles/aunts Population
% incid
ence
100%
Identical Fraternal Siblings Parents
3
glutamatergic synaptic populations in dorsolateral prefrontal cortex (DPFC) (Lewis, 2000; Lewis and
Gonzalez-Burgos, 2000; Mirnics et al., 2000) and changes in cortical dopaminergic innervation (Akil et
al., 1999).
The strongest evidence for the implication of the dopaminergic system, in particular the
dopamine receptor 2 (D2R), in schizophrenia arises from the observation that all clinically effective
anti-psychotic drugs act as antagonists for D2R and that their abilities to block D2R correlate with their
antipsychotic efficacy (Creese et al., 1976; Seeman et al., 1976; Seeman, 1987; Snyder, 1976). This has
been confirmed in vivo by positron emission tomography (PET) and single photon emission computed
tomography (SPECT), which has shown a strong correlation between D2R occupancy and the clinical
efficacy of antipsychotic drugs (Miyamoto et al., 2005; Remington and Kapur, 1999). This is true for
both the older drugs and for the newer “atypical” antipsychotics (Seeman, 2002). The dopamine theory
of schizophrenia is further supported by the fact that indirect DA agonists, such as amphetamine, elicit
psychotomimetic effects (Breier et al., 1997; Castaneda et al., 1988). Moreover, one of the only clear
pathophysiological signs associated with schizophrenia is amphetamine-induced increase in synaptic DA
in the striatum (compared to controls), (Breier et al., 1997; Breier et al., 1998; Laruelle et al., 1996;
Laruelle et al., 1997), and this is associated with the emergence or with a worsening of psychotic
symptoms clinically (Laruelle et al., 1996; Moore et al., 1999). The observation that amphetamine
sensitization mimics schizophrenia in healthy people and that schizophrenia patients have abnormally
high DA release in response to amphetamine was used in the development of an amphetamine
sensitization animal model of schizophrenia. Chronic administration of amphetamine followed by a
withdrawal period produces a state of sensitization in animals that has characteristics similar to those of
schizophrenia, including enhanced dopamine release and schizophrenia-like cognitive deficits (Adams
and Moghaddam, 1998; Jentsch et al., 1997; Jentsch and Roth, 1999; Moghaddam et al., 1997).
Evidence also supports the involvement of glutamatergic signaling in schizophrenia because
repeated exposure to phencyclidine (PCP), an N-methyl-D-aspartate receptor (NMDA-R) antagonist,
produces a variety of behaviours resembling schizophrenia, including cognitive deficits, loss of short-
term memory and negative symptoms (apathy, avolition, social withdrawal) (Allen and Young, 1978;
Krystal et al., 1994; Snyder, 1980; Tamminga, 1998).
4
1.2. Search for genes dysregulated in schizophrenia
In order to try to identify genes dysregulated in schizophrenia, we performed a subtraction
between three control and three schizophrenia frontal cortex samples (see p. 31 and Appendix). This
technique enabled us to compare two populations of mRNA of genes that are expressed in one
population but not the other. This experiment yielded fourteen candidate sequences (Table 1).
template sequence match
(current name) Size (bp) Quantitation results GenBank accession #
Nogo A 1450 upregulated AJ251383
16s rRNA 774 no difference X93334
Band 11 (TP53INP2) 645 n/a NM_021202
BAC 367D17 (genomic) 539 n/a AC003971
MBP 515 no difference M13577
CAGH3 (SERPINE2) 390 small change NM_006216
mito. ATPase 6 410 n/a X93334
HEV (SPARCL1) 360 n/a NM_004684
calpactin/annexin 338 no difference M81457
mito. Cyto. Ox. II 273 no difference X93334
CaMKIIβ 192 upregulated HSU50358
ZNF 207 229 upregulated AF046001
mito. NADH 4L 250 n/a X93334
NF-M (NEFM) 173 no difference Y00067
Table 1. List of templates identified through DNA subtraction. List of fourteen mRNA templates identified through subtraction between three control and three schizophrenia frontal cortex samples. Our preliminary tests determined three templates to be upregulated, six showed no significant difference and five were not tested. TP53INP2 is a tumor protein p53 inducible nuclear protein 2. SERPINE2 is serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2. SPARCL1 is SPARC-like 1 (mast9, hevin).
5
Of these mRNA species, the cDNA levels for 16s rRNA, MBP (myelin basic protein), calpactin,
mitochondrial cytochrome oxidase II, CAGH3 and NF-M (Neurofilament-M) showed no difference
between the schizophrenia and control group and were excluded from further analysis. Three templates
did show a consistent difference; CaMKIIβ, Nogo (RTN4) and ZNF207. Two genes, CaMKIIβ, a kinase
involved in neurodevelopment and neural functioning, and Nogo, a neural growth inhibitor, were
selected as the most appropriate candidates for further analysis.
2. CAMKII
CaMKII is part of a family of calmodulin-dependent protein kinases (CaM kinases) These
kinases sense calcium signals through binding to calmodulin complexed to four Ca2+ ions. CaMKII is a
multifunctional serine/threonine kinase found in essentially all neuronal compartments, composing up to
1% of total protein in the forebrain and 2% of total protein in the hippocampus (Erondu and Kennedy,
1985). (Reviewed by (Hudmon and Schulman, 2002b)). Transient rises in intracellular Ca2+
concentration contain information in their amplitude, duration and frequency and CaMKII is a molecular
decoder for such oscillation frequencies (Colbran and Brown, 2004; De Koninck and Schulman, 1998;
Sheng and Lee, 2000). Once activated, it forms a molecular memory, which is an integral part of its
ability to interpret these signals (Colbran, 2004; Soderling, 2000). Sustained activation of CaMKII
localized at the postsynaptic density (PSD) results in phosphorylation of numerous synaptic substrates
including ion channels, other signaling molecules and scaffolding proteins (Soderling and Derkach,
2000). Within minutes, this leads to modulation of synaptic transmission (Soderling, 2000), secretion
and cell shape (Braun and Schulman, 1995; Hardingham and Bading, 1999), as well as formation of new
PSDs and remodeling of old PSDs (Soderling, 2000), modulating virtually all neuronal functions
(Colbran, 2004; Colbran and Brown, 2004; Jourdain et al., 2003). Together with other PSD molecules,
CaMKII is a key player in determining the effects of synaptic glutamate release (Sharma et al., 2006).
CaMKII is encoded by four genes, producing four distinct subunits, α, β, γ and δ. Each CaMKII
holoenzyme is composed of 8 - 14 of these subunits (Rosenberg et al., 2006). The CaMKIIα (CaMK2A)
gene is located at 5q33.1 and produces three splice variants, α, αB (Li et al., 2001a) and αKAP (Bayer et
al., 1998; O'Leary et al., 2006b). The CaMKIIα subunit is the most highly expressed CaMKII isoform in
the brain. The CaMKIIβ (CaMK2B) gene is located at 7p14.3-p14.1. It is one of the most important
CaMKII isozymes in the brain and it produces at least eight known splice variants, CaMK2B-1 (β),
CaMK2B-2, CaMK2B-3 (β4), CaMK2B-4 (βe), CaMK2B-5 (βe’), CaMK2B-6 (β6), CaMK2B-7 (β2,
6
βM) and CaMK2B-8 (β7) (Li et al., 2001a; Wang et al., 2000). The full length CaMKIIβ splice variant
(β) is the most prevalent splice variant in the brain (Tombes et al., 2003). The evolutionary conservation
of the CaMKII gene emphasizes its importance. It is present in species that have existed for 600 million
years, such as Caenorhabditis elegans, which has one gene for CaMKII with several splice variants
(Reiner et al., 1999) and Drosophila melanogaster, which also carries only one gene, as compared to
four genes found in mammals (Griffith and Greenspan, 1993). Remarkably, the catalytic and calmodulin
binding domains of Drosophila CaMKII are 85% identical to that of the rat kinase polypeptides
(Yamauchi, 2005). CaMKIIδ is the most ancestral form of the kinase, while CaMKIIα is an
evolutionarily novel form, unique to only birds and mammals (Tombes et al., 2003).
The CaMKII genes are transcribed from a tissue specific promoter, which is under strong
negative control (Kraner et al., 1992; Mori et al., 1992) via elements in the 5’ upstream region, tightly
regulating their neuron-specific expression at the mRNA level (Donai et al., 2001; Mima et al., 2001;
Mima et al., 2002). Since the primary control of CaMKIIα and CaMKIIβ expression is at the level of
transcription, it is important to analyze the mRNA levels of these genes.
2.1. Activation of CaMKII
Cells typically maintain an intracellular Ca2+ level of 10-7M, which is 104 times lower than
the level outside the cells. In response to external stimuli, the intracellular Ca2+ level rapidly increases up
to 10-4M as a result of Ca2+ influx derived from extracellular and intracellular sources. The predominant
intracellular Ca2+ binding protein is calmodulin (CaM), which binds four Ca2+ ions (Yamauchi, 2005)
and is then in turn bound by CaMKII. Each CaMKII isozyme is composed of highly conserved N-
terminal catalytic domain (~280aa) followed by a regulatory domain (~40aa), and a C-terminal
variable/association domain (150-220aa) (Colbran, 2004). The regulatory domains of CaMKIIα and
CaMKIIβ are about 90% identical (Colbran and Brown, 2004), reflecting the similarity in function of
these subunits. (Figure 2). In its inactive state, CaMKII forms tightly packed autoinhibited assemblies
(Rosenberg et al., 2005). The autoinhibitory subdomain forms a number of interactions through its
pseudosubstrate region with the catalytic domain (Brickey et al., 1994; Yang and Schulman, 1999).
These interactions block the ATP, substrate and CaM binding sites (Mukherji and Soderling, 1995). In
the inactive state, Thr286 (Thr287 in CaMKIIβ), binds to a hydrophobic pocket (T site, Val208 and
Trp237) close to the substrate-binding site (S site) (Giese et al., 1998), orienting the pseudosubstrate
region for its interaction with the catalytic site (Merrill et al., 2005). In this state, the catalytic domain
7
becomes blocked by a coiled-coil strut formed by the calmodulin-responsive regulatory segments
(Rosenberg et al., 2005).
Figure 2. Functional domains of CaMKIIα . Numbers denote the amino acid number of CaMKIIα protein sequence. P = phosphorylation site. (Colbran, 2004; Hudmon and Schulman, 2002a; Merrill et al., 2005; Rosenberg et al., 2006)
Binding of Ca2+/CaM to neighbouring subunits disrupts the autoinhibitory interactions and
initiates intersubunit phosphorylation (Giese et al., 1998; Griffith, 2004; Meador et al., 1993; O'Leary et
al., 2006a; Smith et al., 1992; Thiel et al., 1988; Yang and Schulman, 1999). Ca2+/CaM displaces
Thr286, which is immediately upstream of the pseudosubstrate region, and allows neighbouring subunits
to phosphorylate each other on Thr286, converting the holoenzyme into cluster of loosely tethered and
independent kinase domains. This intersubunit autophosphorylation maintains the kinase in autonomous
active state even following removal of Ca2+/CaM when intracellular Ca2+ concentrations drop to basal
levels, forming a molecular memory of synapse activity (Colbran and Brown, 2004; Hudmon and
Schulman, 2002b; Merrill et al., 2005). CaMKII is not only regulated through the interaction of its
domains and activation by Thr286 phosphorylation, but also by other regulatory autophosphorylation
sites (T253, T305, T306 and S314), which provide further fine tuning of its response (Colbran, 2004)
(Migues et al., 2006).
8
2.2. Fine tuning of CaMKII activity
A series of mechanisms renders the kinase sensitive to the duration, magnitude and frequency of
incoming calcium signals (De Koninck and Schulman, 1998). Upon activation, autophosphorylation
induces a local conformational change that allows formation of additional, stabilizing interactions
between Ca2+/CaM and CaMKII, resulting in a 1000 fold increase in CaMKII affinity for Ca2+/CaM and
in Ca2+/CaM “trapping” (Braun and Schulman, 1995; Griffith, 2004; Rich and Schulman, 1998; Risch,
1990; Saitoh et al., 1987; Waxham et al., 1998). Different degrees of autophosphorylation of the 12 (or
14) subunits in the holoenzyme enables CaMKII to translate Ca2+ spike frequency into finely graded
kinase activity (De Koninck and Schulman, 1998).
The activation of CaMKII increases the targeting and binding of CaMKII to its site of action, the
postsynaptic density (PSD) (Shen and Meyer, 1999; Shen et al., 2000; Strack et al., 1997; Yoshimura
and Yamauchi, 1997). Furthermore, in response to depolarization, CaMKII can undergo
autophosphorylation at Thr253, which enhances its binding to PSDs (Migues et al., 2006). Other
interactions also prolong the autonomous activity of CaMKII, such as binding to the NR2B subunit of
the NMDA-R. This not only increases the binding affinity of Ca2+/CaM to CaMKII, but also allows
CaMKII to retain its autonomous activity due to the interaction with NR2B rather than to the bound
Ca2+/CaM. This interaction suppresses inhibitory autophosphorylation at Thr305/306, which would
promote dissociation of CaMKII from the PSD (Bayer et al., 2001). At the PSD CaMKII interacts with
more than 30 protein targets (Fink and Meyer, 2002; Yoshimura et al., 2000; Yoshimura et al., 2002).
Dephosphorylation of Thr286 by protein phosphatase 1 (PP1), as well as autophosphorylation of
the inhibitory sites on Thr305, Thr306 and Ser314 in response to Ca2+/CaM dissociation, is critical for
terminating the PSD-localized state of CaMKII (Fink and Meyer, 2002; Shen and Meyer, 1999). This
prevents rebinding of Ca2+/CaM and produces inhibited kinase that, paradoxically, requires phosphatase
to regain its ability to be stimulated by Ca2+/CaM (Colbran and Soderling, 1990; Griffith, 2004;
Mukherji et al., 1994; Patton et al., 1990). PP1, in turn, is inhibited by inhibitor-1 and phosphorylated by
cAMP-dependent protein kinase A (PKA) (Blitzer et al., 1998; Brown et al., 2000). Soluble CaMKII,
such as CaMKIIα homomers, is dephosphorylated by cytosolic protein phosphatase 2A (PP2A) (Fink
and Meyer, 2002; Lisman and Zhabotinsky, 2001; Strack et al., 1997; Yoshimura et al., 1999). CaMKII
is also inactivated by CaMKII inhibitors α and β (CaMKIINα, β).
9
2.3. Subunit composition and holoenzyme characteristics.
The subunit composition of CaMKII holoenzymes is very significant. It is controlled by the
relative isoform expression levels (Hudmon and Schulman, 2002a, b). Because intersubunit
phosphorylation at Thr286/287 involves two neighbouring subunits, each acting as a substrate and a
kinase, their kinetic properties become important variables in the reaction. The frequency dependence
for activation of homomers of α or β isoforms is markedly different (De Koninck and Schulman, 1998).
Hence the ratio of α:β and the type of splice variants of α and β present in a heteromer modulates its
kinetic properties and requirements for autophosphorylation. At 20 nM calmodulin, for example, twice
as many subunits are autophosphorylated in heteromeric kinase with an α:β ratio of 2:1 than in a
homomer (Brocke et al., 1999). The subunits show significant differences in their CaM binding affinity
(γ> β > δ > α), CaM dependence for autophosphorylation ( β> γ> δ> α), and substrate phosphorylation
(Gaertner et al., 2004). The type of a neighbour in a holoenzyme modulates the properties of individual
subunits themselves (Brocke et al., 1999). In the forebrain, the calmodulin dependence of β subunit
autophosphorylation is shifted toward the lower sensitivity of the α subunit. This is likely the result of
high α subunit content in the holoenzyme since most of the neighbours of the β subunits, which
determine the conditions of their phosphorylation in these heteromers, are the low affinity α subunits
(half-maximal autophosphorylation of α is achieved at 130 nM calmodulin, while that for β occurs at 15
nM calmodulin) (Brocke et al., 1999). Hence CaMKIIβ has better sensitivity for low level signals
(Thiagarajan et al., 2002) and an increase in β will result in holoenzyme with higher affinity for
Ca2+/CaM and responsiveness to weaker Ca2+ signals.
The ratio of α and β is regulated by neuronal activity, tuning CaMKII to changes in Ca2+ signal
levels (Thiagarajan et al., 2002). CaMKIIα preferentially responds to NMDA-R activity and CaMKIIβ
to the activation of the AMPA-R. CaMKIIα levels increase in response to the activation of NMDA-Rs,
whereas CaMKIIβ levels increase in response to AMPA-R blockade, with CaMKIIβ levels being
insensitive to NMDA-R activity (Thiagarajan et al., 2002). Additionally, CaMKIIβ regulates the levels
of CaMKIIα, but not vice versa. Increase in β results in decrease in α, but α levels have no effect on β
(Thiagarajan et al., 2002). In turn, the changing ratio of α:β significantly influences synaptic
transmission, an increase in β lowers synaptic charge and increases signaling frequency (Thiagarajan et
al., 2002).
10
2.4. CaMKII splicing and expression.
The splicing of each CaMKII isozyme strongly affects the kinetic properties of its activation,
deactivation, substrate specificity and CaMKII localization (Wang et al., 2000). CaMKII splice variant
expression is strongly tissue type and developmentally controlled and undergoes a transition in
expression from undifferentiated to differentiated tissues (Tombes et al., 2003). (Figure 3.) These
concerted influences are responsible for the unique ability of CaMKII to interpret frequency and
amplitude of Ca2+ signals, as well as the signaling history of a synapse, and convert these to cell type-
specific response (De Koninck and Schulman, 1998; Dosemeci and Albers, 1996; Hanson et al., 1994).
With at least eight splice variants, CaMKIIβ has a much greater spectrum of response types than
CaMKIIα. The pattern of alternative splicing of CaMKIIβ is under tight control and depends on tissue
type, developmental status, as well as physiological and pathological conditions (Srinivasan et al., 1994;
Wang et al., 2000). It differs even among individual hippocampal neurons (Bayer et al., 2002) with most
neurons expressing specifically only one type of splice variant (Brocke et al., 1999). In general
CaMKIIβ has much the same catalytic capacity as CaMKIIα, but it also has the unique ability to bind F-
actin, where it docks the CaMKIIα/β heterooligomers. It can, therefore, partially rescue CaMKIIα
knockouts. Hence CaMKIIβ, but not CaMKIIα, knockouts are embryonic lethal (Karls et al., 1992).
CaMKIIα can partially escape co-assembly with CaMKIIβ through local translation of its
mRNA within dendritic spines (Burgin et al., 1990; Mayford et al., 1996b). Depolarization leads to
movement of CaMKIIα mRNA granules into the dendrite (Rook et al., 2000) and to an activity-
dependent translation of its mRNA (Ouyang et al., 1999) initiated by phosphorylation of cytoplasmic
polyadenylation element binding protein (CPEB) by CaMKII (Colbran and Brown, 2004; Huang et al.,
2002; Huang et al., 2003; Wu et al., 1998). In dendritic spines CaMKIIα binds to actin filaments and
governs both the formation of dendritic spines during development and their structural plasticity in
mature synapses (Fischer et al., 2000; Matus et al., 1982; Shi and Ethell, 2006). Dendritic spines, which
appear as small protrusions from dendrites, harbor the great majority of excitatory synapses and contain
both AMPA and NMDA receptors (Harris, 1999; Hering and Sheng, 2001; Sheng, 2001). In
hippocampal neurons, such local translation provides means of rapidly altering the protein composition
in the spines in a highly spatially restricted manner (Schuman et al., 2006; Steward and Levy, 1982).
The morphogenic ability of CaMKIIβ to induce dendritic arborization and increase synaptic
density is thought to be mediated by its specific binding to F-actin (Fink et al., 2003; O'Leary et al.,
2006a; Shen et al., 1998). CaMKIIβ assembles into large complexes with F-actin, including bundles of
11
parallel actin filaments and associates with the actin cytoskeleton. CaMKIIβ crosslinks parallel F-actin
filaments into bundles, dissociates upon stimulation and likely plays a role in Ca2+-regulated actin
dynamics (O'Leary et al., 2006a). Overexpression of CaMKIIβ leads to lower actin turnover and
inhibition of growth (Okamoto et al., 2007). CaMKII phosphorylates the microtubule-associated protein
2 (MAP2) and regulates microtubule-microfilament interaction (Hely et al., 2001), inducing microtubule
disassembly, stabilizing the dendritic arbor (Fink and Meyer, 2002; Wu and Cline, 1998; Yamauchi and
Fujisawa, 1983).
Upon activation CaMKIIα holoenzymes quickly translocate to the PSD of excitatory synapses,
whereas CaMKIIα/β holoenzymes are bound to F-actin through CaMKIIβ and dissociate at a much
slower rate before translocation to the PSD (Fink and Meyer, 2002; Shen and Meyer, 1999). The
translocation time of CaMKIIα/β holoenzymes to the postsynaptic density, a key variable in neuronal
function, is determined by the tightly controlled ratio of CaMKIIα to CaMKIIβ isoforms (Shen and
Meyer, 1999; Srinivasan et al., 1994). The translocation time of CaMKIIα/β hetrooligomers differs
significantly depending on their α:β composition, hence alteration in the α:β ratio results in serious
consequences, as seen in heterozygous CaMKIIα knockouts.
The PSD is a complex macromolecular protein network containing many known CaMKII
substrates and downstream signaling partners. PSD95, AMPA-Rs, NMDA-Rs, synGAP and Ras (Hering
et al., 2003; Suzuki et al., 2001), densin-180 (Strack et al., 2000; Walikonis et al., 2001), α-actinin
(Dhavan et al., 2002; Walikonis et al., 2001) and cyclin-dependent protein kinase 5 (CDK5) (Dhavan et
al., 2002). CaMKII plays a large role in clustering of synaptic proteins via protein-protein interactions in
dendritic spines (Robison et al., 2005), acting not only as a kinase, but also as a structural element of the
PSD. In turn, both NR2B and densin-180 interact with other PSD proteins (Ohtakara et al., 2002; Sheng,
2001) This allows the CaMKII complex to form and stabilize the synaptic localization of CaMKII-
multiprotein synaptic signaling complex (Robison et al., 2005). Once at the postsynaptic sites, CaMKII
also controls the clustering and synaptic targeting of the NMDA receptor subunit (Gardoni et al., 2003).
Phosphorylation of SAP97 by CaMKII drives SAP97 to the PSD and by promoting postsynaptic
clustering of SAP97 and GluR1, and preventing SAP97 binding to NR2A (Gardoni et al., 2003; Mauceri
et al., 2004), CaMKII provides a mechanism for the regulation of synaptic targeting of AMPA and
NMDA receptors (Gardoni et al., 2003). In addition, CaMKII-dependent phosphorylation of PSD-95
regulates the interaction of PSD-95 with the NR2A and NR2B subunits of the NMDA receptor (Gardoni
et al., 2006).
12
CaMKII is also targeted to PSD by a non-NMDA mechanism. For example, activation of PKC
interferes with CaMKII-NMDA-R interaction, dispersing the synaptic NMDA-Rs, but driving CaMKII
to synapses (Fong et al., 2002). Targeting CaMKII to active synapses ensures potentiation of their
postsynaptic response (Merrill et al., 2005). CaMKII clustering at activated synapses results in
phosphorylation of CaMKII targets (Barria et al., 1997). It increase in the ion channel activity of the
AMPA-Rs (Derkach et al., 1999) and mediates postsynaptic AMPA-R accumulation (Derkach et al.,
1999; Rongo and Kaplan, 1999), thereby contributing to LTP. Transgenic mice in which targeting of
CaMKII has been disrupted display behavioral defects (Elgersma et al., 2002; Miller et al., 2002). In
hippocampus, LTP involves not only postsynaptic receptor insertion, but also presynaptic protein
kinases, including presynaptic mechanisms of potentiation through CaMKII (Lu and Hawkins, 2006;
Zhai et al., 2001). Postsynaptic activation of CaMKII induces structural remodeling of presynaptic
inputs and the retention of active presynaptic partners (Pratt et al., 2003).
Presynaptic CaMKII binds to and phosphorylates synapsin I (Benfenati et al., 1992) and syntaxin
1A, promoting synaptic vesicle exocytosis and neurotransmitter release (Ohyama et al., 2002) (Nomura
et al., 2003; Ohyama et al., 2002). Phosphorylation of CaMKII plays an important role in the conversion
of preexisting but silent presynaptic terminals to functional ones during glutamate-induced plasticity
(Antonova et al., 2001; Colbran, 2004; Du et al., 2004).
2.5. Developmental expression
CaMKII is a major determinant of neuronal growth and synaptogenesis (Zou and Cline, 1996).
Given that its splice variants have very specific characteristics and that holoenzymes with even a small
variation in the ratio of the α and β subunits will respond differently to Ca2+ signals, expression of
CaMKII must be under strict control. CaMKII controls important neuronal developmental milestones
(Zou and Cline, 1996) (Hanson and Schulman, 1992). In early development, it is a master regulator
whose activity drives and possibly coordinates several events of egg activation, including recruitment of
maternal mRNA (Ducibella et al., 2006). Later on in development it is essential for neuronal growth,
branching, synaptic density and maturation (Borodinsky et al., 2002; Rongo and Kaplan, 1999; Wu and
Cline, 1998).
Full length CaMKIIβ is found throughout the adult brain, but is particularly enriched in the
cerebellum (McGuinness et al., 1985; Miller and Kennedy, 1985; Takeuchi et al., 2002). Other splice
variants, such as β3 and βM, are expressed in the rodent endocrine and muscle tissue, with β’ (β1), βe
13
(β4) and β2 found in the adrenal, pituitary and in pancreatic β cells (Bayer et al., 1998; Bayer et al.,
2002; Breen and Ashcroft, 1997; Gloyn and Ashcroft, 2001; Rochlitz et al., 2000; Urquidi and Ashcroft,
1995). A number of isozymes, in particular β’, βe, and βe’ are embryonic (Brocke et al., 1995;
Urushihara and Yamauchi, 2001). During embryonic development the CaMKIIβe and CaMKIIβe’
replace the function of both CaMKIIβ and CaMKIIα, which have a later onset of expression. The
embryonic forms show reduced Ca2+/CaM affinity (Brickey et al., 1994; Brocke et al., 1999) and are
cytoplasmic (O'Leary et al., 2006a), resembling more closely the characteristics of CaMKIIα. In order to
mediate the necessary morphological changes of early postnatal brain development, CaMKIIβe
predominates because it promotes motility of filopodia and neuritic branching, leading to enhanced
arborization, as shown in developing hippocampal neurons.
I II III IV V VI VII X
α Brain, enriched in FC
αB Striatum, midbrain
αKAP Cardiac, skeletal muscle
β Brain, enriched in cerebellum
β', β1 Brain, endocrine tissue
βe, β4 Embryonic, endocrine tissue
β'e Embryonic
βM X3 Endocrine, muscle membrane
β3 X2 Pancreatic β-cell
β2 Endocrine
Figure 3. Tissue specificity of variable domain utilization. (Tombes et al., 2003). The four genes of CaMKII are highly homologous and draw upon a similar pool of variable domains, but not all domains are present in all CaMKII genes. CaMKIIα and CaMKIIβ utilize different variable domains to generate an array of splice variants with specific tissue expression patterns.
CaMKIIα is expressed later in development because of its ability to stabilize the already developed
dendritic arbor structures (Colbran and Brown, 2004; Fink et al., 2003; Wu and Cline, 1998). The ratio
of α:β in the forebrain changes during maturation, from 1:1 in a 10 day old mice to 3:1 in the adult.
14
(McGuinness et al., 1985; Miller and Kennedy, 1985). Once CaMKIIα is expressed after birth, its levels
increase, correlating with the start of dendritic maturation and increased synapse formation. Its
expression seems to be limited to cells that are no longer dividing (Bayer et al., 1999; Menegon et al.,
2002). CaMKII controls one of the main milestones of neuronal maturation, the switch from LTD to
LTP. This developmental loss of capacity for LTD depends on age-dependent decrease in Ca2+-
independent activity of CaMKII. Furthermore, CaMKII plays an important role in the development of
central glutamatergic synapses, as well as in determining their density (Rongo and Kaplan, 1999; Wu
and Cline, 1998). Transgenic animals, in which the CaMKII kinase is 20 to 30x more active in the
absence of Ca2+ than the wild type enzyme, do not lose the capacity for LTD in adulthood (Dudek and
Bear, 1992; Mayford et al., 1995).
Mice deficient in CaMKIIα show neurodevelopmental defects severely affecting about 50% of
the animals (Gordon et al., 1996), these include disturbances in behaviour and cognition (Chen et al.,
1994; Mayford et al., 1996a; Silva et al., 1992a; Silva et al., 1992b). Recently, phosphorylation of
Thr305 on CaMKII was demonstrated to be increased in Angelman mental retardation syndrome
(Weeber et al., 2003). This phosphorylation results in the inactivation of the kinase and in the reduction
of CaMKII presence at the PSD (Albrecht et al., 1997; Weeber et al., 2003).
2.6. Synaptic plasticity and memory formation
Changes in synaptic strength can occur by presynaptic mechanisms such as neurotransmitter
release or postsynaptically, through LTP and LTD. The initiation of postsynaptic LTP or LTD requires
calcium entry through the NMDA-Rs which activate CaMKII (Choi et al., 2000; Renger et al., 2001;
Zakharenko et al., 2001). CaMKII triggers the synaptic insertion of AMPA-Rs in the case of LTP
(Hayashi et al., 2000), or the removal of AMPA-Rs in the case of LTD (Beattie et al., 2000). For a
review see (Malinow and Malenka, 2002).
NMDA-Rs form tetrameric complexes assembled from two obligatory NR1 and two modulatory
NR2 subunits. The different NR2 subunits (NR2A-NR2D) possess distinct gating and pharmacological
properties. In adult hippocampus and neocortex, NR2A and NR2B are the predominant NR2 subunits
(Cull-Candy et al., 2001). CaMKII is part of the NMDA-R-containing macromolecular complex. It
binds to the NR2A and NR2B subunits of the NMDA-R (Leonard et al., 1999; Strack and Colbran,
1998), albeit with a lower affinity for NR2A than NR2B (Leonard et al., 1999). CaMKII is also known
to bind the NR1 (Leonard et al., 1999; Omkumar et al., 1996). Binding to NR2B in turn increases the
15
binding affinity of CaMKII for Ca2+/CaM, which prolongs the autonomous activity of CaMKII and it
also suppresses inhibitory autophosphorylation, further lengthening its presence at the PSD (Ehlers et
al., 1996). In turn, CaMKII Phosphorylates NR2B (Strack et al., 2000) and NR2A, positively regulating
NMDA-R currents (Liao et al., 2001; Lieberman and Mody, 1994; Rostas et al., 1996; Wang and Salter,
1994), increasing the NMDA-R conductance and modulating synaptic strength (Gardoni et al., 1999;
Leonard et al., 1999; Omkumar et al., 1996; Shen and Meyer, 1999).
During puberty NMDA receptors mature, the NR2B subunits are replaced with NR2A, for which
CaMKII has lower affinity, decreasing the formation of LTP (Barria and Malinow, 2005) This NMDA
receptor maturation process coincides with the development of dopaminergic innervation in the
prefrontal cortex (Goldman-Rakic, 1996) when key corticolimbic afferent systems projecting into the
hippocampal formation are actively maturing (Benes, 2000). It also coincides with the age of onset for
schizophrenia (Keshavan, 1999). Activation of CaMKII is essential for normal NMDA-R mediated
formation of LTP in the hippocampus and its associated functions such as spatial learning, memory
(Fink and Meyer, 2002; Lisman et al., 2002) and plasticity of the central nervous system (Fang et al.,
2002; Garry et al., 2003; Hardingham et al., 2003). Reviewed by (Colbran and Brown, 2004). The
involvement of CaMKII is critical, since synaptic plasticity is blocked by both NMDA antagonists
(Engert and Bonhoeffer, 1999) and by CaMKII inhibitors (Soderling, 2000). Depending on a
developmental stage and type of signal, activation of NMDA receptors and subsequent activation of
CaMKII can cause rapid elongation of filopodia (Maletic-Savatic et al., 1999), formation of novel
dendritic spines (Engert and Bonhoeffer, 1999), or shrinkage of existing ones, resulting in neuron
pruning (Segal et al., 2000).
During neurodevelopment, newly formed synapses are silent unless sufficient depolarization by
NMDA-Rs is provided by coincident activity that could activate postsynaptic CaMKII, resulting in the
appearance of AMPA responses (Ben-Ari et al., 1997; Fukunaga and Miyamoto, 1999; Soderling, 1993;
Wu et al., 1996). This response is mediated by CaMKII phosphorylation of AMPA receptors and
resulting membrane insertion of previously silent AMPA receptors (Ben-Ari et al., 1997; Swope et al.,
1999; Wu et al., 1996). Although NMDA receptors are essential in LTP initiation, expression of LTP is
mediated primarily by AMPA receptors (Soderling, 2000; Soderling and Derkach, 2000). (For review
see (Malinow and Malenka, 2002)). CaMKII phosphorylates GluR1 (Barria et al., 1997; Derkach et al.,
1999; Fukunaga et al., 1996; Roche et al., 1996; Vinade and Dosemeci, 2000; Yakel et al., 1995) and
GluR4 subunits of AMPA receptors (Carvalho et al., 1999; Vinade and Dosemeci, 2000). By
16
phosphorylating the AMPA-Rs, CaMKII mediates plasticity at glutamatergic synapses by increasing
single-channel conductance of existing functional AMPA-Rs, stabilizing their higher conductance state
(Vinade and Dosemeci, 2000) and recruiting new high-conductance-state AMPA-Rs to the PSD
membrane and by modulating the NMDA-R response (Derkach et al., 1999; Leonard et al., 1999;
Omkumar et al., 1996). GluR1 expression can be influenced by a number of pathways. In mature
neurons, NR2A promotes, whereas NR2B inhibits GluR1 surface expression (Kim et al., 2005). GluR1
trafficking can also be increased by protein kinase A (PKA) phosphorylation of GluR1 (Esteban et al.,
2003).
Because CaMKII modulates both the NMDA and GABA systems, it plays an important role in
neonatal neurodevelopment. Developing neurons require certain degree of membrane depolarization
with a consequent rise in intracellular calcium to stimulate neurite outgrowth. The GABAergic network,
which develops prior to the glutamatergic one, appears to provide this depolarization (Ben-Ari et al.,
1994). Paradoxically, GABA, the principal inhibitory neurotransmitter in the adult CNS, plays the role
of main neurotransmitter of excitation in the embryonic and neonatal hippocampus (Leinekugel et al.,
1999; Swope et al., 1999). This membrane depolarization is sufficient to remove the voltage-dependent
Mg2+ block of NMDA receptors (Ben-Ari, 2002; Leinekugel et al., 1999; Lu et al., 1999; Stein et al.,
2004) and increase intracellular calcium concentrations, a stimulus necessary for the initial development
of AMPA responses (Ben-Ari et al., 1997; Fukunaga and Miyamoto, 1999; Soderling, 1993; Wu et al.,
1996).
In mature neurons, CaMKII phosphorylates the GABAA receptors (Macdonald and Olsen, 1994),
increasing their substrate binding (Churn and DeLorenzo, 1998) as well as their currents (Wang et al.,
1995), contributing to the enhancement of inhibitory synaptic transmission (Houston and Smart, 2006;
Wang et al., 1995). CaMKII phosphorylates MeCP2 and hence influences dendritic patterning, spine
morphogenesis and the induction of BDNF transcription (Klose and Bird, 2006; Lewis et al., 1992;
Zhou et al., 2006). Mutations in MeCP2 result in defects in dendritic branching, spine density, synaptic
plasticity, learning and memory, and the maturation of inhibitory circuits and leads to Rett Syndrome
(RTT) (Amir et al., 1999; Katz and Shatz, 1996; Zhou et al., 2006).
2.7. Sensitization
Endogenous amphetamine-like compounds, such as β-phenethylamine, are produced by the brain
(Inwang et al., 1973). These compounds modulate affective behaviours such as excitement and alertness
17
and have been shown to be decreased in depression (Sabelli and Mosnaim, 1974). As compared to
amphetamine, these compounds are metabolized quickly and do not appear to be stored (Berry, 2004)
because of the absence of the α-methyl group which inhibits monoamine oxidase (MAO) (Sulzer et al.,
2005). Like amphetamine, β-phenethylamine releases dopamine (Bergman et al., 2001), but unlike
amphetamine it is metabolized quickly. Psychostimulants such as amphetamine and cocaine can elicit
psychotic symptoms in normal subjects and worsen symptoms in patients with schizophrenia
(Lieberman et al., 1997). It is, therefore, likely that the pathway involved in psychostimulant signaling is
abnormal in schizophrenia and this provides a rare and important clue to the etiology of this elusive
disease. Many of the effects of psychomotor stimulants like amphetamine and cocaine seem to be
mediated by similar pathways (Robinson and Berridge, 1993; Wise and Bozarth, 1987). Repeated
treatment of rats with AMPH or cocaine leads to sensitization and an enhanced release of DA from areas
such as the striatum and nucleus accumbens in response to stimuli (Castaneda et al., 1988; Kantor et al.,
1999; Pierce and Kalivas, 1997; Rawls and McGinty, 2000; Robinson and Becker, 1982; Robinson et
al., 1985).
Amphetamine sensitization is mediated by CaMKII, activation of which is essential for the
enhanced dopamine release in sensitized animals (Jones et al., 1998; Raiteri et al., 1979; Warburton et
al., 1996). The enhanced dopamine release component specific to sensitized animals is independent of
dopamine vesicular storage and dependent on Ca2+ and is transporter-mediated. This is different from
depolarization-mediated dopamine release upon acute administration of amphetamine, which is
cytosolic, vesicular and independent of Ca2+ (Jones et al., 1998; Kantor et al., 1999; Pierce and Kalivas,
1997; Raiteri et al., 1979). Both amphetamine sensitization and later enhanced amphetamine release in
sensitized animals depend on CaMKII. Inhibition of hippocampal CaMKII impairs amphetamine
conditioning in rats (Tan, 2002) and attenuates dopamine release in amphetamine sensitized rats (Kantor
et al., 1999; Pierce and Kalivas, 1997). One of the key components of amphetamine treatment is the
development of amphetamine-induced conditioned place preference in the animals, this also is
dependent on CaMKII activation and is abolished by inhibiting CaMKII (Tan, 2002). Furthermore,
phosphorylation of NMDA-Rs by CaMKII-induced stimulation of D1R and D2R contributes to
enhancement in striatal NMDA sensitivity (Oh et al., 1999).
In humans, amphetamine mimics the positive symptoms of schizophrenia, whereas
phencyclidine (PCP) reproduces the schizophrenia-like psychosis such as positive symptoms, negative
symptoms, and cognitive deficits, closely mimicking schizophrenia symptoms (Javitt and Zukin, 1991;
18
Rainey and Crowder, 1975). In rodents, chronic PCP administration activates the mesolimbic dopamine
pathway and impairs prefrontal cortical function (Jentsch and Roth, 1999). Mice treated with PCP
displayed an impairment of latent learning and a decrease of learning-associated phosphorylation of
CaMKII and NR1 in the prefrontal cortex even after drug withdrawal (Mouri et al., 2007). This supports
the involvement of the CaMKII-NMDA-R pathway in schizophrenia.
2.8. Involvement of the D2 receptors
The D2R is one of the key elements implicated in schizophrenia, as all the antipsychotic drugs are
D2R antagonists and show perfect correlation between their effective antipsychotic efficacy dose and
80% D2R occupancy (Creese et al., 1976; Seeman et al., 1976; Seeman, 1987). D2Rs are enriched in
dendritic spine shafts and heads that are contacted by corticostriatal glutamatergic afferents to form
synapses throughout the striatum (Hersch et al., 1995; Yung and Bolam, 2000).
A direct interaction between D2R and NR2B is critical for modulating NMDA-R-mediated
currents and behavioral responsiveness to cocaine (Liu et al., 2006). Acute cocaine administration
results in interaction of D2Rs with the NR2B and disruption of the NR2B-CaMKII binding, weakening
the CaMKII influence over the phosphorylation site on NR2B and leading to reduced phosphorylation
(Liu et al., 2006). D2Rs are also able to activate the nuclear form of CaMKII and stimulate gene
expression through Ca2+ signaling (Takeuchi et al., 2002). In turn, upregulation of CaMKII was shown,
together with the ERK pathway, to positively upregulate the D2R promoter (Takeuchi et al., 2002).
3. NOGO
The reticulon family constitutes of four members, RTN1, RTN2, RTN3, and RTN4/Nogo (Table
2), all of which are expressed in neurons, with RTN3 and Nogo being expressed most prominently in
white matter (Lauren et al., 2006). Only Nogo possesses the unique ability of inhibiting axonal
regeneration (GrandPre et al., 2000). For review see (Oertle and Schwab, 2003). The representative
feature of RTN proteins is their association with membranes of the endoplasmic reticulum (ER) (van de
Velde et al., 1994) and a conserved carboxy terminal (C terminal) region of approximately 200 amino
acid (aa), called the reticulon homology domain (RHD, Pfam PF02453, http://www.sanger.ac.uk/cgi-
bin/Pfam/getacc?PF02453). The RHD consists of two transmembrane segments, separated by a domain
of approximately 66 aa, involved in protein-protein interactions (Oertle et al., 2003a; Oertle et al.,
2003b; Yan et al., 2006).
19
Nogo was first identified through research in spinal cord regeneration as a neural growth
inhibitor produced by oligodendrocytes and associated with myelin (Chen et al., 2000b; GrandPre et al.,
2000; Prinjha et al., 2000; Spillmann et al., 1998). In 1998 it was also identified by our lab, as an
initially unknown gene, Band 9, elevated in the frontal cortex of schizophrenia patients (Novak et al.,
2002). Together with myelin associated glycoprotein (MAG) (Barton et al., 1987) and oligodendrocyte
myelin glycoprotein (OMgp, arretin) (Kottis et al., 2002), it belongs to a family of myelin associated
neural growth inhibitors, or myelin associated inhibitors (MAIs) (McKerracher and Winton, 2002). The
gene for Nogo has been mapped to chromosome position 2p13-14 (Yang et al., 2000) and the gene for
its receptor, RTN4-R/NgR1, is located on chromosome 22q11 (Fournier et al., 2001). It is of interest
that both the Nogo loci and the NgR1 loci are within important regions linked to schizophrenia, 2p15-
p12 (Coon et al., 1998; Shaw et al., 1998; Straub et al., 2002) and 22q11 (Bassett et al., 1998;
Karayiorgou et al., 1995), respectively. Three mutant NgR1 alleles were detected in schizophrenia
patients, two of which were in neuroleptic resistant patients (Sinibaldi et al., 2004).
Alternative naming, location Reference
RTN1 NSP, s-Rex, 14q21-q22 (Kools et al., 1994; Ninkina et al., 1997)
RTN2 NSPL1, 19q13.3 (Geisler et al., 1998; Roebroek et al., 1998)
RTN3 NSPL2, 11q13 (Moreira et al., 1999)
RTN4
Nogo, ASY, RTN-X, RTN-
xL/xS, NI-220/NI-250, NI-35,
VP20, foocen, KIAA0886,
2p13-14
(Buffo et al., 2000; Chen et al., 2000b; GrandPre et al.,
2000; Morris et al., 1999; Nagase et al., 1998; Prinjha et
al., 2000; Qi et al., 2003; Schwab, 1990; Spillmann et
al., 1998; Tagami et al., 2000).
Table 2. Alternative names and chromosomal location of the reticulon family members.
The Nogo gene consists of 14 exons (Oertle et al., 2003a; Oertle et al., 2003b), which could
theoretically produce many splice variants, but only three main isoforms have been identified, Nogo A,
B and C (Oertle et al., 2003a) (Figures 4 and 5). Generally, diversity is generated by the RTNs through
multiple promoters present in the first large intron, rather than alternative splicing. This strategy
produces isoforms with varied amino terminal (Nt) regions, but with conserved carboxyl terminal (Ct)
region containing the RHD (Oertle et al., 2003a; Oertle et al., 2003b; Roebroek et al., 1996; Yan et al.,
20
2006). The (Nt) variation produces tissue-specific isoforms. (Yan et al., 2006). In Nogo, the Nogo-A/B
and the Nogo-C isoforms use alternate promoters, P1 and P2, respectively (Oertle et al., 2003a).
There are three isoform specific domains present in human Nogo, an N terminal region
containing sequence common to Nogo-A and B (aa 1-185), a region unique to Nogo A (amino-Nogo-A,
aa 186-1004) and an C terminal region common to all three splice variants and which contains the RHD
sequence (aa 1005-1192, also known as Nogo66, Pfam PF02453) (Figure 5) (Oertle et al., 2003c).
(Although the N terminal 11 aa’s unique to Nogo C may be classified as another possible functional
region.)
Figure 4. The RTN4/Nogo gene. The alternative use of nine exons (1A - 9) and two promoters (P1 and P2) (A.) gives rise to three brain-expressed splice variants of Nogo (B.) (Oertle et al., 2003a).
The first Nogo exon, 1A, contains the 5’UTR, and the first N-terminal 172 aa’s common to
Nogo-A and Nogo-B (Oertle et al., 2003a). This Nogo-A/B region is rich in proline, it is similar to SH3
ligands and contains a negatively charged cluster which may function as a low-affinity binding site for
Ca2+ (Oertle et al., 2003a). This section is involved in the inhibition of fibroblast spreading and shows a
general inhibitory activity (Fournier et al., 2001) but does not significantly inhibit neural outgrowth
A.
P1 P21Aa 1A 1D 1E 1F 1G 2 3 1C 4 5 6 7 8 9
Promoters and exons only used in testis
Exons used in brain splice variants
B.
P11A 2 3 4 5 6 7 8 9
Nogo - A
P1
1A 4 5 6 7 8 9
Nogo - B
P2
1C 4 5 6 7 8 9
Nogo - C
21
(Oertle et al., 2003c). The next set of exons, 1Aa, 1Ab, 1D, 1E, 1F, 1G and 2, form minor splice variants
only detected in testis (Oertle et al., 2003a). Exon 1C is expressed via the P2 promoter and contains the
5’ end unique to Nogo-C, encoding its N-terminal 11 amino acid residues. Exon 3 encodes the Nogo-A-
specific region (amino-Nogo-A), which is unusually long (2400 bp), highly conserved, and contains
consensus sequences for PEST (protein tyrosine phosphatase) domains (Oertle et al., 2003a). The
amino-Nogo-A region (aa 186-1004) has been shown to inhibit both spreading of non-neuronal cells and
of axon growth and mediate the collapse of neuronal growth cones (Chen et al., 2000b; Fournier et al.,
2001; Oertle et al., 2003c). A section of the amino-Nogo-A, aa 567-748, was shown not to bind to
NgR1, but is nonpermissive as a substrate for multiple cell types and inhibits neural outgrowth,
suggesting that there might exist a distinct receptor mediating this amino-Nogo-A inhibition (Oertle et
al., 2003c). The amino-Nogo-A region also contains a non-inhibitory binding site for the Nogo receptor
located at aa 995-1018 (Hu et al., 2005). The combination of NgR1 binding through its primary target,
the Nogo-66 domain, as well as the amino-Nogo domain creates a substantially higher-affinity NgR1
ligand (Hu et al., 2005). Exons 4 through 9 encode the part common to all the Nogo isoforms and the
part of the protein that is homologous to the reticulon family members (Oertle et al., 2003a). The protein
ends with a common carboxyl-terminal of 188 amino acids (aa 1005-1192) shared by all three isoforms
of Nogo, which contains the RHD with its two transmembrane domains (Watari and Yutsudo, 2003) and
a central loop of 66 amino acids (Nogo-66), which was used to identify the Nogo receptor (NgR1)
(Fournier et al., 2001). Nogo-66 has been shown to be extracellular and has an axon growth inhibitory
effect and growth cone collapsing function on its own (Fournier et al., 2001; GrandPre et al., 2002;
Oertle et al., 2003c). It strongly interacts with the NgR1 through its leucine rich repeat (LRR) domain
(Lauren et al., 2006).
Exon 4 encodes the first large hydrophobic domain (aa 1017-1052) of the RHD. This region
shares significant homology (38% identity) with the GABA-receptor first transmembrane domain
(Oertle et al., 2003a). Exons 6 and 7 encode the second putative transmembrane domain (aa 1118 –
1154) with a leucine-zipper-like motif. The transmembrane domains determine the topology of Nogo.
Because of their large size, the transmembrane domains seem to have the ability to span the membrane
once or twice and so direct the topology of the loop to the cytosolic or, as in differentiated
oligodendrocytes, to the extracellular space (Dodd et al., 2005; GrandPre et al., 2000; Oertle et al.,
2003c). In differentiated oligodendrocytes, both Nogo-66 and amino-Nogo can be extracellular (Oertle
et al., 2003c). In the ER, both the N- and C-termini of RTNs face the cytoplasm and all the hydrophilic
22
segments are on the same side of the membrane (GrandPre et al., 2000; Oertle and Schwab, 2003;
Voeltz et al., 2006). The transmembrane domains also play a role in subcellular localization of Nogo,
the disruption of the second transmembrane segment has been shown to affect the localization of RTN4
in the ER (Oertle et al., 2003c). Exons 8 and 9 encode the C terminus of Nogo, with exon 9 encoding the
last 13 aa of Nogo protein and the 3’UTR, which contains a typical di-lysine ER membrane retention
signal and a potential nuclear localization signal (Oertle et al., 2003a).
Figure 5. Functional domains of Nogo/RTN4 (Oertle et al., 2003c).
3.1. The expression of Nogo
The P2 promoter of Nogo-C contains E-box which can bind muscle-specific transcription
factors, which is consistent with the expression of Nogo-C in muscle (Oertle et al., 2003a; Oertle and
Schwab, 2003). A negative control element upstream of open reading frames (uORF) likely functions as
negative regulator of Nogo-C translational level (Kozak, 2000). Increased calcium levels lead to
activation of calcineurin and to dephosphorylation and hence activation of NF-AT (Crabtree, 1999).
aa 1Nogo-A
N Camino-Nogo-A Nogo-66, RHD
Nogo-B
TM1 TM2
Nogo-C
inhibition of fibroblast spreading (aa 57-185) but not neural outgrowth general inhibitory activity
inhibits spreading of non-neuronal cells and of axon growth mediates the collapse of neuronal growth cones aa 995-1018 (Nogo-A-24), a non-inhibitory binding site for NgR1
Reticulon Homology Domain (RHD)Central loop of 66 amino acids (Nogo-66), extracellular, axon growth inhibitory
growth cone collapsing, primary binding region for NgR1TM2 - localization of Nogo in the ER
aa 1192185 1004
23
The three Nogo isoforms (Nogo-A, B and C) show different developmental and morphological
expression patterns. Nogo-A is mainly found in oligodendrocytes, (and it is the only Nogo variant
expressed by these cells), as well as Schwann cells (Pot et al., 2002), neurons in the brain and by myelin
sheath of the CNS (Wang et al., 2002c). In adult it is absent in non-neuronal cells such as astrocytes and
microglia (Josephson et al., 2001; Marklund et al., 2006). It is particularly strongly expressed by
oligodendrocytes during fetal development, when it is also broadly expressed by the CNS, cerebral
cortex, hippocampus pyramidal cell bodies, but not by Schwann cells in the PNS (Buss et al., 2005).
Nogo B is far less abundant, but it is expressed quite ubiquitously throughout the brain, with an
expression pattern analogous to a housekeeping gene (Oertle et al., 2003a). Nogo C is expressed in the
brain, but it is also expressed in skeletal muscle (Geisler et al., 1998). Because expression of these splice
variants is also developmentally controlled, they likely play a role in development as well as tissue
specificity and differentiation (Oertle et al., 2003a). For example, Nogo A is most strongly expressed in
fully differentiated, postmitotic neurons, and again decreases in the cells of aged animals (Al Halabiah et
al., 2005; Trifunovski et al., 2006). Within the cell, all isoforms concentrate in the ER, but low levels of
Nogo A are also present on the surface of oligodendrocytes and fibroblasts (GrandPre et al., 2000) and
Nogo B on endothelial cells (Acevedo et al., 2004; Dodd et al., 2005; GrandPre et al., 2000; Voeltz et
al., 2006). A somewhat unique presence is the localization of Nogo A in the Golgi (GrandPre et al.,
2000).
3.2. Nogo-A in neurodevelopment
Paradoxically, Nogo-A is localized to growth cones of growing axons in the developing nervous
system, hence under some circumstances it likely also plays a role in neuronal growth, rather than
inhibition (Tozaki et al., 2002). In the hippocampus, Nogo-A is expressed early in development
(Mingorance-Le Meur et al., 2007). Nogo-A expression by cortical neurons follows their gradient of
positioning and maturation in the cerebral cortex (Mingorance-Le Meur et al., 2007). In particular, Nogo
plays an important role in directing the migration of early cohorts of tangentially migrating GABA-ergic
neurons from the ganglionic eminence. The expression pattern of Nogo-A changes when these neurons
change from migratory to resting. It is targeted to their leading processes and its absence results in
alterations in the migration pattern, increase in axon branching, early polarization and delay in the
migration of interneurons toward the neocortex (Mingorance-Le Meur et al., 2007). Although low levels
of the N-terminus of Nogo-A are present at the neuronal surface, Nogo-A is primarily associated with
24
intracellular compartments (ER) and cytoskeletal proteins such as microtubules in the central region of
growth cones (Mingorance-Le Meur et al., 2007). As compared to Nogo-A, neuronal NgR1 expression
in the neocortex does not start until late prenatal and early postnatal stages (Josephson et al., 2002;
Mingorance et al., 2004; Wang et al., 2002c), indicating that during most embryonic development the
functions of cell surface Nogo-A are not mediated by NgR1 (Mingorance-Le Meur et al., 2007).
NgR-1 serves as a receptor for all three neural growth inhibitor proteins associated with myelin,
Nogo, MAG and OMgp and activates the Rho signaling pathway (Fournier et al., 2001; Fournier et al.,
2003; Liu et al., 2002; McGee and Strittmatter, 2003; Wang et al., 2002b). NgR-1 also binds other
members of the reticulon family, namely RTN2 and RTN3, but not RTN1 (Lauren et al., 2006).
Furthermore, the most predominant proteins that interact with Nogo-A are Nogo-B and Nogo-C (Dodd
et al., 2005) and this interaction is not limited only to splice forms of the same gene, since Nogo B was
shown to interact with another member of the reticulon family, RTN3, through its RHD domain (Qi et
al., 2003). It is likely that clustering of the receptors and targets may increase the efficiency of signaling,
as NogoA-MAG clustering has already shown to potentiate NgR1 signaling (Niederost et al., 2002).
Nogo acts through a number of signaling pathways, but the Nogo-NgR1 pathway is possibly the
most studied. Nogo binds to the NgR1 receptor, which, because of its lack of transmembrane domain to
elicit signal, requires co-receptors (Woolf and Bloechlinger, 2002). Three membrane proteins, which
form a transmembrane receptor complex with NgR1, have been identified as its co-receptors: p75NTR (a
low-affinity neurotrophic factor) (Dechant and Barde, 2002; Hasegawa et al., 2004), a membrane
protein LINGO-1 (leucine-rich repeat and immunoglobulin domain-containing, Nogo receptor
interacting protein) (Mi et al., 2004); and TROY (also known as TAJ, TNF receptor superfamily,
expressed in CNS) (Park et al., 2005; Shao et al., 2005; Wang et al., 2002a; Wong et al., 2002). The
localization of the receptors and their co-receptors adds another layer of spatial control, determining
which signaling pathway will be activated (Ding et al., 2001).
3.3. The Gi protein – PKC / IP3 pathways
The Nogo-NgR1- p75NTR complex also activates two Rho independent, closely connected,
pathways, balance of which determines whether the signal will result in neural growth or inhibition
(Hasegawa et al., 2004). By activating the inhibitory guanine nucleotide binding protein (Gi) (Cai et al.,
1999), Nogo triggers the intracellular elevation of Ca2+ as well as the activation of PKC (Hasegawa et
al., 2004). The Gi protein activates PLC (α, β and β2) (Hasegawa et al., 2004), which in turn mediates
25
PIP2 conversion to IP3 and DAG (Berridge, 1998). Binding of IP3 to the IP3 receptor results in
intracellular elevation of Ca2+, while DAG activates PKC. The PKC pathway mediates growth cone
collapse, while the IP3 pathway mediates growth cone extension (Hasegawa et al., 2004). Thus, it is the
balance between PKC and IP3 which is important for the regulation of axon regeneration or inhibition by
Nogo (Hasegawa et al., 2004). Inhibition of PKC results in neurite extension, while inhibition of IP3
receptor enhances neurite retraction (Hasegawa et al., 2004). Dysregulation of these pathways was
shown to be associated with disease or aberrant neural functioning. High levels of PKC in the prefrontal
cortex induced by stress impair working memory and disrupt prefrontal cortical regulation of behaviour
and thought, resulting in impaired judgment, impulsivity and thought disorder (Birnbaum et al., 2004).
This may be of particular importance when considering the effects of Nogo upregulation in
schizophrenia, as increased Nogo signaling may result in similar dysfunction and contribute to the
disease phenotype. Nogo-NgR1 pathways converge to impart the inhibition of actin cytoskeleton
reorganization and increase the stabilization of formed F-actin, which results in stress fibre formation
and neurite retraction (Maekawa et al., 1999). These pathways have been identified as being involved in
axon pathfinding in development (Lee et al., 1994; McQuillen et al., 2002).
3.4. Other pathways involved in Nogo signaling
NgR1 is not always the receptor responsible for mediating Nogo signaling. Although NgR1 is
highly expressed in gray matter where Nogo A is expressed by oligodendrocytes, it is not expressed in
white matter where Nogo is also present (Fournier et al., 2001). Furthermore, in addition to not always
being expressed at the same time or in the same location as NgR1 and Nogo do not always play a neural
growth inhibitory role (Al Halabiah et al., 2005). The reticulons seem to play an important function in
microtubule stability, especially in the ER. Reticulons primarily localize to the tubular ER and interact
with DP1 (Deleted in Polyposis Locus Protein 1)/ Yop1p (S. cerevisiae homologue of DP1), a conserved
integral membrane protein of the peripheral ER. The unique topology of the large transmembrane
domains of Nogo and its interaction with DP1 may help control membrane curvature in the
endomembrane system (Voeltz et al., 2006). Furthermore, the formation of ER tubular network requires
Nogo A (Voeltz et al., 2006) and Nogo-A has been found to co-immunoprecipitate with α-tubulin,
which also suggests that it is involved in microtubule stabilization (Taketomi et al., 2002).
There is evidence of Nogo interacting with multiple signaling and metabolic pathways. Nogo-B
and Nogo-C proteins interact with β-secretase (BACE1, for β-site APP cleaving enzyme / Asp2 /
26
memapsin 2) (He et al., 2004; Murayama et al., 2006; Yan et al., 2006). The β-amyloid peptide (Aβ) is
released from a large amyloid precursor protein (APP) through sequential cleavage by two
endopeptidases: β-secretase and γ-secretase. The accumulation of Aβ is known to cause the formation of
amyloid plaques in Alzheimer’s disease which lead to damage and neuronal death in human brain
(Hardy and Selkoe, 2002). Increased interaction in the ER compartment between RTNs via their C-
terminal RHD and BACE1 sequesters BACE1 from accessing the APP substrate and reduces the
conversion of APP to Aβ (He et al., 2004; Yan et al., 2006).
Nogo-B (ASY, RTN-Xs) has been shown to have diverse functions from apoptosis (Qi et al.,
2003; Tagami et al., 2000) to vascular remodeling and chemotaxis in smooth muscle cells (Acevedo et
al., 2004), which also agrees with its widespread expression pattern (Huber et al., 2002). In addition, it
was isolated as a novel human apoptosis-inducing protein without any known apoptosis-related motifs
(Li et al., 2001b; Watari and Yutsudo, 2003). Nogo-B overexpression was shown to induce ER stress
and precipitate apoptosis. Nogo-B was shown to interact with Bcl–2 and Bcl-XL (Bcl-XL also binds
RTN1-C) and reduce their anti-apoptotic activity (Tagami et al., 2000). Increased interaction of Bcl-2
with Nogo-B increases apoptosis by preventing the translocation of Bcl-2 to the mitochondria from the
ER. Not surprisingly, an increased expression of RTN proteins in certain cells causes changes in their
cellular response to ER stress (Qi et al., 2003). Recently it has been demonstrated that Fluoxetine, a
widely used antidepressant compound, up-regulated expression of Bcl-2 and Bcl-XL (Chiou et al.,
2006). Nogo is located close to a locus that has been associated through linkage with increased risk of
suicide attempts in families with recurrent, early-onset, major depression (Zubenko et al., 2004). Bcl-XL
is another bcl-2 family protein and may be involved in modulation of synaptic stability (Jonas et al.,
2003). Nogo-B also mediates chemotaxis of endothelial cells (Miao et al., 2006) and may play a key role
in triggering the production of pro-inflammatory cytokines and other inflammatory mediators, as well as
in the cellular response to environmental stress. Furthermore, Nogo-B may play important role in the
stabilization of α-tubulin (Rousseau et al., 2005).
Nogo C is transcribed from an alternate promoter (P2) than one used for Nogo-A and Nogo-B
transcription (P1) (Oertle et al., 2003a). In addition to a growth cone collapsing function, mediated
through Nogo-66 binding to the NgR1, Nogo-C may also act similarly to RTN1-C, a protein of the same
family with close similarity to Nogo-C (Oertle et al., 2003b; Yan et al., 2006), which is involved in
neuronal differentiation (Hens et al., 1998). As all main Nogo isoforms, is it expressed in an ER pattern.
It was shown to mediate cell apoptosis by inducing caspase-3 and p53 activation through the JNK-c-Jun-
27
dependent pathway (Chen et al., 2006).
4. DEPRESSION
CaMKII plays an important role in the serotonin signaling pathway. The activation of the
serotonin 1a receptor (5-HT1A) reduces CaMKII autophosphorylation, resulting in decreased GluR1
phosphorylation and smaller AMPA-R mediated currents (Cai et al., 2002; Colbran and Brown, 2004).
The decrease in CaMKII activity also inhibits the induction of LTP by suppressing NMDA receptor
function in postsynaptic neurons. This is achieved by the inhibition of PKA and ensuing activation of
PP1 (Cai et al., 2002), resulting in cognitive deficits and impaired memory retention (Edagawa et al.,
1999). The actions of antidepressants also converge on the CaMKII pathway. Selective serotonin
reuptake inhibitors (SSRIs) (paroxetine and fluvoxamine) upregulate CaMKII activity (Popoli et al.,
1995). They induce presynaptic CaMKII activity and autophosphorylation and increase Ca2+/CaM
dependent phosphorylation of synaptotagmin (with less effect on synapsin) in hippocampus (Popoli et
al., 1997). Furhtermore, synapsin I and synaptotagmin are two major CaMKII presynaptic substrates
(Fukunaga et al., 1996; Popoli et al., 1997). 5-HT1A antagonists also cause a rapid increase in
phosphorylated CaMKII, followed by an enhanced membrane expression of AMPA receptor subunits,
especially GluR1, phosphorylated at the CaMKII site (Schiapparelli et al., 2005).
28
RESULTS
Study No. 1: Schizophrenia and Nogo: elevated mRNA in cortex, and high prevalence of a
homozygous CAA insert.
1. Abstract for study No. 1
Schizophrenia is a major psychiatric disorder which is hypothesized to result from abnormal
neurodevelopment, and may be associated with altered gene expression. To search for genes
overexpressed in schizophrenia, cDNA library subtractive hybridization experiments between post-
mortem human frontal cerebral cortices from schizophrenia individuals and neurological controls were
carried out. One of the genes over-expressed in schizophrenia was identified as Nogo (also known as
reticulon 4, RTN4, NI 250, or RTN-X), a myelin-associated protein which inhibits the outgrowth of
neurites and nerve terminals. The elevated expression of Nogo mRNA in schizophrenia was confirmed
by quantitative reverse transcription-polymerase chain reaction studies using samples from the National
Neurological Research Specimen Bank in Los Angeles and from the Canadian Brain Tissue Bank in
Toronto. We found 16.5 pg Nogo cDNA/µg total RNA in schizophrenia, and 10.2 pg Nogo cDNA/µg
total RNA in controls (n = 7; p = 0.01, t-test for n<30). To identify possible polymorphisms in this gene,
the Nogo nucleotide sequence was determined in a series of schizophrenia and control samples. The
Nogo mRNA was found to contain a CAA insert polymorphism in the 3'-untranslated region. The
prevalence of individuals homozygous for this CAA insert was significantly higher in schizophrenia
compared to controls in genomic DNA samples extracted from post-mortem brain and blood samples:
17/81 or 21% in schizophrenia and 2/61 or 3% in controls (p = 0.0022, Chi-square and Fisher’s Exact
Tests). Because the 3'-untranslated regions of eukaryotic genes are known to regulate gene expression,
the increased frequency of the Nogo CAA insert in schizophrenia may contribute to abnormal regulation
of Nogo gene expression, and may indicate a role for Nogo in disturbed neurodevelopment in
schizophrenia.
2. Introduction for study No. 1
Although schizophrenia is a heritable disorder, the genes responsible for this disease remain to
be identified (Fuller Torrey and Yolken, 2000; Waterwort et al., 2002). It has been hypothesized that
schizophrenia may arise from abnormal neurodevelopment due to aberrant neuron formation and
29
migration, or changes in regenerative/repair capacity in adulthood (Benes, 2000; Bunney and Bunney,
2000). One such critical protein may be Nogo (also known as reticulon 4, RTN4, NI 250 or RTN-X),
which has been shown to be highly expressed by oligodendrocytes in CNS myelinated tissues during rat
fetal development and in the adult rat (Huber et al., 2002; Josephson et al., 2001). Myelin develops in
vivo at about the same time as the CNS loses plasticity, and myelination appears to be synchronized
with the gradual loss of neuronal regenerative ability (Skaper et al., 2001). Nogo has an important role
in regenerative and repair processes in the central nervous system because of its ability to inhibit the
outgrowth of neurites and nerve terminals (Chen et al., 2000b; GrandPre et al., 2000; Prinjha et al.,
2000). The effects of Nogo may be mediated by, or occur in association with, GAP43, CAP23, LIF
kinase, or other neuronal transcription factors, such as c-Jun and JunD, which are associated with
neuronal growth (Zagrebelsky et al., 1998). Nogo has also been shown to inhibit neuronal sprouting of
injured and uninjured Purkinje neurons in the adult rat cerebellum (Buffo et al., 2000). Although Nogo
is also expressed by neurons during development, there is no known functional role for neuronal Nogo
(Huber et al., 2002; Josephson et al., 2001).
The Nogo gene encodes for three alternatively spliced variants, Nogo-A, Nogo-B, and Nogo-C,
which share a common C-terminal domain of 188 amino acids containing two putative transmembrane
domains, and an endoplasmic reticulum retention motif (Skaper et al., 2001). It is not yet clear where the
neurite inhibitory domain resides. Two studies have suggested a Nogo-A-specific region (Chen et al.,
2000b; Prinjha et al., 2000), while a third demonstrated inhibitory activity in a domain common to all
three isoforms (GrandPre et al., 2000). Nogo, therefore, functions as a developmentally regulated
(Huber et al., 2002; Josephson et al., 2001) tonic inhibitor of neuronal growth through negative,
constitutive down-regulation of growth-associated genes (Brittis and Flanagan, 2001; Huber and
Schwab, 2000; Zagrebelsky et al., 1998). Since Nogo has an important role in synaptic plasticity and
neuronal migration, the altered expression of this gene in schizophrenia may contribute to the abnormal
neuronal organization in schizophrenia. At present, there are only a few studies on Nogo expression in
mouse, rat and human fetal tissues (Huber and Schwab, 2000; Josephson et al., 2001), with none
reported on degenerative diseases or schizophrenia.
To search for genes with altered mRNA expression in schizophrenia brain, we carried out cDNA
library subtractive hybridization experiments using RNA extracted from schizophrenia and control post-
mortem frontal cortices (Diatchenko et al., 1996). The brain region selected for study was the prefrontal
cortex, important for memory and cognition. These faculties are often impaired in schizophrenia
30
(Goldman-Rakic and Selemon, 1997), suggesting that there may be changes in gene expression and/or
neuronal organization. These studies identified Nogo as being potentially over-expressed in
schizophrenia. The gene expression of Nogo was, therefore, measured in seven schizophrenia and seven
control frontal cortex samples by quantitative reverse transcription-polymerase chain reaction
(Haberhausen et al., 1998). To identify possible polymorphisms in this gene, the Nogo nucleotide
sequence was determined in a series of schizophrenia and control samples. The Nogo mRNA was found
to contain a CAA insert polymorphism in the 3´-untranslated region, 669 bases beyond the coding
region. The prevalence of individuals homozygous for the CAA insert was significantly higher in
schizophrenia compared to controls.
3. Materials and Methods for study No. 1
3.1. Post-mortem human tissues.
Post-mortem human frontal cortical brain tissues (Brodmann Area 10) were provided by the
Stanley Foundation Neuropathology Consortium (Bethesda, MD) from a collection of 60 brains, 15 each
from individuals diagnosed (DSM-IV criteria) with either schizophrenia, bipolar disorder, non-psychotic
depression or neurologically normal (Torrey et al., 2000). Post-mortem human brain frontal cortices
were also dissected by, and obtained from, the Canadian Brain Tissue Bank (Toronto, ON, Canada) and
the National Neurological Research Specimen Bank (West Los Angeles VA Medical Center, Los
Angeles, CA). The diagnosis of schizophrenia was made by two psychiatrists independently examining
the case records and using DSM-IV criteria (American Psychiatric Association, 1987) (American
Psychiatric Association, 1994).
3.2. Blood samples.
Additional control genomic DNA samples of 34 anonymous Caucasian individuals were
obtained from relatives of rheumatic arthritis patients (laboratory of Dr. K. Siminovitch, Mount Sinai
Hospital, Toronto, ON). Genomic DNA was also obtained from blood samples from individuals
diagnosed with schizophrenia from Dr. J. Lieberman (Hillside Hospital, NY; 15 samples), Dr. C.
Shammi and Dr. C. Hudson (Centre for Addiction and Mental Health, Clarke site, Toronto, ON; 32
samples). Additional genomic DNA samples were obtained from Dr. B. O´Dowd (Toronto, ON; 72
samples) from individuals diagnosed with alcohol addiction (as well as substance abuse in some cases).
31
3.3. PCR-select cDNA subtractive hybridization.
The purpose of using subtractive hybridization in this project was to obtain clones of genes that
were differently expressed in schizophrenia cerebral cortex compared to control tissues. The general
procedure was to convert both mRNA populations in the control and diseased tissues into cDNA. The
schizophrenia (or tester) cDNA was hybridized with the control (or driver) cDNA. The hybrids were
removed, and the remaining unhybridized cDNAs represented genes expressed in schizophrenia but
absent or different from control mRNA.
The method used was the Clontech PCR-Select™ procedure, using the cDNA Subtraction Kit
(Catalog K1804-1; Clontech Laboratories, Palo Alto), and the following outline is adapted from the
procedure provided by Clontech. To obtain preliminary data on this aspect of the project, we used post-
mortem human frontal cortex from two control individuals (who died of non-neurological illnesses) and
from two individuals who died with schizophrenia. Total RNA was extracted from the post-mortem
frontal cortex tissues, using Trizol® Reagent (monophasic solution of phenol and guanidine
isothiocyanate; Life Technologies (Gibco-BRL Products), Burlington, Ontario). The messenger RNA or
Poly A+ RNA was then prepared, using Oligotex™ resin (Qiagen, Mississauga, Ontario). cDNA was
then prepared for each tissue, using 2 mg of poly A+ RNA. The schizophrenia and control cDNAs were
then digested with RsaI (yielding blunt ends). The schizophrenia cDNA was divided into two parts, each
part being ligated with a different cDNA adaptor.
Two hybridizations were now done. The first hybridization was done with an excess of control
cDNA added to each sample of schizophrenia cDNA. The samples were heat denatured and permitted to
anneal. The concentration of high-abundance and low-abundance sequences became equal for the
single-stranded type of molecules (so-called type a molecules in the Clontech procedure). At the same
time, however, the type a molecules were enriched for differentially expressed sequences, while the
cDNAs which were not differentially expressed formed type c molecules (Clontech) with the control
cDNA. In the second hybridization, the two primary hybridization samples were mixed together without
denaturing. Hence, only the remaining equalized and subtracted single-stranded schizophrenia cDNAs
reassociated and formed hybrids (so-called type e in the Clontech procedure). These new hybrids were
double-stranded schizophrenia molecules with different ends, which corresponded to the sequences of
adaptors 1 and 2R. Fresh, denatured control cDNA was added (without denaturing the subtraction mix)
to further enrich fraction e for differentially expressed sequences. After filling in the ends by DNA
polymerase, the type e molecules (which were the differentially expressed schizophrenia sequences) had
32
different annealing sites for the nested primers on their 5´ and 3´ ends. The entire population of
molecules was then subjected to PCR (polymerase chain reaction) in order to amplify the desired
differentially expressed sequences. During PCR, type a and d molecules were missing primer annealing
sites, and were not amplified. Type e molecules, which had two different adaptors, were amplified
exponentially, and these were the equalized, differentially expressed sequences.
We searched for differentially expressed genes in schizophrenia by analysing the type e
molecules directly. This was done by PCR amplification of the type e molecules using many, up to 35,
cycles. This number of cycles amplified the more abundant species of cDNA in the Schizophrenia-
minus-Control cDNAs and in the Control-minus-Schizophrenia cDNAs. These bands, representing the
most abundant species of cDNA, were dissected out, and the cDNAs extracted and cloned (using the
Original TA Cloning® Kit: Box #1 for ligation: Catalog K2001-01; and box #2 for transformation, using
the INVaF´ One Shot™ Kit of E. coli cells: Catalog C2020-03; Invitrogen, San Diego, CA). This
approach yielded many clones, the cDNAs of which were then sequenced and the genes identified
through Genbank.
3.4. Quantitative RT-PCR.
Total RNA was extracted from brain tissue samples using TRIzol® Reagent (Life
Technologies/Invitrogen) according to the manufacturer´s guidelines. Complementary DNA was
prepared from 2 mg of total RNA using the Superscript™ Preamplification System for First Strand
cDNA Synthesis (Life Technologies/Invitrogen). An internally truncated standard for competitive PCR
experiments was prepared with a 75-bp deletion (bases 4460–4534, AF148537) using a reverse primer
with sequences flanking the desired deletion
(5´-TACAGCTTAAACCACAATGGTAGCATTAGATTCAGTCC-3´, complementary to bases 4556–
4535, 4459–4444) and a forward primer (5´-GCATCAGGCACAGATAGATC-3´, bases 3612–3630).
The truncated Nogo PCR product was amplified using Qiagen Taq™ Polymerase, purified using the
Qiaex II Gel Extraction kit (Qiagen, Mississauga, Ontario), and quantitated by A260 UV absorbance.
The competitor template had identical primer annealing sites and similar base composition as the Nogo
cDNA under study, resulting in accurate estimates of transcript levels. The primers used for PCR
amplification were the above forward primer (bases 3612–3630) and a reverse primer
(5´-TACAGCTTAAACCACAATGGTA-3´, complementary to bases 4556–4535), yielding predicted
PCR products of 945 bp from Nogo cDNA and 870 bp from the competitor template. The primers were
33
designed to amplify all three splice variants of Nogo: Nogo-A, Nogo-B, and Nogo-C. To quantitate
Nogo gene expression in frontal cortex cDNA, several dilutions of the competitor template were co-
amplified in PCR reactions with a constant amount of cDNA. The amplified DNA was size-fractionated
by electrophoresis on agarose gels containing ethidium bromide. The amount of stained DNA amplified
(endogenous Nogo and competitor) from each sample was quantified using the Bio-Rad Gel Doc
Imaging System. The amount of endogenous Nogo cDNA in each reaction was determined by
comparison with the level of the coamplified Nogo competitor DNA. That is, the ratios for each dilution
of competitor template input and endogenous Nogo was compared to determine the point of
equivalence.
3.5. Genomic DNA extraction.
Total DNA was extracted from brain tissue samples using TRIzol® Reagent (Life
Technologies/Invitrogen) according to the manufacturer´s guidelines. Genomic DNA was extracted
from blood samples by standard methods. Briefly, 3 ml of blood were mixed with 9 ml of a solution
containing 155 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA. After centrifugation at 3000× g for 10
min at 4 °C, the remaining tissue was digested for 3 h at 55 °C with proteinase K (125 mg/ml) in 3 ml of
nucleus-lysing buffer (75 mM NaCl, 25 mM EDTA, 1% SDS). After centrifugation (3000× g for 10
min), the DNA was precipitated with ethanol, dried, and dissolved in 500 ml of buffer (10 mM Tris, 1
mM EDTA; pH 8) and stored at 4 °C.
3.6. Amplification and sequencing of the Nogo DNA template.
Genomic DNA was PCR-amplified using the Qiagen Taq™ Polymerase Kit (Qiagen). Primers
used for amplification of the Nogo gene, AF148537, were forward primer
(5´-TTACCTGTCTTGACTGCC-3´, bases 3819–3836) and reverse primer
(5´-TACAGCTTAAACCACAATGG-3´, complementary to bases 4556–4537). PCR products were
analyzed using agarose gel electrophoresis before sequence analysis using the Sequenase Version 2.0
PCR Product Sequencing Kit (product # 70170, USB) and the above described reverse primer.
4. Results of study No. 1
The preliminary findings from the subtractive hybridization work revealed the following genes
or gene segments to be over-expressed in schizophrenia: segment within chromosome 13q12–18; G-
34
protein-coupled receptor-7; EST clone 1457813; two unidentified cDNA sequences (400 bp; 800 bp);
and Nogo. A preliminary set of under-expressed genes or gene segments were mitochondrial 16s rRNA;
cytochrome oxidase subunit II; ATPase 6; cDNA with long CAG trinucleotide repeats; antiadhesive
extracellular matrix protein; calcium-dependent membrane-binding protein; protein kinase II;
intermediate neurofilament; five ESTs and five unidentified cDNA sequences (300–1000 bp). Although
some of these genes may be relevant to the many hypotheses of schizophrenia, the Nogo gene appeared
to be the most attractive and interesting for a detailed and precise analysis for the present study.
An internally deleted standard was prepared and used to quantitate the total amount of Nogo
transcript (composed of Nogo-A, Nogo-B, and Nogo-C) in each sample. The clinical summaries and the
values for Nogo gene expression are presented in Table 3. The mean expression in controls was
10.24±1.69 pg Nogo cDNA/ µg total RNA, and in schizophrenia was 16.49±1.83 pg Nogo cDNA/ µg
total RNA, indicating a 60% increase in disease ( P=0.01, t-test for n<30). These data were presented at
the 31st Annual Meeting of the Society for Neuroscience (San Diego, CA, USA, 2001).
Nucleotide sequence analyses of schizophrenia and control DNA samples revealed a
polymorphism in the Nogo cDNA, a single CAA insert in the 3´-untranslated region (bases 4386–4388,
Genbank AF148537), as indicated in Figure 6. The frequency of the CAA insert polymorphism was
examined in neurological controls and in samples from individuals who had schizophrenia. The
prevalence of individuals homozygous for the CAA insert in the schizophrenia samples was 17 out of 81
or 21%, in contrast to 2 out of 61 or 3% in the control samples (Table 4). This difference was found to
be statistically highly significant, using the X2- and Fisher´s exact-tests ( P=0.0022 for both tests). If
only genomic DNA extracted from post-mortem brain samples is considered, the difference increases to
38% in schizophrenia ( n=32) versus 0% in controls ( n=27), and is highly significantly different
(P=0.00058; Fisher´s exact-test for small samples).
35
3661 aaagatgcta tggctaaaat ccaagcaaaa atccctggat tgaagcgcaa agcTGAatga
3721 aaacgcccaa aataattagt aggagttcat ctttaaaggg gatattcatt tgattatacg
3781 gatctttatt tttagccatg cactgttgtg aggaaaaatt acctgtcttg actgccatgt
3841 gttcatcatc ttaagtattg taagctgcta tgtatggatt taaaccgtaa tcatatcttt
3901 ttccTATCtg aggcactggt ggaataaaaa acctgtatat tttactttgt tgcagatagt
3961 cttgccgcat cttggcaagt tgcagagatg gtggagctag aaaaaaaaaa aaaaaagccc
4021 ttttcagttt gtgcactgtg tatggtccgt gtagattgat gcagattttc tgaaatgaaa
4081 tgtttgttta gacgagatca taccggtaaa gcaggaatga caaagcttgc ttttctggta
4141 tgttctaggt gtattgtgac ttttactgtt atattaattg ccaatataag taaatataga
4201 ttatatatgt atagtgtttc acaaagctta gacctttacc ttccagccac cccacagtgc
4261 ttgatatttc agagtcagtc attggttata catgtgtagt tccaaagcac ataagctaga
4321 agaagaaata tttctaggag cactaccatc tgttttcaac atgaaatgcc acacacatag
4381 aactcCAAca acatcaattt cattgcacag actgactgta gttaattttg tcacagaatc
4441 tatggactga atctaatgct tccaaaaatg ttgtttgttt gcaaatatca aacattgtta
4501 tgcaagaaat tattaattac aaaatgaaga tttataccat tgtggtttaa gctgtactga
4561 actaaatctg tggaatgcat tgtgaactgt aaaagcaaag tatcaataaa gcttatagac
4621 ttaaaaaaaa aa
Figure 6. Nogo cDNA sequence. (GenbankAF148537) The CAA insert polymorphism in the 3’-untranslated region is located at bases 4386–4388 and is almost always co-inherited with a deletion of the TATC sequence, as shown here, as compared to the full “TATCTATC” sequence. TGA marks the end of the coding region
36
Brain/Age/sex P.M.
delay (h) Death
Diagnosis,
symptoms
Duration
(psychosis) Antipsychotic Rx
pg Nogo cDNA per
µg total RNA±S.E.
Schizophrenia tissues:
T1176/19/m 52 Suicide Depression 1 year none 19.36 ± 4.33
T1149/22/m 18 Suicide Paranoid 5 years Flupenthixol 9.68 ± 1.77
T1293/29/m 4 Drowning Halluc., depr. 13 years Risperidone 21.84 ± 7.06
L707/28/m 41 Suicide Depression 2 years Off fluphenazine 4years 11.91 ± 3.62
L695/30/m 32 Cardiac Delusions 9 years Thioridazine 20.35 ± 6.44
L1755/74/m 14 Cancer Depression 30 years Trifluperazine 20.35 ± 6.44
L477/82/f 16 Cardiac Delusions 36years Fluphenazine 11.91 ± 2.82
Average: 16.49 ± 1.83*
Control tissues:
L451/25/m 14 Drowning Control None Phencyclidine abuser 16.88 ± 1.98
T1225/31/m 11 Car accident Control None 19.36 ± 4.33
L480/66/m 19 Cardiac infarct Control None 16.38 ± 6.09
L1768/71/m 25 Cardiac; cancer Control None 4.07 ± 1.61
L475/77/f 19 Car accident Control None 7.94 ± 2.79
T1367/80/m 16 Cancer Control None 2.82 ± 0.90
T1214/92/m 3 Tachycardia Control None 4.22 ± 1.43
Average: 10.24 ± 1.69
Table 3. Quantitative RT-PCR of Nogo gene expression and clinical summaries of samples. Abbreviations: L indicates brain tissue from the National Neurological Research Specimen Bank in Los Angeles; T indicates brain tissue from the Canadian Brain Tissue Bank in Toronto. Standard error (SE) for samples based on standard deviation of each sample. SE for average based on standard deviation of the whole (control or schizophrenia) group. * Statistically significantly different from controls (p=0.01, T-test for n<30).
37
Total number
of samples
Homozygous
for CAA insert
Heterozygous
for CAA insert
Schizophrenia:
Stanley Bank 15 6 ** 3
Other Banks 17 6 ** 6
Blood DNA 49 5 24
Total 81 17 (21% of 81) * 33
Controls:
Stanley Bank 15 0 3
Other Banks 12 0 7
Blood DNA 34 2 13
Total 61 2 (3% of 61) 23
Table 4. Frequency of CAA insert in schizophrenia and controls. *Significantly different from controls (P50.0022, χ2- and Fisher’s exact tests). **Combined schizophrenia brain bank tissues significantly different from control brain bank tissues (P50.00058, Fisher’s exact-test).
Almost all the DNA samples were from Caucasians. Of the 12 individuals homozygous for the
CAA insert, two were Asian (one schizophrenia patient of Asian descent was not homozygous for the
insert). The overall CAA allele frequency was 0.41 in schizophrenia and 0.22 in controls. In the well-
matched Stanley brain bank samples, the CAA allele frequency was 0.5 in schizophrenia and 0.1 in
controls. This was in contrast to the blood samples, where the CAA allele frequency was 0.35 in
schizophrenia and 0.25 in controls. These data suggest there are population differences for this Nogo
polymorphism which will require a large set of well-matched samples to elucidate. The CAA insert is in
most cases co-inherited with a TATC deletion at base 3905, 188 bases 3´ of the stop codon (Figure 6).
All individuals homozygous for the CAA insert were also homozygous for the TATC deletion, except
for one schizophrenia sample and one control sample. Three individuals, one each from the bipolar,
major depression, and schizophrenia samples were homozygous for the TATC deletion, but were not
homozygous for the CAA insert.
38
To search for additional polymorphisms, the complete Nogo cDNA sequence was determined for
nine individuals: four schizophrenia, one major depression, and four control samples. No additional
common polymorphisms were identified, and the three polymorphisms listed for Nogo in the NCBI SNP
database (C→T, 1343; C→G, 2850; C→A, 3751), were not present in the nine samples completely
sequenced. Furthermore, two of the three NCBI-listed polymorphisms at positions 1343 and 3751 were
not present in an additional independent set of 10 samples studied. We did identify two additional
polymorphisms, each observed in only one sample, a GTTT deletion (bases 4473 - 4476) and an ATT
insertion at position 4508.
We examined the data for any relation to age of onset. The Stanley Brain Bank samples (n=15)
revealed that none of the five schizophrenia individuals aged 13–19 years were homozygous for the
CAA insert, while six of the 10 schizophrenia individuals with disease onset at age 20–42 years were
homozygous for the CAA insert. We also identified 2/15 (15%) bipolar and 1/15 (7%) major depression
samples to be homozygous for the CAA insert.
In a group of African and Afro-American samples, the prevalence of the CAA insert was three out
of 60, or 5%, in schizophrenia DNA samples, in contrast to two out of 111 or 1.8% in control DNA
samples.
39
Study No. 2: Increased Expression of Calcium/calmodulin-dependent protein kinase IIβ in
Frontal Cortex in Schizophrenia and Depression.
1. Abstract for study No. 2
In searching for genes dysregulated in schizophrenia, we measured the expression of the two
splice variants of calcium/calmodulin-dependent protein kinase II (CaMKIIα and CaMKIIβ) in post-
mortem frontal cerebral cortex tissues from patients who had died with schizophrenia, bipolar disorder,
or severe depression. The mRNA levels of expression of these two splice variants were measured by
Real–Time Quantitative PCR using an Mx4000 instrument. The values for the expression of CaMKIIα
and CaMKIIβ were normalized by the expression of β-glucuronidase in the tissues. The expression of
CaMKIIα was significantly elevated in the depression tissues by 29%. The expression of CaMKIIβ was
significantly elevated in the schizophrenia tissues by 27%, and in the depression tissues by 36%.
Because CaMKIIβ influences the expression of many neuroreceptors and influences neural outgrowth
and pruning, its altered expression in the cerebral cortex in schizophrenia or depression may contribute
to these diseases.
2. Introduction for study No. 2
Inheritance and twin studies indicate that schizophrenia has a genetic component and that about
50% of individuals carrying these genes develop the disease. Furthermore, the risk of schizophrenia is
similar in the offspring of both affected and unaffected monozygotic twins. The fact that the healthy
twins have the same susceptibility genes for schizophrenia but do not express the phenotype indicates
that other factors must contribute to the etiology of schizophrenia. The inheritance pattern in families
indicates that schizophrenia cannot be due to a single disease gene but is likely a result of
polymorphisms in multiple interacting gene loci (Cardno and Gottesman, 2000) and altered expression
of several genes (Buckland et al., 2004). In order to search for genes with altered expression in
schizophrenia, previous work, using subtractive hybridization between post-mortem frontal cortex
samples from control and schizophrenia patients, had identified two genes with elevated expression in
the schizophrenia tissues, calcium-calmodulin-dependent protein kinase IIβ and Nogo (Novak et al.,
2000; Novak et al., 2002). Using an increased number of post-mortem human samples, and testing for
both CaMKII subunits, CaMKIIα and CaMKIIβ, the present study extends the earlier work on
schizophrenia tissues (Novak et al., 2000).
40
The gene for the α subunit of CaMKII is located at 5q32, a locus which has been linked to
schizophrenia (Lewis et al., 2003; Paunio et al., 2001; Sklar et al., 2004). CaMKIIα is highly expressed
in neurons, where it mediates many aspects of neurone function (Colbran, 2004; Colbran and Brown,
2004). The translocation time of CaMKII to the postsynaptic density, a key variable in neuronal
function, is determined by the ratio of CaMKIIα to CaMKIIβ isoforms (Shen and Meyer, 1999). At the
postsynaptic density, CaMKII acts as a decoder of Ca2+ oscillation frequencies (Colbran, 2004;
Soderling, 2000) and a molecular memory of Ca2+ signals (Shen et al., 2000). Other CaMKII splice
variants are targeted to the nucleus and influence gene transcription (Hardingham and Bading, 1999;
Sun et al., 1994). During early development, CaMKII is essential for synaptic density and maturation
(Borodinsky et al., 2002; Rongo and Kaplan, 1999; Wu and Cline, 1998). Schizophrenia usually has an
onset in early adulthood, although it may be present during puberty. This may correspond to the time
when the CaMKII-Ca2+ decoding parameters change, and an absence of this change may cause neurons
to remain immature (Mayford et al., 1995). CaMKIIα mutations can cause neural defects possibly
related to some aspects of schizophrenia (Gordon et al., 1996), such as disturbed behavior and cognition
(Chen et al., 1994; Giese et al., 1998; Mayford et al., 1996a; Silva et al., 1992a; Silva et al., 1992b).
Therefore, because CaMKII has a role in neurodevelopment, because some aspects of disturbed
behavior that occur with CaMKII mutations may relate to schizophrenia, and because our earlier work
on subtractive hybridization had indicated alterations in CaMKII isoforms, we measured the levels of
the two major CaMKII transcripts in post-mortem frontal cortex tissues. The present study found
CaMKIIβ to be elevated in schizophrenia tissues, and also, surprisingly, in tissues from severely
depressed individuals.
3. Materials and Methods for study No. 2
Postmortem tissues were donated by the Stanley Foundation Brain Consortium (Bethesda, MD),
courtesy of Drs. Llewellyn B. Bigelow, Juraj Cervenak, Mary M. Herman, Thomas M. Hyde, Joel E.
Kleinman, José D. Paltn, Robert M. Post, E. Fuller Torrey, Maree J. Webster, and Robert H.Yolken
(Torrey et al., 2000). A total of 60 human frontal cerebral cortex tissues (Brodmann area 10) were
provided by the Stanley Foundation Brain Consortium (Bethesda, MD) from a collection of 60 brains,
fifteen each from individuals diagnosed (DSM IV criteria) with either schizophrenia, bipolar disorder,
non-psychotic depression, or neurological controls (Table 5). The diagnosis of schizophrenia was made
41
by at least two psychiatrists independently examining the case records and using DSM-IV criteria
(American Psychiatric Association, 1994).
3.1. Extraction of total RNA from tissues
TRIzoL® reagent (Invitrogen, Burlington, ON) was used according to the manufacturer’s
guidelines to extract total RNA from the tissues. The tissue was homogenized (Polytron, PT-10 probe,
Brinkmann Instruments, Westbury, NY) in TRIzoL® Reagent (1 ml per 50 mg of tissue) and incubated
at room temperature for 5 min before chloroform (0.2 ml per ml of TRIzoL® Reagent) was added, and
the capped tubes were shaken for 15 s. The samples were incubated at 25°C for 2 min and later
centrifuged at 12,000 x g at 4°C for 15 min. To isolate the total RNA, the aqueous upper phase was
transferred to a new tube. The RNA was precipitated using isopropyl alcohol (0.5 ml per ml of TRIzoL®
Reagent). The samples were incubated at 25°C for 10 min and centrifuged at 12,000 x g at 4°C for 10
min. The RNA pellet was washed with 75% ethanol (1 ml per ml of TRIzoL® Reagent), mixed, and
centrifuged at 12,000 x g at 4°C for 15 min. The RNA pellet was air dried for 5 min, dissolved in RNA
Storage Solution (Ambion), and incubated at 60°C for 10 min. A housekeeping gene, β-glucuronidase,
was used to assess the quality and abundance of RNA for each tissue sample. Although our preliminary
experiments also examined whether β-actin would be a useful housekeeping gene, we found that the
absolute values of β-actin in the tissues were an order of magnitude higher than the expression of
CaMKIIα and CaMKIIβ. Only those tissues were used which showed abundant β-glucuronidase gene
expression and which yielded specific products by means of real-time PCR. Four samples with
anomalously low expression of β-glucuronidase or CaMKIIα and CaMKIIβ cDNA (less than two
standard deviations below average) may have been inadvertently thawed during storage and were,
therefore, excluded.
42
Table 5. Clinical data and findings on post-mortem frontal cerebral cortex tissues.
CaMKII! CaMKII"
disorder hours years
pg CaMKII! /pg
"-glucuronidase
pg CaMKII" /pg "-
glucuronidase
4 34 m Suicide: jumped 23 15 Risperidone, valproate, venlafaxine 5.1 10.0
6 54 m Subdural hematoma 39 14 Lithium, carbamazepine 1.5 4.7
9 30 m Malnutrition, dehydration 45 16 Valproate, bupropion 4.9 7.7
13 30 m Pneumonia, myocarditis 31 8 Lithium, clozapine 2.6 4.9
18 61 f Suicide: overdose 60 43 Fluoxetine, valproate 4.6 5.2
21 31 m Suicide: jumped 28 10 Haloperidol, trazadone, trihexiphenidyl 1.5 2.6
22 25 f Suicide: hanged 24 6 Thiothixene, carbamazepine, lithium, trazodone 5.3 10.9
30 48 m Suicide: immolation 13 21 Untreated for over 20 yrs 3.8 6.5
31 37 f Suicide: overdose 29 23 Lithium, bupropion, !clonazepam, lorazepam 3.4 6.8
32 50 f Malnutrition, dehydration 18 16 None, untreated for several months 1.7 5.3
42 57 m Cardiac 19 27 Haloperidol, diphenhydramine 1.5 3.1
44 50 m Suicide: jumped 19 23 Valproate, clozapine, flurazepam, benzotropine 3.0 6.6
54 50 f Pulmonary emboli 62 25 Valproate, clomipramine 2.4 5.7
Av ± s.e. = 43 ± 3 Av ± s.e. = 32 ± 4
Depression
5 53 f Alcohol intoxication 40 42 Lithium, trazodone 5.3 10.9
7 51 m Suicide: gunshot 26 1 Nefazadone, hydroxyzine 1.7 4.7
23 65 m Cardiac 19 20 No medication for 5 yrs. 2.8 4.7
33 42 m Suicide: hanged 7 10 Temazepam but off medications for > 2 weeks 6.7 9.5
34 43 m Cardiac 43 13 Trimipramine 4.4 4.7
36 32 f Suicide: overdose 47 1 Imipramine, amitriptyline, !nortriptyline, clonazepam 2.3 4.2
37 42 f Cardiac 25 3 Fluoxetine, lithium 2.9 4.5
38 52 m Cardiac 12 6 No medication for 6 yrs. 5.3 8.9
43 56 m Cardiac 23 4 Sertraline 7.3 9.9
46 39 m Suicide: carbon monoxide 23 22 Never treated 2.3 7.7
49 44 f Suicide: overdose 32 17 Fluoxetine, imipramine, lorazepam 2.3 7.2
52 47 m Cardiac 28 20 Fluoxetine, nefazadone 5.5 10.7
55 30 f Suicide: overdose 33 11 None 5.6 10.4
Av ± s.e. = 46 ± 3 Av ± s.e. = 28 ± 3
Control
2 35 f Cardiac 23 None 5.8 8.9
3 53 m Cardiac 28 None 2.0 4.8
8 29 f Motor vehicle accident 42 Saw counselor for weight control 3.9 5.5
16 52 m Cardiac 8 None 4.7 6.7
20 68 f Pulmonary embolus 13 None 3.8 5.4
25 52 m Cardiac 28 None 3.8 5.3
27 41 m Pulmonary embolus 11 None 3.5 5.2
28 57 f Motor vehicle accident 26 None 2.6 3.6
41 52 m Cardiac 22 None 1.9 3.1
51 58 m Cardiac 27 None 3.6 6.4
53 44 f Cardiac 25 None 4.3 8.2
57 42 m Cardiac 27 None 2.9 7.9
58 44 m Cardiac 10 None 0.8 2.7
60 35 f Pulmonary embolus 40 None 1.7 3.8
Av ± s.e. = 47 ± 3 Av ± s.e. = 24 ± 3
Schizophrenia
1 25 m Suicide: hanged 32 5 Risperidone, paroxetine 5.5 9.9
11 52 m Cardiac 61 32 None; untreated for over 20 yrs. 4.4 5.5
12 30 f Suicide: jumped 60 8 Thiothixene, desipramine 5.2 6.8
15 44 m Cardiac 50 27 Haloperidol, carbamazepine, fluoxetine, clonazepam 1.1 2.4
17 30 m Pneumonia 32 17 Risperidone,thioridazine 5.4 5.6
35 31 m Suicide: jumped 14 13 Clozapine 2.0 3.1
39 60 f Cardiac 40 45 None; had ECT 5.7 10.3
40 56 f Suicide: overdose 12 32 Haloperidol, lithium, diphenhydramine, chloral hydrate 4.7 9.8
48 60 m Accidental drowning 31 33 Thioridazine, amitriptyline 1.6 6.6
50 62 f Motor vechicle accident 26 24 None; untreated for several months 3.2 8.1
56 32 m Acute alcohol intoxication 19 5 Clozapine 3.3 9.7
59 49 f Cardiac 38 24 Haloperidol, clozapine, clonazepam 2.0 6.6
Av ± s.e. = 44 ± 4 Av ± s.e. = 35 ± 5
Illness MedicationBipolar Age, m,f Death
P.M.
interval
43
The samples were not treated with DNAase because it would have resulted in further dilution of
the RNA. Instead, the primers used in the Quantitative Real-Time PCR were designed to span across
intron boundaries to prevent amplification of any genomic DNA. Furthermore, a series of preliminary
PCR reactions were done under various conditions to ensure that no mispriming occurred. Optical
density readings during the Real-Time Quantitative PCR runs indicated whether mispriming or whether
primer dimers occurred. The final total RNA concentration was 5 µg of RNA per 2 to 6 µL of water, as
determined by optical density. This solution was further diluted with RNAase-free water to 5 µg RNA
per 11 µL water for cDNA synthesis. Because mRNA only comprises approximately 10% of total RNA
and is highly susceptible to degradation, the quality of the mRNA was important to determine. This was
done by analyzing the 3’ to 5’ ratio of β-glucuronidase. Although all the samples were standardized to
contain the same amount of total RNA, the mRNA levels could vary depending on the extent of mRNA
degradation. The results show that this effect was minimized by standardizing the expression values
with β-glucuronidase. No non-normalized data were used. All the data presented are normalized to β-
glucuronidase, resulting in the dimensionless unit of expression for the CaMKII variants as (pg/µg total
RNA)/(pg β-glucuronidase /µg total RNA). The 3’/5’ ratio also provided an indication of the degree of
completion of the reverse transcription reaction, especially when using oligo-dT primers in the present
study.
3.2. First-strand cDNA synthesis
The SUPERSCRIPT IIITM First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad,
California) was used for cDNA synthesis using oligo (dT)12-18 and 2 µg total RNA. The samples were
incubated at 50°C for 50 min to allow synthesis of the cDNA, and the reaction was terminated at 85°C
for 5 min, chilled on ice for 5 min, and treated with E. coli RNase H (37 ˚C, 20 min). The resultant 21 µl
cDNA samples were stored at -20°C for later use.
3.3. Real-time Quantitative Polymerase Chain Reaction
The method used to quantitate cDNA was the Quantitative Real-Time PCR method. The levels
of cDNA were determined via fluorescence by the thermal cycler during the PCR cycle, and the
instrument’s software calculated the final data. These data were downloaded into an Excel spreadsheet.
Thus, there is no representative image of the PCR output reactions.
44
Primers were designed using the Primer Express software (Perkin-Elmer Applied Biosystems,
Foster City, California) and Oligo 4.0 (National Biosciences, Plymouth, Minnesota), and synthesized by
Sigma-Genosys (Oakville, Ontario). PCR amplification, real-time quantitation and analysis were done
using the Mx4000TM Multiplex Quantitative PCR System (Stratagene, La Jolla, California). A standard
was prepared for each target gene by conventional PCR, using the same primer pairs later used in real-
time quantitative PCR. The PCR products were excised and extracted from the gel using QIAEX II ®
DNA Extraction Kit (Qiagen, Inc., Santa Clarita, California). The DNA sample (standard) was diluted
ten-fold for optical density measurements at 260 nm. The concentration of the standard was determined
from its optical density (Ausubel, 1995).
The reaction mixture for real-time PCR was prepared using the Brilliant™ SYBR® Green QPCR
Core Reagent Kit (Stratagene, La Jolla, California). The company protocol was followed for a reaction
mix containing 1.5 mM MgCl2. The total reaction volume of 25 ml contained 0.25 µl of forward primer
(10 mM) and 0.25 µl (10mM) of reverse primer. The β-glucuronidase forward primer (5’ to 3’) at the 5’
end of the mRNA was CTCTGACAACCGACGCC and the reverse primer (5’ to 3’) was
TACCACACCCAGCCGAC. The β-glucuronidase forward primer (5’ to 3’) at the 3’ end of the mRNA
was GCCGATTTCATGACTGAACAG and the reverse primer (5’ to 3’) was
TATCTCTCTCGCAAAAGGAACGC. The CAMKIIα forward primer (5’ to 3’) was
GGGGGAAACAAGAAGAGC and the reverse primer (5’ to 3’) was GTGCTCTCTGAGGATTC. The
CAMKIIβ forward primer (5’ to 3’) was GTCCACCGCGGCCTC and the reverse primer (5’ to 3’) was
TTTTGGTGCTATTCGTCTGGG.
The SYBR® Green dye was diluted 1:2000 and the Rox reference dye was diluted 1:500. 3 ml of
1/10 diluted cDNA sample (equivalent to 28.6 ng of total RNA) was used per reaction. The cycling
parameters were 95°C for 10 min to activate the SureStartTM Taq, followed by 40 cycles of 95°C
denaturation for 30 s, 58°C reannealing for 30 s, and extension at 72˚ C for 40 s. In the analysis, to
minimize the effect of background noise and well-to-well variation on the results observed, our data
were normalized against the Rox reference dye and the algorithm of adaptive baseline. Each reaction
was repeated at least four times, using two independent cDNA preparations.
3.4. Determination of mRNA degradation.
The degree of degradation of the mRNA was determined by real-time quantitative PCR as the
ratio of the 5’-end template to the 3’-end template amount of β-glucuronidase. In absence of
45
degradation, the ratio should be equal to 1. Of the total number of four samples which had been
excluded in this study, two samples showed a degradation ratio greater than two standard deviations
from average.
4. Results of study No. 2
The amount of the CaMKIIα transcript in the control human frontal cortices was 3.2 ± 0.4
(pg/µg total RNA)/(pg β-glucuronidase/µg total RNA), the units of which are essentially dimensionless
(see later). The corresponding value for the fifteen schizophrenia tissues was 3.7 ± 0.5, not significantly
different from the control value (Figure 7). This increase in CaMKIIα was not statistically significant
(TDIST, p = 0.26) (Table 6). We found a statistically significant increase in the CaMKIIα transcript in
samples from people who had suffered with depression (4.2 ± 0.5, an increase of 29%) (TDIST, p =
0.02) (Figure 7). There was no change in samples from patients with bipolar disorder (-2%).
The amount of the CaMKIIβ transcript in the control human frontal cortices was 5.5 ± 0.7. The
value for the twelve schizophrenia tissues was 7.0 ± 0.8, an increase of 27% (Figure 8). This increase
was statistically significant using a t test for small samples (TDIST, p = 0.01). There was also a
significant increase in the CaMKIIβ levels in samples from patients with depression; the value was 7.6 ±
0.8, an increase of 36% (TDIST, P = 0.002) (Figure 8), as compared with that of controls. There was no
statistically significant difference between controls and samples from patients with bipolar disorder.
As noted above, all values are given as pg transcript per µg total RNA, divided by pg β-glucuronidase
per µg total RNA. The values, therefore, represent dimensionless units of pg transcript per pg β-
glucuronidase transcript. This was done because standardization to a housekeeping gene, β-
glucuronidase, markedly reduced the effect of mRNA degradation. Without this standardization, the
levels of CaMKIIα transcript per total RNA decreased with increasing 3’ to 5’ transcript ratio (measured
for β-glucuronidase, Figure 9, top). Expressing CaMKIIα transcript level as a ratio of a housekeeping
gene, β-glucuronidase, which is also subject to degradation, minimized the effect of the degradation
(Figure 9, bottom).
46
Figure 7. CaMKIIα in schizophrenia and depression. The amounts of CaMKIIα transcripts in the postmortem human frontal cortices from control individuals and from those who had died with schizophrenia or major depression.
47
Figure 8. CaMKIIβ in schizophrenia and depression. The amounts of CaMKIIβ transcripts in the postmortem human frontal cortices from control individuals and from those who had died with schizophrenia or major depression. Solid lines indicate mean; dashed lines indicate s.e.
48
CaMKII transcript adjusted by
β-glucuronidase % difference from controls
CaMKIIα CaMKIIβ CaMKIIα CaMKIIβ
Normal 3.2 ± 0.4 5.5 ± 0.5 — —
Bipolar disorder 3.2 ± 0.4 6.1 ± 0.7 -2% 11%
Depression 4.2 ± 0.5 7.6 ± 0.8 29%a 36% b
Schizophrenia 3.7 ± 0.5 7.0 ± 0.8 13% d 27% c
Table 6. Levels of CaMKIIα and CaMKIIβ transcripts in a control and three disease groups and percent change in disease group levels as compared to healthy controls. a. TDIST p=0.02; b. TDIST p=0.002; c. TDIST p=0.01; d. TDIST p=0.26
It was necessary to determine whether the administration of antipsychotic medications during the
lifetime of the individuals altered the amount of CaMKIIβ expression. The expression of CaMKIIβ was
6.35 ± 1.4 (mean ± s.e.; n = 6) for the antipsychotic-treated bipolar patients (see Table 5) and 6.0 ± 0.4
(mean ± s.e.; n = 7) for the non-antipsychotic-treated bipolar patients; there was no statistically
significant difference between these values. Moreover, the expression of CaMKIIβ was 6.57 ± 0.8
(mean ± s.e.; n = 15) for the antipsychotic-treated schizophrenia and bipolar patients (see Table 5) and
6.58 ± 0.55 (mean ± s.e.; n = 10) for the non-antipsychotic-treated schizophrenia and bipolar patients;
there was no statistically significant difference between these values.
It was further necessary to determine whether suicide significantly altered the amount of
CaMKIIβ expression. The expression of CaMKIIβ was 7.1 ± 0.6 (mean ± s.e.; n = 17) for those
individuals who died by suicide (see Table 5) and 6.3 ± 0.6 (mean ± s.e.; n = 20) for all those who did
not so die (omitting the controls); the difference of 12% was not statistically significant. Moreover,
considering only those patients who died with depression, the expression of CaMKIIβ was 7.3 ± 1.0
(mean ± s.e.; n = 6) for those depressed individuals who died by suicide (see Table 5) and 7.8 ± 1.1
(mean ± s.e.; n = 7) for those who did not so die; the difference of 6% was not statistically significant.
Although no Bonferroni correction was applied to these data, there were four diagnostic groups and two
splice variants, making potentially twelve individual t-tests; thus, using a Bonferroni correction would
be overly stringent.
49
Finally, there was no relation between the post-mortem interval or patient ages (Table 5) with the
elevation of either splice variant. For example, the ages of the patients averaged 43 ± 3 y, 46 ± 3 y, 47 ±
3 y and 44 ± 4 y, for the bipolar patients, the depressed individuals, the controls, and those with
schizophrenia, respectively; these values were not statistically significantly different from each other.
Furthermore, the post-mortem intervals were 32 ± 4 h, 28 ± 3 h, 24 ± 3 h, and 35 ± 5 h, respectively.
While the control value of 24 ± 3 h was statistically significantly lower than that for the bipolar and
schizophrenia individuals, this control value was not statistically significantly different from the value of
28 ± 3 h for the depressed individuals who had the most elevated expression of CaMKIIα and CaMKIIβ
(Figures 7 and 8). Although tissue pH is a common measure for post-mortem tissue, insufficient tissue
remained for this measurement. The values for the expression of β-glucuronidase did not correlate with
the post-mortem interval (data not shown).
50
Figure 9. Effect of mRNA degradation was minimized by normalization with a housekeeping gene. Solid lines indicate mean. Top: Without standardization, the levels of CaMKIIα template per total RNA decreased with increasing 3’ to 5’ β-glucuronidase template ratio. An increase in the 3’ to 5’ ratio was a reflection of an increase in mRNA degradation. Bottom: Expressing the CaMKIIα template level as a ratio of a housekeeping gene, β-glucuronidase, minimized the effect of mRNA degradation.
51
Study No. 3: Nogo A, B and C Expression in Schizophrenia, Depression and Bipolar Frontal
Cortex, and correlation of Nogo expression with CAA/TATC polymorphism in 3’UTR.
1. Abstract for study No. 3
Schizophrenia may result from altered gene expression leading to abnormal neurodevelopment.
In a search for genes with altered expression in schizophrenia, our previous work on human frontal
cerebral cortex found the mRNA of Nogo, a myelin associated protein which inhibits the outgrowth of
neurites and nerve terminals, to be overexpressed in schizophrenia. Because those earlier results did not
examine tissues for the separate Nogo A, B and C splice variants from age- and sex-matched
individuals, we repeated the study for all three splice variants, using a new set of tissues from matched
individuals, and using the more accurate method of quantitative real-time PCR (polymerase chain
reaction). We found Nogo C to be overexpressed by 26% in the schizophrenia tissues, which is in
accordance with our earlier results. The expression of Nogo B was statistically significantly reduced by
17% in the frontal cortices from individuals who had been diagnosed as having had severe depression.
Furthermore, we show that there is a direct correlation between the expression of Nogo A and C and the
presence of alleles with a CAA insert, irrespective of disease status. While upregulation of Nogo C
expression may play a role in schizophrenia, altered Nogo B may contribute to the clinical condition of
depression. Nogo A showed a statistically non-significant increase in expression in schizophrenia.
2. Introduction for study No. 3
Schizophrenia may arise from abnormal neurodevelopment due to aberrant neuron formation or
altered expression of proteins for brain development (Bunney and Bunney, 2000; Goldman-Rakic and
Selemon, 1997). One such critical protein may be Nogo (also known as reticulon 4, RTN4, NI 250 or
RTN-X), which is highly expressed in CNS during neurodevelopment (Josephson et al., 2001). Nogo
inhibits the outgrowth of neurites and nerve terminals (Chen et al., 2000a; Prinjha et al., 2000), and may
have an important role in regulating neuronal migration and plasticity. The Nogo gene encodes three
alternatively spliced variants, Nogo A, Nogo B, and Nogo C, which share a common C-terminal domain
of 188 amino acids containing two putative transmembrane domains (Skaper et al., 2001). All three
forms of Nogo have neuron inhibitory activity (GrandPre et al., 2000). Hence, any significantly altered
expression of this gene may contribute to abnormal neuronal organization in schizophrenia.
Using competitive PCR, our previous work detected an increase in Nogo mRNA in seven post-
52
mortem frontal cerebral cortices from individuals who had schizophrenia (Novak et al., 2000). However,
the individuals in this earlier study were not age- and sex matched and the study did not examine tissues
for the separate Nogo A, B and C splice variants. The present study was done for all three splice variants
on a new larger sample of age- and sex-matched individuals, using the more accurate method of real-
time quantitative PCR. The data from individuals with schizophrenia were compared to healthy controls,
depressed individuals and individuals suffering from bipolar disorder. Nogo expression levels were also
compared with the polymorphic status of the CAA/TATC insert in the 3’UTR of the Nogo gene (Novak
et al., 2002).
3. Materials and Methods for study No. 3
Postmortem tissues were donated by the Stanley Foundation Brain Consortium (Bethesda, MD),
courtesy of Drs. Llewellyn B. Bigelow, Juraj Cervenak, Mary M. Herman, Thomas M. Hyde, Joel E.
Kleinman, José D. Paltn, Robert M. Post, E. Fuller Torrey, Maree J. Webster, and Robert H.Yolken
(Torrey et al., 2000). A total of 60 human frontal cerebral cortex tissues (Brodmann area 10) were
provided by the Stanley Foundation Brain Consortium (Bethesda, MD) from a collection of 60 brains,
fifteen each from individuals diagnosed (DSM IV criteria) with either schizophrenia, bipolar disorder,
non-psychotic depression, or neurological controls (Table 8). The diagnosis of schizophrenia was made
by at least two psychiatrists independently examining the case records and using DSM-IV criteria
(American Psychiatric Association, 1994).
3.1. Extraction of total RNA from tissues
TRIzoL® reagent (Invitrogen, Burlington, ON) was used according to the manufacturer’s
guidelines to extract total RNA from the tissues. The tissue was homogenized (Polytron, PT-10 probe,
Brinkmann Instruments, Westbury, NY) in TRIzoL® Reagent (1 ml per 50 mg of tissue) and incubated
at room temperature for 5 min before chloroform (0.2 ml per ml of TRIzoL® Reagent) was added, and
the capped tubes were shaken for 15 s. The samples were incubated at 25°C for 2 min and later
centrifuged at 12,000 x g at 4°C for 15 min. To isolate the total RNA, the aqueous upper phase was
transferred to a new tube. The RNA was precipitated using isopropyl alcohol (0.5 ml per ml of TRIzoL®
Reagent). The samples were incubated at 25°C for 10 min and centrifuged at 12,000 x g at 4°C for 10
min. The RNA pellet was washed with 75% ethanol (1 ml per ml of TRIzoL® Reagent), mixed, and
53
centrifuged at 12,000 x g at 4°C for 15 min. The RNA pellet was air dried for 5 min, dissolved in RNA
Storage Solution (Ambion), and incubated at 60°C for 10 min.
Two housekeeping genes were analyzed, β-actin and β-glucuronidase. Our experiments indicated
that the absolute values of β-actin in the tissues were an order of magnitude higher than the expression
of Nogo, making it less suitable as a housekeeping gene. A gene closer in magnitude of expression to
Nogo, β-glucuronidase, was used to assess the quality and abundance of mRNA for each tissue sample.
Only those tissues were used which showed abundant β-glucuronidase gene expression and which
yielded specific products by means of real-time PCR. The samples were not treated with DNase to avoid
further dilution of the RNA. Instead, the primers used in the Quantitative Real-Time PCR were designed
to span across intron boundaries to prevent amplification of any genomic DNA. A series of preliminary
PCR reactions were done under various conditions, followed by optical density readings during the
Real-Time Quantitative PCR runs to confirm absence of mispriming. The final total RNA concentration
was 5 µg of RNA per 2 to 6 µL of water, as determined by optical density. This solution was diluted
with RNase-free water to 5 µg RNA per 11 µL water for cDNA synthesis.
3.2. First-strand cDNA synthesis
The SUPERSCRIPT IIITM First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad,
California) was used for cDNA synthesis using oligo (dT)12-18 and 2 µg total RNA. The samples were
incubated at 50°C for 50 min to allow synthesis of the cDNA, and the reaction was terminated at 85°C
for 5 min, chilled on ice for 5 min, and treated with E. coli RNase H (37 °C, 20 min). The resultant 21 µl
cDNA samples were stored at -20°C for later use.
3.3. Real-time Quantitative Polymerase Chain Reaction
The method used to quantitate cDNA was the Quantitative Real-Time PCR method. The levels
of cDNA were determined via fluorescence by the thermal cycler during the PCR cycle, and the
instrument’s software calculated the final data. These data were downloaded into an Excel spreadsheet,
representing the final output of the PCR reactions. We found it very important to include test samples of
known concentration to confirm the accuracy of the analysis within the concentration range of the
samples being analyzed. This is rarely done in other studies, which only rely on secondary indicators of
54
validity of their data, such as slope and reaction efficiency, which we deemed insufficient from our
experience. The target value is for the detected concentration of the test samples to be within 5% of the
known concentration.
Primers were designed using the Primer Express software (Perkin-Elmer Applied Biosystems,
Foster City, California) and Oligo 4.0 (National Biosciences, Plymouth, Minnesota), and synthesized by
Sigma-Genosys (Oakville, Ontario). PCR amplification, real-time quantitation and analysis were done
using the MX4000TM Multiplex Quantitative PCR System (Stratagene, La Jolla, California). A standard
was prepared for each target gene by conventional PCR, using the same primer pairs later used in real-
time quantitative PCR. The PCR products were excised and extracted from the gel using QIAEX II ®
DNA Extraction Kit (Qiagen, Inc., Santa Clarita, California). The DNA sample (standard) was diluted
ten-fold for optical density measurements at 260 nm. The concentration of the standard was determined
from its optical density (Ausubel, 1995).
The reaction mixture for real-time PCR was prepared using the Brilliant™ SYBR® Green QPCR
Core Reagent Kit (Stratagene, La Jolla, California). The company protocol was followed for a reaction
mix containing 1.5 mM MgCl2. The total reaction volume of 25 µl contained 0.25 µl of forward primer
(10 µM) and 0.25 µl (10µM) of reverse primer. The forward primers (5’ to 3’) were
CCGATACAGAAAAAGAGGACAG for Nogo A, GGGCTCAGTGGTTGTTGACC for Nogo B and
CGTGACAAGAGATGGACGG for Nogo C. The reverse primer (5’ to 3’) for Nogo A, B and C was
AGGCTGGCACCAAACACC. The β-glucuronidase forward primer (5’ to 3’) at the 5’ end of the
mRNA was CTCTGACAACCGACGCC and the reverse primer (5’ to 3’) was
TACCACACCCAGCCGAC. The β-glucuronidase forward primer (5’ to 3’) at the 3’ end of the mRNA
was GCCGATTTCATGACTGAACAG and the reverse primer (5’ to 3’) was
TATCTCTCTCGCAAAAGGAACGC.
The SYBR® Green dye was diluted 1:2000 and the Rox reference dye was diluted 1:500. 3 µl of
1/10 diluted cDNA sample (equivalent to 28.6 ng of total RNA) was used per reaction. The cycling
parameters were 95°C for 10 min to activate the SureStartTM Taq, followed by 40 cycles of 95°C
denaturation for 30 s, 58°C reannealing for 30 s, and extension at 72° C for 40 s. In the analysis, to
minimize the effect of background noise and well-to-well variation on the results observed, our data
were normalized against the Rox reference dye and the algorithm of adaptive baseline. Each reaction
was repeated at least four times, using two independent cDNA preparations.
55
3.4. Determination of mRNA degradation.
The degree of degradation of the mRNA was determined by real-time quantitative PCR as the
ratio of the 3’-end to the 5’-end of the template of β-glucuronidase. High ratio represents high degree of
mRNA degradation. Samples with degradation ratio greater than three standard deviations from average
were excluded. The 3’/5’ ratio also provided an indication of the degree of completion of the reverse
transcription reaction, especially when using oligo-dT primers in the present study.
3.5. Detection of a CAA / TATC insert
Data and methods were described in Study No. 1 (Novak et al., 2002).
3.6. Statistical methods.
T distribution (TDIST) analysis was done for all sample groups. It returns the Percentage Points
(probability) for the Student t-distribution where a numeric value (x) is a calculated value of t for which
the Percentage Points are to be computed. The t-distribution is used in the hypothesis testing of small
sample data sets. Standard error has been calculated for all results by the formula SE=SD/SQRT(n) and
presented as value ± SE (Ausubel, 1987).
56
4. Results of study No. 3
All values are expressed as pg template per µg total RNA, all divided by pg β-glucuronidase per
µg total RNA. The values, therefore, possess no units other than the pg template per pg β-glucuronidase.
This was done because standardization to a housekeeping gene, β-glucuronidase, has greatly reduced the
effect of mRNA degradation (Figure 10). Without this adjustment, expressing values as pg per total
RNA would be misleading, because a volume of total RNA contains different amounts of mRNA due to
its high susceptibility to degradation. Standardization to β-glucuronidase has also eliminated
introduction of errors arising from variation in cDNA synthesis efficiency or pipetting during sample
manipulation and dilutions.
We have analyzed the level of mRNA degradation by determining the ratio of β-glucuronidase at
the 3’ end and at 5’ end (Figure 10). High degradation is represented by a high 3’ to 5’ ratio as mRNA is
known to primarily degrade from the 5’ end (Meyer et al., 2004). Samples with degradation levels
higher than three standard deviations from average were excluded, because adjustment by β-
glucuronidase was ineffective (the slope remained high) in these highly degraded samples and the
template levels reflected a decrease due to degradation rather than true template levels. If
standardization by glucuronidase is effective, the slope of the graph of template level (measured at the 3’
end) versus level of degradation (3’/5’) should approach zero, as level of degradation should no longer
have effect on the level of template measured. (Figure 10b.) The following samples were excluded at
different stages of the analysis. Samples 26, 24 contained no RNA. Samples 5, 10, 12, 45 failed to
amplify β-glucuronidase within levels reliably measurable by our instrument, making degradation
analysis impossible. This was an indication of very low mRNA content; furthermore, all these samples
were lower in expression than the group average less 2SD. The following samples, 6, 9, 11, 14, 19, 29,
32, 47, 48, 49, and 59 were both low in β-glucuronidase and highly degraded. These latter samples had
degradation levels higher than group average +3.2 SD and were, therefore, omitted, consistent with
Chauvenet’s criterion (Geigy, 1959). Standardization with β-glucuronidase was ineffective for these
degraded samples and they showed a high correlation of decreasing mRNA levels with the level of
degradation even after standardization.
57
a.
b.
Figure 10. Standardization eliminates effects of degradation. Effect of mRNA degradation was minimized by normalization with a housekeeping gene. (a) Before standardization and removal of degraded samples. (b) After standardization and removal of degraded samples. Expressing the Nogo A template level as a ratio of a housekeeping gene, β-glucuronidase, and discarding highly degraded samples (> average + 3SD), minimized the effect of mRNA degradation. Note that the units before standardization are pg template per µg total RNA. After standardization the units become pg Nogo A per pg β-glucuronidase.
58
The amount of Nogo A transcript in the control human frontal cortices was 7.7 ± 0.4 (pg/µg total
RNA) / (pg of β-glucuronidase/µg total RNA) and in schizophrenia tissues it was 8.6 ± 0.8, this 12%
increase was not statistically significant (TDIST, p<0.3) (Table 7, Figure 11). Nogo A did not
significantly change in bipolar disorder (7.0 ± 0.4, -9%, TDIST, p<0.1) or depression (7.0 ± 0.5, -9%,
TDIST, p<0.2).
The Nogo B transcript showed a significant decrease in depression and, to a lesser degree, in
bipolar disorder. In the control human frontal cortices the level was 2.9 ± 0.1, in depression it was 2.4 ±
0.1, a 17% decrease (TDIST, p<0.002), and in bipolar disorder it was 2.6 ± 0.2, a 10% decrease (TDIST,
p<0.15) (Table 8, Figure 12). It did not significantly change in schizophrenia (2.8 ± 0.2, -3%, TDIST,
p<0.5).
Nogo C expression was significantly higher in schizophrenia than in the control group (Table 7).
The Nogo C level in control group was 27 ± 3 and in schizophrenia it was 34 ± 5, a 26% increase
(TDIST, p<0.03) (Figure 13). It did not change considerably in bipolar disorder (25 ± 4, an 8% decrease;
TDIST, p<0.6) or depression ( 29 ± 4, a 9% increase, TDIST, p<0.6).
Table 7. Levels and percent change of Nogo A, B and C templates in three disease groups as compared to controls. Amount of transcript is expresses as pg template per pg of β-glucuronidase.
Nogo A Nogo B Nogo C Nogo A Nogo B Nogo C
Control 7.7 ± 0.5 2.9 ± 0.1 27 ± 3 — — —
Bipolar dis. 7.0 ± 0.4 2.6 ± 0.2 25 ± 4 -9%, (P<0.1) -10%, (p<0.15) -8%, (p<0.6)
Depression 7.0 ± 0.5 2.4 ± 0.1 29 ± 4 -9%, (P<0.2) -17%, (p<0.002) +9%, (p<0.6)
Schizophrenia 8.6 ± 0.8 2.8 ± 0.2 34 ± 6 +12%, (P<0.3) -3%, (p<0.5) +26%, (p<0.05)
Amount of Transcript % difference from controls
59
Table 8. Clinical summaries for postmortem brain tissues. CAA insert: 0 homozygous for no insert; 1 heterozygous for CAA insert; 2 homozygous for CAA insert. Nogo A, B and C levels are in pg template per pg β-glucuronidase. m, f - male and female, respectively. PM interval - postmortem interval. SE = SD/SQRT(n).
disorder hours years !-glucuronidase !-glucuronidase !-glucuronidase insert
4 34 m Suicide: jumped 23 15 Risperidone, valproate, venlafaxine 9.39 2.97 39.4 1
13 30 m Pneumonia, myocarditis 31 8 Lithium, clozapine 4.73 1.58 16.9 2
18 61 f Suicide: overdose 60 43 Fluoxetine, valproate 6.21 1.85 20.1 1
21 31 m Suicide: jumped 28 10 Halperidol, trazadone, trihexiphenidyl 6.12 3.31 11.9 0
22 25 f Suicide: hanged 24 6 Thiothixene, carbamazepine, lithium, trazodone 8.32 2.50 46.9 0
30 48 m Suicide: immolation 13 21 Untreated for over 20 yrs 7.77 2.97 27.0 2
31 37 f Suicide: overdose 29 23 Lithium, bupropion, !clonazepam, lorazepam 7.31 2.11 25.2 0
42 57 m Cardiac 19 27 Haloperidol, diphenhydramine 6.66 3.36 10.7 0
44 50 m Suicide: jumped 19 23 Valproate, clozapine, flurazepam, benzotropine 6.56 2.79 30.2 0
54 50 f Pulmonary emboli 62 25 Valproate, clomipramine 6.81 2.71 18.8 0
mean ± S.E. 42 ± 4 7.0 ± 0.4 2.6 ± 0.2 24.7 ± 3.7
Depression
7 51 m Suicide: gunshot 26 1 Nefazadone, hydroxyzine 7.46 3.22 19.5 1
23 65 m Cardiac 19 20 No medication for 5 yrs. 6.24 2.56 19.7 2
33 42 m Suicide: hanged 7 10 Temazepam but off medications for > 2 weeks 7.78 2.53 44.7 1
34 43 m Cardiac 43 13 Trimipramine 3.22 1.59 18.1 0
36 32 f Suicide: overdose 47 1 Imipramine, amitriptyline, !nortriptyline, clonazepam 6.37 2.40 11.1 1
37 42 f Cardiac 25 3 Fluoxetine, lithium 7.03 2.73 24.3 1
38 52 m Cardiac 12 6 No medication for 6 yrs. 8.60 2.13 44.3 1
43 56 m Cardiac 23 4 Sertraline 6.90 2.08 31.2 0
46 39 m Suicide: carbon monoxide 23 22 Never treated 6.00 2.41 22.9 1
52 47 m Cardiac 28 20 Fluoxetine, nefazadone 7.93 2.22 40.3 1
55 30 f Suicide: overdose 33 11 None 9.38 2.61 44.3 0
mean ± S.E. 45 ± 3 7.0 ± 0.5 2.4 ± 0.1 29.1 ± 3.7
Control
2 35 f Cardiac 23 None 10.01 2.62 48.2 0
3 53 m Cardiac 28 None 6.60 2.65 23.8 0
8 29 f Motor vehicle accident 42 Saw counselor for weight control 9.12 4.07 29.7 1
16 52 m Cardiac 8 None 8.11 2.81 36.0 1
20 68 f Pulmonary embolus 13 None 7.53 2.83 25.8 0
25 52 m Cardiac 28 None 7.60 2.81 26.3 1
27 41 m Pulmonary embolus 11 None 7.78 2.48 27.4 0
28 57 f Motor vehicle accident 26 None 7.35 3.68 13.0 0
41 52 m Cardiac 22 None 6.61 3.13 14.5 0
51 58 m Cardiac 27 None 7.08 2.67 30.5 0
53 44 f Cardiac 25 None 9.33 3.01 48.5 0
57 42 m Cardiac 27 None 10.35 3.02 32.0 0
58 44 m Cardiac 10 None 5.90 2.83 12.0 0
60 35 f Pulmonary embolus 40 None 4.29 2.16 7.7 0
mean ± S.E. 47 ± 3 7.7 ± 0.4 2.9 ± 0.1 26.8 ± 3.3
Schizophrenia
1 25 m Suicide: hanged 32 5 Risperidone, paroxetine 11.27 2.96 9.5 2
15 44 m Cardiac 50 27 Haloperidol, carbamazepine, fluoxetine, clonazepam benzotropine6.31 3.22 12.6 0
17 30 m Pneumonia 32 17 Risperidone,thioridazine 8.55 2.51 33.3 0
35 31 m Suicide: jumped 14 13 Clozapine 4.36 2.99 37.4 0
39 60 f Cardiac 40 45 None; had ECT 9.76 1.84 39.1 1
40 56 f Suicide: overdose 12 32 Haloperidol, lithium, diphenhydramine, chloral hydrate 9.54 3.04 43.9 1
50 62 f Motor vechicle accident 26 24 None; untreated for several months 10.06 2.90 44.2 2
56 32 m Acute alcohol intoxication 19 5 Clozapine 8.95 3.04 50.8 2
mean ± S.E. 42 ± 5 8.6 ± 0.8 2.8 ± 0.2 33.9 ± 5.3
CAA Illness Medication Nogo A per Nogo B per Nogo C perBipolar Age Death
P.M.
intervalm,f
60
Figure 11. Nogo A in schizophrenia. The amount of Nogo A transcript in the control human frontal cortices and in the schizophrenia tissues. The average was 7.7 ± 0.4 pg Nogo A per pg of β-glucuronidase, and 8.6 ± 0.8 pg Nogo A per pg of β-glucuronidase, respectively. This was an increase of 12%, which was not statistically significant (TDIST, P= 0.3).
61
Figure 12. Nogo B in depression. The average amount of Nogo B transcript in the control human frontal cortices and in samples from patients suffering from depression was 2.9 ± 0.1 and 2.4 ± 0.1 pg Nogo B per pg of β-glucuronidase, respectively. This was a decrease of 17.3% in depression (from 2.91 to 2.41), which was statistically significant (TDIST, P= 0.002).
62
Figure 13. Nogo C in schizophrenia. The average amount of Nogo C transcript in the control human frontal cortices and in the schizophrenia tissues was 27 ± 3 and 34 ± 6 pg Nogo C per pg of β-glucuronidase, respectively. This represents an increase of 26% (TDIST, P= 0.03).
63
Figure 14. Nogo A, B and C levels versus the presence of CAA insert in the 3′-UTR of the Nogo gene, irrespective of disease status. The no insert group represents only individuals homozygous for the allele carrying no insert (n= 23). The insert group represent individuals heterozygous or homozygous for the CAA insert (n= 20). Expression of Nogo A was 13% higher in the insert group (no insert 7.1 ± 0.4; insert 8.0 ± 0.4; TDIST, P= 0.01). Expression of Nogo B was unaffected by the presence or absence of the CAA insert (no insert 2.65 ± 0.12; insert 2.73 ± 0.10; decrease of 3%; TDIST, P= 0.5). Nogo C was 27% higher in individuals carrying at least one CAA insert allele (no insert 25.0 ± 2.7; insert 31.9 ± 2.6; TDIST, P= 0.009).
64
Supplemental Results
1. Effects of antipsychotics.
In a small study we have tested the effect of antipsychotics on the expression of Nogo mRNA.
Even though the results were of low statistical significance because of the small sample size (n=5), the
levels of Nogo mRNA were decreased in the haloperidol treated rats, as expected.
Figure 15. Nogo mRNA levels in antipsychotic treated rats. Nogo mRNA levels in haloperidol treated rats (n=5) as compared to a control group (n=5) of saline treated animals. The error bars represent the standard error for each group. (TDIST, P= 0.14 and P= 0.03 for PFC and striatum, respectively).
65
Nogo A Nogo B Nogo C CaMKIIα CaMKIIβ
antipsychotics 7.4±0.5 2.7±0.1 28.7±3.6 6.8±0.8 3.8±0.5
non-antipsych. 7.4±0.5 2.5±0.1 29.1±3.5 7.1±0.6 3.8±0.5
Table 9. Nogo A, B, C, CaMKIIα and CaMKIIβ levels in patients treated with antipsychotics. Nogo A, B, C, CaMKIIα and CaMKIIβ levels in patients treated with antipsychotics (Clozapine, Fluoxetine, Haloperidol, Risperidone, etc.; n=16) versus patients treated with non-antipsychotic medications (Imipramine, Clonazepam, Clomipramine, etc., or untreated; n=15).
None of the genes showed significant change in expression in patients treated with antipsychotics as
compared to patients treated with non-antipsychotic medication or untreated patients. Therefore it is
plausible that the effects of antipsychotics are exerted downstream of the CaMKII pathway. This would
explain why not all effects of CaMKII upregulation would be alleviated by these drugs and why these
drug treatments are associated with a number of side effects.
2. The mRNA levels of CaMKIIα and CaMKIIβ and their correlation with the presence of CAA
insert in the 3’UTR of the Nogo gene.
Even though the CAA insert in the 3’UTR of Nogo gene is on a different chromosome than
CaMKIIα or CaMKIIβ and hence should have no direct effect on the expression of CaMKII, I have
found a direct correlation between the presence of a CAA insert in the 3’UTR of the Nogo gene and the
mRNA levels of both CaMKIIα and CaMKIIβ isozymes. The Nogo gene is found on chromosome 2,
while the CaMKIIα and β genes are found on chromosomes 5 and 7, respectively. In individuals
carrying one or two copies of the Nogo gene with a CAA insert (n=23), compared to individuals
homozygous for a Nogo gene without an insert (n=29), CaMKIIα levels were increased by 25.5%
(TDIST, P= 0.004) and CaMKIIβ levels were increased by 23%, with high statistical significance
(TDIST, P= 0.002).
66
Figure 16. CaMKIIα mRNA levels and their correlation with the presence of the CAA insert in 3’UTR of the Nogo gene. The mRNA levels were standardized to β-glucuronidase. CaMKIIα levels in individuals with no insert were 3.2±0.3 pg CaMKIIα/pg β-glucuronidase and in individuals carrying at least one CAA insert the levels were 4.0±0.3 pg CaMKIIα/pg β-glucuronidase. The error bars represent standard error. This represents a 25.5% increase (TDIST, P= 0.004).
67
Figure 17. CaMKIIβ mRNA levels and their correlation with the presence of the CAA insert in 3’UTR of the Nogo gene. The mRNA levels were standardized to β-glucuronidase. CaMKIIβ levels in individuals with no insert were 5.9±0.5 pg CaMKIIα/pg β-glucuronidase and in individuals carrying at least one CAA insert the levels were 7.3±0.5 pg CaMKIIα/pg β-glucuronidase. The error bars represent standard error. This represents a 23% increase (TDIST, P= 0.002).
68
DISCUSSION
Discussion for study No. 1
Because early-onset schizophrenia is generally a more severe form of schizophrenia than late-
onset schizophrenia, we examined the data for any relation to age of onset. The data suggest that
individuals with early-onset schizophrenia (before 20 years of age) were much less likely to carry the
CAA insert. However, because of the limited number of samples in this group, supplied by the National
Neurological Research Specimen Bank in Los Angeles and by the Canadian Brain Tissue Bank in
Toronto, these findings are only suggestive, and a much more extensive series of samples need to be
examined. We also analyzed the prevalence of the CAA insert in bipolar and depressed individuals
(Cantor-Graae et al., 2001). It has been hypothesized that schizophrenia and affective disorders may
share some genetic susceptibility (Berrettini, 2000; Maier et al., 1993). We found a slightly increased
prevalence of the CAA insert in these affective disorders. However, the sample size was too small to
comment on the possible role of the CAA insert. In a group of African and Afro-American samples,
there was a 2.8-fold increased prevalence of the homozygous CAA insert polymorphism in
schizophrenia. However, the overall prevalence of the CAA insert in the population was only 3%. These
frequency data were too low for statistical analysis, but do indicate a much lower prevalence of the CAA
insert in African and Afro-American population.
The 5´- and 3´-untranslated regions (UTR) of eukaryotic mRNAs have been shown to regulate
several aspects of gene expression. In particular, the 3´-UTR is involved in the regulation of mRNA
stability (Mitchell and Tollervey, 2001), translation initiation (Gray and Wickens, 1998), and mRNA
subcellular localization (Jansen, 2001). In neurons, several mRNAs have been demonstrated to be
targeted to dendrites via 3´-UTR motifs (Kiebler and DesGroseillers, 2000; Mohr, 1999). Mutations in
the 3´-UTR are associated with a number of diseases, including neuroblastoma, myotonic dystrophy and
α-thalassemia (Conne et al., 2000). The functional role of the CAA insert in the Nogo 3´-UTR is
unknown, and it does not match any known 3´-UTR functional motifs (Pesole et al., 2002). However,
polymorphisms in the Nogo 3´-UTR could contribute to altered expression of the Nogo gene in
schizophrenia.
In order to see whether there is any relation between the prevalence of the CAA insert and the
extent of Nogo expression in the post-mortem human frontal cortex, a separate series of tissues will need
to be examined. This is because we only had sufficient post-mortem tissue from six of the seven
69
schizophrenia tissues in order to obtain both the expression and the DNA sequence. Although all but one
of these schizophrenia tissues revealed a CAA insert (compared to three out of eight post-mortem
control samples), only one schizophrenia sample was homozygous for the CAA insert. Moreover, using
a specific antibody to the Nogo protein, the abundance of the Nogo protein itself will need to be
examined as well in the post-mortem tissue. (At the time, additional or sufficient post-mortem
schizophrenia tissues were not available from the brain banks.)
The gene for Nogo is located on chromosome 2p13–14 (Nagase et al., 1998; Yang et al., 2000),
and genome scans have found schizophrenia to be associated or linked to this region, 2p15–p12 (Coon et
al., 1998; Shaw et al., 1998). The present findings, therefore, offer new support to this chromosomal
region as a site of schizophrenia susceptibility. Increased Nogo mRNA expression in schizophrenia, as
described above, may lead to elevated levels of Nogo protein expression, and may, therefore, result in
increased Nogo receptor stimulation. The Nogo receptor is located on chromosome 22q11 (Fournier et
al., 2001), a region strongly associated with schizophrenia (Bassett et al., 1998; Karayiorgou et al.,
1995). Nogo and its receptor may, therefore, be of considerable relevance to the biological basis of
schizophrenia.
Obviously, the data needs to be confirmed in a much larger series of samples. However, if the
high prevalence of the Nogo CAA insert in schizophrenia holds in a more extensive study, then
individuals homozygous for the Nogo CAA insert would have a significantly elevated risk for
developing schizophrenia. Furthermore, increased Nogo gene expression in schizophrenia also suggests
Nogo may have a role in the disturbed neurodevelopment and plasticity in schizophrenia, since Nogo is
hypothesized to regulate neuronal migration during development (Skaper et al., 2001) and plasticity in
the adult (Buffo et al., 2000).
Discussion for study No. 2
The main finding was that the CaMKIIβ transcript was significantly elevated in the post-mortem
frontal cerebral cortex tissues of individuals who died with schizophrenia or depression (tissue supplied
by the Stanley Foundation Brain Consortium). This finding may be relevant to the development of
schizophrenia or depression. For example, it is known that neural maturation is related to the ratio of α
and β isoforms of CaMKII (Shen and Meyer, 1999). Thus, there are three aspects of neurodevelopment
in which dysregulation of CaMKIIβ may contribute to the development of schizophrenia.
70
First, CaMKII controls many molecular aspects of postnatal dendritic growth and maturation,
including the phosphorylation of GABA-A receptor subunits (Churn and DeLorenzo, 1998; Macdonald
and Olsen, 1994; Wang et al., 1995), and indirectly may assist in the activation of NMDA and AMPA
receptors in early development. (Ben-Ari et al., 1994; Swope et al., 1999). Phosphorylation of the NR2B
subunit of the NMDA receptor by CaMKII is critical for axon guidance and for establishment and
maturation of dendritic spines during neurodevelopment (Rakic et al., 1986). Elevating CaMKII activity
levels in young neurons to levels of mature neurons slows their dendritic growth (Shen et al., 1998). The
increased CaMKIIβ levels observed in schizophrenia patients, if found during early development, may
play a role in abnormal neurodevelopment and subsequent predisposition to schizophrenia.
Second, during puberty, CaMKII plays a key role in CNS maturation, controlling ingrowth of
dopaminergic fibers and pruning of neurons (Soderling, 2000). The timing of schizophrenia onset
coincides with a change in CaMKII subunit composition and its Ca2+ decoding parameters at puberty.
This shift determines the pre- and post-pubertal response characteristics of neurons. Absence of this
maturation, induced by upregulation of CaMKII in mutant animals, leads to neuropathology and
behavioural defects in adulthood (Dudek and Bear, 1992; Keshavan et al., 1994; Mayford et al., 1995).
Third, in an important animal model of schizophrenia, methamphetamine sensitization (Suemaru et al.,
2000), there is an excess of the β isoform of CaMKII, relative to the α isoform (Greenstein et al., 2007;
Lou et al., 1999). This is consistent with our present data showing a much greater increase in CaMKIIβ
than CaMKIIα in schizophrenia.
In addition, CaMKII modulates catecholamine metabolism via phosphorylation of tyrosine
hydroxylase; thus, a disturbance in CaMKII may be compatible with a possible abnormality in dopamine
signaling in schizophrenia (Burt et al., 1976; Seeman et al., 1975; Seeman et al., 1976). Given the
important role CaMKII plays in neuronal development, maturation and signaling, deviations from its
normal level of expression may have clinically significant implications. However, if CaMKIIβ is truly
important for the pathophysiology of schizophrenia, the increase of 27% in the schizophrenia tissues is
surprisingly less than that of the 36% increase found in the tissues from the depressed individuals.
Naturally, the relative or absolute magnitude of these differences will vary considerably depending on
the clinical mix in the patient populations. Although the expression of CaMKIIβ was also elevated in the
post-mortem tissues from severely depressed individuals (Figure 8), the clinical relevance of this finding
is not clear. One of the current hypotheses in the field of antidepressant research is that the
antidepressants may alter the number of synaptic spines. While this may be possible, the present data did
71
not reveal any effect of the clinical antidepressants on the expression of CaMKIIβ; the expression of
CaMKIIβ was 7.5 ± 1.1 (mean ± s.e.) in seven depressed patients who had been on antidepressants,
essentially similar to the value of 7.7 ± 0.8 (mean ± s.e.) in six depressed patients who had not been on
antidepressants. Therefore, if antidepressants alter synaptic spine sprouting, there is no reason to
consider CaMKIIβ as a contributing factor.
Discussion for study No. 3
Because mRNA only comprises approximately 10% of total RNA and is highly susceptible to
degradation, the quality of the mRNA was important to determine. This was done by analyzing the 3’ to
5’ ratio of β-glucuronidase. Although all the samples were standardized to contain the same amount of
total RNA, the mRNA levels could vary greatly depending on the extent of mRNA degradation Figure
10a). The results show that this effect was minimized by standardizing the expression values with β-
glucuronidase (Figure 10b). No non-normalized data were used. All the data presented are normalized to
β-glucuronidase, resulting in the dimensionless unit of expression for the Nogo variants as (pg Nogo
variant /µg total RNA) per (pg β-glucuronidase/µg total RNA).
As noted above, earlier work (Novak et al., 2002) found Nogo mRNA (which consisted of Nogo
A + B + C) to be overexpressed in seven unmatched schizophrenia frontal cortices in tissues from the
National Neurological Research Specimen Bank in Los Angeles and from the Canadian Brain Tissue
Bank in Toronto. The present data, using a different group of age- and sex-matched tissues from the
Stanley Foundation Brain Consortium (Bethesda, MD) and a more accurate method, indicate that Nogo
C is overexpressed in schizophrenia (26% increase) (Figure 13). A novel observation is that Nogo B is
significantly underexpressed in depression (-17%) (Figure 12). Nogo A did not show a significant
change in any of the disease groups examined (Figure 11). The reason for the relative overexpression of
Nogo in the earlier schizophrenia cortices (Novak et al., 2002) stemmed from the fact that Nogo C
constitutes by far the largest fraction of the Nogo mRNA (75%) and is overexpressed in schizophrenia.
Therefore, our current results are in accordance with our previous results.
The gene for Nogo is at chromosome position 2p13-14 (Yang et al., 2000), and genome scans
have found schizophrenia to be associated with this region, 2p15-p12 (Coon et al., 1998; Shaw et al.,
1998; Straub et al., 2002). Thus, altered Nogo mRNA expression in schizophrenia may lead to altered
levels of Nogo protein expression and altered stimulation of the Nogo receptor. The gene for the Nogo
receptor is located on chromosome 22q11 (Fournier et al., 2001), a region also associated with
72
schizophrenia (Bassett et al., 1998; Karayiorgou et al., 1995). Thus, Nogo and its receptor may be
relevant to the biological basis of schizophrenia (Nogo C) and other disorders, such as depression (Nogo
B). In a previous publication we have identified a CAA and a TATC insertion in the 3’UTR of the Nogo
gene (Novak et al., 2002). There is a direct correlation between the expression of Nogo A and C and the
number of alleles with CAA insert and individual carries, irrespective of the disease status (Figure 14).
The inverse is true for TATC insert, as it is generally not inherited with the CAA insert on the same
allele. Generally, an allele has either the CAA insertion or the TATC insertion, but not both. People
carrying a CAA insert on at least one of their Nogo alleles have a 13% higher Nogo A expression and a
27% higher Nogo C expression than people homozygous for alleles without an insert. (If the samples are
sorted based on the presence of TATC insert, the TATC insert samples show statistically nonsignificant
decrease in Nogo C expression).
The 5’- and 3’- untranslated regions (UTR) of eukaryotic mRNAs have been shown to regulate
several aspects of gene expression. The 3’-UTR is involved in the regulation of mRNA stability
(Mitchell and Tollervey, 2001), translation initiation (Gray and Wickens, 1998) and targeting (Kiebler
and DesGroseillers, 2000). Mutations in 3’-UTR are associated with a number of diseases, including
neuroblastoma, myotonic dystrophy and α-thalassemia (Conne et al., 2000). I remains to be shown
whether the 3’UTR CAA insert in Nogo directly results in upregulation of Nogo A and C or if it is
coinherited with another regulatory region, although after sequencing the Nogo mRNA from a number
of individuals, we did not detect any other sequence variants (Novak et al., 2002).
The roles Nogo A, B or C may play in the etiology of schizophrenia or depression are hard to
predict, because we still do not know how these splice variants differ in function. We do know that all
three Nogo splice variants show different developmental and morphological expression patterns (Oertle
et al., 2003a) and, therefore, likely play different roles determined by both developmental stage and
tissue specificity. The difference between the splice variants lies in their N terminal, which determines
their protein interactions (Hu et al., 2005). There are at least three different functional domains present
in Nogo. The N-terminal region common to Nogo A and B is involved in the inhibition of fibroblast
spreading, a central region present only in Nogo A that restricts neurite outgrowth, cell spreading and
induces growth cone collapse, and a C-terminal region common to all splice variants which has a growth
cone collapsing function (Oertle et al., 2003c). Change in the levels of individual splice variants would
then be expected to produce different outcomes.
73
Nogo is a strong inhibitor of neurite outgrowth (Buffo et al., 2000; Chen et al., 2000b; GrandPre
et al., 2000; Prinjha et al., 2000). It is highly expressed by oligodendrocytes in CNS myelinated tissues
during fetal development (Huber et al., 2002; Josephson et al., 2001) and it is hypothesized to suppress
gene expression of neuronal transcription factors, e.g., c-Jun and JunD, which are associated with
neuronal growth (Zagrebelsky et al., 1998). Nogo is not surprisingly also involved in other
neurodegenerative diseases. Autoantibodies to Nogo-A have been detected in MS patients (Reindl et al.,
2003), with Nogo as an important modulator of the immune response to autoimmune-mediated
demyelination (Karnezis et al., 2004). Analogous to our results in schizophrenia, the relative levels of
Nogo splice variants were shown to be differentially affected in other disease states as well. In ALS
levels of Nogo-A protein are elevated and correlate with disease severity and degree of muscle atrophy,
while Nogo-C levels are deceased (Dupuis et al., 2002; Jokic et al., 2005). The splice variant we
identified as significantly elevated in schizophrenia tissues, Nogo-C, in addition to a growth cone
collapsing function, may also act similarly to RTN1-C, a protein of the same family with close similarity
to Nogo-C (Oertle et al., 2003b; Yan et al., 2006), which is involved in neuronal differentiation (Hens et
al., 1998).
Increased levels of Nogo C in schizophrenia may influence neuronal differentiation, neuronal
migration during embryogenesis and synaptic pruning during adolescence. Synaptic pruning is thought
to be associated with schizophrenia and occurs at the time of the disease onset (Feinberg, 1990;
Hickmott and Constantine-Paton, 1997; Keshavan et al., 1994). Observations of maldistribution of
interstitial neurons in prefrontal white matter (Akbarian et al., 1996), as well as enlarged lateral and
third ventricle and smaller anterior hippocampi (Suddath et al., 1990b) do point to a possible
developmental failure, such as abnormal neuronal migration (Suddath et al., 1990).
Nogo-B, which we found significantly decreased in depression, has been shown to have diverse
functions from apoptosis (Qi et al., 2003; Tagami et al., 2000) to vascular remodeling (Acevedo et al.,
2004). Nogo B (RTN-Xs) was shown to interact with Bcl–2 and Bcl-Xl and reduce their anti-apoptotic
activity (Tagami et al., 2000). Bcl-2 was shown to be part of the route by which chronic stressors could
impact depressive state (Hayley et al., 2005). Bcl-Xl is another bcl-2 family protein, which may be
involved in modulation of synaptic stability (Jonas et al., 2003). Recently it has been demonstrated that
Fluoxetine, a widely used antidepressant compound, up-regulated expression of Bcl-2, Bcl-xL (Chiou et
al., 2006). Nogo B is located close to a locus that has been associated through linkage with increased
risk of suicide attempts in families with recurrent, early-onset, major depression (Zubenko et al., 2004).
74
General Discussion
To date, no single gene or pathway has been able to account for all the hallmark signs of
schizophrenia. With the discovery of the involvement of CaMKII, we may be closer to explaining the
full range of pathophysiological findings in schizophrenia. We know that the disease has a strong
genetic component and likely involves more than one gene. We also know that the phenotype is
expressed only in 50% of individuals carrying such genes (Cardno et al., 1999; Risch, 1990) with adult
onset (Kendler et al., 1987).
From a molecular point of view, the identification of CaMKII as a gene key to schizophrenia
explains the involvement of the NMDA, AMPA, GABA and DA signaling pathways (Carlsson et al.,
2001; Harrison et al., 2003; Lewis, 2000; Meador-Woodruff and Healy, 2000; Seeman, 1987).
Furthermore, it explains the observed pathophysiology, which includes abnormal neuronal migration
(Akbarian et al., 1996; Benes, 2000; Kovalenko et al., 2003), anomalous synaptic plasticity and
connectivity (Frankle et al., 2003) and exaggerated, transporter dependent, release of dopamine (DA)
(Breier et al., 1997; Laruelle et al., 1996).
As a corollary, if CaMKII is a key element in the etiology of schizophrenia, perturbation of the
CaMKII pathway should then be able to mimic symptoms of schizophrenia. The following discussion
will focus on the individual pathways affected by upregulation of CaMKII and correlate the effects with
observed pathology in schizophrenia.
1. CaMKII plays an important role in age of onset of schizophrenia
CaMKII mediates synaptic pruning during adolescence, which occurs at the time of onset of
schizophrenia and is thought to be associated with the disease (Feinberg, 1990; Hickmott and
Constantine-Paton, 1997; Keshavan et al., 1994; Teicher et al., 1995). During normal development,
hippocampus shows a developmental loss of capacity for LTD during adolescence. CaMKII controls this
switch from LTD to LTP (Mayford et al., 1995; Mayford et al., 1996a; Muller et al., 1989). This
coincides with the development of dopaminergic innervation in the prefrontal cortex and is abolished in
CaMKII mutant mice (Goldman-Rakic, 1996; Mayford et al., 1995). In transgenic mice with
hyperactivated CaMKII, an animal model of CaMKII upregulation, the capacity for LTD is not lost in
adulthood. High expression of constitutively active CaMKIIα not only shifts the threshold from LTP
toward LTD, but also triggers a number of compensatory mechanisms, such as upregulation of the
inhibitory neuropeptide Y, possibly reducing the LTP induction threshold, and an increase in potassium
75
currents through increase of Kv4.2 channel expression, which decreases excitability. Overall, these
changes lead to the enhancement of some neural connections and elimination of others (reviewed by
(Elgersma et al., 2004)) and would corroborate the neuropathological findings in schizophrenia and
support the abnormal pruning hypothesis of schizophrenia (Feinberg, 1990; Keshavan et al., 1994;
Mayford et al., 1995; Segal et al., 2000; Soderling, 2000). Furthermore, CaMKII mice carrying a
hyperactive form of the enzyme show increased sensitivity to NMDA-R antagonists at concentrations
that do not affect wild type mice. This also parallels the symptoms of schizophrenia (Ohno et al., 2002).
Given the role CaMKII plays during the transition from immature to adult neuronal function, it is
not surprising that CaMKIIα mutant mice show adult onset phenomena (Elgersma et al., 2004). Yet,
interestingly, CaMKIIα null mutants also show neurodevelopmental defects severely affecting about
50% of the animals (Gordon et al., 1996), as seen in schizophrenia. These defects include disturbance in
behaviour such as decreased fear phenomena and high aggression, as well as defects in cognition and
neocortical plasticity (Chen et al., 1994; Elgersma et al., 2004; Mayford et al., 1995; Mayford et al.,
1996a; Silva et al., 1992a; Silva et al., 1992b). This is similar to responses seen in hippocampus-
lesioned animals, another animal model of schizophrenia (Elgersma et al., 2004; Silva et al., 1992a).
Normal learning has been shown in several non-hippocampus dependent learning tasks, an indicator that
this is a good animal model of the disease (Elgersma et al., 2004).
2. Dysregulation of the CaMKII pathway can explain key pathophysiological signs of
schizophrenia.
Developmentally, at the molecular level, CaMKIIβ, which at first is expressed in the absence of
CaMKIIα, determines the establishment of the AMPA, NMDA and GABA signaling pathways (Ben-Ari
et al., 1997). The α and β isoforms drive neuronal differentiation in opposite directions. While
CaMKIIα enhances synaptic strength, CaMKIIβ promotes branching and development of new synapses
(Fink et al., 2003). Overexpression of CaMKIIβ increases dendritic arborization, motility, filopodia
extension, and synapse number. Developing hippocampal neurons, where CaMKIIβ is highly expressed,
show high arborization of dendrites (Fink et al., 2003). Furthermore, the α:β expression ratio of CaMKII
plays a particularly important role in the development and density determination of glutamatergic
synapses (Burgin et al., 1990; Rongo and Kaplan, 1999; Wu and Cline, 1998). This would be in
accordance with the developmental theory of schizophrenia and with findings of abnormal neuronal
migration and plasticity. It would also account for the involvement of the glutamatergic and GABAergic
76
molecular pathways, as well as the dopaminergic pathway since glutamatergic neurons then interact with
dopaminergic neurons via inhibitory GABAergic interneurons (Lieberman et al., 1997).
Schizophrenia is also said to be a disease of the synapse (Frankle et al., 2003; Mirnics et al.,
2001a). The proper levels of CaMKIIα and β are key in normal functioning of the synapse, as well as its
structure and localization (Fink and Meyer, 2002). Deviation from correct α:β ratio results in functional
consequences on synaptic transmission, with overexpression of β rendering these synapses oversensitive
to low Ca2+ signals and easier to trigger (Ahmed et al., 2006; Thiagarajan et al., 2002; Ullian et al.,
2001). CaMKII overexpression also leads to abnormal increases in ion channel activity in activated
synapses (Derkach et al., 1999). In addition, CaMKII activity controls the retention of active presynaptic
partners, which may lead to aberrant retention of synaptic contacts (Pratt et al., 2003). This again is in
accordance with the current theory of schizophrenia neuropathology.
Upregulation of CaMKII also explains the increased sensitivity of schizophrenia patients to
psychostimulants such as amphetamine (Benes, 2000). This is also true in amphetamine sensitized
animals, which is another model of the disease. This unique type of DA release is transporter-mediated,
rather than vesicular (Jones et al., 1998; Kantor et al., 1999; Pierce and Kalivas, 1997; Raiteri et al.,
1979) and is mediated by CaMKII, whose activation is essential for the enhanced DA release (Jones et
al., 1998; Raiteri et al., 1979; Warburton et al., 1996). This dopamine release is mediated by the
phosphorylation of the dopamine transporter (DAT) by CaMKII which results in a reversal of transport
through DAT and dopamine efflux. It is this non-vesicular DA release which is induced by amphetamine
sensitization and present in schizophrenia (Fog et al., 2006; Sulzer et al., 2005). Furthermore, the
function of DAT is controlled by its direct contact with D2Rs. Disruption of this contact results in
hyperlocomotion, analogous to DAT mutant mice (Lee et al., 2007) or CaMKIIα deficient mice (Chen
et al., 1994; Silva et al., 1992a).
As expected, upregulation of CaMKII results in NMDA-R antagonist sensitivity analogous to the
one seen in schizophrenia (Ohno et al., 2002). Correspondingly, inhibition of hippocampal CaMKII
impairs amphetamine conditioning (Tan, 2002) and attenuates amphetamine induced DA release
sensitized rats (Kantor et al., 1999; Pierce and Kalivas, 1997). Studies of opioid tolerance suggest that it
is upregulation of the CaMKIIβ isozyme which is key in this response, as withdrawal of morphine
results in CaMKIIβ overshoot, but does not affect α (Fan et al., 1999; Lou et al., 1999).
The dopaminergic theory is central to the etiology of schizophrenia and therefore it is important
that upregulation of CaMKII can explain the involvement of the DA pathway. There are many
77
interactions between the CaMKII and D2R pathways. The D2R disrupts the interaction between NR2B
and CaMKII, leading to reduced phosphorylation of the NMDA-Rs (Liu et al., 2006). In turn,
upregulation of CaMKII positively upregulates the D2R promoter (Takeuchi et al., 2002). CaMKII also
influences catecholamine synthesis through phosphorylation of tyrosine hydroxylase and tryptophan
hydroxylase (Ohyama et al., 2002; Yamauchi, 2005).
3. Environmental factors
CaMKII plays an important role in abnormal synaptic function, especially in adaptation to the
factors which precipitate schizophrenia. Stress, hypoxia and glutamate receptor agonists have long been
known to be able to elicit psychosis (Tsuang, 2000).
Hippocampus is a major target of stress induced changes (Kim and Diamond, 2002; McEwen
and Sapolsky, 1995). After exposure to stress, hippocampal neurons show changes in cellular
morphology and dendritic remodeling, including atrophy and decreased length of the dendritic branches
and loss of synapses (Du et al., 2004; McEwen, 2003; Sapolsky, 2000). These stress induced alterations
arise from increased glutamatergic neurotransmission through increased levels of glutamate in the
hippocampus and stimulation of the NMDA-Rs and can be prevented by NMDA antagonists (Du et al.,
2004; McEwen, 2003; Sapolsky, 2000) or by AMPA antagonists (Suenaga et al., 2004). Since CaMKII
regulates these pathways, it may have major contribution to the stress induced pathophysiology (Molnar
et al., 2003; Xing et al., 2002). Acute stress upregulates CaMKII and in turn also the AMPA and
NMDA receptors in the hippocampus, while chronic stress results in inhibition of CaMKII and decrease
in BDNF (Aleisa et al., 2006; Suenaga et al., 2004). It is plausible that it is the inability to properly
downregulate CaMKII during chronic stress that may predispose individuals to the disease. In fact, in
chronically stressed rats, nicotine has been shown to counter some of the effects of chronic stress by
increasing the basal levels of BDNF protein. This activated CaMKII through the TrkB/ PLCg pathway
(Minichiello et al., 2002).
During hypoxia, oxygen and glucose supplies are disrupted, resulting in dysregulation of ionic
gradients and depolarization of the plasma membrane. The resulting glutamate release leads to
overstimulation of AMPA and NMDA receptors and large increase in Ca2+ influx, which overactivates
CaMKII (Lee et al., 1999; Merrill et al., 2005). Such insult to an already predisposed system may also
contribute to the development of schizophrenia. By inhibiting CaMKII activity, ischemia-induced
78
accumulation of CaMKII at the PSD is greatly reduced and such drugs may be therapeutically useful
(Meng and Zhang, 2002; Merrill et al., 2005).
In summary, CaMKIIβ upregulation in schizophrenia leads to CaMKII holoenzymes containing
higher β:α ratio will be more responsive to smaller, lower frequency Ca2+ spikes (Brocke et al., 1999).
This will lead to abnormal signal decoding, resulting in altered neuronal response to a given Ca2+ signal.
While CaMKIIα enhances synaptic strength, overexpression of β results in filopodia extension,
increased branching and in development of new synapses (Fink et al., 2003). Expression of CaMKIIβ
also has effect on CaMKIIα expression, downregulating the expression of α, while α has no effect on β
(Thiagarajan et al., 2002). This results in altered maturation, with neurons of an immature phenotype
(Shen and Meyer, 1999) and in abnormal dendritic growth (Shen et al., 1998). The interaction with other
pathways is also be altered. Increase in β lowers synaptic charge and increases signaling frequency, as
well as responsiveness to NMDA-R activity and enhanced inhibition by AMPA-R activity (Thiagarajan
et al., 2002). The overall resulting immature phenotype of neurons with synapses easily triggered by
lower than normal Ca2+ concentrations, abnormal glutamatergic signaling and increased outflow of DA
through the DAT are all in accordance with the findings in schizophrenia.
4. Depression
CaMKII plays a critical role in serotonergic regulation of PFC neuronal activity, which may
partially explain the neuropsychiatric behavioral phenotypes of increased aggression seen in CaMKII
knockout mice (Cai et al., 2002). Long term administration of antidepressants, such as the tricyclic
antidepressant desipramine and norepinephrine reuptake inhibitor reboxetine, was shown to significantly
increase the kinase activity in presynaptic vesicles of frontal and prefrontal cortex (Celano et al., 2003;
Tiraboschi et al., 2004). The SSRI paroxetine increased membrane levels of activated CaMKII which
phosphorylated the AMPA-R and enhanced AMPA-R conductance and receptor delivery (Martinez-
Turrillas et al., 2007). Similar effect was produced by the 5-HT and noradrenaline (NA) reuptake
inhibitor venlafaxine or by a repeated electroconvulsive shock (ECS). Both treatments induced a large
increase in the activity of bound kinase and a decrease in the activity of soluble kinase (Du et al., 2004;
Popoli et al., 2002). This may reflect an increase in β, as β docks the CaMKII α/β holoenzymes. This is
in accordance with our findings, since CaMKIIα, the soluble variant of CaMKII, was found elevated in
depression and would be decreased by the treatment. Because of the similar effects of so many different
79
types of treatments for depression on CaMKII, it is possible that CaMKII may be an important target in
the treatment of depression (Du et al., 2004; Popoli et al., 2002).
CaMKII also plays an important role in catecholamine and serotonin synthesis. Tyrosine
hydroxylase and tryptophan hydroxylase are phosphorylated by CaMKII and then activated by activator
protein (14-3-3 protein). Furthermore, binding of CaMKII to syntaxin is an important step in the
regulation of the exocytosis pathway (Ohyama et al., 2002). Reviewed by (Yamauchi, 2005).
5. Nogo and its role in schizophrenia
Nogo plays an important role in neuronal migration and maturation of the GABAergic neurons.
Aberrant Nogo expression results in improper axon branching, polarization and alterations in migration
patterns (Mingorance-Le Meur et al., 2007). All three Nogo-NgR1 triggered pathways increase the
stabilization of formed F-actin (Maekawa et al., 1999), which is bound by CaMKIIβ and serves as a
reservoir for the inactivated form of the kinase. Hence upregulation of Nogo would impact the balance
between the pools of active and inactive kinase.
Hypoxia upregulates Nogo A/B (Schmidt-Kastner et al., 2006; Wang et al., 2006; Zhou et al.,
2003) and it has been observed that prenatally experienced hypoxia may play a role in predisposition to
schizophrenia (Kietzmann et al., 2001). Hypoxia activates genes involved in vascular development,
which are important players in neurodevelopment, because neurogenesis is greatly depends on
angiogenesis (Carmeliet, 2003; Palmer et al., 2000). This would be in agreement with the role Nogo-B
plays in vascular remodeling (Acevedo et al., 2004).
The Nogo and CaMKII pathways are also coupled via PKC. Like the D2Rs, the NgR couples to
the inhibitory guanine nucleotide binding protein (Gi) (Cai et al., 1999) and triggers intracellular
elevation of Ca2+ as well as the activation of PKC (Hasegawa et al., 2004). Activation of PKC interferes
with CaMKII-NMDA-R interaction, dispersing the synaptic NMDA-Rs, but driving CaMKII to
synapses (Fong et al., 2002). High PKC levels induced by stress were shown to trigger thought disorder
(Johnson et al., 1989).
The exact roles Nogo A, B or C may play in the etiology of schizophrenia or depression are hard
to predict, because we still do not know the many different functions of these splice variants. An
example of this is its involvement in numerous pathological states. We do know that all three Nogo
splice variants show different developmental and morphological expression patterns (Oertle et al.,
2003a) and, therefore, likely play different roles determined by both developmental stage and tissue
80
specificity. Change in the levels of individual splice variants would then be expected to produce
different phenotypic outcomes. Nogo is highly expressed by oligodendrocytes in CNS myelinated
tissues during fetal development (Huber et al., 2002; Josephson et al., 2001) and it is hypothesized to
suppress gene expression of neuronal transcription factors, which are associated with neuronal growth
(Zagrebelsky et al., 1998).
The splice variant we identified as significantly elevated in schizophrenia tissues, Nogo-C, may
also act similarly to RTN1-C, a protein of the same family with close similarity to Nogo-C (Oertle et al.,
2003b), which is involved in neuronal differentiation (Hens et al., 1998). Increased levels of Nogo C in
schizophrenia may influence neuronal migration during embryogenesis. Observations of maldistribution
of interstitial neurons in prefrontal white matter (Akbarian et al., 1996) do point to a possible
developmental failure, such as abnormal neuronal migration (Suddath et al., 1990a).
Schizophrenia and depression may have some etiology in common, especially considering rates of
comorbid depression disorders in patients with schizophrenia (Association, 2000), with nearly 60% of
patients with schizophrenia experiencing a depressive syndrome at some time during the course of their
illness (Lewandowski et al., 2006; Martin et al., 1985). Nogo- B, which we found significantly
decreased in depression, has been shown to have diverse functions from apoptosis (Qi et al., 2003;
Tagami et al., 2000) to vascular remodeling (Acevedo et al., 2004). Nogo B (RTN-Xs) was shown to
interact with Bcl–2 and Bcl-Xl (Tagami et al., 2000). Bcl-2 was shown to be part of the route by which
chronic stressors could impact depressive state (Hayley et al., 2005). Bcl-Xl is another bcl-2 family
protein, which may be involved in modulation of synaptic stability (Jonas et al., 2003). Recently it has
been demonstrated that Fluoxetine, a widely used antidepressant compound, up-regulated expression of
Bcl-2 and Bcl-xL (Chiou et al., 2006). In addition, Nogo is located close to a locus that has been
associated through linkage with increased risk of suicide attempts in families with recurrent, early-onset,
major depression (Zubenko et al., 2004).
Nogo is involved in a number of neurodegenerative diseases. Autoantibodies to Nogo-A have
been detected in multiple sclerosis (MS) patients (Reindl et al., 2003), because it is an important
modulator of the immune response to autoimmune-mediated demyelination (Karnezis et al., 2004).
Nogo-A is elevated in amyotrophic lateral sclerosis (ALS), and correlate with disease severity, while
Nogo-C levels are decreased (Dupuis et al., 2002; Jokic et al., 2005). Nogo was shown to be markedly
up-regulated in temporal lobe epilepsy (TLE), associated with extensive neurodegeneration of
hippocampal neurons and hippocampal reorganization (Bandtlow et al., 2004). Nogo-A was also found
81
to be increased in pyramidal cells in the hippocampus in Alzheimer’s disease (AD) (Gil et al., 2006).
Brain NgR interacts with amyloid precursor protein (APP) through its amyloid-β (Aβ) peptide domain
and reduces Aβ, a protein accumulating in AD plaques, by blockade of α/β secretase or by increased
clearance of (Aβ) protein (Park et al., 2006).
6. The link between CaMKIIβ expression and the CAA insert in the Nogo gene.
The mode of inheritance of schizophrenia suggests the involvement of a number of genes with
mild mutations or possible mutations in regulatory elements of these genes. This implies that
polymorphisms in regulatory elements (“cis-acting variations”) could play a significant role (Bray et al.,
2003). The background in which these genes are expressed may also be important. Neves-Pereira et al.
(2005) studied a polymorphism in the BDNF gene as a risk factor for schizophrenia and showed that the
risk for a disease may depend upon the haplotypic background on which a gene variant is carried
(Neves-Pereira et al., 2005).
Both the gene for Nogo and for its receptor, NgR, lie in chromosomal hotspots linked to
schizophrenia. Nogo is located at 2p13-14 (Yang et al., 2000), which was identified as a possible
susceptibility locus (2p15-p12) (Coon et al., 1998; Shaw et al., 1998; Straub et al., 2002) and NgR is
located on 22q11 (Fournier et al., 2001), a schizophrenia susceptibility locus 4 (SCZD4) 22q11.2
(Bassett et al., 1998; Karayiorgou et al., 1994; Karayiorgou et al., 1995; Online Mendelian Inheritance
in Man, 2007; Pulver et al., 1994). The NgR has recently been identified by Hsu et al. (2007) as possibly
modulating the genetic risk or clinical expression of schizophrenia (Hsu et al., 2007). The gene for the α
subunit of CaMKII is located at 5q32, a locus which has been linked to schizophrenia (SCZD1, 5q23.2-
q34) (Bassett et al., 1988; Lewis et al., 2003; Online Mendelian Inheritance in Man, 2007; Paunio et al.,
2001; Sklar et al., 2004). CaMKIIβ gene is located at 7p14.3-p14.1, a locus that has not yet been
identified as being linked to schizophrenia. It is possible that either a change in the CaMKIIβ gene,
leading to CaMKIIβ dysregulation, is of minor effect and depends strongly on the genetic background in
which it is expressed, or this dysregulation is of secondary effect, primary cause perhaps being an insert
in a regulatory element of a gene such as Nogo. This insert may cause the dysregulation of the Nogo
pathway, which interacts with the CaMKII pathway. CaMKII subunit expression is in turn controlled by
a feedback mechanisms within the CaMKII pathway, perturbation of which would alter the subunit
expression. Another possibility is that Nogo alters the expression of the CaMKII genes directly, through
activation of transcription factors, as Nogo is known to interact with GAP43, CAP23, LIF kinase, c-Jun
82
JunD, and other neuronal transcription factors (Zagrebelsky et al., 1998). In either case we should see a
direct correlation between the presence of CAA insert and high CaMKII expression levels, which is in
fact what I have observed (Figures 16 and 17). Nogo upregulation may lead to an aberrant neuronal
migration, maturation and function, affecting many signaling pathways, such as Kv channels, Gi protein
signaling, the Rho kinase pathway and PKC. This may in turn affect CaMKIIβ signaling, particularly
through BDNF or via Ca2+ currents induced through Gi pathway and through PKC. Since CaMKII plays
highly important role during maturation, especially at puberty, it is quite likely that the preexisting
abnormality manifests itself during this key period of neuronal pruning and functional neural shift, both
of which seem to be controlled by CaMKII.
It is of great interest that new evidence is emerging and confirming the role of CaMKII in
schizophrenia and other psychiatric illnesses, including drug addiction (Li et al., 2008).
We may not be able to correct an insert in the Nogo gene, but the use of CaMKII inhibitors may
perhaps prevent development of the disease in susceptible individuals who show an upregulation of
CaMKII. Further studies are necessary to determine whether CaMKII upregulation is present before
adolescence and whether CaMKII inhibitors would be effective in preventing the development of this
disease.
83
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