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pecialized roles of neurofilament proteins in synapses: Relevance toeuropsychiatric disorders
idong Yuana,b,∗, Ralph A. Nixona,b,c,∗
Center for Dementia Research, Nathan Kline Institute, Orangeburg, New York, 10962, United StatesDepartments of Psychiatry, New York University School of Medicine, New York, NY, 10016, United StatesDepartment of Cell Biology, New York University School of Medicine, New York, NY, 10016, United States
r t i c l e i n f o
rticle history:eceived 11 July 2016eceived in revised form 2 September 2016ccepted 3 September 2016vailable online 5 September 2016
eywords:eurofilament subunit
a b s t r a c t
Neurofilaments are uniquely complex among classes of intermediate filaments in being composed of foursubunits (NFL, NFM, NFH and alpha-internexin in the CNS) that differ in structure, regulation, and func-tion. Although neurofilaments have been traditionally viewed as axonal structural components, recentevidence has revealed that distinctive assemblies of neurofilament subunits are integral componentsof synapses, especially at postsynaptic sites. Within the synaptic compartment, the individual subunitsdifferentially modulate neurotransmission and behavior through interactions with specific neurotrans-mitter receptors. These newly uncovered functions suggest that alterations of neurofilament proteins
ynapseendritic spineeuropsychiatric disease
not only underlie axonopathy in various neurological disorders but also may play vital roles in cog-nition and neuropsychiatric diseases. Here, we review evidence that synaptic neurofilament proteinsare a sizable population in the CNS and we advance the concept that changes in the levels or post-translational modification of individual NF subunits contribute to synaptic and behavioral dysfunctionin certain neuropsychiatric conditions.
Neurofilaments (NFs), the intermediate filaments of matureeurons, are among the most abundant proteins in brain. Unlikehe intermediate filaments of other cell types, which are usually
∗ Corresponding authors at: Center for Dementia Research, Nathan Kline Institute,rangeburg, New York, 10962, United States
homopolymers, NFs in the CNS are hetero-polymers composed ofNFL, NFM, NFH and alpha-internexin subunits (Yuan et al., 2006).Although structurally distinctive, these four NF subunits share abasic tripartite domain structure consisting of a conserved cen-tral �-helical rod region, a short variable head domain at the
amino-terminal end and a tail of highly variable length at the C-terminal end. The short head domain is rich in serine and threonineresidues and contains consensus sites for O-linked glycosylationand phosphorylation (Yuan et al., 2012a). The central rod domain,
hich is relatively conserved among intermediate filament familyembers, contains long stretches of hydrophobic heptad repeats
avoring formation of �-helical coiled-coil dimers. The C-terminalomains contain glutamic- and lysine-rich stretches of varying
ength that mainly establish the size range (58–200 kDa on SDSels) of the four NF subunits. Although all intermediate filamentypes serve roles as structural scaffolds, NF subunit heterogeneitylso confers specialized structural properties to NF, in axons wherehe filaments are extremely long and are often arranged in paral-el with uniform spacing conferred by the long C-terminal tails ofFM and NFH extending perpendicularly from the filament core
Rao et al., 2003, 2002). These unique space filling properties of NFacilitate their well-established role in caliber expansion of large-iameter myelinated axons of peripheral nerves, which is criticalor effective nerve conduction (Zhu et al., 1997). NFs also exten-ively cross-link with other cytoskeletal elements along axons toorm a large metabolically stable stationary NF network (Nixon andogvinenko, 1986; Yuan et al., 2015a, 2009) that is critical to axonaliber expansion (Friede and Samorajski, 1970; Hoffman et al.,987; Ohara et al., 1993) and organelle distribution along axonsRao et al., 2011).
Besides these unique structural and functional features, NF sub-nits are distinguished from other intermediate filament proteinsy the complex regulation of their head and tail domains by phos-horylation, especially those of NFM and NFH, which involvesctions of multiple protein kinases and phosphatases at manyolypeptide sites (Pant and Veeranna, 1995). Although certainhosphorylation events are known to control tail extension andubunit assembly and slow turnover, the purpose of such complexnd dynamically changing phosphate topography on NF subunitsde Waegh et al., 1992; Nixon and Sihag, 1991; Pant and Veeranna,995) has remained puzzling, given the mainly static structuralupport roles ascribed to NF. Both the complex hetero-polymerictructure and dynamically changing phosphate topography of NFroteins suggests that individual NF proteins might serve addi-ional biological roles although there has been relatively littlexploration of this issue until recently.
NF gene mutations are well recognized as causes of severaleurological disorders mainly involving degeneration of periph-ral nerve fibers in accordance with the prominent function of NFn supporting large-diameter myelinated axons (Brownlees et al.,002). Notably, however, NF proteins are abundant in grey matterNS regions as well as white matter (Chan et al., 1997) but influ-nce caliber expansion much less dramatically in most populationsf CNS axons (Dyakin et al., 2010). These observations and evidencehat NF subunits can be axonally transported in various minimallyssembled forms (including as heterodimers) suggest a broader dis-ribution and range of assembly forms of NF subunits within CNSeurons, including substantial populations in synapses. In light ofhese findings, alterations of a particular NF subunit as seen inpecific brain regions in psychiatric and neuropsychiatric disor-ers (Cairns et al., 2003; Clinton et al., 2004; Garcia-Sevilla et al.,997) may reflect an alteration within synapses which influenceshe clinical phenotype.
Psychiatric diseases, affecting an estimated 54 million Amer-cans yearly, cause mild to severe disturbances in thought orehavior usually in the absence of known changes in axonal
ntegrity. Nevertheless, changes in levels and phosphorylation ofF subunits have consistently been noted in certain psychiatricisorders although the location of these changes within neurons isoorly understood. Psychiatric diseases prominently involve alter-tions of synaptic transmission. The highly specialized composition
f synapses includes not only the well characterized vesicular androtein receptor machinery supporting neurotransmission but also
specialized cytoskeleton important for delivering, inserting, andecycling synaptic components. Like other domains in a neuron,
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the cytoskeleton in synapses is composed of microtubules, actinfilaments, the spectrin-rich membrane skeleton, and as recentlyshown, NF assemblies (Yuan et al., 2015b, 2015c). Evidence is alsoemerging that the cytoskeleton, and especially the NF scaffold, actsas a docking platform to organize the topography of organelleswithin different neuronal compartments. Rearrangements of thistopography are dynamically coordinated at least in part by cellularsignals regulating the phosphorylation state of the binding part-ners (Perrot and Julien, 2009; Rao et al., 2011; Styers et al., 2004;Yuan et al., 2015b). Such evidence suggests a range of possibili-ties for understanding the newly recognized roles of individual NFsubunits in modulating synaptic function. In this review, we con-sider the properties of synaptic NF assemblies in the CNS and, inthis context, raise the possibility that certain changes in levels orphosphorylation of NF subunits reported in neuropsychiatric dis-orders (Table 1) disrupt synaptic signaling or, in some cases, reflectadaptive or maladaptive responses to these synaptic disruptions.
2. NF proteins in synapses
2.1. Distinctive assemblies of NF subunits in synapses
Synapses have long been considered to be degradative sites forNF reaching terminals by axonal transport (Roots, 1983). WhenNF proteins have been detected in synaptic fractions and boundto synaptic proteins in vitro, they have previously been viewedas contaminating axonal NF proteins (Matus et al., 1980) andtheir possible role in synapses has rarely been entertained. Usingmultiple independent approaches, however, Yuan et al. recentlyprovided definitive evidence that all four CNS NF subunits are inte-gral resident proteins of synapses and have distinct roles in synapticfunction and behavior (Fig. 1)(Yuan et al., 2015b, 2015c). The NFassemblies isolated from synapses are distinctive as compared tothose in other parts of the neuron in terms of their morphologyand in having higher proportions of alpha-internexin, a loweredphosphorylation state of NFM, and a higher NFH phosphorylationsstate. This unique population of synaptic NF proteins is more abun-dant in the postsynaptic area than in adjacent dendritic areas orpresynaptic terminals and exhibits a different response to subunitperturbation than the axonal population. For example, when NFM isdeleted from mice, NFH phosphorylation and the ratio of NFH to NFLsubunit increase significantly in the synaptic pool in comparison tothe total NF pool (Yuan et al., 2015b). Because NF subunit monomersare inefficiently transported along axons (Yuan et al., 2003, 2006,2015b), NF proteins in synapses are at least oligomeric in form andcan be transported as such in axons, providing one possible basisfor their synaptic location. It is also possible that some NF proteinin postsynaptic boutons is synthesized locally because mRNAs ofNFL (Crino and Eberwine, 1996; Paradies and Steward, 1997) andalpha-internexin (Villace et al., 2004) are reported to be present indendrites and polyribosomes are preferentially localized under thebase of dendritic spines (Steward and Levy, 1982). In this regard,evidence suggests that NF proteins identified in nerve terminalsisolated from squid brain are produced by local protein synthesis(Crispino et al., 1993; Martin et al., 1990). In light of these findings,aforementioned reports noting the presence of NF in synaptic frac-tions and interactions of NF proteins with known synaptic proteinscan be viewed as further support for a substantial synaptic pool ofNF proteins in the brain.
Remarkably, previous proteomic analyses of synaptic fractionshave consistently identified NF proteins but these findings were not
followed up with additional proof of a synaptic localization or func-tion. During the study of activity-regulated proteins in postsynapticdensity fractions by two-dimensional gel electrophoresis and massspectrometry, Satoh et al. identified a total of 90 spots contain-
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Table 1Neurofilament Subunit Expression in Human and Rodent Models of Psychiatric Disease.
Disease NF subunit (gene symbol) Model system Regulation
Protein level Phosphorylation Brain region Method Reference
Schizophrenia NEFL Human Decrease DLPFC WB (Kristiansen et al., 2006)Human Decrease ACC white matter Proteomic (Clark et al., 2007)Human Decrease CC Proteomic (Sivagnanasundaram et al., 2007)Human Decrease DLPFC Proteomic (Pennington et al., 2008)Human Decrease DLPFC white matter Proteomic (English et al., 2009)
NEFM Human Decrease CC Proteomic (Sivagnanasundaram et al., 2007)NEFH Human Decrease DLPFC white matter Proteomic (English et al., 2009)INA Human Increase ACC white matter Proteomic (Clark et al., 2007)
Human Increase CC Proteomic (Sivagnanasundaram et al., 2007)Bipolar disorder NEFL Human Decrease DLPFC Prpteomic (Pennington et al., 2008)
Human Decrease DLPFC white matter Proteomic (English et al., 2009)NEFM Human Increase DLPFC Proteomic (Pennington et al., 2008)
Human Increase DLPFC white matter (English et al., 2009)NEFH Human Decrease DLPFC white matter Proteomic (English et al., 2009)INA Human Decrease DLPFC Proteomic (Pennington et al., 2008)
Drug addiction NEFL Rat Decrease VTA WB (Beitner-Johnson et al., 1992)Rat Decrease VTA IHC (Bunnemann et al., 2000)Human Decrease Frontal cortex WB (Garcia-Sevilla et al., 1997)Neuronal culture Decrease Hippocampus IHC (Saunders et al., 1997)
NEFM Rat Decrease Increase VTA WB (Beitner-Johnson et al., 1992)Rat Decrease VTA IHC (Bunnemann et al., 2000)Neuronal culture Decrease Hippocampus IHC (Saunders et al., 1997)
NEFH Rat Decrease Increase VTA WB (Beitner-Johnson et al., 1992)Rat Decrease VTA IHC (Bunnemann et al., 2000)Neuronal culture Decrease Hippocampus IHC (Saunders et al., 1997)
Alzheimer disease NEFL Human Decrease Occipital cortex WB (Bajo et al., 2001)NEFM Human Decrease Decrease Sciatic nerve WB (Holzer et al., 1999)
Human Increase Hippocampus IHC (Sternberger et al., 1985)Human Increase Hippocampus WB (Vickers et al., 1994)Human Increase Frontal cortex WB, IHC & iTRAQ (Rudrabhatla et al., 2010)
NEFH Human Decrease Decrease Sciatic nerve WB (Holzer et al., 1999)Human Increase Hippocampus IHC (Sternberger et al., 1985)Human Increase Hippocampus WB (Vickers et al., 1994)Human Increase Frontal cortex WB, IHC & iTRAQ (Rudrabhatla et al., 2010)Human Increase Temporal cortex IHC (Thangavel et al., 2009)
INA Human Increase Cortex Proteomic & WB (Zhou et al., 2013)
Note: DLPFC, dorsolateral prefrontal cortex; ACC, anterior cingulate cortex; CC, corpus callosum; WB, Western blotting; IHC, immunohistochemistry; iTRAQ, isobaric peptide tags for relative and absolute quantification.
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Fig. 1. Functional NF subunit assemblies in synapses. Left panel: Immunogold labeled antibodies against the NFM subunit decorating synaptic structures in a linear pattern(immunogold particles outlined in blue) suggesting the presence of short NFs and protofilament/protofibril or unit length filament assemblies. In the upper inset, a filamentwithin a postsynaptic bouton is decorated by immunogold antibodies to both NFL (large gold dots) and NFH (small gold dots). Morphometric analysis indicates a higherdensity of immunogold labeling in postsynaptic boutons than in preterminal dendrites or presynaptic terminals (graph inset). Middle panel: Ultrastructural image of ahuman synapse depicts membranous vesicles, many of which appear to be associated with a loose network of short 10 nm filaments in the post-synaptic region. Right panel:E alizeds endosP
imiateifpp2pNtaatopt2twemadtNot(bu(ose
vidence supports a biological mechanism whereby D1 dopamine receptors internubunit assemblies (outlined in blue) where they are readily available to recycle on
sychiatry 2015; 20:915 with permission).
ng 47 different protein species in the PSD fractions isolated fromouse forebrain (Satoh et al., 2002). Among these spots, alpha-
nternexin and NFM accounted for 8 and 4 spots, respectively. Withn improved proteomic method involving nanoflow HPL coupledo electrospray tandem mass spectrometry (LC–MS/MS), Jordant al. identified all four CNS NF proteins (NFL, NFM, NFH and alpha-nternexin) as abundant components of biochemically purified PSDractions from rat or mouse brain(Jordan et al., 2004). A subsequenthospho-proteomic analysis of PSD proteins, identified 42 phos-hoproteins in PSD preparations from mouse brains (Trinidad et al.,005). Out of 90 phosphorylated peptides derived from these 42hosphoproteins, NF proteins accounted for 23 (NFL 1, NFM 5 andFH 17), indicating the considerable abundance of NF proteins in
he PSD. NF proteins are also glycosylated and in a glyco-proteomicnalysis of PSD preparations from mouse brain, NFL, NFM andlpha-internexin were among 18 identified GlcNAc-modified pro-eins (Vosseller et al., 2006). A limited idea of how NF proteins arerganized within the PSD has been achieved using a novel solidhase and chemical crosslinking approach to distinguish proteinshat reside either at the surface of the PSD or in its interior (Liu et al.,006). The analysis suggested that NFM, NFH and alpha-internexinogether with tubulin subunits reside in the interior of the PSDhile actin resides primarily on the surface. Recently, Moczulska
t al. performed precise quantification of the mouse synaptoso-al proteome during postnatal maturation (from 3 − 8 weeks of
ge) using peptide-based iTRAQ labeling and high-resolution two-imensional peptide fractionation (Moczulska et al., 2014). Amonghe 3422 identified proteins with complete quantifications, NFL,FM, NFH and alpha-internexin were established to be componentsf the synaptosomes and their levels increased during brain postna-al maturation, a period when synapse formation sharply increasesAghajanian and Bloom, 1967). Alpha-internexin had long beenelieved to form a separate filament system from NF triplet proteinsntil it was shown to be the fourth subunit of NFs in the mature CNS
Yuan et al., 2006) although somewhat over-represented among thether NF subunits present in synapses (Yuan et al., 2015b). Ben-on et al. detected alpha-internexin immunoreactivity in dendritesxtending into dendritic spines of cultured hippocampal neurons
on endosomes from the postsynaptic surface (red asterisks) dock on synaptic NFomes to the surface in response to ligand stimulation (adapted from Yuan et al. Mol
(Benson et al., 1996). This immunoreactivity receded from spinesand remained at the base of dendritic protrusions when cells weretreated with latrunculin A to disrupt actin filaments (Zhang andBenson, 2001).
Besides their association with the PSD, NF subunits have beennoted to interact with other synaptic proteins. The synapse-specificPSD95-associated protein SAPAP interacts via its N-terminal regionwith all 4 NF subunits, co-immunoprecipitates with NF proteinsfrom rat brain, and co-localizes with NFs in transfected COS cells(Hirao et al., 2000). Spinophilin, a synaptic protein highly enrichedin dendritic spines (Allen et al., 1997), regulates the formation andfunction of the spines (Feng et al., 2000). Using a shotgun pro-teomic approach, Baucum et al. showed that spinophilin interactsvia its N-terminal domain with NFL, NFM, and alpha-internexin inmouse striatum (Baucum et al., 2010, 2012; Hiday, 2015). More-over, the association increased significantly during mouse brainmaturation (Baucum et al., 2012) during the period of synapto-genesis (Aghajanian and Bloom, 1967) and was dependent on thephosphorylation state of either spinophilin or NF protein (Hiday,2015). The NFL subunit also directly binds to tau (Miyata et al.,1986), a protein initially considered to localize in dendritic spinesonly under pathological conditions (Hoover et al., 2010), but isnot believed to have a physiological role in dendritic functioning(Frandemiche et al., 2014; Ittner et al., 2010). Knockdown of tauusing specific shRNA significantly decreased the spine density ofcultured hippocampal neurons (Chen et al., 2012) and deletion oftau in mice abolished long-term depression (Kimura et al., 2014).Thus the interaction between NFL and tau in dendritic spine couldbe important for normal synaptic transmission.
NF polymers are highly abundant in myelinated axons as esti-mated by conventional electron microscopy but 10 nm filamentshave also been noted in the perikarya and dendrites of neurons,including ones lacking a myelinated axon (Dyakin et al., 2010;Hirokawa et al., 1984; Kong et al., 1998; Zhang et al., 2002). In
1977, Blomberg et al. reported 10 nm filaments as major con-stituents of the partially broken up PSD isolated from dog cerebralcortex and assumed that they were composed of NF proteins(Blomberg et al., 1977). Using a rapid-freezing technique, Landis
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nd Reese described infrequent 9–10 nm filaments in dendriticpines of mouse brain but interpreted these structures as actin fila-ents due to their periodicity (Landis and Reese, 1983). Yuan et al.
lso identified in some synapses that short 10 nm filaments wereecorated by immunogold antibodies to different NF subunits indi-ating that at least some synaptic NF subunits exist in polymerizedorm (Yuan et al., 2015b, 2015c). Although NF proteins are presentn both pre- and postsynaptic areas, 10 nm NF polymers are notbundant in synaptic areas compared to myelinated axons, esti-ated by conventional electron microscopy. The infrequency of
onventional long intermediate-sized polymers in synapses indi-ates that most NF subunits in synapses are probably in the formf oligomeric structures (protofilaments or protofibrils). Consistentith this interpretation, transport of NFM in non-filamentous, but
ligomeric form, has been reported (Terada et al., 1996; Yabe et al.,999; Yuan et al., 2003). The organization of oligomeric NF assem-lies in synapses, quite different from those parallel arrays withpacing controlled by C-terminal extensions of NFH/MFM in axons,ight be closely associated with the synaptic actin network since
isruption of such actin filaments also remarkably affects the dis-ribution of NF assemblies (Zhang and Benson, 2001). The relativebundance of NF proteins in synapses is synapse type-dependent,.g., NF proteins are not present at the postsynaptic sites of neuro-uscular junctions. Instead, another intermediate filament protein
esmin is highly concentrated near the AChR-rich crests of the junc-ional folds at the neuromuscular junctions (Sealock et al., 1989).
.2. NF subunit-specific modulation of synaptic functions andehavior
N-methyl-d-aspartate (NMDA) receptors are excitatory neuro-ransmitter receptors critical for synaptic plasticity and neuronalevelopment in the mammalian brains (Li and Tsien, 2009). Theseeceptors are highly concentrated in postsynaptic membranes oflutamatergic synapses. Yeast-two-hybrid screening revealed thatFL interacts through its rod domain directly with the cytoplasmic-terminal domain of NR1 (Ehlers et al., 1998) suggesting that NFsay anchor or localize NMDA receptors within the neuronal plasmaembrane. Co-expression of NFL with either NR1 or NR2B subunits
n HEK293 cells did not increase surface expression of these sub-nits whereas expression of all three components increased surfacebundance of NR1 by 20% and concomitantly increased NMDA-ediated cytotoxicity (Ratnam and Teichberg, 2005). The NR1
ubunit is ubiquitinated in HEK293 cells and the co-expression withFL antagonizes this ubiquitination process, suggesting anotheray that the interaction of NFL with NR1 may stabilize the NMDA
eceptor. NFL also binds to protein phosphatase-I (PP1), a pro-ein/serine/threonine phosphatase in the PSD (Terry-Lorenzo et al.,000). NFL-bound PP1 could regulate the phosphorylation statesf NF subunits and NMDA NR1 receptors the cellular distributionf which is regulated by the phosphorylation of specific serinesn the C1 exon cassette (Ehlers et al., 1995). On the one hand,FL may modulate synaptic plasticity through interactions withR1, PP1 or 14-3-3 (Ehlers et al., 1998; Miao et al., 2013; Terry-orenzo et al., 2000). On the other hand, the phosphorylation statef NFL is regulated by synaptic plasticity events such as long-termotentiation (LTP) (Hashimoto et al., 2000b) and long-term poten-iation depression (LTD) (Hashimoto et al., 2000a). NFL selectivelynfluences dendritic spine and glutamate receptor function sinceFL-null mice not only have abnormal spines but also dysfunc-
ion of hippocampal-dependent spatial memory, NMDA receptorrotein expression and synaptic plasticity (Yuan, Veeranna et al.
npublished data). LTP is a persistent increase in synaptic strengthollowing high-frequency stimulation of a chemical synapse ands generally considered one of the major cellular mechanisms thatnderlie learning and memory (Bliss and Collingridge, 1993). Yuan
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et al. recently determined LTP in the Schaffer collateral path-way of the hippocampal slices prepared from wild-type controland IHL-TKO mice lacking alpha-internexin, NFH and NFL sub-units (Yuan et al., 2015b). The tetanic stimulation evoked a typicalLTP of fEPSP in slices from wild-type mice. These responses werestable for 120 min. However, tetanic stimulation evoked a signifi-cantly reduced fEPSP slopes in IHL-TKO mice, indicating NF proteinsare, therefore, required for synaptic plasticity. Because LTP in theSchaffer collateral pathway of the hippocampus is NMDA receptor-dependent, altered LTP may be associated with reduced levelsof NMDA-NR1 receptor (Yuan, Veeranna et al. unpublished data).Yuan et al. also conducted a 5-trial social memory assay to deter-mine if IHL-TKO mice have a social memory defect. In this test,the subject mouse was given four brief exposures (trials 1–4) in itshome cage to the same stimulus mouse (intruder). In the 5th trial,the subject mouse encountered an entirely novel stimulus mouse(novel intruder). Wild-type control mice displayed normal socialmemory, as demonstrated by a marked habituation (decreasedexploration) during the first 4 trials and a striking dishabituation(increased exploration) on the presentation of a novel animal on the5th trial. In contrast, IHL-TKO mice showed no significant habitua-tion during the 4 exposures to the stimulus mouse or dishabituationto the novel stimulus mouse. Consistent with reduced LTP, IHL-TKOshowed social interaction deficits, indicating that mice lacking NFproteins failed to develop normal social memory.
Dopamine receptors are G-protein-coupled receptors thatmediate functions of dopamine ranging from voluntary move-ment and reward to hormonal regulation and hypertension (Giraultand Greengard, 2004). These receptors are highly concentrated inthe postsynaptic membrane of dopaminergic synapses. Yeast-two-hybrid screening has shown that the C-terminal tail of NFM directlyinteracts with the cytoplasmic loop of dopamine D1receptor (Kimet al., 2002) and that their co-expression in HEK-293 cells resultedin more than 50% reduction of receptor binding. Recently, the rele-vance of this interaction at synapses was established in geneticallymodified mice. Deletion of NFM, but not deletion of any of the otherNF subunits, greatly amplified dopamine D1-receptor-mediatedmotor responses to cocaine while redistributing postsynaptic D1-receptors from endosomes to the plasma membrane (Fig. 2)(Yuanet al., 2015b). In wild-type mice, NFM colocalized with the D1receptor in synaptic boutons by immunogold electron microscopy.Deletion of NFM also significantly increased D1R-stimulated hip-pocampal LTP further indicating an in vivo interaction betweenNFM and D1R (Yuan et al., 2015b). Depressed hippocampal LTPinduction is NF subunit-selective: maintenance of LTP is deficientin NFH-null mice while basal neurotransmission and induction ofLTP are normal in NFM-null mice. Eliminating all NF proteins fromthe CNS profoundly disrupted synaptic plasticity and social mem-ory without altering the structural integrity of synapses (Yuan et al.,2015b).
Peripherin, a subunit of peripheral nerve NFs, is almost exclu-sively expressed in the neurons of PNS and in the CNS only indefined subsets of neurons directly projecting to the periphery(Brody et al., 1989; Yuan et al., 2012b). However, brain injuries suchas stab lesions and focal ischemia can trigger peripherin expres-sion in CNS neurons normally silent for this gene (Beaulieu et al.,2002). Although definitive evidence for the presence of periph-erin in synapses in the CNS is lacking, electrophysiological studiesrevealed synaptic plasticity was markedly altered in transgenicmice over-expressing the normal peripherin gene under its ownpromoter (Kriz et al., 2005). LTP was 50% greater in CA3 and 50%diminished in CA1 of hippocampus of the mutant mice than in wild-
type controls. These data indicate a role for peripherin as a mediatorof plasticity of hippocampal synapses potentially relevant to cogni-tive diseases. Peripherin was recently shown to be a strong bindingpartner of Rab-7A mutations of which are capable of altering the
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Fig. 2. Model of D1R-containing endosomes anchored on NFM-containing cytoskeletal assemblies. Based on collective findings on NF scaffolding functions and our D1R dataon NF subunit null mice, we propose a model by which NFM acts in synaptic terminals to anchor D1R-containing endosomes formed after agonist-induced internalization ofm apse wr 1R ago2
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atio of soluble and insoluble peripherin in vitro and are causativef CMT2B (Cogli et al., 2013), indicating that peripherin may alsoave a role in vesicular transport. More recent studies involvingeast two-hybrid screens established interactions of peripherinnd SNAP25-interacting protein SIP30 through coiled-coil domainsGentil et al., 2014) further suggesting a potential important role oferipherin in regulating vesicular trafficking at the synapses.
. Alterations of NF proteins in psychiatric disorders
.1. Schizophrenia and bipolar disorder
Schizophrenia, a severe chronic brain disorder affecting ∼1%f the population (Perala et al., 2007), is characterized by abnor-al interpretation of reality and social behavior. The behavioral
yndrome reflects a combination of positive symptoms (eg. hal-ucinations, delusions, and disordered thinking and behavior),egative symptoms (eg. reduced expression, feelings, and speechroduction), and cognitive deficits (eg. poor executive function-
ng, working memory, and attention). The causes of schizophreniare unknown. Various alterations of neuronal and glial functionsave been reported although loss of neurons or neurodegenera-ive change is not considered fundamental to disease development.ostmortem brains from individuals with schizophrenia are usu-lly characterized by reduced dendritic spine density (Garey et al.,998; Glantz and Lewis, 2000; Sweet et al., 2009).
Pharmacological and biochemical evidence supports an impor-ant role of NMDA receptor hypofunction in the pathophysiologyf schizophrenia (Balu et al., 2013; Coyle, 2006). Many genetic riskactors for schizophrenia have been reported, including inheritedommon single nucleotide polymorphisms, copy number variants,are single nucleotide variants and rare de novo variants. De novoenomic copy number variants known to substantially increaseusceptibility to schizophrenia are enriched for members of theMDA receptor postsynaptic signaling complex and are signifi-antly more frequent in individuals with schizophrenia than inontrols (Kirov et al., 2012). Fromer et al. confirmed these find-ngs and further implicated abnormalities of synaptic cytoskeletonegulation and glutamatergic neurotransmission (Fromer et al.,
014). The fact that NFL, NFM and NFH genes map to chromoso-al regions (8p21, 8p22 and 22q12, respectively) that are strongly
mplicated in schizophrenia suggests the involvement of NF pro-eins in this disease (Badner and Gershon, 2002; Lewis et al., 2003).s earlier noted, NFL in synapses (Yuan et al., 2015b) binds the
ould favor desensitization to D1R stimulation: in the absence of NFM, the greaternists, as observed in our in vivo studies (adapted from Yuan et al. Mol Psychiatry
NR1 receptor and may stabilize its presence in the cell mem-brane by preventing its ubiquitination and subsequent degradation(Ehlers et al., 1998; Ratnam and Teichberg, 2005). Besides bind-ing NR1 to influence NMDA receptor function, NFL has also beenshown to interact in a phosphorylation-dependent manner with14-3-3 gamma (Miao et al., 2013), another protein implicated inschizophrenia (Middleton et al., 2005; Toyooka et al., 1999).
Of further potential relevance to NMDA receptor dysfunctionin schizophrenia is a consistent body of evidence that points toselectively reduced levels of NFL subunits in brain regions essentialfor the cognitive and behavior functions affected in schizophre-nia (Kristiansen et al., 2006). In dorsolateral prefrontal cortex(DLPFC), NFL transcript expression is significantly increased acrossall cortical isodense layers in DLPFC from elderly individuals withschizophrenia (mean patient age 80y) while levels of NFL pro-tein are decreased about 50%, collectively suggesting possibly thatturnover of NFL is abnormally high. Proteomic analysis of theDLPFC in schizophrenia revealed synaptic and metabolic abnor-malities, including significantly reduced NFL (1.5-fold) and NFMlevels (1.2-fold) without changes in NFH and alpha-internexin pro-teins (Pennington et al., 2008). Reduced NFL and NFM levels werealso found in study of DLPFC deep white matter in schizophrenia(English et al., 2009) and in the ACC (anterior cingulate cortex)white matter (Clark et al., 2007) where a 3-fold increase (p < 0.05)in alpha-internexin was also measured. Similar changes in NFLand alpha-internexin levels were seen in another proteomic anal-ysis of schizophrenic corpus callosum (Sivagnanasundaram et al.,2007). Although NFL transcription was significantly decreased inthe thalamus of younger adult schizophrenics (mean age 43y), itwas increased 25%-30% relative to controls, along with NFM levels,in elderly schizophrenics (mean age 70y) (Clinton and Meador-Woodruff, 2004). Collectively, these results implicate regionalabnormalities of NFL transcription and/or accelerated NFL degra-dation in schizophrenia. NFL protein has been shown to be morevulnerable than NFM and NFH subunits to calcium-dependent pro-teases (Zimmerman and Schlaepfer, 1982). The changes observedin NFs may be part of the dynamic cytoskeletal remodeling in thepathogenesis of schizophrenia. Dramatic changes in the levels of NFproteins were also observed in axotomized motor and sensory neu-rons (Hoffman et al., 1987; Tetzlaff et al., 1996; Wong and Oblinger,1990). In light of evidence linking NFL to the functional activity of
the NMDA receptor, the lowered levels of NFL could be a poten-tial contributor to the NMDA hypofunction proposed to underlievarious phenotypic features of schizophrenia.
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Bipolar disorder is a brain disease that causes extreme moodwings that include emotional highs and lows. The exact causef bipolar is unknown. Imaging and cellular studies have identi-ed dysfunction of the dorsolateral prefrontal cortex in bipolarisease (Drevets et al., 1997; Rajkowska et al., 2001; Strakowskit al., 2002). NFL transcripts are reported significantly reduced,long with NFM and alpha-internexin proteins (Pennington et al.,008), in adults with bipolar disorder (mean age 42y), but not inajor depressive disorder (Clinton and Meador-Woodruff, 2004).
xamining the differential protein expression in the deep whiteatter from DLPFC from individuals with bipolar disorder and con-
rols, English et al. identified differences in 15% of proteins classifieds cytoskeletal proteins which included increased NFM (1.66 fold,
= 0.03)(English et al., 2009).Given that most studies of psychiatric disorders focusing on
ytoskeletal changes analyzed brain areas enriched in white matterxons and the alterations of NF proteins may well reflect disturbedunction of the axonal compartment. Given the ubiquity and abun-ance of synapses in CNS, it is also possible that even white matternalyses capture alterations of the significant synaptic NF proteinool in neuropsychiatric disorders. It is also reasonable to expecthat future deeper analyses that include terminal regions of theseame vulnerable brain regions and target additional relevant greyatter regions enriched with synaptic boutons, may identify dys-
unction of the synaptic cytoskeletal network, including NF subunitnteractions, more sensitively.
.2. Drug addiction
Drug addiction is a chronic brain disorder that causes com-ulsive drug seeking and use despite adverse consequences. Theesolimbic pathway, which connects the ventral tegmental area
VTA) to the nucleus accumbens, has been implicated in commonechanisms of drug addition (Pierce and Kumaresan, 2006). Theesolimbic pathway releases dopamine into the nucleus accum-
ens, where it affects motivation for rewarding stimuli. Drugsbused by humans such as morphine, cocaine, ethanol and nico-ine have very different chemical structures but all preferentiallyncrease synaptic dopamine concentrations in the mesolimbic sys-em (Di Chiara and Imperato, 1988). This is believed to be the neuralubstrate mediating the reinforcing properties of drugs of abuse.iven their diverse primary sites of action on cell surface recep-
ors, these addictive drugs have to act indirectly on intracellularroteins as a convergence point to exert similar effects on mesolim-ic dopamine function. Beitner-Johnson et al. first reported that NFroteins NFL, NFM and NFH were decreased 15- 50% in the VTAy chronic administration of morphine or cocaine in rats (Beitner-ohnson et al., 1992). The lowering of NF protein levels by theserugs was selective since the levels of 8 other major cytoskeletal orytoskeletal-associated proteins did not change. Chronic exposureo psychotropic drugs lacking reinforcing properties did not alterF levels. In separate studies, morphine administration caused aecrease of NFL immunoreactivity in rat cerebral cortex, which wasntagonized completely by concurrent administration of naloxoneBoronat et al., 2001). Similar antagonism was achieved in threeeparate knockout mice of opioid receptors (mu or delta or kappa)Garcia-Sevilla et al., 2004), implicating all opioid receptor sub-ypes in these effects of morphine. Chronic morphine and cocainelso increased phosphorylation of NFH and NFM tail domains, anffect ablated by knockout of cortical mu-opioid receptors (Garcia-evilla et al., 2004). Interestingly, chronic cocaine, but not chronic
orphine, lowered alpha-internexin levels in the VTA, hinting at
omewhat different mechanisms at play for these two drugs, whichre known to act at different receptors (Beitner-Johnson et al.,992).
Bulletin 126 (2016) 334–346
Rats inbred for alcohol preference expressed lower levels of NFsubunits, suggesting that NF proteins in the VTA may influence pref-erence for drugs (Guitart et al., 1992, 1993). Examining its influenceon NF proteins, GFAP and NMDA receptors, Ortiz et al. reported thatchronic ethanol treatment significantly decreased NF protein lev-els in VTA (by 14%-37%) while increasing GFAP (by 40%) and NMDANR1 subunit (by 27%) (Ortiz et al., 1995). Similar to other drugsof abuse, nicotine activates the mesolimbic pathway by increasingcell firing of dopaminergic neurons in the VTA via nicotinic recep-tors (McGehee et al., 1995). Nicotine microinfusions into the VTAwhich resulted in sensitization of dopamine release (Balfour et al.,1998), also reduced NFL (by 34%, p < 0.05), NFM (by 42%, p < 0.01),and NFH levels (by 38%, p < 0.05) in the VTA but not in the substantianigra in rats (Bunnemann et al., 2000; Sbarbati et al., 2002) withoutaltering neuron numbers. The similar reductions of NF proteins inthe VTA by the chronic treatment of different drugs of abuse withreinforcing properties suggest that common mechanisms underliethese addictive states.
Other brain targets, such as frontal cortex (Busquets et al., 1995;Simonato, 1996), also show lowered NFL levels (47%, p < 0.001) inbrains of addicts dying from opiate overdose (Garcia-Sevilla et al.,1997). Similar NFL reductions (49%, p < 0.001) are also seen in thefrontal cortex of rats after chronic morphine treatment. Narayanaet al. reported immunohistochemical evidence for a significantdecrease in NFH expression in the fimbria and internal capsule ofrats after chronic cocaine administration (Narayana et al., 2014).Analysis of NF phosphorylation in the prefrontal cortex of humanopioid addicts uncovered significantly reduced nonphosphory-lated forms of NFH, NFM, and NFL and corresponding increasesin phosphorylated forms (Ferrer-Alcon et al., 2000, 2003). Similarchanges are seen in brain regions of rats chronically treated withcocaine (Liu et al., 2003). Acute morphine exposure also inducesa marked increase in phosphorylated NFH whereas chronic mor-phine administration followed by opiate withdrawal results in atime-dependent decline in phosphorylated NFH (Cao et al., 2010;Jaquet et al., 2001). A recent study found chronic morphine causespersistent nitration of NFL in cortex and subcortex in mice evenduring abstinence (Pal and Das, 2015), although its significanceremains unclear.
Since PP2A has been shown to be the most effective in the ner-vous system in dephosphorylating the sites in NFH known to bephosphorylated by cdk5 (Veeranna et al., 1995), this phosphatasetogether with cdk5 were also examined. Surprisingly, the levelsof PP2A were found unchanged while the levels of cdk5 and itsneuron-specific activator p35 in the prefrontal cortex of humanopioid addicts were decreased by 18% and 26–44%, respectively(Ferrer-Alcon et al., 2003). In these brains, phosphorylated NFHsignificantly correlated with p35 but not with cdk5. Further stud-ies showed that acute treatment of rats with morphine increasedthe density of cdk5 by 35% in the cerebral cortex. In contrast tothe acute effects, chronic morphine induced a marked decreasein cdk5 by 40% and p35 by 47% in rat brain (Ferrer-Alcon et al.,2003). It therefore appears that, despite the possible stimulatingeffect of the deadly opiate overdose on cdk5, the reduced expres-sion of the cdk5/p35 complex in brains of opioid addicts is thenet result of a chronic opiate effect. These results indicate thatopioid addiction is associated with down regulation of cdk5/p35levels in the brain and the hyperphosphorylation of NFH is notthe result of a reduced dephosphorylation process. Although theabundance of cdk5 was decreased in the brains from chronic opi-oid abusers, Narita et al. showed that the level of phosphorylatedcdk5 (serine-159) immunoreactivity in the frontal cortex was sig-
nificantly increased by chronic morphine treatment (Narita et al.,2005) and phosphorylation of cdk5 at serine-159 dramaticallyincreases cdk5 activation (Sharma et al., 1999). They also demon-strated that the treatment of selective cdk5 inhibitor, roscovitine
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A. Yuan, R.A. Nixon / Brain Res
nd a half deletion of the cdk5 gene caused a significant inhibitionf the rewarding effect of morphine. In addition, chronic cocaine ororphine administration also activates extracellular signal- reg-
lated kinase (ERK), a known NF protein kinase (Veeranna et al.,998), by promoting its phosphorylation (Berhow et al., 1996;aljent et al., 2000). Altogether, these data indicate the hyperphos-horylation of NF proteins under these conditions may be relatedo the up-regulation of phosphorylated cdk5 and increased ERKctivity.
D1R and NMDA NR1 receptors are assembled as an oligomericomplex and are functionally interdependent (Fiorentini et al.,003; Lee et al., 2002). Both receptors bind to NF proteins (Ehlerst al., 1998; Kim et al., 2002) and are also involved in drug addic-ion (Comings et al., 1997; Trujillo and Akil, 1991). Cocaine has beenhown to reduce the D1R-NR1 physical interaction and may influ-nce intracellular signaling (Sun et al., 2009). On the one hand, D1Rs a primary target of stimulant drugs and chronic exposure to drugsf abuse lower levels of NF proteins and alter their phosphoryla-ion state (Beitner-Johnson et al., 1992). On the other hand, NMDAeceptor inhibition increases the phosphorylation state and the sol-bility of NFM protein (Fiumelli et al., 2008). Glutamate has beenhown to inhibit NF transport, which can be reversed by NMDAeceptor blocker MK-801 (Ackerley et al., 2000). Over-activation ofrimarily NMDA receptors by glutamate leads to rapid disruptionf NF proteins in cultured neurons (Chung et al., 2005). The basis forhese NF changes and their relationship to synaptic function remainlusive. As discussed above, our recent studies demonstrated thateletion of NFM in mice amplifies D1R-mediated motor responseso cocaine while redistributing postsynaptic D1R from endosomeso the plasma membrane, indicating a role of this NF subunit in drugddiction (Yuan et al., 2015b).
. Alterations of NF proteins in Alzheimer’s disease
Alzheimer’s disease is an irreversible, degenerative brain dis-rder that progressively destroys memory and other mentalunctions. Early striking loss of synaptic connections within brainegions involved in memory and thinking skills is followed by loss ofeurons of many types. Accompanying neurodegenerative changes
s the development of defining neuropathological hallmarks of AD,euritic plaques, and neurofibrillary tangles, along with extracel-
ular deposition of ß-amyloid. The causes of the most commonate-onset forms of AD (more than 95% of all cases) are not fullynderstood: the much less common early-onset familial forms (lesshan 5% of all cases) are caused by mutations of either of three genes,myloid precursor protein (APP) and presenilins 1 and 2(PSEN1 andSEN2) (Hardy and Selkoe, 2002; Van Cauwenberghe et al., 2016).hese causative genes and other risk factors influence the produc-ion and clearance of APP metabolites that mediate APP signalingascades and may also have cytotoxic properties.
An important feature of AD is the disruption of the neuronalytoskeleton leading to formation of neurofibrillary tangles (NFTs)Buee et al., 2000; Krstic and Knuesel, 2013) numbers of whichorrelate well with neurodegeneration and severity of cognitiveecline in AD (Arriagada et al., 1992; Bierer et al., 1995; Braaknd Braak, 1995). The main constituents of tangles are pairedelical filaments (PHF), initially believed to be composed of NFroteins (Anderton et al., 1982; Dahl et al., 1982; Ihara et al.,981) and later shown to be mainly fibrillar aggregates of tau pro-ein (Kosik et al., 1986; Ksiezak-Reding et al., 1987; Nukina et al.,987). Although NF proteins were somewhat ignored as tangle
onstituents, data has amply confirmed that NF proteins are inte-ral components of the NFTs (Perry et al., 1985). Most recently,ass spectrometry analysis by Pant and colleagues confirmed the
resence of NFM and NFH in purified NFTs from AD brain inde-
Bulletin 126 (2016) 334–346 341
pendently of antibody cross-reaction between NF protein and tau(Rudrabhatla et al., 2011). The NFTs are far more complex than thatof tau − PHFs. They also contain abundant NF proteins, vimentin,phosphorylated MAP1, phosphorylated MAP2, nonphosphorylatedMAP1B, nonphosphorylated MAP2, nonphosphorylated MAP4 andnonphosphorylated MAP6. The question of whether the formationof tau-PHF is the initiating event in NFT formation has not beenthoroughly addressed and the possibility that another NFT con-stituent, like NF protein, may initiate the process has not beenexcluded. In this regard, Morrison and colleagues reported that, cer-tain populations of cortico-cortical pyramidal neurons that containabundant perikaryal NF in relatively low states of phosphorylationare highly vulnerable to neurodegeneration through NFT forma-tion (Hof et al., 1990; Hof and Morrison, 1990; Morrison et al.,1987; Vickers et al., 1996, 1994). More recent studies have pro-vided independent evidence for loss of SMI32 immunopositiveneurons in temporal cortical areas of AD brain (Thangavel et al.,2009). The loss of SMI32 immunoreactivity on cortical regions ofAD brain is often paralleled by increase in NFTs and AT8-positivetau immunoreactivity in neurons (Morrison et al., 1987), indicat-ing nonphosphorylated NF proteins may play a protective role inpreventing the formation of NFTs. In fact, it is suggested that thedevelopment of neurofibrillary pathology may begin with abnor-mal NF accumulations in damaged distal processes followed byreactive perikaryal cytoskeletal changes that ultimately lead to tauhyperphosphorylation and NFT formation (Vickers et al., 1996). Theco-localization of caspase-3 cleaved spectrin with nonphosphory-lated NF-immunoreactive neurons in AD suggests that caspase-3activation may be a pathological event responsible for the loss ofthese vulnerable neurons (Ayala-Grosso et al., 2006).
NFL mRNA is significantly reduced in AD cortex (Clark et al.,1989; McLachlan et al., 1988) (Kittur et al., 1994) and CA1 and CA2regions of hippocampus (Somerville et al., 1991). Like NFL, NFMmRNA was also significantly decreased while NFH mRNA was unal-tered in AD temporal cortex as compared to controls (Kittur et al.,1994). Also significantly increased in AD are NFH (by 1.7-fold), NFM(by 1.5-fold) and NFL proteins (by 1.6-fold) and the degree of phos-phorylation of NFH (by 1.6-2.7-fold) and NFM (by 1.3-1.9-fold) ascompared to Huntington disease (Wang et al., 2001). Proteomicstudies revealed increases of alpha-internexin and NFM proteinsin the postsynaptic densities isolated from AD cortex as comparedto controls with the increase of alpha-internexin being validatedby Western blots (Zhou et al., 2013). Using quantitative phospho-proteomic methodology, Pant and colleagues recently reported 13hyperphosphorylated sites in the C-terminal domain of NFM and10 hyperphosphorylated sites of that of NFH in the frontal cortexof AD brain and all of these sites are in a higher state of phos-phorylation in AD (4–8-fold higher) compared with control brains(Rudrabhatla et al., 2010). Surprisingly, the abundance and numberof KSP repeat hyperphosphorylation of NFM are significantly highercompared with NFH, even though potential phosphorylation sitesin NFH are more numerous. Based on the NF stoichiometry [4:2:2:1(NFL:alpha-internexin:NFM:NFH)] (Yuan et al., 2006), there are 2-fold more KSP repeat sites hyperphosphorylated in NFM comparedwith NFH, indicating that NFM might contribute more to aberrantNF phosphorylation in AD compared with NFH.
Additional abnormalities of NF have also been observed inAD. Significantly decreased O-GlcNAcylation of NFM was observedin AD cortex as compared to controls (Deng et al., 2008) whilecarbonyl-related posttranslational modification of NFH protein wasalso present in the AD NFTs (Smith et al., 1995). Chapman et al.showed that sera of AD patients contain antibodies that bind specif-
ically to NFH of Torpedo cholinergic neurons (Chapman et al., 1989).AD patients also have elevated intrathecal synthesis of anti-NFHantibody (Bartos et al., 2012). Behavioral tests revealed that ratsimmunized with cholinergic NFH for 12 month performed signif-
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cantly worse than controls in T-maze alternation test (Chapmant al., 1991). This prolonged immunization of rats with NFH resultsn cognitive impairment, IgG accumulation in the septum, hip-ocampus and entorhinal cortex with marked loss of neurons in theeptum (Oron et al., 1997). Interestingly, this cognitive impairmentan be reversed by the acetylcholinesterase inhibitor physostig-ine administered 30 min prior to testing (Michaelson et al., 1991).
hese findings suggest that NFH may play a role in the pathogenesisf AD neurons.
Further support for NF protein involvement in AD pathogene-is comes from genetic studies. Deletion of NFL or NFH subunits in44 mice overexpressing htau resulted in a dramatic decrease inhe total number of tau-positive spheroids in the spinal cord andrainstem, indicating a role of NF proteins in the pathogenesis ofeurofibrillary tau pathology (Ishihara et al., 2001). Deletion of NFL
n APP/PS1 transgenic mice, unexpectedly, increased neocortical-amyloid deposition, synapse vulnerability and microgliosis sur-ounding ß-amyloid plaques, suggesting a protective role of this NFubunit in AD (Fernandez-Martos et al., 2015).
The abundance of NFs in axons and the frequency of axonalnjury in neurodegenerative disease has led to the use of NF protein
easurements as a clinical index of brain injury in various neuro-ogical disorders. Higher than normal levels of NFL and NFH in CSFave been reported in AD (Brettschneider et al., 2006; Hu et al.,002) and correlate with disease severity in a recent study of fron-otemporal dementia (Scherling et al., 2014). CSF NFL concentrations increased by the early clinical stage of AD, correlates with cog-itive deterioration and structural changes over time (Zetterbergt al., 2015) and may be a useful marker of disease progression inD (Bacioglu et al., 2016).
Although amyloid plaques and tau tangles are two hallmarksf Alzheimer’s disease, synaptic loss is more robustly correlatedhan these pathological lesions with cognitive deficits (DeKoskynd Scheff, 1990; Terry et al., 1991). Synaptic loss is more pro-ounced in the hippocampus (Scheff et al., 2006) than in temporalnd frontal cortex (Davies et al., 1987) and disturbance of synapticntegrity occurs very early on in the process of AD (Masliah et al.,001). Ultrastructural stereological studies on rapid postmortemutopsy samples showed an 18% synapse loss in the hippocampalA1 region of MCI patients that progressed to a 55% synapse loss
n mild AD (Scheff et al., 2007). Since oligomeric NF proteins areresent in synapses where they interact with synaptic proteins,
oss of synapses in early AD could affect the level of this synapticF pool and may thus contribute to the increased NF protein levels
n CSF and blood (Bacioglu et al., 2016). Alterations of NF proteins inarly AD could also reflect synaptic reorganization while changes ofF subunits in late AD may suggest reorganization of both synapticnd axonal pools.
. Conclusion
The etiology of major neuropsychiatric disorders criticallynvolves dysfunction of synapses and, in late-onset dementias,he ultimate loss of synapses early in the disease. Relatively littlettention, however, has been paid to the dynamics of cytoskeletalroteins at synaptic terminals and particularly the possible synap-ic involvement of NF proteins. Until recently, the functioning of NFrotein assemblies within synapses has been unappreciated andhe focus has been on their axonal roles (eg. radial growth) andisruption in axonopathies involving mainly neurons with longyelinated axons that contain abundant NFs. In the CNS, how-
ver, even axons in major fiber tracts like the corpus callosumhange minimally in caliber in NF triple knockout mice nearly com-letely lacking all 4 CNS NF subunits. This finding underscoresrowing evidence that NF proteins in the CNS have important func-
Bulletin 126 (2016) 334–346
tions beyond caliber expansion. Emerging evidence shows thatNF proteins are present and functional in synapses. The synapticpopulation of NF proteins is distinctive in phosphorylation state,likely present in unconventional stoichiometries and assemblyforms including oligomeric structures, and possibly more plasticin exchanging subunits and altering form in response to signals.Further evidence that individual subunits differentially modulateneurotransmission and behavior through interactions with specificneurotransmitter receptors heightens expectations that alterationsof NF proteins could influence synaptic function in psychiatric dis-orders and dementias. Despite the very frequent occurrence of NFprotein changes in multiple major psychiatric states, most of theanalyses have been on white matter tracts and the changes seen,often involving levels of only one or two subunit, have been difficultto interpret mechanistically from a perspective of changes in theaxonal NF cytoskeleton. The newly uncovered sizable populationof synaptic NF proteins should encourage more directed attentionto regional analyses that interrogate this synaptic NF protein pooland will potentially yield valuable new insights into the significanceof NF subunits in neuropsychiatric disease.
Acknowledgements
This work was supported by Grant 5R01AG005604 from theNational Institutes on Aging.
The authors declare that there are no conflicts of interest.
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