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Pharmacological Inhibition of Cyclophilin Ameliorates
Experimental Allergic Encephalomyelitis
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science
in Physiology & Biophysics at Virginia Commonwealth University
By
ZI LING HUANG University of Virginia, B.A. Biology, 2014
Principal Investigator: UNSONG OH, M.D. VCU School of Medicine, Neurology
Virginia Commonwealth University Richmond, Virginia,
April, 2015
1
Acknowledgements
First and foremost, I would like to express my sincerest gratitude to my principal
investigator and research advisor, Dr. Unsong Oh. My interest in biological research
was sparked by a thirdyear course in my undergraduate career, Introduction to
Neurobiology. However, as a thirdyear transfer student with no research experience, I
was unsuccessful in my attempts at performing undergraduate research. I am extremely
thankful for the opportunity to learn from and work under Dr. Unsong Oh. Despite his
busy schedule as a research scientist and practicing neurologist, he worked with me
sidebyside and personally taught me all relevant research protocol, animalhandling,
and experimental techniques that I’ve conducted in the lab. He was alway available,
regardless of the hour, to answer my questions even if it meant a two hour phone
conversation late into the night. I am grateful for his immense knowledge, guidance,
patience, and selfless dedication to my academic development.
I would also like to thank Dr. Carlos A. Villalba Galea and Dr. Margaret C. Biber
for serving as members of my advisory committee. Their feedback, insight and
suggestions have been integral to the development of my thesis.
Finally, I am forever indebt to my parents for their everlasting love and support in
the pursuit of my own dreams and ambitions.
2
Table of Contents
Acknowledgements…………………………………………………………. 1
Abbreviations………………………………………………………………… 4
Abstract……………………………………………………………………….. 6
Chapters
Introduction…………………………………………………………… 8
1.1 Multiple Sclerosis……………………………………….. 8
1.2 Etiology…………………………………………………… 8
1.3 Clinical Course…………………………………………… 9
1.4 Pathophysiology Neuroinflammation………………… 10
Neuroinflammation Immune Activation
Neuroinflammation Leukocyte Trafficking
Neuroinflammation CNS Inflammatory Cascade
Neuroinflammation Demyelination
1.5 Pathophysiology Neurodegeneration 17
Neurodegeneration Axon damage
Neurodegeneration Perturbation to AxonalIonic Homeostasis
Neurodegeneration Mitochondrial Dysfunction
Neurodegeneration Mitochondrial Permeability Transition
1.6 Cyclophilins as targets for MS therapy NIM811 21
3
Cyclophilin D
Cyclophilin A
1.7 EAE………………………………………………………. 24
Hypothesis….………………………………………………………... 26
Specific Aims……………………………………………………....… 27
Materials and Methods…………………………………………….. 28
Results……………………………………………………………….. 34
Discussion/Future directions………………………………………. 43
Literature Cited………………………………………………………………. 46
Vita…………………………………………………………………………….. 51
4
Abbreviations
CypA: Cyclophilin A
CypD: Cyclophilin D
CsA: Cyclosporin
EAE: Experimental Allergic (Autoimmune) Encephalomyelitis
NIM811: Nmethyl4isoleucinecyclosporin
MS: Multiple Sclerosis
RRMS: RelapsingRemitting Multiple Sclerosis
SPMS: Secondary Progressive Multiple Sclerosis
GWAS: Genome Wide Association Studies
Th: T helper
Treg: T regulatory
BBB: Blood Brain Barrier
TJ: Tight Junction
AJ: Adherens Junction
PBMC: Peripheral Blood Mononuclear Cells
MMP: Matrix Metalloproteinases
EMMPRIN: Extracellular Matrix Metalloproteinases Inducer
TIMP: Tissue Inhibitors of MMPs
5
VDAC: VoltageDependent Anion Channels
NCLX: Isoform of Sodium Calcium Exchanger
ROS: Reactive Oxygen Species
RNS: Reactive Nitrogen Species
mPT: Mitochondrial Permeability Transition
mPTP: Mitochondrial Permeability Transition Pore
FAD: Focal Axonal Degeneration
PPIase: Peptidyl Prolyl cistrans Isomerase
CFA: Complete Freund’s Adjuvant
MOG: Myelin Oligodendrocyte Glycoprotein
DMSO: Dimethyl Sulfoxide
PEG: Polyethylene Glycol
BSA: Bovine Serum Albumin
6
Abstract
Pharmacological Inhibition of Cyclophilin Ameliorates Experimental Allergic Encephalomyelitis
By Zi Ling Huang
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Physiology & Biophysics at Virginia Commonwealth University
Virginia Commonwealth University, 2015
Principal Investigator: Unsong Oh, M.D. VCU School of Medicine, Neurology
A subset of cyclophilins have been implicated in mechanisms of neuroinflammation and
neurodegeneration that contributes to the pathogenesis of Multiple Sclerosis.
Mitochondrial dysfunction leading to mitochondrial permeability transition plays a pivotal
role in axonal damage and disease progression in Multiple Sclerosis. Cyclophilin D
(CypD) is a crucial regulatory component of the mitochondrial permeability transition
7
pore and it was demonstrated that the cyclophilin D knockout animals showed reduced
experimental allergic encephalomyelitis (EAE) clinical disease severity and axonal
injury. We investigated the effect of Nmethyl4isoleucinecyclosporin (NIM811), a
nonimmunosuppressive and nonselective cyclophilin inhibitor, on the course and
severity of EAE. EAE mice treated with NIM811 showed a significant reduction in
clinical disease severity compared to vehicle treated mice. FACS analysis performed
with the dissociated thoracolumbar spine showed that NIM811 treatment was
associated with a reduction in CNS macrophages but does not alter Thelper lineage
frequencies. In addition, we demonstrated NIM811’s effect on crude mitochondrial
fraction obtained from brain and liver homogenates of both wild type and CypD
knockout mice in order to determine drug specificity. Benefits observed from the
pharmacological inhibition of cyclophilin may lead to a novel MS therapy but NIM811’s
exact mechanism of action has yet to be elucidated.
8
Introduction
1.1 Multiple Sclerosis
Multiple Sclerosis (MS) is the leading nontraumatic neurodegenerative disease of the
central nervous system in young adults characterized by inflammation, axonal
damage, and demyelination. WHO recognizes MS as a global disease with 2.5 million
individuals affected worldwide [49] [10]. In the United States alone, MS affects more
than 350,000 individuals with an annual average cost of $47,215 per patient, mounting
to a national cost of $1.7 billion per year [11].
1.2 Etiology
The precise cause of MS is currently unknown. Numerous populationbased studies
comparing concordance rates between monozygotic and dizygotic twins suggest that
susceptibility to MS is genetically influenced [12]. Genomewide association studies
(GWAS) and concordance ratios from twin studies both suggest a polygenic nature for
the genetic susceptibility of MS [10] [12]. HLA DRB1 1501 is partially responsible for
coding the major histocompatibility class II molecules and confer the highest genetic risk
of MS but more than 103 other distinct genetic regions have been implicated with this
disease [13]. Additionally, other factors including ethnicity, geographic location, cigarette
smoking and even exposure to sunlight have been shown to influence risk of MS [14]
[15] [7]. A current hypothesis is that MS develops in individuals who are genetically
susceptible but the penetrance of disease is determinant on environmental factors [16].
9
1.3 Clinical Course
MS has a heterogeneous presentation in patients with 85% of them initially experiencing
a relapsingremitting course of MS (RRMS) [4]. Patients with RRMS will undergo
intervals of alternating disability and recovery. Within 25 years, 90% of patients
diagnosed with RRMS will develop into a secondaryprogressive phase (SPMS)
characterized by a permanent and gradual decline in neurological function (Figure A)
[4]. MS is currently viewed as a CNS disease involving both neuroinflammatory as well
as neurodegenerative mechanisms. In fact, there is still much debate between the
importance of the two general categories of mechanisms on the initiation and course of
disease [17]. Generally, neuroinflammatory mechanisms associated with plaque
formation and demyelination are thought to be responsible for the the
relapsingremitting phase of MS whereas neurodegenerative mechanisms involved in
axonal damage results in the permanent neurological defects seen in secondary
progressive MS [18].
10
Figure A Common presentation of MS in patients [16]
1.4 Pathophysiology Neuroinflammation
Inflammation is associated with MS at all stages of the disease, especially during acute
and relapsingremitting stages of MS. The overall sequence of neuroinflammatory
events in MS includes immune activation, leucocyte trafficking, CNS inflammation, and
the resulting CNS demyelination that results in neurological symptoms.
11
Leucocyte infiltration into the CNS is a characteristic feature of MS and defines lesional
disease activity in MS [8]. Activated autoimmune T cells have been known to play an
important role in the pathogenesis of MS but they are not the sole cause of
demyelination. The importance of CD4+ T helper cells (Th), microglia, oxidative stress,
and cell death in causing demyelination and neurodegeneration have all been
highlighted in a recent comparative gene expression analysis [9]. Therefore, other
adaptive immune responses and innate immune responses also play an integral role in
the pathogenesis of MS [19].
❖ Neuroinflammation Immune Activation
Whether the initial trigger for MS happens in the periphery or the CNS is debated. The
CNSextrinsic model supports the idea that autoreactive T cells are first activated in the
periphery and then infiltrates into the CNS along with other leukocytes. As part of their
maturation, there is negative selection for autoreactive T cells in the medulla of the
thymus. This process, however, is not perfect and autoreactive T cells could still escape
into circulation. Regulatory T cells (Treg) are essential in suppressing autoreactive T cells
since rampant autoimmunity is observed in individuals with dysfunctional Treg i.e. IPEX
(Immunodysregulation polyendocrinopathy enteropathy xlinked) syndrome. Therefore,
differentiated T cells can lose their peripheral tolerance due to intrinsic factors
(molecular mimicry, bystander activation, or T cell receptor coexpression with varying
specificities) and/or extrinsic factors such as regulatory T cell (Treg) dysfunction [19].
12
On the other hand, the CNSintrinsic model may also explain disease development and
a CNS inflammatory cascade where the infiltration of activated leukocytes occur as a
secondary effect [16]. This could be due to a CNS viral infection or exposure to toxins.
CNS injury results in release of pathogen or damageassociated molecular patterns
which elicit an immune response as suggested in Alzheimer’s and Parkingson’s
disease [20]. In the CNSintrinsic model, the CNS injury, whether from a viral infection
or toxic exposure, is the initial trigger and the inflammation is a response to injury.
Supporting the CNSextrinsic model, Alzheimer’s and Parkingson’s disease have very
little shared genetic associations with MS [21] [22] and candidate genes implicated in
multiple sclerosis are mostly immunological sharing a considerable overlap of
associated regions with other autoimmune diseases [13]. Additionally, the CNSextrinsic
model of disease initiation is adopted in the active induction of experimental
autoimmune encephalomyelitis, an animal model of MS.
❖ Neuroinflammation Leukocyte Trafficking
The leakage or breakdown of the bloodbrain barrier (BBB) is a hallmark of multiple
sclerosis where increased permeability of BBB is necessary for the CNS infiltration of
activated leukocytes [17]. Peripheral activation and infiltration of Thelper cells,
specifically autoreactive Th1 and Th17 cells, are integral in the pathogenesis of MS and
are suggested as key players in BBB disruption [19]. Th1 and Th17 can be respectively
distinguished by their signature cytokines of IFNᵯ� and IL17A with a subset of lesional
13
CD4+ Th cells having an intermediate phenotype with expression of both biomarkers
[23].
Soluble factors such as inflammatory cytokines, reactive oxygen species, and enzymes
can facilitate migration of leukocytes into the CNS by modulating BBB function.
Proinflammatory cytokines such as IFNᵯ� affect cellular distribution of tight junctions (TJ)
and adherens junctions (AJ) to increasing paracellular permeability of the BBB
endothelial cells [Larochelle et al]. IL17 down regulates occludin (part of the TJ
complex) and disrupts zona occludens 1 (responsible for anchoring the TJ complex) to
increase BBB permeability [Larochelle et al]. Additionally, the initial CNS infiltration of
leukocytes will enhance permeability in favor of subsequent migration through the BBB
[Larochelle et al].
Peripheral blood mononuclear cells (PBMCs) are known to express and secrete
extracellular MMP inducer (EMMPRIN aka CD147) [24]. Matrix metalloproteinases
(MMPs) are endopeptidases responsible for the degradation of components in the ECM.
MMPs are initially expressed as inactive zymogens regulated by TIMPs (tissue
inhibitors of MMPs). Both activated T cells and macrophages are known to secrete
MMPs in response to inflammatory cytokines.
After the diapedesis of CNS infiltrating leukocytes across the endothelium, there are two
basement membranes that must be crossed before entering the CNS parenchyma
(Figure B) [24]. In patients with RRMS, subgroups of MMP mRNAs are elevated while
TIMP levels remain similar to control [Larochelle et al]. MMP activity is known to
14
degrade junctional complex proteins and cleave anchoring proteins of the parenchymal
basement membrane [Larochelle et al].
Figure B Basement membranes after diapedesis [25]
While the endothelial basement membrane can be readily crossed, the proteolytic
activity of MMPs are required to transmigrate the glia limitans [24]. The interaction of
extracellular cyclophilin A with EMMPRIN will result in the activation of MMPs. The
inhibition of EMMPRIN have been shown to attenuate disease severity seen in EAE
mice [Figure E].
❖ Neuroinflammation CNS inflammatory cascade
CNS infiltrating immune cells are detected in early lesions of MS. The most abundant of
these CNS infiltrating immune cells are macrophages, followed by CD8 T cells, CD4 T
cells, B cells, and plasma cells [16]. At this stage, apart from the focal lesions, damage
to the CNS is relatively minimal. With disease progression, more inflammatory T and B
cells will infiltrate into the CNS along with the activation of microglia and astrocyte,
15
resulting in myelin and axonal damage with a noticeable atrophy of white and grey
matter [26].
Among CNS infiltrating leukocytes, the ratio of B cells and plasma cells will increase
with disease progression but T cell composition remains relatively similar while
microglia and macrophages are chronically activated [27]. Not only does the number of
CNS infiltrating CD8+ cytotoxic T cells exceed that of CD4+ Th cells, their prevalence is
strongly correlated with the extent of axonal damage [27]. Myelinspecific CD8+ T cells,
with the help of monocytederived dendritic cells, can also be readily activated by
epitope spreading in the CNS [29]. In addition to the direct cytotoxic function of CD8+ T
cells on myelin and associated oligodendrocytes, these T cells can exacerbate CNS
inflammation by the production of IFNy and IL7 cytokines in the presence of apoptotic
epitopes [30].
CD20 is the Blymphocyte antigen which increases in concentration on maturing B cells
until final differentiation into a plasma cell. Phase II clinical trials of CD20 specific
monoclonal antibody treatment (rituximab or ocrelizumab) reduces the rate of relapse of
MS [16]. This treatment do not affect terminally differentiated plasma cells but do
deplete other subsets of B cells, suggesting an important contribution of B cells to the
pathogenesis of MS possibly through production of proinflammatory IL6 cytokine and
B cell mediated antigen presentation [31].
There are supporting evidence for functionally distinct, origindependent populations of
macrophages. Microglia and macrophages are both mononuclear phagocytes. They are
16
the dominant immune cell types located in lesions associated with MS. As observed in
EAE, the monocytederived macrophages are attributed to the destruction of myelin,
release of proinflammatory cytokines, and MMP activation whereas the
microgliaderived macrophages promote recovery and clear up myelin and other debris
[32]. In the presence of constant inflammation implicated by proinflammatory T cells as
well as the complement system, microglia will adopt a proinflammatory phenotype [19].
Activity and progression in MS lesions could be identified by phagocyte content,
specifically the degraded myelin products [33].
❖ Neuroinflammation Demyelination
CNS function is dependent on the network of neuronal cells that communicate through
an electrochemical process. A primary function of myelin is to increase the effectiveness
and speed of electrical signaling by saltatory propagation instead of the slower,
continuous impulse that takes place in unmyelinated fibers. Demyelination in MS occurs
as a result of the autoimmune response against myelin and nonmyelin antigen
mediated by CNS infiltrating leukocytes [18]. Aside from T cell and B cell mediated
mechanisms of demyelination, macrophages and activated microglia release
proinflammatory cytokines including nitric oxide and tumor necrosis factora that have
been shown to play an integral role in demyelination and oligodendrocyte death in vitro
[34]. Once demyelination occurs, the axon’s ability to propagate action potential is
impaired. The associated axon would then be at risk for damage and degeneration [4].
Demyelination results in a wide spectrum of neurological disorders consistent with the
signs and symptoms of MS patients [35]. Lesions seen in MS patients are the
17
visualization of focal demyelination in white and grey matters of the CNS [34]. While the
histopathological features of white matter lesions and grey matter lesions differ, they
can both be identified by the presence of focal demyelination [36].
1.5 Pathophysiology Neurodegeneration
In the presence of chronic antigen exposure, central tolerance and peripheral tolerance
may result in immune cell exhaustion [Peterson]. The phenomenon of exhaustion is
seen in MS where, regardless of therapy, CNS infiltrating leukocytes will eventually
decrease [16]. However, the chronic neurodegeneration and progression of MS will
continue even in the absence of CNS infiltrating leukocytes, highlighting the importance
of neurodegeneration in the context of disease progression [16].
❖ Neurodegeneration Axon damage
Axonal damage and loss in MS was reported as early as 1936 [4]. More recently,
identification and investigation of axonal swelling, transection, and wallerian
degeneration have resulted in a paradigm shift in MS research [4]. At the onset of
disease in MS, axon transection and damage will occur on demyelinated axons
identifiable by neurofilament protein dephosphorylation as well as the accumulation of
transport proteins, Ntype calcium channels, and metabotropic glutamate receptors [4].
Phagocytes are found in close proximity to damaged axons with a positive correlation
between the number of phagocytes and extent of acute axonal damage [33]. While
neurofilaments can be seen in these phagocytes, it is unclear whether they are
attacking the axon or merely clearing debris [4]. However, activated microglia and
18
macrophages contribute to oxidative stress, which is implicated as a damage
mechanism for both grey and white matter pathology in MS [33]. After initial axon
transection, Wallerian degeneration of the damaged axon will occur distal to the initial
damage, but the empty myelin will remain forming ovoids [4]. New cortical lesion
formations are more likely to occur in areas connected to previous white matter damage
[37]. It is now recognized that axonal degeneration is the major determinant for
permanent neurological dysfunction in MS patients [4]. However, axonal damage and
degeneration could remain “silent” until neurodegenerative mechanisms exacerbate the
damage and/or the CNS is unable to compensate for physiological defects [37].
❖ Neurodegeneration Perturbation to axonalionic homeostasis
Oligodendrocytes and associated myelin is essential for the longterm health and
survival of axons. After demyelination, as a compensatory response for the restoration
of action potential conduction, sodium channels clustered at the Nodes of Ranvier will
be scattered diffusely along the demyelinated segments to restore neurological function
[4]. In inflammatory white matter lesions, there is a reduction in axoplasmic ATP
production [37]. The ion gradient necessary for conduction of neuronal signaling is
maintained by the sodiumpotassium ATPase. Having the largest consumption of ATP
in the CNS, the normal function of the sodiumpotassium ATPase is reduced in areas of
lesion[38]. Elevated levels of intraaxonal sodium will reverse the normal function of the
sodiumcalcium exchanger [38]. Consequently, the elevation in intraaxonal calcium will
could impair normal mitochondrial function and reduced energy production. This “virtual
hypoxia” or the imbalance between the demand and supply of energy would result a
19
perpetual cycle of degeneration from reduced energy production, axonal transport, and
elevated intraaxonal calcium levels [38].
❖ Neurodegeneration Mitochondrial dysfunction
Mitochondrial dysfunction is central to the current hypothesis of molecular mechanisms
leading to neurodegeneration and disease progression in MS [5]. A major function of
mitochondria is its role in cell survival, partially by the regulation of calcium homeostasis
[5]. Mitochondria will sequester intracellular calcium when it exceeds a certain
threshold; voltagedependent anion channels (VDAC) and a uniporter are responsible
for calcium import through the outer membrane and the sodiumcalcium exchanger
allows for calcium import through the inner membrane [39]. Calcium is slowly released
once intracellular calcium levels returns to normal possibly through an isoform of
sodiumcalcium exchanger (NCLX) [39]. In the environment of chronic inflammation
presented in MS, the production of reactive oxygen and nitrogen species (ROS and
RNS) are suggested to negatively impact mitochondrial function by the accumulation of
mitochondrial DNA mutations [28] and the inhibition of mitochondrial electron transport
[5]. Normally, axonal function and transport has a high energy demand. The
compensatory ion channel redistribution on demyelinated axons in the presence of
excess glutamate released during neuronal injury could place excessive stress on
mitochondrial functions [40]. When ATP production could not keep up with demand,
intraaxonal calcium levels will increase and mitochondrial dysfunction could occur.
❖ Neurodegeneration Mitochondrial permeability transition
20
Excessive intramitochondrial calcium could increase ROS production, release
cytochrome c, inhibit ATP synthesis and induce mitochondrial permeability transitions
[41]. The mitochondrial permeability transition (mPT) is characterized by a sudden
increase in membrane permeability of ions and solutes due to opening of a
nonselective megachannel termed mitochondrial permeability transition pore (mPTP)
[41]. Evidence from in vitro studies suggest that the mPT results in a water flux across
the inner membrane due to osmotic balance resulting in swelling, outer membrane
rupture and release of cytochrome c [42]. Enlarged mitochondria and focal axonal
degeneration (FAD) preceding demyelination has been observed in EAE mice [4]. While
the molecular components of mPTP is still under investigation, cyclophilin D has been
recognized as a component of mPTP that controls calcium dependent opening of the
transition pore and the inhibition of which may influence mitochondrial dysfunction, and
consequently, axonal injury in MS and EAE [6].
mPT was viewed as an invitro artifact due to the large estimated pore radius of 1.4 nm
and its implication in principles of chemiosmosis, that is, until the discovery that
cyclosporin A (CsA) inhibits mPT [42]. CsA is is an immunosuppressive agent that
targets cyclophilins, a family of peptidylprolyl cistrans isomerases. Cyclophilin D
(CypD) is a unique class of cyclophilins found in mammals coded by the Ppif gene in
mice [42]. CypD is believed to serve as a calcium sensitive regulator of mPTP by
decreasing the threshold of intramitochondrial calcium necessary for the opening of
mPTP and mPT. CsA binds matrix cyclophilin D (CypD) and this inhibition corresponds
to an inhibition of mPT by increasing relative calcium insensitivity [Bernardi et al].
21
Mitochondrial permeability transition marked by the opening of mPTP will result in,
among other molecules, the release of sequestered intramitochondrial calcium, serving
to further aggravate mechanisms of neurodegeneration. The elevated calcium levels
could trigger the activation of calciumdependent proteases (i.e. calpain) to eventually
result in neuronal cell death and release of associated inflammatory agents [4].
1.6 Cyclophilins as targets for MS therapy NIM811
Cyclophilins make up a highly conserved subgroup of immunophilins, named by their
binding to immunosuppressant molecules such as cyclosporin A [43]. Most cyclophilins
exhibit diverse cellular locations and possess a peptidyl prolyl cistrans isomerase
(PPIase) activity (Figure C) with an active role in protein folding, trafficking, and
modulating protein functions [43]. As aforementioned, among the different classes of
cyclophilins, cyclophilin A and D are respectively implicated in mechanisms of
neuroinflammation and neurodegeneration in the context of multiple sclerosis.
22
Figure C peptidyl prolyl cistrans isomerase activity of cyclophilins [43]
Nmethyl4isoleucinecyclosporin (NIM811) is a structural and functional analogue of
cyclosporin that retains its functional property and pharmacokinetic profile with similar
oral bioavailability similar to CsA (Figure D) [44]. However, it’s modification makes it
nonimmunosuppressive, significantly decreasing the toxicity of CsA [44].
Figure D Structural formulas for Cyclosporin A and NIM811 [Hopkins et al]
❖ Cyclophilin D Mitochondrial dysfunction
Contemporary therapies target the relapsingremitting or inflammatory phase of MS by
means of immunomodulatory drugs including Natalizumab and Fingolimod. Their
mechanism of function is focused on the impediment of leukocyte infiltration into the
CNS [47] [48]. These therapies are effective in reducing the rate of relapse and new
MRI lesion formation, but have broad sideeffects and cannot effectively prevent the
progression of disease [1] [2]. Therefore, investigating neurodegenerative mechanisms
responsible for permanent neurological defects of progressive MS is crucial for the
development of an effective therapy.
23
Effect of NIM811 in the background of EAE have not been investigated but Forte et al
demonstrated that cyclophilin D knockout mice showed markedly reduced clinical
disease severity in EAE (Figure F). If NIM811 could inhibit the activity of cyclophilin D
and increase the threshold for calcium dependent activation of mitochondrial
permeability transition, it may ameliorate mechanisms of neurodegeneration and serve
as a potential therapy against progressive MS.
❖ Cyclophilin A Leukocyte trafficking
Extracellular cyclophilin A functions as a chemotactic factor for CNS leukocyte
infiltration via interaction with EMMPRIN and the subsequent activation of MMPs [24].
Intraperitoneal injection of an antiEMMPRIN functionblocking antibody was shown to
attenuate EAE disease severity in mice (Figure E). Alternatively, the inhibition of
cyclophilin A by NIM811 could exhibit a similar effect on the reduction of MMP activation
to limit leukocyte infiltration and ameliorate disease severity in EAE mice.
Figure E Inhibition of CD147 ameliorates EAE severity [24]
24
1.7 EAE
Experimental autoimmune encephalomyelitis (EAE) is the most common and
wellstudied animal model of MS [46]. EAE could be induced in many species of varying
genetic backgrounds through an active immunization against a self CNSderived
antigen or a passive transfer of autoimmune T cells [46]. In mice, EAE is characterized
by an ascending paralysis starting from the tail and progressing through the hindlimbs to
an eventual forelimb paralysis. Much like MS, EAE is also a complex disease with
interactions between mechanisms of neurodegeneration and immunopathology to allow
for the approximation of key pathological features seen in MS [45]. In Figure F, the
course of EAE is dissected into three phases, marked by A, B, and C. Segment A
corresponds to immune activation while neuroinflammation and leukocyte trafficking
occurs in segment B. Neurodegeneration, demyelination, axonal injury and
mitochondrial dysfunction could be observed in segment C. This general
compartmentalization allows for the approximation of efficacy by specific therapeutic
agents targeting distinct mechanisms contributing to MS pathology. For instance, in
figure F, cyclophilin D is believed to contribute to the neurodegenerative mechanisms of
mitochondrial dysfunction implicated in progressive MS. As observed, knocking out
cyclophilin D did not affect the onset and initial severity of disease but there was a
significant attenuation of EAE clinical disease severity in segment C. Conversely, in
figure E, inhibition of EMMPRIN activity is thought to impede lymphocyte infiltration and
a corresponding drop in clinical disease severity is seen at the onset and initial severity
of EAE.
25
Figure F Course of EAE in WT vs CypD KO mice [5]
26
Hypothesis
Cyclosporin A was shown to inhibit mechanisms of mitochondrial dysfunction by direct
inhibition of cyclophilin D and increasing the threshold of intramitochondrial calcium
levels necessary for mPT. Knocking out cyclophilin D in EAE mice reduced their clinical
disease severity [5]. Preliminary data from our lab indicated that administering a
cyclophilin inhibitor (NIM811) in wild type EAE mice also reduced EAE clinical disease
severity. We hypothesized that NIM811’s mechanism of action is on the direct inhibition
of cyclophilin D and may mitigate process of neurodegeneration in MS. If that is the
case, we should see increased calcium retention capacity in mitochondria when NIM811
is applied in vitro and in vivo. Additionally, it should have no effect on the course of EAE
in cyclophilin D knockout mice.
27
Specific Aims
S.1 Define the effect of NIM811 on a cyclophilin Ddependent function
Hypothesis: The cyclophilin inhibitor NIM811 reduces severity of EAE through a
cyclophilin Ddependent mechanism.
EAE will be induced in wild type and cyclophilin D knockout animals to verify the
reduction in clinical disease severity. Specific function of NIM811 on cyclophilin D will be
investigated in vitro and ex vivo through mitochondrial calcium retention capacity
assays. Cyclophilin D will be quantified by western blots. The effect of NIM811 on
cyclophilin D knockout animals in the context of EAE will be investigated.
S.2 Investigate the effect of NIM811 on a cyclophilin Adependent mechanism
Hypothesis: The cyclophilin inhibitor NIM811 reduces severity of EAE through a
cyclophilin Adependent mechanism.
Leukocyte infiltration into the CNS will be assess by flow cytometry. Effect of NIM811 on
cyclophilin A knockout mice in the context of EAE will be investigated.
28
Materials and Methods
Experimental Autoimmune Encephalomyelitis
Preparation
C57BL/6 mice were prepared for active induction after maturing to 6 to 10 weeks
of age. Pairs of mice were established based on sex, age, and kinship. Cohorts of
NIM811 or VC treatment were then determined by random chance (i.e. coin flip). Mice
were handled on at least three separate days prior to induction. Animal handling
included scoring for clinical disease severity and measurement of body weight.
Complete Freund’s Adjuvant (CFA) was prepared with 100mg of heatkilled
myobacterium (M. Tuberculosis H37 RA: Difco #263910 6x10 ml) in 10ml of Incomplete
Freund’s Adjuvant (Difco #231141 6x100mg). Myelin Oligodendrocyte Glycoprotein
(MOG3555, MEVGWYRSPFSRVVHLYRNGK, Anaspec. 5mg. Cat# 601305) was diluted
with sterile PBS to 4mg/ml. Emulsification equipment was first lubricated with 200ul of
CFA. Equal parts of MOG and CFA were then manually emulsified using glass syringes
and emulsifying needles until viscous and white in color. 1 ml syringes were filled with
emulsifications and 25G needles were attached. Final concentration of MOG/CFA was
2mg/ml MOG3555 and 5mg/ml M. Tb/CFA. Pertussis toxin (Cat# 180. 50 µg. List
Biological Laboratories, Inc.) was diluted with sterile PBS to 100µg/ml. Working solution
of 1.5 µg/ml were diluted with sterile PBS and a 27G needle was attached to syringe.
29
Induction
Mice was anesthetized by placement into a chamber with isoflurane soaked
nestlet/cotton gauge. 100ul of MOG/CFA emulsion were administered subcutaneously
over the shoulder for a total of 300ug of MOG3555 and 500ug of CFA. Then, 300ng of
pertussis toxin in 200ul of PBS was administered intraperitoneally. Pertussis toxin
injection is repeated 48 hours later.
Scoring
From day five postinduction, EAE clinical disease severity and weight
measurements were monitored daily. After onset of disease, mice were handled gently,
especially avoiding swollen areas. Clinical manifestations of EAE were scored on a
scale of 05.
0 No clinical signs
1 Limp tail or loss of righting reflex
2 Limp tail and loss of righting reflex
3 Partial hindlimb paralysis
4 Complete hindlimb paralysis
5 Forelimb paralysis and death
Daily injections of NIM811 (100mg/kg) or VC were administered intraperitoneally
after onset of disease. NIM811 crystals were reconstituted in dimethyl sulfoxide (DMSO,
SigmaAldrich) and diluted with polyethylene glycol (PEG400, SigmaAldrich) in a 1:1
ratio. Vehicle control consisted of DMSO and PEG400 in a 1:1 ratio. Once EAE mice
30
reached a clinical score of 3 or higher, moist mice chow were placed inside cage and
1ml of 0.9% NaCl were administered daily.
Mitochondrial Calcium Retention Capacity Assay
Mitochondria isolation
Brain and liver were extracted from mice and manually homogenized in 5 mL of
mitochondrial isolation buffer (0.2M mannitol, 50mM sucrose, 1mM EDTA, and 10mM
HEPES, pH 7.4). Homogenate was then transferred into 2 mL microcentrifuge tubes
and centrifuged at 1,300 x g for 10 minutes at 4oC. Supernatant was recovered and
transposed into clean microcentrifuge tubes for repeated centrifugation at 1,300 x g for
5 minutes at 4oC to ensure absence of pellet fragmentation.
Resulting supernatant from second centrifugation was collected into 50 mL
conical tubes. Samples were balanced to +/ 0.01g and centrifuged at 17,000 x g for 15
minutes at 4oC. Supernatant was aspirated to isolate pellet containing mitochondria
fraction. Pellet was then resuspended in 5 mL KCl buffer without EDTA/EGTA for
calcium retention assay (130mM KCl, 2 mM KH2PO, 3 mM HEPES, 2 mM MgCl2) and
underwent final centrifugation at 17,000 x g for 15 minutes at 4oC. Supernatant was
once again aspirated and pellet was resuspended in 250 mL of KCL buffer for
concentrated mitochondrial suspension.
Following crude mitochondrial isolation, protein concentration was measured
using BioRad protein assay kit (BioRad, cat. no. 5000006) according to
31
manufacturer’s protocol. Assays were executed in triplicates and compared to bovine
serum albumin standards (BSA).
Calcium retention capacity
KCl buffer with 0.1% BSA (25m M KCl, 0.1% BSA, 20 mM HEPES, 2 mM MgCl2,
and 2.5 m M KH2 PO4 , pH 7.2) was prepared with the following additives: 8 mM
succinate (complex II substrate), 1 µM rotenone (complex I inhibitor), 2 µg/ml
oligomycin (ATP synthase inhibitor), 0.2mM ADP, 2 µM thapsigargin, and 1µM CaG5N,
a fluorescent calcium indicator. The KCl (0.1% BSA) buffer with additives was then
incubated at 37oC, and mitochondria were resuspended and diluted in prepared buffer
to 0.2 mg/mL. Samples were then dispensed into 200 µL aliquots in a 96black well
assay microplate and quantified in quadruplicates.
The Synergy HTX MultiMode Reader and Gen5 Data Analysis Software were
utilized to monitor the calcium uptake and opening of the PTP through a fluorescence
kinetic run. Prior to dispensing Ca2+ into mitochondria samples, the Biotek plate reader
was preheated to 37oC and syringes were primed with 0.2mM CaCl2. Each well received
sequential nanomolar injections of 0.2mM CaCl2 every 3 minutes until the point of the
PTP was reached and an efflux of Ca2+ was expressed through the fluorescent signaling
at the termination of the kinetic run.
Values produced from fluorescence kinetic run were expressed in arbitrary
fluorescent units. Nanomoles of calcium injected were qualitatively determined by
upward spikes in calcium signaling, and the opening of the PTP was established as a
continuous increase in calcium signaling. Summation of nanomoles of calcium per
32
milligram of mitochondrial protein was calculated as the threshold of calcium uptake
before the induction of the mitochondria PTP.
Flow Cytometry
Mice was euthanized by CO2 inhalation and transcardiac perfusion was then
performed with a rate controlled pump using 0.9% NaCl. Spinal cord was extracted by
applying hydraulic pressure through the spinal column with a 19G needle and syringe
filled with cold PBS. CNS tissue was manually minced with razor blade on ice and
resuspended in 1mL of preheated (37°) RPMI media containing collagenase D (2.5
mg/ml Roche, catalog number: 11088858001) and DNaseI (20 ug/ml SigmaAldrich,
catalog number: D4263). CNS tissue suspension was incubated for 45 minutes at 37°
with slow rotation and then passed through a 70 µm filter into a 50ml conical tube by
pipette. Filter was then washed with 10 mls of RPMI media to maximize yield.
Filtered CNS suspension was centrifuged at 300 x g for 10 minutes at 4°C and
supernatant was aspirated by pipette. Remaining pellet was resuspended in 5mL of
30% Percoll and transferred to a 15 mL conical tube. Resuspended pellet was
centrifuged at 500 x g for 10 minutes at room temperature with slower deceleration
settings (Sorvall legend XTR deceleration deceleration at 7 or lower). The top
lipid/myelin layer will appear white in color and is aspirated completely by vacuum
suction. Remaining CNS cells are resuspended in 5 mL RPMI10 media and centrifuged
at 300 x g for 5 minutes. Remaining cell pellet was resuspended in 1mL of RPMI10
33
media and was aliquoted into into two FACS tubes (BD Biosciences, 5mL polystyrene
roundbottom tube, Ref# 352052).
2mL of FACS buffer (PBS, 0.1% NaN3, 1% FBS) is added to each tube and
centrifuged at 300 x g for 5 minutes. Supernatant was then decanted and blotted.
5µl of Fc block (BD Biosciences, Mouse BD Fc Block, Cat# 553142) was added to each
tube, vortexed, and incubated for 5 minutes at 4℃. Relevant antibodies were then
added, vortexed and incubated for 20 to 30 minutes at 4℃ without light. Cells were then
resuspended in 2mL of FACS buffer, centrifuged at 300 x g for 5 minutes and
supernatant was decanted.
Finally, 25µL of countbright bead microspheres (Life technologies, CountBright
beads 0.54x10^6 beads per 50µL, Ref# C36950) were added into each tube and
content was analyzed by flow cytometry. The sample volume, microsphere volume and
the number of cell events are known. Volume of sample analyzed can be calculated
from the number of microsphere events and is used to determine cell concentration.
34
Results/Discussion
R.1 Effect of NIM811 on the course of EAE
The effect of a cyclophilin inhibitor, NIM811, in the context of MS and EAE was yet
unknown. We induced EAE in wild type C57B/6 mice and provided daily injections of
NIM811 vs vehicle after onset of signs of disease and scored them over time.
NIM811treated EAE mice showed a sustained reduction in clinical disease severity
compared to vehicle treated mice (Figure 1, left). The mean cumulative clinical score
was significantly reduced in NIM811treated EAE mice compared to vehicletreated
EAE mice, indicating a reduction in severity and duration of clinical illness (Figure 1,
right).
Figure 1. NIM811 reduces clinical severity of EAE
Mice (C57BL/6) were actively induced to undergo EAE by subcutaneous injection of
35
MOG3555 in complete Freund’s adjuvant and intraperitoneal (IP) injections of pertussis
toxin. Left: Animals were administered daily IP injections of NIM811 (100 mg/kg) or
vehicle (DMSO:PEG400) after onset of clinical disease (days 12 to 14 post induction)
until the day of sacrifice. N = 20 for NIM811treated and N = 24 for vehicletreated
animals (VC). Mean +/ SEM. Right: Cumulative clinical scores (cumulative score) at
day 20 post induction for vehicle (VC) and NIM811 (100 mg/kg)treated animals.
Median and interquartile range. Unpaired Ttest.
R.2 Effect of NIM811 on mitochondrial calcium retention capacity
We investigated the effect of NIM811 on mitochondrial calcium retention capacity (MT
CRC). As expected, external calcium levels displayed a rapid rise after the addition of
calcium, but was gradually absorbed by mitochondria resulting in the spikes seen in
CRC assays (Figure 2). Eventually, the mitochondrias lose the ability to uptake calcium
and the intramitochondrial calcium will actually leak into the buffer, indicating the
occurrence of mitochondrial permeability transition (MPT). MPT is indicated at the point
where fluorescence levels no longer decrease after the addition of external calcium.
The calcium retention capacity of mitochondria isolated from CypD KO mice is greater
than that of WT mice (Figure 2A). Figure 2B shows the effect of in vitro addition of
NIM811 on the CRC of WT mice. There was a distinct increase in CRC for WT
mitochondria that received a dose of NIM811 (1µM) vs those that did not, indicating the
efficacy of NIM811 in increasing CRC of MT in vitro.
36
Figure 2C compares the CRC of isolated MT from CypD KO mice with and without the
addition of NIM811 (1µM). No difference in CRC was observed, indicating the specificity
of NIM811 for cyclophilin D.
Combining Figure 2 A, B, and C, NIM811 demonstrated its specificity and efficacy
against CypD in vitro where, in the context of CRC, the effect of CypD inhibition due to
NIM811 mimicked that of knocking out CypD.
We then investigated the in vivo effect of NIM811 on CypD. Daily injections of either
NIM811 or VC were administered in WT mice for four days. On Day four, 1 hour after
injections, mitochondria was isolated from brain and CRC assays were performed.
There was no significant difference in CRC between the two cohorts (NIM811 vs VC)
(Figure 2D). Unlike our in vitro assays, NIM811 did not increase CRC in vivo.
In vivo CRCs were repeated with mitochondria isolated from liver tissue to test the
possibility that NIM811 was unable to penetrate CNS tissue due to an intact BBB. No in
vivo effect of NIM811 on CRC of liver mitochondria was observed (data not shown).
37
Figure 2. Mitochondrial calcium retention capacity is increased by NIM811 in vitro
but not in vivo.
Mitochondrial calcium retention capacity was measured by serial additions of 5 µM
calcium ion to crude mitochondrial fraction in buffer containing Calcium Green5N as
fluorescent indicator of extracellular calcium and thapsigargin to inhibit endoplasmic
reticulum uptake of calcium. A) Mitochondrial calcium retention capacity in crude
mitochondrial fraction from wild type (WT) and cyclophilin D knockout (CypD KO)
animals. B) Calcium retention capacity in wild type crude mitochondrial fraction without
and with 1 µM NIM811. C) Calcium retention capacity in crude mitochondrial fraction
38
from CypD KO animals without and with 1 µM NIM811. Fig 2, A B and C are all
representative data from three independent experiments. D) Healthy C57BL/6 animals
were administered daily intraperitoneal injections of NIM811 (100 mg/kg) or vehicle
control for 4 days. Crude mitochondrial fractions were obtained from brains of NIM811
and vehicle treated animals to assess mitochondrial calcium retention capacity. Median
with interquartile range. MannWhitney test.
R.3 Effect of NIM811 on the course of EAE in cyclophilin D knockout mice.
We induced EAE in cyclophilin D knockout and wild type C57B/6 mice to verify the
previously reported reduction in the clinical severity of EAE in CypD KO mice. Daily
clinical score averages of the two cohorts are represented in Fig 2. Both cohorts of mice
were followed for 34 days and scored daily, no injections were administered. A
reduction in EAE clinical disease severity is observed (Figure 3, top).
Active induction of EAE was then performed in CypD KO mice where intraperitoneal
injections of NIM811 vs VC were administered daily after the onset of disease. Clinical
score averages of the two cohorts are shown in Figure 3, bottom left. Cumulative clinical
scores for the two cohorts are presented in Figure 3, bottom right, showing significance.
NIM811 significantly reduced the course and severity of EAE in CypD KO mice,
indicating a mechanism of action independent of cyclophilin D (Figure 3).
39
Figure 3. NIM811 reduces severity of EAE in cyclophilin D knockout mice.
Top: Wild type and cyclophilin D knockout mice (C57/B6) were actively induced to
undergo EAE. Clinical course of EAE in cyclophilin D knockout (KO) animals were
compared to wild type (WT) littermates. N = 6 for cyclophilin D knockout animals and N
= 6 for wild type littermates. Mean +/ SEM. No injections administered over the course
of EAE. Bottom left: Clinical course of cyclophilin D knockout animals administered
40
NIM811 (100 mg/kg) or vehicle (VC) during EAE. N = 7 for NIM811treated and N = 7
for vehicletreated animals. Mean +/ SEM. Bottom right: Cumulative clinical scores for
NIM811treated and vehicletreated cyclophilin D knockout EAE animals. Median with
interquartile range. Unpaired ttest.
R.4 Effect of NIM811 on CNS infiltrating leucocytes
Flow cytometry was performed on dissociated thoracolumbar spine of EAE mice to
compare the relative amount of CNS infiltrating leukocytes in NIM811 vs VC treated
animals. EAE mice was first perfused and the thoracolumbar spine was then extracted,
dissociated, and labeled with monoclonal antibodies specific for CD11b, CD45, and
CD3.
Figure 4, upper panel shows a representative fluorescenceactivated cell sorting
(FACS) analysis. On the very left, gating for countbrightbeads and live cells are
displayed. The middle and left of the upper panel respectively represents the cell
populations from vehicle control and NIM811 treated EAE mice. Compared to vehicle
treated mice, there was a decrease in CD11b+CD45hi cells in the NIM811 treated
animal, representing significant reduction in CNS infiltrating macrophages.
Absolute counts of total leukocytes, T cells, macrophages, and microglia were
compared between the NIM811 vs VC treated cohorts of EAE mice (Figure 4, lower
panel). A significant reduction in total CNS infiltrating leukocytes was observed,
consistent with the significant reduction in CNS infiltrating macrophages. CNS T cells
and microglia populations were not significantly influenced by treatment of NIM811.
41
Figure 4. CNS infiltrating leucocytes in EAE are reduced by NIM811
Thoracolumbar spine from EAE mice treated with NIM811 and vehicle were dissociated,
labeled with monoclonal antibodies specific for CD11b, CD45 and CD3. Microglia were
identified as CD11b+CD45lo cells. Macrophage were CD11b+CD45hi cells.
42
CNSinfitrating T cells were CD3+. Total CNS Leucocytes were CD45+. Top panels
shows representative FACS analysis. Lower panels show data with median and
interquartile range. Unpaired Ttest.
43
Discussion and Future Direction
Multiple sclerosis is a complex, wellstudied disease involving both
immunopathological as well as neurodegenerative mechanisms with extensive interplay
and crosstalk between the central nervous system and the peripheral blood [17]. As a
nonimmunosuppressive, nonselective cyclophilin inhibitor, NIM811 could inhibit two
relevant targets with the possibility of executing a dual effect. Cyclophilin D is implicated
in mechanisms of neurodegeneration associated with progressive MS while the
extracellular chemotactic effect of cyclophilin A is associated with early leukocyte
infiltration into the CNS. The beneficial effect of NIM811 on the course and severity of
EAE was first established in WT mice (Figure 1) and its mechanism of action was then
tested with cyclophilin D knockout mice.
Cyclophilin D in EAE
Since cyclophilin D has been identified as a regulatory component of mPTP, the
inhibition of cyclophilin D may influence mitochondrial dysfunction, and consequently,
axonal injury in MS and EAE [5]. Forte el al demonstrated a decrease in clinical disease
severity of EAE in cyclophilin D knockout mice. This experiment was repeated with
cyclophilin D knockout vs wildtype mice from our lab, where a similar trend was
observed (Figure 3 Top).
Calcium retention capacity assays were performed with isolated mitochondria
from mice brain to investigate the cyclophilin D specific mechanism of NIM811. While
NIM811 did have an specific inhibitory effect on cyclophilin D in vitro (Figure 2 ABC), the
44
increase in calcium retention capacity of isolated mitochondria was not observed in vivo
(Figure 2D). Healthy mice was used for our CRC assays instead of EAE mice because
the animal model of EAE does result in mitochondrial dysfunction and mitochondrial
permeability transition, which could confound our CRC data. However, due to the lack of
an active EAE induction, these WT mice will have an intact blood brain barrier which is
normally broken down by pertussis toxin. The intact blood brain barrier could impede
drug penetration into the CNS. Therefore, we repeated the in vivo calcium retention
capacity assays with liver tissue and similar results were obtained decreasing the
possibility of poor CNS penetration by NIM811.
To verify that NIM811’s mechanism of action in the context of EAE was
independent of cyclophilin D, the effect of NIM811 was studied in EAE cyclophilin D
knockout mice. As expected, EAE cyclophilin D knockout mice treated with NIM811 had
significantly reduced clinical disease severity compared to VC (Figure 3 Bottom).
Cyclophilin A in EAE
NIM811 is a structural analogue of cyclosporin that retains the nonselective
inhibitory property against proteins in the cyclophilin family. As a nonselective
cyclophilin inhibitor, there is another target candidate for NIM811 that could explain its
observed beneficial effect on EAE mice cyclophilin A. Cyclophilin A has a multitude of
functions and is highly abundant, found intracellularly and extracellularly. Extracellularly,
cyclophilin A functions as a leukocyte chemotactic factor by activating matrix
metalloproteinases after binding to CD147 aka EMMPRIN. Matrix metalloproteinases
are necessary for leukocytes to escape periventricular cuffing and enter the CNS
45
parenchyma. Intraperitoneal injection of an antiEMMPRIN functionblocking antibody
was shown to attenuate EAE disease severity in mice [24]. Alternatively, the inhibition of
cyclophilin A and its subsequent extracellular chemotactic function is a possible
mechanism of action for NIM811 in vivo.
Using flow cytometry, subsets of CNS infiltrating leukocytes were compared
between wild type EAE mice receiving NIM811 vs VC treatment. We observed a
significant reduction in total CNS infiltrating leukocytes, consistent with a significant
reduction in macrophages (Figure 4). In order to investigate this cyclophilin
Adependent mechanisms of NIM811, course and biology of EAE in cyclophilin A
knockout (CypA KO) mice should be studied.
If NIM811’s mechanism of action is cyclophilin A dependent, then NIM811 should
have no effect on cyclophilin A knockout mice in the context of EAE. Additionally, EAE
cyclophilin A knockout mice would exhibit a similar reduction in macrophages seen in
Figure 4. Further complicating the matter, CypA has a multitude of functions. An
intracellular signaling mechanism of CypA and its repression of Th2 responses may
also be the target of NIM811. In order to distinguish between extracellular and
intracellular mechanisms, a cellimpermeable cyclophilin inhibitor will be adopted.
46
Literature Cited
[1] Wagner CA, Goverman JM. Novel Insights and Therapeutics in Multiple Sclerosis. F1000Res. 2015 Aug 7;4(F1000 Faculty Rev):517. [2] Ebers GC, Heigenhauser L, Daumer M, Lederer C, Noseworthy JH. Disability as an outcome in MS clinical trials. Neurology 2008; 71:624–31. [3] Nikić I, Merkler D, Sorbara C, Brinkoetter M, Kreutzfeldt M, Bareyre FM, Brück W, Bishop D, Misgeld T, Kerschensteiner M. A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis. Nat Med. 2011 Apr;17(4):4959. [4] Dutta R, Trapp BD. Pathogenesis of axonal and neuronal damage in multiple sclerosis. Neurology 2007; 68: S22–31.discussion S43–54. [5] Forte M, Gold BG, Marracci G, Chaudhary P, Basso E, Johnsen D, Yu X, Fowlkes J, Rahder M, Stem K, Bernardi P, Bourdette D. Cyclophilin D inactivation protects axons in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Proc Natl Acad Sci U S A. 2007 May 1;104(18):755863. [6] Basso E, Fante L, Fowlkes J, Petronilli V, Forte MA, Bernardi P. Properties of the permeability transition pore in mitochondria devoid of Cyclophilin D. J Biol Chem. 2005 May 13;280(19):1855861. [7] Hedström AK, Olsson T, Alfredsson L. The Role of Environment and Lifestyle in Determining the Risk of Multiple Sclerosis. Curr Top Behav Neurosci. 2015;26:87104. [8] Lucchinetti CF, Popescu BF, Bunyan RF, Moll NM, Roemer SF, Lassmann H, Brück W, Parisi JE, Scheithauer BW, Giannini C, Weigand SD, Mandrekar J, Ransohoff RM. Inflammatory cortical demyelination in early multiple sclerosis. N Engl J Med. 2011 Dec 8;365(23):218897. [9] Fischer MT, Wimmer I, Höftberger R, Gerlach S, Haider L, Zrzavy T, Hametner S, Mahad D, Binder CJ, Krumbholz M, Bauer J, Bradl M, Lassmann H. Diseasespecific molecular events in cortical multiple sclerosis lesions. Brain. 2013 Jun;136(Pt 6):1799815.
47
[10] Compston A. Genetic epidemiology of multiple sclerosis. Journal of Neurology, Neurosurgery, and Psychiatry. 1997;62(6):553561. [11] Kobelt G, Berg J, Atherly D, Hadjimichael O. Costs and quality of life in multiple sclerosis: a crosssectional study in the United States. Neurology. 2006 Jun 13;66(11):1696702. [12] Sadovnick AD, Armstrong H, Rice GP, Bulman D, Hashimoto L, Paty DW, Hashimoto SA, Warren S, Hader W, Murray TJ, et al. A populationbased study of multiple sclerosis in twins: update. Ann Neurol. 1993 Mar;33(3):2815. [13] Beecham AH et al. International Multiple Sclerosis Genetics Consortium (IMSGC). Analysis of immunerelated loci identifies 48 new susceptibility variants for multiple sclerosis. Nat Genet. 2013 Nov;45(11):135360. [14] Lutton JD, Winston R, Rodman TC. Multiple sclerosis: etiological mechanisms and future directions. Exp Biol Med (Maywood). 2004 Jan;229(1):1220. [15] Bäärnhielm M, Hedström AK, Kockum I, Sundqvist E, Gustafsson SA, Hillert J, Olsson T, Alfredsson L. Sunlight is associated with decreased multiple sclerosis risk: no interaction with human leukocyte antigenDRB1*15. Eur J Neurol. 2012 Jul;19(7):95562. [16] Dendrou CA, Fugger L, Friese MA. Immunopathology of multiple sclerosis. Nat Rev Immunol. 2015 Sep 15;15(9):54558. [17] Macchi B, MarinoMerlo F, Nocentini U, Pisani V, Cuzzocrea S, Grelli S, Mastino A. Role of inflammation and apoptosis in multiple sclerosis: Comparative analysis between the periphery and the central nervous system. J Neuroimmunol. 2015 Oct 15;287:807. [18] Lee S, Xu L, Shin Y, Gardner L, Hartzes A, Dohan FC, Raine C, Homayouni R, Levin MC. A potential link between autoimmunity and neurodegeneration in immunemediated neurological disease. J Neuroimmunol. 2011 Jun;235(12):5669. [19] Yadav SK, Mindur JE, Ito K, DhibJalbut S. Advances in the immunopathogenesis of multiple sclerosis. Curr Opin Neurol. 2015 Jun;28(3):20619.
48
[20] Heneka MT, Kummer MP, Latz E. Innate immune activation in neurodegenerative disease. Nat Rev Immunol. 2014 Jul;14(7):46377. [21] Lambert JC et al. Metaanalysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease. Nat Genet. 2013 Dec;45(12):14528. [22] Nalls MA et al. Largescale metaanalysis of genomewide association data identifies six new risk loci for Parkinson's disease. Nat Genet. 2014 Sep;46(9):98993. [23] Kebir H, Ifergan I, Alvarez JI, Bernard M, Poirier J, Arbour N, Duquette P, Prat A. Preferential recruitment of interferongammaexpressing TH17 cells in multiple sclerosis. Ann Neurol. 2009 Sep;66(3):390402. [24] Agrawal SM, Silva C, Wang J, Tong JP, Yong VW. A novel antiEMMPRIN functionblocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis. J Neuroinflammation. 2012 Apr 5;9:64. [25] Agrawal SM, Williamson J, Sharma R, Kebir H, Patel K, Prat A, Yong VW. Extracellular matrix metalloproteinase inducer shows active perivascular cuffs in multiple sclerosis. Brain. 2013 Jun;136(Pt 6):176077. [26] Popescu BF, Lucchinetti CF. Pathology of demyelinating diseases. Annu Rev Pathol. 2012;7:185217. [27] Frischer JM, Bramow S, DalBianco A, Lucchinetti CF, Rauschka H, Schmidbauer M, Laursen H, Sorensen PS, Lassmann H. The relation between inflammation and neurodegeneration in multiple sclerosis brains. Brain. 2009 May;132(Pt 5):117589. [28] Fischer MT, Sharma R, Lim JL, Haider L, Frischer JM, Drexhage J, Mahad D, Bradl M, van Horssen J, Lassmann H. NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury. Brain. 2012 Mar;135(Pt 3):88699. [29] Ji Q, Castelli L, Goverman JM. MHC class Irestricted myelin epitopes are crosspresented by TipDCs that promote determinant spreading to CD8⁺ T cells. Nat Immunol. 2013 Mar;14(3):25461. [30] Lolli F, Martini H, Citro A, Franceschini D, Portaccio E, Amato MP, Mechelli R, Annibali V, Sidney J, Sette A, Salvetti M, Barnaba V. Increased CD8+ T cell responses
49
to apoptotic T cellassociated antigens in multiple sclerosis. J Neuroinflammation. 2013 Jul 27;10:94. [31] Barr TA, Shen P, Brown S, Lampropoulou V, Roch T, Lawrie S, Fan B, O'Connor RA, Anderton SM, BarOr A, Fillatreau S, Gray D. B cell depletion therapy ameliorates autoimmune disease through ablation of IL6producing B cells. J Exp Med. 2012 May 7;209(5):100110. [32] Yamasaki R, Lu H, Butovsky O, Ohno N, Rietsch AM, Cialic R, Wu PM, Doykan CE, Lin J, Cotleur AC, Kidd G, Zorlu MM, Sun N, Hu W, Liu L, Lee JC, Taylor SE, Uehlein L, Dixon D, Gu J, Floruta CM, Zhu M, Charo IF, Weiner HL, Ransohoff RM. Differential roles of microglia and monocytes in the inflamed central nervous system. J Exp Med. 2014 Jul 28;211(8):153349. [33] Hemmer B, Kerschensteiner M, Korn T. Role of the innate and adaptive immune responses in the course of multiple sclerosis. Lancet Neurol. 2015 Apr;14(4):40619. [34] Olsen JA, Akirav EM. Remyelination in multiple sclerosis: cellular mechanisms and novel therapeutic approaches. J Neurosci Res. 2015 May;93(5):68796. [35] Chari DM. Remyelination in multiple sclerosis. Int Rev Neurobiol. 2007;79:589620. [36] Prins M, Schul E, Geurts J, van der Valk P, Drukarch B, van Dam AM. Pathological differences between white and grey matter multiple sclerosis lesions. Ann N Y Acad Sci. 2015 Sep;1351:99113. [37] Mallucci G, PeruzzottiJametti L, Bernstock JD, Pluchino S. The role of immune cells, glia and neurons in white and gray matter pathology in multiple sclerosis. Prog Neurobiol. 2015 Apr;127128:122. [38] Trapp BD, Stys PK. Virtual hypoxia and chronic necrosis of demyelinated axons in multiple sclerosis. Lancet Neurol. 2009 Mar;8(3):28091. [39] Giorgi C, Agnoletto C, Bononi A, Bonora M, De Marchi E, Marchi S, Missiroli S, Patergnani S, Poletti F, Rimessi A, Suski JM, Wieckowski MR, Pinton P. Mitochondrial calcium homeostasis as potential target for mitochondrial medicine. Mitochondrion. 2012 Jan;12(1):7785.
50
[40] Friese MA, Schattling B, Fugger L. Mechanisms of neurodegeneration and axonal dysfunction in multiple sclerosis. Nat Rev Neurol. 2014 Apr;10(4):22538. [41] Sullivan PG, Rabchevsky AG, Waldmeier PC, Springer JE. Mitochondrial permeability transition in CNS trauma: cause or effect of neuronal cell death? [42] Bernardi P, Rasola A, Forte M, Lippe G. The Mitochondrial Permeability Transition Pore: Channel Formation by FATP Synthase, Integration in Signal Transduction, and Role in Pathophysiology. Physiol Rev. 2015 Oct;95(4):111155. [43] Kumari S, Roy S, Singh P, SinglaPareek SL, Pareek A. Cyclophilins: proteins in search of function. Plant Signal Behav. 2013 Jan;8(1):e22734. [44] Readnower RD, Pandya JD, McEwen ML, Pauly JR, Springer JE, Sullivan PG. Postinjury administration of the mitochondrial permeability transition pore inhibitor, NIM811, is neuroprotective and improves cognition after traumatic brain injury in rats. J Neurotrauma. 2011 Sep;28(9):184553. [45] Constantinescu CS, Farooqi N, O'Brien K, Gran B. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol. 2011 Oct;164(4):1079106. [46] Procaccini C, De Rosa V, Pucino V, Formisano L, Matarese G. Animal models of Multiple Sclerosis. Eur J Pharmacol. 2015 Jul 15;759:18291. [47] Yednock TA, Cannon C, Fritz LC, SanchezMadrid F, Steinman L, Karin N. Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha 4 beta 1 integrin. Nature. 1992 Mar 5;356(6364):636. [48] Matloubian M, Lo CG, Cinamon G, Lesneski MJ, Xu Y, Brinkmann V, Allende ML, Proia RL, Cyster JG. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature. 2004 Jan 22;427(6972):35560. [49] Atlas: Multiple Sclerosis Resources in the World 2008. Geneva, Switzerland: World Health Organisation; 2008. Available at: http://www.msif.org/aboutms/publicationsandresources/. Accessed April 2, 2016
http://www.msif.org/about-ms/publications-and-resources/
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VITA
Zi L. Huang was born on February 11, 1992, in the province of Hubei, China. At the age
of nine, he immigrated with his family to the United States of America and resided in the
state of Virginia. In 2010, Zi began his postsecondary education at Northern Virginia
Community College while working parttime jobs. Graduating Summa Cum Laude with
an Associates of Science Degree in 2012, he transferred to the University of Virginia
where he graduated with distinction in 2014 with a Bachelors of Arts degree in Biology.
Zi completed the CERT program at Virginia Commonwealth University in 2015 and
continued his graduate studies under the mentorship of Dr. Unsong Oh. While pursuing
a Master of Science in Physiology and Biophysics, he was a teaching assistant for the
undergraduate physiology course, volunteer leader at HandsonRVA, served at the
Goochland Food Pantry on weekends, and volunteered his free time at the Goochland
Free Clinic. Zi was accepted into VCU School of Medicine and will begin coursework
toward a M.D. degree in the Fall of 2016.
Virginia Commonwealth UniversityVCU Scholars Compass2016
Pharmacological Inhibition of Cyclophilin Ameliorates Experimental Allergic EncephalomyelitisZi L. HuangDownloaded from
tmp.1463376995.pdf.SLhoL