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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

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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

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Pharmacological Inhibition of Cyclophilin Ameliorates Experimental Allergic ... · 2017. 1. 13. · A thesis submitted in partial fulfillment of the requirements for the degree of