Projects Funded - Preclinical

MSA Coalition Grant #2021-09-001 – $50,000

No disease-modifying treatment is available for multiple system atrophy (MSA). The cytopathological hallmark of MSA is the accumulation of a-synuclein (aSyn) protein aggregates forming glial cytoplasmic inclusions. The aggregated form of aSyn plays a central role in the pathogenesis of MSA. Thus, there is the hope that drugs that prevent or revert aSyn aggregation might alter MSA progression by preventing, blocking, or slowing neurodegeneration. We identified SynuClean-D (SC-D), a small molecule that interrupts aSyn aggregation, disentangles mature fibrils, and hampers the propagation of aggregated aSyn. The agglutination of all these synergic properties in a single scaffold endorses SC-D with significant neuroprotective activity. This project aims to evolve this candidate into a drug able to penetrate the brain efficiently and potentially prevent the progression of neurological damage. In a subsequent stage of the project, the molecule’s activity will be validated in a preclinical model of MSA that reproduces the specific pathology of this disorder in the mouse brain. If successful, this preclinical proof of concept could form a basis for clinical trials in people living with MSA.

RESULTS

MSA Coalition Grant #2021-09-003 – $50,000

Real-time quaking-induced conversion (RT-QuIC) is an ultrasensitive detection technology for misfolded proteins and has greater sensitivity and specificity compared to conventional approaches such as immunohistochemistry. As demonstrated in peer-reviewed publications, Dr. Kondru has implemented the RTQuIC assay to study various protein misfolding diseases and implemented to detect seeding α-syn in the brain and cerebrospinal fluid (CSF) as a biomarker in α- synucleinopathies and has a sensitivity and specificity level ranging from 95-100%. Recent evidence based on animal models of α-synucleinopathies and cryo-electron microscopy (cryo-EM) studying synuclein derived from human autopsy brain report compelling evidence that demonstrates distinct pathological synuclein strains between disease types, which can be used as a molecular biomarker to distinguish between diseases. This was recently validated in a study whereby the RT-QuIC assay was able to discriminate between samples of CSF from patients diagnosed with PD and MSA, with 95% sensitivity. This technology has yet to be explored for the drug discovery. This proposal leverages the recent advances in the distinction of seeding α-synuclein strains in MSA using RT-QuIC technology. We propose to extend this application for drug discovery to specifically target the MSA α-synuclein species by using prescreened and validated compounds that are a result of high-throughput screen.

RESULTS

MSA Coalition Grant #2021-09-005 – $50,000

Toxic α-synuclein aggregates in synucleinopathies are hypothesized to spread through the nervous system templating native α-synuclein into the aggregated form in a prion-like fashion. Evidence shows that extracts from MSA brains are more toxic upon inoculation in A53T-h-α-synuclein transgenic mic (M83 line) than extracts from PD brains and behaved as a prion [1]. These results suggest that the aggregated species in MSA are functionally different from the aggregates in PD and dementia with Lewy bodies as recently demonstrated at the structural level [2] . Characteristically of MSA, α-synuclein aggregate-containing inclusions are predominantly found in oligodendrocytes. The cellular milieu of oligodendrocytes have been implicated as a factor in strain formation, as they are able to convert one α-synuclein strain into another [3]. To demonstrate that oligodendroglial factors directly can make α-synuclein aggregates more toxic we demonstrate the α-synuclein-aggregate stimulating oligodendroglial protein p25α causes the formation a α-synuclein aggregate strain that upon inoculation kills M83 mice faster than control preformed fibrils (PFF) (Fig. 1; [4]). These results show that differences in aggregate structure are crucial factors in disease progression and toxicity. The prion-like property of aggregates seeding native α-synuclein into an aggregated α-synuclein species has allowed seed amplification techniques to generate patient derived aggregates from patient cerebrospinal fluid (CSF) for functional and structural studies [5], [6]. MSA and PD α-synuclein aggregates derived from CSF were demonstrated to differ in structure and function [6]. We obtained from Claudio Soto, PD and MSA aggregates derived from CSF and can reamplify them for further studies without perturbing their structure (Fig. 2). Our preliminary data demonstrates different functional effects on cellular inclusion formation and impacts on cellular functions in our oligodendroglial cells and organotypic brain slice culture models (Fig 3 & 4). Strikingly, PD fibrils induces stronger inclusion cytopathology in the oligodendrocyte cell line model than the MSA fibrils whereas the opposite was observed in the brain tissue model. The mechanisms behind these differences will be the focus of the present project but may involve different impacts on cellular homeostatic systems e.g. the ability of cells to sequester pathological aggregates in incluisions.

We hypothesize that intracellular α-synuclein aggregates contributes to cellular degeneration by activation of the ER calcium pump SERCA, leading to increased Ca2+ load in the ER lumen and reduced Ca2+ levels in the cytosol. This α-synuclein induced Ca2+ dysregulation precedes cell death, and we have shown that by counteracting the Ca2+ dysregulation with the SERCA inhibitor cyclopiazonic acid (CPA) we are able to increase cell viability in cellular and in vivo models of neuro- and oligodendroglial degeneration [7]. Another way of counteracting the ER Ca2+ overload while simultaneously normalizing cytosolic Ca2+ concentration, is by activation of the ER resident calcium channel Ryanodine Receptor (RyR). Caffeine is a known agonist of RyR, and indeed studies have shown association between coffee consumption and reduced risk of PD [8]. Additionally, chronic caffeine treatment has been shown to reduce inclusion formation and reestablish autophagic activity in a PFF mouse model [9]. Our unpublished data also show increased survival with caffeine treatment in a mouse model of prion-like spreading (Fig. 5). Notably, although caffeine affects multiple cellular pathways, our knowledge of α-synuclein induced Ca2+ dysregulation inclines us to believe that caffeines agonistic effect on RyR is the determining factor in reducing aggregate induced stress because the adenosine receptor agonist istradefylline did not have any beneficial effect in the mouse model (data not shown). Moreover, we hypothesize that Ca2+ overload in the ER will increase the Ca2+ flux into the mitochondria leading to neurodegeneration as inhibiting the ER-mitochondrial flux protects α-synuclein aggregate models [10], which corroborate our hypothesis of dysregulation of ER Ca2+ homeostasis in synucleinopathies (Fig 6).

We propose to determine if MSA aggregate-dependent toxicity in our models displays sensitivity to the protective effect of the calcium modulating compounds as demonstrated for SERCA inhibitor cyclopiazonic acid (CPA) [7], ryanodine receptor agonist caffeine (Fig. 6), and IP3R antagonist 2-APB [10] and will compare this to PD aggregates. Such insight will be decisive for identifying potential candidate compounds for MSA selective targets. We hypothesize that restoring calcium homeostasis will normalize cellular function, prevent cell death and thus prolong life and enhances quality of life for MSA patients.

RESULTS

MSA Coalition Grant #2021-09-006 – $50,000

Pathological hallmarks of multiple system atrophy (MSA) include deposits of a protein called alpha-synuclein in neurons and support cells called oligodendroglia. But MSA also involves a process called neuroinflammation in which brain cells known as microglia and astrocytes respond to neuronal injury. Neuroinflammatory responses may dictate whether certain brain regions degenerate or are protected in the course of the disease. Recently, Biohaven Pharmaceuticals instituted a clinical trial to test whether verdiperstat, an inhibitor of the inflammatory enzyme myeloperoxidase, could halt MSA progression. In parallel, we tested whether verdiperstat could alter neuroinflammation in the brain of patients who had previously been tracked with a PET scan that measures neuroinflammation. Our study has enrolled 20 patients, and 10 so far have received the drug. The postmortem brain of an advanced-stage MSA patient who had been imaged for ~3years (and participating in our trial on compassionate basis) now provides a unique opportunity to examine the brain responses to verdiperstat. In this case, there was a strikingly different response among distinct brain regions. In parallel to ongoing PET investigations of other patients treated with verdiperstat, we propose here to examine in detail the neuroinflammatory gene-expression changes among different brain regions in this patient and compare this to other untreated MSA patients and control subjects. We will do this by examining gene changes at the single-cell level in these brains, as well as employing brain-tissue markers of neuroinflammation. Finally, we will generate induced pluripotent stem cells (iPSC) from this and other patients. Using iPSC models, we will in the future test whether different astrocyte and microglial cell populations have differing responses to verdiperstat. Our study, using verdiperstat as a probe of inflammation, promises to shed light into this important process in MSA and how it might be appropriately targeted for therapeutic benefit.

RESULTS

MSA Coalition Grant #2020-05-003 – $50,000

The proposed study aims to verify whether methylphenidate (MPH) “Ritalin”, a drug used to treat attention deficit hyperactivity disorder (ADHD) and narcolepsy, has a neuroprotective effect on patient-derived in vitro models of MSA. A-synuclein (asyn) is the pathogenic protein of MSA which aggregates and accumulates in glia and neurons, especially in oligodendrocytes and that constitutes the pathologic hallmark of MSA. We have recently found that Synapsin III (Syn III), a protein composing Lewy body insoluble fibrils of a-syn in the brain of Parkinson’s disease (PD) patients, can be also found within these glial cytoplasmic inclusions (GCI) in the postmortem brains of MSA patients (preliminary results). Our studies in the last few years have disclosed that Syn III controls a-syn aggregation as well as the related neurotoxcitiy and that MPH efficiently restores motor functions in a PD animal model by modulating the pathological a-syn/Syn III interplay in experimental models of PD. Our working hypothesis is that, since Syn III also composes GCI, MPH may also target these pathological aggregates in the brain of MSA patients and it can lead to the prevention or reduction of a-syn aggregation, resulting in neuroprotection. To test this hypothesis, we will generate and differentiate in vitro models of MSA and we will test if MPH rescue their phenotype.

RESULTS

MSA Coalition Grant #2016-09-010 – $35,000

No disease-modifying treatment is available for multiple system atrophy (MSA). Nilotinib, a drug used to treat leukemia, inhibits the enzyme activity of a protein tyrosine kinase called Abelson (c-Abl). Recent studies in preclinical models of Parkinson’s disease (PD) suggest that inhibition of c-Abl activity by nilotinib might help increase the clearance of alpha-synuclein, reducing neuroinflammation and provide neuroprotection. A pilot clinical trial in PD and Dementia with Lewy Body (DLB) patients further suggests that nilotinib may prove useful as a disease-modifying drug in neurodegenerative diseases associated with alpha-synuclein aggregation. A larger, randomized, placebo-controlled trial is expected to start in 2017. Whether this strategy may work in MSA is unknown. This study aims at assessing the effects of nilotinib on motor symptoms, alpha-synuclein aggregation and surrogate markers of neurodegeneration and neuroinflammation in a transgenic mouse model of MSA. If positive, this preclinical proof of concept study will pave the way for validating nilotinib as a candidate neuroprotective drug to be tested in a clinical trial in MSA patients.

RESULTS

1. Publication (April 2020): Nilotinib Fails to Prevent Synucleinopathy and Cell Loss in a Mouse Model of Multiple System Atrophy.

2. A poster abstract was presented at the International Congress for Parkinson’s Disease and Movement Disorders in Hong Kong, October 2018:

P. Guerin, M. Lopez-Cuina, E. Bezard, W. Meissner, P-O. Fernagut.
Nilotinib for treating MSA: A preclinical proof of concept study [abstract].
Mov Disord. 2018; 33 (suppl 2).

3. Research update by Pierre-Olivier Fernagut (August 2018)

No disease-modifying treatment is available for multiple system atrophy (MSA). Nilotinib, a drug used to treat leukemia, inhibits the enzyme activity of a protein tyrosine kinase called Abelson (c-Abl). Recent studies in preclinical models of Parkinson’s disease (PD) suggest that inhibition of c-Abl activity by nilotinib might help increasing the clearance of alpha-synuclein, reducing neuroinflammation and providing neuroprotection. A pilot clinical trial in PD and dementia with Lewy bodies patients has not identified issues with safety or tolerability. Larger, randomized, placebo-controlled trials have started in 2017. Whether this strategy may work in MSA is unknown.

This study investigated the effects of 2 doses of nilotinib on motor symptoms, alpha-synuclein aggregation and surrogate markers of neurodegeneration in a transgenic mouse model of MSA to determine if nilotinib may be a candidate neuroprotective drug to be tested in a clinical trial in MSA.

Nilotinib daily intraperitoneal administration during 3 months was well tolerated by the mice. Motor tests did not show any behavioral modification induced by nilotinib and neuropathological analyses indicated no beneficial effects of nilotinib on dopaminergic neurons degeneration in the mouse model of MSA, whereas the highest dose of nilotinib led to decreased levels of alpha-synuclein and c-Abl in the striatum.

Altogether, these results indicate that nilotinib may not be a robust candidate for a disease-modifying treatment in MSA.

3. Research update by Pierre-Olivier Fernagut (August 2017)

No disease-modifying treatment is available for multiple system atrophy (MSA). Nilotinib, a drug used to treat leukemia, inhibits the enzyme activity of a protein tyrosine kinase called Abelson (c-Abl). Recent studies in preclinical models of Parkinson’s disease (PD) suggest that inhibition of c-Abl activity by nilotinib might help increasing the clearance of alphasynuclein, reducing neuroinflammation and providing neuroprotection. A pilot clinical trial in PD and dementia with Lewy bodies patients further suggests that nilotinib may prove useful as a disease-modifying drug in neurodegenerative diseases associated with alpha-synuclein aggregation. A larger, randomized, placebo-controlled trial is expected to start in 2017. Whether this strategy may work in MSA is unknown.

This study aims at assessing the effects of nilotinib on motor symptoms, alpha-synuclein aggregation and surrogate markers of neurodegeneration and neuroinflammation in a transgenic mouse model of MSA. If positive, this preclinical proof of concept study will pave the way for validating nilotinib as acandidate neuroprotective drug to be tested in a clinical trial in MSA.

So far daily intraperitoneal administration of nilotinib during 3 months proved to be safe and well tolerated by mice. Motor tests did not show any behavioral modification in mice treated with nilotinib compared with placebo. Post-mortem analysis of alpha-synuclein accumulation and neuronal loss is ongoing.

 

MSA Coalition Grant #2015-04-006 -$48,746

MSA Coalition Grant #2016-09-005 – $50,000

Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disorder that currently lacks efficient therapy. We will test a novel drug candidate that blocks formation of toxic α-synuclein aggregates, which are believed to have a causative role in the pathogenesis of MSA. The drug has shown promising results in pre-clinical models of Alzheimer’s and Parkinson’s disease. As a necessary step towards human clinical trials, we will now test the drug in a pre-clinical model of
MSA. In a proof-of-concept study supported by the MSA Coalition, we demonstrated that when delivered directly into the brain, the drug reduced the density of toxic α-synuclein aggregates in the brains of MSA mice.

The current follow-up project has the goal to expand these studies and test the ability of the new drug to prevent and/or reverse the formation of the α-synuclein aggregates when applied subcutaneously – a route of administration with higher
relevance to future clinical applications. The mouse studies will be conducted in collaboration between the Stefanova laboratory at the Medical University of Innsbruck, Austria, and the Bitan laboratory at UCLA, using a well-established preclinical model of MSA that reproduces the specific pathology of this disorder in the mouse brain.

RESULTS

1. Published article (July 2019): The molecular tweezer CLR01 reduces aggregated, pathologic, and seeding-competent α-synuclein in experimental multiple system atrophy.

2. Presentation (January 2019): Promising results in a new proof-of-concept pre-clinical study of the drug candidate CLR01

3. Research Update by Nadia Stefanova (August 2018)

Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disorder that currently lacks efficient therapy. We are testing a novel drug candidate that blocks formation of toxic protein clumps, or aggregates, made of the protein αsynuclein, which are believed to have a causative role in the pathogenesis of MSA. The drug has shown promising results in pre-clinical models of Alzheimer’s and Parkinson’s disease. As a necessary step towards human clinical trials, we are
now testing the drug in a pre-clinical model of MSA. In a previous proof-of-concept study supported by the MSA Coalition, we demonstrated that when delivered directly into the brain, the drug reduced the density of toxic α-synuclein aggregates
in the brains of MSA mice.

The current follow-up project has the goal to expand these studies and test the ability of the drug to prevent and/or reverse the formation of the α-synuclein aggregates when applied subcutaneously – a route of administration with higher relevance to future clinical applications. The mouse studies are conducted in collaboration between the Stefanova laboratory at the Medical University of Innsbruck, Austria, and the Bitan laboratory at UCLA, using a well-established pre-clinical model of MSA that reproduces the specific pathology of this disorder in the mouse brain. Previously, such studies were done using devices called osmotic minipumps, which deliver a steady dose of the drug into the body for up to five weeks, but are limited in the amount of drug they can deliver and for studies longer than 5 weeks. We found out about a company that provides custom-made pellets for subcutaneous, continuous delivery of drugs, which could potentially overcome the dose and timelimit restrictions of the osmotic minipumps, and decided to test this attractive approach. We performed surgery to implant the subcutaneously, pellets which were designed to deliver three different doses of the drug, or placebo, over a period of two months. Each group included 5 male and 5 female MSA mice. Blood sampling was performed after one and two months of treatment to define the levels of the drug in the blood. The effect of the treatment on the motor deficits of the mice was tested at the end of the treatment period. We are in the process of testing the effect also on the brain pathology. Furthermore, during the treatment, we collected additional blood samples to measure α-synuclein levels in the brain of the mice using a novel diagnostic test. We discovered that the pellets did not work quite as promised and found the company to be quite rigid and unsupportive, which limited the efficacy of the drug delivery system. Nonetheless, our initial analysis shows the first evidence of efficient reduction of brain pathology and the novel blood test showed a trend toward dose-dependent decrease in αsynuclein in the brain of the mice, suggesting that if the problem with the delivery system can be overcome, the treatment will reduce the offending brain protein aggregates efficiently. Further studies are needed to refine the delivery system of CLR01 and provide continuously stable levels of the drug over long time periods.

4. Research Update by Nadia Stefanova (December 2017)

Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disorder that currently lacks efficient therapy. We are testing a novel drug candidate that blocks formation of toxic protein clumps, or aggregates, made of the protein αsynuclein, which are believed to have a causative role in the pathogenesis of MSA. The drug has shown promising results in pre-clinical models of Alzheimer’s and Parkinson’s disease. As a necessary step towards human clinical trials, we are now testing the drug in a pre-clinical model of MSA. In a previous proof-of-concept study supported by the MSA Coalition, we demonstrated that when delivered directly into the brain, the drug reduced the density of toxic α-synuclein aggregates in the brains of MSA mice.

The current follow-up project has the goal to expand these studies and test the ability of the drug to prevent and/or reverse the formation of the α-synuclein aggregates when applied subcutaneously – a route of administration with higher relevance to future clinical applications. The mouse studies are conducted in collaboration between the Stefanova laboratory at the Medical University of Innsbruck, Austria, and the Bitan laboratory at UCLA, using a well-established pre-clinical model of MSA that reproduces the specific pathology of this disorder in the mouse brain. We have completed the low-dose treatments and currently perform the high-dose treatment with the drug. We are also using a novel blood test that allows getting a glimpse into the brain biochemistry using a simple blood test and our initial results using this test suggest that the drug is effective in lowering the levels of brain α-synuclein. Follow-up analysis will include functional assessment of mouse behavior as well as neuropathological and biochemical analysis of the brain to detect changes in the α-synuclein load.

2015 Grant:

MSA Coalition Grant #2015-04-006 – $48,746

Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disorder that currently lacks efficient therapy. We will test a novel drug candidate that blocks formation of toxic alpha-synuclein aggregates, which are believed to have causative role in the pathogenesis of MSA. The drug has shown promising results in preclinical models of Alzheimer’s and Parkinson’s disease. As a necessary step towards human clinical trials, we will now test the drug in a pre-clinical model of MSA. The project has the goal to test the ability of the new drug to prevent and/or reverse the formation of the alpha-synuclein aggregates that are believed to play a major role in the disease process of MSA. The mouse studies will be conducted in collaboration between the Stefanova laboratory at the Medical University of Innsbruck, Austria and the Bitan laboratory at UCLA using a well-established pre-clinical model of MSA which reproduces the specific pathology of this disorder in the mouse brain. These studies will establish the dose-dependent effects of the drug and its efficacy in MSA-like neurodegeneration. The data of this proof-of-concept study will be followed by behavioral studies and thorough characterization of neuroprotection effects generating a preclinical rationale for a future interventional trial in MSA.

RESULTS

1. Research Update by Nadia Stefanova (November 2015):

Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disorder that currently lacks efficient therapy. We are testing a novel drug candidate that blocks formation of toxic alpha-synuclein aggregates, which are believed to have a causative role in the pathogenesis of MSA. The drug has shown promising results in pre-clinical models of Alzheimer’s and Parkinson’s disease. As a necessary step towards human clinical trials, we now test the drug in a pre-clinical model of MSA.

The project has the goal to test the ability of the new drug CLR01 to prevent and/or reverse the formation of the alpha-synuclein aggregates that are believed to play a major role in the disease process of MSA.

MSA transgenic mice that have alpha-synuclein aggregates in oligodendrocytes and replicate the pathological hallmark of the human disease were used. The MSA mice were divided into treatment groups as follows: group one, receiving vehicle; group two, receiving the drug CLR01 at low concentration; and group three, receiving the drug CLR01 at high concentration over a period of one month. To ensure that the drug reached the CNS, it was delivered by an intracerebroventricular catheter.

Our first results indicate that the treatment with the drug CLR01 is not related to indicative of toxicity, i.e. body weight dynamics and survival in the three groups did not differ when monitored during the in vivo observation period. Behavioral analysis at the end of the treatment period indicated significant functional improvement in a dose-dependent manner in MSA mice that received CLR01 therapy back to levels of healthy mice lacking alpha-synuclein aggregates in oligodendrocytes.

Currently we are performing neuropathological and biochemical analysis of the brains to define the pathological substrate of the observed functional improvement.

Based on our interim data we have identified a promising therapeutic strategy to provide functional amelioration in MSA mice. Importantly, the beneficial effect of CLR01 therapy was achieved in MSA mice with a clear phenotypic dysfunction, which corresponds to a stage of clinically overt disease in patients. Therefore the results suggest that CLR01 treatment may be efficient to provide disease modification in MSA patients even when initiated after the clinical onset of symptoms.

Also see results of the followup grant to Stefanova and Bitan

MSA Coalition Grant #2015-04-008 – $30,000

Developing neuroprotective and disease-modifying treatments for multiple system atrophy (MSA) is an urgent unmet need. As of late, interest has been given to insulin and insulin like growth factor-1 (IGF-1) in the neurodegenerative disorder field as both mediate numerous pro-survival actions through their brain receptors. Studies in animal models and postmortem human brain tissue of patients suffering from Alzheimer’s disease (AD) and Parkinson’s disease (PD) have found impaired insulin/IGF-1 signaling and insulin resistance. Moreover, several FDA-approved anti-diabetics have shown positive effects on behavior and surrogate markers of neurodegeneration in preclinical models of AD and PD. These encouraging findings have motivated the conduction of several pilot studies in patients with AD and PD. In this view, a clinical trial assessing the effects of the glucagon-like peptide-1 agonist exendin-4 in PD patients reported beneficial effects on motor symptoms and cognition. A follow up study showed persisting positive effects one year after the end of the study. Interestingly, patients with multiple system atrophy (MSA) show a significant increase in insulin/IGF-1 serum levels suggesting that insulin/IGF-1 signaling may also be impaired in MSA. Our preliminary findings in postmortem brain tissue of MSA patients suggest impaired insulin/IGF-1 signaling and brain insulin resistance. This study aims at assessing the effects of exendin-4 on motor symptoms and surrogate markers of neurodegeneration in a transgenic mouse model of MSA. In case of positive results, our ultimate goal is to test this compound in MSA patients, in line with our major objective to develop disease-modifying or neuroprotective treatments for MSA.

RESULTS

1. Published article (May 2017): Insulin resistance and exendin-4 treatment for multiple system atrophy

2. MSA Coalition Blog (March 2017): Report on MSA Coalition Funded Research: Exendin-4

3. Final report by Wassilios Meissner (January 2017):

Developing neuroprotective and disease-modifying treatments for multiple system atrophy (MSA) is an urgent unmet need. As of late, interest has been given to insulin and insulin like growth factor-1 (IGF-1) in brain disorders as both mediate numerous positive actions through their brain receptors. Studies in animal models and postmortem tissue of patients suffering from Alzheimer’s disease (AD) and Parkinson’s disease (PD) have found impaired insulin/IGF-1 signaling and insulin resistance. Moreover, several approved anti-diabetic drugs have shown positive effects in animal models of AD and PD. These encouraging findings have motivated the conduction of several clinical pilot studies in patients with AD and PD.
Interestingly, MSA patients show a significant increase in insulin/IGF-1 serum levels suggesting that insulin/IGF-1 signaling may also be impaired in MSA. Our findings confirm the presence of insulin resistance in postmortem brain tissue of MSA patients compared to healthy controls. MSA mice likewise show brain insulin resistance, which is reversed by exendin-4, an approved anti-diabetic drug that acts on glucagon-like peptide-1 (GLP-1) receptors. Exendin-4 treatment further preserved dopamine neurons and limited the accumulation of alphasynuclein,
a protein that cumulates in the brains of MSA patients. Taken together, our study provides a rationale for the development of GLP-1 agonists as a potential treatment for MSA.

MSA Coalition Grant #2015-04-004 – $50,000

Multiple system atrophy (MSA) is a rare and devastating neurological disease that impairs body functions and that shares many Parkinson’s disease symptoms. Sadly, there is no treatment that stops the evolution of MSA and current therapies are focused on managing its symptoms. At the molecular level, MSA is characterized by the abnormal accumulation of the protein alpha-synuclein within brain cells, and it is believed that this accumulation is behind the cell death and inflammation observed in the brain. Therefore, our objective is to explore the potential use of the combination of two therapies in order to restore normal alpha-synuclein and inflammation levels. These therapies are immunization against alpha-synuclein, in order to reduce its levels, together with the use of a potent anti-inflammatory drug, lenalidomide, aimed to reduce pathological brain inflammation. We believe that the combined use of both therapies will have synergistic protective effects in an animal model of MSA, and that such approach could be applied to the treatment of MSA patients.

RESULTS

1. Published article (January 2017): Combination of alpha-synuclein immunotherapy with anti-inflammatory treatment in a transgenic mouse model of multiple system atrophy

2. Video: Presentation by Eliezer Masliah at the MSA Coalition Annual Patient & Family Conference (September 2015)

3. Research update by Eliezer Masliah (December 2015):

The main objective of this project is to determine the behavioral and neuropathological effects of immunotherapy against α-syn plus pharmacological reduction of neuroinflammation in a MSA model, and investigate the mechanism of action of such combination of therapies. For this purpose we selected the use of a single-chain variable fragment antibody against alpha-synucleincontaining the ApoB motif (sD5-apoB), and the anti-inflammatory and immunomodulatory drug lenalidomide.

During this first year we have performed animal experiments in the MBP-hα-syn tg mouse Line 1 (MBP1-hα-syn) and are ready to proceed to the neuropathological, biochemical and mechanistic analysis of those brain samples. Moreover, we have also validated the use of both lenalidomide and α-syn-targeted therapies using the ApoB motif in the MBP1-hα-syn mouse model of MSA, both necessary steps to confirm the feasibility of our proposed research. A summary of the research performed this first year is as follows:

1. Combination therapy using immunotherapy against α-syn plus pharmacological reduction of neuroinflammation in MBP1-hα-syn transgenic mice

For the combination therapy experiments we have used MBP1-hα-syn transgenic mice, which express medium levels of alpha-synucelein in oligodendrocytes. Non-tg and tg animals were divided in 4 groups each: vehicle injected with lentivirus (LV) control, vehicle injected with LV-sD5-apoB, lenalidomide injected with LV control, and lenalidomide injected with LV-sD5-apoB (n=8 per group). The lentiviral preparations were injected intra-peritoneally at 10 month/old, one week before lenalidomide treatment in order to avoid a possible inhibitory effect of lenalidomide on antibody production. Subsequent lenalidomide treatment (100 mg/kg) was performed orally by gavage daily for 4 weeks. Mice were behaviorally analyzed before and after the treatments (11 month/old).

We are currently analyzing behavioral data and performing the initial neuropathological analysis (Aim 1). For that purpose, we will perform immunohistochemistry using antibodies against alpha-synucelein, neuronal markers (NeuN, MAP2), glial markers (GFAP, Iba-1) and synaptic markers (synaptophysin). We will also use immunoblot and ELISA analysis for the quantitative and semi-quantitative analysis of alpha-synucelein degradation and protein levels, to confirm and implement immunohistochemical results. We will also analyze the expression level of genes involved in autophagy (Becn1, Atg3, Atg7, Atg12, Map1lc3), proteasomal degradation (Uba1, Uba2, Uba3, Smurf1, Nedd8) and unfolded protein response (Edem1, Eif2a, Herpud1) by PCR array, to obtain mechanistic insight on the effect of the combined therapy on this MSA mouse model.

2. Lenalidomide reduces α-syn accumulation and microgliosis in the striatum of MBP1-hα-syn transgenic mice

During this first year we have also analyzed the effects of the immunomodulatory drug lenalidomide in the MBP1-hα-syn mouse model of MSA. Non-tg and MBP1-hα-syn tg mice were treated with either vehicle or lenalidomide at 100 mg/kg for 4 weeks, as previously described (Valera et al., 2015). Neuropathologically, we observed a reduction in the number of alpha-synucelein positive cells in striatum after lenalidomide treatment (Figure 1A, 1B), together with a normalization of the pro-inflammatory microglial phenotype as measured by Iba1 staining in the same brain region. Our preliminary results suggest that the pro-inflammatory activation of microglia in MBP1-hα-syn tg animals might impair microglial functioning, and the reduction in this microglial activation induced by lenalidomide could lead to an improvement in the phagocytic activity of these cells towards extracellular alpha-synuclein. We have previously observed a similar reduction in pro-inflammatory microglial activation by lenalidomide in a transgenic mouse model of PD (Valera et al., 2015).

3. A brain-targeted (ApoB), modified α-syn-degrading enzyme (neurosin) reduces α-syn accumulation in MBP1-hα-syn transgenic mice

Neurosin (kallikrein-6) is a serine protease capable of cleaving alpha-synuclein in the CNS, and we had previously shown that LV vector delivery of neurosin into the brain of a mouse model of Parkinson’s disease reduces the accumulation of alpha-synuclein and improves neuronal synaptic integrity (Spencer et al., 2013). Therefore, in this first year we have investigated the ability of a modified, systemically delivered neurosin to reduce the levels of alpha-synuclein in oligodendrocytes and reduce the cell-to-cell spread of alpha-synuclein to glial cells in the MBP1-hα-syn mouse model of MSA. We engineered a viral vector that expresses a neurosin genetically modified for increased half-life (R80Q mutation) that also contains a the brain-targeting sequence ApoB for delivery into the CNS. Peripheral administration of the LV-neurosin-ApoB to the MBP1-hα-syn tg model resulted in accumulation of neurosin-ApoB in the CNS, reduced accumulation of alpha-synuclein in oligodendrocytes and astrocytes, improved myelin sheath formation in the corpus callosum and behavioral improvements (Spencer et al., 2015). These results validate the use of brain-targeted therapies aimed at reducing alpha-synuclein as potential therapeutic tools for MSA.

 

MSA Coalition Grant #2015-04-009 – $50,000

Multiple system atrophy (MSA) is a rapidly progressing brain disease with no treatment or cure. My laboratory is studying an FDA-approved drug that is used worldwide to treat patients with multiple sclerosis, a brain disorder that like MSA damages the myelinating cells of the brain. MSA, like MS, tends to strike a younger

population than those who develop a more common movement disorder Parkinson’s disease (PD). MSA and PD have limited overlapping pathology and symptoms, and both may benefit by treatments that protect the brain’s signaling and support cells. In this grant, we will test an FDA-approved drug and two new compounds based on the FDA-approved drug in animal and cellular models of MSA. This will allow us to assess their ability to protect both the signaling neurons and the supporting myelinating cells. If data from this preclinical assessment are supportive, the FDA-approved drug can be rapidly repurposed for MSA, providing the first therapy that may slow disease progression and improve the quality of life.

RESULTS

1. Video: Presentation by Ruth Perez at the MSA Coalition Annual Patient & Family Conference (October 2016)

2. Slides and Handouts from Presentation by Ruth Perez at the MSA Coalition Annual Patient & Family Conference (October 2016)

3. Published article (September 2016): FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice: CONTRIBUTIONS OF PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR (PRO-BDNF) AND MATURE BDNF

4. Final Report by Ruth Perez (February 2017)

My team and I are working to make a difference for everyone affected by MSA. We received MSA Coalition seed grant funding in which we are seeking to identify therapies to slow or stop the relentless progression of MSA. I presented some of my laboratory’s findings (using parkinsonian mice) to patients and caregivers at the MSA Family Conference in New Orleans last October. That research had been just published in Journal of Biological Chemistry (Vidal-Martinez et al, 2016), and showed the benefits of a potential novel therapy. With MSA Coalition funds, we began evaluating the repurposing potential of that same drug, which is already approved by the Food and Drug Administration (FDA) to treat the demyelinating disorder, multiple sclerosis. The FDA approved drug, FTY720 (also known as
Fingolimod or Gilenya), has now been evaluated in MSA cell models. In data just published in Neuropharmacology (Segura-Ulate et al, 2017), we show that FTY720 increases the levels of a highly beneficial protective factor that improves the health of brain cells. FTY720 protects neuronal communicating cells and also the myelinating oligodendroglia cells that help optimize neuronal communication. We began evaluating two novel FTY720-derivative compounds that we synthesized in collaboration with our medicinal chemist colleague, who was also supported on this MSA Coalition grant. In a recently published PlosOne paper (Enoru et al, 2016) we show that both new FTY720-derivatives: (i) rapidly enter brain after being injected into mice, (ii) form non-toxic-by-products similar to the FDA approved parent drug FTY720, but (iii) are not metabolically modified like FTY720 in a way that can impair the immune system’s ability to fight infection. We just published these newest findings in Journal of Pharmacological Sciences (Segura-Ulate et al, 2017 in press), showing these beneficial properties of our derivatives. This suggests that these novel derivatives may be even better for treating aging disorders like MSA. We initiated preclinical assessment of the FTY720-compounds in MSA mice, which were generously provided by Dr. Virginia Lee of the University of Pennsylvania. However, continuing those studies will require additional funds.

5. Research Update by Ruth Perez (January 2016)

We established a Material Transfer Agreement with Dr. Virginia Lee at the University of Pennsylvania to obtain her CNPase promotor MSA mice to establish a breeding colony in El Paso. Due to unforeseen problems with her live mice, we had to make arrangements to rederive the CNPase mice at the Jackson Laboratories. This began in November and we were recently notified that mice have been born. Mice will be genotyped prior to shipping to us by the end of February.
In the meantime, we have been evaluating FTY720/Fingolimod/Gilenya in cell models and have obtained data for a paper that is currently in preparation. The data in cells show that FTY720 is turning on neurotrophic factor expression and has the capacity to reverse pathology induced by toxic α-synuclein (aSyn) accumulation in oligodendroglia cells (OLGs). This suggests that a novel protective property for MSA could occur by stimulating neurotrophic factor expression in OLGs by using FTY720. These findings further encourage us to evaluate the potential benefits of the drug in vivo in the CNPase mice to begin soon.
We have also been working with Dr. Jeffrey Arterburn to create new versions of the drug for further analysis, and recently received a shipment of drug from him.
Funding has been used to cover the cost of reagents, supplies, salaries and mice. As we proceed with the newly rederived MSA mice, we will require additional funding.

 

MSA Coalition Grant #2013-12-004 – $50,000

Multiple system atrophy (MSA) is a rapidly progressive, incurable neurodegenerative disease characterized by parkinsonism and dysautonomia, associated with the accumulation of alpha-synuclein (α-syn) within oligodendroglial cells. The accumulation of α-syn within oligodendrocytes impairs the ability of these cells to produce trophic factors that support neurons, resulting in neurodegeneration. To date, there are no effective treatments for MSA and there is an urgent need for therapeutic alternatives that prevent or revert pathological symptoms. In this context, the main objective of this application is to explore the potential therapeutic use of enhancing α-syn degradation in oligodendrocytes by peripherally delivering a brain-penetrating enzyme that catabolises α-syn. For that purpose, our central hypothesis is that peripheral delivery of the extracellular α-syn degrading enzyme Neurosin , targeted to the CNS by means of the Apolipoprotein B (ApoB) LDL receptor-binding domain, could induce significant α-syn degradation in brain to be neuroprotective and improve MSA pathology in a transgenic (tg) animal model.

Specific Aims

Aim 1: To determine the effect of peripheral delivery of neurosin on α-syn pathology in a MSA model. For this purpose MBP-hα-syn tg mice – a model of MSA – will receive intra-peritoneal injections of a lentiviral vector expressing neurosin or control vector at 6 mo of age. Neurosin will be linked to the ApoB LDL receptor-binding domain for targeted delivery to the CNS, and the effect of neurosin in MSA pathology will be analyzed.

Aim 2: To determine the effect of peripheral neurosin in α-syn degradation pathways in a MSA model. In order to analyze how enhanced levels of extracellular α-syn degradation by neurosin in brain might modulate other α-syn degradation pathways, we will analyze the components of main α-syn degradation cascades at gene expression and protein levels.

Background and significance

The main pathological feature of MSA is the presence of glial cytoplasmic inclusions (GCIs) in oligodendrocytes. A major constituent of GCIs is α-syn, an abundant synaptic protein, which has also been implicated in the pathogenesis of other parkinsonian disorders, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Recent studies suggest that abnormal accumulation of α-syn in neurons and glia leads to cellular dysfunction and neurodegeneration. In the search for new therapies that target the pathogenesis of MSA, increased α-syn degradation would reduce α-syn burden and thus the neurodegeneration events associated with it. Although the majority of intracellular α-syn is degraded via autophagy and the proteasome, the presence of α-syn in the extracellular space suggests that another route of protein degradation may be proteases secreted extracellularly. One these proteases is Neurosin (Kallikrein 6), which is active extracellularly and capable of cleaving α-syn in the NAC region and to a lesser extent in the c-terminus. We have previously shown that intra-cerebral injection of neurosin reduces α-syn accumulation in a mouse model of PD and that addition of the ApoB LDL receptor-binding domain enhances CNS trafficking.

It is increasingly evident that the toxicity of extracellular α-syn might be related to it ability to be taken up by neighboring cells and act in prion-like manner, and this behavior could be a key event in the origin and progression of the disease. Compounds that reduce intracellular α-syn aggregation have received significant attention in recent years, but the possibility of reducing α-syn spreading from cell to cell by degrading extracellular α-syn has not been explored in such depth. The innovation of this proposal is that we will increase the brain levels of an extracellular α-syn degrading enzyme by means of peripheral gene therapy and targeted delivery to the CNS, increasing degradation of extracellular α-syn and potentially resulting in the activation intracellular degradation pathways as well. Therefore, delivery of brain-targeted neurosin into the CNS might represent a potential therapeutic option for neurodegenerative disorders.

RESULTS

1. Published article (September 2015): A brain-targeted, modified neurosin (kallikrein-6) reduces α-synuclein accumulation in a mouse model of multiple system atrophy

2. Video: Presentation by Eliezer Masliah at the MSA Coalition Annual Patient & Family Conference (September 2015)

3. Research update by Eliezer Masliah (December 2015):

A brain-targeted (ApoB), modified α-syn-degrading enzyme (neurosin) reduces α-syn accumulation in MBP1-hα-syn transgenic mice

Neurosin (kallikrein-6) is a serine protease capable of cleaving alpha-synuclein in the CNS, and we had previously shown that LV vector delivery of neurosin into the brain of a mouse model of Parkinson’s disease reduces the accumulation of alpha-synuclein and improves neuronal synaptic integrity (Spencer et al., 2013). Therefore, in this first year we have investigated the ability of a modified, systemically delivered neurosin to reduce the levels of alpha-synuclein in oligodendrocytes and reduce the cell-to-cell spread of alpha-synuclein to glial cells in the MBP1-hα-syn mouse model of MSA. We engineered a viral vector that expresses a neurosin genetically modified for increased half-life (R80Q mutation) that also contains a the brain-targeting sequence ApoB for delivery into the CNS. Peripheral administration of the LV-neurosin-ApoB to the MBP1-hα-syn tg model resulted in accumulation of neurosin-ApoB in the CNS, reduced accumulation of alpha-synuclein in oligodendrocytes and astrocytes, improved myelin sheath formation in the corpus callosum and behavioral improvements (Spencer et al., 2015). These results validate the use of brain-targeted therapies aimed at reducing alpha-synuclein as potential therapeutic tools for MSA.