Projects Funded - Preclinical

EVALUATING POTENTIAL NEW TREATMENTS IN THE LAB - "PRECLINICAL"

Before a new investigational therapy is ever administered to MSA patients, governments require that it first be tested in laboratory models to ensure safety and efficacy.

There is a possibility that existing drugs may be effective against multiple system atrophy and might slow or halt the progression of the illness. Identification and testing of these drugs is needed both in preclinical models of MSA and in human patients. At the same time, as more is learned about the illness, new medications targeting the underlying cause need to be developed, including therapies utilizing the immune system.

The MSA Coalition supports preclinical laboratory research in order to enable researchers to gather the evidence needed to move promising therapies towards clinical trials in MSA patients. The estimated price tag to cover the costs of testing of existing medications and initial development of new medications in the lab is $10 million.

Although we currently can only invest a small amount towards this line of research, The MSA Coalition is proud of the work we have inspired. Several groundbreaking articles have been published in highly prestigious medical journals like Movement Disorders, Brain and Nature. There has also been news media coverage and our funded researchers have been given awards for excellence.

Learn more about the status of our preclinical laboratory projects by exploring the links below.

**UPDATED RESULTS** “Nilotinib for treating MSA: a preclinical proof of concept study”: Pierre-Olivier Fernagut, Ph.D. (University of Bordeaux, France) and Wassilios Meissner, M.D., Ph.D. (University of Bordeaux, France)

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.

 

**UPDATED RESULTS** “Targeting alpha-synuclein pathology with the molecular tweezer CLR01 in MSA: optimization of drug delivery and biochemical analysis”: Nadia Stefanova, MD, PhD (Medical University of Innsbruck, Austria) and Gal Bitan, PhD (University of California Los Angeles, CA)
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.

**NEW** "Myeloid cell depletion as therapeutic target in multiple system atrophy":Juergen Winkler (University Hospital Erlangen, Germany) & Johannes Schlachetzki (UC San Diego, USA)

MSA Coalition Grant #2020-05-005 – $50,000

Multiple system atrophy is a rare neurodegenerative disease with a rapid and devastating disease progression. At present, no therapy is available which stops or even slows disease progression of MSA patients. The immune response within the brain, so-called neuroinflammation, is an important feature of neurodegenerative diseases. In MSA patients, a severe immune response of microglia, the brain resident immune cells, has been described. Depletion of microglia showed beneficial effects in animal models of other neurodegenerative diseases such as Alzheimer’s disease or amyotrophic lateral sclerosis. In order to target the neuroinflammatory response in MSA, the main objective of this project is to reduce the number of microglia in a mouse model of MSA. Therapeutic effects will be investigated on a cellular and behavioral level. To reduce microglial numbers, we will use a compound (PLX5622) that has already been tested in a phase Ib clinical trial for treatment of rheumatoid arthritis. PLX5622 inhibits the colony-stimulating factor 1 receptor (CSF1R), which is expressed on microglia and plays a role in survival and maintenance of these cells. In case our preclinical study achieves a successful outcome, we aim to translate our results into a clinical trial in order to investigate its efficacy in MSA patients.

**NEW** "Testing MPH as a novel neuroprotective therapy in pre-clinical models of Multiple System Atrophy":Alessio DiFonzo (University of Milan, Italy) & Arianna Belluci (University of Brescia, Italy)

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.

“Can ceruloplasmin or metal chelation be used as a therapeutic agent in an animal model of MSA”: David Finkelstein, PhD (Florey Institute, Melbourne, Australia)

MSA Coalition Grant #2017-10-009 – $26,000

Some metals such as iron, copper and zinc are essential dietary nutrients that are necessary for many biological processes. When these metals are imbalanced (too much or too little) or incorrectly located within the body, they can start to function incorrectly and cause damage to cells and organs. The brain is an organ that has a high requirement for these metals but it is also particularly vulnerable to imbalances.

Changes in these metals in the brain have been implicated in a number of conditions including Parkinson’s disease and Alzheimer’s disease and many researchers have begun to investigate if these metals can be targeted by drugs to treat the disease.

Multiple system atrophy is a rare disease of largely unknown origin and with currently no cure. The disease shares many chemical similarities with Parkinson’s disease (in terms of the pathology). Because of these similarities, we can take advantage of the large amount of research that has been conducted into
Parkinson’s disease to form hypotheses about multiple system atrophy. While Parkinson’s disease also has no cure, much more is known about the processes happening in the brain in people with the disease. We have used this approach to hypothesise that metals are imbalanced in the brain of people with multiple system atrophy and that imbalances are associated with specific proteins that bind these metals.

We will use this seed funding to test this hypothesis using post-mortem brain tissue donated from multiple system atrophy patients and a mouse model of the disease. We will also use this mouse model to test if targeting these metal imbalances can prevent the development of the disease. If successful, this project
could form a basis for clinical trials in people living with MSA.

RESULTS

1. Presentation (August 2019): Exploring the Role of Iron in Multiple System Atrophy

2. Research update by David Finkelstein (August 2018)

The objective of the planned research project is to investigate the role of ceruloplasmin dysfunction in the formation of alpha-synuclein aggregates – the pathological hallmark of MSA – and if this dysfunction represents a valid target for the development of therapies. To achieve this, the specific aims of the project
are:
1. To investigate the levels and protein binding of copper (CU) and iron (Fe) in human MSA post-mortem brain tissue compared with controls and in PLP-aSyn mice compared with non-transgenic littermates
2. To investigate activity and copper-binding of ceruloplasmin in human MSA post-mortem brain tissue compared with controls and in PLP-aSyn mice compared with non-transgenic littermates
3. To investigate if ceruloplasmin or metal chelation can reduce the number of alpha-synuclein inclusions and the amount of aggregated alpha-synuclein in the PLP-aSyn mouse model of MSA

We have completed all experiments and are currently analysing the data.The data from the ceruloplasmin activity assay was found to be too variable to be used at this time. Apart from the ceruloplasmin assay, all other experiments were successful and appear to be sufficiently statistically powered to show significant differences. That is, they were technically successful. We are currently writing two manuscripts and intend to submit them by the first quarter of 2019.

“Peripheral delivery of brain-targeted neurosin - an alpha-synuclein degrading enzyme - as a novel treatment for MSA”: Eliezer Masliah, MD (University of California San Diego, CA)

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. LINK COMING SOON 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.

 

"Preclinical Evaluation of Novel Therapeutic for MSA": Ruth Perez, Ph.D. (Texas Tech University Health Sciences Center)

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. LINK COMING SOON 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.

 

“Glucagon like peptide-1 agonists for treating MSA: a preclinical proof of concept study”: Wassilios Meissner, M.D, Ph.D. (University of Bordeaux, France)

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.

“Combination of immunotherapy against alpha-synuclein and anti-inflammatory treatment for Multiple System Atrophy”: Eliezer Masliah, MD (University of California San Diego, CA)

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. LINK COMING SOON 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.

 

“Targeting alpha-synuclein pathology with the molecular tweezer CLR01 in MSA “: Nadia Stefanova, MD, PhD (Medical University of Innsbruck, Austria)

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

“GRK2 targeted knock-down as therapy for multiple system atrophy”: Erwan Bezard, Ph.D. (University of Bordeaux, France)

MSA Coalition Grant #2016-09-006 – $50,000

Developing neuroprotective and disease-modifying treatments for multiple system atrophy (MSA) is an urgent unmet need. Insulin and related factors act as neuromodulators in the brain. Neurodegenerative diseases are characterized by impaired insulin/insulin like growth factor-1 (IGF-1) signaling and insulin resistance (i.e. decreased insulin/IGF-1 signaling). We recently demonstrated that such insulin resistance is a key feature of (MSA) in the brain of MSA patients as well as in in the gold-standard transgenic mice model of MSA, the PLPsynuclein mouse. Counteracting such insulin resistance thus bears the potential of being neuroprotective and of improving autonomic failure such as orthostatic hypotension. The insulin signaling is notably transduced by an integrator protein named G protein–coupled receptor kinase 2 (GRK2). Both mouse models of insulin resistance and humans with metabolic syndromes display increased levels of GRK2. Even more interestingly, a recent attempt to knock-down the peripheral expression of GRK2 resulted into a reversal of diet-induced obesity and insulin resistance in a model of diabetis. We will test the hypothesis that knocking-down the expression of GRK2 in the whole body or specifically in the brain has positive effects on insulin resistance, cell survival and α-synuclein pathology in the gold-standard transgenic mice model of MSA, the PLP-synuclein mouse.

If successful, we would then move this strategy to our newly developed non-human primate model of MSA for finalizing the preclinical validation of the GRK2 target.

 

RESULTS

1. Research update by Erwan Bezard (July 2018)

Developing neuroprotective and disease-modifying treatments for multiple system atrophy (MSA) is an urgent unmet need. Insulin and related factors act as neuromodulators in the brain. Neurodegenerative diseases are characterized by impaired insulin/insulin like growth factor-1 (IGF-1) signalling and insulin resistance (i.e. decreased insulin/IGF-1 signalling). We recently demonstrated that such insulin resistance is a key feature of (MSA) in the brain of MSA patients as well as in in the
gold-standard transgenic mice model of MSA, the PLP-synuclein mouse. Counteracting such insulin resistance thus bears the potential of being neuroprotective and of improving autonomic failure such as orthostatic hypotension. The insulin signalling is notably transduced by an integrator protein named G protein–coupled receptor kinase 2 (GRK2). Both mouse models of insulin resistance and humans with metabolic syndromes display increased levels of GRK2. Even more interestingly, a recent attempt to knock-down the peripheral expression of GRK2 resulted into a reversal of diet-induced obesity and insulin resistance in a model of diabetes. We will test the hypothesis that knocking-down the expression of GRK2 in the whole body or specifically in the brain has
positive effects on insulin resistance, cell survival and α-synuclein pathology in the gold-standard transgenic mice model of MSA, the PLP-synuclein mouse.
If successful, we would then move this strategy to our newly developed non-human primate model of MSA for finalizing the preclinical validation of the GRK2 target. So far, the GRK2 knock down chosen approach is successful in vitro and is
currently being tested in vivo.

Building Hope Through Research


Since 2013, the Multiple System Atrophy Coalition has funded 42 MSA focused research projects for a total of $2 Million.

Explore the links below to learn more about our research goals and the outcomes of our funded projects.

The 42 projects cover four major themes:

We are making an impact!