Projects Funded - Pathogenesis

UNCOVERING THE CAUSE OF MSA - "PATHOGENESIS"

We know that multiple system atrophy, like Parkinson’s disease and Lewy body disease, is a synucleinopathy, leaving deposits of the protein alpha-synuclein in nerve cells within the brain and spinal cord. How these deposits develop, how they damage nerve cells, and how they spread that damage to other cells remain unanswered questions.

$10 million is needed to work toward understanding the causes of multiple system atrophy, including but not limited to determining how and why alpha-synuclein is deposited in the cells of the brain and how this drives disease development and progression. In addition, determining other causative and contributing factors (e.g. genetic mutations, environmental factors) is critical.

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 pathogenesis projects by exploring the links below.

 

**UPDATED RESULTS** “Towards translational animal models of MSA”: Jeffrey Kordower, PhD (Rush University Medical Center, Chicago, IL)

MSA Coalition Grant #2017-10-002 – $50,000

Multiple system atrophy (MSA) is an unrelenting progressive neurodegenerative disease, characterized by autonomic nervous system failure and a movement disorder similar to Parkinson’s disease, called MSA-P or motor dysfunction with cerebellar ataxia, called MSA-C. Typical autonomic symptoms are low blood pressure, postural hypotension and bladder control difficulties. Currently there are no valuable symptomatic or disease modifying treatments for this devastating disease. Examination of the brain of MSA patients reveals widespread cytoplasmic alpha-synuclein-rich protein inclusions in the glial cells called oligodendroglia. MSA is therefore classified as an alpha-synucleinopathy together with Parkinson’s disease and dementia with Lewy bodies. The frequency of glial cytoplasmic inclusions in MSA correlates with neuronal cell loss and symptom severity. In contrast, physiological functions of alpha-synuclein in neurons are believed to be involved in neuronal homeostasis and neurotransmitter release. Cellular inclusions take the form of aggregates that are thought to be pathological. Alpha-synuclein inclusions interrupt the normal supportive functions of oligodendroglia; myelin sheath formation and production of trophic factors. Neurons are also predisposed to degeneration and neuronal death by various other mechanisms.

Understanding disease pathogenesis as well as interventions targeting either parkinsonism and/or autonomic nervous system failure are urgently needed in MSA. The aim of this project is to establish novel rat models of MSA-P and MSA-C by overexpressing alpha-synuclein protein in the brain oligodendrocytes. This will be achieved by using a novel oligodendrocyte-selective adeno-associated viral vector, which will be injected in the rat brain. Our preliminary evidence supports the usefulness of this vector in rats and monkeys. We aim to create models of both forms of MSA to help us better understand its pathophysiology and to be able to test with validity new, urgently needed, treatment possibilities that stop, delay or reverse the disease progression.

RESULTS

1. Publication (January 2021): Viral-based rodent and nonhuman primate models of multiple system atrophy: Fidelity to the human disease

2. Research update by Jeffrey Kordower (July 2018)

Multiple system atrophy (MSA) is an unrelenting progressive neurodegenerative disease, characterized by autonomic nervous system failure and a movement disorder similar to Parkinson’s disease, called MSA-P or motor dysfunction with cerebellar ataxia, called MSA-C. Examination of the brain of MSA patients reveals widespread cytoplasmic alpha-synuclein-rich protein inclusions in the glial cells called oligodendroglia. The accumulation of alpha-synuclein causes dysfunction in oligodendrocytes, leading to demyelination, loss of trophic support for neurons, and neuroinflammation, with secondary loss of neurons. The frequency of glial cytoplasmic inclusions in MSA correlates with neuronal cell loss and symptom severity. Currently there are no valuable symptomatic or disease modifying treatments for this devastating disease. The aim of this project is to establish novel rat models of MSA-P and MSA-C by overexpressing alpha-synuclein protein in the brain oligodendrocytes. This will be achieved by using a novel oligodendrocyte-selective adeno-associated viral vector, which will be injected in the rat brain. Our preliminary evidence supports the usefulness of this vector in rats and monkeys. We aim to create models of both forms of MSA to help us better understand its pathophysiology and to be able to test with validity new, urgently needed, treatment possibilities that stop, delay or reverse the disease progression.

Graduate student David Marmion will be presenting an update on the progress of this grant at the 2018 Synuclein conference in Lausanne Switzerland in September.

 

**NEW** "The role of repeat expansions in Multiple Systems Atrophy (MSA)": John Hardy, PhD & Viorica Chelban, MD, PhD (University College of London, UK)

MSA Coalition Grant #2020-05-004 – $50,000

MSA is a difficult diagnosis, especially in the early stages with overlap with other causes of ataxia and parkinsonism. Based on the current diagnostic criteria only 60% of possible and probable MSA meet the pathological criteria in a recent brain bank series. There is particularly significant early overlap with MSA-C and late onset idiopathic cerebellar ataxia (ILOCA), which is often mild with frequent autonomic features due to a repeat expansion in the RFC1 gene. The other clinical overlap is with patients with neuronal intracytoplasmic inclusion disease (NIID) and NOTCH2NLC repeat expansions where the clinical features are heterogeneous but often include ataxia and parkinsonism. Due to the overlap in some clinical features of MSA with RFC1 and NIID expansions, particularly during the early stages of disease and the notorious difficulty in diagnosing MSA, we hypothesize that the repeat expansion in NOTCH2NLC and RFC1 may be implicated in a group of patients that fit the diagnostic criteria for suggestive MSA, but with atypical symptoms or unusual disease trajectories. In addition, novel repeats may play a role in the cause of more typical MSA and could explain the homogenous neuropathology but varied clinical features through variable size, interruptions and penetrance. We have collected over the years through extensive collaborations with brain banks around the world a cohort of over 300 pathologically diagnosed MSA cases and 590 clinically diagnosed MSA patients and matched controls. In these samples we will analyze the NOTCH2NLC and RCF1 repeat expansions using a 3-stage analysis approach and confirmation with Southern blotting. In 20 typical MSA patients, diagnosed clinically and pathologically confirmed in which we have matched blood and brain-derived DNA we will investigate MSA using long read sequencing with the PacBio Sequenl II from brain and blood samples for repeat expansions.

**NEW** "Exosomal alpha-synuclein and TPPP/p25α: Potential culprits in MSA pathogenesis?": Maria Xilouri (Academy of Athens, Greece)

MSA Coalition Grant #2020-05-001 – $50,000

Multiple system atrophy (MSA) is a rare neurological disorder characterized by the presence of cytoplasmic inclusions filled with the neuronal protein alpha-synuclein within oligodendrocytes, the glial brain cells that create the myelin sheath surrounding the neuronal axons. Accumulation of alpha-synuclein and the oligodendrocyte-specific protein TPPP/p25α, leads to oligodendroglial degeneration that precedes neuronal demise. Our published data suggest that oligodendroglial alpha-synuclein (expressed at miniscule amounts) and p25α cooperate to form pathological alpha-synuclein forms in MSA-like experimental models. Since alpha-synuclein is predominantly expressed in neurons, the prevailing hypothesis is that oligodendrocytes take-up neuronally-derived alpha-synuclein, secreted partly via exosomes, from the extracellular space.

Exosomes are small vesicles suggested to play a major role in the intercellular communication through the transport of multilevel information and cell-to-cell spreading of toxic molecules. However, the role of oligodendroglial-derived exosomes in the development and spread of MSA pathology remains unexplored. Our preliminary data suggest that alpha-synuclein and p25α are secreted by oligodendroglial exosomes and that addition of pathological alpha-synuclein conformations (fibrils) augments the exosomal cargo of aSyn and p25α.Moreover, brain exosomes from a mouse model of MSA are enriched in pathological alpha-synuclein and p25α, further suggesting that oligodendrocytes may transmit aSyn and/or p25α pathology between them via exosomes.

To investigate this hypothesis, we propose herein to perform a comprehensive analysis of exosomes isolated from oligodendroglial cells incubated with pathological forms of alpha-synuclein derived from human MSA brains, animal models of MSA and human post-mortem material from MSA brains. We will also assess the potential pathogenic propensity of these nanovesicles by injecting them in neural cultures and in the mouse brain. This line of research is important in uncovering the contribution of oligodendroglial-derived exosomes in disease pathogenesis and in pinpointing the factors that mediate the communication between neurons and oligodendrocytes, which may represent potential therapeutic targets for MSA.

**UPDATED RESULTS** “Defining novel pathways for multiple system atrophy”: Manu Sharma, PhD (University of Tubingen, Germany)

MSA Coalition Grant #2017-10-006 – $50,000

Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disorder characterized by variable combinations of parkinsonism, cerebellar ataxia, autonomic failure and pyramidal signs. The prevalence is 4.4 per 100,000. The clinical manifestation is influenced by an ethnic background, with parkinsonism more commonly seen in Caucasians and cerebellar features more frequently observed in the East Asian population. So far, emerging evidence indicates that alpha-synuclein – the most common risk factor for Parkinson diseases (PD) – is central to disease pathogenesis, and thus indicating common underlying pathways for PD and MSA. Despite the progress made in understanding the pathophysiology of disease; there has been a limited success in defining novel biomarkers for MSA. The advancement in genomic technologies has provided an opportunity to process large samples of patients for various diseases including neurodegenerative diseases to identify novel genetic makers, which can be used as a genetic-readout to understand the disease progression for various complex diseases including MSA.
In the current proposal, “Defining novel pathways for multiple system atrophy (DEFPATH-MSA)”, for the first time, using a newly designed NeuroChip; we will screen the European Multiple System Atrophy Study Group (EMSA-SG) cohort, a consortium of investigators from academic centres across Europe and Israel committed to perform research studies aimed at improving the treatment of MSA and advancing knowledge about the aetiology and pathogenesis of MSA, to identify novel clinical biomarkers for MSA. The screening of MSA cohort will enable at an affordable cost to routinely perform diagnostic screening for MSA patients, and also help clinicians/researchers to identify at-risk subjects. The longitudinal evaluation of at-risk cohort will help to understand the underlying pathogenetic mechanism for MSA.

RESULTS

1. Publication (October 2020): Shared Genetics of Multiple System Atrophy and Inflammatory Bowel Disease

2. Research update by Manu Sharma (September 2018)

The project, Defining novel pathways for multiple system atrophy (DEFPATH-MSA), aims to develops a comprehensive clinical-genetic cohort by processing customized genotyping array, NeuroChip2, using European Multiple System Atrophy study group registry (EMSA-SG). The applicability of using a customized genotyping platform comprising putative risk variants identified by exome sequencing, and targeted resequencing of top loci identified in genoGWAS using population based samples, as well as those derived from literature review for the complex diseases such as NeuroX chip is now routinely used to allow rapid screening for genetic mutations and risk factors associated with associated with complex neurodegenerative diseases. Our study aims to develop an in-depth genetic catalogue for MSA patients. This will help to define novel pathways and targets for therapeutic interventions.
Currently, most of the sites are preparing a fresh aliquot of DNA samples for the shipment. We aim to start the genotyping of our cohort latest in coming weeks.

**PRESS COVERAGE ** “Pathological alpha-Synuclein in glial cytoplasmic inclusions represent a unique alpha-Synuclein strain”: Chao Peng, PhD (University of Pennsylvania, Philadelphia, PA)

MSA Coalition Grant #2017-10-003 – $50,000

Alpha-Synuclein (α-Syn) is the disease protein that misfolds and accumulates as aggregates with deleterious consequences in neurodegenerative disorders known as α-Synucleinopathies. These include multiple system atrophy (MSA), Parkinson’s disease (PD) without and with dementia (PDD), dementia with Lewy bodies (DLB) and ~50% of Alzheimer’s disease (AD) patients. Pathologically, MSA is distinct
because it is characterized by the accumulation of misfolded α-Syn in oligodendrocytes as glial cytoplasmic inclusions (GCIs), while in all other α-Synucleinopathies, α-Syn aggregates in neurons as Lewy bodies and neurites. Clinically, MSA is far more aggressive with a much shorter disease duration
than PD (6-9 years for MSA compared with ~12 years for PD) although the age of onset for both is similar (~60 years). It is still unknown why there are such dramatic clinical and pathological differences between MSA and other α-Synucleinopathies. We hypothesis that pathological α-Syn in MSA could have unique structure and properties that contribute to the distinct phenotypes of MSA. By preparing
pathological α-Syn from brains of MSA and other α-Synucleinopathy patients, we will analyze their structure and biological activity using multiple biochemical methods as well as cell and animal models. With this detailed analysis of MSA brain derived pathological α-Syn, we will get critical insights into the mechanism why MSA is so different from other α-Synucleinopathies. Furthermore, we will also
systematically analyze the post-translational modifications (PTMs) such as phosphorylation, acetylation, ubiquitination and methylation on misfolded α-Syn from MSA patients and compare with those from other α-Synucleinopathy patients to test whether unique PTMs in MSA brain derived α-Syn contributes to its distinct biological activity. These studies will dramatically improve our understating of the etiology of MSA and help us develop better models and therapeutic strategies for MSA.

RESULTS

This groundbreaking research study supported by a grant from the MSA Coalition was published in the prestigious journal “Nature” and covered in the press in May 2018.

1. Presentation (July 2019): Pathological alpha-synuclein in MSA is Unique and Different from All the Other Neurodegenerative Diseases

2. Press coverage (May 2018) Penn Medicine News: Diverse Parkinson’s-related Disorders May Stem from Different Strains of Same Disease Protein, According to Penn Study

3. Press coverage (May 2018) Science Daily: Diverse Parkinson’s-related disorders may stem from different strains of same protein

4. Published article (May 2018) Nature: Cellular milieu imparts distinct pathological α-synuclein strains in α-synucleinopathies

5. Research update by Chao Peng (November 2018)

During the past year, we demonstrated that the pathological alpha-Synuclein (α-Syn) in MSA patients (GCI-α-Syn) is very unique and different from those (LB-α-Syn) in all the other neurodegenerative diseases such as Parkinson’s disease, Dementia with Lewy bodies and also some of the Alzheimer’s disease patients. GCI-α-Syn forms a more compact structure compared with LB-α-Syn. More importantly, GCI-α-Syn is about 1000 fold more efficient than LB-α-Syn in inducing the misfolding of normal α-Syn, which likely contribute to the aggressive nature of MSA. Moreover, our research demonstrated that the unique intracellular environment of oligodendrocytes leads to the generation of this highly aggressive GCI-α-Syn. To further explore the molecular basis of the unique properties of GCI-α-Syn, we systematically analyzed the post-translational modifications such as phosphorylation, acetylation and methylation on GCI-α-Syn and compared with LB-α-Syn.

**AWARD WINNING** “Stem cell-based Therapeutics Platform for MSA”: Vikram Khurana, MD, PhD (MIT Whitehead Institute, Boston MA)

MSA Coalition Grant #2013-12-002 – $50,000

This project aims to develop new models of Multiple System Atrophy by utilizing stem cell technology to generate human patient-derived stem cell models of the disease. This will enable the study of the biology of an individual patient’s disease in the dish by creating their neurons and oligodendrocytes and then looking for signatures of alpha-synuclein toxicity. If alpha-synuclein toxicity signatures are identified it will further assess whether it’s possible to reverse this toxicity with genes and small molecules. This work may lead to a new way of testing and discovering potential therapies for MSA beyond the current mouse models of the disease.

RESULTS

Dr. Vikram Khurana was bestowed with the Bishop Dr. Karl Golser Award for this groundbreaking research resulting from one of the very first seed grants awarded by the MSA Coalition.

1. June 2018: Bishop Dr. Karl Golser Foundation Awards Inaugural Research Prize For Atypical Parkinsonism

2. Award Announcement (February 2018) Khurana Wins Bishop Dr. Karl Golser Award

3. Published article (February 2017) Genome-scale networks link neurodegenerative disease genes to alpha-synuclein through specific molecular pathways

4. Video presentation: MSA Coalition Patient & Family Conference Crystal City, MD (October 2014)

DR. VIK KHURANA: “PLANNING A NATIONAL STEM CELL BANK FOR MULTIPLE SYSTEM ATROPHY”

5. Research Update by Vikram Khurana (December 2015):

It is now possible to convert cells from our patients (like blood or skin cells) into embryonic-like stem cells that can be coaxed into forming cells from complex tissue like the human brain. So, for the first time in history we can take, say, a skin cell from a patient with MSA and make that same patient’s brain cells in the dish. Just a few years ago no one would have dreamed this was possible – to get brain cells from patients we had to wait until after death. The stem cell-based approach allows us to study in the lab the abnormalities associated with a living patient’s disease in the lab, and find ways to reverse it.

The MSA Coalition seed grant allowed me to build on my existing work in Parkinson’s disease to create stem cell models from my patients with MSA. We have started to identify abnormalities in these cells and to test potential therapies that can reverse them. We have a long way to go before these findings in the dish reach the clinic, but, thanks to the MSA Coalition, we have made a good start. For a disease like MSA – where there are no clear gene mutations – it is vital that we collect more stem cell lines from patients, and we are beginning to do that. We have high hopes to generate a national stem cell bank for the disease that will provide a critical research tool for the MSA research community and speed up the tempo of drug discovery for this devastating disorder.

“Unravelling the mechanism of alpha-synuclein seeding in oligodendrocytes”: Maria Xilouri, PhD (Biomedical Research Foundation of the Academy of Athens, Greece)

MSA Coalition Grant #2016-09-003 – $50,000

Multiple system atrophy (MSA) is a neurological disorder associated with the degeneration of nerve cells in specific brain areas. MSA is characterized by the accumulation of cytoplasmic inclusions filled with the neuronal protein alpha-synuclein within oligodendrocytes. Oligodendrocytes are a selective type of glial cells that provide support and insulation to axons, creating the myelin sheath around them. Accumulation of alpha-synuclein together with the oligodendrocyte-specific protein TPPP/p25α, which may result from a failure of proteolytic systems, leads to oligodendroglial degeneration. Since oligodendrocytes do not normally express alpha-synuclein, a prevailing hypothesis is that the protein is entering
oligodendroglial cells following its release by neurons that normally express alpha-synuclein. Up to date the precise species of alpha-synuclein responsible for the formation of these inclusions and the proteolytic machineries capable of their removal remain unknown. We plan to identify the mechanisms that control the transmission of alpha-synuclein in oligodendrocytes and to assess the role of p25α in this process. To investigate this, we will use various forms of alpha-synuclein produced in bacteria (recombinant) or produced in neurons, apply them to oligodendrocytes and assess their uptake and turnover, using selective
pharmacological and molecular means to inhibit the major proteolytic systems inside the cell. Moreover, we will do the same experiments in primary oligodendrocytes isolated from a mouse model of MSA that overexpresses alpha-synuclein only in oligodendrocytes, and in mice that normally do not express alpha-synuclein in any cell (knock-out mice) or mice that express the protein normally in neurons. This line of research is important in pinpointing the factors that regulate the transfer, accumulation and clearance of alpha-synuclein within oligodendrocytes and the role of p25α in these processes. The major outcome will be the identification of the mechanisms that can clear abnormal forms of alpha-synuclein in oligodendrocytes that might represent potential therapeutic targets for MSA.

RESULTS

1. Presentation (May 2019): It takes two to tango: Alpha-synuclein and p25-alpha in MSA pathogenesis

2. Published article (April 2019): Endogenous oligodendroglial alpha-synuclein and TPPP/p25α orchestrate alpha-synuclein pathology in experimental multiple system atrophy models.

3. Research update by Maria Xilouri (March 2019)

Multiple system atrophy (MSA) is a neurological disorder associated with the degeneration of nerve cells in specific brain areas. The disease is characterized by the accumulation of cytoplasmic inclusions within oligodendrocytes, which are a selective type of glial cells that provide support and insulation to axons, creating the myelin sheath around them. The major constituents of these inclusions are the neuronal protein alpha-synuclein and the oligodendrocyte-specific protein TPPP/p25a. Given that oligodendrocytes do not normally express alpha-synuclein, a prevailing hypothesis is that the protein is entering oligodendroglial cells following its release by neurons that normally express alpha-synuclein. Up to date the precise species of alpha-synuclein responsible for the formation of these inclusions and the proteolytic machineries capable of their removal remain unknown. The aim of the current proposal was to elucidate the mechanisms that control the transmission of alphasynuclein in oligodendrocytes and to evaluate the role of p25a in this process. Given that alpha-synuclein can normally obtain various conformations inside the cell (monomers, oligomers, fibrils), we have applied such conformations of the human protein produced in bacteria (recombinant) or in neuronal cells (associated with
small vesicles termed exosomes) to oligodendroglial cells and assessed which of these forms exert the highest propensity to induce pathology. Our results suggest that all forms of alpha-synuclein can be uptaken by oligodendrocytes, but only fibrillar alpha-synuclein evokes the formation of very insoluble toxic species that
further recruit the rat endogenous protein, thus seeding alpha-synuclein pathology. Interestingly, p25a overexpression accelerated the recruitment of the endogenous rat protein and augmented the formation of very insoluble toxic alpha-synuclein species. The exogenously added alpha-synuclein accumulated in specific
cellular organelles, the mitochondria and the lysosomes. In addition, our data so far suggest that both the proteasome and the lysosome, the two main proteolytic systems inside the cell, are recruited to clear the excess burden of oligodendroglial alpha-synuclein. Furthermore, phosphorylation of alpha-synuclein, an event closely
related to the spread of alpha-synuclein pathology, was detected only when alpha-synuclein or p25a was overexpressed. Most importantly, the findings obtained from the immortalized cell lines, were further confirmed in primary oligodendrocytes isolated from a mouse model of MSA that overexpresses alpha-synuclein only in
oligodendrocytes, and in mice that express the protein normally in neurons. In this more physiological setting, only fibrillar alpha-synuclein seeded the endogenous oligodendroglial protein to aberrant assemblies that were also positive for p25a. These effects were accompanied by a cytoskeletal collapse and a redistribution
of p25a from the myelin sheath to the cell soma, thus recapitulating the main features of MSA. Collectively, our results reveal, for the first time, the seeding of the endogenous oligodendroglial alphasynuclein upon uptake of misfolded conformations of the protein and suggest that like in the human disease,
only a minute amount of endogenous oligodendroglial alpha-synuclein at baseline is sufficient for this effect. Furthermore, p25a actively participates in the formation of such aberrant alpha-synuclein species. In future studies, we seek to develop novel MSA models by manipulating the levels of both alpha-synuclein and p25a, in
which early events leading up to the formation of the glial cytoplasmic inclusions could be monitored. By using such models, we seek to identify molecular targets that could be exploited to inhibit alpha-synuclein deposition in oligodendrocytes, providing potential therapeutic avenues for MSA therapy.

“Spreading of alpha-synuclein pathology in multiple system atrophy”: Johannes Brettschneider, PhD (University of Ulm, Germany) and John Trojanowski, MD, PhD (University of Pennsylvania,Philadelphia, PA) – Joint Grant with CurePSP

MSA Coalition Grant #2014-04-001 – $30,000

The main neuropathological findings in multiple system atrophy (MSA) are aggregates of the protein alpha-synuclein in oligodendrocytes, the cells that produce the insulating myelin sheath surrounding axons or nerve fibers known as oligodendrocytes and also in specific nerve cells of the central nervous system (CNS). Similar aggregates of alpha-synuclein have been observed in other neurodegenerative synucleinopathies, including sporadic Parkinson’s disease and dementia with Lewy bodies, and the progressive regional spreading of such protein aggregates from a focal onset to widespread areas of the CNS is now considered a key aspect characterizing the pathology of many neurodegenerative diseases. To date, however, there are no studies that have attempted to determine if a regional spreading of alpha-synuclein aggregates might also underlie disease progression in MSA. Furthermore, it is unclear what characterizes the cells that are vulnerable and those that are resistant to alpha-synuclein pathology in MSA, and what determines pathways of disease progression. We aim to analyze neuropathological evidence for a potential spreading of alpha-synuclein pathology in a cohort of n=60 clinically well-characterized autopsy cases of MSA from patients studied clinically during life. We will analyze neuroanatomical characteristics of cells that are vulnerable and resistant to alpha-synuclein pathology, and we shall determine pathways by which alpha-synuclein pathology could propagate. In so doing, we can lay the essential groundwork for strategies aiming to prevent the propagation of alpha-synuclein in MSA, which would constitute a fundamentally novel approach to its treatment. For the development of potential therapeutic agents, for example using immune therapy, insights into propagation pathways, potential mechanisms of dissemination, and determinants of vulnerability of specific cellular types to alpha-synuclein propagation are essential.

RESULTS

1. Published article (June 2017) Progression of alpha‐synuclein pathology in multiple system atrophy of the cerebellar type

2. Research Update by Johannes Brettschneider (December 2014):

We aim to establish a sequential order in which aggregates consisting of the protein alpha-synuclein spread within the nervous system of patients with multiple system atrophy (MSA). Such a staging will be valuable not only for neuropathologists, but also to monitor disease progression in living patients once imaging markers of alpha-synuclein – which are currently developed by our group and others – become available. Then, the knowledge of the sequential order in which different areas of the brain are affected by alpha-synuclein pathology could help to determine the effectiveness of new therapeutic agents in clinical trials. To achieve that aim, we are currently analyzing a clinically well-defined cohort of 47 autopsy cases with MSA. We use special neuropathological techniques that allow a detailed analysis of anatomical structures that are involved by this alpha-synuclein pathology. We correlate our findings to detailed clinical data that is available from a comprehensive database at our center. In a first step, we focus on pathological changes in the cerebellum (a part of the brain important for movement coordination) and its connections in the brainstem and spinal cord, which show characteristic changes in MSA due to alpha-synuclein aggregation and neuronal loss.

“Mechanisms of Selective Neuronal Death in MSA: Focus on blood pressure controlling areas “: Eduardo Benarroch, MD (Mayo Clinic Rochester, MN)

MSA Coalition Grant #2015-04-007 – $50,000

Despite extensive research in multiple system atrophy (MSA), the cause of death of neurons (nerve cells) controlling movement, blood pressure and other functions is yet unknown. Neuronal death may be due to their abnormal interaction with oligodendrocytes, the cells that produce myelin and provide nutrition to the neuronal portion called axons. Some neurons are affected early in the disease, but what makes these neurons vulnerable is still uncertain. Both neurons and oligodendrocytes accumulate the protein αlpha-synuclein (α-SYN), and this abnormal accumulation may propagate along connections between neurons. Therefore, it is important to determine what causes neuronal loss and whether this propagates along vulnerable pathways. Over the past several years we have studied brain areas controlling blood pressure in MSA, since a fall of blood pressure during standing (called orthostatic hypotension) is one of the most disabling MSA symptoms. We found that loss of one group of neurons controlling blood pressure is associated with α-SYN accumulation in surrounding oligodendrocytes and indices of abnormal iron metabolism. We hypothesize that there is a reciprocal interaction between neurons and oligodendrocytes leading to loss of both types of cells, including abnormal iron metabolism and nutrient deprivation. We plan to study four different brain areas controlling blood pressure; two containing cells that are potentially more vulnerable and two that may be more resistant but share similar connections. We will combine techniques to identify these neurons, and relate the severity of their loss with that of accumulation of α-SYN in surrounding oligodendrocytes, markers of iron metabolism, and nutrient transfer. The identification of the cause and interactions that make some neurons selectively vulnerable may help to develop treatments that protect vulnerable neurons and slow disease progression.

RESULTS

1. Published article (August 2015): Brain: Expanding the spectrum of neuronal pathology in multiple system atrophy

“Selective Cell Vulnerability in MSA: Insights from cases with associated Lewy Body Disease”: Eduardo Benarroch, MD (Mayo Clinic Rochester, MN) – Joint Grant with CurePSP

MSA Coalition Grant #2014-04-002 – $25,000

Despite extensive research, the cause of neuronal loss in multiple system atrophy (MSA) remains elusive. ln particular, there is yet no clear explanation for the mechanism that causes the abnormal accumulation of the protein alpha-synuclein in the oligodendrocytes (the cells that produce the myelin around the axons of neurons in the central nervous system), called glial cytoplasmic inclusions (GCls) and the impact that this has in survival of the neurons. This is particularly relevant in areas of the central nervous system that control vital functions such as blood pressure and breathing, which are frequently affected and cause disability and risk of death in MSA patients. These abnormalities are more severe than in Parkinson’s disease and other so-called Lewy body disorders (LBD), in which alpha-synuclein accumulates in neurons instead of oligodendrocytes. Our laboratory has the unique opportunity to address these questions, as we have availability of brains obtained at autopsy from patients with clinical diagnosis of either MSA or DLB but that show the pathological changes seen in both conditions. The study of these cases may help to understand why neurons controlling vital functions are so much more vulnerable in MSA than in LBD. One potentially important question is how the different types of cells in the central nervous system handle iron in these two types of disorders, as iron is potentially toxic to both oligodendrocytes and neurons. Approaches aimed to reduce abnormal iron accumulation may protect cells in vital brain areas from damage in MSA. Our laboratory has all the technical capabilities and expertise to undertake these studies. We expect to obtain enough preliminary information to apply for a NIH grant to further investigate this important issue.

RESULTS

1. Published article (July 2015) Histaminergic tuberomammillary neuron loss in multiple system atrophy and dementia with Lewy bodies

 

“Mechanisms of Excessive Daytime Sleepiness and Sleep Related Respiratory Dysfunction in MSA”: Eduardo Benarroch, MD (Mayo Clinic Rochester, MN)

MSA Coalition Grant #2013-12-003 – $50,000

Excessive daytime sleepiness and sleep related respiratory disorders such as sleep apnea and laryngeal stridor are prominent symptoms in patients with Multiple System Atrophy. This study aims to uncover the underlying mechanisms of these sleep disorders through pathological studies. Understanding the underlying causes of sleep disorders associated with MSA can provide rationale for development of pharmacological approaches for treatment of these conditions. This can potentially lead both to improvement of quality of life and prevention of premature death in MSA patients.

RESULTS

1. Published article (December 2016): Medullary neuronal loss is not associated with α-synuclein burden in multiple system atrophy.

2. Research update by Eduardo Benarroch (December 2015):

As part of our studies on the mechanisms of neuronal loss and its relationship to oligodendrocyte pathology in in multiple system atrophy (MSA) we determined the relationship between loss of medullary adrenergic and serotonergic neurons and the degree of total α-synuclein accumulation in both neurons and oligodendrocytes. We used studied autopsy tissue from seven MSA and six control individuals. We performed stereologic quantitation in immunocytochemically sections of the medulla, with focus in areas controlling sympathetic and respiratory functions and known to be affected in MSA. Whereas adrenergic (C1) neurons appeared to be more vulnerable than serotonergic medullary raphe neurons, the degree of neuronal loss did not correlate with the magnitude of α-synuclein burden. This indicates that other factors in addition to α-synuclein accumulation in oligodendrocytes (glial cytoplasmic inclusions) or neurons must have a role in determining neuronal death. This provides the basis for follow-up studies on the contribution of impaired iron homeostasis and energy transfer between glial cell and neurons in the pathodynamics of MSA.

Our results were presented the combined 2015 meeting of the International Society for Autonomic Neuroscience held in conjunction with the American Autonomic Society, the European Federation of Autonomic Societies and the Japanese Society for Neurovegetative Research held in Stresa Italy from 26th-29th September 2015. A manuscript entitled “Medullary Neuronal Loss and α-synuclein Burden in Multiple System Atrophy” has been submitted to the Annals of Neurology and is currently under review.

 

“COQ2 mutation and methylation dysfunction leading to alpha-synuclein pathology”: W. Scott Kim, PhD (University of Sydney, Australia), Glenda Halliday, PhD (University of Sydney, Australia) and Poul Jensen, MD, PhD (University of Aarhus, Denmark)

MSA Coalition Grant #2016-09-004 – $50,000

Multiple system atrophy (MSA) is a rapid-onset brain disorder impacting on multiple functions of the body, including blood pressure, heart rate, balance and muscle movement. The cause of MSA is unknown, no specific risk factors have been identified, and there is no cure or effective treatment. Autopsies of MSA brains show deposits of a protein called α-synuclein in oligodendrocyte cells, which are the support cells of the brain. Alpha-synuclein deposits are believed to be toxic to oligodendrocytes. Without the proper functioning oligodendrocytes, neurons, the nerve cells of the brain, will eventually all die. The cause of α-synuclein deposition in oligodendrocytes remains poorly understood. Recent reports indicate that variants of the COQ2 gene are associated with an increased risk for MSA in certain populations. The known function of COQ2 is in the production of coenzyme Q10 (anti-oxidant) and ATP (energy storage molecule). However, the role of COQ2 in MSA context is unknown. Our preliminary data showed that both COQ2 and ATP levels were significantly decreased in disease-affected regions of MSA brain, providing strong evidence for impairment of COQ2 synthesis or function in MSA brain. We therefore proposed a hypothesis that impaired function of COQ2 contributes to α-synuclein pathology in MSA. In this project we will determine if the COQ2 gene is altered in Caucasian MSA patients. We will also determine if coenzyme Q10 treatment prevents or reduces alpha-synuclein deposition in oligodendrocytes. Our study will reveal pathways to control alpha-synuclein deposition that may lead to developing treatments for MSA.

RESULTS

1. Research update by W. Scott Kim (May 2018)

The pathological hallmark of multiple system atrophy (MSA) is the presence of abnormal deposition of a protein called alpha-synuclein in the degenerating cells of the brain. The cause of alpha-synuclein deposition remains poorly understood. Recent reports indicated that variants of the COQ2 gene are associated with an increased risk for MSA in certain populations.We have previously shown thatCOQ2 level is specifically decreased in disease-affected regions of MSA brain, providing evidence for perturbation of COQ2 function in MSA brain. We therefore proposed a hypothesis that impaired function of COQ2 contributes to α-synuclein pathology in MSA. The primary aim of our project was to identify any changes in thetotal DNA, including COQ2, of MSA brain. We isolated DNA from brain tissues of MSA cases and compared it to that of controls. We have found a few yet significant changes in the DNA sequence of MSA brain, and some chemical modifications to the DNA. Our data has provided new insights into understanding the disease mechanism of MSA.

“Towards new strategies for effective immunotherapy in Multiple System Atrophy”: Bente Pakkenberg, PhD (University of Copenhagen, Denmark)

MSA Coalition Grant #2017-10-008 – $40,000

The protein α-synuclein (α-syn) is involved in Multiple System Atrophy (MSA) and Parkinson’s disease (PD), both progressive neurological disorders where accumulation and propagation of toxic α-syn aggregates is believed to drive disease pathology and death of brain cells. Currently, there is no disease-modifying treatment for MSA and PD. Immunotherapy, the use of components of the immune system to achieve therapeutic goals, is currently being intensively explored as promising disease-modifying treatment for neurodegenerative diseases. Our very recent data indicate that healthy individuals possess specific autoantibodies with high binding properties against α-synuclein and that such antibodies are sparse in PD and almost absent in MSA patients (Manuscript under revision). First responder antibodies are decreased to the same degree. These very surprising observations suggest that α-syn aggregates may not effectively be cleared by the immune system in PD and MSA. Therefore, we want to evaluate the α-syn specific antibodies and the antibody producing cells in healthy individuals and MSA/PD patients. Further, we will fully characterize, at the molecular levels, the anti-α-syn antibodies produced in healthy individuals and elucidate the immunological signatures in MSA and PD. These aims will be achieved by evaluation of immune cells (T- and B-lymphocytes) and cytokine profiling, and detection of α-syn antigen-specific memory B-cells, followed by gene analyses of B-cell receptors among α-syn positive memory B-cells, generation of fully human α-syn specific antibodies and analyses of specificity of the generated antibodies. By these means, our main aim is to clarify the “root” source of the immune decline observed in PD and MSA patients. The project will use well-established and advanced equipment in collaboration between the University of Copenhagen and neurological departments in Copenhagen, Odense and Aarhus, Denmark.

RESULTS

1. Research update by Bente Pakkenberg (July 2018)

Our recently published results indicate that individuals have specific autoantibodies in the blood (antibodies that react against host proteins) binding to a-syn and those antibodies binds more strongly to a-syn in healthy individuals, while the binding is reduced in PD patients, and almost absent in patients with MSA. Further, we have showed that the primarily responding antibodies in the clearance pathway are highly reduced both in MSA and PD patients. Similar phenomena were further observed in a new, validation cohort of PD and MSA patients from who samples of plasma and cerebrospinal fluid (CSF) were collected at the same time from each subject. Both levels and binding efficiency of anti-a-syn antibodies were reduced in plasma and CSF. This may indicate deficiency of the clearance system that results in accumulation of a-syn.

Based on these results, we hypothesize that the immune system is imbalanced in MSA and PD, reflected both in the antibody producing cells and in other components of the immune system. Therefore, we aim to evaluate the antibody producing cells in MSA and PD patients in comparison to healthy controls. Further, we will fully characterize, at the molecular levels, the anti-a-syn antibodies produced by individual cells to obtain the autoantibody profile of each individual and compare it with the disease status. To evaluate the interplay between the different branches of the immune system, we will additionally assess different populations of immune cells (T-and B-lymphocytes) and their active state. We will re­evaluate the results in a follow-up samples to describe the progression in MSA and PD at the immune system level. This will determine how central immune-based processes impact the disease picture, but also constitute a new source for predictive biomarkers and template-based vaccine designs in MSA and PD.

We have now completed the first round of recruitment and collected blood samples from 25 MSA, 39 PD patients and 46 healthy controls. Around 70 percent of on year follow-up samples have been accomplished. To start with we have evaluated a-syn levels in the blood in all samples collected thus far, and we found that MSA and PD patients have increased levels of a-syn compared to controls.

Using a novel method, we have successfully isolated individual single cells which produce antibodies specific for a-syn. The next step is to characterize the antibodies by sequence analyses. When the sequence analysis is optimized we will run it on all samples to look for potential disease differences and immune characteristics. This is planned to be completed in the fall 2018. The main results will be submitted in 2019.

Regarding the complex, immune system dynamics in the diseases, we are going to evaluate around SO different populations of cells and further characterize their activity state. This part of the project is believed to be completed in August/September 2018 and submitted in 2019. Later in 2019 the same analyses are going to be carried out on a 1-1.5 years follow-up samples to gain a picture of disease progression at the immune level.

“Proteomic analysis of glial cytoplasmic inclusions in MSA”: Thomas Wisniewski, MD (New York University, New York, NY)

MSA Coalition Grant #2017-10-007 – $50,000

Multiple system atrophy (MSA) is a rare neurodegenerative disorder caused by the abnormal accumulation of the protein alpha-synuclein predominantly in brain glial cells, a type of cell that provides protection and support for neurons. The accumulation of alpha-synuclein causes dysfunction and death of these cells and, eventually, also kills neurons. In glial cells, alpha-synuclein accumulates forming clumps referred to as glial cytoplasmic inclusions (GCI), which can be observed under the microscope and are used to confirm the diagnosis of MSA at autopsy. In addition to alpha-synuclein, these GCI contain other proteins, such as tau, p25-
alpha, and beta-III tubulin. Depending on their predominant symptoms, patients with MSA are classified as parkinsonian (MSA-P), when they present with muscle rigidity and shuffling gait, or cerebellar (MSA-C) when they present with slurred speech or staggering gait. There are other differences between patients with MSA-P and MSA-C, which raises the question whether these presentations are actually different disorders. To determine potential differences between MSA-P and MSA-C we will study the composition of the GCI from brain samples of patients with both
types of MSA. To do so we will use our novel proteomic techniques that allow use of formalin fixed, paraffin embedded tissue, to define the specific and detailed composition of the GCI from brain samples obtained at autopsy of patients with MSA-P and MSA-C. Our findings may have important implications given that, if significant differences are found, patients with either type may respond differently to potential new medications that can halt or slow the progression of the disease.

RESULTS

1. Research update by Thomas Wisniewski (July 2018)

Multiple system atrophy (MSA), the most rapidly progressive synucleinopathy, is characterized by the abnormal accumulation of the protein α-synuclein predominantly in oligodendroglia, forming glial cytoplasmic inclusions (GCI). Patients with MSA present with either a cerebellar (MSA-C) or parkinsonian (MSA-P) phenotype. We hypothesize that the differences between MSA-P and MSA-C are due, at least in part, to different composition of the GCI resulting in different “species” of aggregated α-synuclein. Here we propose a pilot study using novel proteomic techniques to determine potential differences in the composition of the CGI in patients with MSA-P versus MSA-C. This is an important research question as both phenotypes may respond differently in clinical trials of potential disease-modifying drugs.

Our studies are on-going as planned. We have done optimization studies using our proteomic methods on tissue with GCI. Using our optimized method GCI have been isolated from tissue of cases with either MSA-C or MSA-P. Full results and analyses are in progress and will be submitted as a manuscript for publication.

“Toward the in situ proteome of normal and pathologic alpha-synuclein in human neurons and glial cells”: Vikram Khurana, MD, PhD (Brigham and Women’s Hospital, Boston MA)

MSA Coalition Grant #2016-09-009 – $50,000

Multiple System Atrophy (MSA) is an incurable and devastating neurodegenerative disease. It is known as a “synucleinopathy” because its hallmark pathology is the misfolding and aggregation of a small protein known as alpha-synuclein within distinct cell­ types of the brain. More common synucleinopathies include Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). While alpha-synuclein aggregates predominantly in distinct populations of neurons (nerve cells) in PD and DLB, the characteristic pathology of MSA is strikingly different. In MSA, alpha-synuclein characteristically aggregates in both neurons and in glial cells known as oligodendrocytes. Oligodendrocytes “insulate” and increase the speed of electrical conduction along nerve fibers in the brain. How alpha-synuclein triggers dysfunction and death of distinct cell ­types is poorly understood. Recently, we hypothesized that if we could map where alpha­-synuclein was in a cell, and which other proteins it interacts with, we could develop insights into how it was harming those cells. We optimized a novel mapping method for proteins within living rodent neurons, and created such a map for alpha-synuclein. We found that alpha-synuclein interacted with proteins that were critical in mediating its cellular toxicity. Now, in this proposal, we seek to apply a similar strategy for human neurons and oligodendrocytes from MSA patients. We plan to develop such alpha-synuclein maps within oligodendrocytes generated from MSA patient stem cells. Furthermore, we make use of MSA patient brain tissue to capture the distinct types of misfolded alpha-synuclein implicated in this disease. Knowledge gained from these studies will enable us in time to understand disease mechanisms within distinct cell types of the brain, and open up the possibility of patient ­targeted therapies for MSA.

RESULTS

Coming soon

"Gene expression & methylation as a route to MSA biomarkers and drug targets”: Henry Houlden, PhD (University College of London, UK)

MSA Coalition Grant #2015-04-002 – $50,000

Multiple system atrophy (MSA) is a neurodegenerative disorder with an overall prevalence of 2-4/100,000 people. MSA is characterized by abnormal movements (parkinsonism), unsteadiness (ataxia) as well as alterations in blood pressure control and urinary problems (autonomic failure). In the initial stages of the disease, MSA can be difficult to distinguish from Parkinson’s disease and some forms of ataxia. However, MSA has additional clinical features and an aggressive progression, with an average time to death being 8-9 years. The brain of MSA patients presents accumulation of a protein called alpha-synuclein, and loss of neurons in specific regions of the brain. The causes of MSA are largely unknown. There are small families with this condition but the majority of cases are not known to be inherited from generation to generation (sporadic disease). An increased risk of MSA has been proposed as associated with variations in certain genes – SNCA and COQ2. This study aims to investigate how genes make products that are needed for cells, and which variations those products show that can be related to the disease. This will eventually identify candidate genes and measurable indicators (biomarkers) of the disease, and reveal target pathways for therapeutic intervention.

RESULTS

1. Published article (open access – November 2017): Multiple system atrophy: genetic risks and alpha-synuclein mutations.

2. Research update by Henry Houlden (December 2015):

This study aims to investigate how genes make products that are needed for cells, and which variations those products show that can be related to multiple system atrophy (MSA). For that we need to analyze blood samples and tissue from specific regions of the brain of individuals with and without the disease.

We have already collected blood samples from approximately 20 individuals, and are continuing the collection of volunteer individuals at our MSA specialized clinic at the National Hospital for Neurology and Neurosurgery (NHNN, London, UK).

Because the brain tissue we had collected previously was not of enough quality for the analyses we need to do, we have requested and awaiting additional brain tissue from Queen Square Brain Bank (London, UK).

To obtain more reliable results, we need to analyze in the lab the samples from all individuals at the same time. Therefore the main experiments in the lab will only start by early 2016.

“Understanding the degradation of alpha-synuclein protein in MSA”: Janice Holton, MD, PhD (University College of London, UK)

MSA Coalition Grant #2015-04-010 – $45,456

In multiple system atrophy (MSA) a protein called alpha-synuclein sticks together in oligodendrocytes to form glial cytoplasmic inclusions (GCIs). Oligodendrocytes are specialized brain cells that are crucial for supporting neurons. There is no increase in the production of alpha-synuclein in MSA, so we believe that the breakdown of alpha-synuclein from the brain may not be working properly. We will look at 2 substances, known as proteases, called cathepsin D (CTSD) and kallikrein 6 (KLK6), which breakdown alpha-synuclein. CTSD and KLK6 are particularly relevant to MSA because they are found in neurons, oligodendrocytes and inside GCIs. Alpha-synuclein also clumps together in Parkinson’s disease (PD) where nerve cells are the main target. CTSD and KLK6 may have reduced activity in PD contributing to the accumulation of alpha-synuclein in neurons and damaging them. This strongly suggests a role for dysfunction of CTSD and KLK6 in MSA. We will analyze the activity of CTSD and KLK6 in 10 regions of 20 MSA and 20 control brains and determine how this relates to the number of GCIs. Next we will grow oligodendrocytes and control their levels of CTSD and KLK6 to see if we can improve the breakdown of alpha-synuclein. As the first study to examine proteases in MSA we will improve understanding of alpha-synuclein breakdown and help to find new avenues for research and potential treatments.

RESULTS

1. Published article (June 2018): Exploring the putative role of kallikrein-6, calpain-1 and cathepsin-D in the proteolytic degradation of α-synuclein in multiple system atrophy.

2. Research update by Janice Holton (February 2017)

We know that a sticky protein called alpha-synuclein builds up in specific areas of the brain in diseases such as Parkinson’s disease (PD), dementia with Lewy bodies and multiple system atrophy (MSA). We also know that no extra alpha-synuclein is made in brains with these diseases and that in a healthy brain any alpha-synuclein that were to accumulate would be cleaned out by specialised cleaning enzymes. It is for this reason that we believe that in MSA brain these cleaning enzymes are not working properly. In collaboration with our colleagues Professor Seth Love and Dr Scott Miners, based at Bristol University, we aimed to examine the activity and expression levels of these enzymes in MSA post mortem brain and also to use cells grown in a dish as a model of MSA to see whether we could improve the clear out of alpha-synuclein by these enzymes.

Our results have shown that the activity of all three enzymes analysed is increased in several regions of the brain that are severely affected by MSA. We have also shown that the expression levels of two enzymes are particularly increased in the putamen which is severely affected in Parkinson’s type MSA and mixed MSA. Interestingly one of these enzymes breaks down alpha-synuclein in a non-toxic way while the other breaks it down to form toxic fragments. It is this second enzyme which is also increased in the cerebellar white matter which is severely affected in cerebellar type MSA and mixed MSA.

We have presented this data in poster format at the 5th International Congress on Multiple System Atrophy 2016 in Salerno, Italy and will be presenting these data in an oral presentation at the 13th International Conference on Alzheimer’s disease and Parkinson’s disease and Related Neurological Disorders in Vienna, Austria in March this year.

3. Research update by Janice Holton (December 2015):

In multiple system atrophy (MSA) and similar diseases like Parkinson’s disease and dementia with Lewy bodies, we know that the sticky protein alpha-synuclein builds up in areas of the brain and is likely responsible for the damage which occurs in the brain. However, our work and the work of others have shown that no extra alpha-synuclein is created in brains with these diseases compared to healthy brain. We also know that in a healthy brain specialized enzymes break down any alpha-synuclein that builds up before it can be a problem. For this reason, we believe that the alpha-synuclein which accumulates in the brains of people with MSA is not being properly cleared out. This is why the aim of this study is to analyze whether these enzymes are working in MSA brains and see if we can improve their efficiency in a cell culture system.

We are happy to say that we are on schedule with regards to the time line which we outlined in the original grant proposal. To date we have sampled tissue from six brain regions from twenty non-diseased and twenty MSA brains as well as two cases which have a G51D mutation and one case with an A53E mutation of the SNCA alpha-synuclein gene. Samples have been sent to Prof Seth Love’s group in Bristol where they are currently training a new research technician to perform assay which will allow us to determine whether these enzymes are working at the sample efficiency in MSA as in non-diseased brain. Meanwhile, at the Queen Square Brain Bank, I have been analyzing the quantity of the enzymes in the same brain regions compared to non-diseased control. I have also been preparing samples of paraffin embedded MSA and non-diseased brain tissue to mount on to glass slides and to stain for enzymes and alpha-synuclein. These samples will be viewed using a specialized fluorescent microscope and we will be able to see whether the enzymes are in the right location to target alpha-synuclein and perform effectively.

Finally we are currently growing and maturing oligodendrocyte cells which we will use in the New Year to test whether we can fine tune the level of enzymes so that we can improve the breakdown of any type of alpha-synuclein we choose to introduce to the cell dish, be it normal alpha-synuclein or the more complex G51D mutant alpha-synuclein.

“The role of hemoglobin overexpression in molecular pathology of MSA”: Michael Janitz, MD, PhD (University of New South Wales, Australia) and Ronald Melki, PhD (Paris-Saclay Institute of Neuroscience, Paris, France)

MSA Coalition Grant #2016-09-002 – $48,500

Multiple system atrophy (MSA) is a distinct member of the group of neurodegenerative diseases called α-synucleinopathies whereby the fibrillar protein α-synuclein aggregates in oligodendroglia. Although well defined clinically the molecular pathophysiology of MSA has not yet been elucidated. We recently discovered that hemoglobin (HB) genes are highly expressed in the MSA white matter (WM) in the cerebral cortex, which is the primary target structure for MSA-specific neurodegeneration. Hemoglobin is the largest source of peripheral iron in the human body and it may play a role in iron homeostasis throughout the brain. The correlation among iron, hemoglobin and neurodegeneration is further supported by the finding that high levels of α-synuclein are present in blood, specifically in red blood cells. We hypothesize that the overexpression of HB
causes oxidative stress which leads to impairment of oligodendrocyte functionality, which is WM major cellular component. Moreover increased levels of HB proteins might have a direct impact on α-synuclein aggregation. In this project two, internationally recognized, research teams will combine their expertise
in brain transcriptome and proteome analysis to elucidate a mechanism through which HB overexpression leads to MSA pathology. This ambitious goal will be achieved through determination of cell types involved in this pathology, physiological effects of HB overexpression in oligodendrocytes and interaction between HB and α-synuclein deposits. This project, for the first time, will establish a link between WM pathology in MSA, iron homeostasis and α-synuclein metabolism. This collaborative proposal is significant because it will not only provide insights into
MSA pathology but also will lead to identification of new molecular targets for MSA early diagnosis and therapeutic intervention.

RESULTS

1. Research update by Michael Janitz (October 2017)

This project led to establishment of foundations for further analysis of the role of haemoglobin overexpression in oligodendrocytes and thus paved the way towards understanding of the iron metabolism in pathology of multiple system atrophy. Although further experimental work will be required to fully achieve the original project objectives this proposal helped to initiate important research to further understand the molecular pathology of multiple system atrophy.

Individual project aims:

Aim 1. Determine specific cell types in the MSA human cortex, which overexpress HB genes. We have completed preparation of RNA expression plasmid constructs for HBA1, HBA2 and HBB genes and tested their capacity to produce RNA probes for in situ hybridization. We however encountered significant challenges in optimisation of in situ hybridization experiments, in particular in gaining sufficient stability of tissue sections. We therefore could not achieve conclusive results from this set of experiments.

Aim 2. Determine the impact of HB overexpression on oligodendrocyte maturation.
We completed design and optimization of RT-­‐qPCR experiments aiming to assess expression levels of oligodendrocyte maturation markers. Specifically, we isolated total RNA from the CG4 cell line and used it as a template for testing qPCR primers for individual marker genes. Next, we isolated the PCR products and confirmed their specificity using Sanger sequencing. As a preparation to differentiation experiments we also transfected the OPCs with HBA and HBB plasmid expression constructs. Using Western blotting assay we however were not able to detect overexpression of the specific proteins. To address these difficulties we re-­‐establish cell cultures, cloned new expression constructs however without success. We will be continuing the analysis in order to achieve the aim objectives in the future using alternate cells and expression constructs.

Aim 3. Determine the influence of HB on induction of oxidative stress in oligodendrocytes. These experiments will be performed once we confirm HB overexpression using Western blotting. Although this analysis will not be performed within this project we are determined to accomplish this work package using alternative resources.

Aim 4. Establish a relationship between HB overexpression and the aggregation of α­‐synuclein in oligodendrocytes.

Our previous anticipation to commence these experiments earlier this year has been compromised due to intensification of experiments, involving multiple repetitions and protocol modifications, to achieve Aims 1 and 2. Due to budgetary constraints we decided not to pursue the Aim 4 further and focus on estimation of effects of HB overexpression on cell maturation.

Together, the project resulted in building foundations for further analysis of the biological effects of HB overexpression in oligodendrocytes. We will be continuing the work towards achievement of anticipated project objectives using alternative resources.

2. Research update by Michael Janitz (April 2017)

Multiple system atrophy (MSA) is a distinct member of the group of neurodegenerative diseases called α-synucleinopathies whereby the fibrillar protein α-synuclein aggregates in oligodendroglia. Although well-defined clinically the molecular pathophysiology of MSA has not yet been elucidated.

We recently discovered that hemoglobin (HB) genes are highly expressed in the MSA white matter (WM) in the cerebral cortex, which is the primary target structure for MSA-specific neurodegeneration. Hemoglobin transports oxygen throughout our tissues. It is the largest source of peripheral iron in the human body and it may play a role in iron homeostasis throughout the brain. We hypothesize that the overexpression of HB causes oxidative stress, which leads to impairment of oligodendrocyte functionality, which is WM major cellular component. Moreover, oxidative stress, together with increased levels of HB proteins, might have a direct impact on α-synuclein aggregation. This proposal therefore aims to elucidate a mechanism through which HB overexpression leads to MSA pathology.

We are happy to report that we are progressing with the research according to the time line outlined in the original grant proposal. Regarding the first two aims of the project, namely (1) determination of specific cell types in the MSA human cortex, which overexpress HB genes and (2) assessment of the impact of HB overexpression on oligodendrocyte maturation, we generated all necessary plasmid constructs, optimized polymerase chain reaction (PCR) assays and initiated cell cultures and tissue preparations. As for the third aim, which is determination of the influence of HB on induction of oxidative stress in oligodendrocytes, we completed construction of HB protein expression plasmids for oxidative stress measurements. Together, the project is well on track and we estimate its completion within the scheduled time frame.

 

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!