SYNUCLEINOPATHIES: DEFINING & UNDERSTANDING THEIR HETEROGENEITY
- Genetic and spatial maps of alpha-synucleinopathy in distinct CNS cell types and genetic backgrounds. We have developed novel stem-cell derived glial and neuronal models that rapidly produce the hallmark protein pathologies of neurodegeneration. Ongoing projects involve unbiased proteome-scale screening methods (including CRISPR-Cas9 genome editing and APEX-based in situ proteome labeling) to identify genes that modify neurodegenerative phenotypes in these cells and proteins that interact with alpha-synuclein.
- Cellular pathologies associated with distinct biophysical forms (“strains”) of alpha synuclein in stem cell-derived neurons and glia (Figure). Recently, considerable interest has arisen around the spread of alpha-syuclein from cell to cell in a “prionoid” fashion. Distinct synucleinopathies arise from different biophysical forms, or “strains”, of misfolded alpha-synuclein. We are capturing and amplifying strains from brain, skin and spinal fluid samples of hundreds of patients in our clinic who have different synucleinopathies and testing these strains for distinct biological effects in patient-derived stem cells. Genetic manipulation and single-cell longitudinal imaging are being used to understand and track the consequences of alpha-synuclein aggregation.
- Novel small-molecule probes as candidate diagnostic and therapeutics for synucleinopathies (Figure). With collaborators at MassGeneral (including Drs Changing Wang and Stephen Gomperts) we have performed a DEL screen of billions of small molecules to identify those that bind alpha-synuclein fibrils rather than other amyloid fibrils. The first of these screen hits is being tested in the clinic as candidate radiotracers for visualizing alpha-synuclein. We are on the cusp of beginning to test them now as candidate therapeutic molecules.
- The effects of alpha-synuclein on mRNA processing and translation. We have discussed novel functions of alpha-synuclein in the regulation of gene expression. A variety of model systems are used in these investigations, from in vitro systems to yeast to patient-derived neurons and glia to better understand how alpha synuclein’s interactions with mRNA binding proteins reflects its intrinsic biology and pathology. We believe this biology is centrally involved in cellular responses to membrane stress, including from environmental toxicants and viruses.
- Neuroinflammatory signaling and microbiome. Increasingly neuroinflammatory processes are considered to be critical in the progression of neurodegenerative diseases, but the specific ways in which neuroimmune signaling is engaged as a result of protein misfolding is unclear. We are investigating how alpha-synuclein may specifically engage pathologic glial and immune responses when it accumulates or misfolds. We have optimized a variety of stem-cell protocols that enable us to capture neurons and different glial cells in the correct ratio to capture inflammatory and immune signaling. We are collecting the microbiome of patients who all have suceptibility to Parkinson’s disease but have very different disease outcomes. In a matched mouse model we are studying how the microbes from these patients may be impact neuroinflammation in the brain to alter disease outcome.
- Functional genomics. We are developing biologically driven tools to help genomic analysis and patient stratification. Since the misfolding of alpha-synuclein by definition is the central pathology of synucleinopathies, regardless of the cause, we take our molecular network maps as a starting point for understand why specific individual succumb to these diseases (and others escape), and why some patients progress far more quickly than others. Methods are diverse, from statistical genetics approaches (a close collaboration with Dr Shamil Sunyaev) and machine-learning methods (a close collaboration with Dr Marinka Zitnik). This project utilizes genomic sequencing data from individual patients, multi-generation kindreds we follow with dominant alpha-synuclein mutations and larger scale genomic datasets. The Harvard Biomarkers Study 2.0 centered in our lab is a major source of clinical and genomic information for our study. Our ultimate aim is to develop therapeutics that rationally target biologically defined subsets of patients.
- Gene-environment interactions. In a collaboration with Drs Beate Ritz (UCLA) we are systematically analyzing the effects of pesticides known to be associated with Parkinson’s disease in dopamine neurons engineered in our lab to purity. In this project, in collaboration with the Studer lab at Memorial Sloan Kettering, we are also working to generate stem cells from an entire population of PD patients in the same dish so population-level gene-environment interactions of many patients can be analyzed and studied simultaneously. This technology will also enable us i to perform increasingly larger clinical trials “in the dish.”
CEREBELLAR ATAXIAS
We have generated iPSC from patients who harbor CAG tract (polyglutamine-encoding) expansion mutations that lead to neurodegenerative ataxias, including SCA-1, SCA-2, SCA-3, SCA-7, SCA-8 and DRPLA. Beginning with SCA-3 and DRPLA, we are interested in understanding the biological consequences of misfolding of the encoded proteins and the effects on the multi-protein complexes in which they below.
- Gene therapies. Therapeutically, we are interested in whether genetic correction of these expansions is equivalent to knockdown, a project that has consequences for understanding the potential (and also the potential limitations) of antisense gene therapy for these diseases. We have an active project in which we are testing a gene therapy for DRPLA in stem-cell models matched to patients prior to introducing them in a clinical context. The idea is to better understand on- and off-target effects of ASOs, biologics and small-molecule therapies “in the dish” before clinical trial.
CLINICAL RESEARCH
For clinically oriented and translational research please, and information about major biobanking (Harvard Biomarkers Study 2.0) and MyTrial projects, please find relevant information here: “Clinical Research Questions and Approaches.” Also, see these sections on clinical research.
Figure: Rapid alpha-synucleinopathy stem-cell models. We have optimized approaches to amplify alpha-synuclein (seed amplification assay; SAA) from our patients and introduce them into matched stem-cell models. Briefly, each for each participating patient we take a body fluid or tissue collection that enables us to both capture alpha-synuclein and generate a pluripotent stem cell. We can then create a personalized model by introducing a patient’s alpha-synuclein strain into neurons and glial cells derived from that same patient. We use these models for testing diagnostic radiotracers and candidate therapies, but also for understanding the protein-aggregation pathology in a human cellular context and specific genetic background.