Neurodegeneration and Cell Death

In this session we will discuss drug development in neurodegenerative diseases: challenges and opportunities.

Accurate modeling of protein aggregation in diverse cell systems, such as fluorescently tagged tau or alpha-synuclein (aSyn)-expressing cell lines or 'biosensor' cells, is crucial for understanding the mechanisms underlying neurodegenerative processes. This includes examining the early stages of amyloid fibril formation and their localization within these cells, a critical yet underexplored aspect in studying neurodegenerative diseases. This study aimed to analyze the structure and formation of tau fibrils in fluorescently tagged tau-expressing cell lines and induced pluripotent stem cell (iPSC)-derived human neurons, comparing their resemblance to amyloid structures in tauopathies and exploring early fibril formation stages. 

We employed a suite of advanced imaging techniques, including cryo-confocal microscopy, cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), and cryo-focused ion beam (cryo-FIB) milling, to study tau fibrils in these cell models seeded with brain extracts from tauopathy cases and recombinant tau. We also showed fibrils within these cell models, both extracted and in situ, using correlative light and electron microscopy (CLEM) and cryo-ET, and analyzed their subcellular localization. Our findings demonstrate that these cell lines can form amyloid structures similar to neurofibrillary tangles observed in tauopathies. Cryo-EM and cryo-ET analyses revealed the presence of cross-β sheets and protein chains spaced 4.7 Å apart. Preliminary data using cryo-FIB and cryo-ET provided insights into the early stages of fibril formation and localization within the cells.

These findings support the use of fluorescently tagged tau-expressing cell lines as valid models for studying tau fibril formation in tauopathies, laying a foundation for further understanding the early development of these conditions in cell models. The immediate next steps include the detailed characterization of the biochemical pathways involved in tau fibril formation and the identification of specific molecular targets for therapeutic intervention. Additionally, our workflow can be applied to studying amyloid formation in other cell models, such as those relevant for synucleinopathies, by adapting the imaging and analytical techniques to alpha-synuclein-expressing cell lines. This approach will facilitate the investigation of early amyloidogenic events and the development of targeted therapies for a broader range of neurodegenerative diseases.

This research was generously supported by the National Institutes of Health (NIH), National Institute on Aging (NIA) and the National Institute of Neurological Disorders and Stroke (NINDS).

The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. We developed a suite of iPSC-based ‘inclusionopathy’ models including 60 iPSC & hESC lines, that allow us to rapidly induce CNS cells and express aggregation-prone proteins at brain-like levels utilizing piggyBac or targeted transgenes. We engineered cortical neuron inclusionopathy models that recapitulate known fibril- and lipid-rich inclusion subtypes seen in Parkinson’s brain, through exogenous seeding or a-synuclein mutation. Using a combination of longitudinal single-cell tracking and cross-sectional analyses, we identified multiple inclusion classes with distinct biological implications for neuronal survival. Genetic-modifier and protein-interaction screens pinpointed RNA-processing and actin cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. By incorporating patient-specific CNS cells and proteinaceous strains amplifiable from patient tissue and biofluids, this technology now offers a path to a personalized stem cell model from individual patients amenable to testing new diagnostic and therapeutic strategies.

Neuroinflammation plays an increasingly important role in neurodegenerative diseases. However, most studies have identified a principal role for inflammatory cells such as microglia in these processes. Based on the fundamental idea that neurons accumulate DNA damage over a lifetime, we tested whether major damage-initiated pathways were present and active within neurons. Using a combination of human stem cell and mouse models, as well as postmortem tissue from individuals with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, we found concordant upregulation of neuron-intrinsic inflammatory signals via the stimulator of interferon genes (STING) pathway. Moreover, the activation of the STING pathway was restricted to the most vulnerable neuronal populations, thus relating the process to the underlying question of cell-type specificity in neurodegenerative diseases. These findings raise the question of how neuron-intrinsic inflammatory signals contribute to neuroinflammation and neurodegeneration.

Parkinson disease (PD) is a neurodegenerative movement disorder marked by progressive motor and non-motor symptoms that lead to profound disability. Neurodegeneration in PD relates to toxic aggregation of alpha-synuclein (asyn), and mounting evidence shows that asyn can be degraded through the conserved pathway of autophagy. However, available methods to modulate autophagy fail to confer clinical benefits due to intrinsic resistance of neurons to these methods. This resistance stems in part from MTMR5 (myotubularin-related phosphatase 5), a potent autophagy suppressor that we found to be selectively enriched in neurons. To investigate how and to what extent MTMR5 manipulation modifies asyn toxicity, we established a novel human induced pluripotent stem cell (iPSC)-derived neuron model of PD expressing fluorescently labeled autophagy effectors and asyn. We found that knockdown of MTMR5 significantly augmented autophagic clearance of asyn and mitigated neuronal death. Our findings attest to the neuroprotective effects of targeting MTMR5 for asyn proteostasis.

Many progressive, and fatal neurodegenerative disorders have severe gastrointestinal (GI) symptoms in addition to their better studied central nervous system (CNS) disease. These GI symptoms markedly impair quality of life and are often attributed to CNS disease or medication side effects. We have been exploring the basis of these GI symptoms in the neuronal ceroid lipofuscinoses (NCLs or Batten disease) a group of inherited lysosomal storage disorders. We have revealed an underappreciated impact of lysosomal dysfunction upon the enteric nervous system (ENS), the intrinsic nervous system of the bowel that controls most aspects of bowel function. In mouse models of CLN1, CLN2 and CLN3 disease we have found that although the ENS develops normally, ENS neurons and glia progressively degenerate throughout the small bowel and colon, resulting in bowel dysmotility. These can be prevented by neonatal administration of AAV-mediated gene therapy.