Neurodegeneration and Cell Death*
Date/Time: Sunday, September 10, 2023 - 3:30 PM – 5:00 PM
Track: Cross-Cutting Special Interest Group (SIG)
Room: Franklin Hall 13 (4th Floor)
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Description:
Session Evaluation Form: https://myana.org/form/ana2023-session-evaluationneuro
Chair: Alice Chen-Plotkin, MD, PhD, FANA
Co-Chair: Vikram Khurana, MD, PhD
Neurodegenerative diseases represent one of the greatest unmet needs in medicine and neurology. The SIG on Neurodegeneration and Cell Death brings together ANA members with an interest in this area, thus providing a forum for discussion of recent science in this area and offering trainees an opportunity to learn about this area of our field.
Learning Objectives:
- Mechanisms underlying common neurodegenerative diseases.
- Cellular interactions that increase neuronal resilience.
Somatic mutation profiling and pathogenesis in Alzheimer’s disease by single-neuron genome sequencing
Speaker: Michael Miller, MD, PhD
Alzheimer’s disease (AD) is characterized pathologically by deposition of misfolded amyloid-beta and tau proteins. However, protein-directed therapeutic strategies have shown modest clinical benefit, pointing to the need to examine pathogenesis from a broader lens. Neurons each harbor somatic single nucleotide variants (sSNV) in their genomes, which increase with age, at a rate of ~15 sSNV per year. In AD, DNA damage is increased, with potentially significant effects on the genome of each cell. We performed single-cell whole-genome sequencing on neurons from postmortem brain tissue from humans with AD and age-matched controls, using two independent genome amplification methods (MDA and PTA), and analyzed the burden of somatic mutations. We also performed mutational signature analysis of the nucleotide changes and trinucleotide context to assess for mutagenic patterns. We found significantly increased sSNV in AD, with each neuron carrying hundreds of additional somatic mutations, with a distinct mutation pattern. AD neurons show an increase in Signature C, which contains distinct nucleotide changes including C>A variants. We found elevated 8-Oxoguanine DNA lesions, evidence that these mutations may result from oxidative damage to DNA. Mutations also show a mechanistic role for gene transcription in the generation of sSNV. Somatic mutations are predicted to produce deleterious effects on the neuron, including gene inactivation and neoantigen-stimulated immune attack. Somatic mutations accumulate abundantly in Alzheimer’s disease, distributed across the genome. Our findings implicate multiple mutagenic forces in sSNV generation in AD, illuminating multiple upstream components of disease pathogenesis including DNA oxidation and transcription-coupled DNA repair. Furthermore, elevated somatic mutation levels appear to produce a toxic cellular state, positioning neurons for dysfunction and death. These findings therefore identify somatic mutation accumulation as a novel process in neurodegeneration, through which we may further dissect the cascade of events in Alzheimer’s disease pathogenesis.
Astrocyte-neuron interactions in health and disease
Speaker: Nicola Allen, PhD
Our work investigates how neuronal synapses are regulated throughout life: from the formation of synapses during development, to the remodeling of synapses in the adult in response to experience, to the loss of synapses in aging. We approach this by asking how non-neuronal glial cells, specifically astrocytes, regulate synapse number and synaptic function. This has led to identification of proteins secreted by developing astrocytes that are sufficient to induce immature synapses to form, and additional signals secreted by adult astrocytes that induce synapse maturation and limit synaptic plasticity. We have further identified altered protein secretion from astrocytes in genetic neurodevelopmental disorders, and determined which of these alterations is responsible for negatively impacting neuronal development. We are now asking if manipulation of synapse-regulating factors in astrocytes is sufficient to delay progression of synaptic dysfunction in aging and neurodegeneration.
The brain transcriptome in aging and Alzheimer’s disease
Speaker: Joshua Shulman, MD, PhD
In Alzheimer’s disease (AD), changes in the brain transcriptome are hypothesized to mediate the impact of neuropathology on cognition. Gene expression profiling from postmortem brain tissue is a promising approach to identify causal pathways; however, there are challenges to definitively resolve the upstream pathologic triggers along with the downstream consequences for AD clinical manifestations. We have functionally dissected AD-associated gene expression modules using a cross-species strategy in Drosophila melanogaster models. First, integrating longitudinal RNA-sequencing and fly behavioral phenotyping, we interrogated unique and shared transcriptional responses to amyloid beta (Aβ) plaques, tau neurofibrillary tangles, and/or aging, along with potential links to progressive neuronal dysfunction. Our results highlight hundreds of conserved, differentially expressed genes mapping to AD regulatory networks. Strikingly, there was a ~70% overlap between age- and tau-induced changes. Whereas aging has a broad impact across most brain cell types, tau-triggered changes are strongly polarized to excitatory neurons and glia. In order to confirm causal modules and pinpoint AD network drivers, we performed systematic in vivo genetic manipulations of hundreds of conserved, prioritized targets, validating modifiers of Aβ- and/or tau-induced neurodegeneration. We discover an up-regulated, causal network, significantly enriched for both AD risk variants and markers of immunity / inflammation, and which promotes AD neurodegeneration. By contrast, a promising synaptic regulatory network is strongly downregulated in human AD and is enriched for loss-of-function suppressors of Aβ/tau, consistent with a potential compensatory response to glutamatergic excitotoxic brain injury. In sum, our cross-species, systems genetic approach establishes a putative causal chain linking AD pathology, large-scale gene expression perturbations, and ultimately, neurodegeneration.
Transdifferentiation: A Novel Tool for Disease Modeling and Translational Applications in Alzheimer's Disease
Oral Abstract Presenter: Ching-Chieh Chou, PhD
Background: A majority of Alzheimer's disease (AD) cases are sporadic and manifest symptoms after age 65, suggesting that advanced age is the prominent risk factor. A tool for modeling aged human neurons, the cell type mostly impacted by AD, is still lacking, which hampers our understanding of disease mechanisms and therapeutic approaches. This study aims to develop a new cell model that overcomes cellular rejuvenation caused by stem cell reprogramming for aging and AD. As inspired by our preliminary proteomics data, we hypothesize that the transdifferentiated neurons (tNeurons) from human somatic cells retain aging signatures and are amenable to detailed biological characterization of mechanisms underlying proteostasis and endosome-lysosomal deficits in AD. Methodology: We leveraged a powerful direct reprogramming paradigm by combining proneural transcription factors and small molecules to transdifferentiate human adult fibroblasts into neurons. Human fibroblasts were derived from healthy controls differing in age and patients with genetic and sporadic forms of AD matched with gender and APOE genotypes. We used quantitative proteomics, high-throughput imaging and biochemical analysis of cell phenotypes, supported by histological validation in APP-transgenic mouse and patient brain tissue. We further integrated CRISPR genome engineering and drug discovery techniques to provide pharmacological strategies that protect against endosome-lysosomal defects, protein aggregation and neurodegeneration in AD. Result: Transdifferentiation approach yielded >85% of tNeurons as cortical glutamatergic neurons after 35-42 days in culture. We showed that tNeurons exhibit changes to DNA repair and histone modifications and the presence of amyloid-β and hyper-phosphorylated tau deposits related to AD. Quantitative proteomics and trajectory analysis revealed neuronal proteome markers associated with AD risk from GWAS analysis and unexpectedly linked lysosomal quality control (LQC) pathway to AD. We molecularly defined LQC deficits and inflammatory responses in AD tNeurons and accumulations of LQC markers containing LAMP1/2-postive lysosomes, proteostasis factors and amyloid-β inclusions in AD mouse and human brain tissue. The treatment of our newly discovered drug that specifically targets lysosomal v-ATPases successfully ameliorated these AD pathologies. Conclusion: This study generates and characterizes the next generation of neuronal models for late-onset AD. We demonstrate that tNeurons are a tractable system and predictive model for disease mechanism exploration and therapeutics development. The outcomes aligning with the pathophysiological features of AD provide novel insights into LQC deficits as the underlying mechanism of AD pathogenesis.
Amyloid Beta Fibrils Induce Microglial Biosynthesis of Heparan Sulfate Proteoglycans Leading to Increased Tau Phagocytosis and Seeding
Oral Abstract Presenter: Brandon Holmes, MD, PhD
Emerging evidence now establishes a central role for microglia in Alzheimer’s disease. The microglia cell-surface proteome, or surfaceome, is a critical hub that enables neuroprotective, neurotoxic, and neuroinflammatory signaling in the diseased brain. Targeting the microglia surfaceome with selective pharmacologic agents may allow for the manipulation of diverse microglia states and function and therefore holds tremendous experimental and therapeutic potential. Precise microglia targeting requires broad knowledge of how the surfaceome remodels in the disease environment and this biology has not been systematically explored. To elucidate how microglia remodel their surfaceome in the context of Alzheimer’s disease pathology, I performed mass spectrometry-based surfaceome profiling of human iPSC microglia after exposure to Aβ fibrils. My data reveals a robust upregulation of heparan sulfate proteoglycans (HSPGs) and proteins that promote phagocytosis. These Aβ-primed microglia increase their capacity to bind and phagocytose tau aggregates via HSPGs. Specifically, I have identified the glypican family of HSPGs as the key core proteins responsible for this pro-phagocytic phenotype. Using immunohistochemistry, I have demonstrated that these glypicans are specifically enriched in AD-associated microglia and are not found in healthy aged-matched control human brain. Finally, I have developed a Drosophila model of amyloid-induced tau spread. Knockdown of microglial glypicans results in reduced tau spread as well as rescue of locomotion deficits and early lethality. Taken together, this data demonstrate that Aβ alters the microglia surfaceome to promote tau uptake and spread through the brain and provides a mechanism to link Aβ and tau pathology through microglia and HSPGs.
A CRISPR Library Approach to Identify Microglial Genes That Regulate Uptake and Endolysosomal Trafficking of Aggregated Alpha-Synuclein
Oral Abstract Presenter: Albert Davis, MD, PhD
Accumulation of misfolded alpha-synuclein (aSyn) protein is a hallmark of Parkinson (PD) disease and related illnesses termed “synucleinopathies” and likely contributes to their pathogenesis. aSyn is abundantly expressed in neurons and accumulates primarily in neurons in Parkinson disease and dementia with Lewy bodies. Misfolded forms of aSyn can induce aggregation of normal aSyn, and they can be released into the extracellular space where they can be taken up by neighboring neurons, a process that is thought to accelerate prion-like spreading of aSyn pathology across brain networks. In addition to neurons, other cell types including microglia are also known to take up aggregated aSyn from the extracellular space. As the resident macrophage-lineage immune cells of the brain, microglia participate in phagocytosis and clearance of multiple pathogens and aggregated self-proteins, including aSyn. This process may serve a critical function in limiting the propagation of misfolded aSyn in neural networks. Because the molecular determinants of microglial clearance of aSyn aggregates are incompletely understood, we performed a CRISPR library screen in a BV2 microglial cell line to identify gene candidates that modulate uptake and endolysosomal trafficking of aSyn aggregates. We added aSyn fibrils incorporating the pH-sensitive dye pHrodo to the culture medium of BV2 cells transduced with mouse CRISPR Brie lentiviral libraries targeting approximately 20,000 genes. Following incubation, we harvested the cells and analyzed the pHrodo fluorescence by flow cytometry. By sorting the cells corresponding to the bottom 3% and the top 3% of pHrodo fluorescence intensity and sequencing the guide RNAs present in those pools, we identified candidate genes that either promote or inhibit uptake and endolysosomal accumulation of aSyn, respectively. Gene ontology analysis confirmed enrichment of multiple canonical endolysosomal genes in addition to several novel candidates. Pairing these results with human genetic data related to PD risk will inform an integrated approach to validation of potential disease-modifying targets for synucleinopathies.