protein aggregation
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Linking Single-Cell Transcriptomic, Morphological, and Temporal Signatures of Vulnerability in Neurodegeneration
Neurodegeneration involves complex cellular phenotypes and molecular changes that vary widely among the cells of the nervous system. Current methodologies permit either detailed molecular profiling (e.g., single-cell transcriptomics) or functional phenotyping (e.g., live imaging of neuronal activity), but not both in the same cells. Thus, it is difficult to directly link a neuron's functional state or fate with its gene expression profile. To address this limitation, we developed an innovative technology, VISTA-FISH (Video Imaging with Spatial- Temporal Analysis by FISH), that couples prospective live-cell imaging with high-resolution spatial transcriptomic profiling of the same cells. This approach enables in situ comparisons of gene expression in neurons that exhibit divergent behaviors or outcomes. Using VISTA-FISH, we will profile iPS-derived human neurons to link single-cell gene expression, morphology, and temporal phenotypes to study molecular pathways driving resilience as well as susceptibility. After exposing neurons carrying TDP43 and C9orf72 mutations to a stimulus inducing TDP43 aggregation, we will jointly record TDP43 localization and neuron activity using live-cell microscopy, then measure single-cell gene expression of the same cells (Aim 1). We will also combine live-cell measurements of TDP43 half-life with CRISPR screening and single-cell gene expression (Aim 2). These rich datasets will enable us to determine transcriptomic changes associated with differences in protein aggregation, protein synthesis, and protein degradation in individual cells, providing an unprecedented molecular perspective on factors responsible for vulnerability and resilience to neurodegeneration.
Addressing C-F bonds and amyloid-formation in biological systems
The ingestion, pulmonary inhalation, and dermal infiltration of C-F bond-containing compounds, most commonly found in the form of per- and polyfluoroalkyl organic acids, causes oxidative stress, inflammation, DNA damage, and developmental defects in infants and adults. These chemicals accumulate in the brain, disrupt neurological function and compromise cognitive and locomotory behavior. Yet, we lack a high-resolution road-map of the interactions between C-F bonds and biomolecular assemblies driving the trajectory towards neurodegenerative outcomes. This gap constitutes a significant barrier to advancing measures designed to mitigate C-F chemistry-associated neurotoxicity. Emerging experimental and computational data from our laboratory reveals that perfluorooctanoic acid, perfluorodecanoic acid and perfluorosulfonic acid corrupt biomolecular structures through C-F:side-chain interactions in tested soluble, globular proteins found in milk and tissues (matrices where C-F chemistries have been detected). Furthermore, they impaired the physiological function in these proteins through displacement of physiological ligands or by compromising the binding of co-factors. The neuroblastoma-derived SHSY-5Y cell line insulted with the said C-F moieties displayed altered gene expression corresponding to reactive oxygen species (ROS), protein ubiquitination, inflammation along with compromised cytoskeletal integrity. C-F bond ingestion ablated dopaminergic (DA) neurons in the nematode C. elegans and induced locomotory deficits in a manner mimicking paraquat. Based on these findings, we propose to gather data towards our hypothesis that C-F bond exposure perturbs biomolecular, cellular and organismal assemblies to onset neurodegeneration-linked trajectories. In Aim 1, we will determine whether organic fluoroacids alter mRNA levels in differentiated SHSY-5Y cells and in neuroprotective gut bacteria (Lactobacillus rhamnosus, Bifidobacterium lactis and Lactobacillus acidophilus). We will examine whether the neuroblastoma cell line exposed to C-F chemistry displays readouts designed to inform the onset of neurodegeneration-associated trajectories (including α-synuclein aggregation). In Aim 2, we will further address in a preclinical model whether C-F burden induces protein aggregation (α-synuclein, amyloid β, mHTT), interferes with dopaminergic neuronal assembles and induces locomotory deficits. Completion of the proposed work will complement ongoing experimental biophysical, structural (crystallographic, NMR) and computational (docking, molecular dynamics simulations) mapping of the interactions between these anthropogenic “forever” chemicals and amyloid-forming proteins potentially resulting in a soluble-to-toxic transformation. It will prepare the stage for vertebrate testing. The findings from this relatively understudied area likely exposes interventional targets for C-F chemistry associated neurotoxicity, spurs therapeutic efforts and can also guide the development of more biocompatible alternatives.
The glymphatic system in motor neurone disease
Neurodegenerative diseases are chronic and inexorable conditions characterised by the presence of insoluble aggregates of abnormally ubiquinated and phosphorylated proteins. Recent evidence also suggests that protein misfolding can propagate throughout the body in a prion-like fashion via the interstitial or cerebrospinal fluids (CSF). As protein aggregation occurs well before the onset of brain damage and symptoms, new biomarkers sensitive to early pathology, together with therapeutic strategies that include eliminating seed proteins and blocking cell-to-cell spread, are of vital importance. The glymphatic system, which facilitates the continuous exchange of CSF and interstitial fluid to clear the brain of waste, presents as a potential biomarker of disease severity, therapeutic target, and drug delivery system. In this webinar, Associate Professor David Wright from the Department of Neuroscience, Monash University, will outline recent advances in using MRI to investigate the glymphatic system. He will also present some of his lab’s recent work investigating glymphatic clearance in preclinical models of motor neurone disease. Associate Professor David Wright is an NHMRC Emerging Leadership Fellow and the Director of Preclinical Imaging in the Department of Neuroscience, Monash University and the Alfred Research Alliance, Alfred Health. His research encompasses the development, application and analysis of advanced magnetic resonance imaging techniques for the study of disease, with a particular emphasis on neurodegenerative disorders. Although less than three years post PhD, he has published over 60 peer-reviewed journal articles in leading neuroscience journals such as Nature Medicine, Brain, and Cerebral Cortex.
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