Biology subjects

Chen-Plotkin, A. S.

Publications and source records attributed to Chen-Plotkin, A. S..

6 recordsLinked to original sources

bioRxiv2026

Single-Molecule Imaging Reveals Differential Stability of Alpha-Synuclein Aggregates

-Synuclein (-syn) aggregation is central to Parkinsons disease (PD), yet measurements in biofluids are confounded by the coexistence of monomeric and aggregated species. Using Syn-IMAGR, a single-molecule imaging platform with sub-femtomolar sensitivity, we show that purified -syn aggregates undergo dilution-induced disassembly, revealing a concentration-dependent equilibrium. Applied to postmortem brain lysates, Syn-IMAGR distinguishes physiological -syn multimers, which are dimmer and readily dissociate upon dilution, from PD-associated aggregates, which remain detectable and exhibit greater structural resistance to disruption. These results indicate that -syn assemblies occupy distinct stability regimes, with PD-associated aggregates representing a more persistent and less dilution-sensitive structural state. Syn-IMAGR thus provides a quantitative framework for resolving -syn species and for probing their concentration-dependent equilibrium.

neuroscience↗

bioRxiv2026

Microglial reactivity in the hippocampal CA2 is associated with advanced neuronal α-synulceinopathy

Lewy body diseases are thought to evolve by the spread of intraneuronal -synuclein pathology. However, staging models of -synuclein pathology in the human brain seldom evaluate regions with direct synaptic connectivity to model microglial processes in disease. Here, we address this gap by testing the hypothesis that, within the well-defined synaptic connectivity of the intrahippocampal circuit, neuronal -synuclein pathology is associated with microglial reactivity. We selected a cohort of autopsy-confirmed Lewy body disease patients with hippocampal neuronal -synuclein pathology and minimal age-related co-pathologies (n=62) and as a control for other intraneuronal hippocampal pathology, we used a cohort of cognitively healthy patients with focal hippocampal tau accumulation with minimal amyloid plaques, termed primary age-related tauopathy (n=12). We immunostained consecutive hippocampal sections for -synuclein pathology and established markers of microglial reactivity, Iba1, HLA-DR, and CD68. With validated digital histology methods, we measured percent area occupied in 6 hippocampal subfields to compare the percent area occupied within subfields between patient cohorts, correlate the percent area occupied within and between subfields, and use linear-mixed effects models to compare the percent area occupied across subfields while co-varying for demographics. We also constructed two groups of either low-level hippocampal -synuclein pathology restricted to the cornu ammonis 2-3 subfields (Focal Subtype) or widespread -synuclein pathology within additional subfields (Widespread Subtype) to model hypothesized -synuclein pathological spread within the intrahippocampal circuit. Lewy body disease patients exhibited increased HLA-DR and CD68 in most hippocampal subfields compared with primary age-related tauopathy. In Lewy body disease patients, staining for all microglial markers was highest in the cornu ammonis field 2. Neuronal -synuclein pathology in the cornu ammonis field 2 consistently correlated with only HLA-DR and CD68 but not Iba1. Patients classified as the Widespread Subtype had worse cognitive impairment and increased HLA-DR and CD68 within the cornu ammonis field 2. Furthermore, HLA-DR and CD68 in the cornu ammonis field 2 correlated with distal neuronal -synuclein pathology only in subfields with retrograde connectivity. We found increased hippocampal microglial abnormalities in Lewy body disease patients compared with cognitively normal controls with primary age-related tauopathy, suggesting a relatively specific microglial/inflammatory response to neuronal -synuclein pathology. Our data is consistent with predominantly retrograde transmission of neuronal -synuclein pathology across hippocampal subfields, where the focal microglial response in the cornu ammonis field 2 may influence clinical outcomes and -synuclein pathological spread. These data suggest that microglial processes can help refine Lewy body disease histopathological staging.

neuroscience↗

bioRxiv2023

Assessment of extracellular vesicle protein cargo as neurodegenerative disease biomarkers

Extracellular vesicles (EVs) are released by all cells and hold great promise as a class of biomarkers. As EVs represent a way of capturing molecular information about the proteins inside of cells, EVs from biofluids could be used to better understand and diagnose disease from difficult to access organs such as the brain. This promise has led to increased interest in measuring EV proteins from both total EVs as well as brain-derived EVs isolated from the blood. However, the measurement of cargo proteins in EVs has been challenging because EVs are present at low levels and EV isolation methods are imperfect at separating EVs from free proteins. Thus, it is difficult to know whether a protein measured after EV isolation is truly inside EVs. In this study, we developed methods to measure whether a protein is inside EVs and quantify the ratio of a protein in EVs relative to total plasma. To achieve this, we combined a high-yield size exclusion chromatography (SEC) protocol with an optimized protease protection assay and Single Molecule Array (Simoa) digital ELISA assays for ultrasensitive measurement of proteins inside EVs. We applied these methods to analyze key proteins involved in neurodegenerative diseases: -synuclein, Tau, A{beta}40, and A{beta}42. We found that -synuclein and Tau are present in plasma EVs at a small fraction of the levels in total plasma, whereas A{beta}40 and A{beta}42 are undetectable in plasma EVs. This work provides a framework for determining the levels of proteins in EVs and represents an important step in the development of EV diagnostics for diseases of the brain, as well as other organs.

neuroscience↗

bioRxiv2021

TMEM106B modifies TDP-43 pathology in human ALS brain and cell-based models of TDP-43 proteinopathy

The neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TAR DNA-binding protein-43 (TDP-43) inclusions (FTLD-TDP) share the neuropathological hallmark of aggregates of TDP-43. However, factors governing the severity and regional distribution of TDP-43 pathology, which may account for the divergent clinical presentations of ALS and FTLD-TDP, are not well-understood. Here, we investigated the influence of genotypes at TMEM106B, a locus associated with risk for FTLD-TDP, and hexanucleotide repeat expansions in C9orf72, a known genetic cause for both ALS and FTLD-TDP, on global TDP-43 pathology and regional distribution of TDP-43 pathology in 899 postmortem cases from a spectrum of neurodegenerative diseases. We found that, among the 110 ALS cases, minor (C)- allele homozygotes at the TMEM106B locus sentinel SNP rs1990622 had more TDP-43 pathology globally, as well as in select brain regions. C9orf72 expansions similarly associated with greater TDP-43 pathology in ALS. However, adjusting for C9orf72 expansion status did not affect the relationship between TMEM106B genotype and TDP-43 pathology. In order to elucidate the direction of causality for this association, we directly manipulated TMEM106B levels in an inducible cell system that expresses mislocalized TDP-43 protein. We found that partial knockdown of TMEM106B, to levels similar to what would be expected in rs1990622 C allele carriers, led to development of more TDP-43 cytoplasmic aggregates, which were more insoluble, in this system. Taken together, our results support a causal role for TMEM106B in modifying the development of TDP-43 proteinopathy.

neuroscience↗

bioRxiv2020

L1CAM is not Associated with Extracellular Vesicles in Human Cerebrospinal Fluid or Plasma

Neuron-derived extracellular vesicles (NDEVs) present a tremendous opportunity to learn about the biochemistry of brain cells in living patients. L1CAM is a transmembrane protein expressed in neurons that is presumed to be found on NDEVs in human biofluids. Previous studies have used L1CAM immuno-isolation from human plasma to isolate NDEVs for neurodegenerative disease diagnostics. We developed a panel of ultrasensitive Single Molecule Array (Simoa) assays for known EV markers, as well as L1CAM, and applied it to study EVs in human plasma and cerebrospinal fluid (CSF). We fractionated plasma and CSF by size exclusion chromatography (SEC) and density gradient centrifugation (DGC) to separate EVs from free proteins. We observed that L1CAM did not elute in the EV fractions, but rather eluted in the free protein fractions. We found that L1CAM is present as a free protein in human plasma and CSF, possibly due to proteolytic cleavage and/or alternative splicing. We further demonstrate that the isoforms found in CSF and plasma are different. These data collectively establish that L1CAM in plasma is not EV associated and should therefore not be used for NDEV isolation. Importantly, the framework and tools described herein will allow for evaluation of other potential candidate markers for isolation of NDEVs.

neuroscience↗

bioRxiv2018

AAV-mediated progranulin delivery to a mouse model of progranulin deficiency causes T cell-mediated hippocampal degeneration

Adeno-associated virus (AAV)-mediated gene replacement is emerging as a safe and effective means of correcting single-gene mutations, and use of AAV vectors for treatment of diseases of the CNS is increasing. AAV-mediated progranulin gene (GRN) delivery has been proposed as a treatment for GRN-deficient frontotemporal dementia (FTD) and neuronal ceroid lipofuscinosis (NCL), and two recent studies using focal intraparenchymal AAV-Grn delivery to brain have shown moderate success in histopathologic and behavioral rescue in mouse FTD models. Here, we used AAV9 to deliver GRN to the lateral ventricle to achieve widespread expression in the Grn null mouse brain. We found that despite a global increase in progranulin throughout many brain regions, overexpression of GRN resulted in dramatic and selective hippocampal toxicity and degeneration affecting both neurons and glia. Histologically, hippocampal degeneration was preceded by T cell infiltration and perivascular cuffing, suggesting an inflammatory component to the ensuing neuronal loss. GRN delivery with an ependymal-targeting AAV for selective secretion of progranulin into the cerebrospinal fluid (CSF) similarly resulted in T cell infiltration as well as ependymal hypertrophy. Interestingly, overexpression of GRN in wild-type animals also provoked T cell infiltration. These results call into question the safety of GRN overexpression in the CNS, with evidence for both a region-selective immune response and cellular proliferative response following GRN gene delivery. Our results highlight the importance of careful consideration of target gene biology and cellular response to overexpression in relevant animal models prior to progressing to the clinic.\n\nSignificance StatementGene therapies using adeno-associated viral (AAV) vectors show great promise for many human diseases, including diseases that affect the central nervous system (CNS). Frontotemporal dementia (FTD) and neuronal ceroid lipofuscinosis (NCL) are neurodegenerative diseases resulting from loss of one or both copies of the gene encoding progranulin (GRN), and gene replacement has been proposed for these currently untreatable disorders. Here, we used two different AAV vectors to induce widespread brain GRN expression in mice lacking the gene, as well as in wild-type mice. Unexpectedly, GRN overexpression resulted in T cell infiltration, followed by marked hippocampal neurodegeneration. Our results call into question the safety of GRN overexpression in the CNS, with wider implications for development of CNS gene therapies.

neuroscience↗