Biology subjects

Rousseaux, M. W. C.

Publications and source records attributed to Rousseaux, M. W. C..

7 recordsLinked to original sources

bioRxiv2024

A stress-dependent TDP-43 SUMOylation program preserves neuronal function

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are overwhelmingly linked to TDP-43 dysfunction. Mutations in TDP-43 are rare, indicating that the progressive accumulation of exogenous factors - such as cellular stressors - converge on TDP-43 to play a key role in disease pathogenesis. Post translational modifications such as SUMOylation play essential roles in response to such exogenous stressors. We therefore set out to understand how SUMOylation may regulate TDP-43 in health and disease. We find that TDP-43 is regulated dynamically via SUMOylation in response to cellular stressors. When this process is blocked in vivo, we note age-dependent TDP-43 pathology and sex-specific behavioral deficits linking TDP-43 SUMOylation with aging and disease. We further find that SUMOylation is correlated with human aging and disease states. Collectively, this work presents TDP-43 SUMOylation as an early physiological response to cellular stress, disruption of which may confer a risk for TDP-43 proteinopathy.

neuroscience↗

bioRxiv2024

Identification of stress specific autophagy regulators from tandem CRISPR screens

Autophagy is a conserved degradative process that promotes cellular homeostasis under stress conditions. Under nutrient starvation autophagy is largely non-selective, promoting the indiscriminate breakdown of cytosolic components. Conversely, selective autophagy is responsible for the specific turnover of damaged organelles including endoplasmic reticula, lysosomes, mitochondria, and peroxisomes. The mechanisms of selective autophagy are best understood through the activity of cargo-specific receptors called autophagy receptors, which facilitate the engulfment of the targeted cargo within autophagosomes, leading to subsequent degradation. We hypothesized that selective autophagy may be regulated by distinct upstream signaling from starvation induced autophagy, providing an additional layer of regulatory control to targeted autophagic degradation. To comprehensively address this question we conducted kinome-wide CRISPR screens to identify distinct signaling pathways responsible for the regulation of basal autophagy, starvation-induced autophagy, and two types of selective autophagy, ER-phagy and pexophagy. These parallel screens identified established and novel autophagy shared regulators under these conditions, as well as kinases specifically required for ER-phagy or pexophagy. More specifically, CDK11A and NME3 were further characterized to be selective ER-phagy regulators. Meanwhile, PAN3 and CDC42BPG were identified as activator or inhibitor of pexophagy, respectively. Collectively, these datasets provide the first comparative description of the kinase signaling specificity, separating regulation of selective autophagy and bulk autophagy. HighlightsO_LIParallel pooled kinome genetic knockout screens reveal known and novel regulators of autophagy under basal conditions, nutrient starvation, ER stress, and peroxisomal stress C_LIO_LISelective ER and peroxisomal autophagy both have unique activators and inhibitors that distinguish them from bulk autophagy C_LIO_LICDK11A and NME3 specifically induce and inhibit ER-phagy, respectively C_LIO_LIPAN3 and CDC42BPG specifically induce and inhibit pexophagy, respectively C_LI

cell biology↗

bioRxiv2023

A topographical atlas of alpha-Synuclein dosage and cell-type expression in the mouse brain and periphery

Parkinsons disease (PD) is the second most common neurodegenerative disease worldwide and presents pathologically with Lewy pathology and dopaminergic neuron loss. Lewy pathology contains aggregated Synuclein (Syn), a protein encoded by the SNCA gene which is also mutated or duplicated in a subset of familial PD cases. Due to its predominant presynaptic localization, immunostaining for the protein results in diffuse signal, providing little insight into the types of cells expressing Syn. As a result, insight into Syn expression-driven cellular vulnerability has been difficult to ascertain. Using a combination of knock-in mice that target Syn to the nucleus of cells (SncaNLS) and in situ hybridization of Snca in wild-type mice, we systematically map the topography and cell types expressing Syn in the mouse brain, spinal cord, retina, and gut. We find a high degree of correlation between Syn protein and RNA levels across multiple brain regions and further identify cell types with low and high Syn. We found that Syn is highly expressed in neurons, particularly those involved in PD and to a lower extent in non-neuronal cell types, notably those of oligodendrocyte lineage. We also find that Syn is devoid in certain neuron types (e.g. ChAT-positive motor neurons), and that all enteric neurons express Syn to a certain degree. Taken together, this atlas provides much-needed insight into the cellular topography of Syn, and provides a quantitative map to test assumptions about the role of Syn in network vulnerability in PD and other Synucleinopathies.

neuroscience↗

bioRxiv2022

Characterizing the differential distribution and targets of Sumo paralogs in the mouse brain

SUMOylation is an evolutionarily conserved and essential mechanism whereby Small Ubiquitin Like Modifiers, or SUMO proteins (Sumo in mice), are covalently bound to protein substrates in a highly dynamic and reversible manner. SUMOylation is involved in a variety of basic neurological processes including learning and memory, and central nervous system development, but is also linked with neurological disorders. However, studying SUMOylation in vivo remains challenging due to limited tools to study Sumo proteins and their targets in their native context. More complexity arises from the fact that Sumo1 and Sumo2 are [~]50% homologous, whereas Sumo2 and Sumo3 are nearly identical and indistinguishable with antibodies. While Sumo paralogues can compensate for one anothers loss, Sumo2 is highest expressed and only paralog essential for embryonic development making it critical to uncover roles specific to Sumo2 in vivo. To further examine the roles of Sumo2, and to begin to tease apart the redundancy and similarity between key Sumo paralogs, we generated (His6-)HA epitope-tagged Sumo2 knock-in mouse alleles, expanding the current Sumo knock-in mouse tool-kit comprising of the previously generated His6-HA-Sumo1 knock-in model. Using these HA-Sumo mouse lines, we performed whole brain imaging and mapping to the Allen Brain Atlas to analyze the relative distribution of the Sumo1 and Sumo2 paralogues in the adult mouse brain. We observed differential staining patterns between Sumo1 and Sumo2, including a partial localization of Sumo2 in nerve cell synapses of the hippocampus. Combining immunoprecipitation with mass spectrometry, we identified native substrates targeted by Sumo1 or Sumo2 in the mouse brain. We validated select hits using proximity ligation assays, further providing insight into the subcellular distribution of neuronal Sumo2-conjugates. These mouse models thus serve as valuable tools to study the cellular and biochemical roles of SUMOylation in the central nervous system.

neuroscience↗

bioRxiv2022

Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy

Parkinsonas disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of -Synuclein (-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the -Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress -Syn replicate several features of PD. Decreasing total -Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of -Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of -Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the -Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical -Syn pathology, indicated by reduced numbers of pS129 -Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic -Syn species. We further validated these findings on pS129 -Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated -Syn in human neurons, in PFF- challenged rat neurons and in the brains of -Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

neuroscience↗

bioRxiv2021

Constitutive nuclear accumulation of endogenous alpha-synuclein in mice causes motor dysfunction and cortical atrophy, independent of protein aggregation.

BackgroundA growing body of evidence suggests that nuclear alpha-synuclein (Syn) plays a role in the pathogenesis of Parkinsons disease (PD). However, this question has been difficult to address as controlling the localization of Syn in experimental systems often requires protein overexpression, which affects its aggregation propensity. MethodsWe engineered SncaNLS mice which localize endogenous Syn to the nucleus. We characterized these mice on a behavioral, histological, and biochemical level to determine whether the increase of nuclear Syn is sufficient to elicit PD-like phenotypes. ResultsSncaNLS mice exhibit age-dependent motor deficits and altered gastrointestinal function. We found that these phenotypes were not linked to Syn aggregation or phosphorylation. Through histological analyses, we observed motor cortex atrophy in the absence of midbrain dopaminergic neurodegeneration. We sampled cortical proteomes of SncaNLS mice and controls to determine the molecular underpinnings of these pathologies. Interestingly, we found several dysregulated proteins involved in dopaminergic signaling, namely Darpp-32, which we further confirmed was decreased in cortical samples of the SncaNLS mice compared to controls via immunoblotting. ConclusionsThese results suggest that chronic endogenous nuclear Syn can elicit toxic phenotypes in mice, independent of its aggregation. This model raises key questions related to the mechanism of Syn toxicity in PD and provides a new model to study an underappreciated aspect of PD pathogenesis.

neuroscience↗

bioRxiv2018

CRISPRCloud2: A cloud-based platform for deconvoluting CRISPR screen data

The simplicity and cost-effectiveness of CRISPR technology have made high-throughput pooled screening approaches available to many. However, the large amount of sequencing data derived from these studies yields often unwieldy datasets requiring considerable bioinformatic resources to deconvolute data; a feature which is simply not accessible to many wet labs. To address these needs, we have developed a cloud-based webtool CRISPRCloud2 that provides a state-of-the-art accuracy in mapping short reads to CRISPR library, a powerful statistical test that aggregates information across multiple sgRNAs targeting the same gene, a user-friendly data visualization and query interface, as well as easy linking to other CRISPR tools and bioinformatics resources for target prioritization. CRISPRCloud2 is a one-stop shop for labs analyzing CRISPR screen data.

bioinformatics↗