Clarke, J. H. | Search bioRxiv

bioRxiv2026

Cross-platform multi-laboratory screening identifies HUWE1, USP33 and USP20 as negative-regulators of autophagy or mitophagy with relevance to neurodegeneration

Autophagy and mitophagy are essential cellular processes implicated in neurodegenerative diseases, yet few tractable targets have been robustly validated for therapeutic modulation of these pathways. In an industry-academia consortium we carried out an extensive literature review and expert curation, leading to selection of 29 genes previously reported to enhance autophagy upon genetic or pharmacological modulation. These genes were classified based on whether downregulation or overexpression induced autophagic activity. Using siRNA knockdown, small-molecule modulators, and transient transfection approaches, we systematically screened these targets in parallel across HeLa and HEK-293 cell lines using high- and low-content phenotypic imaging. Promising candidates were further evaluated in induced pluripotent stem cell (iPSC)-derived neurons and in Drosophila models. Three targets emerged as top candidates: HUWE1 downregulation consistently enhanced autophagy and aggregate clearance, while USP33 downregulation promoted mitophagy. We also identified USP20, a close homologue of USP33, as an inhibitor of mitophagy, a role not previously assigned to this protein. These effects on autophagy/mitophagy were corroborated in Drosophila, highlighting their potential functional relevance across species. This cross-platform, multi-laboratory study establishes a framework for identifying and validating regulators of autophagy and mitophagy and provides a route which enables a comprehensive reassessment of the current target literature in this field. The validation of HUWE1, USP20, and USP33 as candidate therapeutic targets offers new opportunities for intervention in neurodegenerative diseases.

neuroscience↗

bioRxiv2023

Tau phosphorylated at serine 356 is associated with Alzheimer's disease pathology and can be lowered in mouse and human brain tissue using the NUAK inhibitor WZ4003

Tau hyperphosphorylation and aggregation is a common feature of many dementia-causing neurodegenerative diseases. Tau can be phosphorylated at up to 85 different sites, and there is increasing interest in whether tau phosphorylation at specific epitopes, by specific kinases, plays an important role in disease progression. The AMP-activated protein kinase (AMPK) related enzyme NUAK1 been identified as a potential mediator of tau pathology, whereby NUAK1-mediated phosphorylation of tau at Ser356 prevents the degradation of tau by the proteasome, further exacerbating tau hyperphosphorylation and accumulation. This study provides a detailed characterisation of the association of p-tau Ser356 with progression of Alzheimers disease pathology, identifying a Braak stage-dependent increase in p-tau Ser356 protein levels and an almost ubiquitous presence in neurofibrillary tangles. We also demonstrate, using sub-diffraction-limit resolution array tomography imaging, that p-tau Ser356 co-localises with synapses in AD post-mortem brain tissue, increasing evidence that this form of tau may play important roles in AD progression. To assess the potential impacts of pharmacological NUAK inhibition in an ex vivo system that retains multiple cell types and brain-relevant neuronal architecture, we treated postnatal mouse organotypic brain slice cultures from wildtype or APP/PS1 littermates with the commercially available NUAK1/2 inhibitor WZ4003. Whilst there were no genotype specific effects, we found that WZ4003 results in a culture-phase dependent loss of total tau and p-tau Ser356, which corresponds with a reduction in neuronal and synaptic proteins. By contrast, application of WZ4003 to live human brain slice cultures results in a specific lowering of p-tau Ser356, alongside increased neuronal tubulin protein. This work identifies differential responses of postnatal mouse organotypic brain slice cultures and adult human brain slice cultures to NUAK1 inhibition that will be important to consider in future work developing tau-targeting therapeutics for human disease.

neuroscience↗