Biology subjects

Allan, D. W.

Publications and source records attributed to Allan, D. W..

9 recordsLinked to original sources

bioRxiv2026

Context-dependent toxicity of human Tau isoforms in a Drosophila tauopathy model

Tauopathies are characterised by progressive deterioration of brain regions due to abnormal accumulation of the microtubule-associated protein tau (MAPT). Alternative splicing of MAPT pre-mRNA results in six tau isoforms, which are classified into two groups depending on the number of microtubule-binding domain repeats (3R vs 4R). Although many tauopathies are 3R or 4R-specific, the relative contributions of individual isoforms to neurotoxicity remain incompletely understood. To systematically characterise differences in tau isoform toxicity, we created a novel set of Drosophila lines expressing equivalent amounts of the six human tau isoforms (hTau) at levels sufficient to induce visible phenotypes. Using a variety of assays including survival, negative geotaxis and tissue-level or cell-type-specific degeneration, we found that hTau isoform toxicity is not uniform across different biological contexts. Despite generally higher toxicity of 4R isoforms compared to 3R, the effects of individual hTau isoforms varied with the temporal window of expression, tissue type, and neuronal identity. Restricting hTau expression to small homogeneous neuronal populations enabled detailed analysis of isoform-specific degeneration. Neurons previously observed to be vulnerable or resilient to hTau toxicity exhibited differences in the onset and progression of degeneration, suggesting that resilience may be an early and transitory state, with most or all neurons eventually succumbing to tau toxicity over time. Notably, these differences in toxicity were not readily explained by variations in hTau abundance and phosphorylation. Together, our findings demonstrate that tau toxicity is highly context-dependent, clearly isoform-specific, and shaped by interactions between tau and its cellular environment.

neuroscience↗

bioRxiv2026

A Cell-Based Assay to Categorize Variants of Human Succinate Semialdehyde Dehydrogenase Associated with Autism Spectrum Disorder

Many genes are implicated in autism spectrum disorder (ASD) as a result of sequencing the genomes of individuals with ASD, however in most cases it remains unclear which genes are playing causative roles. ALDH5A1, which encodes the enzyme succinate semialdehyde dehydrogenase (SSADH), an important regulator of GABA metabolism, is causative in the syndrome SSADH deficiency and is also implicated in ASD. However, it is unknown how variants found in ASD affect SSADH function. We developed a yeast growth assay that models SSADH deficiency to quantify the functional impact of seven ALDH5A1 variants found in ASD. In this assay, expression of human ALDH5A1 partially complemented the growth defect caused by deletion of the ALDH5A1 ortholog UGA2. Using growth rate measurements, we calculated functional scores for 27 variants divided into calibration and ASD test variant groups. Functional scores for benign and pathogenic calibration variants segregated accordingly, validating the assay, while ASD variants displayed a range of activities from complete loss to normal function. Comparisons with published enzymatic assays and computational predictions showed broad agreement, while also identifying some limitations of these approaches. SUMMARY STATEMENTWe introduce a simple yeast assay to measure how autism-linked gene variants in ALDH5A1, which encodes the enzyme succinate semialdehyde dehydrogenase, affect its function, helping clarifying its role in autism.

neuroscience↗

bioRxiv2025

The Drosophila wing is a high-throughput and versatile screening tool for Tau-mediated disease mechanisms and drug discovery.

Tau protein contributes to microtubule stability, which is disrupted in Alzheimers disease and other Tauopathies. In these diseases, Tau molecules become hyperphosphorylated, misfolded and aggregated, propagating pathology across the brain. Studies dissecting disease mechanisms or screening disease-modifying therapies rely on animal models that unveil pathogenic events in vivo but also take several weeks or months to complete. Here we describe a versatile experimental paradigm that yields results in days and yet offers all the advantages of a genetically tractable in vivo system: the Drosophila wing disc. Mimicking neurotoxicity, human Tau expression causes cell death in the wing disc leading to quantifiable phenotypes in the adult wing. The neuroprotective peptide NAP ameliorates Tau toxicity in this system, validating it as a cost-effective drug screening tool. Phenocopying adult neurons, Tau toxicity in the wing disc is exacerbated by simulating hyper-phosphorylation and prevented by suppressing aggregation. Additionally, we show that the wing disc can dissect disease mechanisms that underpin clinically relevant Tau variants. Thus, the wing disc offers an in vivo experimental paradigm for fast and efficient exploration of disease mechanism and screening.

neuroscience↗

bioRxiv2024

Aggregation promoting sequences rather than phosphorylation are essential for Tau-mediated toxicity in Drosophila

BackgroundDisease-modifying therapies for tauopathies like Alzheimers disease have targeted Tau hyperphosphorylation and aggregation, as both pathological manifestations are implicated in Tau-mediated toxicity. However, the relative contributions of these pathology-linked changes to Tau neurotoxicity remain unclear. MethodsLeveraging the genetic tractability of Drosophila, we generated multiple inducible human Tau transgenes with altered phosphorylation status and/or aggregation propensity. Their individual and combined impact was tested in vivo by quantifying Tau accumulation and neurodegenerative phenotypes in the aging fly nervous system. ResultsWe report that phospho-mimicking Tau (hTau2N4RE14) induced profound neurodegeneration, supporting a neurotoxic role for phosphorylation. However, when we rendered hTau2N4RE14 aggregation incompetent, by deleting the 306VQIVYK311 motif in the microtubule-binding region, neurotoxicity was abolished. Moreover, a peptide inhibitor targeting this motif efficaciously reduced Tau toxicity in aging Drosophila. ConclusionNeurodegeneration mediated by Tau hyperphosphorylation is gated via at least one aggregation-mediating motif on the protein. This highlights the primacy of blocking Tau aggregation in therapy, perhaps without the need to clear phosphorylated species.

neuroscience↗

bioRxiv2024

A multi-model approach defines function altering MECP2 missense variants identified in individuals with autism spectrum disorder

Pathogenic variants in MECP2 commonly lead to Rett syndrome, where MECP2s function as a DNA cytosine methylation reader is believed critical. MECP2 variants are also catalogued in individuals with autism spectrum disorder (ASD), including nine missense variants which had no known clinical significance at the start of this study. To assess these nine variants as risk alleles for ASD, we developed MECP2 variant functional assays using budding yeast and Drosophila. We calibrated these assays with known pathogenic and benign variants. Our data predict that four ASD variants are loss of function and five are functional. Protein destabilization offers insight into the altered function of some of these variants. Notably, yeast and Drosophila lack DNA methylation, yet all Rett pathogenic and ASD variants located in the methyl DNA binding domain that we analyzed proved to be loss of function, suggesting a clinically-relevant role for non-methyl DNA-binding by MECP2.

neuroscience↗

bioRxiv2024

Mimicking Human EED Variants in Drosophila: A Promising Strategy to Analyze Human EED Variant Function

The Polycomb Repressive Complex 2 is an epigenetic reader/writer that methylates histone H3K27. Rare germline partial loss of function (pLoF) variants in core members of the complex (EZH2, EED, SUZ12) cause overgrowth and intellectual disability syndromes, whereas somatic variants are implicated in cancer. However, up to 1% of the general population will have a rare variant in one of these genes, most of which would be classified as variants of uncertain significance (VoUS). Towards screening these VoUS for partial LoF alleles that may contribute to disease, here we report functional assays in Drosophila to interrogate EED missense variants. We mimicked the amino acid change(s) of EED variants into its Drosophila ortholog, esc, and tested their function. Known likely benign variants functioned wildtype and known pathogenic variants were LoF. We further demonstrate the utility of this calibrated assay as a scalable approach to assist clinical interpretation of human EED VoUS.

genetics↗

bioRxiv2022

ADULT EXPRESSION OF SEMAPHORINS AND PLEXINS IS ESSENTIAL FOR MOTONEURON SURVIVAL

A role for axon guidance genes in the adult nervous system has not been fully elucidated. We performed an RNAi screen against guidance genes in the adult Drosophila melanogaster nervous system and identified fourteen genes required for adult survival and normal motility. Additionally, we show that adult expression of Semaphorins and Plexins in motoneurons is necessary for neuronal survival, indicating that guidance genes have critical functions in the mature nervous system.

neuroscience↗

bioRxiv2019

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PHTS, we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occurred across the protein, resulting in partial to complete loss of function (LoF), which was well correlated across models. However, assays were selectively sensitive to variants located in substrate binding and catalytic domains, which exhibited complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

genetics↗

bioRxiv2018

IRES-mediated ribosome repositioning directs translation of a +1 overlapping ORF that enhances viral pathogenicity

RNA structures can interact with the ribosome to alter translational reading frame maintenance and promote recoding that result in alternative protein products. Here, we show that the internal ribosome entry site (IRES) from the dicistrovirus Cricket paralysis virus drives translation of the 0-frame viral polyprotein and an overlapping +1 open reading frame, called ORFx, via a novel mechanism whereby a subset of ribosomes recruited to the IRES bypasses downstream to resume translation at the +1-frame 13th non-AUG codon. A mutant of CrPV containing a stop codon in the +1 frame ORFx sequence, yet synonymous in the 0-frame, is attenuated compared to wild-type virus in a Drosophila infection model, indicating the importance of +1 ORFx expression in promoting viral pathogenesis. This work demonstrates a novel programmed IRES-mediated recoding strategy to increase viral coding capacity and impact virus infection, highlighting the diversity of RNA-driven translation initiation mechanisms in eukaryotes.

biochemistry↗