Biology subjects

Tacconelli, S.

Publications and source records attributed to Tacconelli, S..

3 recordsLinked to original sources

bioRxiv2026

Differential TDP-43 interactomes between the cortex and cerebellum in the mouse

TAR DNA binding protein 43 (TDP-43) is the core pathogenic protein across a spectrum of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, but its pathological deposition shows regional selectivity, with abundant TDP-43 positive aggregates in the frontal cortex and spinal cord, and a much lower burden in the cerebellum. In health, TDP-43 expression in the cerebellum is higher than in cortex, hence what underpins this differential vulnerability to TDP-43 aggregation is unclear. Here we demonstrate that in healthy C57Bl/6J mice not only is TDP-43 expression higher in the cerebellum than the cortex, but that this expression difference is driven primarily by differences in cytoplasmic load. Mass spectrometry analysis of TDP-43 pull downs from the cortex and cerebellum of healthy mice identified TDP-43 interactors across a number of core functional pathways, with numerous differences between the two brain regions. Data are available via ProteomeXchange with identifier PXD062532. Notably, there were more interactors identified in both nuclear and cytoplasmic fractions within the cortex than the cerebellum. Putative interactions with four core paraspeckle proteins; SFPQ, NONO, FUS and PSPC1 were confirmed using immunoprecipitation and western blot analysis. Follow up validation using proximity ligation assay showed abundant perinuclear cytoplasmic interactions between TDP-43 and all four paraspeckle proteins in both large motor cortex neurons and purkinje cells, with significantly reduced nuclear interactions detected in the motor cortex for SFPQ, FUS and PSPC1. These findings suggest that TDP-43-protein interactions markedly differ between the TDP-43 pathogenesis vulnerable cortex and relatively resistant cerebellum and exploring these differences may yield new insight into disease mechanisms within ALS/FTD.

neuroscience↗

bioRxiv2025

A validated panel of commercial antibodies for reliable detection of FET proteins.

The FET protein family comprises the highly conserved RNA-binding proteins FUS, EWS and TAF15 which are implicated in RNA metabolism and neurodegenerative diseases such as ALS and FTLD. Despite their structural similarity, reliable detection of individual FET proteins remains challenging due to antibody cross-reactivity and inconsistent localisation patterns reported in the literature. To address this, we systematically evaluated 23 commercially available antibodies for specificity and performance across western blotting, immunocytochemistry, and immunohistochemistry. Using single and double shRNA knockdowns in HeLa cells, we confirmed target engagement and identified antibodies which were specific as well as those with significant cross-reactivity. Immunofluorescence in rat primary neurons and HeLa cells showed antibody and cell type dependent variations in nuclear and cytoplasmic localisation; from these, we identified a subset that demonstrated high-quality, region-specific staining in postmortem human brain tissue. Our findings highlight substantial variability in antibody performance and underscore the need for rigorous validation to ensure reproducibility in FET protein research. We present a validated panel of antibodies suitable for diverse applications, providing a critical resource for studies of FET protein biology and pathology.

neuroscience↗

bioRxiv2025

CRISPR/Cas9-mediated generation of a de novo C9ORF72 knock-in isogenic iPSC cell bank to model ALS and FTD

BackgroundThe pathogenic G4C2 repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies focused on delineating the underlying perturbed mechanisms resulting from this genetic mutation are often confounded by the heterogeneity present in current disease models, such as patient-derived iPSC lines, with estimations of up to 50% of the variation in iPSC cell phenotypes resulting from inter-individual differences. Isogenic models, in which the pathogenic mutation is introduced into a defined genetic background, offer a powerful approach to isolating mutation-specific effects and enable high-resolution comparison across distinct ALS/FTD-associated mutations. Such models are essential for uncovering convergent disease mechanisms and improving reproducibility in ALS/FTD research. MethodsA two-step scarless CRISPR/Cas9 genome editing strategy was used to generate isogenic human iPSC lines carrying a de novo knock-in of a disease-length G4C2 repeat expansion in the C9ORF72 locus. The resulting lines underwent thorough quality control and were differentiated into lower motor neurons and assessed for the presence of key ALS/FTD pathologies, including changes to C9ORF72 mRNA and protein expression, RNA foci and dipeptide repeat proteins. ResultsTwo C9ORF72 knock-in iPSC lines were generated with 631 and 600 G4C2 repeats, alongside an isogenic genome editing control line. The C9ORF72 G4C2 repeat expansion knock-in iPSC lines exhibit both loss-of-function and gain-of-function pathological features characteristic of ALS/FTD. Compared to the parental wild-type KOLF2.1J line and isogenic (wild-type) CRISPR control line, these exhibit a significant reduction in C9ORF72 mRNA and protein levels, the presence of RNA foci accumulation, and a marked increase in poly(GA) and poly(GP) dipeptide repeat protein levels in iPSCs and motor neurons. ConclusionsThis is one of the first reports of a successful knock-in of the pathogenic C9ORF72 G4C2 repeat expansion into a human iPSC line, establishing a genetically defined and physiologically relevant model of ALS/FTD. These isogenic lines recapitulate both key loss- and gain-of-function disease pathologies, providing a crucial complement to existing patient-derived iPSC banks. By eliminating confounding genetic background variability, these cell lines will enable more precise interrogation of C9ORF72-linked pathomechanisms and offer a robust platform for comparative studies across the ALS and FTD spectrum, mechanistic investigations, and future therapeutic targeting with enhanced translational relevance.

neuroscience↗