Cross-Species Single-Cell Analysis Reveals Sex Differences in Mitochondrial Dysfunction as a Conserved Feature of Tau Pathology

Tau pathology represents one of the main hallmarks of Alzheimer's disease (AD), with previous studies reporting significant sex differences in tau accumulation and disease progression. While the Tau P301L zebrafish model has emerged as a promising tool for studying tauopathy, it has not been systematically compared to human AD at the single-cell level, particularly regarding sex-dependent molecular alterations that could validate its translational relevance. This study aimed to characterize the molecular and cellular pathogenesis of the Tau P301L zebrafish model using single-cell transcriptomics to reveal cell-type-specific and sex-dimorphic responses to tau pathology and confirm its relevance through comparison to human AD and the THY-Tau22 mouse model. We performed single-nucleus RNA sequencing on brain tissue from 6-month-old Tau P301L transgenic and wild-type zebrafish, analyzing differential gene expression across major brain cell types with sex-specific resolution. Cross-species comparisons with single-cell mouse and human AD datasets were performed through gene, pathway, and network-level analyses. Our analysis revealed sex-dimorphic patterns of mitochondrial dysfunction, where electron transport chain genes showed opposite tau-associated expression alterations between males and females across multiple cell types. The comprehensive single-cell characterization of the Tau P301L zebrafish supports the utility of this model for translational AD research and demonstrates that sex-specific mitochondrial responses represent cross-species conserved pathological mechanisms with important implications for personalized therapeutic strategies. Overall design: We used the Tg(TauP301L-dsRed) transgenic zebrafish line, which expresses human mutant tau (P301L) under the control of the HuC/D promoter, ensuring neuron-specific expression. This model recapitulates key features of human tauopathy, including tau hyperphosphorylation, neurofibrillary tangle formation, and progressive neurodegeneration. Wild-type AB zebrafish served as controls. We used two biological replicates for each condition (transgenic and wildtype control), with two technical replicates for each biological replicate. All zebrafish procedures were conducted under authorization from Luxembourg's Grand-Ducal Decrees (20 January 2016 and 26 January 2023) and adhered to European regulations (Directive 2010/63/EU). Wild-type AB and Tg(TauP301L-dsRed) zebrafish were maintained under standard conditions at 28.5 ± 0.5°C with a 14h light/10h dark cycle. Adult fish (6 months old) were housed at 4-6 fish per liter in 3.5L tanks with controlled water parameters (pH 7.5, conductivity 700-900 µS/cm). Fish were euthanized using buffered tricaine methanesulfonate (MS-222; 250 mg/L) for 15 minutes, and brains were dissected under stereomicroscopic guidance using sterilized tools, immediately flash-frozen in liquid nitrogen, and stored at -80°C until processing. Nuclei isolation was performed using the Minute Single Nucleus Isolation Kit (BN-020, Invent Biotechnologies) with modifications including addition of Protector RNase Inhibitor (3335399001, Roche) at 0.4U/µl before homogenization. Isolated nuclei from two zebrafish brains were pooled per condition, resuspended in PBS with 4% BSA and 0.2U/µl RNase inhibitor, filtered through 20µm filters (04-004-2335, CellTrics), centrifuged, and resuspended in PBS with 2% BSA and 0.2U/µl RNase inhibitor to achieve 1300-1700 nuclei/µl. Single-nuclei RNA sequencing libraries were prepared using the Chromium GEM-X Single Cell 3' Kit v4 (1000686, 10X Genomics) following the manufacturer's protocols with approximately 17,000 nuclei loaded per sample for GEM generation. Libraries were quantified using Qubit Fluorometer 4.0 (ThermoFisher Scientific), size distribution was assessed on Fragment Analyzer 5200 (Agilent), and equimolar pooled libraries were sequenced on NextSeq2000 (Illumina) in the Luxembourg Centre for Systems Biomedicine (LCSB) Genomics platform (RRID: SCR_021931).