Gottfried, I. | bioRxiv Science

bioRxiv2025

Super-Resolved Spatial Transcriptomics Reveals Early Changes in RNA Localization in the 5xFAD Hippocampus

Cell-type-specific changes in gene expression and RNA localization are hallmarks of Alzheimers disease (AD) and other neurodegenerative disorders, yet spatial dysregulation in early disease stages remains poorly defined. Here, we applied Expansion Sequencing (ExSeq) to map the spatial distribution of 101 genes at super-resolution in the hippocampus of 4-week-old 5xFAD and wild-type (WT) mice, prior to overt pathology. We uncovered early alterations in RNA spatial organization and gene expression, including 23 genes showing altered localization without changes in abundance in the 5xFAD hippocampus. Using spatial expression analysis and single-cell neighborhood analysis, we identified cell-type- and region-specific molecular programs associated with synaptic function, neuroinflammation, and metabolic stress that differed between 5xFAD and WT mice. Spatial RNA velocity further revealed state differences influenced by local cell to cell interactions. Together, these results suggest that RNA positioning and transcriptional programs are perturbed at early disease stages. Finally, we provide the full super-resolution ExSeq dataset as an open resource for spatial and cell-type-specific analyses in early Alzheimers disease research. HighlightsO_LISuper-resolved transcriptomic profiling of the hippocampus at early disease stages C_LIO_LIIdentification of 23 genes with altered spatial localization without changes in abundance C_LIO_LIEarly alterations in single-cell neighborhood organization in the 5xFAD hippocampus C_LIO_LISpatial RNA velocity reveals cell-type-specific cell state differences shaped by cell-cell proximity C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/678295v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1b46cf2org.highwire.dtl.DTLVardef@5cc55eorg.highwire.dtl.DTLVardef@a1ade4org.highwire.dtl.DTLVardef@9f833e_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Biochemical and neurophysiological effects of deficiency of the mitochondrial import protein TIMM50

TIMM50, an essential TIM23 complex subunit, is suggested to facilitate the import of [~]60% of the mitochondrial proteome. In this study, we characterized a TIMM50 disease causing mutation in human fibroblasts and noted significant decreases in TIM23 core protein levels (TIMM50, TIMM17A/B, and TIMM23). Strikingly, TIMM50 deficiency had no impact on the steady state levels of most of its putative substrates, suggesting that even low levels of a functional TIM23 complex are sufficient to maintain the majority of TIM23 complex-dependent mitochondrial proteome. As TIMM50 mutations have been linked to severe neurological phenotypes, we aimed to characterize TIMM50 defects in manipulated mammalian neurons. TIMM50 knockdown in mouse neurons had a minor effect on the steady state level of most of the mitochondrial proteome, supporting the results observed in patient fibroblasts. Amongst the few affected TIM23 substrates, a decrease in the steady state level of components of the intricate oxidative phosphorylation and mitochondrial ribosome complexes was evident. This led to declined respiration rates in fibroblasts and neurons, reduced cellular ATP levels and defective mitochondrial trafficking in neuronal processes, possibly contributing to the developmental defects observed in patients with TIMM50 disease. Finally, increased electrical activity was observed in TIMM50 deficient mice neuronal cells, which correlated with reduced levels of KCNJ10 and KCNA2 plasma membrane potassium channels, likely underlying the patients epileptic phenotype.

biochemistry↗