TDP-43 dysregulation impairs cholesterol metabolism linked with myelination defects

Abstract

TDP-43 is a nuclear protein encoded by the TARDBP gene, which forms pathological aggregates in various neurodegenerative diseases, collectively known as TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These diseases are characterized by multiple pathological mechanisms, with disruptions in lipid regulatory pathways emerging as a critical factor. However, the role of TDP-43 in the regulation of the brain lipid homeostasis and the potential connection of TDP-43 dysfunction to myelin alterations in TDP-43 proteionopathies remain poorly understood, despite the fact that lipids, particularly cholesterol, comprise nearly 70% of myelin. To investigate the causal relationship between TDP-43 dysfunction and disruptions in brain cholesterol homeostasis, we conducted multi-omics analyses (lipidomics, transcriptomics, and functional splicing) on the frontal cortex from the Tardbp^M323K/M323K knock-in mouse model. Lipidomic analysis revealed alterations in lipid pathways related to membrane composition and lipid droplet accumulation, particularly affecting cholesterol-related species. We found higher lipid droplet accumulation in primary fibroblasts derived from these mice, as well as in the brain of the mutant mice. Similarly, the immunohistochemical detection of a lipid droplet marker was higher in the postmortem frontal cortex, gray matter, and white matter of FTLD-TDP patients compared to non-neurological controls. Transcriptomic analyses showed that TDP-43 pathology led to transcriptional dysregulation of genes essential for myelin production and maintenance. We identified impaired cholesterol metabolism, mainly through the downregulation of endogenous cholesterol synthesis, alongside upregulated cholesterol transport pathways, which we further replicated in FTLD-TDP patients transcriptomic datasets. Collectively, our findings suggest that TDP-43 dysfunction disrupts brain cholesterol homeostasis, potentially compromising myelin integrity.

Keywords: Cholesterol metabolism; Lipid droplets; Lipidomics; Myelin; TDP-43 proteinopathies; Transcriptomics.

Fig. 1

Reduction of myelin proteins and oligodendrocyte-related gene expression in Tardbp^M323K/M323K mice. a Representative images of PLP1 immunostaining in the frontal cortex of Tardbp^+/+ and Tardbp^M323K/M323K mice, with quantification of total fluorescence intensity (right). Scale bar: 100 µm. Experimental n per group: 4 Tardbp^+/+ and 4 Tardbp^M323K/M323K. b Western blot analysis of PLP1 protein levels in frontal cortex samples with quantification relative to wild-type at 3 months. Experimental n per group: 3 Tardbp^+/+ and 3 Tardbp^M323K/M323K of 3 months and 4 Tardbp^+/+ and 4 Tardbp^M323K/M323K of 12 months. c Relative expression of myelin- and oligodendrocyte-related genes (Plp1, Mbp, Qki, Myrf, Olig2) normalized to Hprt in frontal cortex samples from Tardbp^+/+ and Tardbp^M323K/M323K mice. Experimental n per group: 4 Tardbp^+/+ and 5 Tardbp.^M323K/M323K of 3 months. All are females. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01

Fig. 2

Tardbp altered lipid metabolism in the frontal cortex. a Partial least squares discriminant analysis (PLS-DA) of samples lipidome. b Volcano plot analysis on lipids. Red dots indicate significantly upregulated genes, blue dots indicate significantly downregulated genes, and gray dots indicate genes with no significant change. c Enrichment analysis of the frontal cortex in the “ranking mode” The gray vertical lines indicate the cut-off value of significant enrichments (q < 0.05). Bar colors are scaled with the enrichment (− log q-values). FDR: false-discovery rate. d Lipid Ontology (LION) enrichment analysis with upregulation in lipid droplets and cholesterol esters, together a downregulation in membrane components e Ratio of cholesteryl ester:free cholesterol:total glycerophospholipids and cholesteryl ester:total glycerophospholipids. Male mice at 9 months of age, n = 6 Tardbp^+/+ n = 6 Tardbp^M323K/M323K in lipidomics data. f Western blot analysis of SREBP2 protein levels in frontal cortex samples at 12 months of age, with quantification relative to wild-type (WT). Female mice, n = 4 Tardbp^+/+ n = 4 Tardbp^M323K/M323K. Data are presented as mean ± SEM. **p < 0.01

Fig. 3

Increased lipid droplet accumulation and PLIN2 expression in Tardbp^M323K/M323K mice and FTLD-TDP43 patients. a Representative images of lipid droplets in fibroblast cultures from Tardbp^+/+ and Tardbp^M323K/M323K mice under basal conditions and after oleic acid (O.A.) treatment. Cells were stained with Hoechst (nuclei, blue), BODIPY (lipid droplets, green), and PLIN2 (red). Merged images highlight co-localization. Quantification of lipid droplet number per cell (bottom). Scale bar = 20 µm. Experimental n per group: 4 Tardbp^+/+ and 4 Tardbp^M323K/M323K. b Flow cytometry analysis of lipid content in fibroblasts from Tardbp^+/+ and Tardbp^M323K/M323K mice, with quantification of the ratio of mean fluorescence intensity (MFI) normalized to wild-type (WT) (bottom). Experimental n per group: 5 Tardbp^+/+ and 6 Tardbp^M323K/M323K. c Representative images of Plin2 expression in frontal cortex sections from Tardbp^+/+ and Tardbp^M323K/M323K mice, with quantification (bottom). Experimental n per group: 5 Tardbp^+/+ females and 5 Tardbp^M323K/M323K females. Scale bar = 20 µm. d Representative images of PLIN2 expression in brain sections from control and FTLD-TDP43 patients. Sections were stained with DRAQ5 (nuclei, blue), IBA1 (microglia, green), and PLIN2 (red). Quantification of PLIN2 total intensity in gray and white matter (bottom). Scale bar = 20 µm. Experimental n per group: 6 controls and 6 FTLD-TDP43 patients. Data are presented as mean ± SEM. *p < 0.05

Fig. 4

PLIN2 immunoreactivity in the cerebellum of Tardbp^M323K/M323K mice as well as in the cerebellum and frontal cortex of FTLD-TDP43 and Pick’s patients. a Immunofluorescence for TDP-43 in the cerebellum from Tardbp^+/+ and Tardbp^M323K/M323K mice showed no significant change in nuclear localization of TDP-43 (n = 4 per group). b Immunofluorescence for PLIN2 in the cerebellum of these mice. c Representative immunohistochemistry for TDP-43 in the cerebellum of control and FTLD-TDP patients. d Immunofluorescence for PLIN2 in human control and FTLD-TDP cerebellums. Quantification reveals no differences in PLIN2 signal in FTLD-TDP samples with the control group (n = 4–7 per group). e Triple immunofluorescence staining for IBA1 (microglia), PLIN2, and DRAQ5 in human control, FTLD-TDP, and Pick’s disease brain samples. High-magnification (ZOOM) images show PLIN2 signals localized within microglia. Quantification of PLIN2 intensity relative to nuclear area (PLIN2/DRAQ5) reveals a significant increase in FTLD-TDP compared to control and Pick’s disease cases (n = 4–7 per group). Data are presented as mean ± SEM. p < 0.05, one-way ANOVA or unpaired t test as appropriate. Scale bars: 100 μm (A, overview), 20 μm (B–E, and A, insets)

Fig. 5

Transcriptomic and functional analysis of disrupted cholesterol metabolic processes in the frontal cortex of TDP-43 pathology. a Overrepresentation analysis (ORA) of the most significantly deregulated biological processes in the frontal cortex of 9-month-old male Tardbp^M323K/M323K mice, based on transcriptomic data. The size of the dots represents the number of genes involved in each pathway, while the color indicates the statistical significance level (adjusted p value). Experimental n per group: 4 Tardbp^+/+ and 4 Tardbp^M323K/M323K male mice. b Venn diagram showing the overlap of differentially expressed genes (DEGs) in the frontal cortex with lipid-related genes. The lipid gene list was obtained by merging genes annotated under the Gene Ontology (GO) terms “lipid metabolic process” (GO:0006629) and “lipid transport” (GO:0006869). c Protein–protein interaction (PPI) network of differentially expressed lipid-related genes. A total of 120 lipid-related genes were identified and classified into 3 key biological processes based on GO terms: membrane lipid metabolism (green), cholesterol metabolism (red), and myelination (blue). d ORA of the most significantly deregulated biological processes, considering only the 124 lipid-related DEGs, analyzed according to their fold change. e Venn diagram depicting the overlap between genes with at least one splicing event (FDR < 0.05, Inc level > 0.5) and genes associated with myelination or cholesterol metabolism. f Sashimi plots illustrating alternative splicing events in Dhcr7 and Lpcat3. g Comparative fold-change analysis of genes involved in cholesterol synthesis and transport between the frontal cortex of Tardbp^M323K/M323K mice and wild-type mice, as well as between FTLD Type A, B, or C patients and controls