TMEM106B core deposition associates with TDP-43 pathology and is increased in risk SNP carriers for frontotemporal dementia

Abstract

Genetic variation at the transmembrane protein 106B gene (TMEM106B) has been linked to risk of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) through an unknown mechanism. We found that presence of the TMEM106B rs3173615 protective genotype was associated with longer survival after symptom onset in a postmortem FTLD-TDP cohort, suggesting a slower disease course. The seminal discovery that filaments derived from TMEM106B is a common feature in aging and, across a range of neurodegenerative disorders, suggests that genetic variants in TMEM106B could modulate disease risk and progression through modulating TMEM106B aggregation. To explore this possibility and assess the pathological relevance of TMEM106B accumulation, we generated a new antibody targeting the TMEM106B filament core sequence. Analysis of postmortem samples revealed that the TMEM106B rs3173615 risk allele was associated with higher TMEM106B core accumulation in patients with FTLD-TDP. In contrast, minimal TMEM106B core deposition was detected in carriers of the protective allele. Although the abundance of monomeric full-length TMEM106B was unchanged, carriers of the protective genotype exhibited an increase in dimeric full-length TMEM106B. Increased TMEM106B core deposition was also associated with enhanced TDP-43 dysfunction, and interactome data suggested a role for TMEM106B core filaments in impaired RNA transport, local translation, and endolysosomal function in FTLD-TDP. Overall, these findings suggest that prevention of TMEM106B core accumulation is central to the mechanism by which the TMEM106B protective haplotype reduces disease risk and slows progression.

Fig. 1.. TMEM106B core antibody detects TMEM106B filaments and a glycosylated 29-kDa species.

(A) Schematic illustration of the TMEM106B filament structure solved by cryo-EM, with the epitope for the TMEM106B core antibody we developed colored in blue. The rs3173615 coding variant, with p.T185 as the reference sequence, is colored in red. Experimentally detected glycosylation (gly-) sites are also noted, with N and C termini (at residues 120 and 254, respectively) colored in yellow. (B) TMEM106B-positive filaments were detected in the sarkosyl-insoluble fraction from patients with FTLD-TDP and AD by iEM. Scale bars, 100 nm. (C) Sarkosyl-insoluble fractions from FTLD-TDP cases (n = 8) with cryo-EM confirmed TMEM106B filaments were evaluated by SDS– polyacrylamide gel electrophoresis (PAGE). Immunoblotting with the TMEM106B core antibody detected a 29-kDa species and full-length TMEM106B (FL) in some cases. (D) Sarkosyl-insoluble fractions from FTLD-TDP [n = 2, both also shown in (B) and (C)] and AD (n = 2) were incubated in the presence or absence of PNGase (peptide N-glycosidase F) to induce deglycosylation, followed by immunoblotting for TMEM106B to demonstrate that the 29-kDa TMEM106B species is glycosylated.

Fig. 2.. TMEM106B core deposition associates with risk haplotype.

(A) The sarkosyl-insoluble fraction was extracted from the frontal cortices of patients with FTLD-TDP (n = 292 total: 129 CC, 136 CG, and 27 GG) and separated by SDS-PAGE (top). In addition, the radioimmunoprecipitation assay (RIPA)–soluble fraction from the frontal cortices of FTLD-TDP patients (n = 16 total: 8 CC and 8 GG) was separated by SDS-PAGE using cold conditions to visualize both dimeric and monomeric forms of TMEM106B (bottom). Immunoblotting was performed using TMEM106B and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (RIPA-soluble fraction only) antibodies. (B) Quantification was performed on the 29-kDa TMEM106B-positive fragment (A, top). Data presented as means ± SEM, with statistical analysis performed using a one-way ANOVA with Dunn’s multiple comparisons (****P < 0.0001). (C) Quantification was performed on full-length TMEM106B dimer (left) and monomer (right), using GAPDH to control for protein loading [immunoblots presented in (A, bottom)]. Data presented as means ± SEM, with statistical analysis performed using a Student’s t test (**P < 0.01). ns, not significant; A.U., arbitrary units. (D) Schematic diagram of the TMEM106B protein illustrating the N-terminal domain, transmembrane domain (TM), and C-terminal fibril core. Location of peptide sequences identified by mass spectrometry are depicted. (E and F) Quantification of N-terminal (E) or C-terminal (F) peptides mapped to the TMEM106B protein demonstrate abundance in the sarkosyl-insoluble fraction of FTLD-TDP cases stratified by rs3173615 genotype. Fifty-four cases were processed for mass spectrometry. The N-terminal peptide was detected in 19 CC, 15 CG, and 6 GG cases and was not detected in 14 cases. The C-terminal core domain peptide was detected in all cases (n = 24 CC, 22 CG, and 8 GG). Statistical analysis performed using the Kruskal-Wallis test with Dunn’s multiple comparisons (*P < 0.05). (G and H) Regression analysis of N-terminal (G) or C-terminal (H) peptides with 29-kDa TMEM106B fragment quantification by immunoblot.

Fig. 3.. Insoluble TMEM106B interactome gives mechanistic insight into the role of TMEM106B deposition in disease pathogenesis for FTLD-TDP.

(A) Volcano plot of significantly enriched proteins that coimmunoprecipitated with TMEM106B from the sarkosyl-insoluble P3 fraction of nine FTLD-TDP CC/TT185 cases. Those with an adjusted P < 0.05 (cutoff marked by dashed line) are highlighted in red. (B and C) Top 10 GO Biological Process (B) and KEGG pathways (C) of significant hits in (A), sorted by fold enrichment. (D) STRING diagrams depicting proteins in the “endocytosis,”“ribosome,” and “pathways of neurodegeneration” KEGG pathways. Markov clustering (granularity/inflation parameter = 3) was used to visualize proteins with well-characterized functional relations in each pathway.