TDP-43 proteinopathy occurs independently of autophagic substrate accumulation and underlies nuclear defects in Niemann-Pick C disease

Abstract

Aims: Neuronal cytoplasmic inclusions of TAR-DNA binding protein of 43 kDa (TDP-43) are a pathological hallmark of diverse neurodegenerative disorders, yet the processes that mediate their formation and their functional significance remain incompletely understood. Both dysfunction in autophagy and neuroinflammation have been linked to TDP-43 mislocalisation. Here, we investigate TDP-43 proteinopathy in Niemann-Pick type C disease (NPC), an autosomal recessive lysosomal storage disease (LSD) distinguished by the accumulation of unesterified cholesterol within late endosomes and lysosomes. NPC is characterised by neurodegeneration, neuroinflammation and multifocal disruption of the autophagy pathway.

Methods: We utilised immunohistochemistry, confocal microscopy, electron microscopy and biochemical and gene expression studies to characterise TDP-43 pathology and autophagic substrate accumulation in Npc1-deficient mice.

Results: In the NPC brain, cytoplasmic TDP-43 mislocalisation was independent of autophagic substrate accumulation. These pathologies occurred in distinct neuronal subtypes, as brainstem cholinergic neurons were more susceptible to TDP-43 mislocalisation, whereas glutamatergic neurons exhibited hallmarks of autophagic dysfunction. Furthermore, TDP-43 mislocalisation did not co-localise with markers of stress granules or progress to ubiquitinated aggregates over months in vivo, indicating a stable, early stage in the aggregation process. Neither microgliosis nor neuroinflammation were sufficient to drive TDP-43 proteinopathy in the NPC brain. Notably, cytoplasmic TDP-43 co-localised with the nuclear import factor importin α, and TDP-43 mislocalised neurons demonstrated nuclear membrane abnormalities and disruption of nucleocytoplasmic transport.

Conclusion: Our findings highlight the relationship between LSDs and TDP-43 proteinopathy, define its functional importance in NPC by triggering nuclear dysfunction, and expand the spectrum of TDP-43 pathology in the diseased brain.

Keywords: Niemann-Pick type C; TDP-43; autophagy; lysosomal diseases; nucleocytoplasmic transport.

Figure 1.. Npc1 deficiency triggers age-dependent accumulation of swollen axons containing autophagic cargo

(a) Axonal spheroids in the brainstem, quantified from 4-5 fields, N=3 mice per genotype and age. (b) Electron micrographs of the brainstem taken from 16-week Npc1^flox/−, Syn-Cre⁺ mice. Top: An axonal spheroid surrounded by normal sized axons. Scale bar: 2 μm. Bottom: Higher magnification shows accumulation of vesicular cargo. Scale bar: 500 nm. (c) Brain sections from 16-week Npc1^flox/+, Syn-Cre⁺ and Npc1^flox/−, Syn-Cre⁺ mice were stained with the indicated markers and imaged by confocal microscopy. Scale bar: 10 μm. Data are shown as mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 by (a) one-way ANOVA with Tukey’s multiple comparisons (a) F=10.04 (top); F=566.1 (bottom)

Figure 2.. Independent accumulation of autophagic cargo and cytoplasmic TDP-43 mislocalisation

(a-c) The brainstem from 4-week Npc1^+/+ and Npc1^−/− mice was stained for the indicated markers and imaged by confocal microscopy. Scale bar: 10 μm. (d) Left: The percentage of TDP-43 mislocalised cells co-localised with importin α or p62. Right: The percentage of p62+ cells co-localised with cytoplasmic TDP-43. Twenty to thirty TDP-43+ or p62+ cells were quantified per mouse, N=3 mice. Data are shown as mean ± s.e.m.

Figure 3.. TDP-43 cytoplasmic mislocalisation persists in vivo for months, up to the end-stage in Npc1^−/− mice

(a-c) The brainstem from 11-week Npc1^+/+ and Npc1^−/− mice was stained with the indicated markers and imaged by confocal microscopy. Scale bar: 10 μm. (d-e) Left: The percentage of TDP-43 mislocalised cells co-localised with importin α or p62. Right: The percentage of p62+ cells co-localised with cytoplasmic TDP-43. Twenty to thirty TDP-43+ or p62+ cells were quantified per mouse, N=3 mice per genotype. (f) Top: The brainstem from 11-week Npc1^−/− mice was stained with the indicated markers and imaged by confocal microscopy. Scale bar: 5 μm. Bottom: The percentage of importin α+ or p62+ that were ChAT+. Fifteen to twenty-five cells were quantified per mouse, N=3 mice per genotype. (g) Left: The brainstem from 11-week Npc1^−/− mice was stained with the indicated markers and imaged by confocal microscopy. Scale bar: 5 μm. Right: The percentage of VGLUT1+ cells that were p62+ or importin α+. Fifteen to twenty-five cells were quantified per mouse, N=3 mice per genotype. (h) Brainstem from 11-week Npc1^−/− mice was stained with the indicated markers and imaged by confocal microscopy. Scale bar: 5 μm. Data are shown as mean ± s.e.m. **P ≤ 0.01 by (f-g) Student’s t-test (f) t=5.388 (g) t=7.429

Figure 4.. Progressive accumulation of autophagic cargo in Npc1-I1061T mice

(a-c) The brainstem from Npc1-I1061T mice at 3.7, 7 and 10-weeks of age was stained with the indicated markers and imaged by confocal microscopy. Scale bar: 10 μm. (d) The timeline summarises the age-dependent accumulation of cholesterol and autophagic substrates in I1061T mice. (e) Importin α+ cells in the brainstem, quantified from N=3 mice per genotype. Data are shown as mean ± s.e.m.

Figure 5.. Microgliosis and complement activation in NPC mice

(a) The brainstem from 4-week Npc1^+/+ and Npc1^−/− mice, 11-week WT and Npc1-I1061T mice, and 11-week Npc1^+/+ and Npc1^−/− mice was stained with Iba-1 and imaged by confocal microscopy. Scale bar: 10 μm. Right: Iba1+ cells per field, quantified from 4-5 fields, N=3 mice per genotype. (b) Relative expression of C1qa and C3 mRNA in brainstem by qPCR. N=4-5 mice per genotype. Data are shown as mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 by (a-b) one-way ANOVA with Tukey’s multiple comparisons (a) F=64.48; (b) F=40.57 (C1qa) and F=22.51 (C3)

Figure 6.. The immunoproteasome is induced in NPC mice

(a-b) Relative expression of immunoproteasome 20S core subunits (a) and alternative lid (b) was determined in the cerebellum (CB) and brainstem (BS) of 12-week WT and Npc1-I1061T mice by qPCR. N=5 mice per genotype. (c) The relative abundance of immunoproteasome subunits in the cerebellum of 12-week WT and Npc1-I1061T mice was determined by western blot. N=3 mice per genotype. Quantified at right. (d) Relative expression of immunoproteasome, alternative lid and constitutive proteasome subunits was determined by qPCR in the brainstem of 11-week Npc1^+/+ and Npc1^−/− mice. N=4 mice per genotype. Data are shown as mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001 by (a-d) Student’s t-test (a) t=5.833 (CB PSMB8); t=6.27 (BS PSMB8); t=4.23 (CB PSMB9); t=3.088 (BS PSMB9); t=3.864 (CB PSMB10); t=1.083 (BS PSMB10) (b) t=5.893 (CB PSME1); t=2.556 (BS PSME1); t=2.853 (CB PSME2); t=2.077 (BS PSME2) (c) t=8.039 (PSMB8); t=4.454 (PSMB10) (d) t=8.351 (PSMB8); t=8.067 (PSMB9); t=2.077 (PSMB10); t=8.281 (PSME1); t=3.216 (PSME2); t=2.05 (PSMB5)

Figure 7.. Importin α mislocalisation marks neurons with disrupted poly(A) RNA export and nuclear membrane morphology

(a) The brainstem from 11-week Npc1^−/− mice was stained with p62 or importin α. Poly(A) RNA was detected by RNA fluorescence in situ hybridization (FISH) with an oligo(dT) probe and imaged by confocal microscopy. Scale bar: 5 μm. Poly(A) RNA N:C ratio quantified from 20-30 p62+ or importin α+ cells per mouse, with N=6 mice. (b) The brainstem from 11-week Npc1^−/− mice was stained with the indicated markers and imaged by confocal microscopy. Scale bar: 5 μm. Circularity of Lamin B1 staining was quantified from 20-30 p62+ or importin α+ cells per mouse, with N=6 mice. Data are shown as mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, by (a-b) Student’s t-test (a) t=2.603 (b) t=4.53