Tau deletion exacerbates the phenotype of Niemann-Pick type C mice and implicates autophagy in pathogenesis

Abstract

Hyperphosphorylation and aggregation of the microtubule-binding protein tau characterize a diverse array of neurodegenerative disorders. Most of these lack mutations in the encoding MAPT gene, and the role of tau in disease pathogenesis remains controversial. Among these tauopathies is Niemann-Pick type C disease (NPC), a lysosomal storage disorder characterized by progressive neurodegeneration and premature death, most often caused by an inherited deficiency in the intracellular lipid trafficking protein NPC1. To determine the extent to which tau affects NPC pathogenesis, we generated Npc1-/- mice deficient in tau. Unexpectedly, NPC1/tau double null mutants are generated in markedly smaller litters, exhibit an enhanced systemic phenotype and die significantly earlier than NPC1 single null mutants. As autophagy is up-regulated in NPC and protein degradation through this pathway depends on movement along microtubules, we knocked down MAPT expression in NPC1-deficient human fibroblasts and examined effects on this pathway. We show that an acute reduction of tau expression in a cellular model of NPC decreases induction and flux through the autophagic pathway. Our data establish that MAPT deletion exacerbates the NPC phenotype through a mechanism independent of tau protein aggregation and identifies a critical role for tau in the regulation of autophagy in NPC1-deficient cells.

Figure 1.

Mapt deletion decreases litter size of NPC1-deficient mice. (A) Litter sizes (mean±SEM) generated by Npc1+/− mice, stratified by parental Mapt genotype. Npc1+/− and Mapt−/− breedings generated fewer pups on average (P = 0.0002 by ANOVA with Newman–Keul's multiple comparison test) than mice with at least one functional Mapt allele (n, number of litters). (B) Npc1 null allele distribution in pups generated by NPC1 haploinsufficient mice, stratified by parental Mapt genotype. The percentage of NPC1/tau double null pups generated by Npc1+/−; Mapt−/− breeding was not significantly different from expected (P = 0.2 by χ² analysis) (n, number of mice). (C) Litter size (mean±SEM) of wild-type (black bar) and Mapt−/− (white bar) mice. N, number of litters; n.s., not significant (P > 0.05) by unpaired Student's t-test.

Figure 2.

Decreased weight and early death of NPC1/tau double null mutant mice. (A) Weight (mean±SEM) of 5-week-old female (left panel) and male (right panel) mice. A significant difference between NPC1 single nulls (Npc1−/− and Mapt+/+), and NPC1 nulls haploinsufficient or deficient in tau was observed in females, whereas all Npc1−/− males were observed to have decreased weights when compared with wild-type and Mapt−/− controls. *, ** and *** signify P-values of <0.05, <0.01 and <0.001, respectively, by ANOVA with Newman–Keul's multiple comparison test. (B) Survival curve. Npc1−/−; Mapt +/− (green line) and Npc1−/−; Mapt−/− (blue line) were significantly different (P < 0.0001 by log-rank test) from wild-type, Mapt null (black line) and Npc1−/−; Mapt +/+ (red line) mice. (C) Age at death for Npc1−/−; Mapt +/+ (red triangles), Npc1−/−; Mapt +/− (blue squares) and Npc1−/−; Mapt−/− (black triangles) mice.

Figure 3.

Systemic phenotypes associated with NPC1/tau double null mice. (A) Double null mice (Npc1−/−; Mapt−/−) demonstrate shortened snout and kyphosis when compared with control mice (Npc1+/+; Mapt−/−). (B) Still images of video reveal abnormal gait of double null mice (Npc1−/−; Mapt−/−, bottom panel). (C) Footprint analysis demonstrates toe walking gait of 4-week-old double null mice (Npc1−/−; Mapt−/−) when compared with NPC1 single null (Npc1−/−; Mapt +/+) and control mice (Npc1+/+; Mapt−/−) of the same age and sex. (D) Nearly, all NPC1/tau double null males that survive 5 weeks or longer develop penile prolapse, whereas NPC1 single null males and WT males rarely exhibit this phenotype (P = 0.038 by ANOVA).

Figure 4.

LC3 and p62 expressions in NPC1 and NPC1/tau null mice. (A) Immunohistochemical staining demonstrates frequent LC3-positive vacuoles in liver of 10-week-old double null mutant (Npc1−/−; Mapt−/−) when compared with single null mutant (Npc1−/−; Mapt +/+) or wild-type mice (original magnification, 1000×). (B) Liver lysates from 10-week-old mice were probed by western blot for expression of LC3. Both double null (Npc1−/−; Mapt−/−) and NPC1 single null mice (Npc1−/−; Mapt +/+) demonstrate increased levels of LC3-II compared WITH controls. GAPDH serves as a loading control. (C–E) Lysates from the telencephalon (C) and cerebellum (D and E) of NPC1 single null, and NPC1/tau double null mice demonstrate increased levels of LC3-II and SDS-insoluble p62 compared with controls.

Figure 5.

Tau knockdown decreases autophagic induction and flux in NPC1-deficient cells. (A) Relative MAPT mRNA levels (mean±SEM) 72 h post-treatment with non-targeted (black bars) or MAPT siRNAs (white bars), as determined by qPCR. Targeted siRNAs significantly reduced endogenous MAPT mRNA expression in control and NPC1-deficient primary human fibroblasts by unpaired Student's t-test. (B) Human fibroblast lysates from control (lanes 1 and 2) and NPC1-deficient (lanes 3 and 4) cells were analyzed by western blot for the expression of LC3 (top) and GAPDH (bottom) 72 h post-treatment with non-targeted (lanes 1 and 3) or MAPT siRNAs (lanes 2 and 4). (C) Degradation of long-lived proteins in control (left panel) and NPC1-deficient human fibroblasts (right panel), following transfection with non-targeted (black bars) or MAPT siRNAs (white bars). Data (mean±SEM) are reported relative to non-targeted siRNA-transfected cells at 72 h. Relative proteolysis is significantly decreased in NPC1-deficient cells (P = 0.016 by unpaired Student's t-test), but not in controls (P > 0.05).

Figure 6.

The ubiquitin–proteasome pathway is intact in NPC1-deficient animals. (A) Activity of the 20S proteasome, assayed in liver lysates of 10-week-old mice (mean±SEM). P > 0.05 by ANOVA. (B) Western blot analyses of liver lysates from 10-week-old mice demonstrate equivalent expression of hsp40, stress-inducible hsp70, hsp90 and 20S proteasome subunits. GAPDH serves as a loading control.

Figure 7.

Model of the relationship between NPC1 deficiency, autophagy and tau. Functional deficiency of NPC1 impairs intracellular lipid trafficking and leads to the accumulation of unesterified cholesterol, sphingolipids and complex gangliosides in the late endosomal/lysosomal network. Pathological changes in NPC include progressive neurodegeneration associated with the accumulation of hyperphosphorylated tau (depicted at right). In NPC model systems and patient fibroblasts, defects in lipid transport stimulate a Beclin-1-dependent increase of basal autophagy, which we propose exerts a protective effect by facilitating the re-use of critical cellular components. The deletion of endogenous tau abrogates this response and exacerbates the NPC phenotype. We speculate that similar effects may be triggered by tau hyperphosphorylation, as occurs in NPC patients.