Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are characterized by cytoplasmic protein aggregates in the brain and spinal cord that include TAR-DNA binding protein 43 (TDP-43). TDP-43 is normally localized in the nucleus with roles in the regulation of gene expression, and pathological cytoplasmic aggregates are associated with depletion of nuclear protein. Here, we generated transgenic mice expressing human TDP-43 with a defective nuclear localization signal in the forebrain (hTDP-43-ΔNLS), and compared them with mice expressing WT hTDP-43 (hTDP-43-WT) to determine the effects of mislocalized cytoplasmic TDP-43 on neuronal viability. Expression of either hTDP-43-ΔNLS or hTDP-43-WT led to neuron loss in selectively vulnerable forebrain regions, corticospinal tract degeneration, and motor spasticity recapitulating key aspects of FTLD and primary lateral sclerosis. Only rare cytoplasmic phosphorylated and ubiquitinated TDP-43 inclusions were seen in hTDP-43-ΔNLS mice, suggesting that cytoplasmic inclusions were not required to induce neuronal death. Instead, neurodegeneration in hTDP-43 and hTDP-43-ΔNLS-expressing neurons was accompanied by a dramatic downregulation of the endogenous mouse TDP-43. Moreover, mice expressing hTDP-43-ΔNLS exhibited profound changes in gene expression in cortical neurons. Our data suggest that perturbation of endogenous nuclear TDP-43 results in loss of normal TDP-43 function(s) and gene regulatory pathways, culminating in degeneration of selectively vulnerable affected neurons.

Figure 1. Generation and biochemical characterization of inducible TDP-43 Tg mice.

(A) Generation of conditional Tg mice. Camk2a-tTA mice were crossed with tetO-TDP-WT or tetO-TDP-ΔNLS mice. In bigenic mice, Dox inhibits tTA binding to the tetracycline-responsive promoter element (tetO), repressing hTDP-43 expression. (B) Dox was removed at weaning (P28) followed by analysis at the indicated time points. (C) Expression of hTDP-43 in Tg mice. Immunoblot of hTDP-43 or total TDP-43 (h+mTDP-43) in cortical RIPA extracts (1 month off Dox) of nTg, tTA, tTA/TDP-WT (WT4, WT8, WT5 and WT12), and tTA/TDP-ΔNLS mice (ΔNLS4 and ΔNLS19) showed variable expression of hTDP-43. GAPDH is a loading control. Fold expression relative to nTg and tTA control mice is shown, as described in Methods. (D) Regional expression of hTDP-43. RIPA extracts from different brain regions of tTA/TDP-WT or tTA/TDP-ΔNLS mice (1 month off Dox) were immunoblotted for hTDP-43, h+m TDP-43, and GAPDH. Ctx, cerebral cortex; Hp, hippocampus; OB, olfactory bulb; Cb, cerebellum; Rest, brain stem plus subcortical gray regions; SC, spinal cord. (E) Low levels of insoluble hTDP-43. Sequential cortical extracts from tTA/TDP-WT and tTA/TDP-ΔNLS mice of increasing time off Dox were immunoblotted for hTDP-43 showing low levels of TDP-43 in urea fractions. Urea lysates were loaded at 4× relative to RIPA lysates so that the low level of insoluble TDP-43 could be seen. R, RIPA; U, urea. n = at least 3 animals per group analyzed in C–E.

Figure 2. Neuron loss and cerebral atrophy in tTA/TDP-WT mice.

(A–L) Expression of hTDP-43 and time-dependent cell loss in tTA/TDP-WT12 mice. (A–D) H&E sections of nTg (A) and tTA/TDP-WT12 (B–D) brain revealed time-dependent hippocampal neuron loss in bigenic mice. Note the loss of DG neurons as indicated by arrowheads. (E–L) Nuclear expression of hTDP-43. IHC for hTDP-43 (E–H) and h+mTDP-43 (I–L) showed mosaic nuclear expression of hTDP-43 in tTA/TDP-WT12 and a severe thinning of the DG cell layer after 3 to 6 months off Dox. Insets in E–L display higher magnification of DG neurons. Scale bars: 500 μm (A–D); 200 μm (E–L). (M) Loss of DG neurons in tTA/TDP-WT12 mice. Quantification of DG neurons shows progressive neuron loss in tTA/TDP-WT12 mice. (N) Time-dependent brain atrophy in tTA/TDP-WT12 mice. There was a progressive reduction in total brain weight in tTA/TDP-WT12 mice. Data in M and N represent mean ± SEM (n = 3–4 per group). *Significantly different from nTg (P < 0.001); ^###significantly different from 1 month off Dox (P < 0.001); ^#significantly different from 1 month off Dox (P < 0.05); ^‡significantly different from 3 months off Dox (P < 0.05).

Figure 3. Cytoplasmic hTDP-43 expression and progressive neuronal loss in tTA/TDP-ΔNLS mice.

(A–D) Progressive neuron loss in tTA/TDP-ΔNLS4 mice. Hippocampal H&E sections of nTg (A) and tTA/TDP-ΔNLS (B) mice show a progressive DG neuron loss evident from 1 month off Dox (arrowhead highlights DG degeneration). (E–J) Cytoplasmic TDP-43 expression in tTA/TDP-ΔNLS hippocampus. IHC for hTDP-43 (E–G) or h+m TDP-43 (H–J) was performed on hippocampal sections from bigenic mice 1 week off Dox showing predominantly cytoplasmic expression of TDP-43. Insets show nTg mice with no hTDP-43 expression and nuclear h+mTDP-43 expression. Scale bars: 500 μm; (A–D, E and H); 200 μm (F, G, I, and J). (K) Loss of DG neurons in tTA/TDP-ΔNLS4 mice. Quantification of DG neurons shows progressive neuronal loss in tTA/TDP-ΔNLS mice. Data shown represent mean ± SEM (n = 3–4 per group). *Significantly different from nTg (P < 0.001). (L) Immunoblot analysis of enriched cytoplasmic (C) and nuclear (N) fractions isolated from cortical extracts of monogenic tTA and 2-month-old bigenic tTA/TDP-ΔNLS mice 10 days off Dox showing the presence of hTDP-43 in both fractions in tTA/TDP-ΔNLS. Note endogenous mTDP-43 is predominately nuclear. Nuclear hnRNP A2/B1 and cytoplasmic HSP90 indicated that the fractions are well separated.

Figure 4. TDP-43 aggregates are phosphorylated and ubiquitinated in tTA/TDP-ΔNLS mice.

(A–H) TDP-43 aggregates in cortex and hippocampus of tTA/TDP-ΔNLS mice. IHC of cortex from tTA/TDP-ΔNLS4 (A) and tTA/TDP-ΔNLS19 mice (B) at 1 month off Dox with p409/410 TDP-43 antibody shows relatively few aggregates in tTA/TDP-ΔNLS4 mice and higher number of inclusions in tTA/TDP-ΔNLS19 mice. Inclusions in the DG (C–E) and cortex (F–H) of tTA/TDP-ΔNLS19 mice 1 week off DOX were immunopositive for p409/410 TDP-43 (C and F), p403/404 TDP-43 (D and G), and ubiquitin (Ub) (E and H) antibodies. (I–N) Colocalization of pathologic epitopes in TDP-43 aggregates. IF staining with antibodies to ubiquitin (green) and p409/410 TDP-43 (red) demonstrated extensive colocalization within pathological cytoplasmic aggregates (arrows) in cortex (I–N) from tTA/TDP-ΔNLS19 mice 1 week off Dox. Scale bars: 50 μm (A and B); 20 μm (C–E).

Figure 5. Motor dysfunction and CST degeneration in tTA/TDP mice.

(A) Prevalence of abnormal limb-clasping motor phenotype. tTA/TDP-ΔNLS4 mice showed an early and time-dependent increase in abnormal clasping behavior, with all mice showing motor impairment within 3 weeks off Dox. A lower proportion of tTA/hTDP-WT mice (aggregate data for all WT lines) showed motor impairment only after several months off Dox. This clasping behavior corresponds to spasticity that occurs in PLS and ALS. (B) Axonal loss and gliosis of the CST after 1 month off Dox. IHC analysis of the cervical spinal cord using antibodies against phosphorylated NF-H (top row: RMO217) and phosphorylated NF-H+M (second row: TA51) showed loss of axons in the CST (ventral portion of the dorsal columns, top rows). Higher-power view of this region stained with a rabbit anti-NFL antibody in nTg mice showed numerous puncta corresponding to axons in cross-section, which is reduced in tTA/TDP-ΔNLS4 mice (third row). GFAP IHC demonstrated moderate gliosis in tTA-TDP-WT (both WT5 and WT12) and severe gliosis in tTA/hTDP-ΔNLS4 mice. H&E sections of the proximal and distal forelimb and hind limb showed no evidence of atrophy or grouping. Representative sections of biceps are shown. Scale bars: 100 μm (top 2 rows); 10 μm (third row); 50 μm (bottom 2 rows).

Figure 6. Downregulation of endogenous nuclear mTDP-43 in neurons overexpressing cytoplasmic or nuclear hTDP-43.

(A–I) hTDP-43 downregulates mTDP-43. IF for hTDP-43 (green) or mTDP-43 (red) in DG of tTA/TDP-WT12 (A–C) or tTA/TDP-ΔNLS4 (D–F) 1 month off Dox showed neurons expressing hTDP-43 with loss of mTDP-43 staining (insets: higher magnification). (G–I) IF for hTDP-43 (red) or mTDP-43 (green) of cortex from tTA/TDP-WT12 mice 1 month off Dox showed loss of mTDP-43 expression in neurons expressing hTDP-43 (arrows). (J) Intranuclear mTDP-43 aggregates. IHC of tTA/TDP-ΔNLS19 cortex for mTDP-43 showed rare intranuclear aggregates (insets: higher magnification of boxed regions). Note the absence of mTDP-43 in neurons with aggregates (*) compared with a cell with reduced (**) or normal (***) mTDP-43 expression. Scale bars: 200 μm (A–F); 20 μm (G–I); 50 μm (J). (K) Reduced mTDP-43 protein in tTA/TDP mice. Immunoblot for hTDP-43, mTDP-43, and h+mTDP-43 of cortical RIPA extracts from tTA/TDP-WT12 or tTA/TDP-ΔNLS4 at various times off Dox showed increased hTDP-43 and reduced mTDP-43 protein relative to nTg (–/–) and tetO-TDP monogenic (+/–) mice. GAPDH is a loading control. (L) Quantification of immunostained sections of DG from tTA/TDP-WT12, tTA/TDP-ΔNLS4 mice, and control nTg and monogenic (tTA only or tetO-TDP only) mice (n = 29). The percentage of neurons with inclusions (green, p409/410 stained sections), percentage of neurons with reduced mTDP-43 expression (red, anti-mTDP43 stained sections) and percentage of neuronal degeneration (blue, H&E sections as in Figures 2 and 3) are shown as mean ± SEM versus time off Dox (t = 0 represents control mice).

Figure 7. Analysis of global mRNA expression.

(A) Principal component (PC) analysis revealed a distinct global gene expression signature for tTA/TDP-ΔNLS (tTA/TDP-ΔNLS4, blue, n = 5), while other groups (nTg n = 8, tTA/TDP-WT n = 4, and monogenic ΔNLS n = 4) were relatively admixed. Data shown as PC1 versus PC2 for each individual mouse. (B) Hierarchical cluster analysis of the genes differentially expressed between tTA/TDP-ΔNLS and nTg mice (FDR < 0.05, with absolute value of FC > 2, shown here, n = 197 genes) corroborated PC analysis. Genes dysregulated in tTA/TDP-ΔNLS mice were evenly split between those downregulated (blue) and upregulated (red). Columns on heat map correspond to individual animals designated as follows: tTA/TDP-ΔNLS (blue), nTg (green), tTA/TDP-WT (purple), and monogenic ΔNLS (red). Rows correspond to individual probe sets, with blue indicating lower, gray indicating intermediate, and red indicating higher expression. (C) Biological pathway analysis of genes dysregulated in tTA/TDP-ΔNLS mice revealed many enriched pathways. The top 5 enrichment scores (negative log₁₀ χ² P value) are shown (higher score indicates greater enrichment). (D) Within the second-most-enriched pathway (macromolecular complex assembly, blue bar in C), most of the genes were involved in DNA-protein complex assembly. (E) Microarray and qRT-PCR results showed good concordance (r² of 0.7959, and no discordant directions of change), as shown by plotting log₂ FC values for microarray data (x axis) versus qRT-PCR results (y axis). The 6 genes that differed significantly between tTA/TDP-ΔNLS and nTg littermates (red squares) and 8 genes that did not differ significantly between groups (black circles) are shown (see Table 2). A negative value denotes lower expression in the tTA/TDP-ΔNLS mice.