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. 2012 Jan;122(1):107-18.
doi: 10.1172/JCI59130. Epub 2011 Dec 12.

Mutant TDP-43 in motor neurons promotes the onset and progression of ALS in rats

Cao Huang  1 , Jianbin Tong, Fangfang Bi, Hongxia Zhou, Xu-Gang Xia
Affiliations

Mutant TDP-43 in motor neurons promotes the onset and progression of ALS in rats

Cao Huang et al. J Clin Invest. 2012 Jan.

Abstract

Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron degeneration, which ultimately leads to paralysis and death. Mutation of TAR DNA binding protein 43 (TDP-43) has been linked to the development of an inherited form of ALS. Existing TDP-43 transgenic animals develop a limited loss of motor neurons and therefore do not faithfully reproduce the core phenotype of ALS. Here, we report the creation of multiple lines of transgenic rats in which expression of ALS-associated mutant human TDP-43 is restricted to either motor neurons or other types of neurons and skeletal muscle and can be switched on and off. All of these rats developed progressive paralysis reminiscent of ALS when the transgene was switched on. Rats expressing mutant TDP-43 in motor neurons alone lost more spinal motor neurons than rats expressing the disease gene in varying neurons and muscle cells, although these rats all developed remarkable denervation atrophy of skeletal muscles. Intriguingly, progression of the disease was halted after transgene expression was switched off; in rats with limited loss of motor neurons, we observed a dramatic recovery of motor function, but in rats with profound loss of motor neurons, we only observed a moderate recovery of motor function. Our finding suggests that mutant TDP-43 in motor neurons is sufficient to promote the onset and progression of ALS and that motor neuron degeneration is partially reversible, at least in mutant TDP-43 transgenic rats.

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Figures

Figure 1
Figure 1. Restricted overexpression of mutant human TDP-43 (hTDP-43) in neurons is achieved in rats.
(A) Immunoblotting showed that the hTDP-43 transgene was expressed in the spinal cord (SP) and hippocampus (HP) of NEF-tTA/TRE–TDP-43M337V double-transgenic rats soon after Dox was withdrawn (Dox–). The rats were deprived of Dox at the age of 60 days. Equal loading was confirmed by probing the same membrane with an antibody to GAPDH. (B–J) Human TDP-43 immunoreactivity was examined in the spinal cord (B–D), cortex (E–H), and hippocampus (I and J) of NEF-tTA/TRE–TDP-43M337V double-transgenic (C, D, and G–J) and NEF-tTA single-transgenic (B, E, and F) rats. Coronal sections of neocortex were counterstained lightly with hematoxylin to display the nuclei (E–H). (K–P) Double-label fluorescence staining revealed that human TDP-43 (green, K, M, N, and P) was colocalized with the neuronal marker NeuN (red, L and M), but was not colocalized with the astrocyte marker GFAP (red: O and P) in the cortex. Rats were examined 10 days after Dox withdrawal. Scale bars: 200 μm (B, C, and I); 100 μm (E and G); 20 μm (D, F, H, and J–P).
Figure 2
Figure 2. Overexpression of mutant TDP-43 in neurons causes progressive paralysis in rats.
(A) The grip strength of forepaws (f) and hind paws (h) was measured for NEF-tTA/TRE–TDP-43M337V double-transgenic (M337V) and NEF-tTA single-transgenic rats. Data are means ± SD (n = 7). (B) Open-field assay revealed an unrecoverable reduction of mobility in M337V rats after TDP-43 transgene was turned on. Data are means ± SD (n = 7). (C) M337V rats rapidly developed paralysis after the transgene was turned on. Within 2 to 3 days, disease progressed from paralysis stage to end stages. (D–I) Cresyl violet staining revealed the structure of frontal cortex (D and E), hippocampus (F and G), and the ventral horn of the lumbar spinal cord (H and I). The tissues were taken from M337V rats at disease end stages (E, G, and I) or from NEF-tTA rats at matched ages (D, F, and H). (J and K) Stereological cell counting revealed the number of motor neurons (>25 μm in diameter) in lumbar cords (L3–L5) and revealed the number of neurons in dentate gyrus. Data are means ± SD (n = 5). *P < 0.05. All rats used were females and were constantly given Dox in drinking water (50 μg/ml) until they were 60 days old. Scale bars: 100 μm (D–E, H, and I); 200 μm (F and G).
Figure 3
Figure 3. Motor units are remodeled in diseased rats after mutant TDP-43 is removed.
(A–C) H&E staining of gastrocnemius muscle revealed normal structure in NEF-tTA transgenic rats (A), grouped atrophy (arrows) in NEF-tTA/TRE–TDP-43M337V double-transgenic rats at disease end stages (B, M337V), and regenerated muscle cells of varied sizes in diseased M337V rats after Dox treatment (C, M337V + Dox). (D–F) Histochemistry for nonspecific esterase revealed accumulated muscle fibers of varied sizes, but with similar staining (arrowheads), in M337V rats after Dox treatment. (G–I) Staining for ATPase (pH 4.6) revealed regenerated muscle cells of varied sizes in M337V rats after Dox treatment (I). (J–O) Toluidine blue staining revealed the structure of L3 ventral (J–L) and dorsal (M–O) roots. Scale bars: 30 μm. (P) Myofibers with or without centered nucleus were quantified on the cross sections of gastrocnemius stained with H&E. (Q) The cross areas of myofibers were quantified with ImageJ on the cross sections of gastrocnemius stained for ATPase. Quantification was done on 3 photos of the cross section of gastrocnemius, and each group contained 4 rats (P and Q). Data are means ± SD (n = 4). (R) Unbiased cell counting revealed the number of motor neurons (>25 μm) in the lumbar cords (L3–L5). Data are means ± SD (n = 7). *P < 0.05. All rats were deprived of Dox to induce disease at 60 days of age. Dox-untreated rats were examined at disease end stages, and Dox-treated rats were examined 2 months after Dox treatment (Q and R).
Figure 4
Figure 4. Suppressing mutant TDP-43 expression results in partial recovery of motor function in rats.
(A) Immunoblotting revealed that Dox treatment (Dox+) suppressed the expression of mutant TDP-43 transgene (hTDP-43) as early as 5 days after Dox was administered. Equal loading was probed with an antibody to GAPDH. Tissues: FB, forebrain; SM, skeletal muscle. (B–D) Representative photographs show a M337V rat at paralysis stage (B) and at 10 days (C) and 2 months (D) after Dox treatment. (E) Open-field assay revealed a quick recovery of mobility in M337V rats after the transgene was turned off by Dox treatment (Dox+). (F) The grip strength of hind paws was partially recovered in M337V rats after the transgene was turned off. NEF-tTA denotes NEF-tTA single-transgenic rats, and M337V denotes NEF-tTA/TRE–TDP-43M337V double-transgenic rats. Data are means ± SD (n = 7).
Figure 5
Figure 5. Restricted overexpression of mutant TDP-43 in motor neurons is achieved in transgenic rats.
(A–C) X-gal staining revealed that the positive cells were restricted to the ventral horns of the lumbar cord in ChAT–tTA-9/TRE-LacZ double-transgenic (B and C), but not in TRE-LacZ single-transgenic (A), rats. Cross sections of lumbar cords were first stained with X-gal and then were counterstained with fast red. (D and E) Immunohistochemistry revealed that human TDP-43 immunoreactivity was restricted in large cells in the ventral horns of the lumbar cord. ChAT–tTA-9/TRE–TDP-43M337V transgenic rat at 70 days old was examined for human TDP-43 expression at 10 days after Dox withdrawal. (F–L) Double-label immunofluorescence staining revealed that human TDP-43 (red, F, G, I, J, and L) was not colocalized with APC (a marker of oligodendrocytes; F, green) or GFAP (a marker of astrocytes; H and I, green), but was colocalized with ChAT (a marker of motor neurons; K and L, green). Scale bars: 100 μm (A, B, and D); 50 μm (C); 20 μm (E–L).
Figure 6
Figure 6. Restricted overexpression of mutant TDP-43 in motor neurons results in progressive paralysis and substantial motor neuron death.
(A) A diagram shows the course of gene induction and disease progression. ChAT–tTA-9/TRE–TDP-43M337V double-transgenic (M337V) rats were deprived of Dox (Dox–) at 60 days of age. (B) Immunoblotting revealed induction of mutant TDP-43 transgene (hTDP-43) after Dox withdrawal (Dox–). (C) Open-field assay measured mobility within 20 minutes. ChAT-tTA denotes ChAT–tTA-9 (line 9) transgenic rats. (D) The grip strength of 2 hind paws was measured daily. Data are means ± SD (C and D, n = 10). (E and F) Graphs show the probability of disease onset and rat mortality. Rats were counted at death when they reached disease end stages. 10 rats of equal sex composition were assessed in E and F. (G) Stereological cell counting revealed a profound loss of motor neurons (>25 μm in diameter) in the L3–L5 cords of M337V rats at end stages as compared with ChAT-tTA rats at matched ages. Data are means ± SD (n = 8). *P < 0.01. (H–K) Representative photos of low (H and J) and high (I and K) magnification show the lumbar cord of a M337V rat at disease end stage (J and K) or a ChAT-tTA rat at matched age (H and I). Scale bars: 200 μm (H and J); 60 μm (I and K). (L and M) Graphs show the average size of motor neurons in L3–L5 cords and the total volume of the ventral horns of L3–L5 cord. Data are means ± SD (n = 8).
Figure 7
Figure 7. Suppressing mutant TDP-43 expression prevents disease from progression in rats.
(A) A diagram shows induction (Dox withdrawal) and suppression (Dox addition) of mutant TDP-43 (hTDP-43) transgene. (B) Immunoblotting revealed gene suppression after Dox addition (Dox+). (C) Open-field assay measured mobility within 20 minutes. ChAT-tTA denotes ChAT–tTA-9 rats, and M337V denotes ChAT–tTA-9/TRE–TDP-43M337V rats. (D) The body weight of an individual rat at 66 days of age was used as the base for calculating alteration in body weight. Data are means ± SD (C and D, n = 8). (E–M) H&E staining (E–G) and histochemistry for nonspecific esterase (H–J) and ATPase (K–M) revealed grouped atrophy of skeletal muscle in Dox-untreated (F, I, and L) and varied sizes of muscle fibers in Dox-treated (G, J, and M) rats. Arrowheads point to grouped atrophy, and arrow points to a neuromuscular junction. (N–S) Toluidine blue staining showed L3 ventral (N–P) and dorsal (Q–S) roots. All scale bars: 30 μm. (T) Myofibers were quantified on cross sections of gastrocnemius stained with H&E. (U) The cross areas of myofibers were quantified with ImageJ on cross sections of gastrocnemius stained for ATPase. Quantification was done on 3 photos of the cross sections (T and U). Data are means ± SD (n = 4 rats). *P < 0.05. (V) The number of motor neurons (>25 μm) is not different between Dox-treated and -untreated M337V rats. The group of Dox-untreated M337V rats is identical to the M337V group of Figure 6G. Dox-treated rats were terminated at 90 days of age. Data are means ± SD (n = 8).
Figure 8
Figure 8. Astrocytes and microglia react to motor neuron death in rats.
(A–L) Immunohistochemistry revealed marked activation of astrocytes (GFAP) and microglia (Iba1) in the spinal cords of mutant TDP-43 transgenic rats. (M–R) Immunofluorescence staining showed the distribution of astrocytes (GFAP) and NG2 glia in the ventral horns of lumbar cords. ChAT–tTA-9/TRE–TDP-43M337V double-transgenic (M337V) rats were deprived of Dox at 60 days of age and were completely paralyzed (end stage) by 78 days of age. ChAT–tTA-9 single-transgenic rats were terminated at matched ages. Randomly chosen M337V rats were treated with Dox (M337V + Dox) to prevent mutant TDP-43 from further expression after disease onset. Dox-treated rats were terminated at 90 days of age. Scale bars: 200 μm (A–C and G–I); 100 μm (D–F, J–L, and M–R).
Figure 9
Figure 9. Ubiquitin accumulates in the motor neurons of ALS rats.
(A–F) Immunostaining revealed a marked accumulation of ubiquitin in the motor neurons of mutant TDP-43 transgenic rats. (G–I) Confocal microscopy revealed that ubiquitin aggregates accumulated in the motor neurons expressing mutant human TDP-43 (hTDP-43). ChAT–tTA-9/TRE–TDP-43M337V double-transgenic (M337V) rats were deprived of Dox at 60 days of age and were paralyzed at the age of 78 days. ChAT–tTA-9 single-transgenic (ChAT-tTA) rats were terminated at matched ages. Some M337V rats were treated with Dox (M337V + Dox) to prevent mutant TDP-43 from further expression after disease onset. Dox-treated rats were terminated at 90 days of age. Scale bars: 200 μm (A–C); 50 μm (D–F); 10 μm (G–I).
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