Mutant TDP-43 in motor neurons promotes the onset and progression of ALS in rats (original) (raw)
Overexpression of mutant TDP-43 in neurons and muscles causes progressive paralysis in rats. We previously showed that ubiquitous overexpression of mutant TDP-43 in rats causes early onset and rapid progression of paralysis reminiscent of ALS (3). To improve the rat model, we intended to overexpress mutant TDP-43 selectively in neurons and chose the regulatory elements (promoter) of the human neurofilament heavy chain (NEF) gene to direct transgene expression (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI59130DS1). In our TDP-43 transgenic rats (3, 29), tetracycline response element (TRE) drives TDP-43 transgene, whose expression fully depends on the activity of tetracycline-controlled transactivator (tTA). A short fragment (8.5 kb) of human NEF promoter synthesizes transgene in differentiated neurons in mice (30). We used a long fragment (18 kb) of the NEF promoter to drive tTA transgene in rats. We established a single-copy transgenic line that exhibits X chromosome–linked transmission of the NEF-tTA transgene (Supplemental Figure 1). To avoid gene insertion–caused mutation, we only used female rats heterozygous for NEF-tTA in the following studies. By crossing NEF-tTA transgene onto a TRE–TDP-43M337V transgenic background (line 16), we detected a substantial Dox-regulated expression of human TDP-43 transgene (Figure 1, A–J). Expression of the transgene was restricted in neurons (Figure 1, K–M) and was not detected in astrocytes (Figure 1, N–P) — the largest population of nonneuronal cells in the CNS. Unexpectedly, skeletal muscles also expressed human TDP-43 transgene (Supplemental 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in the spinal cord (SP) and hippocampus (HP) of NEF-tTA/TRE–TDP-43M337V double-transgenic rats soon after Dox was withdrawn (Dox–).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).
To induce disease phenotypes in adult rats, we deprived NEF-tTA/TRE–TDP-43M337V double-transgenic rats of Dox at the age of 60 days and observed a full activation of mutant TDP-43 gene in rats by the age of 70 days (Figure 1A). Two weeks after mutant TDP-43 was expressed, the mutant rats began losing grip strength and mobility (Figure 2, A and B), displaying the early signs of paralysis (disease onset). Within 3 weeks after mutant TDP-43 activation, rats lost the ability to retract hind legs (Figure 2, A–C, and Supplemental Video 1). To accurately assess motor neuron death in rats, we used unbiased stereological cell counting. In our publications (3, 31), we counted the number of motor neurons in 1 spinal segment (L3). To increase the accuracy of cell counting, here we counted the number of motor neurons in a long segment of lumbar cords (L3–L5). Stereological cell counting revealed a moderate, but significant, loss of neurons in the spinal cord and dentate gyrus of transgenic rats at disease end stages (Figure 2, D–K). In mutant TDP-43 transgenic rats at paralysis stages, toluidine blue staining and electromicroscopy revealed degenerating axons in the ventral, but not in the dorsal, roots (Figure 3, K–N, and Supplemental Figure 2, C and D). Silver staining revealed degenerating neurons in the ventral horn of the lumbar cord (Supplemental Figure 2, A and B). At paralysis stages, mutant TDP-43 transgenic rats displayed grouped atrophy of skeletal muscles revealed by H&E staining (Figure 3B) and by histochemistry for nonspecific esterase (Figure 3E) and ATPase (Figure 3H). In contrast, NEF-tTA single-transgenic rats did not display any of these pathological changes (Figures 2 and 3; Supplemental Figure 2). Restricted overexpression of mutant TDP-43 in neurons and in muscles induced motor neuron degeneration and denervation atrophy of muscles, reproducing the key features of ALS in rats.
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).
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).
Ubiquitinated TDP-43 inclusion is a characteristic of sporadic ALS and FTLD (2). We next examined TDP-43 inclusion, but did not observe typical TDP-43 inclusion in mutant TDP-43 transgenic rats at paralysis stages (Figure 1, D and H). We detected rare ubiquitin aggregates in the cortex (Supplemental Figure 3), but not in the spinal cord (data not shown), of paralyzed rats. Our finding confirmed that TDP-43 inclusion is not essential to neuron death, at least in rodent models (3, 7).
Removal of mutant TDP-43 partly restores motor function in ALS rats expressing the disease gene in neurons and muscles. Restricted expression of mutant TDP-43 in neurons and in muscles caused severe damage to motor axons and to skeletal muscles (Figure 3 and Supplemental Figure 2), but only caused a moderate loss of motor neurons in the spinal cord (Figure 2). We determined the potential of functional recovery in the diseased rats. Since expression of mutant TDP-43 in rats was subject to Dox regulation (Supplemental Figure 1), we treated the rats with Dox to prevent the mutant TDP-43 transgene from further expression after the rats developed paralysis (Figure 4). To repress disease gene expression quickly, Dox was subcutaneously injected and was simultaneously given in drinking water (Figure 4A). Paralyzed rats were provided Dox-soaked soft food on the cage floor. By the fifth day of Dox treatment, mutant TDP-43 was efficiently suppressed (Figure 4A). Suppressing the mutant TDP-43 gene led to restoration of motor function in rats (Figure 4, B–D, and Supplemental Videos 1–4). Dox-treated transgenic rats began to retract hind legs by the seventh day and to totter around the cage by the ninth day of Dox treatment. Open-field assay revealed a gradual recovery of motor function in the Dox-treated rats (Figure 4E). Even 2 months after Dox treatment, grip strength recovered partially (Figure 4F). Functional recovery was dramatic but incomplete in the rats expressing mutant TDP-43 in neurons and in skeletal muscles.
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).
Remodeling of motor units contributes to the functional recovery in Dox-treated transgenic rats. We examined the anatomical basis of functional recovery in Dox-treated transgenic rats. As expected, stereological cell counting revealed no difference in the number of motor neurons between Dox-treated and Dox-untreated ALS rats (Figure 3R). Morphological analyses showed that degenerating motor axons were eliminated in Dox-treated ALS rats (Figure 3, J–Q). H&E staining revealed that damaged muscle fibers in the gastrocnemius muscle were cleared and that muscle fibers of varied sizes were regenerated (Figure 3, A–C). Regenerating myofibers displayed centered nuclei and increased in size (Figure 3, P and Q).
While the histochemistry for nonspecific esterase and ATPase revealed normal myofibers with uniform sizes, polygonal shapes, and eccentric nuclei (Figure 3, D and G), these stainings revealed denervated myofibers with small sizes and angulated shapes (Figure 3, E and H). Nonspecific esterase staining displayed normal myofibers with pale yellow to brown color (Figure 3D), but displayed denervated or newly regenerated myofibers with red-brown color (Figure 3, E and F). ATPase staining (pH 4.6) can distinguish 3 types of myofibers, with light to dark colors. ATPase staining revealed a mosaic distribution of different myofibers in normal muscles (Figure 3G), but it revealed the grouping of myofibers of the same types in denervated or newly regenerated muscles (Figure 3, H and I). Histological analyses suggested that motor units were remodeled in Dox-treated transgenic rats.
Novel tTA transgenic rats are created to express transgenes restrictedly in motor neurons. To determine whether motor neuron death is a cell-autonomous process in TDP-43–related ALS, we developed multiple transgenic rat lines that express tTA transgene restrictedly in motor neurons (Figure 5, Supplemental Figures 4 and 5). Previous studies showed that the promoter of the choline acetyltransferase (ChAT) gene directs synthesis of transgenes selectively in spinal motor neurons in mice (32). We isolated mouse ChAT promoter from a BAC clone and used it to drive tTA transgene in rats. We obtained 3 expression lines that carry 2, 5, or 9 copies of the tTA gene (Supplemental Figure 4). Individual lines were designated as ChAT–tTA-2, -5, or -9. It is a technical challenge to measure the expression levels of transgene in a single cell population in animals. Thus, we crossed each ChAT-tTA transgenic line with TRE–TDP-43M337V (line 16) rats and purified motor neurons from the double-transgenic embryos (embryonic day 14) using immunopanning (33). We then determined the expression levels of the TDP-43M337V transgene in purified motor neurons and detected varying expression of human TDP-43 in individual ChAT-tTA lines (Supplemental Figure 4, E–J). Compared with the NEF-tTA line, ChAT-tTA lines 5 and 9, but not 2, expressed the transgene at higher levels (Supplemental Figure 4, G, I, and J).
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).
To facilitate determination of the transgene expression profile, we created a tTA reporter line that carries the LacZ transgene under control of the TRE promoter (Supplemental Figure 5A). We established a 3-copy TRE-LacZ line and crossed it with a ChAT–tTA-9 line to determine tTA expression profile (Figure 5, A–C, and Supplemental Figure 5, B–E). In ChAT–tTA-9/TRE-LacZ double-transgenic rats, expression of the LacZ gene was restricted to the ventral horn of the spinal cord (Figure 5, B and C). Double-label immunostaining revealed that LacZ immunoreactivity was restricted in ChAT-positive cells and that more than 60% of motor neurons expressed LacZ in the ChAT–tTA-9 line (Supplemental Figure 5, B–E). In contrast, about 40% of motor neurons expressed LacZ in the ChAT–tTA-2 and -5 lines. We then focused our studies on the ChAT–tTA-9 line.
Motor neurons undergo cell-autonomous death in TDP-43 transgenic rats. We crossed ChAT-tTA lines (ChAT–tTA-5 and ChAT–tTA-9) with TDP-43M337V transgenic rats (line 16) to examine disease induction, and we focused our studies on the ChAT–tTA-9 line, as this line expresses transgenes in the most motor neurons (Supplemental Figure 5). Similar to the reporter gene LacZ (Figure 5, B and C, and Supplemental Figure 5), mutant human TDP-43 was expressed restrictedly in the ventral horn of the spinal cord in ChAT–tTA-9/TDP-43M337V rats (Figure 5, D and E). Double-label immunostaining revealed that human TDP-43 was colocalized with the motor neuron marker ChAT (Figure 5, J–L), but was not colocalized with the oligodendrocyte marker APC (Figure 5F) or the astrocyte marker GFAP (Figure 5, G–I). The results suggest that expression of TDP-43M337V is restricted in spinal motor neurons.
To induce disease phenotypes in adult rats, we gave transgenic rats Dox in drinking water until the rats were 60 days old (Figure 6A and Supplemental Figure 6A). Sprague Dawley rats are sexually mature at the age of 70 days. In ChAT–tTA-9/TDP-43M337V transgenic rats, human TDP-43 was undetectable in the presence of Dox, but was detected by 3 days off Dox (Figure 6B). Human TDP-43 was not detectable in the skeletal muscles (Figure 6B). Soon after expression of mutant TDP-43, rats began losing grip strength and motor activity, as revealed by open-field assay and grip-strength measure (Figure 6, C and D). By the age of 70 days (10 days off Dox), most mutant rats developed the early signs of paralysis (Figure 6E). Rats reached disease end stages within a week after disease onset (Figure 6F). On average, the duration of disease progression was less than 2 weeks (Figure 6, E and F). Unbiased stereological counting revealed that more than 60% of spinal motor neurons were lost in mutant TDP-43 transgenic rats at disease end stages (Figure 6, G–K). Interestingly, the size of surviving motor neurons and the volume of ventral lumbar cords were unaltered at disease end stages (Figure 6, L and M). Toluidine blue staining revealed that axons in the ventral roots (motor axons), but not in the dorsal roots (sensory axons), were damaged (Figure 7, N–S). As a result of motor neuron death, groups of skeletal muscle fibers were atrophied, as revealed by H&E staining (Figure 7, E and F) and by histochemistry for nonspecific esterase (Figure 7, H and I) and ATPase (Figure 7, K and L). Restricted overexpression of mutant TDP-43 in motor neurons caused a severe loss of motor neurons and motor axons and caused a denervation atrophy of skeletal muscles in ChAT–tTA-9/TDP-43M337V transgenic rats.
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).
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).
We next characterized ChAT–tTA-5/TDP-43M337V transgenic rats (Supplemental Figure 6). When Dox was withdrawn at the age of 60 days, rats displayed disease onset by the age of 85 days (Supplemental Figure 6, A and B), but did not reach disease end stages by the age of 150 days. The ChAT–tTA-5 line directed transgene expression in less than 50% of spinal motor neurons (Supplemental Figure 5E), and thus, ChAT–tTA-5/TDP-43M337V rats would not lose enough motor neurons to cause full paralysis. Indeed, only about 30% of motor neurons were lost in the rats by 150 days of age (Supplemental Figure 6C). Accordingly, fewer motor axons in the ventral roots were lost in ChAT–tTA-5/TDP-43M337V rats compared with ChAT–tTA-9/TDP-43M337V rats (Supplemental Figure 6, H and I, and Figure 7, N–P). Overexpression of mutant TDP-43 at a low level and in fewer motor neurons caused moderate paralysis phenotypes in rats (Supplemental Figure 6).
Removal of mutant TDP-43 prevents disease from progression in rats expressing the disease gene restrictedly in motor neurons. Compared with NEF-tTA/TDP-43M337V rats (Figure 2), ChAT–tTA-9/TDP-43M337V rats displayed a more severe loss of motor neurons, but displayed a similar progression of paralysis phenotype (Figure 6). We determined whether disease progression could be halted by stopping mutant TDP-43 expression after disease onset in ChAT–tTA-9/TDP-43M337V rats. When Dox was withdrawn from ChAT–tTA-9/TDP-43M337V rats at the age of 60 days (Figure 6A), the rats developed an early paralysis phenotype by 70 days of age and reached disease end stages by 78 days of age (Figure 6, C–F). Dox was thus withdrawn from rats that were 60 days old and was added back 12 days later, so that mutant TDP-43 was expressed in the rats from 63 to 77 days of age (Figure 6B and Figure 7B). We refer to restoring Dox as a treatment. Dox-untreated and Dox-treated rats all developed paralysis at comparable speeds before Dox produced an effect on disease gene expression (Figure 7C). Soft food was provided to rats with paralysis. While the disease rapidly progressed to end stages in Dox-untreated rats, the disease stopped progressing at paralysis stages in Dox-treated rats (Figure 7, C and D). Within a few days, Dox-treated rats began to gain body weight and to gain partial recovery of motor activity (Figure 7, C and D). Dox-untreated rats were terminated at disease end stages (by age of 80 days), and Dox-treated rats were terminated at 90 days of age.
In Dox-treated transgenic rats, H&E staining of gastrocnemius revealed remodeled motor units: some muscle fibers with centered nuclei, muscle fibers of varied sizes, and absence of atrophied muscle fibers (Figure 7, E–G, and T). Histochemistry for nonspecific esterase (Figure 7, H–J) and ATPase (Figure 7, K–M) showed that muscle cells of the same types, but with varied sizes, were grouped, suggesting a remodeling of motor units. During functional recovery, muscle fibers increased in size (Figure 7U). Toluidine blue staining revealed that most degenerated motor axons were cleared in the ventral roots (Figure 7, N–P). Interestingly, stereological cell counting revealed that the number of spinal motor neurons (L3–L5) between Dox-treated and Dox-untreated transgenic rats was similar (Figure 7V). These findings suggest that degenerating motor neurons cannot be rescued and that survived motor units can be remodeled.
Distribution of reactive astrocytes and microglia differs in the spinal cord of ALS rats. Glial cells play an important role in motor neuron degeneration (34–36). We examined glial reaction to motor neuron death in ChAT–tTA-9/TDP-43M337V transgenic rats. Astrocytes and microglia were activated in the spinal cords of paralyzed rats (Figure 8). Mutant TDP-43 transgenic rats lost a large quantity of motor neurons at disease end stages (Figure 6G). In rats with active disease, reactive astrocytes distributed across the whole spinal cord and were not restricted to the ventral horns, where motor neurons expressing mutant TDP-43 were dying (Figure 8, B, E, and N), but microglia were activated around diseased motor neurons in the ventral horns (Figures 8, H and K). NG2-positive glia were also activated around diseased motor neurons (Figure 8, P–R). When disease progression was halted by Dox treatment (Figure 7, A–D), microglial reaction was quenched (Figure 8, I and L) and reactive astrocytes were restricted to the ventral horns where damaged motor neurons resided (Figure 8, C, F, and O). Varied response of glial cells may suggest a different role for astrocytes and microglia in the propagation and repair of neuronal damage in the disease.
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).
Ubiquitin accumulates in motor neurons expressing mutant TDP-43 in rats with paralysis. Similar to NEF-tTA/TDP-43M337V rats, ChAT–tTA-9/TDP-43M337V rats did not develop TDP-43 inclusion in diseased motor neurons (Supplemental Figure 7). As the disease progressed, mutant TDP-43 accumulated in the cytoplasm of motor neurons in transgenic rats (Supplemental Figure 7). In NEF-tTA/TDP-43M337V rats at disease end stages, rare ubiquitin aggregates were formed only in cortical neurons (Supplemental Figure 3). In contrast, ubiquitin accumulated in diseased motor neurons in ChAT–tTA-9/TDP-43M337V rats (Figure 9 and Supplemental Figure 8). Accumulated ubiquitin delineated the cell body and neurites of diseased motor neurons expressing mutant TDP-43 (Figure 9E and Supplemental Figure 8, B and C). Motor neurons with ubiquitin aggregates were few at disease onset, but were remarkably increased at paralysis stages (Supplemental Figure 8G). Accumulated ubiquitin was detectable only in rats with active disease (Figure 9, B, E, and G–I), but not in rats with disease halted by Dox treatment (Figure 9, C and F). The results suggest that accumulated ubiquitin may be cleared after the disease is halted and that ubiquitin accumulation may be an accompanying event of motor neuron death in the disease caused by TDP-43 mutation.
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).