(A) Schematic diagram shows the structure of the human TDP transgene extracted from a BAC clone. The M337V mutation was introduced into the TDP transgene construct using a recombineering technique. (B) PCR identified transgenic founders carrying the normal (WT) or mutant (TDP43^M337V) human TDP gene. NT: nontransgenic littermate. (C) Immunoblotting detected a robust expression of the TDP transgene in the forebrain of TDP43^M337V transgenic founders and TDP43^WT transgenic offspring (first generation). Membranes were probed with an antibody against human TDP-43 (generated in-house) and then with an antibody against GAPDH. NT: a nontransgenic littermate. (D–F) Immunohistochemistry revealed that human TDP-43 was expressed in the normal (E) and mutant (F) human TDP transgenic rats, but not in nontransgenic littermates (D) at the age of 29 days. Transverse sections through the L4 spinal cord were stained for human TDP-43 immunoreactivity and then counterstained with haematoxylin. Micrographs show the ventral horn of the lumbar spinal cord.

(A–C) Transverse sections of lumbar spinal cord were immunostained for human TDP-43 (red) and ChAT (green). Scale bars: 30 µm. (D–F) Cresyl violet staining revealed motor neurons in the ventral horn of L3 spinal segments. Scale bars: 100 µm. (G–I) Toluidine blue staining shows axons of L3 ventral roots. (J,K) Transmission electron microscopy (EM) shows the structure of axons in the L3 ventral roots. Degenerating axons were shrunken and had collapsed myelin (arrows). Scale bars: 5 µm. (L) The number of large neurons (>25 µm in diameter) in the ventral horn of L3 spinal segments was estimated by stereological cell counting. Data are expressed as the mean ± SD (n = 9–15). *p<0.05. (M) Axons of L3 ventral roots were visualized by EM, and 60 axons (>4 µm in diameter) of each animal were examined for integrity. Data are expressed as mean ± SD (n = 8 or 9). *p<0.01. Tissues were collected from paralyzed transgenic rats of line 7 (age: 45±7 days) conditionally expressing the mutant human TDP gene (M337V: C, F, I, K, L, and M), age-matched nontransgenic littermates (NT: A, D, G, L, and M), and age-matched transgenic rats constitutively expressing the normal human mini TDP gene (WT: B, E, H, J, L, and M).

(A,B) Bielschowski silver staining revealed degenerating neurons in the lumbar spinal cord of a paralyzed TRE-TDP-43^M337V transgenic rat (line 7) (B), but not in the spinal cord of a nontransgenic littermate (A). (C,D) TUNEL staining shows motor neurons in the spinal cord undergoing apoptosis (D: arrow) in a paralyzed TRE-TDP-43^M337V transgenic rat ( line 7) (D), but not in its nontransgenic littermate (C). Transverse sections of the L3 spinal segment were stained with ChAT antibody to visualize motor neurons and were labeled using a TUNEL staining kit to visualize apoptosis.

(A–F) FD silver staining shows degeneration of non-motor neurons in paralyzed tTA/TRE-TDP-43^M337V bigenic rats (line 7; age: 45 days) (B,D,F), but not in a tTA transgenic littermate (A,B,C). Degenerating neurons were outlined by deposits of silver particles (arrows). Scale bars: 20 µm.

(A) Schematic diagram shows the structure of the inducible mutant TDP transgene (TRE-TDP-43^M337V). Expression of the mutant TDP transgene depends on tTA activation and can be suppressed by Dox, which binds to tTA and renders it inactive. (B) Immunoblotting detected a robust expression of the transgene in the spinal cord of P20 rats. Membranes were probed sequentially with antibodies against human TDP-43 (generated in-house), human and rat TDP-43 (ProteinTech), and rat GAPDH. M337V: transgenic rats carrying the conditional mutant TDP (TRE-TDP-43^M337V) and CAG-tTA transgenes; WT: transgenic rat carrying the mini normal human TDP transgene (miniTDP-43^WT); NT: nontransgenic littermate of the WT transgenic rat; *, a weak nonspecific band. (C) Photo of a mutant TDP transgenic rat (line 7) paralyzed at P40. (D) Graphs show the probability of disease onset, defined as an unrecoverable reduction in running time on a Rotarod. Disease onset for line 16 was not plotted, since these animals experienced early paralysis with rapid progression and accurate definition of disease onset was technically difficult. (E) Survival analysis revealed that lifespan was remarkably reduced in the mutant TDP transgenic rats. Rats were euthanized and counted as dead when two or more legs became paralyzed. Male and female rats of line 16 were combined as one group because disease progression between each gender was indistinguishable. Definition of symbols in (D,E): ▪, normal TDP transgenic rats carrying the miniTDP-43^WT transgene (line 4; n = 9); • and ○, mutant male (•, n = 10) and female (○, n = 12) transgenic rats carrying the TRE-TDP-43^M337V (line 7) and CAGtTA transgenes; ♦, mutant TDP transgenic rats carrying the TRE-TDP-43^M337V (line 16) and the CAGtTA transgenes (n = 14). All breeding female rats were given Dox in drinking water (50 µg/ml) until 4 days before delivery.

(A–C) The neuromuscular junction (NMJ) in the gastrocnemius muscles was examined by confocal microscopy to reconstruct the focal structure. Compared to the NMJ in a control rat (A), the NMJ in TRE-TDP-43^M337V transgenic rats (line 7) was partially denervated at disease onset (B) and severely denervated at disease end stages (C). Axon terminals were visualized by immunostaining for synaptophysin and neurofilament (NF), while postsynaptic nicotinic receptors were visualized with Alexa fluor 555-conjugated α-bungarotoxin. (D) Quantification of NMJ denervation in nontransgenic control rats (NT) and TRE-TDP-43^M337V transgenic rats at disease onset or the paralysis stage. Twenty NMJs were examined for each animal (n = 4 or 5). (E) Electromyography of the gastrocnemius muscles revealed that frequent fibrillation potentials (arrows) were present in paralyzed TRE-TDP-43^M337V transgenic rats (M337V), but not in a nontransgenic littermate (NT). (F,G) EM revealed that intramuscular axons underwent degeneration in a mutant transgenic rat (line 7, age: 32 days) at disease onset (G), but not in an age-matched nontransgenic littermate (F). The arrow indicates a cluster of aggregates that accumulated in the axon of a mutant rat (G). SM: skeletal muscle; a: axon; N: the nucleus of a Schwann cell. Scale bars: 2 µm. (H,I) Group atrophy of the gastrocnemius muscle in paralyzed TRE-TDP-43^M337V transgenic rats was detected by H&E staining (H, arrows) and by histochemistry for nonspecific esterase (I).

(A,B) Double immunofluorescence staining shows an accumulation of astrocytes around motor neurons in a paralyzed TRE-TDP-43^M337V transgenic rat (line 7) (B), but not in a nontransgenic littermate (A). (C,D) Double immunofluorescence staining shows an activation of microglial cells in a paralyzed TRE-TDP-43^M337V transgenic rat (line 7) (D), but not in a nontransgenic littermate (C). Transverse sections of the L3 spinal cord were immunostained for ChAT (red; motor neuron marker), GFAP (green; astrocyte marker), or cd68 (green; microglia and macrophage marker).

(A) Immunohistochemistry revealed phosphorylation and cytoplasmic localization of TDP-43 (arrows) in the brains of FTLD patients, but not in control subjects. Paraffin-embedded sections were stained with a polyclonal antibody against TDP-43 phosphorylated at aa409/410. (B,C) Immunostaining revealed that human TDP-43 was present in both the nucleus and cytoplasm of cells from the mutant (M337V: line 7) and normal (WT) TDP transgenic rats, but not in the cells of a nontransgenic rat (NT). TDP-43 inclusions (arrow) were rarely detected, being present in only the cortex of paralyzed M337V transgenic rats. Cryopreserved sections of the rat brain and spinal cord were stained with a polyclonal antibody specific to human TDP-43. (D) Immunostaining shows a cytoplasmic accumulation of phosphorylated TDP-43 in mutant (M337V) and normal (WT) TDP transgenic rats. A weak signal for phosphorylated TDP-43 was also detected in the nuclei of cells from a nontransgenic rat (NT). Cryopreserved sections of the spinal cord were stained with a polyclonal antibody against TDP-43 phosphorylated at aa409/410. All scale bars: 20 µm.