Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43

Abstract

Accumulation of phosphorylated cytoplasmic TDP-43 inclusions accompanied by loss of normal nuclear TDP-43 in neurons and glia of the brain and spinal cord are the molecular hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the role of cytoplasmic TDP-43 in the pathogenesis of these neurodegenerative TDP-43 proteinopathies remains unclear, due in part to a lack of valid mouse models. We therefore generated new mice with doxycycline (Dox)-suppressible expression of human TDP-43 (hTDP-43) harboring a defective nuclear localization signal (∆NLS) under the control of the neurofilament heavy chain promoter. Expression of hTDP-43∆NLS in these 'regulatable NLS' (rNLS) mice resulted in the accumulation of insoluble, phosphorylated cytoplasmic TDP-43 in brain and spinal cord, loss of endogenous nuclear mouse TDP-43 (mTDP-43), brain atrophy, muscle denervation, dramatic motor neuron loss, and progressive motor impairments leading to death. Notably, suppression of hTDP-43∆NLS expression by return of Dox to rNLS mice after disease onset caused a dramatic decrease in phosphorylated TDP-43 pathology, an increase in nuclear mTDP-43 to control levels, and the prevention of further motor neuron loss. rNLS mice back on Dox also showed a significant increase in muscle innervation, a rescue of motor impairments, and a dramatic extension of lifespan. Thus, the rNLS mice are new TDP-43 mouse models that delineate the timeline of pathology development, muscle denervation and neuron loss in ALS/FTLD-TDP. Importantly, even after neurodegeneration and onset of motor dysfunction, removal of cytoplasmic TDP-43 and the concomitant return of nuclear TDP-43 led to neuron preservation, muscle re-innervation and functional recovery.

Keywords: Amyotrophic lateral sclerosis (ALS); Frontotemporal dementia (FTD); Frontotemporal lobar degeneration (FTLD); Motor neuron; Mouse model; Neurodegeneration; Spinal cord; TDP-43.

Fig 1

Expression of hTDP-43ΔNLS in brain and spinal cord of rNLS8 and rNLS9B mice. a Schematic for Dox-regulatable expression of hTDP-43ΔNLS in bigenic mice under the control of the NEFH promoter. Expression of hTDP-43 and total (h+m) TDP-43 protein in olfactory bulb (Ob), cerebellum (Cb), hippocampus (Hp), brainstem and remainder of the brain (Bs), cortex (Cx), spinal cord (Sc), spleen (Sp), kidney (Kd) and liver (Lv) of b rNLS8 and c rNLS9B mice at 4 weeks off Dox. Approximate molecular weight markers in kDa are shown on the left and GAPDH is a loading control. Representative immunoblots of n = 3. Representative images at 1 week off Dox show widespread expression of hTDP-43ΔNLS in d Hp, Cx, and e Sc of rNLS8 mice. IHC in rNLS8 motor cortex showing cytoplasmic TDP-43 inclusions (arrows) detected with both f an N-terminal (Nt)-TDP-43 antibody, and g a C-terminal (Ct)-TDP-43 antibody, at 2 weeks off Dox. h, i There is widespread hTDP-43 expression (green) in NeuN⁺ neurons (red), shown at 4 weeks off Dox. Arrowhead in i indicates rare non-neuronal hTDP-43-positive glial cell, and nuclei are shown by DAPI (blue). Scale bars d 500 μm, e 200 μm, f, g 50 μm, h, i 50 μm

Fig 2

Accumulation of insoluble pTDP-43 over time in brain and spinal cord, and formation of pTDP-43 inclusions in rNLS mice. a RIPA-soluble hTDP-43ΔNLS, mouse (m) TDP-43 and total (h+m) TDP-43 in rNLS8 cortex at various time points compared to non-transgenic (nTg) and monogenic (tTA only) controls at 1 week off Dox. Quantification is shown below each representative immunoblot with n = 3 mice for nTg, tTa and 1, 2, 4 and 6 weeks off Dox, and n = 9 for end stage mice (7–18 weeks); see also Fig. S2. Approximate molecular weight markers in kDa are shown on the left and GAPDH is shown as a loading control. b RIPA-insoluble hTDP-43ΔNLS, mTDP-43 and h+mTDP-43 from 1 week off Dox in rNLS8 cortex. Higher molecular weight and phosphorylated (p) TDP-43 are detected from 2 weeks off Dox. Extract from a human FTLD patient is shown as a positive control. Asterisks indicate pTDP-43, arrowhead indicates 43 kDa TDP-43, and hash mark indicates 20–25 kDa C-terminal TDP-43 fragments. c RIPA-insoluble pTDP-43 in rNLS8 spinal cord from 4 weeks off Dox. Time points of 7–18 weeks in a–c are disease end stage. pTDP-43 pathology was detected with antibodies to both pS409/410- and pS403/404-TDP-43 in rNLS8 at 4 weeks off Dox in d motor cortex and f spinal cord (Sc), rNLS8 at 6 weeks off Dox in h striatum, in rNLS9B at 4 weeks off Dox in e motor cortex, g Sc, and i striatum, and in the j motor cortex and k Sc of human ALS patients. Arrows indicate examples of large cytoplasmic inclusions, and asterisks in e and i indicate small pTDP-43-positive puncta. Scale bar d–k 50 μm

Fig 3

rNLS8 mice develop progressive cortical atrophy, muscle denervation, MN loss and muscle atrophy. a, b Representative brain sections from nTg and rNLS8 mice 18 weeks off Dox at Bregma 1.10, stained with haemotoxylin and eosin (H&E). Thickness is measured from the edge of the brain section to the white matter below (indicated with a bar). c Measurement of cortical thickness in nTg and rNLS8 mice at different ages, n = 3 per time point. d Brain mass of rNLS8 mice compared to littermate nTg or tTA monogenic controls at disease end stage of 10–18 weeks off Dox, n = 9 per group. e–h Representative images and quantification of the overlap of VAChT-positive motor terminals (red) with acetylcholine receptors stained using BTX (green) as an indicator of innervated motor endplates in the tibialis anterior (TA) muscle of rNLS8 mice. The TA muscle showed marked denervation at 6 and 8 weeks off Dox (f–g, vacated NMJs noted with asterisks), n = 4 mice per time point. i–k IF for MN marker VAChT (red) with nuclear marker DAPI (blue) revealed loss of MNs in the lumbar spinal cord at 6 and 8 weeks off Dox in rNLS8 mice. I Quantification of number of VAChT-positive lumbar MNs in rNLS8 mice at different times off Dox. n = 4 mice per time point. m, n H&E staining of TA muscle showed gross muscle atrophy in rNLS8 mice at disease end stage compared to littermate nTg control. Examples of central nuclei are shown by an arrow and atrophic fibers by an asterisk. o, p TA and gastrocnemius muscle masses of rNLS8 mice compared to littermate nTg or tTA monogenic controls at disease end stage of 10–18 weeks off Dox, n = 7 per group. Scale bars a, b 500 μm, e–g 50 μm, i–k, m–n 100 μm; *p < 0.05, **p < 0.01, ***p < 0.001 versus control by one-way ANOVA with Bonferroni's post hoc test; ^###p < 0.001 by paired two-tailed t test

Fig 4

Expression of hTDP-43ΔNLS results in dramatic motor impairments, weight loss, and death in rNLS8 mice. Onset of a hindlimb clasping, b tremor, and progressive declines in c wirehang and d rotarod performance in rNLS8 mice, n = 10 controls, n = 14 bigenic. e rNLS8 mice also showed significant weight loss relative to controls beginning 3 weeks after Dox removal, n = 19 per group. f Kaplan–Meier survival curve for rNLS8 mice and littermate controls shows a marked decrease in survival time in rNLS8 mice, n = 19 per group

Fig 5

Schematic showing the time course of major neurodegenerative events in the rNLS8 mouse model of ALS/FTLD-TDP. The earliest events are the accumulation of cytoplasmic hTDP-43 and down-regulation of mTDP-43. Next, mice begin to show motor impairments including clasping, tremor, and impairments in motor tasks, and in some cells hTDP-43 begins to aggregate and becomes phosphorylated at pathological sites. By 4 weeks after initiation of hTDP-43ΔNLS expression, there is a significant brain atrophy and marked axonal dieback from hindlimb muscle. Cell loss and axonal dieback, and their functional consequences, continue to progress until disease end stage

Fig 6

Soluble and insoluble TDP-43 are rapidly cleared from brain and spinal cord of rNLS8 mice upon re-introduction of Dox. a Schematic of experimental design. b, c Levels of hTDP-43 and h+mTDP-43 in RIPA-soluble (R) and RIPA-insoluble/urea-soluble (U) protein fractions of rNLS8 mouse cortex and spinal cord at 6 weeks and at subsequent time points back on Dox (+Dox), with tetO-hTDP-43-ΔNLS monogenic (Mono) littermate control. p403/404-TDP-43 and p409/410-TDP-43 were rapidly eliminated from the U fraction. Approximate molecular weight markers in kDa are shown on the left and GAPDH is the loading control. d hTDP-43 was eliminated from spinal cord MNs in rNLS8 mice +Dox, and e p409/410-TDP-43 inclusions were detected in the motor cortex in rNLS8 mice at 6 weeks, but were not detected in rNLS8 mice +Dox (images of 6 weeks off Dox +18 weeks back on Dox are shown). Scale bars d 100 μm, e 50 μm

Fig 7

Endogenous nuclear mTDP-43 rapidly returns in cortex of rNLS8 mice upon re-introduction of Dox. a Nuclear mTDP-43 levels in rNLS8 mice at 6 weeks off Dox and after Dox re-introduction (+18 weeks Dox shown), compared to tetO-hTDP-43-ΔNLS monogenic (Mono) littermate control. b, c Endogenous mTDP-43 levels are decreased in rNLS8 mice at 6 weeks off Dox, but are significantly increased by +2 weeks Dox; ***p < 0.001 versus control by one-way ANOVA with Bonferroni's post hoc test, n = 3 per group. d–f Levels of NFL protein, but not NFM, are significantly decreased in rNLS8 mice at 6 weeks off Dox. Representative immunoblots are shown; **p < 0.01 and ***p < 0.001 versus control by one-way ANOVA with Bonferroni's post hoc test, n = 3 per group, +Dox indicates +18–32 weeks back on Dox. Scale bar a 50 μm

Fig 8

Suppression of hTDP-43ΔNLS expression rescues motor phenotypes and restores lifespan in rNLS8 mice. a Hindlimb splaying phenotype of nTg control, rNLS8 mouse after 6 weeks of hTDP-43ΔNLS expression and rNLS8 mouse back on Dox after 6 weeks of hTDP-43ΔNLS expression (+Dox, 2 weeks back on Dox shown). b Weight loss reached a nadir at 6 weeks in rNLS8 mice back on Dox (n = 11 rNLS8 +Dox and n = 8 littermate nTg/monogenic controls). c MN number in lumbar SC, and d, e analysis of NMJ innervation in rNLS8 mice after 6 weeks of hTDP-43ΔNLS expression and at 3–4 months back on Dox (n = 3–4 per group), *p < 0.05 versus 6 weeks off Dox, image shows rNLS8 mouse at 6 weeks off Dox +12 weeks back on Dox. f Wirehang performance of rNLS8 mice back on Dox at 6 weeks, p < 0.001 from 3 weeks back on Dox versus 6 weeks off Dox, and g rotarod performance of rNLS8 mice back on Dox at 6 weeks, p < 0.05 at 1 week back on Dox and p < 0.001 from 2 weeks back on Dox versus 6 weeks off Dox, by repeated measures ANOVA with Bonferroni's post hoc test, n = 8. h Survival in rNLS8 mice back on Dox at 6 weeks (rNLS8 + Dox—blue line, n = 14, littermate nTg/monogenic controls—black line, n = 11) and in rNLS8 off Dox (rNLS—dotted red line, n = 19, also included in Fig. 4f). Triangles/circles indicate censored animals either killed for analysis or remaining alive