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Selective Spatiotemporal Vulnerability of Central Nervous System Neurons to Pathologic TAR DNA-Binding Protein 43 in Aged Transgenic Mice

  • Annika van Hummel
    Affiliations
    Motor Neuron Disease Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
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  • Gabriella Chan
    Affiliations
    Motor Neuron Disease Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
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  • Julia van der Hoven
    Affiliations
    Dementia Research Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
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  • Marco Morsch
    Affiliations
    Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University Sydney, Sydney, New South Wales, Australia
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  • Stefania Ippati
    Affiliations
    Dementia Research Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
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  • Lisa Suh
    Affiliations
    Dementia Research Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
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  • Mian Bi
    Affiliations
    Dementia Research Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
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  • Prita R. Asih
    Affiliations
    Dementia Research Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
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  • Wei S. Lee
    Affiliations
    Motor Neuron Disease Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
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  • Troy A. Butler
    Affiliations
    Motor Neuron Disease Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
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  • Magdalena Przybyla
    Affiliations
    Dementia Research Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
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  • Glenda M. Halliday
    Affiliations
    Brain and Mind Centre, University of Sydney, Sydney, New South Wales, Australia

    Central Clinical School, University of Sydney, Sydney, New South Wales, Australia
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  • Olivier Piguet
    Affiliations
    Brain and Mind Centre, University of Sydney, Sydney, New South Wales, Australia

    School of Psychology, University of Sydney, Sydney, New South Wales, Australia

    ARC Centre of Excellence in Cognition and its Disorders, Sydney, New South Wales, Australia
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  • Matthew C. Kiernan
    Affiliations
    Brain and Mind Centre, University of Sydney, Sydney, New South Wales, Australia
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  • Roger S. Chung
    Affiliations
    Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University Sydney, Sydney, New South Wales, Australia
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  • Lars M. Ittner
    Affiliations
    Dementia Research Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia

    Neuroscience Research Australia, Sydney, New South Wales, Australia
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  • Yazi D. Ke
    Correspondence
    Address correspondence to Yazi D. Ke, Ph.D., Motor Neuron Disease Unit, Room 232, Level 2, Wallace Wurth Building, University of New South Wales, Sydney, NSW 2052, Australia.
    Affiliations
    Motor Neuron Disease Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
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Open ArchivePublished:March 22, 2018DOI:https://doi.org/10.1016/j.ajpath.2018.03.002
      Amyotrophic lateral sclerosis (ALS) is a rapidly progressing and fatal disease characterized by muscular atrophy because of loss of upper and lower motor neurons. Histopathologically, most patients with ALS have abnormal cytoplasmic accumulation and aggregation of the nuclear RNA-regulating protein TAR DNA-binding protein 43 (TDP-43). Pathogenic mutations in the TARDBP gene that encode TDP-43 have been identified in familial ALS. We have previously reported transgenic mice with neuronal expression of human TDP-43 carrying the pathogenic A315T mutation (iTDP-43A315T mice), presenting with early-onset motor deficits in adolescent animals. Here, we analyzed aged iTDP-43A315T mice, focusing on the spatiotemporal profile and progression of neurodegeneration in upper and lower motor neurons. Magnetic resonance imaging and histologic analysis revealed a differential loss of upper motor neurons in a hierarchical order as iTDP-43A315T mice aged. Furthermore, we report progressive gait problems, profound motor deficits, and muscle atrophy in aged iTDP-43A315T mice. Despite these deficits and TDP-43 pathologic disorders in lower motor neurons, stereological analysis did not show cell loss in spinal cords. Taken together, neuronal populations in aging iTDP-43A315T mice show differential susceptibility to the expression of human TDP-43A315T.
      Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative condition of the motor nervous system affecting frequently individuals in their prime. Although progressive muscle weakness and degeneration are the main symptoms of ALS, the disease shares symptoms, neuropathology, and genetics with frontotemporal lobar degeneration as part of a disease continuum.
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      We have previously reported early-onset motor and behavioral deficits in young transgenic mice expressing human TDP-43 with the pathogenic familial A315T mutation in central nervous system neurons (iTDP-43A315T).
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      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      In this report, we extend our earlier findings of motor deficits and neuropathology to aged iTDP-43A315T mice, revealing selective vulnerability of central nervous system neurons to pathologic TDP-43A315T expression in vivo.

      Materials and Methods

      Mice

      Transgenic mice with expression of neuronal transactivator [line mThy1.2-tTA(6)] and human A315T mutant TDP-43 under control of the tetracycline-responsive element promoter [line pTRE-TDP-43A315T(13)] have been described previously.
      • Ke Y.D.
      • van Hummel A.
      • Stevens C.H.
      • Gladbach A.
      • Ippati S.
      • Bi M.
      • Lee W.S.
      • Kruger S.
      • van der Hoven J.
      • Volkerling A.
      • Bongers A.
      • Halliday G.
      • Haass N.K.
      • Kiernan M.
      • Delerue F.
      • Ittner L.M.
      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      • Delerue F.
      • White M.
      • Ittner L.M.
      Inducible, tightly regulated and non-leaky neuronal gene expression in mice.
      Mice were maintained on a C57Bl/6 background and housed on a 12-hour light/dark cycle with access to standard chow and water ad libitum. Both male and female mice were used throughout this study. All experiments were approved by the University of New South Wales animal care and ethics committee.

      MRI

      Magnetic resonance imaging (MRI) was performed as described in detail before.
      • Ke Y.D.
      • van Hummel A.
      • Stevens C.H.
      • Gladbach A.
      • Ippati S.
      • Bi M.
      • Lee W.S.
      • Kruger S.
      • van der Hoven J.
      • Volkerling A.
      • Bongers A.
      • Halliday G.
      • Haass N.K.
      • Kiernan M.
      • Delerue F.
      • Ittner L.M.
      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      All brains were mapped to the Australian Mouse Brain Mapping Consortium mouse brain atlas with the use of an affine registration (12 DOF).
      • Ullmann J.F.
      • Watson C.
      • Janke A.L.
      • Kurniawan N.D.
      • Reutens D.C.
      A segmentation protocol and MRI atlas of the C57BL/6J mouse neocortex.
      Brain atlas images were rotated, scaled, and sheared to match MRI scanned images. The motor and visual cortices were individually delineated through consecutive coronal images, using this transformed label map as a guide. The caudate putamen and cerebellum are visually distinct areas, and so segmentation was performed manually on these regions. Image registration was performed according to voxel data, which are independent of genotype of mice. All volumes were calculated and rendered in consecutive coronal MRI images with 3D Slicer software version 4.7.0 (NIH, Bethesda, MD; https://hpc.nih.gov/apps/3Dslicer.html).

      Gait Analysis

      Gait of mice was analyzed using a DigiGait system (Mouse Specifics Inc., Boston, MA). Briefly, individual mice (n = 5 to 14 per group) were placed in an enclosed chamber on a transparent, motorized treadmill belt and allowed to acclimatize to the machine at a speed of 6 cm/second. Mice were then recorded for at least four complete strides at a belt speed of 15 to 17 cm/second, with the paws captured by a camera from underneath. Gait analysis was performed in 1-month–old mice [n = 10 (non-tg), n = 28 (single-tg), n = 10 (iTDP-43)], 3-month–old mice [n = 4 (non-tg), n = 12 (single-tg), n = 4 (iTDP-43)], and 8-month–old mice [n = 4 (non-tg), n = 14 (single-tg), n = 10 (iTDP-43)]. A range of spatial and temporal gait parameters was calculated by DigiGait software (DigiGait Analyser version 14.5 and DigiGait Imager version 14; Mouse Specifics Inc.) for each limb to detect differences in gait between transgenic and nontransgenic mice across different ages.

      Motor Testing

      All motor tests were performed as previously described,
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      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
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      Early-onset axonal pathology in a novel P301S-Tau transgenic mouse model of frontotemporal lobar degeneration.
      with 7 (non-tg), 7 (single-tg), and 7 (iTDP-43) mice used.

      Rota-Rod

      Motor performance of mice was determined using a Rotarod (Ugo Basile, Varese, Italy) in acceleration mode (5 to 60 rpm) over 120 seconds. The longest time each mouse remained on the turning wheel of five attempts per session was recorded. Mice were previously recorded daily during 10 days and five trials per day, with no differences observed between mice trained longer or shorter on the Rotarod. As such, only the 3 days of testing were done for the rest of the study.

      Pole Test

      To test strength and coordination, mice were placed at the apex of a vertical pole (47.5 cm length of dowel, diameter 0.8 cm) facing upward. The time taken to turn around, descend the pole, and reach the ground (with all four paws) was measured with a maximum time of 120 seconds. Mice underwent one to two training sessions before the test session, during which the best time was taken of two trials. Mice who were unable to descend the pole (slipped or fell) were given the maximum time.

      Hanging Wire Test

      Mice were allowed to hang on an inverted rectangular wire mesh over a Perspex box for a maximum of 3 minutes, and latency to falling off was recorded (longest time of two attempts).
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      • Bi M.
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      No overt deficits in aged tau-deficient C57Bl/6.Mapttm1(EGFP)Kit GFP knockin mice.

      Grip Strength Test

      A grip strength meter (Ametek Chatillon, Berwyn, PA) was used to measure the force exerted by the forelimbs of the mouse. Mice were placed such that they had a double overhand grip on a thin metal wire attached to the meter and were pulled away from the meter in a horizontal direction until they let go, and a peak force (N) was recorded at the moment when the mice let go. The highest force from five attempts was recorded.

      Histology and Staining

      For immunohistochemistry, tissues were embedded in paraffin and sectioned. Sectioned tissues were stained as previously described.
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      • Ittner L.M.
      Site-specific phosphorylation of tau inhibits amyloid-beta toxicity in Alzheimer's mice.
      The next 12-month–old mice were used for all histologic analysis [n = 9 (non- and single tg) and n = 5 (iTDP-43)]. Primary antibodies were against human TDP-43 (Proteintech, Chicago, IL), choline acetyltransferase (ChAT; Millipore, Burlington, MA), synaptophysin (Abcam, Cambridge, UK), and laminin (Sigma-Aldrich, St. Louis, MO). Sections were imaged on an Olympus BX51 microscope equipped with a DP70 camera (Olympus, Tokyo, Japan). Cortical thickness was determined on sagittal sections by using the measurement tool of the microscope software. Neuromuscular junction (NMJ) staining with bungarotoxin (Sigma-Aldrich) and synaptophysin antibody was done on fresh-frozen sections of tibialis anterior muscle as previously described.
      • Morsch M.
      • Reddel S.W.
      • Ghazanfari N.
      • Toyka K.V.
      • Phillips W.D.
      Muscle specific kinase autoantibodies cause synaptic failure through progressive wastage of postsynaptic acetylcholine receptors.

      Statistical Analysis

      Statistical analysis was performed with the Prism software package version 6.0 (GraphPad Inc., San Diego, CA) by using t-tests for pairwise comparison and analysis of variance for group comparison. All values are presented as means ± SEM.

      Results

      Loss of Upper Motor Neurons in Aged iTDP-43A315T Mice

      Mutant TDP-43–expressing iTDP-43A315T mice were compared with single-transgenic [nonexpressing pTRE-TDP-43A315T(13)] or nontransgenic control littermates throughout the study. We previously reported reduced brain and hippocampal volume in 6-month–old iTDP-43A315T mice compared with controls, without analyzing effects on other brain areas.
      • Ke Y.D.
      • van Hummel A.
      • Stevens C.H.
      • Gladbach A.
      • Ippati S.
      • Bi M.
      • Lee W.S.
      • Kruger S.
      • van der Hoven J.
      • Volkerling A.
      • Bongers A.
      • Halliday G.
      • Haass N.K.
      • Kiernan M.
      • Delerue F.
      • Ittner L.M.
      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      To further analyze this MRI data set, volumetric analysis of different cortical brain areas was performed with brain atlas–guided mapping (Figure 1A). For comparison, corresponding tissue sections were immunohistochemically stained for transgenic TDP-43A315T expression pattern depicted at the same level (Figure 1A). Comparing volume loss with the overall size of the brain, only atrophy of cortical motor areas was significant in iTDP-43A315T mice compared with controls, whereas visual cortex, striatum, and cerebellum showed no relative atrophy (Figure 1B).
      Figure thumbnail gr1
      Figure 1Motor cortex atrophy in iTDP-43A315T mice. A: Representative magnetic resonance imaging (MRI) coronal sections of 6-month–old iTDP-43A315T (iTDP-43) and control (ctr) mice on the left. Brain areas are outlined. Representative staining for human TDP-43 (hTDP-43) in coronal sections of 6-month–old iTDP-43 and ctr mice. B: Volumetric quantification of delineated brain areas showing reduced volumes in iTDP-43 mice compared with ctr mice. C: Motor cortex (MCx; top) and VCx (visual cortex; bottom) sagittal sections of iTDP-43 and ctr mice stained for transgenic hTDP-43 (brown). Neuronal layers II/III (top bracket) and V (bottom bracket) are indicated in iTDP-43 MCx sections. Note the progressive loss of transgenic hTDP-43–expressing cells in layers II/III/V and atrophy of MCx in iTDP-43 mice with no observed differences in layers I, IV, and VI. D: Progressive MCx thickness decrease in iTDP-43 mice compared with ctr mice over 2, 6, and 12 months of age. Comparable VCx thickness in iTDP-43 and ctr mice. E: Representative immunostaining of 12-month–old iTDP-43 mice and ctr motor cortices for CTIP2 (green), a marker for deeper cortical layers V and VI, hTDP-43 (red), and nuclear DAPI (blue). F: Quantification showing reduced numbers of CTIP2-positive neurons and layer V/VI thickness in iTDP-43 mice compared with ctr mice. Data are expressed as means ± SEM (B, D, and F). n = 3 mice (B); n = 6 mice (D). P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 (t-test). Scale bars: 500 μm (A); 200 μm (C and E). Cb, cerebellum; CPu, caudate putamen; M1, primary motor cortex; M2, secondary motor cortex.
      iTDP-43A315T mice had cortical atrophy and loss of transgenic TDP-43A315T–expressing layer V neurons at 4.5 months of age.
      • Ke Y.D.
      • van Hummel A.
      • Stevens C.H.
      • Gladbach A.
      • Ippati S.
      • Bi M.
      • Lee W.S.
      • Kruger S.
      • van der Hoven J.
      • Volkerling A.
      • Bongers A.
      • Halliday G.
      • Haass N.K.
      • Kiernan M.
      • Delerue F.
      • Ittner L.M.
      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      To determine progression of cortical atrophy, the analysis was extended to older mice. Brain area–specific analysis of immunostained brain sections showed that numbers of neurons expressing transgenic TDP-43A315T in the motor cortex decreased mostly by 6 months in iTDP-43A315T mice (Figure 1C). The continuing significant shrinkage of the motor cortex at 12 months of age, however, resulted in higher density of the remaining iTDP-43A315T–expressing transgenic neurons. Accordingly, density of TDP-43A315T–positive neurons per area in layer II/III decreased from 2 to 6 months of age (number of neurons/0.1 mm2: 100.1 ± 5.5 versus 61.3 ± 6.1; n = 4; P = 0.0033), consistent with loss of neurons, but apparently increased from 6 to 12 months (number of neurons/0.1 mm2: 61.3 ± 6.1 versus 139.1 ± 2.5; n = 4; P < 0.0001). However, numbers of TDP-43A315T–positive neurons actually remained comparable between 6 and 12 months of age, when considering the reduced thickness in layer II/III because of atrophy (number of neurons/0.1 mm2: 49.0 ± 4.9 versus 57.9 ± 1.1; n = 4; P = 0.1257), suggesting no further loss of neurons after 6 months of age. Furthermore, ventricles were enlarged in 12-month–old iTDP-43A315T mice compared with littermate controls (data not shown). By 6 months of age, loss of TDP-43A315T–expressing motor cortex neurons was apparent in cortical layers II/III and, in particular, V (Figure 1C). In contrast, the visual cortex of iTDP-43A315T mice was of comparable thickness at 2 and 6 months of age and showed only atrophy at 12 months of age with loss of TDP-43A315T–expressing neurons (Figure 1C). Quantification of cortical thickness confirmed progressive atrophy of the motor cortex in iTDP-43A315T mice compared with controls as they aged, whereas atrophy occurred in the visual cortex only at 12 months of age (Figure 1D). Staining with a deep cortical layer V/VI marker, CTIP2, further confirmed a loss of neurons in these motor cortical layers (Figure 1E). Accordingly, layer width and number of CTIP2-positive neurons were reduced in iTDP-43A315T mice compared with controls at 12 months of age (Figure 1F). Taken together, aged iTDP-43A315T mice had brain atrophy that particularly affected the motor cortex, first layer V, followed by layer II/III neurons.

      Severe Motor Deficits in Aged iTDP-43A315T Mice

      Young iTDP-43A315T mice had early-onset motor deficits at 3 months of age.
      • Ke Y.D.
      • van Hummel A.
      • Stevens C.H.
      • Gladbach A.
      • Ippati S.
      • Bi M.
      • Lee W.S.
      • Kruger S.
      • van der Hoven J.
      • Volkerling A.
      • Bongers A.
      • Halliday G.
      • Haass N.K.
      • Kiernan M.
      • Delerue F.
      • Ittner L.M.
      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      Here, 1-year–old iTDP-43A315T and control mice were subjected to the same motor test paradigms used earlier in adolescent mice. Specifically, the mice were tested using pole, wire, and beam tests as well as accelerating Rotarod. Notably, the aged iTDP-43A315T mice were not able to complete the beam tests, due to severe motor impairments (data not shown). Similarly, iTDP-43A315T mice were not capable of turning or descending when placed on the top of a vertical beam for pole testing, in contrast to control mice which reached the base within 27.9 ± 15.5 seconds (Figure 2A). Although control mice were able to hold onto an inverted wire mesh for 95.9 ± 30.7 seconds, iTDP-43A315T mice dropped off significantly earlier at 11.4 ± 3.9 seconds (Figure 2B). During Rotarod testing, iTDP-43A315T mice failed to stay on the accelerating rod for more than 34.1 ± 3.2 seconds, whereas control animals remained on the rod for significantly longer at 68.8 ± 5.7 seconds (Figure 2C). Notably, control mice improved their performance on the Rotarod significantly over the three testing days, whereas iTDP-43A315T mice showed no improvement. Despite the severe motor deficits, limb paralysis or decreased survival was not observed in the colony of iTDP-43A315T mice, followed up to 1 year of age. Taken together, aged iTDP-43A315T mice had severe motor deficits.
      Figure thumbnail gr2
      Figure 2Severe motor deficits in aged iTDP-43A315T mice. A: Aged control (ctr) mice descend from the vertical pole significantly faster than iTDP-43A315T mice, which all fail to turn and descend within the test cutoff time of 120 seconds. B: iTDP-43A315T mice fall off an inverted wire mesh significantly earlier than ctr mice. C: iTDP-43A315T mice fall off the accelerating rod significantly earlier than ctr mice during Rotarod testing. Ctr mice but not iTDP-43A315T mice improve significantly over the testing period. Data are expressed as means ± SEM. n = 7 mice for each test. P < 0.05, ∗∗P < 0.01 (t-test); P < 0.05, ††P < 0.01, and ††††P < 0.0001 (two-way analysis of variance; Sidak's post hoc test).

      Progressive Gait Problems in Aging iTDP-43A315T Mice

      ALS is characterized by hypokinesia and gait deficits,
      • Moon Y.
      • Sung J.
      • An R.
      • Hernandez M.E.
      • Sosnoff J.J.
      Gait variability in people with neurological disorders: a systematic review and meta-analysis.
      and gait changes have not been previously investigated in iTDP-43A315T mice. Progressive muscle atrophy and motor problems observed in iTDP-43A315T mice would likely affect the overall movement of the animals. Therefore, iTDP-43A315T and control mice at different ages were subjected to digital gait analysis while moving on a treadmill, and different parameters of the gait dynamics and patterns were analyzed. At 8 months of age, the gait pattern of iTDP-43A315T mice was already different from nontransgenic and single transgenic littermate controls, with reduced step length and increased step frequency at the same treadmill speed (Figure 3, A and B ). Digital analysis of individual stride phases revealed significantly reduced stride time (time to complete a full step cycle), stride length (distance covered per step), swing time (time paws are in the air during each step), and propel time (time spent to move forward), together with increased compensatory stride frequency in iTDP-43A315T mice compared with control mice at 8 months of age (Figure 3B). When comparing gait patterns over time, it was found that at 1 month of age, swing time of both front and hind limbs were already significantly reduced in iTDP-43A315T mice (Figure 3C). Similar deficits were detected at 3 and 8 months of age in iTDP-43A315T mice, with the relative deficits for the front limbs becoming larger over time (Figure 3C). Gait symmetry (ratio of fore limb stepping forward to hind limb stepping frequency) typically declines with age, as seen in nontransgenic and single transgenic controls, when comparing 1-month–old mice with 8-month–old mice (Figure 3D). Failure to decline in 8-month–old iTDP-43A315T mice indicated increased stepping frequency of the hind limbs to propel forward. At 1 month of age, paw areas at peak stance were unchanged in iTDP-43A315T mice (Figure 3E). However, at 3 months of age paw areas at peak stance of the hind limbs were significantly smaller in iTDP-43A315T mice, and eventually both hind and front limbs were significantly affected at 8 months of age, indicative of progressive mechanical changes (Figure 3E). This was further supported by a reduced change of paw area over time in the hind limbs of 3- and 8-month–old iTDP-43A315T mice but not in 1-month–old iTDP-43A315T mice, suggesting problems with shifting load on and off the paws during walking (Figure 3F). Taken together, iTDP-43A315T mice displayed significant and progressive gait deficits as they aged.
      Figure thumbnail gr3
      Figure 3Progressive gait problems in aging iTDP-43A315T mice. A: Representative gait pattern of nontransgenic (non-tg) and single-transgenic (single-tg) control and iTDP-43A315T (iTDP-43) mice at 8 months of age. B: Gait parameters of non-tg, single-tg, and iTDP-43A315T mice. C Left: Reduced swing duration of fore and hind limbs in iTDP-43A315T mice compared with non-tg and single-tg controls. Right: Progressive decline in swing duration of fore limbs in iTDP-43A315T mice. D: Gait symmetry in non-tg, single-tg, and iTDP-43A315T mice. E: Progressive decline in peak stance area of hind limbs followed by fore limbs in iTDP-43A315T mice compared with non-tg and single-tg controls. F: Reduced change of paw area in hind limbs of iTDP-43A315T mice compared with non-tg and single-tg controls. Data are expressed as means ± SEM (BF). n = 4 non-tg mice (B); n = 14 single-tg mice (B); n = 10 iTDP-43 mice (B); n = 10 non-tg 1-month–old mice (C, left, and DF); n = 28 single-tg 1-month–old mice (C, left, and DF); n = 10 iTDP-43 1-month–old mice (C, left, and DF); n = 4 non-tg 3-month–old mice (C, left, and DF); n = 12 single-tg 3-month–old mice (C, left, and DF); n = 4 iTDP-43 3-month–old mice (C, left, and DF); n = 4 non-tg 8-month–old mice (Cleft, and DF); n = 14 single-tg 8-month–old mice (C, left, and DF); n = 10 iTDP-43 8-month–old mice (C, left, and DF); n = 10 mice 1 month old (C, right), n = 4 mice 3 months old (C, right); n = 10 mice 8 months old (C, right). P < 0.05l, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 (one-way analysis of variance; Sidak's post hoc test).

      Muscle Atrophy in Aged iTDP-43A315T Mice

      Motor deficits in adolescent iTDP-43A315T mice were accompanied by muscle atrophy.
      • Ke Y.D.
      • van Hummel A.
      • Stevens C.H.
      • Gladbach A.
      • Ippati S.
      • Bi M.
      • Lee W.S.
      • Kruger S.
      • van der Hoven J.
      • Volkerling A.
      • Bongers A.
      • Halliday G.
      • Haass N.K.
      • Kiernan M.
      • Delerue F.
      • Ittner L.M.
      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      With the use of cross sections of the tibialis anterior muscle, fiber diameter distribution was determined in 1-year–old iTDP-43A315T and control mice. Immunostaining of basal membrane laminin showed smaller muscle fibers in iTDP-43A315T mice compared with large diameter fibers in control muscles (Figure 4A). Muscle fiber diameter distribution in iTDP-43A315T mice was left shifted compared with control mice, in line with atrophy (Figure 4B). Consistent with muscle atrophy, forearm grip strength was significantly reduced in aged iTDP-43A315T mice compared with control mice (Figure 4C). Muscles from 5- and 8-month–old iTDP-43A315T mice and controls were stained with bungarotoxin to label postsynaptic acetylcholine receptors and antibodies to presynaptic synaptophysin to visualize the architecture of NMJs. Despite the apparent atrophy of muscles in iTDP-43A315T mice, NMJs presented similarly in control and iTDP-43A315T tibialis anterior muscles at both ages (Figure 4D). Accordingly, postsynaptic acetylcholine receptor distribution was indistinguishable between iTDP-43A315T and control muscles at 5 and 8 months of age. Although presynaptic synaptophysin distribution was comparable in iTDP-43A315T and control muscles at 5 months of age, there was a nonsignificant trend to reduced synaptophysin staining in iTDP-43A315T mice at 8 months of age (Figure 4E). Taken together, aged iTDP-43A315T mice had severe muscle atrophy, reduced grip strength, yet no overt changes to NMJs.
      Figure thumbnail gr4
      Figure 4Substantial muscle atrophy in aged iTDP-43A315T mice. A: Staining of basal membrane laminin (green) of muscle sections from 12-month–old control (ctr) and iTDP-43A315T (iTDP-43) mice. B: Shifted muscle fiber diameter distribution in 12-month–old iTDP-43 mice compared with ctr mice. Reduced mean muscle fiber diameter in 12-month–old iTDP-43 mice compared with ctr mice. C: Reduced grip strength in 12-month–old iTDP-43 mice compared with ctr mice. D: Examples of postsynaptic acetylcholine receptor (AChR; red) and presynaptic synaptophysin (Syp; green) staining of neuromuscular junctions (NMJs) from 5- and 8-month–old ctr and iTDP-43 mice. E: Unchanged postsynaptic AChR and presynaptic Syp distribution and co-localization in NMJs of ctr and iTDP-43 mice. Data are expressed as means ± SEM (B, C, E). n = 7 ctr mice (B and C); n = 5 iTDP-43 mice (B); n = 7 iTDP-43 mice (C); n = 6 ctr mice 5 months old (E); n = 4 iTDP-43 mice 5 months old (E); n = 5 ctr mice 8 months old (E); n = 5 iTDP-43 mice 8 months old (E). P < 0.05 (t-test). Scale bars: 50 μm (A); 10 μm (D).

      TDP-43 Pathologic Disorders in Spinal Cord Motor Neurons of Aged iTDP-43A315T Mice

      Transgenic TDP-43A315T expression in spinal cord motor neurons was associated with TDP-43 pathologic disorders in adolescent iTDP-43A315T mice.
      • Ke Y.D.
      • van Hummel A.
      • Stevens C.H.
      • Gladbach A.
      • Ippati S.
      • Bi M.
      • Lee W.S.
      • Kruger S.
      • van der Hoven J.
      • Volkerling A.
      • Bongers A.
      • Halliday G.
      • Haass N.K.
      • Kiernan M.
      • Delerue F.
      • Ittner L.M.
      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      To determine the degree of pathologic disorder in aged iTDP-43A315T mice, serial sections were stained at different levels of transgenic and control spinal cords with antibodies to human TDP-43 (Figure 5A). At all levels of iTDP-43A315T spinal cords, some large caliper motor neurons expressed transgenic TDP-43A315T with nuclear and abundant cytoplasmic localization. Serial sections stained for the motor neuron marker ChAT and human TDP-43 confirmed the small number motor neurons expressing TDP-43A315T (Figure 5B). Quantification of these stainings revealed comparable numbers of ChAT-positive motor neurons in both control and iTDP-43A315T mice (Figure 5B). Numbers of human TDP-43–positive neurons in spinal cords of iTDP-43A315T mice were of similar numbers to that of ChAT-positive cells; however, only 14.1% ± 4.1% of ChAT-positive motor neurons expressed transgenic human TDP-43, suggesting most of the human TDP-43–positive neurons were ChAT-negative. In addition to motor neurons, a large number of smaller cells also stained positively for transgenic TDP-43A315T, with some cytoplasmic localization. Double-labeling with the neuronal marker NeuN confirmed purely neuronal expression of transgenic TDP-43A315T throughout the gray matter of iTDP-43A315T spinal cords (Figure 5C). Co-staining with parvalbumin showed a near complete overlap with TDP-43A315T, consistent with transgene expression in interneurons (Figure 5D). Astrocytes and microglia did not express TDP-43A315T (data not shown), and no staining of human TDP-43 was observed in control spinal cords. Taken together, transgenic TDP-43A315T was expressed mostly in interneurons and some motor neurons in the spinal cord of iTDP-43A315T mice, where it localized to both the nucleus and cytoplasm, but did not cause overt lower motor neuron loss.
      Figure thumbnail gr5
      Figure 5TDP-43 expression in spinal cord neurons in aged iTDP-43A315T mice. A: Representative sections of spinal cords from 12-month–old control (ctr) and iTDP-43A315T (iTDP-43) mice stained with human TDP-43 (hTDP-43)–specific antibodies (brown). Boxed areas in top panels indicate area shown at higher magnification in lower panels. Some large caliper motor neurons present with abundant cytoplasmic hTDP-43 staining (arrows), as well as several smaller neurons with hTDP-43 expression (arrowheads). B: Representative immunofluorescence images showing co-localization of transgenic TDP-43A315T and the motor neuron marker choline acetyltransferase (ChAT) in the spinal cords of iTDP-43 mice. Quantification of numbers of Chat-, hTDP-43–, and double-positive motor neurons of serial spinal cord section from ctr and iTDP-43 mice. C: hTDP-43–positive cells in the spinal cord of iTDP-43 mice are also NeuN-positive. D: hTDP-43–positive cells in the spinal cord of iTDP-43 mice are also positive for the interneuron marker parvalbumin (parv). Data are expressed as means ± SEM (B). n = 10 ctr mice (B); n = 5 iTDP-43 mice (B). Scale bars: 200 μm (A, top); 100 μm (A, bottom); 50 μm (BD).

      Discussion

      In the present study, the phenotypic presentation of iTDP-43A315T mice was followed with age. At 1 year of age, iTDP-43A315T mice showed severe motor deficits, muscle atrophy, and cytoplasmic TDP-43 accumulation in spinal cord motor neurons. Furthermore, iTDP-43A315T mice developed progressive gait problems with aging.
      We have previously reported a selective and progressive loss of pyramidal layer V neurons in the cortex of young iTDP-43A315T mice, with no change in layer II/III and VI neurons, despite marked expression of transgenic human TDP-43.
      • Ke Y.D.
      • van Hummel A.
      • Stevens C.H.
      • Gladbach A.
      • Ippati S.
      • Bi M.
      • Lee W.S.
      • Kruger S.
      • van der Hoven J.
      • Volkerling A.
      • Bongers A.
      • Halliday G.
      • Haass N.K.
      • Kiernan M.
      • Delerue F.
      • Ittner L.M.
      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      Similarly, there was no loss of spinal cord motor neurons in young iTDP-43A315T mice, but there was a muscle weakness and atrophy, indicating neuronal dysfunction.
      • Ke Y.D.
      • van Hummel A.
      • Stevens C.H.
      • Gladbach A.
      • Ippati S.
      • Bi M.
      • Lee W.S.
      • Kruger S.
      • van der Hoven J.
      • Volkerling A.
      • Bongers A.
      • Halliday G.
      • Haass N.K.
      • Kiernan M.
      • Delerue F.
      • Ittner L.M.
      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      Cortical motor areas were relatively earlier affected by atrophy than other cortical areas in aged iTDP-43A315T mice, whereas there was still no overt loss of spinal cord motor neurons. Interestingly, layer II/III showed a significant degeneration in 12-month–old iTDP-43A315T mice. In contrast, in the visual cortex only layer V neurons were affected at 12 months of age in iTDP-43A315T mice. Distinct effects of transgenic mutant TDP-43 expression on cortical neurons have also recently been reported in independent TDP-43Q331K and TDP-43A315T mouse strains.
      • Fogarty M.J.
      • Klenowski P.M.
      • Lee J.D.
      • Drieberg-Thompson J.R.
      • Bartlett S.E.
      • Ngo S.T.
      • Hilliard M.A.
      • Bellingham M.C.
      • Noakes P.G.
      Cortical synaptic and dendritic spine abnormalities in a presymptomatic TDP-43 model of amyotrophic lateral sclerosis.
      • Handley E.E.
      • Pitman K.A.
      • Dawkins E.
      • Young K.M.
      • Clark R.M.
      • Jiang T.C.
      • Turner B.J.
      • Dickson T.C.
      • Blizzard C.A.
      Synapse dysfunction of layer V pyramidal neurons precedes neurodegeneration in a mouse model of TDP-43 proteinopathies.
      Furthermore, selective neuronal loss associated with TDP-43 pathologic disorders has recently been reported for cranial nerve motor nuclei, with cell loss in the hypoglossal nucleus 8 weeks after expression of a human TDP-43 variant that is excluded from entering the nucleus (TDP-43-ΔNLS) in aged mice, while sparing oculomotor, trigeminal, and facial nuclei.
      • Spiller K.J.
      • Cheung C.J.
      • Restrepo C.R.
      • Kwong L.K.
      • Stieber A.M.
      • Trojanowski J.Q.
      • Lee V.M.
      Selective motor neuron resistance and recovery in a new inducible mouse model of TDP-43 proteinopathy.
      Expression of TDP-43A315T predominantly in parvalbumin-positive spinal cord interneurons and some motor neurons may impair neuronal circuits that control muscle function, thereby contributing to muscle atrophy in aged iTDP-43A315T mice in the absence of motor neuron loss (up to an age of 12 months analyzed here) or changes in NMJs. Lack of corticospinal afferents, due to upper motor neuron loss, may further contribute to the muscular dysfunction. However, the exact mechanism(s) underlying muscle atrophy in iTDP-43A315T mice remains to be shown. Taken together, the differential vulnerability of different neuron types may suggest the intriguing possibility that there are different pathologic TDP-43 species with distinct effects on neurons, and models such as the iTDP-43A315T mice may assist in future identification of these species.
      Degeneration and TDP-43 pathologic disorders in motor areas and muscle disorders affected functional performance of iTDP-43A315T mice. Accordingly, motor testing of aged iTDP-43A315T mice indicated severe deficits, including the inability to perform tasks that young transgenic mice were once able to successfully complete. These findings are in line with our initial report of iTDP-43A315T mice with progression of motor deficits from none at 1 month of age to significant deficits already at 3 months of age.
      • Ke Y.D.
      • van Hummel A.
      • Stevens C.H.
      • Gladbach A.
      • Ippati S.
      • Bi M.
      • Lee W.S.
      • Kruger S.
      • van der Hoven J.
      • Volkerling A.
      • Bongers A.
      • Halliday G.
      • Haass N.K.
      • Kiernan M.
      • Delerue F.
      • Ittner L.M.
      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      Therefore, iTDP-43A315T mice develop significant motor deficits early on, which persist as they age. This parallels the early degeneration of upper motor neuron areas and the progressive development of TDP-43 pathologic disorders in lower motor neurons and muscle wasting.
      Patients diagnosed with ALS have been reported to have abnormal gait parameters.
      • Iancu Ferfoglia R.
      • Heritier Barras A.C.
      • Pollak P.
      • Janssens J.P.
      • Pradat P.F.
      • Allali G.
      Gait performance and use of mental imagery as a measure of disease progression in amyotrophic lateral sclerosis.
      • Xia Y.
      • Gao Q.
      • Lu Y.
      • Ye Q.
      A novel approach for analysis of altered gait variability in amyotrophic lateral sclerosis.
      • Radovanovic S.
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      • Peric S.
      • Basta I.
      • Kostic V.
      • Stevic Z.
      Gait in amyotrophic lateral sclerosis: is gait pattern differently affected in spinal and bulbar onset of the disease during dual task walking?.
      Digital and longitudinal gait analysis has not been done in TDP-43 transgenic ALS mouse models before. iTDP-43A315T mice have early-onset changes in gait parameters, which become more complex over time, including some deficits that progress from initially affecting hind limbs at 3 months of age to eventually impairing both fore and hind limbs at 8 months of age. Homozygous TDP-43M337V mice displayed irregular gait patterns at weaning together with a premature mortality, which precluded a detailed gait analysis over time.
      • Xu Y.F.
      • Zhang Y.J.
      • Lin W.L.
      • Cao X.
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      • Dickson D.W.
      • Lewis J.
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      Expression of mutant TDP-43 induces neuronal dysfunction in transgenic mice.
      Furthermore, abnormal gait has been reported in several mouse models of ALS without transgenic TDP-43 expression. Mancuso et al
      • Mancuso R.
      • Olivan S.
      • Osta R.
      • Navarro X.
      Evolution of gait abnormalities in SOD1(G93A) transgenic mice.
      provided a detailed gait analysis of SOD1G93A–expressing ALS mice before and after onset of overt motor deficits on the Rotarod. Similar to iTDP-43A315T mice, SOD1G93A mice developed progressively increasing gait deficits. Interestingly, analysis of the stride phases showed progressive increase in stride, stance, swing, and propulsion times and reduced stride frequency,
      • Mancuso R.
      • Olivan S.
      • Osta R.
      • Navarro X.
      Evolution of gait abnormalities in SOD1(G93A) transgenic mice.
      as opposed to increased stride frequency and reduced stride phase times found in iTDP-43A315T mice. These differences may be explained because SOD1G93A mice have a rapid progressive hind limb paralysis and loss of spinal cord motor neurons, whereas the iTDP-43A315T mice are characterized by slow progressive muscle atrophy, which limits limb strength and movement, and neuronal loss limited cortical motor areas, but do not develop paralysis. Furthermore, transgenic TDP-43A315T expression was found predominantly in spinal cord interneurons and only a limited number of motor neurons, which may suggest that dysfunction of neuronal circuits that controls rhythm of locomotor movement, including left–right alternation and flexor–extensor equilibrium,
      • Ramirez-Jarquin U.N.
      • Tapia R.
      Excitatory and inhibitory neuronal circuits in the spinal cord and their role in the control of motor neuron function and degeneration.
      rather than loss of spinal cord motor neurons contribute to gait problems in iTDP-43A315T mice. However, Vergouts et al
      • Vergouts M.
      • Marinangeli C.
      • Ingelbrecht C.
      • Genard G.
      • Schakman O.
      • Sternotte A.
      • Calas A.G.
      • Hermans E.
      Early ALS-type gait abnormalities in AMP-dependent protein kinase-deficient mice suggest a role for this metabolic sensor in early stages of the disease.
      found early gait anomalies with reduced stride length in SOD1G93A mice,
      • Vergouts M.
      • Marinangeli C.
      • Ingelbrecht C.
      • Genard G.
      • Schakman O.
      • Sternotte A.
      • Calas A.G.
      • Hermans E.
      Early ALS-type gait abnormalities in AMP-dependent protein kinase-deficient mice suggest a role for this metabolic sensor in early stages of the disease.
      comparable with iTDP-43A315T mice. Furthermore, a polyGA transgenic C9ORF72 model developed progressive gait anomalies and TDP-43 pathologic disorders.
      • Schludi M.H.
      • Becker L.
      • Garrett L.
      • Gendron T.F.
      • Zhou Q.
      • Schreiber F.
      • Popper B.
      • Dimou L.
      • Strom T.M.
      • Winkelmann J.
      • von Thaden A.
      • Rentzsch K.
      • May S.
      • Michaelsen M.
      • Schwenk B.M.
      • Tan J.
      • Schoser B.
      • Dieterich M.
      • Petrucelli L.
      • Holter S.M.
      • Wurst W.
      • Fuchs H.
      • Gailus-Durner V.
      • de Angelis M.H.
      • Klopstock T.
      • Arzberger T.
      • Edbauer D.
      Spinal poly-GA inclusions in a C9orf72 mouse model trigger motor deficits and inflammation without neuron loss.
      Taken together, abnormal gait is found in several ALS mouse models; however, its presentation depends on the underlying phenotype.

      Conclusions

      The present study showed the progression of functional deficits in iTDP-43A315T mice as they age, including a detailed analysis of progressive gait problems. Furthermore, differences in neuronal loss between cortical motor areas and spinal cord provided additional evidence to the concept of selective vulnerability of different neuronal populations to TDP-43 pathologic disorders,
      • Ke Y.D.
      • van Hummel A.
      • Stevens C.H.
      • Gladbach A.
      • Ippati S.
      • Bi M.
      • Lee W.S.
      • Kruger S.
      • van der Hoven J.
      • Volkerling A.
      • Bongers A.
      • Halliday G.
      • Haass N.K.
      • Kiernan M.
      • Delerue F.
      • Ittner L.M.
      Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.
      • Spiller K.J.
      • Cheung C.J.
      • Restrepo C.R.
      • Kwong L.K.
      • Stieber A.M.
      • Trojanowski J.Q.
      • Lee V.M.
      Selective motor neuron resistance and recovery in a new inducible mouse model of TDP-43 proteinopathy.
      and they may assist in future identification of specific toxic TDP-43 species. Finally, differences in phenotype between ALS mouse models, including between TDP-43 transgenic stains, highlight the importance of a thorough characterization of each model to determine their value for investigating pathomechanisms and testing drugs.

      Acknowledgments

      We thank the staff of the Biological Resources Center at the University of New South Wales for animal care.
      Y.D.K. designed and supervised the study; A.v.H., G.C., J.v.d.H., M.M., S.I., L.S., M.B., P.R.A., W.S.L., M.P., and T.A.B. performed experiments; A.v.H., M.M., M.B. R.S.C., L.M.I., and Y.D.K. analyzed data; G.M.H., O.P., M.C.K., and R.S.C. acquired funding support; and L.M.I. and Y.D.K. wrote the manuscript with input from all authors.

      Supplemental Data

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