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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2013 Jan 14;94(1):56–64. doi: 10.1111/iep.12006

Premature death of TDP-43 (A315T) transgenic mice due to gastrointestinal complications prior to development of full neurological symptoms of amyotrophic lateral sclerosis

Mohammad A Esmaeili *, Marzieh Panahi *, Shilpi Yadav *, Leah Hennings †, Mahmoud Kiaei *
PMCID: PMC3575874  PMID: 23317354

Abstract

Abnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP-43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Transgenic mouse lines overexpressing wild-type or mutant TDP-43 exhibit ALS-like symptom, motor abnormalities and early paralysis followed by death. Reports on lifespan and phenotypic behaviour in Prp-TDP-43 (A315T) vary, and these animals are not fully characterized. Although it has been proposed that the approximate 20% loss of motor neurons at end stage is responsible for the severe weakness and death in TDP-43 mice, this degree of neurologic damage appears insufficient to cause death. Hence we studied these mice to further characterize and determine the reason for the death. Our characterization of TDP-43 transgenic mice showed that these mice develop ALS-like symptoms that later become compounded by gastrointestinal (GI) complications that resulted in death. This is the first report of a set of pathological evidence in the GI track that is strong indicator for the cause of death of Prp-hTDP-43 (A315T) transgenic mice.

Keywords: amyotrophic lateral sclerosis, gastrointestinal track, mouse model of ALS, TDP-43

Amyotrophic lateral sclerosis is a progressively lethal degenerative disease that primarily involves motor neurons. The exact mechanism of neuronal death in ALS remains unknown. Several mechanisms have been implicated, including glutamate excitotoxicity (Rothstein 1995; Maragakis & Rothstein 2001), aberrant protein aggregates containing mutant superoxide dismutase 1 (Bruijn et al. 1998; Wood et al. 2003), mislocalization and hypophosphorylation of neurofilaments (Lariviere & Julien 2004), oxidative damage and enhanced free radical formation (Agar & Durham 2003), neuroinflammation (Almer et al. 2001; Raoul et al. 2002; Hensley et al. 2003; Kiaei et al. 2005, 2006), proteasome dysfunction (Kabashi et al. 2004; Urushitani et al. 2004), mitochondrial dysfunction (Xu et al. 2004) and apoptosis (Przedborski 2004; Pasinelli & Brown 2006). TDP-43 aggregation was identified in human ALS spinal cord post-mortem tissues (Neumann et al. 2006), and follow-up studies analysing genetic samples from ALS patients reported mutation in tardbp gene in patients with ALS and FTD-ALS (Arai et al. 2006; Neumann et al. 2006; Sleegers et al. 2008; Kwiatkowski et al. 2009; Vance et al. 2009).

The hypothesis of TDP-43 proteinopathy and its role in neurodegeneration was strengthened by mutations found in patients with familial ALS (Gitcho et al. 2008; Kabashi et al. 2008; Yokoseki et al. 2008). In neurodegenerative diseases, TDP-43 can be found in the cytoplasm associated with ubiquitinated inclusions, where the protein is poorly soluble, hyperphosphorylated and cleaved into smaller fragments (25 and 35 kDa), making TDP-43 aggregates a hallmark pathology of ALS and FTLD-U cases (Neumann et al. 2006). Several transgenic mouse models overexpressing human mutant TDP-43 have been created (Kwiatkowski et al. 2009; Wegorzewska et al. 2009; Shan et al. 2010; Stallings et al. 2010; Wils et al. 2010; Gendron & Petrucelli 2011; Swarup et al. 2011). A rat model overexpressing wild-type TDP-43 using AAV-9 showed ALS-like phenotype (Tatom et al. 2009). Wegorzewska et al. 2009, reported the creation of Prp-TDP-43 (A315T) and shortly after became available from the Jackson Laboratory for use by researchers worldwide. The initial report indicated that TDP-43 mice were normal up to 3 months of age. Then they developed a gait abnormality by 3–4 months of age and began to lose weight around 4.5 months of age (Wegorzewska et al. 2009). TDP-43 mice were initially reported to live 154 ± 19 days and lose about 20% of their spinal motor neurons at the end stage. We think that this level of neuronal loss is sufficient to account for death in these mice at this stage.

The lifespan also appeared to be reduced when these mice were crossed with C57BL6 mice [Jax database, http://jaxmice.jax.org (stock #010700)]. Therefore we imported several breeding pairs from the Jackson Laboratory and established a colony for further observation, characterization and to determine the cause of death. Here, we describe our findings in further characterization of these TDP-43 mice. To the best of our knowledge, this is the first report of a gastrointestinal (GI) abnormality and thus possible cause of death for TDP-43 mice due to the gastrointestinal complications not necessarily due to the spinal motor neuron loss.

Materials and methods

Mouse model of ALS and behavioural studies

Transgenic mice, Prp-hTDP-43 (A315T), were purchased from Jackson Laboratories and a colony was established in the animal vivarium at University of Arkansas for Medical Sciences (UAMS). Animals were maintained and handled using approved protocols according to UAMS IACUC guidelines. The mice were group housed under a 12-h light/dark cycle with access to food and water at all times, and their general heath was regularly checked. The TDP-43 transgenic mice had been generated and transgenic offspring genotyped by PCR assay of DNA obtained from tail tissue as described (Wegorzewska et al. 2009). These TDP-43 transgenic mice had been generated by using a cDNA encoding human TDP-43 with an N-terminal Flag tag and A315T mutation placed in front of prion promoter. This was linearized and microinjected into fertilized eggs from hybrid C57Bl6/JxCBA mice. One founder became the TDP-43 transgenic mice line and was backcrossed to C57Bl6/J for two generations. This line was submitted to the Jackson Laboratory Repository and then became available for purchase (Jax stock No. 010700). Breeder pairs were ordered from Jackson Laboratory (Bar Harbor, ME, USA) to establish a colony, and we established a line and maintained them by breeding transgenic males with wild-type littermate females or breeding transgenic females with wild-type littermate male to maintain the same background (Jax stated that the background is 80% congenic C57Bl6/J) as the founder line. Animals were fed ad lib with a rodent diet called the Teklad Global Diet #2019 (Harlan Laboratories, Houston, Texas, USA), which is a 19% protein extruded rodent diet and is a typical diet for mice. Although we did not measure the food consumption rate in TDP-43 transgenics vs. wild-type mice, TDP-43 mice that developed GI abnormalities lost weight, which is an indication that they may have consumed less food. Based on our previous experience, a mouse that weighs around 25 g will consume about 5 g of food. TDP-43 mice with GI problem had reduced food intake, because the GI problem caused mice to reduce their activity and look frizzled, hunched and unhappy suggesting that they were suffering from abdominal pain.

According to Jackson Laboratories website information on this stock, 100% C57Bl6/J congenic TDP-43 mice had a shorter lifespan (average of 97 ± 11 days) and the difference between male and female persisted. This shorter lifespan can result in a shorter breeding window for males, and hence problems can be encountered in maintaining the line. In our studies we found that transgenic females will breed and their breeding window is much longer than that of males. Therefore, mice bred as 80% C57Bl6/J congenic are more suitable for line maintenance. Motor performance was assessed using a rotarod apparatus (Harvard apparatus, Holliston, MA, USA). The rotarod test measures the capacity of mice to remain upright on a rod rotating at 12 rotations per minute. The time elapsed before a mouse falls off the rotarod measures its competency at the task and provides a reliable indication of motor performance and progression of motor dysfunction. Mice were trained for 1 week on the rotarod and subsequently tested twice per week starting at 60 days of age. Each mouse underwent three trials per session; the best result of three trials was recorded, with the maximum result set at 180 s. Mice were observed each morning and were weighed twice per week. Mice were deemed to have reached the end stage of the disorder when they were no longer able to initiate movement after being gently agitated for 2 min or could not right themselves within 30 s when placed on their backs. When they reached this stage, the animals were euthanized by sodium pentobarbital overdose.

Histology and Immunohistochemical analysis

Mice were perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4. Immediately following perfusion, intestines were removed and placed into 10% neutral buffered formalin. Longitudinal and transverse sections of each segment of the gastrointestinal tract were processed and embedded into paraffin, sectioned at 4 μm and stained with H&E. The ileocecocolic region was embedded in a single block. Sections were examined under a light microscope by a veterinary pathologist (LH). Sections from the spinal cord (L1-L6) were similarly processed and embedded.

Selected spinal cord sections were immunostained using the diaminobenzidine (DAB) immunoperoxidase method. The primary antibodies were rabbit polyclonal TDP-43 (ProteinTech Group, Chicago, IL, USA) and mouse monoclonal Neu-N (Millipore, Billerica, MA, USA), which is a neuron-specific marker. Paraffin-embedded GI tissues and spinal cords were processed, and antigen retrieval was performed in a Decloaking Chamber (Biocare, Concord, CA, USA) using Dako Target Retrieval solution (Dako, Carpinteria, CA, USA) for 20 min. Slides were cooled at room temperature for 30 min. Following retrieval, the sections were rinsed in distilled water and transferred to Tris-buffered saline with tween 20, (TBST, Dako). Endogenous peroxidase activity was blocked using Dako Peroxidase block (Dako) for 10 min, and slides were rinsed with TBST. For TDP-43 antibody, Dako Protein Block (Dako) was applied for 10 min and slides were rinsed with TBST. For Neu-N antibody, slides were blocked in 10% normal goat serum (Vector Laboratories, Burlingame, CA) for 30 min and blotted from the edge of the slides prior to incubation with primary antibody. Primary antibodies were diluted in Dako diluent with background reducing components. Slides were incubated with TDP-43 antibody (1:800) at room temperature for 2 h or Neu-N antibody (1:100) at 4°C, overnight. Biotinylated goat anti-rabbit (TDP-43) or anti-mouse (Neu-N) at 1:400 was applied for 30 min, and slides were rinsed in TBST. Detection was achieved by incubation in DAB+ (Dako) for 3 min. Slides were rinsed in tap water, counterstained with Hematoxylin 2 (Richard-Allen Scientific, Kalamazoo, MI) for 30 sections and cover slipped with permanent mounting media. Magnifications noted in figure legends are the objective times 10× eye piece.

Western blotting

Expression level of TDP-43 was assessed by Western blot analysis of tissue extracted from spinal cord and intestinal (caecum) using standard procedures. Thirty microgram of total protein from each sample was loaded into each lane. Anti-TDP-43 antibody was purchased from ProteinTech Group (ProteinTech Group, Chicago, IL).

Statistical analysis

Kaplan–Meier survival analysis was used for survival comparisons. Repeated measures anova was used for rotarod and weight comparisons. Statistical significance was set at the P < 0.05 level.

Results

Phenotype and motor abnormalities

To study the normal function of hTDP-43 and its role in neurodegeneration, we examined TDP-43 mice in (approximately 80%) C57BL6/J background males and females from birth. TDP-43 mice developed an abnormal hindlimb reflex characterized by retraction of hindlimbs towards the trunk when lifted by their tail, as opposed to wild-type littermates, which showed normal extension of the legs (Figure 1a). This reflex was demonstrated as one of the earliest symptoms of loss of motor control in several ALS mouse models (Duchen et al. 1964). A footprint analysis for TDP-43 mice showed a significant ≍ 2-fold decrease in the stride length of hindlimbs (1.5 cm vs. 3.5 cm, P < 0.05) and forelimb (1.5 cm vs. 3.4 cm, P < 0.05). Moreover, footprint of TDP-43 transgenic mice demonstrated markedly wide-based stance, small stride and frequent offline stumbling compared with wild-type littermates, which showed a normal, narrow-based stance with steady, close-proximity forelimb and hindlimb footprints (Figure 1b,c). Prp-hTDP-43 mice also developed a gait abnormality by approximately 105–110 days of age and developed a characteristic ‘swimming’ gait at age 115–120 days, where they were unable to hold their body off the ground, but could still use their limbs for propulsion to slide on their stomachs (Figure 1d). These observations closely resemble what is reported on Thy-1 wild-type TDP-43 transgenic mice (Wils et al. 2010). Females looked normal in the first 4–5 months, and hindlimb and gait abnormalities were detected approximately 160 days postnatal (data not shown). TDP-43 mice showed motor weakness at around 60 days of age by rotarod testing. These TDP-43 mice took longer to be acquainted and learn to stay on the rotarod in comparison with littermate controls. Non-transgenic littermates were easily acquainted and learned to stay on the rotating rod for several minutes after 2–3 days of rotarod trials in 1 week. TDP-43 mice could only stay on the rotarod for a short amount of time (maximum ≍50 s when rod rotating at constant speed of 12 rpm) (Figure 2a). Slower rotarod speed had longer time spend on the rod, for example, at 6 rpm paradigm for rotarod testing, there was no difference for TDP-43 mice as compared to wild-type litter mates; however, transgenic mice gradually showed reduced time spent on the rotarod (data not shown). Weights were measured twice per week, and we found the onset of weight loss started around 90–95 days for male mice and 180–185 days for female mice (Figure 2b). Weight loss was gradual; however, males reached a peak weight of 28 g at 90 days and then lost about 10 g by the time of death. In females, the peak weight was 25 g at 180 days with loss of only 5 g by the time of death.

Figure 1.

Figure 1

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Disease signs in TDP-43 mice. (a) Abnormal limb reflex in TDP-43 males compared to wild-type mice at different ages. (b) Disturbed footprint pattern in TDP-43 mice (at age 110 days) with markedly wide-based stance, small stride and frequent offline stumbling in transgenic mice. (c) Significant 2-fold decrease in the stride of both forelimb and hindlimb (P < 0.05). (d) Swimming gait at age 120 days and end-stage paralysis in TDP-43 male mice. TDP-43 transgenic mouse was unable to move its hindlimb or right itself when placed on its back.

Figure 2.

Figure 2

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Motor performance and body weight analysis. (a) TDP-43 males and non-transgenic little mate controls motor performance and body mass (n = 15 per group). (b) TDP-43 females and non-transgenic little mate control motor performance and body mass (n = 15 per group). Motor performance was assessed using a rotarod apparatus rotating at constant speed of 12 rpm and mice were scored for the length of time on the rotating rod (max 180 s), and details are described in the material and methods. Values are the mean ± SEM.

Survival analysis

Hemizygous mice for TDP-43(A315T) were originally published on a mixed C57BL/6–CBA genetic background and develop a progressive gait disorder around 3–4 months of age with death around 5 months of age. Previous studies have shown that on average males die almost 1 month earlier than females (Wegorzewska et al., 2009). Due to continued backcrossing to C57BL/6J at the Jackson Laboratory Repository, this strain is now fully congenic on a C57BL/6J background. The lifespan in TDP-43 males in pure C57BL/6 background is significantly shorter than those that retain about 20% CBA background (JAX data base). Therefore, this made is much harder to maintain the colony. It is advisable to maintain at least approximately 20% CBA background. This can be achieved by breeding transgenic males with non-transgenic female littermates. Survival differences between male and female mice are still observed. However, hemizygous males on a C57BL/6J genetic background have an average survival time of approximately 3–5 months (97 ± 11 days); this is thus lethal earlier than is seen in hemizygous male on the mixed C57BL/6–CBA genetic background. On a C57BL/6J genetic background, hemizygous females live significantly longer than males (Jax database, http://jaxmice.jax.org/strain/010700.html). In our study, male and female Prp-hTDP-43(A315T) transgenic mice in C57BL6 background (approximately 80%) were analysed for their lifespan. The lifespan in TDP-43(A315T) males was significantly shorter than females (119 ± 18 days vs. 257 ± 15 days respectively), P < 0.001 (Figure 3). The range in survival was from 101 to 137 days for males and 242 to 272 days for females.

Figure 3.

Figure 3

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Kaplan–Meier survival curve of TDP-43 males (=15) and females (n = 10). Significant differences were determined at P < 0.0001.

Gastrointestinal track pathology

The general well-being of TDP-43 mice was evaluated daily beginning at 60 days postnatal, and animals were monitored for changes in physical appearance and motility. We noticed that transgenic mice go through a gradual reduction in mobility, reduced grooming and hunched posture (Figure 4a). These conditions gradually developed further, and transgenic mice became weak, poorly groomed and increasingly hunched. Mice were still capable of bearing their weight and could walk if forced to do so. Their condition rapidly deteriorated, and occasionally an animal was found dead in the cage overnight.

Figure 4.

Figure 4

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(a) Symptomatic TDP-43 male near end stage appears lethargic, frozen, hunched posture (Kyphosis) and reduced mobility (arrow). (b) Photograph showing gross gastrointestinal tract with swollen intestine, enlarged caecum in TDP-43 mouse at the end stage (arrow).

Gross anatomy of gastrointestinal tract of TDP-43 mice

Examination of the intestinal tract from transgenic mice (Figure 4b) with early lesions revealed intestinal dilation. The caecum and lower ileum were mildly to moderately dilated, with dry, firm faecal material. A fibrous band was present at the ileocecocolic junction that appeared to be circumferential in one animal. No luminal obstructions were noted. In more advanced lesions, dilation was marked and extended into the upper small intestine. Contents of the jejunum and duodenum were watery. Stomachs were empty, indicating anorexia in all cases; normal faecal pellets were present in the colon. Age-matched wild-type controls had no gross or histologic lesions.

Histopathological analysis

Dilation of caecum and ileum was evident in sub-gross sections of the ileocecocolic region (Figure 5). Myocytes within the tunica muscularis were mildly, multifocally vacuolated at early stages, progressing to diffuse and moderate to marked vacuolization (Figure 6). Myenteric plexi were similarly vacuolated, although ganglion cells were present. Oedema of the lamina propria and submucosa was one of the most prominent histologic changes in TPD-43 mice. Moderate to marked oedema was present in the caecum and ileum at all stages. At later stage (Figure 7), mild to moderate oedema was present in the upper small intestine and the proximal colon. In the TDP-43 mice demonstrating late symptoms oedema extended to the serosa, and there was increased collagen at the ileocaecal junction (Figure 8). A small area of mucosal erosion with associated suppurative inflammation was noted in this region. Moderate infiltrate of neutrophils was present in the lamina propria and submucosa of the ileocaecal junction at all stages.

Figure 5.

Figure 5

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Microscopic sub-gross images of Ileocecocolic transgenic mice at end stage and wild-type littermate. Ileum is markedly distended in transgenic animal (a) compared to wild type (b). Lymph node (LN) is enlarged and oedematous. Colon (c) is unaffected. Some caecal contents have been removed from affected animals to facilitate processing and sectioning. H&E stain. Bar=2 mm.

Figure 6.

Figure 6

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Marked diffuse vacuolization of smooth muscle fibres (arrow) in transgenic mouse (a) compared to wild type (b). H&E stain, 400× (40× objective, 10× eyepiece) magnification. Bar =50 μm.

Figure 7.

Figure 7

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Mucosal (lamina propria) and submucosal oedema (arrows) in the caecum (top row) and ileum (bottom row) of transgenic mouse (left column) compared to wild-type control (right column). Caecum (top row) and ileum (bottom row). 200× magnification, bar =100 μm.

Figure 8.

Figure 8

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Microscopic [haematoxylin and eosin (H&E) stain] images of Ileocaecal junction of transgenic mouse with serosal oedema and fibrosis. (a) H&E, 100×. (b) Trichrome, 200×. Arrows indicate collagen fibres. (c) WT control, trichrome, 200×.

Next, we studied spinal cord and intestine of both wild-type and transgenic mice for TDP-43 proteinopathy. Spinal cord sections show abnormal neuronal TDP-43 localization. Ventral horn neurons in the spinal cord of transgenic mice lacked robust nuclear TDP-43 staining. The staining appeared to be diffuse and in some neurons there was a resemblance to skein-like inclusions (Figure 9).

Figure 9.

Figure 9

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Immunohistochemistry for TARDBP protein in spinal cord sections. Strong nuclear TDP-43 staining is present in the spinal cord of wild-type mice (arrows) (a); however, spinal cord neurons from TDP-43 mice (at age 110 days) stained abnormally and diffused. Some neurons have skein-like inclusions (arrow) (b).

In intestinal sections TARDP protein appears to be more abundant in transgenic animals compared to wild-type controls, with abundant labelling of neurons in the myenteric plexus (Figure 10).

Figure 10.

Figure 10

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Immunohistochemistry for TARDBP protein in intestinal sections of transgenic (a) and wild-type mice (b) at age 110 days. Vacuolation of myenteric plexus (stars) in A. Cytoplasm of ganglion cells and myocytes stains darker, with cytoplasmic aggregates (arrow) present in transgenic animal. 1000× magnification. Bar =20 μm. Insert: Negative control.

Staining is more intense in transgenic animals, and there is more cytoplasmic accumulation of protein compared to wild type. To verify that intestinal lesions and death occur during a time when neuronal loss from spinal cord is insufficient to cause death, spinal sections were labelled with Neu-N, a neuronal-specific antibody (Figure 11). Neuronal staining is similar in wild-type and transgenic animals and does not warrant a neuronal cell count using unbiased stereology program.

Figure 11.

Figure 11

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No loss of motor neurons is evident in the spinal cord of transgenic (a) compared to wild-type (b) mice at age 110 days. Immunoperoxidase stain, left: 40× magnification. Right: 400× magnification.

Western blot analysis of TDP-43 level in spinal cord and intestinal tissues (caecum) of wild-type and transgenic mice at end stage confirms expression of TDP-43 protein in total soluble homogenates from both spinal cord and the caecum (Figure 12).

Figure 12.

Figure 12

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(a) Western blot analysis of TDP-43 level in spinal cord and intestinal (caecum) from wild-type and transgenic TDP-43 mice at end stage. Lanes 1 and 3 each contain 30 μg of total lysates from spinal cord of wild-type and transgenic mice respectively, and lanes 2 and 4 each contain 30 μg of total lysates from intestine of wild-type and transgenic mice respectively. (b) The density of TDP-43 bands is measured, and the ratios are calculated in arbitrary units.

Discussion

The mouse prion protein (mPrp) promoter drives expression of transgenes at high levels in the nervous system and has successfully been used to model dominantly inherited forms of neurodegenerative diseases (Borchelt et al. 1997; Wang et al. 2005). Using the mPrp promoter, Baloh’s group reported a transgenic mouse line that expressed a human TDP-43 cDNA transgene with the A315T mutation, carrying an amino terminal Flag tag (Prp-TDP43 A315T). This drove expression in most tissues, but was highest in neurons and glia in the brain and spinal cord, approximately 3-fold over endogenous TDP-43 (Wegorzewska et al. 2009). To characterize TDP-43 mice in more detail and understand its role in neurodegeneration, we examined TDP-43 mice in C57BL6/J background (approximately 80% C57BL6 background) mice. Our data showed that they initially appeared normal and weighted the same as their littermates until adulthood. At about 3 months of age in males and 6 months in females, they started an abnormal gait and weight loss and died.

In ALS, a loss of nuclear TDP-43 staining is commonly observed in affected spinal cord and/or brain neurons (Neumann et al. 2006). Although exogenous TDP-43 in TDP-43 transgenic rodent is primarily nuclear, in some models like TDP-43 mice, a loss of nuclear TDP-43 in selectively vulnerable neurons is observed prior to obvious sign of degeneration (Wegorzewska et al. 2009). These findings are consistent with the possibility that loss of nuclear TDP-43 plays a role in neurodegeneration, although the reason for TDP-43 translocation from the nucleus remains unknown. In addition, the early gait abnormality in this animal model may be due to disruption of descending or ascending pathways in the spinal cord, but is not due to loss of muscle innervations (Wegorzewska et al. 2009). Although it has been shown that the approximate 20% loss of motor neurons at end stage is responsible for the severe weakness and death in TDP-43 mice (Wegorzewska et al. 2009), our investigation showed gastrointestinal tract phenotype and pathology become the dominate problem in these mice and early death precludes the full development of ALS. These mice do not fully develop the paralysis seen in other mouse models of ALS like reported in transgenic SOD1 (G93A, G37R, G85R) mice. NeuN staining revealed there is no obvious neuronal loss; however, neurons in the spinal cord of TDP43 mice appear smaller and perhaps appear dystrophic. Our investigation revealed that these animals are dying due to presumptive reduction in motility originating in the ileocaecal area of the gastrointestinal tract. It is not known how mutant TDP-43 overexpression under the control of prion promoter caused GI tract abnormalities in these mice. However, myenteric neurons may encounter toxicity from TDP-43 overexpression and that may lead to reduced GI tract motility to trigger GI abnormalities. It would be of great interest to test this hypothesis in experiments designed to measure the GI motility and examine the myenteric plexus and carryout nerve conductance in TDP-43 mice intestinal tract. In contrast to G93A SOD1 mice or other SOD1 mutant mice, TDP-43 mice have unusual phenotypes lacking signature characteristics of ALS. However, the observed pathologies in TDP-43 proteinopathies resemble what is seen in ALS patients’ post-mortem spinal cords and brain. The Wegorzewska et al. TDP-43 mice are also different in a phenotypical and perhaps pathological sense from other TDP-43 mutant mice. For example, TDP-43 transgenic mice generated in Dr. Philip Wong's laboratory, where low-expressing TDP-43 (G344S) under the control of Thy 1 promoter (express 2- to 3-folds over endogenous TDP-43) develop full paralysis at 2 years of age (Abstract, 22nd international annual ALS/MND meeting, 2011, Sydney, Australia).

While our manuscript was under review, a report published in Brain Research showed that other laboratories studying the TDP-43 mice from Wegorzweska et al. had made observation similar to ours with respect to GI abnormalities. Their conclusion was that the ‘progressively thinned colon, swollen small intestine and subsequent reduced food intake, may be the cause of death in TDP-43 mice’. They proposed that TDP-43 accumulation in the myenteric plexus may play a role in the pathological changes in the intestinal tract in TDP-43 mice (Guo et al. 2012).

In TDP-43 mice, the mechanism of GI pathology is unclear, although paralytic ileus is the presumptive cause of the observed changes. Although serosal fibrosis was noted in one animal, there was no evidence of luminal obstruction. Vacuolization of smooth muscle fibres within the tunica muscularis suggests atrophy, possibly secondary to neuronal changes or vagal damage. Oedema is considered to be secondary to the stasis. Changes appear to be regional at early time points and focused on the ileocaecal area. Oedema progresses to involve the entire intestinal tract from the proximal colon to the duodenum, although it is unclear whether stasis is present throughout the intestinal tract or is limited to the ileocaecal region. The presence of faecal pellets in the colon suggests that ileus is not total until very late. Differential diagnoses for causes of paralytic ileus include metabolic disorders and electrolyte imbalance, peritonitis or sepsis. However, there is no evidence of peritonitis or sepsis, and occurrence of these conditions in multiple animals widely separated in time would be unlikely.

Conclusions

Our characterization of TDP-43 transgenic mice showed that the GI pathology only manifested after the appearance of ALS-like symptom and the age of GI abnormalities was found always after the ALS onset. These suggest that GI abnormalities must be due to TDP-43 disregulation. Therefore this is the first report of pathological evidence in the GI track that is strong indicator for the cause of death of TDP-43 mice.

Acknowledgments

Funding provided by UAMS and Department of Neurobiology and Developmental Sciences, Center for translational Neurosciences start up fund, Paul Dunn Fund to MK. Authors acknowledge the support by grants from the National Center for Research Resources (5P20RR020146-09) and the National Institute of General Medical Sciences (8 P20 GM103425-09). We thank Kim Tarkington for helping to compile and edit the references. Authors acknowledge valuable technical supports by Jennifer Johnson and Yingni Che. Histology performed in the UAMS Translational Pathology Shared Resource.

Conflict of interest

There is no conflict of interest.

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