https://orcid.org/0000-0002-8299-0233
Figures & data
Table 1. Real-time PCR Primers.
Figure 1. TDP43 mutant mice have less body weight, decreased rotarod test time, slowed intestinal mobility, and altered enteric neuromuscular structure, compared with the age-matched WT mice.
(a) TDP43 mutant mice started to show weight loss from the age of 11 weeks compared to WT mice. Each data point represents the average body weight. Data are expressed as mean ± SD. n = 10, two-way ANOVA test. (b) TDP43 mice had significantly reduced rotarod test time compared to WT mice. At the age of 12 weeks, the mice were subjected to the trial on the accelerating spindle from 4 to 40 rpm for 300 s. Latency to fall was recorded when the mouse fell from the rod. Each mouse was tested in 4 trials per day for 2 consecutive days. The mean times for 8 trials of the tests were calculated for each mouse. Data are expressed as mean ± SD. n = 10, Welch’s t-test. (c) TDP43 mutant mice had significantly increased gut transit time at 12 weeks compared to WT mice. In age-matched WT and TDP43 mutant mice, intestinal mobility was tested using Evans blue marker (5% Evans blue, 5% gum Arabic in drinking water). Data are expressed as mean ± SD. n = 10, Welch’s t-test. (d) At the age of 12 weeks, the expression of α-SMA protein decreased while the expression of GFAP protein increased in intestines of TDP43 mice compared to those in WT mice in western Blotting (WB). Data are shown as mean ± SD. n = 4 per group, Welch’s t-test. (e) Human TDP43 protein (FLAG) was observed in intestinal TDP43 mutant mice compared to the age-matched WT mice by IHC staining. Mouse or rabbit IgG was used as the negative experimental control in all the staining. The percentage of positive TDP43 (FLAG) was quantified with ImageJ IHC profiler by counting 3 images for each sample. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (f) α-SMA expression decreased in intestinal TDP43 mutant mice compared with WT mice, as determined by IF staining. The relative fluorescence intensity was quantified with ImageJ by counting 3 images for each sample. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (g) GFAP expression increased in the intestines of the TDP43 mutant mice compared to the WT mice in IF staining. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. All p-values are shown in the figures.
Figure 2. Augmented intestinal permeability and altered tight junctions (TJ) in the intestine, and spinal cord of the TDP43 mice compared with the age-matched WT mice.
(a) Intestinal permeability increased in TDP43 mutant mice, compared with WT mice. Data are expressed as the mean ± SD, n = 10, Welch’s t-test. (b) ZO-1 and Claudin-5 expression decreased in intestines of TDP43 mice compared to those in WT mice in WB. Data are shown as mean ± SD, n = 4 per group, Welch’s t-test. (c) Decreased expression of ZO-1 in the colon of TDP43 mutant mice, compared with WT mice, as determined by IF staining. The relative fluorescence intensity was quantified with ImageJ by counting 3 images for each sample. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (d) The expression of GFAP protein increased while the expression of Claudin-5 protein decreased in spinal cord of TDP43 mutant mice, compared to WT mice by WB. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. All p values are shown in the figures. (e) Human mutant TDP43 protein (FLAG) was observed in the spinal cord of TDP43 mice, compared to the WT mice by IHC staining. The percentage of positive TDP43 (FLAG) was quantified with ImageJ IHC profiler by counting 3 images for each sample. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (f) GFAP expression increased in the spinal cord of TDP43 mutant mice compared to the WT mice by IF staining. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (g) Claudin-5 expression decreased in spinal cord of TDP43 mutant mice compared with WT mice, as determined by IF staining. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. All p-values are shown in the figures.
Figure 3. Increased human mutant TDP43 and GFAP protein expression while decreased ZO-1 and Claudin-5 protein expression in the brains of TDP43 mutant mice compared with the age-matched WT mice.
(a) HmTDP43 protein (FLAG) was observed in the brains of TDP43 mice, compared to the WT mice by IHC staining. The percentage of positive TDP43 (FLAG) was quantified with ImageJ IHC profiler by counting 3 images for each sample. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (b) Increased GFAP expression in the brains of TDP43 mutant mice by IF staining. The relative fluorescence intensity was quantified with ImageJ by counting 3 images for each sample. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (c) Decreased Claudin-5 in the brains of TDP43 mutant mice compared with WT mice, as determined by IF staining. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (d) Decreased ZO-1 expression in the brains of TDP43 mutant mice, compared with WT mice. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (e) Claudin-3 expression was not changed in the brains of the TDP43 mice compared to WT mice, n = 3 per group. All p-values are shown in the figures.
Figure 4. Butyrate treatment led to enhanced ENS and muscle function in the colon, reduced hmTDP43 mutant and GFAP protein expression, increased TJs proteins expression in the colon, spinal cord, and brain of TDP43 mutant mice.
(a) Body weight changes of TDP43 mutant mice after butyrate treatment. Male or female TDP43 mutant mice were treated with or without 2% butyrate in the drinking water from 9 weeks to 12 weeks. Butyrate treated TDP43 mutant mice show slight weight loss without statistical significance from the age of 11 weeks, compared to the TDP43 mice without treatment. Data are expressed as mean ± SD. n = 13-15, two-way ANOVA test. (b) TDP43 mutant mice with butyrate treatment showed a significantly increased rotarod test time. Data are expressed as mean ± SD. n = 13-15, Welch’s t-test. (c) TDP43 mutant mice with butyrate treatment showed a significantly decreased gut transit time. Data are expressed as mean ± SD. n = 13-15, Welch’s t-test. (d) The butyrate treated TDP43 mutant mice had reduced hmTDP43 and GFAP, and enhanced α-SMA protein expression in the intestines, compared to non-treated TDP43 mutant mice in WB. Data are shown as mean ± SD, n = 4 per group, Welch’s t-test. (e) Butyrate treatment led to decreased hmTDP43 expression in the intestines of TDP43 mutant mice. The percentage of positive TDP43 (FLAG) was quantified with ImageJ IHC profiler by counting 3 images for each sample. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (f) Butyrate treatment led to increased α-SMA expression in the intestines of TDP43 mutant mice. The relative fluorescence intensity was quantified with ImageJ by counting 3 images for each sample. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (g) Butyrate treatment led to decreased GFAP expression in the intestines of TDP43 mutant mice. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (h) Intestinal permeability decreased in TDP43 mutant mice treated with butyrate. Data are expressed as mean ± SD. n = 10, Welch’s t-test. (i) The butyrate treated TDP43 mutant mice had enhanced ZO-1 and Claudin-5 protein expression in the intestines compared to non-treated TDP43 mutant mice in WB. Data are shown as mean ± SD, n = 4 per group, Welch’s t-test. (j) Increased ZO-1 expression in the colon of TDP43 mutant mice with butyrate treatment in IF. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (k) Reduced human TDP43 protein expression, decreased GFAP expression and enhanced Claudin-5 expression in the spinal cord of TDP43 mutant mice treated with butyrate in WB. Data are shown as mean ± SD, n = 4, Welch’s t-test. (l) Butyrate treatment led to decreased hmTDP43 in the spinal cord of TDP43 mutant mice. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (m) Butyrate treatment led to decreased GFAP expression in the spinal cord of TDP43 mutant mice by IF. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (n) Butyrate treatment led to increased Claudin-5 expression in the spinal cord of TDP43 mutant mice, as determined by IF. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (o) Butyrate treatment led to decreased human TDP43 expression in the brains of TDP43 mice. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (p) Butyrate treatment led to decreased GFAP expression in the brains of TDP43 mice in IF staining. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. Butyrate treatment led to increased Claudin-5 (q) and ZO-1 (r) expression in the brains of TDP43 mice, as determined in IF staining. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. All p-values are shown in the figures.
Figure 5. Probiotics treatment led to enhanced ENS and muscle function in the intestines, reduced hmTDP43 and GFAP protein expression, increased TJs proteins expression in the intestine, spinal cord, and brain of TDP43 mutant mice.
(a) Body weight changes of TDP43 mutant mice after probiotics treatment. Male or female TDP43 mice were treated with or without probiotics (VSL#3, 1 × 109CFU) daily by oral gavage starting from 9 weeks to 12 weeks. Probiotic treated TDP43 mice showed slight weight loss without a statistical significance at the age of 12 weeks, compared to the no-treatment TDP43 mutant mice. Data are expressed as mean ± SD. n = 13-15, two-way ANOVA test. (b) TDP43 mutant mice with probiotics treatment showed a significantly increased rotarod test time. Data are expressed as mean ± SD. n = 13-15, Welch’s t-test. (c) TDP43 mutant mice with probiotics treatment showed a significantly decreased gut transit time. Data are expressed as mean ± SD. n = 13-15, Welch’s t-test. (d) The VSL#3 treated TDP43 mutant mice had reduced hm TDP43 and GFAP expression, and enhanced α-SMA in the colon, compared to TDP43 mutant mice without treatment, as determined in WB. Data are shown as mean ± SD, n = 4 per group, Welch’s t-test. VSL#3 treatment led to decreased hmTDP43 (e), increased α-SMA expression (f) and decreased GFAP expression (g) in the colon of TDP43 mutant mice. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (h) Intestinal permeability decreased in TDP43 mutant mice treated with probiotics. Data are expressed as mean ± SD. n = 10, Welch’s t-test. (i) Increased ZO-1 and Claudin-5 protein expression in the intestines of TDP43 mutant mice with VSL#3 treatment in WB. Data are shown as mean ± SD, n = 4 per group, Welch’s t-test. (j) Increased ZO-1 expression in the intestines of TDP43 mutant mice with probiotics treatment in IF. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. (k) Reduced hmTDP43 and GFAP expression, enhanced Claudin-5 expression in the spinal cord of TDP43 mutant mice treated with probiotics. Data are shown as mean ± SD, n = 4, Welch’s t-test. Probiotics treatment led to decreased hmTDP43 (l), decreased GFAP expression (m) and increased Claudin-5 expression (n) in the spinal cord of TDP43 mutant mice, as determined by IHC or IF staining. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. Probiotics treatment led to reduced human TDP43 expression (o), decreased GFAP expression (p), increased Claudin-5 expression (q), and increased ZO-1 expression (r) in the brains of TDP43 mice. Data are shown as mean ± SD, n = 3 per group, Welch’s t-test. All p-values are shown in the figures.
Figure 6. Butyrate or VSL#3 treatment corrected dysbiosis in ALS TDP43 mice, modulating microbiome leads reduced LPS expression in ALS TDP43 mice.
(a) TDP43 mice led to dysbiosis and a shift in the bacterial profile. TDP43 mice decreased butyryl-coenzyme A CoA transferase genes, decreased Butyrivibrio fibrisolvens and increased Bacteroides fragilis, compared with WT mice. Data are expressed as the mean ± SD. N = 6, Welch’s t-test. (b) Butyrate or (c) probiotics treatment increased butyryl-coenzyme A CoA transferase gene, increased Butyrivibrio fibrisolvens and decreased Bacteroides fragilis in the TDP43 mice treated with butyrate or VSL#3. Data are expressed as the mean ± SD. n = 6, Welch’s t-test. (d) LPS were significantly higher in TDP43 mice than that in WT mice, but reduced LPS level was found in TDP43 mice after butyrate or probiotics treatment. Data are expressed as the mean ± SD, n = 6, Welch’s t-test. All p-values are shown in the figures.
Figure 7. Butyrate or probiotic VSL#3 treatment reduced serum IL-6, IL-17, and IFN-γ expression in TDP43 mice.
(a) Serum cytokines, IL-6, IL-17, and IFN-γ, were significantly higher in TDP43 mutant mice. Serum samples were collected from TDP43 mutant mice and age-matched WT mice, and then cytokines were detected by a Luminex detection system. Data are expressed as the mean ± SD, n = 6, Welch’s t-test. (b) Serum IL-6, IL-17, and IFN-γ were significantly lower in TDP43 mice treated with butyrate. Data are expressed as the mean ± SD, n = 6, Welch’s t-test. (c) Serum IL-6, IL-17, and IFN-γ were significantly lower in TDP43 mice treated with VSL#3. Data are expressed as the mean ± SD, n = 6, Welch’s t -test. All p-values are shown in the figures.
Figure 8. Butyrate or probiotics treatment decreased aggregation of hmTDP43 protein, decreased GFAP and IBA1 expression in EGCs isolated from TDP43 mutant mice.
(a) Aggregated hmTDP43 protein and increased GFAP were observed in the TDP43 EGCs. TDP43 protein aggregates were quantified with AggreCount. Data are shown as mean ± SD, n = 5, Welch’s t-test. (b) Aggregated human TDP43 protein and increased IBA1 protein expression were observed in the EGCs from TDP43 mice, compared with the WT EGCs. Data are shown as mean ± SD, n = 5, Welch’s t-test. (c) Butyrate or probiotics treatment reduced hmTDP43 aggregation and decreased GFAP expression in cultured TDP43 EGCs. Data are expressed as mean ± SD, n = 5, one-way ANONA test. (d) Butyrate or probiotics treatment reduced hmTDP43 aggregation and decreased IBA1 in EGCs with hmTDP43. Data are expressed as mean ± SD, n = 5, one-way ANONA test. p-values are shown in the figures.
Figure 9. A working model of modulating microbiome in TDP43 mutant mice through the gut-microbiome-brain axis.
The mechanisms of butyrate or probiotics in protecting TDP43 mice are several folds: cleaning protein aggregation, maintaining TJ proteins, inhibiting inflammation, and restoring beneficial bacteria. TDP43 mice had significantly increased hmTDP43 and GFAP, decreased expression of α-SMA, tight junction proteins (ZO-1 and Claudin-5) in the colon, spinal cord, and brain compared with age-matched WT mice, while butyrate or probiotic treatment facilitates the restoration of beneficial microbiome and barrier functions. TDP43 mice showed dysbiosis and high serum inflammation, while butyrate or probiotics treatment can reduce systemic and neuroinflammation.
Data availability statement
The data that support the findings of this study are available from the corresponding author, J.S., upon reasonable request.