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. 2023 Oct 29;26(11):108362. doi: 10.1016/j.isci.2023.108362

Figure 3.

Figure 3

Homozygous deletion of Tmem106b exacerbates Grn KO behavioral and lysosomal phenotypes

(A) Wire hang time was significantly decreased in Tmem −/−, Grn −/− mice and worsened with age. Mixed-effects model (REML) test revealed an overall significant effect of genotype F (4, 79) = 44.12; p < 0.0001, time F (2.611, 141.9) = 11.64; p < 0.0001 and a genotype × time interaction F (12,163) = 3.322; p = 0.0002. Tukey’s multiple comparisons test revealed that at the 3, 3.5, and 4 months time points, the Tmem −/−, Grn −/− group displayed significantly reduced hang times when compared to each of the other 4 genotypes.

(B) Tmem −/−, Grn −/− mice displayed a severe clasping phenotype. REML test on clasping severity revealed an overall effect of genotype F (4, 80) = 49.58; p < 0.0001, time F (2.214, 125.5) = 20.34; p < 0.0001 and a genotype × time interaction F (12,170) = 25.11; p < 0.0001). Tukey’s multiple comparisons test revealed that at the 3.5 and 4 months time points, the Tmem −/−, Grn −/− group displayed significantly increased clasping severity when individually compared to each of the other 4 genotypes.

(C) When placed on their sides Tmem −/−, Grn −/− mice had delayed righting times. REML test on righting latency revealed an overall significant effect for genotype F (4, 82) = 14.06; p < 0.0001, time F (1.602, 94.01) = 10.29; p = 0.0003 and a genotype, time interaction F (12,176) = 11.54; p < 0.0001. Tukey’s multiple comparisons test revealed that at 4 months the Tmem −/−, Grn −/− mice are the only group that displayed a significantly righting phenotype when individually compared to each of the other 4 genotypes. n = 6–15 animals/genotype for behavioral tests.

(D) A significant effect of genotype on LAMP1 staining was detected in whole section analysis (% area) by brain region: forebrain (FB) p = 0.0004, hindbrain (HB) p < 0.0001, and lumbar spinal cord (SC) p < 0.0001 sections by one-way ANOVA. Tmem −/−, Grn −/− mice show significantly increased LAMP1 in HB and SC when compared Grn −/− alone and to all other genotypes. Tmem −/−, Grn −/− differed from all other groups except Grn −/− mice in FB.

(E) An overall effect of genotype was detected for CD68 staining in whole section analysis (% area) by brain region: FB p = 0.0004, HB p < 0.0001 and SC p < 0.0001 using one-way ANOVA. Tmem −/−, Grn −/− mice show significantly increased levels of CD68 in FB, HB, and SC when compared to all other genotypes.

(F) Representative images of CatD staining in FB, HB, and SC showing increases in Tmem −/−, Grn −/− tissues and decreased CatD in Tmem −/− tissue, specifically in spinal cord (see insert).

(G) An overall effect of genotype was detected for CatD staining in whole section analysis (% area) by brain region: FB p = 0.0162, HB p < 0.0001, and SC p < 0.0001 sections by one-way ANOVA. In FB, Grn −/− mice had increased CatD levels compared to Wt mice. Similar to Grn −/− mice, Tmem +/−, Grn +/−, and Tmem −/−, Grn −/− had elevated CatD levels compared to Wt mice. In HB, Grn−/− mice did not show elevated CatD levels. Tmem +/−, Grn +/− differed significantly from Wt mice and Tmem −/−, Grn −/− mice showed significant increases in CatD compared to both Grn −/− and Wt mice. In SC, CatD levels were elevated in Tmem −/−, Grn −/− mice compared to Grn −/− alone and Wt mice, whereas, Tmem −/− mice had significantly decreased levels when compared to the Wt mice. For histology n = 11–16 animals/genotype and 4–5 sections/animal/tissue area/stain. Student’s t test or Tukey’s post-hoc ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ##p < 0.01 vs. Tmem +/−, Grn +/− and Grn −/− mice. All error bars represent S.E.M.

(H) Representative images of immunofluorescent staining for CatD (green), microglia (Iba1, red), and DAPI (blue).Orange arrow indicate CatD+ve axonal swellings. White arrowhead indicates CatD+ve microglia.