Seth and Jocelynn’s TRENA analysis of striatal transcription in the allelic series mice suggests reduced SMAD3 promoter occupancy in Htt^Q111/+ mice. To confirm this, Jeff Cantle has generated western blots from 4 Htt^+/+ and 4 Htt^Q111/+ mice. Our hypothesis is that SMAD3 signaling is reduced in the striatum of Htt^Q111/+ mice.

TGF-Beta Signaling overview, from Cell Signaling:

Transforming growth factor-β (TGF-β) superfamily signaling plays a critical role in the regulation of cell growth, differentiation, and development in a wide range of biological systems. In general, signaling is initiated with ligand-induced oligomerization of serine/threonine receptor kinases and phosphorylation of the cytoplasmic signaling molecules Smad2 and Smad3 for the TGF-β/activin pathway, or Smad1/5/9 for the bone morphogenetic protein (BMP) pathway. Carboxy-terminal phosphorylation of Smads by activated receptors results in their partnering with the common signaling transducer Smad4, and translocation to the nucleus

The total SMAD3 antibody was validated by Jeff Cantle to have the correct molecular weight. The phospho-SMAD3 antibody used recognizes phospho-serines 423 and 425 on the C-terminus of SMAD3. Jeff Cantle’s development notes are here.

First, graph the raw levels of actin, total SMAD3 and phospho-SMAD3 across genotypes.

For normalization, we normalize both the total SMAD3 and the pSMAD3 to Actin in the same lane.

Next, we consider the statistical summary of the SMAD3 and pSMAD3 levels.

# Summary stats

wb_quant %>%
  group_by(Geno) %>%
  summarise(n=n(),mean_total=mean(Normalized.totalSMAD3),mean_phospho=mean(Normalized.pSMAD3), mean_ratio=mean(Normalized.pSMAD3/Normalized.totalSMAD3))

## # A tibble: 2 x 5
##     Geno     n mean_total mean_phospho mean_ratio
##   <fctr> <int>      <dbl>        <dbl>      <dbl>
## 1     WT     4 0.05028658  0.004989727 0.09955574
## 2   Q111     4 0.04504624  0.004111098 0.09082750

# Given we have a hypothesis we can use a one sided t-test -
# Compare total SMAD3, pSMAD3 and pSMAD3/SMAD3
t.test(Normalized.totalSMAD3~Geno,data=wb_quant,alternative="greater")

## 
##  Welch Two Sample t-test
## 
## data:  Normalized.totalSMAD3 by Geno
## t = 1.5539, df = 4.7481, p-value = 0.09199
## alternative hypothesis: true difference in means is greater than 0
## 95 percent confidence interval:
##  -0.001635785          Inf
## sample estimates:
##   mean in group WT mean in group Q111 
##         0.05028658         0.04504624

t.test(Normalized.pSMAD3~Geno,data=wb_quant,alternative="greater")

## 
##  Welch Two Sample t-test
## 
## data:  Normalized.pSMAD3 by Geno
## t = 1.9717, df = 4.714, p-value = 0.05458
## alternative hypothesis: true difference in means is greater than 0
## 95 percent confidence interval:
##  -3.146908e-05           Inf
## sample estimates:
##   mean in group WT mean in group Q111 
##        0.004989727        0.004111098

t.test(pSmad3ToTotalSmad3~Geno,data=wb_quant,alternative="greater")

## 
##  Welch Two Sample t-test
## 
## data:  pSmad3ToTotalSmad3 by Geno
## t = 1.3521, df = 4.0565, p-value = 0.1234
## alternative hypothesis: true difference in means is greater than 0
## 95 percent confidence interval:
##  -0.004978296          Inf
## sample estimates:
##   mean in group WT mean in group Q111 
##         0.09955574         0.09082750

Interim Conclusion

This provides suggestive evidence for two separate trends:

Total SMAD3 levels may be reduced in the striatum
After correcting for total SMAD3 reductions, phospho-SMAD3 still seems reduced by about 10%

This will require a few more animals to confirm. Jeff Cantle is going to use Jocelynn’s banked striatal tissue to generate another ~4-5 samples per genotype and will run the same type of blot so we can combine the data.

pSMAD3/SMAD3 Western Blot Analysis

Interim Conclusion