Figure 7. PKR-mediated innate immune pathways are activated by mHTT.

(A) Heatmap of expression of known interferon-stimulated genes and antiviral signaling factors related to innate immune signaling (West et al., 2015) in our TRAP data. (B) Interferon ELISA assay results from whole striatal tissue from the R6/2 model at 9-weeks of age (left panel) or the zQ175DN model at 23-months of age (right panel). (C) Western blot analysis of total PKR levels in whole striatal tissue from the R6/2 model at 9-weeks of age (left panel) or the zQ175DN model at 23-months of age (right panel). (D) Western blot analysis of phospho-PKR levels in whole striatal tissue from the R6/2 model at 9-weeks of age (left panel) or the zQ175DN model at 23-months of age (right panel). (E) Indirect immunofluorescent staining against NeuN (red pseudocolor), total PKR (green pseudocolor), and DAPI (blue pseudocolor), in the R6/2 and isogenic control striatum at 9-weeks of age, 40X magnification. Scale bar is 20 μm. (F) Quantitative PCR (qPCR) of mtRNAs associated with PKR after immunopurification of PKR from cytosolic extracts of either R6/2 or isogenic control striatum. (**) denotes p-value < 0.0021; (****) denotes p-value < 0.0001; multiple t-test using the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 1%. (G) Top panel: representative images of indirect immunofluorescent staining against total PKR (red pseudocolor) and DAPI (blue pseudocolor), in cultured day in vitro 14 iPSC-derived induced human neurons treated with DMSO vehicle control, 30 mg/mL interferon-β (a positive control treatment known to induce PKR) or mitochondrial oxidative phosphorylation inhibitors [50 nM Complex I inhibitor rotenone, 10 μM Complex II inhibitor 3-nitropropionic acid (3-NP), and 1 μM Complex III inhibitor antimycin-A] in both normal media containing either glucose-containing media or glucose-free media to increase dependency upon OXPHOS function. Scale bar = 0.45 mm. Bottom panel: quantitation across 8 images in each condition, * = p-value < 0.05, two-tailed Student’s t-test. (H) Model for innate immune activation by mutant HTT (mHTT). mHTT can induce transcriptional dysregulation in several cell types. In iSPNs, mHTT leads to a reduction in oxidative phosphorylation gene expression, which along with other mitochondrial insults leads to mitochondrial damage and release of innate immunogenic mtRNAs into the iSPN cytosol. These released mtRNAs then bind to PKR, triggering a high level of innate immune activation in iSPNs.