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. 2020 Apr 9;21(6):e49783. doi: 10.15252/embr.201949783

Figure 7. Knocking down PRKG2 alleviated demyelination and decreased phosphorylation of MYRF in PLP‐150Q/Cas9 mice.

Figure 7

  1. Construction of AAV‐U6‐gRNA‐CMV‐RFP vector expressing PRKG2 gRNA under the U6 promoter and RFP protein under the CMV promoter, respectively. AAV‐gRNA‐PRKG2 was injected into the corpus callosum (CC, indicated between two dotted lines) in PLP‐150Q/Cas9 mouse at 2 months of age and the injected tissues were isolated 4 weeks after injection (lower panel). Ctx: cortex, Str: striatum, LV: lateral ventricle. Scale bar: 100 μm.
  2. Western blotting analysis of the PLP‐150Q/Cas9 mouse corpus callosum tissues using the antibody to Ser259 (pMYRF), phosphor‐PRKG2, or MBP. Note that reduction of mouse PRKG2 by CRISPR/Cas9 leads to the decreased MYRF phosphorylation and the increased expression of MBP. The ratios of pMYRF to MYRF or MBP to GAPDH obtained from three independent experiments were presented under the blots. Student's t‐test; pMYRF: ***P = 0.00026; MBP; **P = 0.0036. Data are presented as mean ± SEM. fMYRF: full‐length MYRF, nMYRF: N‐terminal MYRF.
  3. Immunohistochemical staining with antibody to Ser259 showed that knocking down PRKG2 reduced MYRF phosphorylation in the corpus callosum of PLP‐150Q mouse. Scale bar: 40 μm.
  4. Quantitative analysis of the pMYRF‐positive cells in each field (40×). Twenty random fields in each section were examined, n = 3 mice in each group. One‐way ANOVA with Tukey's test; ***P = 0.00022. Data are mean ± SEM.
  5. Immunofluorescent staining with antibodies to Ser259 or MBP showed the AAV‐ PRKG2‐gRNA (red) injection increased MBP expression (green) as compared with AAV‐gRNA‐control. Scale bar: 40 μm.
  6. Quantitative analysis of the MBP‐positive cells in the corpus callosum injected with control‐gRNA or PRKG2‐gRNA. Twenty random fields (40×) in each section were examined. n = 3 mice in each group. One‐way ANOVA with Tukey's test; ***P = 4.43 × 10−5. Data are mean ± SEM.
  7. A proposed model for the protective effect of LAQ in HD mice. LAQ increases myelin protein expression by reducing the expression of PRKG2, leading to reduced phosphorylation of MYRF and its dissociation from mutant HTT and therefore increasing its transcriptional activity to express myelin‐associated genes.

Source data are available online for this figure.