Figure 7. Neuronal-specific induction of G34R results in neurodegeneration and cortical atrophy.

A. Schematic representation of the CRISPR G34R-conditional knock-in strategy. An inducible transgene cassette targeting exon 1 of the H3f3a gene was inserted using Cas9:gRNA approach. Loxp sites flanked an adenovirus splice-acceptor (SA), mEGFP, and 3x polyA sequences, followed by the H3.3G34R cDNA. Carrier H3f3a^+/LGFPLG34R mice were crossed with Emx1-Cre or Foxg1-Cre mice to generate heterozygous H3f3a^+/G34R mice with cortical-specific expression of G34R. B. In situ hybridization of Emx1 and Foxg1 genes (Allen Brain Atlas). H3.3G34R IHC staining for representative sagittal section of adult mouse brain from G34R^Emx1 and G34R^Foxg1. CRB: cerebellum. C. Expression levels of Emx1 and Foxg1 in murine developing brain atlas. D. Immunofluorescent co-staining of neuronal marker NeuN (green) and G34R (red) in G34R^Emx1 adult mouse brain (20x). E. G34R^Emx1 shows cortex atrophy. Representative image and brain weights (means ± SE) of WT^Emx1 and the reduced cortical region (arrow) of G34R^Emx1 mice. Student’s t-test. **P < 0.01, **** P < 0.0001. F. Immunohistochemical staining of layer V neuronal marker (Oct6) (top), neurons (NeuN), and astrocytes (GFAP) (bottom) in WT^Emx1 and G34R^Emx1 (20x). Related quantifications, Oct6 (n = 3) and GFAP (n ≥ 4), means ± SD. Student’s t-test. *P < 0.05, **P < 0.01. G. Immunohistochemical staining of microglial marker (Iba1), neuronal postsynaptic protein PSD95, and complement C3 in adult WT^Emx1 and G34R^Emx1 mice (63x). Related quantifications, Iba1 (n ≥ 3) and C3 (n = 3), means ± SD. Student’s t-test. *P < 0.05. H. Model to illustrate dysregulation of epigenetic reprogramming in the postnatal G34R brain, which may account for neuroinflammation and progressive neurodegeneration.