Fig 2: Gja1 deficiency causes reduction of Apoe production and promotes Aß production in neuron/astrocyte cocultures. a. Quantitative PCR analysis of Apoe in wildtype (WT) and Gja1-/- astrocytes. b. Apoe, Cx43, and actin levels in wildtype and Gja1-/- astrocytes treated with the indicated concentration of Aß1–42 oligomers for 48 h were determined by immunoblot analysis of cell lysates. c. Apoe secreted into the conditioned medium of astrocytes in B was quantitatively determined by specific ELISA in quadruplicate wells. d and e. Amyloidß secreted to the conditioned medium of wildtype cortical neurons cocultured with either wildtype or Gja1-/- astrocytes was quantitatively determined by Aß40 and Aß42 specific ELISA

Fig 3: A working hypothesis of GJA1 dysregulation in AD. Progressive upregulation of GJA1 in the brains during LOAD pathogenesis (in response to amyloid accumulation) serves to enhance the coordinated gene network function to orchestrate neuroprotective response by astrocytes including Aß production and clearance (CST3, CLU, CTSB, ECE2, MEGF10, PSENEN and GSAP), APOE production (APOE, ABCA1, CAV1 and SPON1), and supporting neuronal activity (DLG4, NLGN3, GRINA and GRIN2D). However, the enhanced neuronal activity due to chronic and prolonged GJA1 upregulation triggers neuronal wear and eventual death

Fig 4: Inflammatory cytokines downregulates Gja1, Apoe, and other network genes. a. Wildtype primary mouse astrocytes were treated with either IL-1ß (10 ng/ml) or TNFa (10 ng/ml) or in combination for 7 days and levels of Cx43, Apoe expression were analyzed by immunoblot. Representative results from 4 (Gja1) and 3 (Apoe) independent experiments are shown. b. Protein levels of Cx43 (left) and Apoe (right) were quantitatively analyzed for these technical replicates after normalization to Actin. ANOVA followed by Bonferroni post-hoc tests are indicated by asterisks. * p < 0.05, ** p < 0.01, *** p < 0.001. c. Quantitative gene expression analysis in wildtype astrocytes with treatments similar to a) was performed to analyze the other key drivers of the GJA1-centered network. Results representative of two independent experiments are shown. Statistical analysis was performed as in B, and only the comparison between control and IL-1ß/TNFa is shown