Fig. 5: Biochemical changes in PSD fractions from multiple brain regions and diminished release of intracellular calcium in Shank1 R882H-KI HOM mice.

A Immunoblot analyses of PSD fractions from the frontal cortex (FC), hippocampus (HP) and cerebellar cortex (CBC) in WT and Shank1 R882H-KI HOM mice. A, a Representative results for the indicated proteins. Each lane was loaded with protein samples from an individual mouse, with β3-Tubulin as a loading control, and normalized to WT levels (n = 3 mice per group). A, b The levels of Homer family proteins were significantly reduced in three brain regions of R882H-KI mice. A specific member of the Homer family proteins was downregulated in each brain region, with Homer1 downregulated in the HP, Homer2 downregulated in the FC and Homer3 downregulated in the CBC. A, c Only mGluR1 protein levels were decreased in the three regions of R882H-KI mice. No changes in AMPAR (GluA1, GluA2 and GluA4), NMDAR (GluN1 and GluN2A) or PSD-95 were observed. A, d The protein levels of IP3R1 and the calcium signaling-associated molecules p-ERK1/2, Ppp3ca and Ppp3cb were also reduced in three brain regions of R882H-KI mice, while t-ERK1/2 levels in these mice were comparable to those in WT mice. The legend p-ERK1/2 represents the ratio of p-ERK1/2 to t-ERK1/2. All data were obtained from three pairs of mice tested at least in duplicate and are presented as the mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for HOM groups compared with WT, by unpaired Student’s t-test. B Protein levels of Homer1/2/3 in PSD fractions from three brain regions. Homer1 is highly expressed in the HP, Homer2 is highly expressed in the FC, and Homer3 is highly expressed in the CBC in WT mice. The bands are representative of two or three independent experiments. For the statistical results, see Supplementary Fig. 6C. C Flow cytometry was used to determine the concentrations of intracellular calcium in the hippocampal neurons of WT and Shank1 R882H-KI HOM mice (n = 5 for each genotype). C, a, b Effects of stimulation with 50 µM and 100 µM DHPG on the intracellular calcium concentration ([Ca²⁺]_i). Hippocampal neurons in mice of both genotypes exhibited elevated [Ca²⁺]_i (WT: **** for 10 min, 15 min and 30 min vs. vehicle; HOM: *** for 10 min, ** for 15 min and 30 min vs. vehicle). At each time point, [Ca²⁺]_i in HOM mice was always lower than that in WT mice. C, c Effect of stimulation with 100 µM glutamate (Glu) on [Ca²⁺]_i. Hippocampal neurons in mice of both genotypes exhibited elevated [Ca²⁺]_i (WT: **** for 10 min, 15 min and 30 min vs. vehicle; HOM: **** for 10 min and 15 min, * for 30 min vs. vehicle). At each time point, [Ca²⁺]i in HOM mice was always lower than that in WT mice. The value presented is the mean fluorescence intensity induced by Fluo-4. All data were obtained from five independent experiments and are presented as the mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for HOM groups compared with WT, by two-way ANOVA.