Figure 6. RTT astrocytes display abnormal calcium load in the ER, calcium leak from the ER, baseline cytosolic calcium level, and TRPC4-dependent SOCE.

(A) Left, average traces of Fluo4 fluorescence changes in wild type and mutant astrocytes treated with 1 μM TG to release ER calcium. Bath solution contained 0 mM Ca²⁺ plus 2 mM EGTA. Right, Quantification of the peak amplitude of TG induced Ca²⁺ elevations. (B) The rise phase (dots) shown in (A) is fitted with a single exponential curve. (C) Quantification of the time constant of the rise phase in the left panel from congenic mutant and wild type human RTT astrocytes in response to TG. (D) Representative GFP and Rhod-2 images in primary astrocytes from female Mecp2^+/- ± with a GFP transgene on the wild type X chromosome. Scale bars = 50 μm. (E–F) Quantification of Rhod-2 fluorescence intensity in GFP negative and GFP positive primary astrocytes isolated from either female Mecp2^+/- ± with a GFP transgene on the wild type X chromosome (E) or female Mecp2^+/+ mice with a GFP transgene on the wild type X chromosome (F). (G) The average trace of Fluo4 fluorescence changes in response to extracellular Ca²⁺ (2 mM) after depletion of ER Ca²⁺ store using TG. (H) The Western blot result showing the protein level of TRPC4 in WT and MeCP2 mutant astrocytes. (I) Quantification of the Western blot results. (J) Representative traces of current density of TG-induced inward current in wild type and mutant astrocytes held at −70 mV. This current was partially blocked by TRPC4 selective antagonist, ML204. (K) Quantification of ML204 sensitive current from wild type and mutant astrocytes held at −70 mV. (L) Representative traces of current density of TG-induced inward current in mutant astrocytes infected with lentivirus expressing either GFP alone or co-expressing GFP and shTRPC4. (M) Quantification of ML204 sensitive current at −70 mV. The bar graphs in this figure show the mean ±s.e.m. *p<0.05, **p<0.01, ***p<0.001.

Figure 6—figure supplement 1. Mutant human RTT astrocytes exhibit abnormal Ca²⁺ activity in Ca²⁺-free aCSF (0 mM Ca²⁺ plus 2 mM EGTA).

Higher percentage of mutant astrocytes showing spontaneous Ca²⁺ elevations (left, p=0.04). The mutant cells exhibit a significantly higher frequency (middle, p=0.04) and amplitude (right, p=0.02) than wild type cells. 20 WT and 61 MT human astrocytes were analyzed in these experiments. The bar graphs in this figure show the mean ±s.e.m. *p<0.05.

Figure 6—figure supplement 2. Co-staining of TRPC4 and astrocyte marker S100β on hippocampal sections from wild type (WT) and Mecp2 knockout (Mecp2^-/y) mice.

(A) Strong TRPC4 immunoreactivity from Mecp2^-/y (top) but not WT (bottom) mice. Arrows indicate TRPC4 fluorescence co-localized with S100β positive cells. White inserts provide zoomed-in views of the yellow boxed regions. Scale bar = 50 μm. (B) Quantification of relative TRPC4 immunoreactivity in S100β positive cells in the hippocampus of WT and Mecp2^-/y mice (WT = 1). ***p<0.001.

Figure 6—figure supplement 3. ChIP-qPCR analysis of MeCP2 occupancy on the promoter of the TrpC4 gene in primary mouse astrocytes isolated from either Mecp2-Flag mice (Flag) or control mice (ctrl).

4 pairs of primers (TRPC4-P-1 to −4) were used to cover the TrpC4 promoter. MeCP2 binding signal from the ChIP samples was normalize to signal from the input samples. The bar graphs in this figure show the mean ±s.e.m.

Figure 6—figure supplement 4. I-V curve of TG-induced ML204-sensitive current from astrocytes infected with Lenti-GFP or Lenti-GFP-shTRPC4.

Figure 6—figure supplement 5. Mecp2^-/y astrocytes display abnormal ER Ca²⁺ load, Ca²⁺ leakage, store operated Ca²⁺ entry, and TRPC4 expression.

(A) Left, average traces of Fluo4 fluorescence changes in wild type and Mecp2^-/y astrocytes treated with 1 μM TG to release ER calcium. Ca²⁺-free aCSF (0 mM Ca²⁺ plus 2 mM EGTA) was used; right, quantification of the peak amplitude of F/F₀ in response to TG. p=0.001. (B) Left, the rise phase (dots) shown in (a) is fitted with a single exponential curve; right, the time constant of the rise phase from Mecp2^-/y astrocytes in response to TG was smaller than that from wild type cells. p=0.004. (C) The average trace of Fluo4 fluorescence changes in response to 2 mM aCSF after depletion of ER Ca²⁺ store by TG. (D) The quantification of ML204 sensitive current from wild type and Mecp2^-/y astrocytes at −70 mV, illustrating higher TRPC4 mediated current in Mecp2^-/y astrocytes. p=0.04. 9–38 WT and 10–39 Mecp2 null astrocytes were included in these analyses. (E) Western blot analysis showing increased level of TRPC4 protein in both mouse and human mutant astrocytes. The bar graphs in this figure show the mean ±s.e.m. *p<0.05, **p<0.01.