Figure 2.

Endoplasmic reticulum Ca²⁺ homeostasis is dysregulated in cultured motor neurons from SBMA muscular atrophy mice. Primary embryonic motor neurons from wild-type (WT) and AR100 SBMA mice were imaged after 7 days in culture to monitor cytosolic Ca²⁺ levels. (A) The trace illustrates the protocol used to measure basal cytosolic Ca²⁺ and infer endoplasmic reticulum Ca²⁺ and store-operated Ca²⁺ influx and is described in the main text. (B) Basal cytosolic Ca²⁺ levels were measured in primary AR100 and wild-type motor neurons in a recording medium containing 1 mM Ca²⁺. Significantly higher [Ca²⁺] was measured in AR100 compared to wild-type motor neurons both in the absence and presence of 50 nM dihydrotestosterone (DHT) for 72 h (n ≥ 31, P = 0.02 and 0.002 respectively, ANOVA with Bonferroni post hoc analysis; n = number of motor neurons from at least three replicate cultures). (C) Cytosolic Ca²⁺ responses to thapsigargin exposure in wild-type and AR100 motor neurons were established in the absence and presence of dihydrotestosterone. (D) The increase in cytosolic Ca²⁺ levels in AR100 and wild-type motor neurons following thapsigargin application in a Ca²⁺ free recording medium was used to infer levels of endoplasmic reticulum Ca²⁺. A significantly lower level of inferred endoplasmic reticulum Ca²⁺ was present in AR100 motor neurons compared to wild-type controls in the presence of dihydrotestosterone (n ≥ 31, P = 0.002, ANOVA with Bonferroni post hoc analysis). (E) Following addition of Ca²⁺ in the recording media, cytosolic [Ca²⁺] was measured in wild-type and AR100 motor neurons in the absence and presence of dihydrotestosterone treatment. (F) The increase in cytosolic Ca²⁺ levels in primary AR100 and wild-type motor neurons after external Ca²⁺ introduction was measured and was used to infer levels of store-operated Ca²⁺ influx. Significantly higher levels of inferred store-operated Ca²⁺ influx in AR100 motor neurons compared to wild-type in the presence of dihydrotestosteronewere observed (n ≥ 31, P = 0.027, ANOVA with Bonferroni post hoc analysis). Error bars show the SEM. *P < 0.05, **P < 0.01, ***P < 0.001.