Figure 6. Loss of Sig1R or expression of mutant SOD1 compromises the MAM integrity in motor neurons.

• A–I
  Immunofluorescence staining of spinal cords and brains from non‐transgenic (Non‐Tg), SOD1 transgenic, or Sig1R^−/− mice. Transverse sections of mouse spinal cords (A, D–I) or sagittal sections of mouse brains (B, C) were stained using anti‐Sig1R (white), βIII‐tubulin (red), and IP₃R3 (green) antibodies. Note that Sig1R and IP₃R3 are co‐localized in the motor neurons of the anterior horn (A) and the hypoglossal nucleus (B), and IP₃R3 was not expressed in hippocampal neurons (C). Mutant SOD1 induced aggregation of Sig1R and mislocalization of IP₃R3 in anterior horn neurons (D–G), while their abnormalities were not observed in SOD1^WT motor neurons (H). Mislocalization of IP₃R3 was also observed in Sig1R^−/− mouse spinal cords (I). Scale bars: 50 μm.
• J, K
  Representative electron micrographs of the MAM (J) (double‐headed arrows) in motor neurons of 12‐month‐old Non‐Tg, SOD1^G85R, or Sig1R^−/− mice. Note that ER–mitochondria contacted areas were reduced in both SOD1^G85R or Sig1R^−/− mice. Quantification of the mitochondria surface associated with ER was calculated in (K). For quantification, 13–19 motor neurons and 224–316 mitochondria with MAM in each animal (n = 2) were analyzed. Data are expressed as mean ± SEM. **P < 0.0001 versus Non‐Tg; one‐way ANOVA with subsequent post hoc Tukey's test. Scale bars: 300 nm.
• L
  The levels for IP₃R3 and calreticulin were decreased in the MAM fractions of Sig1R^−/− mouse brains. MAM fractions and whole tissue lysates of Sig1R^+/+ or Sig1R^−/− mouse brains were immunoblotted. Representative blots from three independent experiments are shown.

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