Increased ROS-Dependent Fission of Mitochondria Causes Abnormal Morphology of the Cell Powerhouses in a Murine Model of Amyotrophic Lateral Sclerosis

Figure 4

Abnormal fission-related proteins in cervical wobbler spinal cord at p40. (a) mRNA expression levels of Mfn1, Mfn2, Opa1, Oma1, and Dnm1l from the stable phase of wild-type (WT) and wobbler (WR) spinal cords were investigated by qPCR. mRNA levels were significantly reduced in WR except Dnm1l. For relative quantification, the 2^−∆∆Ct method was conducted using GAPDH for normalization. -11 per genotype. (b) Exemplary Western blots of Mfn1 (≈85 kDa), Mfn2 (≈85 kDa), Opa1 (≈100 kDa), and Oma1 (≈50 kDa) in the cervical spinal cord of p40 WT and WR. Actin (≈45 kDa) and calnexin (≈90 kDa) were used as control proteins. Bar charts represent the semiquantitative analysis of protein expression levels. Western blots revealed unchanged expression of Mfn1, Opa1, and Oma1 as well as significantly increased expression of Mfn2 in the cervical spinal cord of wobbler mice. -10 per genotype. (c) Exemplary Western blots of Drp1 (≈85 kDa), p-Drp1 (Ser616; ≈85 kDa), and actin (≈45 kDa) as control protein. Analysis of band intensity is presented in bar charts. Total amount of Drp1 does not differ between the two genotypes; Drp1 is significant more often phosphorylated at Ser616 in cervical spinal cords of wobbler mice. per genotype. (d) Exemplary Western blots of CaMKII (≈55 kDa) and oxidized Ox-CaMKII (Met281/282; ≈55 kDa) in combination with calnexin (≈90 kDa) as control protein from wild-type and wobbler cervical spinal cords. Analysis of band intensity is presented in bar charts and showed equal CaMKII levels; thus, the proportion of oxidized CaMKII at Met281/282 is significantly increased in wobbler spinal cords compared to wild-type. per genotype. All data are presented as the , and Student’s -test was performed for significance testing between WT and WR. Values with were considered to be significant. Significant differences are indicated by , , , , and .

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