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. 2012 Jun;1822(6):862-74.
doi: 10.1016/j.bbadis.2012.02.017. Epub 2012 Feb 23.

Dynamin-related protein 1 heterozygote knockout mice do not have synaptic and mitochondrial deficiencies

Maria Manczak  1 , Hiromi Sesaki, Yusuke Kageyama, P Hemachandra Reddy
Affiliations

Dynamin-related protein 1 heterozygote knockout mice do not have synaptic and mitochondrial deficiencies

Maria Manczak et al. Biochim Biophys Acta. 2012 Jun.

Abstract

The objective of this study was to elucidate the effect of partial reduction of the mitochondrial fission protein, dynamin-related protein 1 (Drp1) on mitochondrial activity and synaptic viability. Recent knockout studies of Drp1 revealed that homozygote Drp1 knockout mice are embryonic lethal due to reduced mitochondrial fission, and that this reduced fission leads to developmental defects in the brain. In contrast, heterozygote Drp1 knockout mice appear to be normal in terms of lifespan, fertility, and viability, and phenotypically these animals are not different from wild-type mice. However, the effects of partial Drp1 reduction on mitochondrial function and synaptic activity are not well understood. In the present study, we sought to characterize synaptic, dendritic and mitochondrial proteins, and mitochondrial function and GTPase enzymatic activity, in Drp1 heterozygote knockout mice. Interestingly, we found no significant changes in synaptic, dendritic, and mitochondrial proteins in the Drp1 heterozygote knockout mice compared to the wild-type mice. Further, mitochondrial function and GTPase enzymatic activity appeared to be normal. However, H(2)O(2) and lipid peroxidation levels were significantly reduced in the Drp1 heterozygote knockout mice compared to the wild-type mice. These findings suggest that partial Drp1 reduction does not affect mitochondrial and synaptic viability and may have therapeutic use in treating patients with Alzheimer's disease and Huntington's disease.

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Figures

Figure 1
Figure 1
Immunoblotting analysis of mitochondrial proteins. A represents representative immunoblots of mitochondrial proteins in Drp1+/+ mice and Drp1+/− mice. Fifty μg of protein lysate was used from cerebral cortex and cerebellum tissues from the Drp1+/+ and Drp1+/− mice. Immunoblotting analyses were performed, using Drp1, Fis1, Mfn1, Mfn2, CypD, and beta actin, as a control for equal loading protein. B represents quantitative densitometric analysis of mitochondrial proteins.
Figure 1
Figure 1
Immunoblotting analysis of mitochondrial proteins. A represents representative immunoblots of mitochondrial proteins in Drp1+/+ mice and Drp1+/− mice. Fifty μg of protein lysate was used from cerebral cortex and cerebellum tissues from the Drp1+/+ and Drp1+/− mice. Immunoblotting analyses were performed, using Drp1, Fis1, Mfn1, Mfn2, CypD, and beta actin, as a control for equal loading protein. B represents quantitative densitometric analysis of mitochondrial proteins.
Figure 2
Figure 2
Immunoblotting analysis of synaptic and dendritic proteins. A represents representative immunoblots of synaptic and dendritic proteins in Drp1+/+ mice and Drp1+/− mice. Fifty μg of protein lysate was used from cerebral cortex and cerebellum tissues from the Drp1+/+ and Drp1+/− mice. Immunoblotting analyses were performed, using synaptophysin, PSD95, MAP2, and beta actin, as a control for equal loading protein. B represents quantitative densitometric analysis of synaptic and dendritic proteins.
Figure 2
Figure 2
Immunoblotting analysis of synaptic and dendritic proteins. A represents representative immunoblots of synaptic and dendritic proteins in Drp1+/+ mice and Drp1+/− mice. Fifty μg of protein lysate was used from cerebral cortex and cerebellum tissues from the Drp1+/+ and Drp1+/− mice. Immunoblotting analyses were performed, using synaptophysin, PSD95, MAP2, and beta actin, as a control for equal loading protein. B represents quantitative densitometric analysis of synaptic and dendritic proteins.
Figure 3
Figure 3
Immunoreactivity of Drp1 in brain sections from Drp1+/+ mice and Drp1+/− mice. (A) represents the hippocampus (10X the original magnification); (B), the cerebral cortex (10X the original magnification); (C), the cerebral cortex (40X the original magnification) and (D) quantification of Drp1 immunoreactivity in the cerebral cortex.
Figure 4
Figure 4
Immunoreactivity of Fis1 in brain sections from Drp1+/+ mice and Drp1+/− mice. (A) represents the hippocampus (10X the original magnification); (B), the cerebral cortex (10X the original magnification); (C), the cerebral cortex (40X the original magnification) and (D) quantification of Fis1 immunoreactivity in the cerebral cortex.
Figure 5
Figure 5
Immunoreactivity of Mfn1 in brain sections from Drp1+/+ mice and Drp1+/− mice. (A) the cerebral cortex (10X the original magnification); (B), the cerebral cortex (40X the original magnification) and (C) quantification of Mfn1 immunoreactivity in the cerebral cortex.
Figure 6
Figure 6
Immunoreactivity of cyclophilin D in brain sections from Drp1+/+ mice and Drp1+/− mice. (A) represents the hippocampus (10X the original magnification); (B), the cerebral cortex (10X the original magnification); (C), the cerebral cortex (40X the original magnification) and (D) quantification of CypD immunoreactivity in the cerebral cortex.
Figure 7
Figure 7
Immunoreactivity of synaptophysin in brain sections from Drp1+/+ mice and Drp1+/− mice. (A) represents the hippocampus (10X the original magnification); (B), the cerebral cortex (10X the original magnification); (C), the cerebral cortex (40X the original magnification) and quantification of synaptophysin immunoreactivity in the cerebral cortex.
Figure 8
Figure 8
Immunoreactivity of PSD95 in brain sections from Drp1+/+ mice and Drp1+/− mice. (A) represents the hippocampus (10X the original magnification); (B), the cerebral cortex (10X the original magnification); (C), the cerebral cortex (40X the original magnification) and (D) quantification of MAP2 immunoreactivity in the cerebral cortex.
Figure 9
Figure 9
Immunoreactivity of MAP2 in brain sections from Drp1+/+ mice and Drp1+/− mice. (A) the cerebral cortex (10X the original magnification); (B), the cerebral cortex (40X the original magnification) and (C) quantification of MAP2 immunoreactivity in the cerebral cortex.
Figure 10
Figure 10
Immunoreactivity of mitochondrial matrix marker, pyruvate dehydrogenase from Drp1+/+ and Drp1+/− mice. (A) represents hippocampal section 9100X the orginal magnification) and (B) represents cerebral cortex (100X the original magnification. White arrows indicate elongated mitochondria.
Figure 11
Figure 11
Mitochondrial functional parameters in Drp1+/+ mice and Drp1+/− mice. (A) represents hydrogen peroxide in cerebral cortex and cerebellum tissues from Drp1+/− mice and Drp1+/+ mice. Significantly reduced hydrogen peroxide production was found in the cerebral cortex of Drp1+/− mice compared to Drp1+/+ mice (P<0.05). (B) represents cytochrome oxidase activity. Cytochrome oxidase activity was unchanged in the cerebral cortex and cerebellum tissues from Drp1+/− mice compared to Drp1+/+ mice. (C) represents lipid peroxidation in cerebral cortex and cerebellum tissues from Drp1+/− mice and Drp1+/+ mice. Significantly reduced lipid peroxidation levels were found in the cerebral cortex of Drp1+/− mice compared to Drp1+/+ mice (P<0.05). (D) represents synaptosomal mitochondrial ATP. Synaptosomal mitochondrial ATP was unchanged in the cerebral cortex and cerebellum tissues from Drp1+/− mice compared to Drp1+/+ mice.
Figure 12
Figure 12
GTPase enzymatic activity in Drp1+/+ mice and Drp1+/− mice. GTPase enzymatic activity was unchanged in the cerebral cortex and cerebellum tissues from Drp1+/− mice compared to Drp1+/+ mice.
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