Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration
Figure 3
Defective mitochondrial morphogenesis and ultrastructure in Phb2-deficient neurons in vivo.
(A) Transmission electron microscopy analysis of the mitochondrial ultrastructure in DG neurons of 6-week-old Phb2NKO and Phb2fl/fl control mice. The enlargements show the double membrane of the mitochondrion and the emergence of one crista. Scale bar: 400 nm. (B) Fragmentation and perinuclear clustering of PHB2-deficient neuronal mitochondria. Primary hippocampal neurons isolated from E18.5 Phb2fl/fl embryos were infected with lentiviruses expressing mitochondrially targeted EGFP and Cre recombinase (NLS-Cre) as indicated. Fixed samples were immunostained with antibodies directed against GFP and neuronal βIII-tubulin followed by DAPI staining. a′, b′ are magnifications of the boxed insets shown in a, b. Scale bars: 10 µm. (C) Quantification of mitochondrial morphology in PHB2-deficient and control primary hippocampal neurons. Cells were infected with lentiviruses expressing Cre recombinase when indicated and processed as described in (B). Cells containing tubular (white bars) or fragmented mitochondria (red bars) were classified. >200 cells were scored in three independent experiments. ***P<0.001. Error bars indicate SEM. (D) Quantification of mitochondria per neurites in PHB2-deficient primary hippocampal neurons. Phb2fl/fl neurons were infected with lentiviruses expressing Cre recombinase when indicated and processed as described in (B). >30 cells were scored in three independent experiments. **P<0.01. Error bars indicate SEM. (E) Immunoblot analysis of hippocampal tissue lysates from Phb2NKO (KO) and Phb2fl/fl (WT) control mice of the indicated age. Lysates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Antibodies directed against VDAC and the 70 kDa subunit of complex II were used to monitor equal gel loading. b/e: long/short OPA1 isoforms.