Ndufs4 ablation decreases synaptophysin expression in hippocampus

Abstract

Altered function of mitochondrial respiratory chain in brain cells is related to many neurodegenerative diseases. NADH Dehydrogenase (Ubiquinone) Fe-S protein 4 (Ndufs4) is one of the subunits of mitochondrial complex I and its mutation in human is associated with Leigh syndrome. However, the molecular biological role of Ndufs4 in neuronal function is poorly understood. In this study, upon Ndufs4 expression confirmation in NeuN-positive neurons, and GFAP-positive astrocytes in WT mouse hippocampus, we found significant decrease of mitochondrial respiration in Ndufs4-KO mouse hippocampus. Although there was no change in the number of NeuN positive neurons in Ndufs4-KO hippocampus, the expression of synaptophysin, a presynaptic protein, was significantly decreased. To investigate the detailed mechanism, we silenced Ndufs4 in Neuro-2a cells and we observed shorter neurite lengths with decreased expression of synaptophysin. Furthermore, western blot analysis for phosphorylated extracellular regulated kinase (pERK) revealed that Ndufs4 silencing decreases the activity of ERK signalling. These results suggest that Ndufs4-modulated mitochondrial activity may be involved in neuroplasticity via regulating synaptophysin expression.

Figure 1

Expression and localization of Ndufs4 in cerebral cortex and hippocampus. (A) Representative image from Western blot for Ndufs4 in brain of WT and Ndufs4-KO mouse. T: total protein, M: protein from isolated mitochondria. (B) Representative image from enzyme-based immunohistochemistry showing the expression of Ndufs4 in cerebral cortex and hippocampus. Insets enlarge the boxed area. Scale for low magnification: 500 µm, for high magnification: 50 µm. (C) Representative image from co-immunofluorescence staining of Ndufs4 (Green) with NeuN, GFAP and MBP (red) in hippocampus of WT mouse. Boxed area is enlarged on right. Scale for low magnification: 500 µm, for high magnification: 40 µm. (D–F) Mitochondrial complex I respiration assay by XF24 analyser using isolated mitochondria from punch out hippocampus of WT (n = 3) and Ndufs4-KO (n = 3) mouse.

Figure 2

Ndufs4 deficiency shows astrocyte reactivity in hippocampus displaying normal GFAP expression and proliferation in primary culture of astrocyte. (A–C) Representative image from immunofluorescence staining of NeuN (A), GFAP (B) and MBP (C) in hippocampus of WT and Ndufs4-KO mouse. Boxed area is enlarged on right. Scale for low magnification: 500 µm, for high magnification: 50 µm. (D) Representative image of Western blot for GFAP, NeuN and MBP in different regions of WT and Ndufs4-KO mouse brain. (E) Representative image of Western blot for GFAP in WT and Ndufs4-KO primary astrocytes. (F) Proliferation assay of WT (n = 6) and Ndufs4-KO (n = 6) astrocytes.

Figure 3

Ndufs4 deficiency shows altered synaptic protein expression without neuronal loss. (A) Representative image of co-immunofluorescence staining of NeuN and Caspase 3 in hippocampus and olfactory bulb (OB) of WT mouse and Ndufs4-KO. Insets enlarge the boxed area. Low magnification green indicates NeuN and red indicates caspase 3, scale bar: 400 µm. In high magnification red indicates caspase 3 with no green stain, scale: 20 µm. (B) Representative images of Western blot for synaptophysin, PSD95, Vglut1, EAAT1, EAAT2, EAAT3 in WT and Ndufs4-KO mouse hippocampus. (C–H) Analysis of band densities by ImageJ (n = 3).

Figure 4

Ndufs4 silencing impairs neurite outgrowth and synaptophysin-positive puncta in differentiated Neuro-2a cell. (A) Representative phase contrast image of neurite outgrowth in Ndufs4 silenced differentiated Neuro-2a cells. Scale bar 50 µm. (B) ImageJ analysis of average neurite length of differentiated cells (n = 6). (C) Representative image of differentiated Neuro-2a cells immunostained for β-III tubulin and synaptophysin. Circles indicate location of synaptophysin in neurites. Scale bar 40 µm (D) ImageJ analysis of average number of synaptophysin in neurites/50 µm (n = 6). (E) Representative image of Western blot for the pERK and ERK in control and Ndufs4 silenced differentiated Neuro-2a cells.