Journal: Frontiers in Molecular Neuroscience

Article Title: Interference With Complex IV as a Model of Age-Related Decline in Synaptic Connectivity

doi: 10.3389/fnmol.2020.00043

Figure Lengend Snippet: Neuron-specific knockdown of Cytochrome C Oxidase subunit 4 (Cox4). (A) Determination of lentiviral transduction efficiency, i.e., percentage of cells positive for both virally expressed EGFP and neuronal marker Microtubule-associated protein 2 (MAP2). Micrographs depict confocal images of a rat primary neuronal culture transduced with Lenti04CmiCox79/SEW (miCox79) and counterstained for MAP2. n = 10 images for all groups. (B,C) Knockdown of rat Cox4 expression as assessed by quantitative real-time PCR (B) and single-cell quantitative immunocytochemistry (C) in lentivirally transduced primary rat neurons in vitro . One-way ANOVA, Dunnett’s multiple comparisons test. *** p < 0.001; ** p < 0.01. n = 6 wells for all groups (B) . Kruskal–Wallis test, Dunn’s multiple comparisons test. *** p < 0.001; ** p < 0.01. n = 25 cells for all groups (C) . (D,E) Determination of cell viability and activated caspase 3/7 (ApoLive-Glo TM Multiplex Assay) in cultures of rat primary neurons after lentiviral transduction with either miCTR, miCox79 or miCox474 expressing lentiviral vectors. One-way ANOVA, Dunnett’s multiple comparisons test. n.s., not significant. n = 6 wells for all groups. (F) Determination of ATP content in primary rat neurons transduced with miCTR or miCox79 expressing lentiviral vectors and treated with picomolar concentrations of rotenone for 30 min before measurement. Multiple t -tests. *** p < 0.001; ** p < 0.01. n = 6 wells for all groups.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Cox4 (Cell Signaling Technology, Danvers, MA, USA, Cat. No. 11967), monoclonal mouse anti-gephyrin (Synaptic Systems, Cat. No. 147021), monoclonal mouse anti-MAP2 (Merck, Cat. No. M1406), polyclonal rabbit anti-MAP2 (Merck, Cat. No. AB5622), monoclonal mouse anti-NeuN (Merck, Cat. No. MAB377), polyclonal rabbit anti-PSD95 (Abcam, Cat. No. ab18258), monoclonal rabbit anti-PSD95 (Cell Signaling Technology, Danvers, MA, USA), polyclonal rabbit anti-VGAT (Synaptic Systems, Cat. No. 131003), monoclonal mouse anti-VGlutI (Synaptic Systems, Cat. No. 135511).

Techniques: Transduction, Marker, Expressing, Real-time Polymerase Chain Reaction, Immunocytochemistry, In Vitro, Multiplex Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Interference With Complex IV as a Model of Age-Related Decline in Synaptic Connectivity

doi: 10.3389/fnmol.2020.00043

Figure Lengend Snippet: Synaptic loss in vitro after knockdown of Cox4. (A) Micrographs of cultured primary rat neurons transduced with lentiviral suspensions for the expression of either miCTR, miCox79, or miCox474 and stained for neuronal markers MAP2, Gephyrin (Geph), and PSD95, respectively. The respective left images (EGFP/Geph, EGFP/PSD95) represent maximum intensity projections (MIP) of confocal z-stacks. The remaining tiles depict neuronal cultures after surface rendering using immunofluorescent signals of marker proteins indicated. Segmentation of Gephyrin and PSD95 positive immunofluorescent signals was carried out on 20 μm segments of primary dendrites (dashed boxes). Scale bars, 20 μm, and 2 μM, respectively. (B,C) Quantification of Gephyrin and PSD95 cluster densities on 20 μm segments of primary dendrites of hippocampal neurons transduced as indicated. n = 39 dendritic segments for all groups. Kruskal–Wallis test, Dunn’s multiple comparisons test. *** p < 0.001; ** p < 0.01. (D) Quantification of Gephyrin cluster densities on 20 μm segments of primary dendrites. Hippocampal neurons were transduced with lentiviral suspensions and treated with pharmacological inhibitors as indicated. SB, SB216763 (GSK3 inhibitor); CC, Compound C (AMPK inhibitor). Kruskal–Wallis test, Dunn’s multiple comparisons test. *** p < 0.001. ns, not significant. n = 40 dendritic segments for all groups.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Cox4 (Cell Signaling Technology, Danvers, MA, USA, Cat. No. 11967), monoclonal mouse anti-gephyrin (Synaptic Systems, Cat. No. 147021), monoclonal mouse anti-MAP2 (Merck, Cat. No. M1406), polyclonal rabbit anti-MAP2 (Merck, Cat. No. AB5622), monoclonal mouse anti-NeuN (Merck, Cat. No. MAB377), polyclonal rabbit anti-PSD95 (Abcam, Cat. No. ab18258), monoclonal rabbit anti-PSD95 (Cell Signaling Technology, Danvers, MA, USA), polyclonal rabbit anti-VGAT (Synaptic Systems, Cat. No. 131003), monoclonal mouse anti-VGlutI (Synaptic Systems, Cat. No. 135511).

Techniques: In Vitro, Cell Culture, Transduction, Expressing, Staining, Marker