Journal: The Journal of Neuroscience

Article Title: Mitochondrial Dynamics and Bioenergetic Dysfunction Is Associated with Synaptic Alterations in Mutant SOD1 Motor Neurons

doi: 10.1523/JNEUROSCI.1233-11.2012

Figure Lengend Snippet: Impaired mitochondrial fusion in the soma and motor axons of G93A SOD1 motor neurons. A, Cell bodies from non-transgenic control and G93A SOD1 motor neurons containing mitoDendra-labeled mitochondria before (−1 min) and after (0 min) photo-activation. Subsets of mitochondria (green and red fluorescence) were followed over time (10, 40, 70, and 100 min) by live imaging microscopy. Note that the appearance of yellow fluorescence, as a result of the mixing between green and red mitochondria (i.e. fusion, indicated by arrows), was delayed in mutant SOD1 motor neurons. Scale bar, 10 μm. B, Fusion rates were obtained in single optical z-sections by measuring the colocalization (in %) of red over green fluorescent mitochondria at the indicated time points. The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. C, In the inset, example of a time-lapse recording of axonal mitochondria (numbers indicate time in min) showing a fusion event (arrow). Scale bar, 5 μm. The graph shows the analysis of fusion (% of fusion events of total moving mitochondria) in control and G93A SOD1 motor axons. n (axons) = 24 non-transgenic, 28 WT and 16 G93A axonal segments. *P<0.05 versus non-transgenic. D, Time-lapse microscopy of mitochondrial transport in the soma of non-transgenic and G93A SOD1 motor neurons. All mitochondria in a ROI of the soma were photo-converted, while only non-photo-converted (green fluorescent) mitochondria were followed over time. Note a decrease of mobile mitochondria towards the ROI in mutant SOD1 motor neurons compared to controls. Scale bar, 10 μm. E, Analysis of the transport of green mitochondria over the photo-activated area (no green mitochondria present). The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. F, Analysis of the transport of red mitochondria over the non-photo-activated area (no red mitochondria present). The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. All data obtained from 3–5 independent experiments. The error bars represent ± SE.

Article Snippet: Colocalization and Integrated Morphometric Analysis applications were from Metamorph software (Universal Imaging Co.).

Techniques: Transgenic Assay, Control, Labeling, Activation Assay, Fluorescence, Imaging, Microscopy, Mutagenesis, Time-lapse Microscopy

Journal: Neuron

Article Title: GRASP1 regulates synaptic plasticity and learning through endosomal recycling of AMPA receptors

doi: 10.1016/j.neuron.2017.02.031

Figure Lengend Snippet: (A) Colocalization (white) of internalized GluA2 (iGluA2, green) and EEA1 (magenta) in cell bodies of hippocampal neurons transfected with a shRNA for GRASP1 knockdown (KD) and either GRASP1 WT or ID mutants (S73N, R822Q). (% iGluA2 colocalization: KD+WT= 8.4 ± 1.0; KD+S73N= 9.3 ± 1.4; KD+R822Q= 12.0 ± 1.5; n= 23–24 neurons/group, p> 0.05, one-way ANOVA).

Article Snippet: Colocalization of internalized AMAPR with each marker was measured in the “colocalization module” in MetaMorph as described (Lee et al., 2004) which calculated % integrated values of internalized GluA2 overlapping with endosomal markers.

Techniques: Transfection, shRNA, Knockdown

Journal: Human Molecular Genetics

Article Title: In vitro and in vivo studies of the ALS-FTLD protein CHCHD10 reveal novel mitochondrial topology and protein interactions

doi: 10.1093/hmg/ddx397

Figure Lengend Snippet: Mutant CHCHD10 overexpression in HEK293 cells. (A) Western blot of crude mitochondrial fractions from control HEK293 cells and cells transfected with WT CHCHD10-FLAG, R15L CHCHD10-FLAG or S59L CHCHD10-FLAG. (B) Co-IP of WT, R15L or S59L CHCHD10-FLAG with FLAG antibody or IgG control. Immunoblot: FLAG, CHCHD2. (C) Quantification of Western blot band intensity following CHX treatment. Data are expressed as percentage of the FLAG intensity at 0 h. n = 4. (D) Representative images of HeLa cells transfected with WT CHCHD10-Myc, R15L CHCHD10-Myc or S59L CHCHD10-Myc and immunostained for Myc (green) or Tom20 (red). Bar = 5 μm. (E) CHCHD10 and Tom20 percentage of colocalization n = 15 cells for WT CHCHD10-Myc, n = 22 cells for R15L CHCHD10-Myc, n = 25 cells for S59L CHCHD10-Myc. (F–G) Oxygen consumption rate (OCR) in cells transfected with empty vector (pcdna) or CHCHD10-FLAG, before or after FCCP treatment (1 μM). n = 3. (H) ATP synthesis in cells transfected with empty vector (pcdna) or CHCHD10-FLAG. n = 3 to 5.

Article Snippet: For CHCHD10 and Tom20 colocalization, ten random fields were imaged and the colocalization of the two fluorochromes was assessed using the colocalization function in MetaMorph.

Techniques: Mutagenesis, Over Expression, Western Blot, Control, Transfection, Co-Immunoprecipitation Assay, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: Lanosterol induces mitochondrial uncoupling and protects dopaminergic neurons from cell death in a model for Parkinson's disease

doi: 10.1038/cdd.2011.105

Figure Lengend Snippet: LSS is redistributed from ER to mitochondria in dopaminergic neurons on addition of MPP+. ( a ) Confocal images of ventral midbrain neurons stained with TH (white), LSS (green), either KDEL (red, first and third panels from the top) or TOMM20 (red, second and fourth panels from the top) of ventral midbrain neurons in control and MPP+ treatment. White boxes show dopaminergic neurons as determined by TH+ staining. Right panels show pixel intensity correlation plots of LSS with either KDEL or TOMM20 in dopaminergic neurons. Scale bar represents 10 μ m. ( b ) Average R 2 values (a measure of colocalization) of two classes of neurons in control and MPP+-treated conditions. In control, R 2 values from co-staining of KDEL-LSS, n =26 and n =27, TOMM20-LSS, n =20 and n =21, were assessed for dopaminergic and non-dopaminergic neurons respectively. In MPP+-treated cells, R 2 values from co-staining of KDEL-LSS, n =20 and n =20, for TOMM20-LSS, n =18 and n =17, were assessed for dopaminergic and non-dopaminergic neurons respectively. In MPP+/lanosterol co-treated cells, R 2 values from co-staining of KDEL-LSS, n =18 and n =23, for TOMM20-LSS, n =19 and n =18, were assessed for dopaminergic and non-dopaminergic neurons respectively. **** P <0.00001

Article Snippet: To quantify mitophagy, the percentage of MitoRed and LC3-positive mitochondria were plotted as a percentage to total MitoRed-positive mitochondria using the Metamorph 7.6 colocalization drop-in.

Techniques: Staining

Journal: Cell Death and Differentiation

Article Title: Lanosterol induces mitochondrial uncoupling and protects dopaminergic neurons from cell death in a model for Parkinson's disease

doi: 10.1038/cdd.2011.105

Figure Lengend Snippet: Lanosterol increases mitophagy in axons. ( a ) Confocal images of hippocamal axons stained with LC3 (top panels, green) and electroporated with MitoRed (middle panels, red). Bottom panels show of LC3 and MitoRed. White arrows represent-colocalization of the two channels. Scale bar represents 20 μ m. ( b ) Graphical plot of average percentage of mitochondria with LC3-positive stains. Error bars represent S.E.M. For each condition, n >40 axons were assessed from three independent experiments. * P <0.05

Article Snippet: To quantify mitophagy, the percentage of MitoRed and LC3-positive mitochondria were plotted as a percentage to total MitoRed-positive mitochondria using the Metamorph 7.6 colocalization drop-in.

Techniques: Staining