Journal: PLoS ONE

Article Title: Activation of FADD-Dependent Neuronal Death Pathways as a Predictor of Pathogenicity for LRRK2 Mutations

doi: 10.1371/journal.pone.0166053

Figure Lengend Snippet: a) Primary rat cortical neurons were co-transfected with WT or mutant LRRK2 as indicated together with pcms-EGFP, and fixed and processed for anti-LRRK2 immunofluorescence together with Hoechst to label nuclei. Representative images are shown depicting anti-LRRK2 in red. b) Primary neurons as in (a) except treated with the LRRK2 kinase inhibitor MLi-2 (10nM). c) For quantification of apoptotic neurons, a minimum of 100 GFP-positive neurons were counted from each of three parallel coverslips per condition in a blinded fashion. Three independent transfections were conducted. Apoptotic neurons were defined as having two or more condensed nuclear fragments within a GFP-expressing neuronal profile. ** p<0.01, compared to WT; *** p<0.001, compared to WT; ### p<0.001, compared to the corresponding vehicle-treated culture.

Article Snippet: Parallel coverslips were double immunostained for GFP (chicken polyclonal; Abcam; Cambridge, UK) and Flag (mouse monoclonal, clone M2; Sigma) or LRRK2 (rabbit monoclonal, clone c41-2; Epitomics/Abcam) to confirm that the majority of GFP-positive cells also over-expressed Flag-LRRK2.

Techniques: Transfection, Mutagenesis, Immunofluorescence, Expressing

Journal: PLoS ONE

Article Title: Activation of FADD-Dependent Neuronal Death Pathways as a Predictor of Pathogenicity for LRRK2 Mutations

doi: 10.1371/journal.pone.0166053

Figure Lengend Snippet: a) HEK293T cells expressing GFP-tagged I2020T-LRRK2 were extracted with 0.1% Triton X-100 for 5 min, fixed and processed for anti-GFP immunostaining with DAPI counterstaining. b) HEK293T cells were transfected and extracted as in (a), and the resulting soluble and insoluble fractions were subjected to SEC. Alternate fractions were analyzed by SDS-PAGE for the presence of GFP-tagged LRRK2. HEK293T cells transiently over-expressing Flag-LRRK2 were (c) lysed normally to produce whole-cell extracts, or (d, e) subjected to mild in situ extraction as in (a). c) Detergent-free total cell extracts of HEK293T cells subjected to SEC were analyzed by dot blot using anti-Flag antibodies to show any shift in elution towards lower or higher molecular weight complexes. Representative blots are shown (of n = 2), demonstrating in a complimentary way the differential oligomeric behavior of the different LRRK2 mutants. The approximate elution point of the native molecular weight standards thyroglobulin and apoferritin are shown as a reference only. d) The amount of LRRK2 (in ng; determined by ELISA using recombinant full-length LRRK2 as a standard) present in the total fraction, or in either the soluble or insoluble fractions was determined by ELISA. Similar levels of expression were observed for each of the mutants tested. The increased ratio of insoluble:soluble LRRK2 for the I2020T mutant (e) reflects its re-organization to extraction-resistant filamentous structures.,None of the other sequence variants significantly altered this profile in comparison to WT-LRRK2, although there was a non-significant trend for reduced solubility for the T1410M variant. ** p<0.01 compared to WT-LRRK2.

Article Snippet: Parallel coverslips were double immunostained for GFP (chicken polyclonal; Abcam; Cambridge, UK) and Flag (mouse monoclonal, clone M2; Sigma) or LRRK2 (rabbit monoclonal, clone c41-2; Epitomics/Abcam) to confirm that the majority of GFP-positive cells also over-expressed Flag-LRRK2.

Techniques: Expressing, Immunostaining, Transfection, SDS Page, In Situ, Dot Blot, Molecular Weight, Enzyme-linked Immunosorbent Assay, Recombinant, Mutagenesis, Sequencing, Solubility, Variant Assay

Journal: PLoS ONE

Article Title: Activation of FADD-Dependent Neuronal Death Pathways as a Predictor of Pathogenicity for LRRK2 Mutations

doi: 10.1371/journal.pone.0166053

Figure Lengend Snippet: a) Primary cortical neurons were co-transfected with an EGFP-pCMS reporter vector, and the LRRK2 mutant together with the FADD dominant negative (HA-tagged leucine-zipper FADD death domain; lzDD) or empty pcDNA vector as a control. The percentage of GFP-positive neurons containing apoptotic nuclear features (defined as above) was determined 72h following transfection. The presence of the dimeric FADD death domain significantly reduced neuronal apoptotic death in cultures expressing each of the LRRK2 variants. b) A representative image of primary neurons expressing Flag-K544E-LRRK2 with the lz-DD fragment, co-stained for ant-GFP and anti-HA (red). *** p<0.001 compared to WT LRRK2; ## p<0.01 compared to pcDNA; ### p<0.001 compared to pcDNA.

Article Snippet: Parallel coverslips were double immunostained for GFP (chicken polyclonal; Abcam; Cambridge, UK) and Flag (mouse monoclonal, clone M2; Sigma) or LRRK2 (rabbit monoclonal, clone c41-2; Epitomics/Abcam) to confirm that the majority of GFP-positive cells also over-expressed Flag-LRRK2.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Dominant Negative Mutation, Expressing, Staining