lrrk2-wt-myc  (Addgene inc)

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: ( A ) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. ( B ) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from LRRK2 WT and KO mouse whole brains were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. ( C ) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from CDGI WT and KO mouse whole brains were subjected to IP with anti-CDGI followed by anti-LRRK2 and anti-CDGI immunoblotting. ( D ) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse striatal lysates. Lysates prepared from LRRK2 WT and KO mouse striatum were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. ( E ) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged F1 (the GEF domain), F2 (the EF-hands domain), F3 (the DAG domain), or full-length (FL) CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag or anti-MYC immunoblotting. A schematic representation of CDGI F1, F2, and F3 domains is shown. ( F ) Co-IP analysis of the interaction between V5-tagged CDGI and flag-tagged LRRK2 fragments in cotransfected HEK 293T cells. Co-IP with anti-V5 was followed by anti-flag or anti-V5 immunoblotting. A schematic representation of the different LRRK2 fragments is shown. ( G ) Co-IP analysis of the interaction between V5-tagged CDGI and MYC-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-V5 immunoblotting. ( H ) Co-IP analysis of the interaction between flag-tagged CDGI-F1 domain and Myc-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. ( I ) Coimmunostaining of CDGI (green), LRRK2 (red), and DARPP32 (magenta) in mouse striatal sections. aa, amino acid.

Article Snippet: LRRK2 -WT-MYC was from the Dawson lab (Addgene plasmid #17609) ( ).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Mutagenesis

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: ( A ) GTP loading analysis of LRRK2 by an in vivo 32 P-orthophosphate labeling in HEK 293T cells overexpressing the indicated plasmids. The migration of GTP and GDP is indicated. The Ca 2+ ionophore A23187 and TPA were applied. ( B ) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). ( C ) GTP loading analysis of LRRK2 R1441C (RC) and G2019S (GS) with CDG-WT or GW by an in vivo 32 P-orthophosphate labeling in HEK 293T cells. TN (T1348N) was a negative control. ( D ) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). ( E ) The levels of LRRK2 bound to GTP were analyzed by pull-down assays with GTP-agarose from HEK 293T cells. ( F ) Quantification of LRRK2 bound to GTP-agarose beads. Data were normalized to LRRK2-WT alone in (B), (D), and (F). ( G ) GTP loading analysis of LRRK2 by an in vivo 32 P-orthophosphate labeling in WT striatal neurons compared to CDGI KO neurons transduced by LV-CDGI-WT or LV-CDGI-GW. ( H ) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). Data were normalized to WT neurons. ( I ) A diagram of the guanine nucleotide exchange assay is illustrated. Free BODIPY-FL-GDP is quenched with a low fluorescence in the solutions while showing increased fluorescence upon binding to GTPases. GEF catalyzes the exchange of preloaded BODIPY-FL-GDP for GTP causing a decrease in fluorescence. ( J ) Recombinant LRRK2 preloaded with BODIPY-FL-GDP was incubated with recombinant GST-CDGI proteins in the presence of excess cold GTP. Nucleotide exchange on LRRK2 was monitored by the fluorescence intensity change with different concentrations of CDGI-WT every 36 s for 15 min. Data are the means ± SEM, n = 3. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. n.s., not statistically significant.

Article Snippet: LRRK2 -WT-MYC was from the Dawson lab (Addgene plasmid #17609) ( ).

Techniques: In Vivo, Labeling, Migration, Binding Assay, Activity Assay, Negative Control, Fluorescence, Recombinant, Incubation

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: ( A to C ) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in HEK 293T cells cotransfected with LRRK2 and CDGI-WT or GW. After 48 hours, cells were harvested and fractionated into cytosol and membrane fractions. Samples were immunoblotted with anti-MYC, V5, LAMP1, Rab10, and phosphor-Rab10. Data were normalized to LRRK2-MYC alone. ( D and E ) Liquid nitrogen freeze-thaw methods assessing LRRK2 membrane association. HEK 293T cells with stably expressed eGFP-LRRK2 were transfected with or without mCherry-CDGI. After 48 hours, cells were permeabilized by liquid nitrogen freeze-thaw to deplete cytosol and then fixed, immunostained with LAMP1, and visualized by confocal microscopy. Scale bar, 20 μM. Data were normalized to eGFP-LRRK2 stable cells transfected with mCherry. ( F and G ) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in CDGI WT striatal neurons compared to CDGI KO neurons. CDGI WT and KO striatal neurons were treated with A23187 and TPA before being harvested and fractionated into cytosol and membrane fractions. Data were normalized to CDGI-WT neurons. Data are the means ± SEM, n = 3. One-way ANOVA followed by Tukey’s post hoc test was used for the data analysis of multiple comparisons, and Student’s t tests (unpaired, two-tailed) were used for the data analysis of two comparisons. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: LRRK2 -WT-MYC was from the Dawson lab (Addgene plasmid #17609) ( ).

Techniques: Cell Fractionation, Membrane, Stable Transfection, Transfection, Confocal Microscopy, Two Tailed Test

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: ( A ) Representative images of eye morphology of 1-week-old flies of the indicated genotypes by light microscopy. Drosophila LRRK2 ( dLRRK ) RNAi knockdown ( dLRRK RI ), human LRRK2 WT ( LRRK2 WT ), and LRRK2 mutant GS or RC ( LRRK2 GS or LRRK2 RC ) were coexpressed with human CDGI WT ( CDGI WT ) or CDGI-GW ( CDGI GW ) in fly eyes by a GMR-GAL4 driver ( GMR > CDGI/LRRK2 ). For each genotype, images were taken from at least 10 flies. Scale bar, 100 μm. ( B ) Representative images of eye morphology of 1-week-old flies of the indicated genotypes by SEM. Scale bar, 100 μm. ( C ) The fly eye size was quantified by ImageJ for each genotype. n = 10. Data are mean ± SEM, one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: LRRK2 -WT-MYC was from the Dawson lab (Addgene plasmid #17609) ( ).

Techniques: Light Microscopy, Knockdown, Mutagenesis

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: ( A ) Representative images of NeuN staining of mouse striatum. Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, the striatal neurons were immunolabeled by a NeuN antibody. Scale bar, 250 μm. ( B ) Quantification of NeuN-positive neurons in the striatum using an unbiased stereological method with stereo investigator software from five animals per group. ( C ) Representative images of DA neuron staining in mouse SNpc. After 9 to 10 months of viral injection, the DA neurons were immunolabeled by a TH antibody. Scale bar, 500 μm. ( D ) Quantification of TH-positive neurons in SNpc using an unbiased stereological method with stereo investigator software from five to seven animals per group. Data are mean ± SEM, two-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: LRRK2 -WT-MYC was from the Dawson lab (Addgene plasmid #17609) ( ).

Techniques: Staining, Injection, Immunolabeling, Software

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, a battery of behavioral tests was performed. ( A ) Open-field test. The total time spent at the center was analyzed. ( B ) Open-field test. The total time spent at the corner was analyzed. ( C ) Rotarod test. The average retention time was analyzed. ( D ) Pole test to monitor behavioral abnormalities. The total time to descend to the bottom was recorded. Data are mean ± SEM, n = 5 to 9 per group, one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05.

Article Snippet: LRRK2 -WT-MYC was from the Dawson lab (Addgene plasmid #17609) ( ).

Techniques: Injection, Battery

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: LRRK2 GTPase cycle is between inactive off GDP-bound state and active on GTP-bound state. CDGI binds to LRRK2 serving as a physiological GEF for LRRK2 to increase LRRK2 GTP binding activity and GDP release, and membrane association and in turn regulates LRRK2-induced striatal and DA neurodegeneration and behavioral deficits.

Article Snippet: LRRK2 -WT-MYC was from the Dawson lab (Addgene plasmid #17609) ( ).

Techniques: Binding Assay, Activity Assay, Membrane

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 1. CDGI interacts with LRRK2. (A) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. (B) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from LRRK2 WT and KO mouse whole brains were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. (C) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from CDGI WT and KO mouse whole brains were subjected to IP with anti-CDGI followed by anti-LRRK2 and anti-CDGI immunoblotting. (D) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse striatal lysates. Lysates prepared from LRRK2 WT and KO mouse stria- tum were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. (E) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged F1 (the GEF domain), F2 (the EF-hands domain), F3 (the DAG domain), or full-length (FL) CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was fol- lowed by anti-flag or anti-MYC immunoblotting. A schematic representation of CDGI F1, F2, and F3 domains is shown. (F) Co-IP analysis of the interaction between V5- tagged CDGI and flag-tagged LRRK2 fragments in cotransfected HEK 293T cells. Co-IP with anti-V5 was followed by anti-flag or anti-V5 immunoblotting. A schematic representation of the different LRRK2 fragments is shown. (G) Co-IP analysis of the interaction between V5-tagged CDGI and MYC-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-V5 immunoblotting. (H) Co-IP analysis of the interaction between flag-tagged CDGI-F1 domain and Myc-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. (I) Coimmunostaining of CDGI (green), LRRK2 (red), and DARPP32 (magenta) in mouse striatal sections. aa, amino acid.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Mutagenesis

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 2. CDGI acts as a potential GEF for LRRK2 to increase LRRK2 GTP binding activity and GDP release. (A) GTP loading analysis of LRRK2 by an in vivo 32P- orthophosphate labeling in HEK 293T cells overexpressing the indicated plasmids. The migration of GTP and GDP is indicated. The Ca2+ ionophore A23187 and TPA were applied. (B) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). (C) GTP loading analysis of LRRK2 R1441C (RC) and G2019S (GS) with CDG-WT or GW by an in vivo 32P-orthophosphate labeling in HEK 293T cells. TN (T1348N) was a negative control. (D) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). (E) The levels of LRRK2 bound to GTP were analyzed by pull-down assays with GTP-agarose from HEK 293T cells. (F) Quantification of LRRK2 bound to GTP-agarose beads. Data were normalized to LRRK2-WT alone in (B), (D), and (F). (G) GTP loading analysis of LRRK2 by an in vivo 32P-orthophosphate labeling in WT stria- tal neurons compared to CDGI KO neurons transduced by LV-CDGI-WT or LV-CDGI-GW. (H) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). Data were normalized to WT neurons. (I) A diagram of the guanine nucleotide exchange assay is illustrated. Free BODIPY-FL-GDP is quenched with a low fluorescence in the solutions while showing increased fluorescence upon binding to GTPases. GEF catalyzes the exchange of preloaded BODIPY-FL-GDP for GTP causing a decrease in fluorescence. (J) Recombinant LRRK2 preloaded with BODIPY-FL-GDP was incubated with recombinant GST-CDGI proteins in the presence of excess cold GTP. Nucleotide exchange on LRRK2 was monitored by the fluorescence intensity change with different concentrations of CDGI-WT every 36 s for 15 min. Data are the means ± SEM, n = 3. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n.s., not statistically significant.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Binding Assay, Activity Assay, In Vivo, Labeling, Migration, Negative Control, Fluorescence, Recombinant, Incubation

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 3. CDGI increases LRRK2 membrane association. (A to C) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in HEK 293T cells co- transfected with LRRK2 and CDGI-WT or GW. After 48 hours, cells were harvested and fractionated into cytosol and membrane fractions. Samples were immunoblotted with anti-MYC, V5, LAMP1, Rab10, and phosphor-Rab10. Data were normalized to LRRK2-MYC alone. (D and E) Liquid nitrogen freeze-thaw methods assessing LRRK2 membrane association. HEK 293T cells with stably expressed eGFP-LRRK2 were transfected with or without mCherry-CDGI. After 48 hours, cells were permeabilized by liquid nitrogen freeze-thaw to deplete cytosol and then fixed, immunostained with LAMP1, and visualized by confocal microscopy. Scale bar, 20 μM. Data were normalized to eGFP-LRRK2 stable cells transfected with mCherry. (F and G) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in CDGI WT striatal neu- rons compared to CDGI KO neurons. CDGI WT and KO striatal neurons were treated with A23187 and TPA before being harvested and fractionated into cytosol and mem- brane fractions. Data were normalized to CDGI-WT neurons. Data are the means ± SEM, n = 3. One-way ANOVA followed by Tukey’s post hoc test was used for the data analysis of multiple comparisons, and Student’s t tests (unpaired, two-tailed) were used for the data analysis of two comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Membrane, Cell Fractionation, Transfection, Stable Transfection, Confocal Microscopy, Two Tailed Test

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 4. CDGI acts upstream of LRRK2 to regulate retinal neurodegeneration. (A) Representative images of eye morphology of 1-week-old flies of the indicated geno- types by light microscopy. Drosophila LRRK2 (dLRRK) RNAi knockdown (dLRRKRI), human LRRK2 WT (LRRK2WT), and LRRK2 mutant GS or RC (LRRK2GS or LRRK2RC) were coex- pressed with human CDGI WT (CDGIWT) or CDGI-GW (CDGIGW) in fly eyes by a GMR-GAL4 driver (GMR > CDGI/LRRK2). For each genotype, images were taken from at least 10 flies. Scale bar, 100 μm. (B) Representative images of eye morphology of 1-week-old flies of the indicated genotypes by SEM. Scale bar, 100 μm. (C) The fly eye size was quantified by ImageJ for each genotype. n = 10. Data are mean ± SEM, one-way ANOVA followed by Tukey’s post hoc test, *P < 0.05, **P < 0.01, and ****P < 0.0001.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Light Microscopy, Knockdown, Mutagenesis

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 5. CDGI acts upstream of LRRK2 to regulate striatal and DA neurodegeneration. (A) Representative images of NeuN staining of mouse striatum. Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, the striatal neurons were immuno- labeled by a NeuN antibody. Scale bar, 250 μm. (B) Quantification of NeuN-positive neurons in the striatum using an unbiased stereological method with stereo investiga- tor software from five animals per group. (C) Representative images of DA neuron staining in mouse SNpc. After 9 to 10 months of viral injection, the DA neurons were immunolabeled by a TH antibody. Scale bar, 500 μm. (D) Quantification of TH-positive neurons in SNpc using an unbiased stereological method with stereo investigator software from five to seven animals per group. Data are mean ± SEM, two-way ANOVA followed by Tukey’s post hoc test, *P < 0.05, ** P < 0.01, and ***P < 0.001.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Staining, Injection, Labeling, Software, Immunolabeling

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 6. CDGI acts upstream of LRRK2 to regulate locomotor behavioral deficits. Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, a battery of behavioral tests was performed. (A) Open-field test. The total time spent at the center was analyzed. (B) Open-field test. The total time spent at the corner was analyzed. (C) Rotarod test. The average retention time was analyzed. (D) Pole test to monitor behav- ioral abnormalities. The total time to descend to the bottom was recorded. Data are mean ± SEM, n = 5 to 9 per group, one-way ANOVA followed by Tukey’s post hoc test, *P < 0.05.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Injection, Battery

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 7. Model of CDGI regulation of LRRK2 function and LRRK2-induced neurodegeneration. LRRK2 GTPase cycle is between inactive off GDP-bound state and active on GTP-bound state. CDGI binds to LRRK2 serving as a physiological GEF for LRRK2 to increase LRRK2 GTP binding activity and GDP release, and membrane association and in turn regulates LRRK2-induced striatal and DA neurodegeneration and behavioral deficits.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Binding Assay, Activity Assay, Membrane

Journal: Frontiers in Molecular Neuroscience

Article Title: The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1

doi: 10.3389/fnmol.2018.00064

Figure Lengend Snippet: Simplified pedigree of the analyzed family. (A) Symbols with black upper corner indicate individuals affected by Parkinson’s disease (PD). Age of onset of PD (years) is reported; in the patients’ relatives, age at last examination or age at death is reported. The result of leucine-rich repeat kinase 2 (LRRK2) genetic screening for the mutation was either wild type (WT) or heterozygous carrier (E193K). Gender of healthy siblings has been masked to protect the anonymity of the families. (B) The table summarizes clinical data of the three E193K carriers. (C) Sequencing of PCR product from exon 6 of WT and mutant alleles. The upper chromatogram of the portion of the sequencing gel shows the wild allele and the lower the heterozygous mutant allele.

Article Snippet: To this aim, we expressed myc-tagged LRRK2 WT or E193K variant together with FLAG-14-3-3ε in N2A cells (Gloeckner et al., ).

Techniques: Mutagenesis, Sequencing

Journal: Frontiers in Molecular Neuroscience

Article Title: The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1

doi: 10.3389/fnmol.2018.00064

Figure Lengend Snippet: E193K variant does not affect LRRK2 kinase activity. (A) Ribbon diagram showing a structural homology model (based on the ARM domain of adenomatous polyposis coli (APC) protein from human, PDB: 3T7U) for the LRRK2 N-terminal region (residues 7-322). N-terminus (N) and C-terminus (C) of the structural model as well as the position of E193 (stick model) are indicated. (B) In vitro radioactive kinase assays of 3x-Flag LRRK2 WT and E193K purified from HEK cells. Five nanomolar of purified Flag-LRRK2 WT or E193K from HEK293T cells were subjected to in vitro radiometric kinase assays and the radioactivity incorporated was quantified by PhosphoImager. Upper panel represents autophosphorylation and lower panel western blotting with anti-flag antibodies to quantify total loading. (C) Quantification of moles of 33 P incorporated by LRRK2. Graphs report mean ± standard error (SE), n = 4. (D) In vitro kinase assays as in (A) . Five nanomolar of purified Flag-LRRK2 WT or E193K from HEK293T cells were also subjected to in vitro non-radioactive kinase assays. Autosphosphorylation levels were measured by western blotting with anti-Ser1292 and anti-Thr2483 antibodies (upper panels). Lower panel represents total protein loading, probed with anti-flag antibodies. (E) Quantification of phosphorylation at Thr2483 and (F) Ser1292, expressed as optical density and normalized vs. total LRRK2 protein amount. Graphs report mean ± SE, n = 4. (G) Purified Flag-LRRK2 WT, E193K or K1906M protein were incubated with Flag-Rab8 (1:15 molar ratio) in the presence or absence of 1 mM ATP and subjected to PhosTag assay to analyze phosphorylation stoichiometry and SDS-PAGE to verify total protein amount. Anti-Flag antibody was used to reveal LRRK2 and Rab8. (H) Quantification of Rab8 phosphorylation, expressed as optical density and normalized vs. total Rab8 (phosphorylated + unphosphorylated band). Data are presented as mean ± SE ( n = 3); ** p < 0.01 vs. -ATP, same LRRK2 variant.

Article Snippet: To this aim, we expressed myc-tagged LRRK2 WT or E193K variant together with FLAG-14-3-3ε in N2A cells (Gloeckner et al., ).

Techniques: Variant Assay, Activity Assay, In Vitro, Purification, Radioactivity, Western Blot, Incubation, SDS Page

Journal: Frontiers in Molecular Neuroscience

Article Title: The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1

doi: 10.3389/fnmol.2018.00064

Figure Lengend Snippet: E193K variant affects LRRK2 phosphorylation at Ser935. (A) Protein lysate prepared from primary fibroblast obtained from healthy individuals or E193K carrier was analyzed by western-blotting to appreciate total LRRK2 level and phosphorylation at Ser-935. (B,C) The graphs report LRRK2 total level, expressed as optical density and normalized vs. β-tubulin amount (B) and P-Ser935 level, expressed as optical density and normalized vs. total LRRK2 amount (C) . Data are shown as mean ± SE, n = 8; *** p < 0.001 vs. WT. (D) N2a cells expressing FLAG-14-3-3ε together with myc-LRRK2 WT or myc-LRRK2 E193K variant were solubilized and processed for FLAG immunopurification. We evaluated the extent of LRRK2 binding to 14-3-3ε by measuring the amount of myc LRRK2 co-precipitating with FLAG 14-3-3ε (E) The graph reports the amount of myc-LRRK2 variant recovered in FLAG immunoprecipitates. Data were normalized to the amount of 14-3-3ε immunoprecipitated and expressed as mean ± SE, n = 4; ** p < 0.01versus WT. (F) N2a cells expressing GFP together with RFP-LRRK2 WT or RFP-LRRK2 E193K variant were processed for imaging purposes. Scale bar = 10 μm.

Article Snippet: To this aim, we expressed myc-tagged LRRK2 WT or E193K variant together with FLAG-14-3-3ε in N2A cells (Gloeckner et al., ).

Techniques: Variant Assay, Western Blot, Expressing, Immu-Puri, Binding Assay, Immunoprecipitation, Imaging

Journal: Frontiers in Molecular Neuroscience

Article Title: The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1

doi: 10.3389/fnmol.2018.00064

Figure Lengend Snippet: E193K variant affects LRRK2 supra-molecular organization. (A) Protein lysate prepared from primary fibroblast obtained from healthy individuals or E193K carrier was separated by size exclusion chromatography (SEC). LRRK2 elution profile was revealed by dot-blot using anti-LRRK2 antibody. Theoretical molecular weight are: V 0 at fraction 8.5, 669 kDa at fraction 12, 449 kDa at fraction 13. (B) The graph reports the intensity of each dot (fraction) normalized by the integrated intensities. Data are shown as mean ± SE, n = 4; * p < 0.05 vs. WT. Column void volume is 7.5 ml. (C) Primary fibroblasts obtained from healthy individuals or an E193K carrier were assayed by filter retardation assay to isolate high molecular weight (HMW) form of LRRK2 on acetate cellulose membrane and total LRRK2 on nitrocellulose membrane. (D) The graph reports LRRK2 HMW amount expressed as fold over total LRRK2. Data are shown as mean ± SE, n = 8; ** p < 0.01 vs. WT.

Article Snippet: To this aim, we expressed myc-tagged LRRK2 WT or E193K variant together with FLAG-14-3-3ε in N2A cells (Gloeckner et al., ).

Techniques: Variant Assay, Size-exclusion Chromatography, Dot Blot, Molecular Weight

Journal: Frontiers in Molecular Neuroscience

Article Title: The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1

doi: 10.3389/fnmol.2018.00064

Figure Lengend Snippet: E193K variant affects LRRK2-DRP1 complex. (A) Extracts of mouse adult forebrain were incubated with anti-LRRK2 antibodies or rat IgG. The immunocomplexes were isolated with protein G-Sepharose and the samples were resolved by SDS-PAGE and analyzed by immunoblotting with anti DRP1 and anti LRRK2 antibodies. Arrowhead indicates unspecific band recognized by anti-rabbit secondary antibody. (B) We isolated on streptavidin resin strep-FLAG-LRRK2 full-length (full-length), strep-FLAG-LRRK2ΔWD40 (ΔWD40) and strep-FLAG-LRRK2ΔN–terminal (ΔN–terminal) protein from HEK293 over-expressing cells. Interacting proteins were resolved by western-blotting. (C) We performed a GST-pull down approach to explore the interactome associated to LRRK2 N-terminal Armadillo domain. GST-fusion proteins corresponding to Armadillo domain of LRRK2 WT and LRRK2 E193K (E193K) were used to retain interactors from adult forebrain lysate. The complexes were isolated with GSH-Sepharose beads, the samples were resolved by SDS-PAGE and analyzed by immunoblotting with anti DRP1 and anti P-Ser616 DRP1 antibodies. (D) We evaluated the extent of DRP1 and P-Ser616 DRP1 bound to WT and E193K Armadillo domain expressed as ratio over WT domain. Graph reports mean ± SE; n = 4; ** p < 0.01 vs. WT. (E) N2A cells expressing Strep-FLAG-LRRK2 WT or Strep-FLAG-LRRK2 E193K variant were treated or not with MPP+ (1 mM, 24 h) solubilized and processed for streptavidin immunopurification. We evaluated the extent of DRP1 binding to LRRK2 by measuring the amount of DRP1 protein co-precipitating with Strep-FLAG LRRK2 variant. (F) The graph reports the amount of DRP1 recovered in FLAG immunoprecipitates. Data were normalized to the amount of LRRK2 variant immunoprecipitated and expressed as mean ± SE, n = 7; * p < 0.05 vs. WT, ## p < 0.01 vs. E193K not treated.

Article Snippet: To this aim, we expressed myc-tagged LRRK2 WT or E193K variant together with FLAG-14-3-3ε in N2A cells (Gloeckner et al., ).

Techniques: Variant Assay, Incubation, Isolation, SDS Page, Western Blot, Expressing, Immu-Puri, Binding Assay, Immunoprecipitation

Journal: Frontiers in Molecular Neuroscience

Article Title: The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1

doi: 10.3389/fnmol.2018.00064

Figure Lengend Snippet: The E193K variant prevents mitochondrial fission upon MPP+ treatment. (A) Under basal conditions mitochondria undergo continuous cycles of fusion and fission. Fission requires the activity of DRP1. LRRK2 binds to DRP1 and increases DRP1 recruitment to mitochondria. (B) Under noxious stimuli, WT cells react via the LRRK2-DRP1 complex that allows mitochondrial fission (1). Eventually, damaged mitochondria are eliminated via autophagy (2) limiting the production of ROS. (C) In the presence of a toxic insult the LRRK2 E193K-DRP1 complex is partially destabilized. This leads to an incomplete fragmentation of mitochondria that prevents the clearance of damaged organelles and increases the production of ROS.

Article Snippet: To this aim, we expressed myc-tagged LRRK2 WT or E193K variant together with FLAG-14-3-3ε in N2A cells (Gloeckner et al., ).

Techniques: Variant Assay, Activity Assay