Journal: Neural Regeneration Research

Article Title: Differential distribution of PINK1 and Parkin in the primate brain implies distinct roles

doi: 10.4103/nrr.nrr-d-23-01140

Figure Lengend Snippet: Figure 1 ｜A newly developed PINK1 monoclonal antibody shows selective expression of PINK1 in primate brain. (A) Schematic diagram of human PINK1 protein structure and the recognition sites of monoclonal PINK1 antibody E7B6. (B) HEK293 cells were transfected with human PINK1 cDNA. Western blot analysis of lysates from the cortex of wild type (WT) and PINK1 mutant (M6) monkeys and PINK1-transfected HEK293 cell lysates showing that E7B6 recognizes the endogenous monkey PINK1 and transfected human PINK1. (C) E7B6 western blot of monkey brain tissue showing various expression levels of PINK1 and Parkin. (D) The ratios of PINK1 to vinculin and Parkin to vinculin in monkey brain tissue of C. (E) E7B6 western blot analysis of human brain and peripheral tissue from a 54-year-old individual showing the selective expression of PINK1 in the human brain. In C and E, Parkin expression levels in various brain regions are different from those of PINK1. (F) The ratios of PINK1 to vinculin and Parkin to vinculin in human tissue from E. BS: Brain stem; Cereb: cerebellum; Ctx: cortex; Hippo: hippocampus; SN: substantia nigra; Str: striatum.

Article Snippet: Table 1 | Main antibodies used in this study Antibody Host Dilution Cat# RRID Supplier Application PINK1 Mouse 1:50 E7B6 NA Homemade antibody by MerryBio, Nanjing China WB, IF, IHC PINK1 Rabbit 1:500 BC100-494 AB_10127658 Novus Biologicals , Centennial, CO, USA WB PINK1 Rat 1:500 MAB4357 AB_2164246 R&D Systems, Minneapolis, MN, USA IF PINK1 Sheep 1:500 S086D AB_2924297 MRC, Scotland, UK WB Parkin Mouse 1:1000 sc-32282 AB_628104 Santa Cruz Biotechnology, Santa Cruz, CA, USA WB, IF Parkin Mouse 1:1000 MAB5512 AB_2267915 Millipore, Temecula, CA, USA IF Vinculin Mouse 1:5000 MAB3574 AB_2304338 Millipore WB (Internal reference) NeuN Rabbit 1:5000 ab177487 AB_2532109 Abcam, Cambridge, MA, USA WB, IF (Neuron) SNAP25 Rabbit 1 μg/mL ab41455 AB_945552 Abcam WB (Synaptosomal) TOM20 Rabbit 1:500 #42406 AB_2687663 Cell Signaling Technolog, Danvers, MA, USA IF (outer mitochondrial membrane) PSD95 Rabbit 1:2000 ab238135 AB_2895158 Abcam WB (Synaptosomal) Calnexin Rabbit 1:1000 ab22595 AB_2069006 Abcam WB (Endoplasmic reticulum) MBP Rabbit 1:3000 ab218011 AB_2895537 Abcam WB (Myelin) Histone H3 Rabbit 1:3000 #9715 AB_331563 Cell Signaling Technology WB (Nuclei) GAPDH Mouse 1:5000 60004-1-Ig AB_2107436 Proteintech, Rosemont, IL, USA WB (Cytosol) VDAC1 Rabbit 1:3000 ab15895 AB_2214787 Abcam WB (Mitochondria) RSP6 Rabbit 1:3000 2217S AB_331355 Cell Signaling WB (Ribosome) GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IF: immunofluorescence; IHC: immunohistochemistry; MBP: myelin basic protein; NeuN: neuronal nuclei; PSD95: postsynaptic density protein-95; RPS6: ribosomal protein S6; SNAP25: 25 kDa synaptosome-associated protein; WB: western blotting.

Techniques: Expressing, Transfection, Western Blot, Mutagenesis

Journal: Neural Regeneration Research

Article Title: Differential distribution of PINK1 and Parkin in the primate brain implies distinct roles

doi: 10.4103/nrr.nrr-d-23-01140

Figure Lengend Snippet: Figure 2 ｜Postnatal expression of PINK1 and PINK1 knockdown in monkey brain. (A) Western blotting of the monkey cortex from WT and PINK1 mutant (M6) monkeys showing that both BC100-494 and S086D antibodies specifically recognize endogenous PINK1 that is knocked down in M6. (B) Western blot of cortical lysates from monkeys at ages postnatal day 1 (P1), P140, 2 years, and 10 years, and human cortex. Three different PINK1 antibodies (E7B6, BC100-494, and S086D) were used to probe the tissue. (C) E7B6 western blotting analysis of lysates from the cortex of WT monkeys (WT1, P1; WT2, 5 years) and PINK1 mutant monkeys (M7, aborted embryo day 139; M1, P7; M2, P7) and a 5-year-old monkey injected with AAV9-PINK1 gRNA/Cas9 to knock down PINK1 (KD). Knocking down PINK1 can cause neuronal loss in developing and adult monkey brains. (D) AAV9 viral expression of CRISPR/Cas9 to target the PINK1 gene in the prefrontal cortex in the 5-year-old WT adult monkey for 6 weeks resulted in a remarkable loss of neuronal cells, which was evident by the reduced number of NeuN-positive cells. E7B6 immunostaining showed a significant reduction of PINK1 protein in the AAV9- PINK1 gRNA/Cas9-injected region compared with that in the control gRNA/Cas9 injected-region. Arrows indicate AAV-infected cells. Green: PINK1 sgRNA-targeted cells; pink: PINK1 (E7B6) staining; red: NeuN staining; blue: nuclear (DAPI) staining. (E) Glial fibrillary acidic protein (GFAP) immunostaining of the cortex from the same brain sections in D showing that the remaining GFP-positive cells in the AAV9-PINK1 gRNA/Cas9-injected region are GFAP-positive astrocytes. Green: AAV-PINK1 gRNA expression; red: GFAP staining; blue: nuclear staining (DAPI). (F) Quantitative assessment of NeuN-positive neurons in D and GFAP-positive astrocytes in E (n = 5 sections for each group). Data shown as mean ± SEM (n = 5). *P < 0.05, ***P < 0.001 (two-tailed Student’s t-test). In D and E, scale bars: 20 μm. For immunocytochemical analysis, the numbers of cells labeled by specific antibodies per image (40×) from at least three independent experiments were quantified. DAPI: 4′,6-Diamidino-2-phenylindole; ns: not significant; PSD95: postsynaptic density protein-95.

Article Snippet: Table 1 | Main antibodies used in this study Antibody Host Dilution Cat# RRID Supplier Application PINK1 Mouse 1:50 E7B6 NA Homemade antibody by MerryBio, Nanjing China WB, IF, IHC PINK1 Rabbit 1:500 BC100-494 AB_10127658 Novus Biologicals , Centennial, CO, USA WB PINK1 Rat 1:500 MAB4357 AB_2164246 R&D Systems, Minneapolis, MN, USA IF PINK1 Sheep 1:500 S086D AB_2924297 MRC, Scotland, UK WB Parkin Mouse 1:1000 sc-32282 AB_628104 Santa Cruz Biotechnology, Santa Cruz, CA, USA WB, IF Parkin Mouse 1:1000 MAB5512 AB_2267915 Millipore, Temecula, CA, USA IF Vinculin Mouse 1:5000 MAB3574 AB_2304338 Millipore WB (Internal reference) NeuN Rabbit 1:5000 ab177487 AB_2532109 Abcam, Cambridge, MA, USA WB, IF (Neuron) SNAP25 Rabbit 1 μg/mL ab41455 AB_945552 Abcam WB (Synaptosomal) TOM20 Rabbit 1:500 #42406 AB_2687663 Cell Signaling Technolog, Danvers, MA, USA IF (outer mitochondrial membrane) PSD95 Rabbit 1:2000 ab238135 AB_2895158 Abcam WB (Synaptosomal) Calnexin Rabbit 1:1000 ab22595 AB_2069006 Abcam WB (Endoplasmic reticulum) MBP Rabbit 1:3000 ab218011 AB_2895537 Abcam WB (Myelin) Histone H3 Rabbit 1:3000 #9715 AB_331563 Cell Signaling Technology WB (Nuclei) GAPDH Mouse 1:5000 60004-1-Ig AB_2107436 Proteintech, Rosemont, IL, USA WB (Cytosol) VDAC1 Rabbit 1:3000 ab15895 AB_2214787 Abcam WB (Mitochondria) RSP6 Rabbit 1:3000 2217S AB_331355 Cell Signaling WB (Ribosome) GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IF: immunofluorescence; IHC: immunohistochemistry; MBP: myelin basic protein; NeuN: neuronal nuclei; PSD95: postsynaptic density protein-95; RPS6: ribosomal protein S6; SNAP25: 25 kDa synaptosome-associated protein; WB: western blotting.

Techniques: Expressing, Knockdown, Western Blot, Mutagenesis, Injection, CRISPR, Immunostaining, Control, Infection, Staining, Two Tailed Test, Labeling

Journal: Neural Regeneration Research

Article Title: Differential distribution of PINK1 and Parkin in the primate brain implies distinct roles

doi: 10.4103/nrr.nrr-d-23-01140

Figure Lengend Snippet: Figure 5 ｜Relative expression levels of PINK1 and Parkin in subcellular fractions isolated from the cortex of three monkeys aged 9, 24, and 26 years. The levels were calculated using ImageJ, and the values are ratios to “Total.” Data are presented as mean ± SEM (n = 3 for each group) and were analyzed by GraphPad Prism 8.0.2 Paired. LP: Layer pellet; LP1: myelin; LP2: other subcellular structures; LP3: synaptosome; LP4: mitochondria; P: pellets; S: supernatant fluid.

Article Snippet: Table 1 | Main antibodies used in this study Antibody Host Dilution Cat# RRID Supplier Application PINK1 Mouse 1:50 E7B6 NA Homemade antibody by MerryBio, Nanjing China WB, IF, IHC PINK1 Rabbit 1:500 BC100-494 AB_10127658 Novus Biologicals , Centennial, CO, USA WB PINK1 Rat 1:500 MAB4357 AB_2164246 R&D Systems, Minneapolis, MN, USA IF PINK1 Sheep 1:500 S086D AB_2924297 MRC, Scotland, UK WB Parkin Mouse 1:1000 sc-32282 AB_628104 Santa Cruz Biotechnology, Santa Cruz, CA, USA WB, IF Parkin Mouse 1:1000 MAB5512 AB_2267915 Millipore, Temecula, CA, USA IF Vinculin Mouse 1:5000 MAB3574 AB_2304338 Millipore WB (Internal reference) NeuN Rabbit 1:5000 ab177487 AB_2532109 Abcam, Cambridge, MA, USA WB, IF (Neuron) SNAP25 Rabbit 1 μg/mL ab41455 AB_945552 Abcam WB (Synaptosomal) TOM20 Rabbit 1:500 #42406 AB_2687663 Cell Signaling Technolog, Danvers, MA, USA IF (outer mitochondrial membrane) PSD95 Rabbit 1:2000 ab238135 AB_2895158 Abcam WB (Synaptosomal) Calnexin Rabbit 1:1000 ab22595 AB_2069006 Abcam WB (Endoplasmic reticulum) MBP Rabbit 1:3000 ab218011 AB_2895537 Abcam WB (Myelin) Histone H3 Rabbit 1:3000 #9715 AB_331563 Cell Signaling Technology WB (Nuclei) GAPDH Mouse 1:5000 60004-1-Ig AB_2107436 Proteintech, Rosemont, IL, USA WB (Cytosol) VDAC1 Rabbit 1:3000 ab15895 AB_2214787 Abcam WB (Mitochondria) RSP6 Rabbit 1:3000 2217S AB_331355 Cell Signaling WB (Ribosome) GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IF: immunofluorescence; IHC: immunohistochemistry; MBP: myelin basic protein; NeuN: neuronal nuclei; PSD95: postsynaptic density protein-95; RPS6: ribosomal protein S6; SNAP25: 25 kDa synaptosome-associated protein; WB: western blotting.

Techniques: Expressing, Isolation