Journal: Science Advances

Article Title: Phosphoglycerate kinase is a central leverage point in Parkinson’s disease–driven neuronal metabolic deficits

doi: 10.1126/sciadv.adn6016

Figure Lengend Snippet: ( A ) Immunostaining against PGK1 (magenta), synapsin I/II (yellow), and DJ-1 (cyan) shows that DJ-1 and PGK1 are both present in nerve terminals (white arrows). Scale bar, 2 μm. Created using Biorender.com ( B ) KD of DJ-1 (black) slows SV endocytosis kinetics compared to 0.1 mM glucose control (blue) vGlutI-pH–transfected cells, when neurons were challenged with 600 APs delivered at 10 Hz (black bar) . PGK1-HALO expression (red) or TZ incubation (green) failed to restore SV kinetics. ( C ) Quantification of the remaining fluorescence 60 s after stimulation [dotted line in (B)]. Means ± SEM. Control, n = 16; DJ-1 KD, n = 12; DJ-1 KD + PGK1-HALO, n = 13; DJ-1 KD + 10 μM TZ, n = 11; (ns) P > 0.05 and * P < 0.05, one-way ANOVA. ( D ) Cumulative histogram of presynaptic PGK1 fluorescence immunostaining intensity in control (gray) and DJ-1 KD nerve terminals (pink). DJ-1 KD, n = 171; nontransfected, n = 500. ( E ) DJ-1 fluorescence immunostaining in control (gray) and PGK1 KD nerve terminals (yellow). PGK1 KD, n = 289; nontransfected, n = 500. Distributions in both KDs are significantly different from their respective controls. P < 0.001, Kolmogorov-Smirnoff test. ( F ) MST identified an in vitro interaction between PGK1 and DJ-1 with a low micromolar affinity (~15 μM).

Article Snippet: The following previously published DNA constructs were used: vGLUT1-pH , DJ-1 (a gift from M. Cookson, Addgene, plasmid #29347, RRID: Addgene_29347, http://n2t.net/addgene:29347 ), synapto-iATPSnFR2-miRFP670nano3 (Addgene, plasmid #209730, RRID: Addgene_209730, http://n2t.net/addgene:209730 ), and synapto-cpsfGFP-miRFP670nano3 (Addgene, plasmid #214926, RRID: Addgene_214926, http://n2t.net/addgene:214926 ).

Techniques: Immunostaining, Control, Transfection, Expressing, Incubation, Fluorescence, In Vitro

Journal: Science Advances

Article Title: Phosphoglycerate kinase is a central leverage point in Parkinson’s disease–driven neuronal metabolic deficits

doi: 10.1126/sciadv.adn6016

Figure Lengend Snippet: ( A ) The kinetics of presynaptic ATP normalized to the prestimulus values when neurons were stimulated with 600 APs at 10 Hz in 0.1 mM glucose (blue) and in the absence of DJ-1 (black) reveal a significantly larger activity-induced drop in ATP and defective poststimulus recovery. Comparison of absolute nerve terminal ATP values before stimulus ( B ) and normalized values at the end of the stimulus ( C ) and 50 s after stimulus ( D ) in control and DJ-1 KD neurons. Means ± SEM. Control, n = 12; DJ-1 KD, n = 11. * P < 0.05 and *** P < 0.001, unpaired t test. ( E ) The kinetics of presynaptic ATP normalized to the prestimulus values when neurons were stimulated with 600 APs at 10 Hz in 0.1 mM glucose with PGK1-HALO and DJ-1 KD (dark red) reveal an inability of PGK1 to accelerate ATP kinetics in absence of DJ-1. Comparison of absolute nerve terminal ATP values before stimulus ( F ) and normalized values at the end of the stimulus ( G ) and 50 s after stimulus ( H ) in PGK1-HALO with and without DJ-1 KD neurons. Means ± SEM. PGK1-HALO, n = 10; PGK1-HALO + DJ-1 KD, n = 5. * P < 0.05 and ** P < 0.01, unpaired t test. ( I ) The kinetics of presynaptic ATP normalized to the prestimulus values when neurons were stimulated with 600 APs at 10 Hz in 0.1 mM glucose in TZ and DJ-1 KD (dark green) reveal an inability of TZ to accelerate ATP kinetics in absence of DJ-1. Comparison of absolute nerve terminal ATP values before stimulus ( J ) and normalized values at the end of the stimulus ( K ) and 50 s after stimulus ( L ) in TZ and TZ with DJ-1 KD neurons. Means ± SEM. TZ, n = 17; TZ + DJ-1 KD, n = 8. * P < 0.05 and *** P < 0.001, unpaired t test.

Article Snippet: The following previously published DNA constructs were used: vGLUT1-pH , DJ-1 (a gift from M. Cookson, Addgene, plasmid #29347, RRID: Addgene_29347, http://n2t.net/addgene:29347 ), synapto-iATPSnFR2-miRFP670nano3 (Addgene, plasmid #209730, RRID: Addgene_209730, http://n2t.net/addgene:209730 ), and synapto-cpsfGFP-miRFP670nano3 (Addgene, plasmid #214926, RRID: Addgene_214926, http://n2t.net/addgene:214926 ).

Techniques: Activity Assay, Comparison, Control

Journal: Science Advances

Article Title: Phosphoglycerate kinase is a central leverage point in Parkinson’s disease–driven neuronal metabolic deficits

doi: 10.1126/sciadv.adn6016

Figure Lengend Snippet: ( A ) An AAV-driving hSyn PGK1-mRuby was injected unilaterally in the substantia nigra 30 days before 6-OHDA was injected in the MFB, with control mice only receiving the 6-OHDA injections. At 30 or 90 days after the 6-OHDA injection, mice were subjected to apomoprhine-induced rotation tests. ( B ) PGK1-mRuby expression significantly suppressed the apomorphine-induced rotations. Means ± SEM. Control, n = 11; AAV PGK1, n = 10. * P < 0.05 and ** P < 0.01, two-way ANOVA. ( C ) Immunostaining of control and AAV PGK1 mid-brain brain slices against 4′,6-diamidino-2-phenylindole (DAPI), mRuby, and TH. Scale bars, 50 μm. ( D ) Quantification of the number of TH-positive cells showed a significant increase in animals receiving the PGK1 AAV. Control, n = 7; AAV PGK1, n = 7. ** P < 0.01, unpaired t test. ( E ) Before immunostaining, the striata of the animals were injected with the retrograde tracer cholera toxin subunit b (Ctb). Scale bars, 100 μm. ( F ) Quantification of the number of Ctb-positive cells also showed a significant increase after lesion in case of the PGK1 AAV. Control, n = 3; AAV PGK1, n = 3. ** P < 0.01, unpaired t test. ( G ) Quantification of the number of DAT-positive cells showed a significant increase in in animals receiving the PGK1 AAV. Control, n = 7; AAV PGK1, n = 7. * P < 0.05, unpaired t test. ( H ) Human brain energetics decline physiologically with age, one of the most important risk factors in PD. Several PARK animal models (red) exhibit deficits in energy homeostasis. Up-regulating PGK1 activity (part of PARK12) and its necessary partner PARK7/DJ-1 has now been shown to rescue at least four different PARK genes (green), proposing a crucial unifying theme of neuronal hypometabolism in PD pathology. (H) created using Biorender.com.

Article Snippet: The following previously published DNA constructs were used: vGLUT1-pH , DJ-1 (a gift from M. Cookson, Addgene, plasmid #29347, RRID: Addgene_29347, http://n2t.net/addgene:29347 ), synapto-iATPSnFR2-miRFP670nano3 (Addgene, plasmid #209730, RRID: Addgene_209730, http://n2t.net/addgene:209730 ), and synapto-cpsfGFP-miRFP670nano3 (Addgene, plasmid #214926, RRID: Addgene_214926, http://n2t.net/addgene:214926 ).

Techniques: Injection, Control, Expressing, Immunostaining, Activity Assay

Journal: bioRxiv

Article Title: Efemp1 modulates elastic fiber formation and mechanics of the extrahepatic bile duct

doi: 10.1101/2021.12.05.471313

Figure Lengend Snippet: (a) Representative images of EFEMP1, elastin and elastic fiber staining in neonatal (P2, 5, 7, 10, 14) and adult mouse extrahepatic bile duct as well as human extrahepatic bile duct from individuals aged 3 days old, 6 months old, 3 years old, 6 years old and 19 years old (adult). White arrows show biliary epithelial cells (BEC) and fibroblasts (fib) expressing EFEMP1. Yellow arrows show EFEMP1 localization under the biliary epithelial cell monolayer. (b) Representative images of elastin staining in mouse samples. In all immunohistochemistry staining, nuclei are stained with DAPI (blue). Yellow arrow shows elastin localization under the biliary epithelial cell monolayer (c) Representative images of elastic fiber staining, with stained elastic fibers in black (teal arrows), nuclei in dark brown and collagen in pink. Yellow arrow shows elastic fiber localization under the biliary epithelial cell monolayer. In all images, dotted lines and L denote lumens. For mice, 3-4 samples were stained for each age. Size bars = 50 μm.

Article Snippet: Heat-mediated antigen retrieval with 10 mM citric acid pH 6.0 in a pressure cooker for 2 hrs was used for staining with antibodies against EFEMP1 (1:100, Thermo Fisher, PA5-29347, Waltham, MA); 0.5% hyaluronidase (Sigma, H3506 type I-S, St-Louis, MO) treatment for 60 min at 37°C was used for staining with antibodies against elastin (1:100, Cedarlane, CL55041AP, Burlington, NC).

Techniques: Staining, Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: Efemp1 modulates elastic fiber formation and mechanics of the extrahepatic bile duct

doi: 10.1101/2021.12.05.471313

Figure Lengend Snippet: (a) Representative staining of elastin in WT and Efemp1 +/- extrahepatic bile ducts and quantification of percent area of staining. P= 0.5264 (b) Representative elastic fiber staining of WT and Efemp1 +/- extrahepatic bile ducts and quantification of the number of elastic fibers per 50μm 2 . (c) Representative images of collagen by SHG imaging in WT and Efemp1 +/- extrahepatic bile ducts, with quantification of percent area of signal. Forward signal in red and backward signal in green. P = 0.1843. Dotted lines and L denote lumens. 6-8 ducts were analyzed per genotype. Data shown are mean ± SEM, significance determined by unpaired T-test.

Article Snippet: Heat-mediated antigen retrieval with 10 mM citric acid pH 6.0 in a pressure cooker for 2 hrs was used for staining with antibodies against EFEMP1 (1:100, Thermo Fisher, PA5-29347, Waltham, MA); 0.5% hyaluronidase (Sigma, H3506 type I-S, St-Louis, MO) treatment for 60 min at 37°C was used for staining with antibodies against elastin (1:100, Cedarlane, CL55041AP, Burlington, NC).

Techniques: Staining, Imaging

Journal: bioRxiv

Article Title: Efemp1 modulates elastic fiber formation and mechanics of the extrahepatic bile duct

doi: 10.1101/2021.12.05.471313

Figure Lengend Snippet: (a) Measured outer diameter of extrahepatic bile ducts at increasing pressures. Physiological and obstruction pressures derived from the literature are indicated on the graph. (b) Calculated stress and stretch of WT and Efemp1 +/- extrahepatic bile ducts under increasing pressure in 2-mm increments from 0-20 mm Hg. Significance shown is for stretch. No difference in stress was observed, p = 0.057. (c) Measured inner radius of unpressurized (unloaded) and pressurized (loaded; around 0.1 mmHg) WT and Efemp1 +/- extrahepatic bile ducts. There is no difference between WT vs Efemp +/- in the unloaded state, p=0.9265 (d) Calculated tangential modulus/stiffness at normal physiological pressure (2 mmHg) and obstruction pressure (12 mmHg) in WT and Efemp1 +/- extrahepatic bile ducts. No significant difference is observed comparing WT vs Efemp1 +/- p = 0.5144 at both normal and obstruction pressures. 6-7 ducts were analyzed per genotype. Data shown are mean ± SEM, two-way ANOVA with a Šidák post-hoc test statistical test.

Article Snippet: Heat-mediated antigen retrieval with 10 mM citric acid pH 6.0 in a pressure cooker for 2 hrs was used for staining with antibodies against EFEMP1 (1:100, Thermo Fisher, PA5-29347, Waltham, MA); 0.5% hyaluronidase (Sigma, H3506 type I-S, St-Louis, MO) treatment for 60 min at 37°C was used for staining with antibodies against elastin (1:100, Cedarlane, CL55041AP, Burlington, NC).

Techniques: Derivative Assay