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pf 475 (MedChemExpress)

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Rating: 93 (8 reviews)

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MedChemExpress pf 475
Pf 475, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pf 475 - by Bioz Stars, 2026-02

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Journal: CNS Neuroscience & Therapeutics

Article Title: Efficacy of a benzothiazole‐based LRRK2 inhibitor in oligodendrocyte precursor cells and in a murine model of multiple sclerosis

doi: 10.1111/cns.14552

Figure Lengend Snippet: The inhibition of LRRK2 by PF‐475 and compound 4 promotes the proliferation of OPCs from rat brain cortices. (A) Representative images of OPCs cultures treated for 24 h with PF‐475 (0.1 μM) and 4 (1 and 5 μM) 6 h with BrdU, the number of BrdU + Olig2 + was measured. (B) Quantification of proliferating OPCs (BrdU + /Olig2 + double staining) was performed in respect to the total number of oligodendroglial cells (Olig2 + ) for LRRK2 inhibitors PF‐475 and compounds 1–5. Values represent the mean ± SEM of three replications in five different experiments. Results of Student's t ‐test are represented as: * p < 0.05 versus control. The scale bar represents 25 μm in A–D.

Article Snippet: The inhibitor PF‐06447475 (PF‐475) was obtained from Sigma‐Aldrich.

Techniques: Inhibition, Double Staining

a. Representative immunoblots for detecting Lrrk2 and Rab proteins in forebrain lysates from Charles Rivers(Sprague Dawley, (CD(SD)) rats treated with PF-475 (3 or 30 mg·kg−1) and vehicle (Veh) controls. Tissue was collected two hours post-dosing. Quantification of b. total Lrrk2 protein levels normalized to HSC70, and c. pS935-Lrrk2 levels normalized to total Lrrk2 protein. Quantification of pT73-Rab10 levels normalized to d. HSC70 and e. total Rab10 protein. Quantification of pS106-Rab12 levels normalized to f. HSC70 and g. total Rab12. Data are graphed as average measures from triplicate western blots for biological replicates (N=3 rats per group). All values are presented as fold changes to the vehicle treated group. Bar graphs show group means ± SEM. Significance between groups was determined by one-way ANOVA with Dunnett’s multiple comparison test with respect to the indicated group and the vehicle treated group mean. *p< 0.05, **p< 0.01. All other comparisons not indicated by asterisks were not significant.

Journal: Brain research

Article Title: Genetic background influences LRRK2-mediated Rab phosphorylation in the rat brain

doi: 10.1016/j.brainres.2021.147372

Figure Lengend Snippet: a. Representative immunoblots for detecting Lrrk2 and Rab proteins in forebrain lysates from Charles Rivers(Sprague Dawley, (CD(SD)) rats treated with PF-475 (3 or 30 mg·kg−1) and vehicle (Veh) controls. Tissue was collected two hours post-dosing. Quantification of b. total Lrrk2 protein levels normalized to HSC70, and c. pS935-Lrrk2 levels normalized to total Lrrk2 protein. Quantification of pT73-Rab10 levels normalized to d. HSC70 and e. total Rab10 protein. Quantification of pS106-Rab12 levels normalized to f. HSC70 and g. total Rab12. Data are graphed as average measures from triplicate western blots for biological replicates (N=3 rats per group). All values are presented as fold changes to the vehicle treated group. Bar graphs show group means ± SEM. Significance between groups was determined by one-way ANOVA with Dunnett’s multiple comparison test with respect to the indicated group and the vehicle treated group mean. *p< 0.05, **p< 0.01. All other comparisons not indicated by asterisks were not significant.

Article Snippet: Representative immunoblots for detecting Lrrk2 and Rab proteins in forebrain lysates from Charles Rivers(Sprague Dawley, (CD(SD)) rats treated with PF-475 (3 or 30 mg·kg −1 ) and vehicle (Veh) controls.

Techniques: Western Blot

a. Representative immunoblots for detecting Lrrk2 and Rab proteins in forebrain lysates from Lrrk2 knockout (KO) and Long Evans (LE) rats treated with a high dose of PF-475 (30 mg·kg−1) or vehicle control. Tissue was collected two hours post-dosing. Quantification of b. total Lrrk2 protein levels normalized to HSC70 and c. pS935-Lrrk2 levels normalized to total Lrrk2 protein. Quantification of pT73-Rab10 levels normalized to d. HSC70 and e. total Rab10 protein. Quantification of pS106-Rab12 levels normalized to f. HSC70 and g. total Rab12. Data are graphed as average measures from triplicate western blots for biological replicates (N=3 rats per group). All values are presented as fold changes to the vehicle treated group. Bar graphs show group means ± SEM. Significance between treated and untreated (vehicle) controls was determined using two-tailed unpaired t-tests. Data in panels d and e were log transformed prior to t-tests to better approximate normal group distributions. *p< 0.05, **p< 0.01, ***p< 0.001, ns is not significant.

Journal: Brain research

Article Title: Genetic background influences LRRK2-mediated Rab phosphorylation in the rat brain

doi: 10.1016/j.brainres.2021.147372

Figure Lengend Snippet: a. Representative immunoblots for detecting Lrrk2 and Rab proteins in forebrain lysates from Lrrk2 knockout (KO) and Long Evans (LE) rats treated with a high dose of PF-475 (30 mg·kg−1) or vehicle control. Tissue was collected two hours post-dosing. Quantification of b. total Lrrk2 protein levels normalized to HSC70 and c. pS935-Lrrk2 levels normalized to total Lrrk2 protein. Quantification of pT73-Rab10 levels normalized to d. HSC70 and e. total Rab10 protein. Quantification of pS106-Rab12 levels normalized to f. HSC70 and g. total Rab12. Data are graphed as average measures from triplicate western blots for biological replicates (N=3 rats per group). All values are presented as fold changes to the vehicle treated group. Bar graphs show group means ± SEM. Significance between treated and untreated (vehicle) controls was determined using two-tailed unpaired t-tests. Data in panels d and e were log transformed prior to t-tests to better approximate normal group distributions. *p< 0.05, **p< 0.01, ***p< 0.001, ns is not significant.

Techniques: Western Blot, Knock-Out, Two Tailed Test, Transformation Assay

$G2019S LRRK2 hippocampal neurons do not have elevated α-synuclein pathology 14 days post-transduction. a Primary hippocampal neurons from NTG or G2019S pups were transduced with 2.5 μg/mL α-synuclein PFFs and allowed to age a further 14 days prior to sequential detergent fractionation. TX-100-insoluble α-synuclein and p62 are similar in both neuron types. b Quantification of soluble proteins shows ~ 25-fold elevation in the expression of LRRK2 and a commensurate ~ 50-fold elevation in pS395 LRRK2, indicative of the elevated LRRK2 kinase activity associated with the G2019S mutation. Soluble α-synuclein levels were equivalent between the cultures. c No significant differences were found between the genotypes in insoluble proteins by an unpaired t-test with Welch’s correction. ( N = 3 biological replicates for each protein). Means + s.e.m.; ** P < 0.01; * P < 0.05 by an unpaired t-test with Welch’s correction for unequal variances. All values are normalized to NTG neurons treated with α-synuclein PFFs and DMSO$

Journal: Acta Neuropathologica Communications

Article Title: LRRK2 activity does not dramatically alter α-synuclein pathology in primary neurons

doi: 10.1186/s40478-018-0550-0

Figure Lengend Snippet: G2019S LRRK2 hippocampal neurons do not have elevated α-synuclein pathology 14 days post-transduction. a Primary hippocampal neurons from NTG or G2019S pups were transduced with 2.5 μg/mL α-synuclein PFFs and allowed to age a further 14 days prior to sequential detergent fractionation. TX-100-insoluble α-synuclein and p62 are similar in both neuron types. b Quantification of soluble proteins shows ~ 25-fold elevation in the expression of LRRK2 and a commensurate ~ 50-fold elevation in pS395 LRRK2, indicative of the elevated LRRK2 kinase activity associated with the G2019S mutation. Soluble α-synuclein levels were equivalent between the cultures. c No significant differences were found between the genotypes in insoluble proteins by an unpaired t-test with Welch’s correction. ( N = 3 biological replicates for each protein). Means + s.e.m.; ** P < 0.01; * P < 0.05 by an unpaired t-test with Welch’s correction for unequal variances. All values are normalized to NTG neurons treated with α-synuclein PFFs and DMSO

Article Snippet: LRRK2 inhibitors PF-475 and PF-360 were synthesized at Pfizer, Inc. MLi-2 was obtained from Tocris Bioscience (5756).

Techniques: Transduction, Fractionation, Expressing, Activity Assay, Mutagenesis

$LRRK2 inhibition does not reduce insoluble α-synuclein in wildtype hippocampal neurons. a Primary hippocampal neurons from CD1 pups were treated with 30 nM LRRK2 inhibitors PF-475, PF-360 or DMSO as a vehicle control, then transduced with 2.5 μg/mL α-synuclein PFFs and allowed to age a further 14 days prior to sequential detergent fractionation. TX-100-insoluble α-synuclein and p62 are similar in both LRRK2 inhibitor treated and untreated neurons. b Quantification of soluble proteins show some reduction of LRRK2 protein levels PF-475 treatment and ~ 75% inhibition of LRRK2 activity (as assayed by pS935 LRRK2) by both PF-475 and PF-360. c Insoluble pS129 α-synuclein was slightly, but significantly elevated by PF-360 treatment, while α-synuclein and p62 were unchanged by one-way ANOVA. ( N = 5 biological replicates for each protein). Means + s.e.m.; * P < 0.05, **** p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparison test. All values are normalized first to GAPDH, as a loading control, then to DMSO-treated neurons$

Journal: Acta Neuropathologica Communications

Article Title: LRRK2 activity does not dramatically alter α-synuclein pathology in primary neurons

doi: 10.1186/s40478-018-0550-0

Figure Lengend Snippet: LRRK2 inhibition does not reduce insoluble α-synuclein in wildtype hippocampal neurons. a Primary hippocampal neurons from CD1 pups were treated with 30 nM LRRK2 inhibitors PF-475, PF-360 or DMSO as a vehicle control, then transduced with 2.5 μg/mL α-synuclein PFFs and allowed to age a further 14 days prior to sequential detergent fractionation. TX-100-insoluble α-synuclein and p62 are similar in both LRRK2 inhibitor treated and untreated neurons. b Quantification of soluble proteins show some reduction of LRRK2 protein levels PF-475 treatment and ~ 75% inhibition of LRRK2 activity (as assayed by pS935 LRRK2) by both PF-475 and PF-360. c Insoluble pS129 α-synuclein was slightly, but significantly elevated by PF-360 treatment, while α-synuclein and p62 were unchanged by one-way ANOVA. ( N = 5 biological replicates for each protein). Means + s.e.m.; * P < 0.05, **** p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparison test. All values are normalized first to GAPDH, as a loading control, then to DMSO-treated neurons

Article Snippet: LRRK2 inhibitors PF-475 and PF-360 were synthesized at Pfizer, Inc. MLi-2 was obtained from Tocris Bioscience (5756).

Techniques: Inhibition, Control, Transduction, Fractionation, Activity Assay, Comparison

LRRK2 inhibition does not reduce pathological α-synuclein in wildtype hippocampal neurons. a Primary cortical neurons were treated with LRRK2 inhibitors PF-475, PF-360, or MLi-2, at 5 DIV and fed with media containing inhibitors each week for 16 days. Cell lysate was run by Western blot to detect LRRK2 and pS935 LRRK2, which is indicative of LRRK2 activity. Images shown are representative of n = 4–12 biological replicates. b Quantification of Western blot of LRRK2 and pS935 LRRK2. LRRK2 levels were not significantly altered by one-way ANOVA, but pS935 levels were reduced to near undetectable levels (* p < 0.05, ** p < 0.01, Kruskal-Wallis test with Dunn’s multiple comparison test). c Primary hippocampal neurons from CD1 pups were transduced with α-synuclein PFFs and allowed to age a further 14 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). The neurons were additionally treated with LRRK2 inhibitors PF-475, PF-360, or MLi-2, 2 days prior to transduction and fed with media containing inhibitors each week thereafter. No large differences can be observed in the type or abundance of α-synuclein pathology. d Quantification of α-synuclein pathology reveals no change in response to LRRK2 inhibition, while PBS-treated neurons have no pathology (**** p < 0.0001 by Dunnett’s multiple comparison test). MAP2 area ( e ) and neuron number ( f ) are also not altered in response to LRRK2 inhibition. No significant response was seen when compared with vehicle-treated neurons by one-way ANOVA with Dunnett’s multiple comparison test ( d ) or by Kruskal-Wallis test followed by Dunn’s multiple comparison test ( e ) and ( f ). ( N = 9 biological replicates). Means + s.e.m.; all values are normalized to neurons treated with α-synuclein PFFs and DMSO. Scale bars = 50 μm

Journal: Acta Neuropathologica Communications

Article Title: LRRK2 activity does not dramatically alter α-synuclein pathology in primary neurons

doi: 10.1186/s40478-018-0550-0

Figure Lengend Snippet: LRRK2 inhibition does not reduce pathological α-synuclein in wildtype hippocampal neurons. a Primary cortical neurons were treated with LRRK2 inhibitors PF-475, PF-360, or MLi-2, at 5 DIV and fed with media containing inhibitors each week for 16 days. Cell lysate was run by Western blot to detect LRRK2 and pS935 LRRK2, which is indicative of LRRK2 activity. Images shown are representative of n = 4–12 biological replicates. b Quantification of Western blot of LRRK2 and pS935 LRRK2. LRRK2 levels were not significantly altered by one-way ANOVA, but pS935 levels were reduced to near undetectable levels (* p < 0.05, ** p < 0.01, Kruskal-Wallis test with Dunn’s multiple comparison test). c Primary hippocampal neurons from CD1 pups were transduced with α-synuclein PFFs and allowed to age a further 14 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). The neurons were additionally treated with LRRK2 inhibitors PF-475, PF-360, or MLi-2, 2 days prior to transduction and fed with media containing inhibitors each week thereafter. No large differences can be observed in the type or abundance of α-synuclein pathology. d Quantification of α-synuclein pathology reveals no change in response to LRRK2 inhibition, while PBS-treated neurons have no pathology (**** p < 0.0001 by Dunnett’s multiple comparison test). MAP2 area ( e ) and neuron number ( f ) are also not altered in response to LRRK2 inhibition. No significant response was seen when compared with vehicle-treated neurons by one-way ANOVA with Dunnett’s multiple comparison test ( d ) or by Kruskal-Wallis test followed by Dunn’s multiple comparison test ( e ) and ( f ). ( N = 9 biological replicates). Means + s.e.m.; all values are normalized to neurons treated with α-synuclein PFFs and DMSO. Scale bars = 50 μm

Article Snippet: LRRK2 inhibitors PF-475 and PF-360 were synthesized at Pfizer, Inc. MLi-2 was obtained from Tocris Bioscience (5756).

Techniques: Inhibition, Western Blot, Activity Assay, Comparison, Transduction, Staining

LRRK2 inhibition does not alter pathology induced by human Lewy body α-synuclein. a A schematic representation of pathological α-synuclein purification from human cortical tissue. b Primary hippocampal neurons from CD1 pups were treated with 100 nM LRRK2 inhibitors or a vehicle control. Two days later, neurons were treated with 40 ng/mL human LB α-synuclein and allowed to age a further 14 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). No large differences can be observed in the type or abundance of α-synuclein pathology. c Quantification of α-synuclein pathology reveals an increase in neurons treated with LB α-synuclein compared to those treated with PBS (** p < 0.01), but no change in response to LRRK2 inhibition. MAP2 area ( d ) and neuron number ( e ) are also not altered meaningfully in response to LRRK2 inhibition. ( N = 11–12 biological replicates treated with LB α-synuclein from 4 separate cases (2 AD, 1 PDD, 1 DLB). Means + s.e.m.; all values are normalized to neurons treated with LB α-synuclein and DMSO. Scale bars = 50 μm

Journal: Acta Neuropathologica Communications

Article Title: LRRK2 activity does not dramatically alter α-synuclein pathology in primary neurons

doi: 10.1186/s40478-018-0550-0

Figure Lengend Snippet: LRRK2 inhibition does not alter pathology induced by human Lewy body α-synuclein. a A schematic representation of pathological α-synuclein purification from human cortical tissue. b Primary hippocampal neurons from CD1 pups were treated with 100 nM LRRK2 inhibitors or a vehicle control. Two days later, neurons were treated with 40 ng/mL human LB α-synuclein and allowed to age a further 14 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). No large differences can be observed in the type or abundance of α-synuclein pathology. c Quantification of α-synuclein pathology reveals an increase in neurons treated with LB α-synuclein compared to those treated with PBS (** p < 0.01), but no change in response to LRRK2 inhibition. MAP2 area ( d ) and neuron number ( e ) are also not altered meaningfully in response to LRRK2 inhibition. ( N = 11–12 biological replicates treated with LB α-synuclein from 4 separate cases (2 AD, 1 PDD, 1 DLB). Means + s.e.m.; all values are normalized to neurons treated with LB α-synuclein and DMSO. Scale bars = 50 μm

Article Snippet: LRRK2 inhibitors PF-475 and PF-360 were synthesized at Pfizer, Inc. MLi-2 was obtained from Tocris Bioscience (5756).

Techniques: Inhibition, Purification, Control, Staining

G2019S LRRK2 hippocampal neurons show mild, reversible elevation in induced α-synuclein pathology 21 days post-transduction. Primary hippocampal neurons from NTG ( a ) or G2019S ( b ) pups were transduced with α-synuclein PFFs and allowed to age a further 21 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). The neurons were additionally treated with LRRK2 inhibitors PF-475 and PF-360 2 days prior to transduction and fed with media containing inhibitors each week thereafter. No large differences can be observed in the type or abundance of α-synuclein pathology. c Quantification of α-synuclein pathology reveals a mild elevation in G2019S neurons, which is reversible with 30 or 120 nM PF-360. * P < 0.05 by Dunnett’s multiple comparison test between NTG and G2019S neurons or Sidak’s multiple comparisons test between G2019S neurons treated with LRRK2 inhibitors. d MAP2 area is reduced with 21 days α-synuclein PFF treatment in both NTG and G2019S neurons, and is not significantly affected by LRRK2 inhibitor treatment. * P < 0.05 by 2-way ANOVA with Dunnett’s multiple comparison test for comparison within genotype. e The number of neurons, as quantified by NeuN number, is reduced with 21 days α-synuclein PFF treatment in both NTG and G2019S neurons, although not significantly by 2-way ANOVA followed by Dunnett’s multiple comparison test and is not significantly affected by LRRK2 inhibitor treatment. ( N = 12 biological replicates). Means + s.e.m.; all values are normalized to NTG neurons treated with α-synuclein PFFs and DMSO. Scale bars = 50 μm

Journal: Acta Neuropathologica Communications

Article Title: LRRK2 activity does not dramatically alter α-synuclein pathology in primary neurons

doi: 10.1186/s40478-018-0550-0

Figure Lengend Snippet: G2019S LRRK2 hippocampal neurons show mild, reversible elevation in induced α-synuclein pathology 21 days post-transduction. Primary hippocampal neurons from NTG ( a ) or G2019S ( b ) pups were transduced with α-synuclein PFFs and allowed to age a further 21 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). The neurons were additionally treated with LRRK2 inhibitors PF-475 and PF-360 2 days prior to transduction and fed with media containing inhibitors each week thereafter. No large differences can be observed in the type or abundance of α-synuclein pathology. c Quantification of α-synuclein pathology reveals a mild elevation in G2019S neurons, which is reversible with 30 or 120 nM PF-360. * P < 0.05 by Dunnett’s multiple comparison test between NTG and G2019S neurons or Sidak’s multiple comparisons test between G2019S neurons treated with LRRK2 inhibitors. d MAP2 area is reduced with 21 days α-synuclein PFF treatment in both NTG and G2019S neurons, and is not significantly affected by LRRK2 inhibitor treatment. * P < 0.05 by 2-way ANOVA with Dunnett’s multiple comparison test for comparison within genotype. e The number of neurons, as quantified by NeuN number, is reduced with 21 days α-synuclein PFF treatment in both NTG and G2019S neurons, although not significantly by 2-way ANOVA followed by Dunnett’s multiple comparison test and is not significantly affected by LRRK2 inhibitor treatment. ( N = 12 biological replicates). Means + s.e.m.; all values are normalized to NTG neurons treated with α-synuclein PFFs and DMSO. Scale bars = 50 μm

Article Snippet: LRRK2 inhibitors PF-475 and PF-360 were synthesized at Pfizer, Inc. MLi-2 was obtained from Tocris Bioscience (5756).

Techniques: Transduction, Staining, Comparison

Neither G2019S LRRK2 expression nor LRRK2 inhibition alters α-synuclein pathology in midbrain neurons. a Primary midbrain/striatum cultures from NTG or G2019S pups were transduced with α-synuclein PFFs and allowed to age a further 14 days prior to fixation and staining for pS129 α-synuclein (magenta) and TH (gray). The neurons were additionally treated with 30 nM LRRK2 inhibitors PF-475, PF-360, or MLi-2 2 days prior to transduction and fed with media containing inhibitors each week thereafter. b Quantification of α-synuclein pathology in TH+ neurons shows no effect of G2019S LRRK2 expression or LRRK2 inhibitor treatments by 2-way ANOVA (** p < 0.01, *** p < 0.001 for PBS- compared to PFF-treated neurons by Dunnett’s multiple comparison test). c The number of TH+ neurons showed no significant response to treatment by Kruskal-Wallis test followed by Dunn’s multiple comparison test. ( N = 6–9 biological replicates). Means + s.e.m.; all values are normalized to NTG neurons treated with α-synuclein LB material and DMSO. Scale bars = 50 μm

Journal: Acta Neuropathologica Communications

Article Title: LRRK2 activity does not dramatically alter α-synuclein pathology in primary neurons

doi: 10.1186/s40478-018-0550-0

Figure Lengend Snippet: Neither G2019S LRRK2 expression nor LRRK2 inhibition alters α-synuclein pathology in midbrain neurons. a Primary midbrain/striatum cultures from NTG or G2019S pups were transduced with α-synuclein PFFs and allowed to age a further 14 days prior to fixation and staining for pS129 α-synuclein (magenta) and TH (gray). The neurons were additionally treated with 30 nM LRRK2 inhibitors PF-475, PF-360, or MLi-2 2 days prior to transduction and fed with media containing inhibitors each week thereafter. b Quantification of α-synuclein pathology in TH+ neurons shows no effect of G2019S LRRK2 expression or LRRK2 inhibitor treatments by 2-way ANOVA (** p < 0.01, *** p < 0.001 for PBS- compared to PFF-treated neurons by Dunnett’s multiple comparison test). c The number of TH+ neurons showed no significant response to treatment by Kruskal-Wallis test followed by Dunn’s multiple comparison test. ( N = 6–9 biological replicates). Means + s.e.m.; all values are normalized to NTG neurons treated with α-synuclein LB material and DMSO. Scale bars = 50 μm

Article Snippet: LRRK2 inhibitors PF-475 and PF-360 were synthesized at Pfizer, Inc. MLi-2 was obtained from Tocris Bioscience (5756).

Techniques: Expressing, Inhibition, Transduction, Staining, Comparison

Experimental design. (A) Brain tissue from the peripheral injury (peri-injury) in the TBI group and from the same location in the sham group was obtained for the assay. (B) Experiment I was designed to demonstrate the expression levels and locations of LRRK2/p38/Drosha over time after TBI and determine a suitable time point for the second experiment. (C) Experiment II was designed to observe the effects of LRRK2 on early brain injury after TBI and explore the potential mechanisms.

Journal: Frontiers in Cellular Neuroscience

Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats

doi: 10.3389/fncel.2018.00051

Figure Lengend Snippet: Experimental design. (A) Brain tissue from the peripheral injury (peri-injury) in the TBI group and from the same location in the sham group was obtained for the assay. (B) Experiment I was designed to demonstrate the expression levels and locations of LRRK2/p38/Drosha over time after TBI and determine a suitable time point for the second experiment. (C) Experiment II was designed to observe the effects of LRRK2 on early brain injury after TBI and explore the potential mechanisms.

Article Snippet: Additionally, LRRK2 inhibitor PF-475 (Sigma, United states) was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.9% saline to a final concentration of <1% DMSO.

Techniques: Expressing

The mRNA and protein expression levels of LRRK2, p38, and Drosha in the peri-injury cortex after TBI. (A) Amplification and melting temperature curves for LRRK2, p38, Drosha (right), and GAPDH (left) were obtained to identify the cycle thresholds and confirm the specificity of real-time PCR amplification. The relative mRNA expression levels of (B) LRRK2, (C) p38, and (D) Drosha were evaluated using the ratio of the number of target mRNAs to the GAPDH mRNA. Western blot was performed to determine the protein levels of endogenous (E) LRRK2, (F) p38, and (G) Drosha in the sham and TBI groups at 6, 12, 24, 48, and 72 h. The relative densities of each protein were normalized to the sham group. The results show that the expression levels of LRRK2 and p-p38 were increased at both the mRNA and protein levels after TBI, whereas Drosha expression exhibited the opposite trend. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. N = 6 for each group per time point. Data are expressed as the mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01 vs. sham.

Journal: Frontiers in Cellular Neuroscience

Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats

doi: 10.3389/fncel.2018.00051

Figure Lengend Snippet: The mRNA and protein expression levels of LRRK2, p38, and Drosha in the peri-injury cortex after TBI. (A) Amplification and melting temperature curves for LRRK2, p38, Drosha (right), and GAPDH (left) were obtained to identify the cycle thresholds and confirm the specificity of real-time PCR amplification. The relative mRNA expression levels of (B) LRRK2, (C) p38, and (D) Drosha were evaluated using the ratio of the number of target mRNAs to the GAPDH mRNA. Western blot was performed to determine the protein levels of endogenous (E) LRRK2, (F) p38, and (G) Drosha in the sham and TBI groups at 6, 12, 24, 48, and 72 h. The relative densities of each protein were normalized to the sham group. The results show that the expression levels of LRRK2 and p-p38 were increased at both the mRNA and protein levels after TBI, whereas Drosha expression exhibited the opposite trend. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. N = 6 for each group per time point. Data are expressed as the mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01 vs. sham.

Article Snippet: Additionally, LRRK2 inhibitor PF-475 (Sigma, United states) was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.9% saline to a final concentration of <1% DMSO.

Techniques: Expressing, Amplification, Real-time Polymerase Chain Reaction, Western Blot

LRRK2, p38, and Drosha expression in neurons of the peri-injury cortex after TBI. Representative double-immunofluorescence staining images of LRRK2 (A) , p38 (B) , and Drosha (C) (green) with NeuN (red)-marked neurons to show expression profiles in the sham and 12-h TBI groups. The nuclei were fluorescently labeled with DAPI (blue). The arrows indicate the colocalization of LRRK2/p-p38/Drosha with neurons. Scale bar = 50 μm. (D–F) The percentage of LRRK2/p-p38/Drosha-positive neurons is shown. Statistical analyses were performed using Student’s t -test; data are expressed as the mean ± SD, n = 6 for each group; ∗ P < 0.05 vs. sham.

Journal: Frontiers in Cellular Neuroscience

Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats

doi: 10.3389/fncel.2018.00051

Figure Lengend Snippet: LRRK2, p38, and Drosha expression in neurons of the peri-injury cortex after TBI. Representative double-immunofluorescence staining images of LRRK2 (A) , p38 (B) , and Drosha (C) (green) with NeuN (red)-marked neurons to show expression profiles in the sham and 12-h TBI groups. The nuclei were fluorescently labeled with DAPI (blue). The arrows indicate the colocalization of LRRK2/p-p38/Drosha with neurons. Scale bar = 50 μm. (D–F) The percentage of LRRK2/p-p38/Drosha-positive neurons is shown. Statistical analyses were performed using Student’s t -test; data are expressed as the mean ± SD, n = 6 for each group; ∗ P < 0.05 vs. sham.

Article Snippet: Additionally, LRRK2 inhibitor PF-475 (Sigma, United states) was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.9% saline to a final concentration of <1% DMSO.

Techniques: Expressing, Double Immunofluorescence Staining, Labeling

Effects of LRRK2 intervention on neurological score, brain edema, and BBB integrity after TBI. (A) Western blot analysis was conducted to assess the efficiencies of LRRK2 intervention in the pericontusional cortex at 12 h after TBI. (B) The brain water content of the bilateral hemispheres of the different groups was measured using the wet–dry method. (C) Western blots showing the levels of albumin extravasation in the pericontusional cortex at 12 h after TBI. (D) Representative neurological behavior scores from the modified Garcia test in the experimental groups at 72 h after TBI are shown. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD, n = 6 animals per group. ∗ P < 0.05, ∗∗ P < 0.01 vs. sham; # P < 0.05, ## P < 0.01 vs. the Vehicle group; & P < 0.05 vs. the Over-Ctr group. Ipsi indicates the ipsilateral injured hemispheres; Contra, contra-lateral uninjured hemispheres.

Journal: Frontiers in Cellular Neuroscience

Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats

doi: 10.3389/fncel.2018.00051

Figure Lengend Snippet: Effects of LRRK2 intervention on neurological score, brain edema, and BBB integrity after TBI. (A) Western blot analysis was conducted to assess the efficiencies of LRRK2 intervention in the pericontusional cortex at 12 h after TBI. (B) The brain water content of the bilateral hemispheres of the different groups was measured using the wet–dry method. (C) Western blots showing the levels of albumin extravasation in the pericontusional cortex at 12 h after TBI. (D) Representative neurological behavior scores from the modified Garcia test in the experimental groups at 72 h after TBI are shown. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD, n = 6 animals per group. ∗ P < 0.05, ∗∗ P < 0.01 vs. sham; # P < 0.05, ## P < 0.01 vs. the Vehicle group; & P < 0.05 vs. the Over-Ctr group. Ipsi indicates the ipsilateral injured hemispheres; Contra, contra-lateral uninjured hemispheres.

Article Snippet: Additionally, LRRK2 inhibitor PF-475 (Sigma, United states) was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.9% saline to a final concentration of <1% DMSO.

Techniques: Western Blot, Modification

Effects of LRRK2 intervention on the p-p38 and Drosha protein levels after TBI. Western blot analysis was conducted to assess the (A) p-p38 and (B) Drosha protein levels after LRRK2 intervention. (C,D) The densities of the protein bands in the representative images were analyzed and normalized to GAPDH. The PF-475 treatment reversed the TBI-induced increase in p-p38 and decrease in Drosha expression, whereas LRRK2 overexpression augmented the TBI-induced changes in p-p38 and Drosha expression. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD, n = 6 animals per group. ∗ P < 0.05, ∗∗ P < 0.01 vs. sham; # P < 0.05, ## P < 0.01 vs. the Vehicle group; & P < 0.05 vs. the Over-Ctr group; $ P < 0.05 vs. the indicated group.

Journal: Frontiers in Cellular Neuroscience

Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats

doi: 10.3389/fncel.2018.00051

Figure Lengend Snippet: Effects of LRRK2 intervention on the p-p38 and Drosha protein levels after TBI. Western blot analysis was conducted to assess the (A) p-p38 and (B) Drosha protein levels after LRRK2 intervention. (C,D) The densities of the protein bands in the representative images were analyzed and normalized to GAPDH. The PF-475 treatment reversed the TBI-induced increase in p-p38 and decrease in Drosha expression, whereas LRRK2 overexpression augmented the TBI-induced changes in p-p38 and Drosha expression. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD, n = 6 animals per group. ∗ P < 0.05, ∗∗ P < 0.01 vs. sham; # P < 0.05, ## P < 0.01 vs. the Vehicle group; & P < 0.05 vs. the Over-Ctr group; $ P < 0.05 vs. the indicated group.

Article Snippet: Additionally, LRRK2 inhibitor PF-475 (Sigma, United states) was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.9% saline to a final concentration of <1% DMSO.

Techniques: Western Blot, Expressing, Over Expression

Effects of LRRK2 intervention on brain cell death and neuronal degeneration after TBI. (A) Representative photomicrographs of TUNEL staining in the experimental groups are shown. Sections were labeled with TUNEL (green) to assess apoptotic brain cells; sections were counterstained with DAPI (blue) to detect the nuclei. The arrows indicate the TUNEL-positive cells. Scale bar = 50 μm. (B) Representative photomicrographs of FJB staining in the experimental groups are shown. The arrows indicate the dead neurons. Scale bar = 50 μm. (C) The percentage of TUNEL-positive cells in the brain is shown (apoptotic cells)/(total cells) × 100%. (D) The neuronal degeneration index is presented as the number of FJB-positive cells per visual field. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are expressed as the mean ± SD, n = 6 animals per group. ∗∗∗ P < 0.001 vs. sham; # P < 0.05, ## P < 0.01 vs. the Vehicle group; & P < 0.05, && P < 0.01 vs. the Over-Ctr group.

Journal: Frontiers in Cellular Neuroscience

Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats

doi: 10.3389/fncel.2018.00051

Figure Lengend Snippet: Effects of LRRK2 intervention on brain cell death and neuronal degeneration after TBI. (A) Representative photomicrographs of TUNEL staining in the experimental groups are shown. Sections were labeled with TUNEL (green) to assess apoptotic brain cells; sections were counterstained with DAPI (blue) to detect the nuclei. The arrows indicate the TUNEL-positive cells. Scale bar = 50 μm. (B) Representative photomicrographs of FJB staining in the experimental groups are shown. The arrows indicate the dead neurons. Scale bar = 50 μm. (C) The percentage of TUNEL-positive cells in the brain is shown (apoptotic cells)/(total cells) × 100%. (D) The neuronal degeneration index is presented as the number of FJB-positive cells per visual field. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are expressed as the mean ± SD, n = 6 animals per group. ∗∗∗ P < 0.001 vs. sham; # P < 0.05, ## P < 0.01 vs. the Vehicle group; & P < 0.05, && P < 0.01 vs. the Over-Ctr group.

Article Snippet: Additionally, LRRK2 inhibitor PF-475 (Sigma, United states) was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.9% saline to a final concentration of <1% DMSO.

Techniques: TUNEL Assay, Staining, Labeling

Simultaneous overexpression of Drosha abolishes the neurotoxic effects of LRRK2 overexpression on neurological impairment, brain edema, and BBB integrity after TBI. (A) Western blot analysis was conducted to assess the Drosha overexpression efficiency and the effect of LRRK2 expression in the pericontusional cortex at 12 h after TBI. The results showed that Drosha overexpression pretreatment effectively raised the Drosha protein level but did not change the expression of LRRK2. Meanwhile, the neurotoxic effects of LRRK2 overexpression on (B) brain edema, (C) albumin leakage, and (D) neurological score were reversed by simultaneous Drosha overexpression. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD, n = 6 animals per group. ∗ P < 0.05, ∗∗ P < 0.01 vs. sham, # P < 0.05, ## P < 0.01 vs. the Over-Ctr group; & P < 0.05, && P < 0.01 vs. the Over-LRRK2 group.

Journal: Frontiers in Cellular Neuroscience

Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats

doi: 10.3389/fncel.2018.00051

Figure Lengend Snippet: Simultaneous overexpression of Drosha abolishes the neurotoxic effects of LRRK2 overexpression on neurological impairment, brain edema, and BBB integrity after TBI. (A) Western blot analysis was conducted to assess the Drosha overexpression efficiency and the effect of LRRK2 expression in the pericontusional cortex at 12 h after TBI. The results showed that Drosha overexpression pretreatment effectively raised the Drosha protein level but did not change the expression of LRRK2. Meanwhile, the neurotoxic effects of LRRK2 overexpression on (B) brain edema, (C) albumin leakage, and (D) neurological score were reversed by simultaneous Drosha overexpression. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD, n = 6 animals per group. ∗ P < 0.05, ∗∗ P < 0.01 vs. sham, # P < 0.05, ## P < 0.01 vs. the Over-Ctr group; & P < 0.05, && P < 0.01 vs. the Over-LRRK2 group.

Article Snippet: Additionally, LRRK2 inhibitor PF-475 (Sigma, United states) was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.9% saline to a final concentration of <1% DMSO.

Techniques: Over Expression, Western Blot, Expressing

Simultaneous overexpression of Drosha abolishes the neurotoxic effects of LRRK2 overexpression on brain cell death and neuronal degeneration after TBI. Representative photomicrographs of (A) TUNEL and (B) FJB staining of the pericontusional cortex in the experimental groups are shown. The arrows indicate the TUNEL-positive or FJB-positive cells. Scale bar = 50 μm. (C) The apoptosis index is expressed as the ratio of (apoptotic cells)/(total cells) × 100%. (D) The neuronal degeneration index is presented as the number of FJB-positive cells per visual field. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD, n = 6 animals per group. ∗∗ P < 0.01 vs. sham; # P < 0.05 vs. the Over-Ctr group; & P < 0.05, && P < 0.01 vs. the Over-LRRK2 group.

Journal: Frontiers in Cellular Neuroscience

Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats

doi: 10.3389/fncel.2018.00051

Figure Lengend Snippet: Simultaneous overexpression of Drosha abolishes the neurotoxic effects of LRRK2 overexpression on brain cell death and neuronal degeneration after TBI. Representative photomicrographs of (A) TUNEL and (B) FJB staining of the pericontusional cortex in the experimental groups are shown. The arrows indicate the TUNEL-positive or FJB-positive cells. Scale bar = 50 μm. (C) The apoptosis index is expressed as the ratio of (apoptotic cells)/(total cells) × 100%. (D) The neuronal degeneration index is presented as the number of FJB-positive cells per visual field. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD, n = 6 animals per group. ∗∗ P < 0.01 vs. sham; # P < 0.05 vs. the Over-Ctr group; & P < 0.05, && P < 0.01 vs. the Over-LRRK2 group.

Article Snippet: Additionally, LRRK2 inhibitor PF-475 (Sigma, United states) was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.9% saline to a final concentration of <1% DMSO.

Techniques: Over Expression, TUNEL Assay, Staining

Mode pattern illustrating the possible mechanisms underlying LRRK2-mediated neurotoxicity after TBI. Briefly, TBI induces a rapid increase in the expression of endogenous LRRK2, which can activate the p38 pathway. Phosphorylated p38 then translocates from the cytoplasm to the nucleus and disrupts the stability of Drosha, promoting its nuclear export and degradation. As a nuclear RNase III enzyme, Drosha deficiency in the nucleus can hinder miRNA maturation, which eventually sensitizes cells to stress and results in neurological injury after TBI.

Journal: Frontiers in Cellular Neuroscience

Article Title: LRRK2 Contributes to Secondary Brain Injury Through a p38/Drosha Signaling Pathway After Traumatic Brain Injury in Rats

doi: 10.3389/fncel.2018.00051

Figure Lengend Snippet: Mode pattern illustrating the possible mechanisms underlying LRRK2-mediated neurotoxicity after TBI. Briefly, TBI induces a rapid increase in the expression of endogenous LRRK2, which can activate the p38 pathway. Phosphorylated p38 then translocates from the cytoplasm to the nucleus and disrupts the stability of Drosha, promoting its nuclear export and degradation. As a nuclear RNase III enzyme, Drosha deficiency in the nucleus can hinder miRNA maturation, which eventually sensitizes cells to stress and results in neurological injury after TBI.

Article Snippet: Additionally, LRRK2 inhibitor PF-475 (Sigma, United states) was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.9% saline to a final concentration of <1% DMSO.

Techniques: Expressing

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