PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy

Defective mitochondrial quality control is shown to be a mechanism for neurodegeneration in some forms of Parkinson's disease

Low Dose of Clonazepam Is Effective in the Treatment of Painless Legs and Moving Toes Syndrome: A Case Report

Introduction: Painless legs and moving toes syndrome (PoLMT) is a rare movement disorder characterized by flexion, extension, abduction, adduction, and torsion of toes in the absence of pain. It is considered a variant of painful legs and moving toes syndrome, which is characterized by similar movements but is accompanied by pain. Although neuropathy, spinal cord compression, brain tumor, cerebral infarction, and Wilson's disease have been reported to be associated with PoLMT, the actual cause, trigger, and mechanism remain unclear. Therefore, a standardized treatment for PoLMT is not established yet. Case Presentation: We describe a 64-year-old Japanese woman with no past medical history who presented with nonrhythmic repetitive involuntary toe movement of the left foot in the absence of pain. She was diagnosed with idiopathic PoLMT and treated with a low dose of clonazepam (0.5 mg/day). The involuntary movement disappeared completely several days after treatment. Conclusion: A low dose of clonazepam is effective in the treatment of PoLMT

p150^glued-Associated Disorders Are Caused by Activation of Intrinsic Apoptotic Pathway

<div><p>Mutations in p150^glued cause hereditary motor neuropathy with vocal cord paralysis (HMN7B) and Perry syndrome (PS). Here we show that both overexpression of p150^glued mutants and knockdown of endogenous p150^glued induce apoptosis. Overexpression of a p150^glued plasmid containing either a HMN7B or PS mutation resulted in cytoplasmic p150^glued-positive aggregates and was associated with cell death. Cells containing mutant p150^glued aggregates underwent apoptosis that was characterized by an increase in cleaved caspase-3- or Annexin V-positive cells and was attenuated by both zVAD-fmk (a pan-caspase inhibitor) application and caspase-3 siRNA knockdown. In addition, overexpression of mutant p150^glued decreased mitochondrial membrane potentials and increased levels of translocase of the mitochondrial outer membrane (Tom20) protein, indicating accumulation of damaged mitochondria. Importantly, siRNA knockdown of endogenous p150^glued independently induced apoptosis via caspase-8 activation and was not associated with mitochondrial morphological changes. Simultaneous knockdown of endogenous p150^glued and overexpression of mutant p150^glued had additive apoptosis induction effects. These findings suggest that both p150^glued gain-of-toxic-function and loss-of-physiological-function can cause apoptosis and may underlie the pathogenesis of p150^glued-associated disorders.</p></div

Abnormal mitochondria accumulate in cells overexpressing mutant p150^glued.

<p>(A) HeLa cells transfected with GFP-empty vector, GFP-tagged wild-type or mutant (G59S or G71R) p150^glued were incubated with MitoTracker Deep Red (100 nM) for 15 min and analyzed using confocal microscopy. Insets show higher magnification of the boxed areas. Bars, 10 μm. (B, C) Twenty-four hours after transfection, GFP-positive HeLa cells were sorted using flow cytometry and analyzed by immunoblotting with antibodies against TOM20 and actin (B). Densitometry analysis of TOM20 levels relative to actin was performed in three independent experiments (C). (D, E) Twenty-four hours after transfection, HeLa cells were incubated with Mitotracker-Red CMXRos (25 nM) for 15 min, and intracellular fluorescence intensity was measured by flow cytometry. The histograms of MitoTracker-Red CMXRos fluorescence in GFP-positive cells (D) and the percentages of GFP-positive cells with reduced mitochondrial potentials (E) are shown. The error bar indicates each standard deviation. Statistics are from three independent experiments: *, p<0.05; **, p<0.01; ***, p<0.001.</p

Depletion of p150^glued accelerates mutant p150^glued-induced cell death.

<p>Forty-eight hours after transfection with control scrambled siRNA or DCTN1 siRNA, HeLa cells were transfected with GFP-empty vector or mutant (G59S or G71R) p150^glued for 24 h. Cells were stained with Annexin V, and GFP-positive cells were analyzed by flow cytometry. The ratios of Annexin V-positive cells relative to Annexin V-negative cells were calculated. The error bar indicates each standard deviation. Statistics are from three independent experiments: *,p<0.05; **,p<0.01; ***,p<0.001.</p

Mutant p150^glued proteins activate intrinsic apoptotic pathways.

<p>(A) HeLa cells transfected with GFP-empty, GFP-tagged wild-type or mutant (G59S or G71R) p150^glued were fixed and stained with DAPI after 24 and 48 h. GFP-positive cells were counted from three independent experiments. The percentage of GFP-positive cells with nuclear abnormalities is shown. (B) Ratio of transfected HeLa cells with nuclear abnormalities after treatment with or without z-VAD (100 μM) for 24 hours. Values are relative to the GFP-empty value, which is set at 1. (C, D) Transfected SH-SY5Y cells after treatment with or without z-VAD (100 μM) for 48 h were stained with Annexin V and PI, and GFP-positive cells were analyzed by flow cytometry. (E) HeLa cells transfected with GFP-tagged wild-type or mutant p150^glued were fixed and stained with an antibody against cleaved caspase-3. Bars, 10 μm. (F) Twenty-four hours after transfection, SH-SY5Y cells were fixed and stained with a cleaved caspase-3 antibody. GFP-positive cells were analyzed by flow cytometry and the mean fluorescent intensity was calculated. (G) HeLa cells were transfected with control scrambled siRNA or caspase-3 siRNA for 72 h and immunoblotting analysis was performed to monitor the knockdown efficiency of the caspase-3 siRNA. (H) Twenty-four hours after transfection with control siRNA or caspase-3 siRNA, HeLa cells were transfected with GFP-empty, GFP-tagged wild-type or mutant p150^glued. Forty-eight hours after transfection, cells were fixed and stained with DAPI, and ratios of GFP-positive cells with nuclear abnormalities were analyzed. Values are relative to the GFP-empty value, which is set at 1. The error bar indicates each standard deviation. Statistics are from three independent experiments: N.S., not significant; *, p<0.05; **, p<0.01; ***, p<0.001.</p

Depletion of p150^glued does not induce damaged mitochondria accumulation.

<p>(A) Control siRNA or DCTN1 siRNA transfected cells were fixed and co-stained with antibodies against LAMP2 (green) and TOM20 (red), and analyzed using confocal microscopy. Bars, 10 μm. (B) Control siRNA or DCTN1 siRNA transfected HeLa cells were analyzed by immunoblotting with antibodies against complex I, TOM20, and actin. (C, D) Densitometry analysis of complex I (C) and TOM20 (D) levels relative to actin was performed. (E, F) DCTN1 siRNA transfected HeLa cells were incubated with Mitotracker-Red CMXRos and intracellular fluorescence intensity was measured by flow cytometry. The histograms of MitoTracker-Red CMXRos fluorescence (E) and the percentages of cells with reduced mitochondrial potentials (F) are shown. The error bar indicates each standard deviation. Statistics are from three independent experiments: N.S., not significant.</p