Prevention of mitochondrial impairment by inhibition of protein phosphatase 1 activity in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of motor neurons (MNs) and subsequent muscle weakness. These pathological features are associated with numerous cellular changes, including alteration in mitochondrial morphology and function. However, the molecular mechanisms associating mitochondrial structure with ALS pathology are poorly understood. In this study, we found that Dynamin-related protein 1 (Drp1) was dephosphorylated in several ALS models, including those with SOD1 and TDP-43 mutations, and the dephosphorylation was mediated by the pathological induction of protein phosphatase 1 (PP1) activity in these models. Suppression of the PP1-Drp1 cascade effectively prevented ALS-related symptoms, including mitochondrial fragmentation, mitochondrial complex I impairment, axonal degeneration, and cell death, in primary neuronal culture models, iPSC-derived human MNs, and zebrafish models in vivo. These results suggest that modulation of PP1-Drp1 activity may be a therapeutic target for multiple pathological features of ALS

Constriction of the mitochondrial inner compartment is a priming event for mitochondrial division

Mitochondrial division is critical for the maintenance and regulation of mitochondrial function, quality and distribution. This process is controlled by cytosolic actin-based constriction machinery and dynamin-related protein 1 (Drp1) on mitochondrial outer membrane (OMM). Although mitochondrial physiology, including oxidative phosphorylation, is also important for efficient mitochondrial division, morphological alterations of the mitochondrial inner-mem-brane (IMM) have not been clearly elucidated. Here we report spontaneous and repetitive constriction of mitochondrial inner compartment (CoMIC) associated with subsequent division in neurons. Although CoMIC is potentiated by inhibition of Drp1 and occurs at the potential division spots contacting the endoplasmic reticulum, it appears on IMM independently of OMM. Intra-mitochondrial influx of Ca2 + induces and potentiates CoMIC, and leads to K+-mediated mitochondrial bulging and depolarization. Synergistically, optic atrophy 1 (Opa1) also regulates CoMIC via controlling Mic60-mediated OMM–IMM tethering. Therefore, we propose that CoMIC is a priming event for efficient mitochondrial division. © The Author(s) 2017.TRU

Drp1-Zip1 interaction regulates mitochondrial quality surveillance system

Cho et al. report that Drp1 that is the executioner for mitochondrial fission can reduce the mitochondrial membrane potential (MMP) during the mitochondrial division, and this new action helps identify bad sectors in the interconnected mitochondria. © 2018 Elsevier Inc.Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn 2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating “bad sectors” in the mitochondrial network, serving as a mitochondrial quality surveillance system. © 2018 Elsevier Inc.1

N-Terminal Truncated UCH-L1 Prevents Parkinson's Disease Associated Damage

<div><p>Ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been proposed as one of the Parkinson's disease (PD) related genes, but the possible molecular connection between UCH-L1 and PD is not well understood. In this study, we discovered an N-terminal 11 amino acid truncated variant UCH-L1 that we called NT-UCH-L1, in mouse brain tissue as well as in NCI-H157 lung cancer and SH-SY5Y neuroblastoma cell lines. <i>In vivo</i> experiments and hydrogen-deuterium exchange (HDX) with tandem mass spectrometry (MS) studies showed that NT-UCH-L1 is readily aggregated and degraded, and has more flexible structure than UCH-L1. Post-translational modifications including monoubiquitination and disulfide crosslinking regulate the stability and cellular localization of NT-UCH-L1, as confirmed by mutational and proteomic studies. Stable expression of NT-UCH-L1 decreases cellular ROS levels and protects cells from H₂O₂, rotenone and CCCP-induced cell death. NT-UCH-L1-expressing transgenic mice are less susceptible to degeneration of nigrostriatal dopaminergic neurons seen in the MPTP mouse model of PD, in comparison to control animals. These results suggest that NT-UCH-L1 may have the potential to prevent neural damage in diseases like PD.</p></div

Localization of NT-UCH-L1 in mitochondria.

<p>(A) HeLa cells stably expressing UCH-L1-myc, NT-UCH-L1-myc and K15/157R NT-UCH-L1-myc were stained with anti-myc and Alexa Fluor 488 secondary antibody (green). Mitochondria and nucleus were stained with Mitotracker (red) and DAPI (blue), respectively. Cells were visualized by confocal microscopy. (B) Cells were fractionated into cytosolic and mitochondrial fractionations. UCH-L1s were immunoblotted using anti-myc antibody and Hsp60 and PRX6, a mitochondrial and cytosolic marker protein, respectively, were immunoblotted using anti-Hsp60 and anti-PRX6 antibodies. The relative distribution of UCH-L1-myc, NT-UCH-L1-myc and K15/157R NT-UCH-L1-myc in the cytosolic and mitochondrial fractions were quantified by measuring their density (C). The mean ± s.d. of three independent experiments is shown. (D) Mitochondrial fractions of HeLa cells expressing NT-UCH-L1 were incubated with various concentrations of proteinase K for 1 h at 50°C and immunoblotted with anti-myc, Tom20, cytochrome C and COX4 antibodies. (E) Cells were treated 10((M CCCP for 0, 1, or 6 h and fractionated into cytosolic and mitochondrial fractionations. UCH-L1s were immunoblotted using anti-myc antibody and Hsp60, a mitochondrial marker protein, was immunoblotted using anti-Hsp60 antibody. (F) Cells were treated with 10((M CCCP in combination with 10((M MG132 (an inhibitor of proteasomal degradation), 10 mM NH4Cl (an inhibitor of lysosomal degradation) or 10 mM 3MA (an inhibitor of autophagy). UCH-L1s were immunoblotted using anti-myc antibody.</p

Monoubiquitination of NT-UCH-L1.

<p>(A, B) NCI-H157 cells transiently transfected with empty vector, pcDNA3.1 UCH-L1-myc or pcDNA3.1 NT-UCH-L1-myc were subjected to immunoprecipitation using anti-myc antibody. Beads bound proteins were resolved in 13% SDS-PAGE and visualized by silver staining (A) and Western blot analysis using anti-myc and anti-ubiquitin antibodies (B). *, non-specific bands. (C) A tandem MS spectrum of a peptide of NT-UCH-L1 containing Lys15. It was sequenced using nanoLC-ESI-q-TOF tandem MS and the GlyGly ubiquitin C-terminus fragment on Lys15 was detected. (D) HeLa cells stably expressing UCH-L1-myc, NT-UCH-L1-myc and K15/157R NT-UCH-L1-myc were examined. Cells were immunostained with anti-myc antibody to confirm the monoubiquitination sites.</p

Generation of hNT-UCH-L1-myc transgenic mice and the protective role of NT-UCH-L1 in the MPTP model of Parkinson's disease.

<p>(A) NT-UCH-L1 was constructed under control of the CAG promoter. (B) Quantification of the mRNAs of the NT-UCH-L1-myc transgene from five transgenic mice lines using quantitative RT-PCR. (C) Immunoprecipitation analysis of hNT-UCH-L1-myc in control and NT-Tg mouse brain using anti-myc antibody. Immunoprecipitants were analyzed by immunoblotting method using anti-myc antibody. 3^rd land, cell lysate of HEK293 cells transiently expressing NT-UCH-L1-myc. (D) Immunohistochemical analysis showing neuroprotective effect of NT-UCH-L1 on nigrostriatal dopaminergic neurons. Mice in each group (non-Tg control (a, b), non-Tg MPTP (c, d), NT-UCH-L1 control (e, f) and NT-UCH-L1 MPTP (g, h)) were sacrificed 7 d after the last MPTP injection (c, d, g, h) or PBS as controls (a, b, e, f) and brain tissues were processed for tyrosine hydroxylase (TH) immunostaining in the substantia nigra pars compacta (SN, a, c, e, g) and striatum (STR, b, d, f, h). Scale bar, a, c, e, g, 100((m; b, d, f, h, 50((m. (E, F) The number of TH-positive cells in the SN (D) and optical density of TH-positive striatal fibers (D) are shown in graphs. n = 3 or 4 for each experimental group.</p

Discovery of NT-UCH-L1 in human lung cancer and neuroblastoma cell lines and its enzymatic and structural differences from UCH-L1.

<p>(A) NCI-H157 (left panels) and SH-SY5Y (right panels) cells were analyzed using 2D-PAGE and visualized by Western blot analysis using anti-UCH-L1 (top panels), anti-N-terminal peptide (against the peptide, ¹MQLKPMEINPE¹¹) antibodies (lower panels), or silver staining (middle panels). Four spots representing UCH-L1 are numbered from 1 to 4. (B) Mouse whole brain tissue was analyzed using 2D-PAGE and visualized by Western blot analysis using anti-UCH-L1 (top panel) and anti-N-terminal peptide antibodies (lower panel). The location of NT was marked by a circle in each panel. (C) A diagram of UCH-L1 and N-terminal 11 amino acid truncated NT-UCH-L1. (D) Ubiquitin hydrolase activities of GST-UCH-L1 and GST-NT-UCH-L1 were measured using Ub-AMC as a substrate. 5 nM of GST-UCH-L1 or GST-NT-UCH-L1 was incubated with 0–1000 nM of Ub-AMC and monitored the release of free AMC at 460 nm. (E) HeLa cells transiently expressing NT-UCH-L1 were subjected to migration assay using transwell coated with Matrigel™. After 24 h of seeding, the number of migrated cells in the lower chamber was counted. The expression of NT-UCH-L1 in HeLa cells were verified by Western blot analysis using anti-myc antibody, and anti-α-tubulin antibody as a loading control.</p

Disulfide bond formation and ROS lowering effect of NT-UCH-L1.

<p>(A) Recombinant UCH-L1 and NT-UCH-L1 were fractionated using FPLC. The fractions were resolved in non-reducing ((−) 2-ME) and reducing ((+) 2-ME) SDS-PAGE gels. UCH-L1 and NT-UCH-L1 were immunoblotted using anti-UCH-L1 antibody. (B) Recombinant UCH-L1, NT-UCH-L1, and its Cys mutants were analyzed in non-reducing SDS-PAGE gel and visualized by silver staining. Arrows indicate position of monomer UCH-L1 and NT-UCH-L1. (C) HeLa cells expressing UCH-L1-myc, NT-UCH-L1-myc, C90S NT-UCH-L1-myc, C132S NT-UCH-L1, and C90/132S NT-UCH-L1-myc were treated with various concentrations of H₂O₂ for 1 h and analyzed using non-reducing ((−) 2-ME) and reducing ((+) 2-ME) gels. UCH-L1s were immunoblotted using anti-myc antibody. (E) HeLa cells stably expressing UCH-L1-myc, NT-UCH-L1-myc, K15/157R NT-UCH-L1-myc and C90/132S NT-UCH-L1-myc were stained with CM-H₂DCFDA and analyzed cellular ROS level using FACS analyzer. Black, mock; blue, UCH-L1; green, NT-UCH-L1; orange, K15/157R NT-UCH-L1; magenta, C90/132S NT-UCH-L1.</p