Hepatitis C virus (HCV) infection remains a major cause of hepatic inflammation and liver disease. HCV triggers NLRP3 inflammasome activation and interleukin-1β (IL-1β) production from hepatic macrophages, or Kupffer cells, to drive the hepatic inflammatory response. Here we examined HCV activation of the NLRP3 inflammasome signaling cascade in primary human monocyte derived macrophages and THP-1 cell models of hepatic macrophages to define the HCV-specific agonist and cellular processes of inflammasome activation. We identified the HCV core protein as a virion-specific factor of inflammasome activation. The core protein was both necessary and sufficient for IL-1β production from macrophages exposed to HCV or soluble core protein alone. NLRP3 inflammasome activation by the HCV core protein required calcium mobilization linked with phospholipase-C activation. Our findings reveal a molecular basis of hepatic inflammasome activation and IL-1β release triggered by HCV core protein. This study deciphers the molecular mechanism of Hepatitis C virus (HCV)-induced hepatic inflammation. HCV triggers NLRP3 inflammasome activation and IL-1β release from hepatic macrophages, thus driving liver inflammation. Using biochemical, virological, and genetic approaches we identified the HCV core protein as the specific viral stimulus that triggers intracellular calcium signaling linked with phospholipase-C activation to drive NLRP3 inflammasome activation and IL-1β release in macrophages. Flaviviridae family [HCV continues as a global health problem causing chronic and progressive liver disease –5. HCV ie family . It is te family . While ce family , 9. DAAse family , 11. Mose family , 13. TheHCV replicates in hepatocytes, the chief parenchymal cell of the liver. During infection HCV also interacts with hepatic macrophages such as the liver-resident Kupffer cells (KCs), which make up 15–20% of the hepatic non-parenchymal cells . KCs areOne of the key intrahepatic inflammatory soluble factors produced by KCs in response to DAMP or PAMP interaction is interleukin-1β (IL-1β) . IL-1β iElevated serum levels of IL-1β and IL-18 are prevalent in patients infected with HCV , 37. FurHCV is a potent inducer of IL-1β production in macrophages and our studies have shown that HCV itself contains all the factors needed to trigger both signal-one and signal-two of NLRP3 inflammasome activation , 33. To Ten proteins are encoded by the HCV virion wherein three of these proteins, Core, E1 and E2, are the viral structural proteins . HCV corThe HCV ion channel p7, is a small transmembrane protein . AlthougWe examined the ability of HCV core protein to stimulate IL-1β production and ASC-speck formation in the reconstituted system. We found the HCV core protein, as similar to the influenza A virus M2 protein (Flu-M2)- a known NLRP3 inflammasome signal-two viral protein stimulus , triggerTo determine if the HCV core protein is a signal-two stimulus for NLRP3 inflammasome activation as occurs in macrophages exposed to HCV , 33, we The HCV core protein is expressed from the viral polyprotein as a 191-amino acid polypeptide , 58. TheWe next tested the ability of four patient-derived HCV core protein constructs to trigger NLRP3 inflammasome activation. Each of the clinical isolates of the core-coding region of HCV was recovered from sera of patients undergoing acute HCV infection with HCV genotype 1 prior to antiviral therapy, with each patient progressing to chronic infection . For patTo determine the mechanism by which HCV core protein triggers IL-1β production, we first assessed the subcellular distribution of the core protein after virion uptake of HCV-treated THP-1 macrophages. We found that within one hour following exposure to HCV, the core protein was present within the cell cytoplasm. HCV core protein was also punctuated around the plasma membrane, likely representing cell surface-bound virion  Fig 7A)Fig 7A). Our findings defines the molecular mechanism by which HCV triggers IL-1β production in macrophages . HepaticCalcium signaling is a complex event that is important for many cellular processes including activation of the NLRP3 inflammasome , 67, 71.Our studies show that KCs, THP-1 macrophages, and primary macrophages produce and secrete inflammatory IL-1β following exposure to HCV  or solubp7 could also play an important role in hepatic inflammation. As previously reported , 50, andHCV employs many innate immune evasion strategies to mediate persistent infection linked with chronic liver disease . In infeOur study underscores a central role of macrophages in HCV pathogenesis. IL-1β is a major mediator of hepatic inflammatory cytokine production from liver cells responding directly to IL-1β signaling . BlockadPichia pastoris and was used at 10-30ug/ml. Nigericin, ATP, D609, u-73343, u-73122, DMSO, and PMA (Sigma). Indo-I-AM calcium flux detection kit was purchased from Calbiochem and LPS from Addipogen. For polyU/UC RNA transfection and/or DNA transfection, Mirus Trans-IT mRNA transfection (for RNA transfection) and Mirus low-toxicity (LT1) (for DNA transfection) were used. The polyU/UC RNA was made as previously described [Antibodies: monoclonal anti-HCV core antibody (ThermoFisher), anti-influenza-M2 and anti-Caspase-1 (Santa Cruz Technologies), anti-Flag (Sigma), anti-actin (Millipore), anti-IL-1β , goat-anti-mouse or goat-anti-rabbit secondary antibodies (ThermoFisher). HCV E2 antibody . Other reagents: Recombinant human TNF and m-CSF were purchased from Peprotech and recombinant HCV core protein (rHCV-core) . The recombinant core protein is produced in escribed . The p7 2. For differentiating THP-1 cells into macrophages, THP-1 monocytes were treated with 40nM of PMA for 24hrs. The following day the cells were washed with PBS and then cultured in fresh media for another 24hrs for resting. Stimulation of THP-1 cells took place after resting .THP-1 cells were obtained from ATCC, U2OS (osteosarcoma cells) were obtained from ATCC, and Huh7 K2040 cells and Huh7.5 cells were described previously ). NLRP3 2 over 1800 seconds). For measuring JFH-1 inhibition by JK3/32, Huh7 cells were cultured and propagated as described previously [in vitro transcription  as per manufacturer’s instructions. RNA was then purified by acid phono/chloroform and isopropanol precipitation. 4x106 Huh7 cells were then electroporated as described previously [4 cells/well in 100ul volume in 96 well plates and incubated for 4hrs prior to addition of inhibitor. Compounds were prepared at 400x in DMSO, diluted 1:20 into media in an intermediate plate, and 1:20 into the final cell plate to yield final 0.25% (v/v) DMSO. All compound treatments were dosed in duplicate. Treated cells were incubated 72hrs before performing 1:4 dilution (50ul) of virus-containing supernatant onto a plate of naïve Huh7 cells (150uls), seeded at 8x103cells/well 6hrs previously. Infected Huh7 cells were incubated 48hrs before washing 3x in PBS and fixing in 4% PFA. Fixed cells were washed in PBS and permeabilized using 0.1% triton X-100 (v/v) in PBS for 10min, room temperature. Following PBS wash, cells were immune-stained for NS5A to quantify infected cells. Sheep anti-NS5A antibody [HCV was propagated and tittered as described previously . Brieflyeviously . Secreteeviously . Brieflyeviously . Electroantibody  was usedantibody , normaliFor the inflammasome components, the cDNA of human proIL-1β, procaspase-1, NLRP3 and ASC were purchased from Origene. These cDNAs were subcloned into pEF vector which contains the human elongation factor-1 alpha (EF1α) promotor to drive ectopic gene expression. The inflammasome components are Flag-tagged for easy detection. Viral proteins were subcloned and expression was detected either by Flag antibody or gene-specific antibody. To amplify the core region from the pSJ   supplemented with protease inhibitor cocktail. Protein from cell lysates were resolved by SDS-PAGE with either commercially purchased precast gels (Biorad) or in lab made-15% or 12.5% gels and then transferred onto PVDF membrane. Probing for the indicated protein was performed using a specific antibody or anti-Flag and actin was used as a loading control. For immunoprecipitation (IP), cells were lysed in RIPA buffer. Cell lysate were incubated with anti-Core antibody or IgG control overnight at 4°C. The following day, IP was performed using protein G dynabeads and then immunoblot was performed as described.RNA from cells was extracted using the RNeasy mini kit (Qiagen). After obtaining RNA, cDNA was made using the Biorad Iscrip Select cDNA synthesis kit. Then real time-PCR (qRT-PCR) was performed using SYBR green PCR master mix. IL-1β and GAPDH (housekeeping gene) primers were used (both purchased from Qiagen).Cells were seeded on cover slips. After stimulation is complete, cells were washed with 1xPBS and then fixed with either 3% paraformaldehyde or ice-cold methanol. After fixation, cells were permeabilized with 0.2% of triton-x. Non-specific binding was blocked with 3% non-fat incarnation milk in 1x PBS. Staining with primary antibody was performed for 30min at room temperature. Alexa-Fluor-594 or alexa-Fluor-488 were used as secondary antibodies. The nucleus is stained with DAPI and wheat-germ agglutinin-594 (ThermoFisher) marks the plasma membrane. Imaging was performed using Nikon Eclipse Ti confocal microscope. Images were taken with 60X oil immersion objective. All images were mounted using the prolong gold antifade reagent. Images were acquired and processed using the NIS advanced research software.Calcium flux was measured in differentiated THP-1 cells. 2-5x10^6 cells were first stained with Indo-I-AM at 1:500 in 200ul-RPMI for 45min at 37°C. after staining with Indo-I, cells were washed with RPMI and either left untreated or stimulated with recombinant core for 10min-1hr. Samples were run on LSRII under the following settings: SS-355, FS-231, DAPI-A-352 and Hoechst-red-A-368. For baseline calcium levels, untreated cells were acquired for 10–30 seconds then stimulated cells were acquired for 2-3min. Calcium flux is represented as a ratio of DAPI to Hoechst-red. Calcium flux represents a shift from 530/30-uv-Hoechst-red  to 405/20-uv-DAPI .Peripheral blood mononuclear cells (PBMCs) of healthy donors were obtained from TRIMA leukoreduction (LRS) chambers . Total mononuclear cells were obtained by centrifugation using ficoll-paque plus (followed manufacturer’s protocol). Briefly, the whole blood was diluted 1:2 with 1xPBS then 35ml of the diluted blood was layered on top of 15ml of Ficoll-paque plus in 50ml conical tube. Centrifugation was performed at room temperature at 300xg for 30-45min with no brake. After centrifugation is complete, 5–10 million total PBMCs were cultured in complete-RPMI containing 40-100ng/ml of human m-CSF. Two days after culturing, non-adherent cells were removed and fresh RPMI containing 40-100ng/ml of m-CSF was added. After five days of culturing, cells were collected by scrapping gently in RPMI then seeded 0.3–0.8 million cells per well of a 12-well plate. The following day cells were treated with different stimuli.The human blood samples were obtained from healthy donors. The samples were purchased from Bloodworks Northwest (Washington). The samples are delivered in TRIMA leukoreduction (LRS) chambers and then processed to isolate PBMCs. Samples were non-identifiable.For ELISA, supernatant from both non-stimulated and stimulated cells were collected. To remove cell debris, cells were spun for 5 minutes at medium speed. ELISA was performed according to the manufacturer’s manual .For statistical analysis, student t-test or one-way Anova (for multiple comparisons) was performed. Any p value less than 0.05 was considered statistically significant. Graphs are represented as the means +/- SD.S1 Fig(A) IL-1β ELISA in THP-1 cells. THP-1 cells were differentiated with PMA then treated with uv-HCV or LPS/Ng in the presence of a pharmacological p7 ion channel inhibitor. Jk3/32 (active inhibitor), R-21 (inactive inhibitor) and DMSO (vehicle control). (B) JHF-1 secretion post incubation in either JK3/32 or DMSO control.(TIF)Click here for additional data file.S2 FigInduction of IL-1β gene expression is normalized to GAPDH.(TIF)Click here for additional data file.S3 FigPatient#1(ID: 4-7-1), patient#2 (ID: 27-9-5), patient#3(ID: 3-9-5), patient#4(ID: 6-8-2) and patient#4-1(ID: 30-4-4).(TIF)Click here for additional data file.S4 Fig(A) Immunoblot showing Co-IP of core with NLRP3 inflammasome components in the reconstituted system. (B) Intracellular calcium in primary human monocyte-derived macrophages upon treatment with rHCV-Core. (C) ELISA of IL-1β in differentiated THP-1 cells stimulated with rTNF and rHCV-core in the presence of D609 inhibitor. For (D) cells were first stimulated with TNF then treated with rHCV-core or ATP in the presence of DMSO or u-73343 or u-73122.(TIF)Click here for additional data file.


Escherichia coli, isolated in January 2013 at King Abdulaziz Medical City (KAMC), Riyadh, Saudi Arabia. The isolate (named SA186) was sequence type 131 (ST131) and belonged to serotype O25b-H4 and clade B (fimH22).We report the complete genome sequence of a colistin-resistant strain of uropathogenic  Escherichia coli, isolated in January 2013 at King Abdulaziz Medical City (KAMC), Riyadh, Saudi Arabia. The isolate (named SA186) was sequence type 131 (ST131) and belonged to serotype O25b-H4 and clade B (fimH22).We report the complete genome sequence of a colistin-resistant strain of uropathogenic  ST131 i1) clone .mcr-1-positive ST131-O25:H4-fimH22 clade UPEC isolate SA186, which was isolated from a 2-year-old female patient with urinary tract infection in January 2013. Susceptibility testing performed using the Vitek II XL system  showed that SA186 was resistant to ampicillin , piperacillin , gentamicin , tobramycin , norfloxacin , and trimethoprim  but susceptible to third-generation cephalosporins and carbapenems. SA186 resistance to colistin  was confirmed by the microbroth dilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) (We determined the complete genome sequence of the The isolate was grown on Columbia agar (Oxoid) at 37°C, and genomic DNA was extracted from an overnight single starting colony culture using the QIAsymphony SP instrument following the manufacturer’s instructions (Qiagen). Genomic DNA (>50 kb) was sheared by passage through a 26-gauge needle, and the size of the fragments thus generated (>30 kb) was checked on a fragment analyzer. Fragmented DNA cleaned with Ampure XP beads (Agencourt) was used for library preparation using the SMRTbell template prep kit version 1.0 (PacBio) according to the manufacturer’s instructions. The complete genome sequence of SA186 was determined using the PacBio RS II single-molecule real-time (SMRT) instrument at the Centre for Genomic Research at the University of Liverpool (United Kingdom).PacBio sequence reads were assembled following the Hierarchical Genome Assembly Process (HGAP) workflow version 2.3.0 with the default settings . Brieflymcr-1 gene and belonged to the incompatibility group IncHI2 and plasmid multilocus sequence typing (pMLST) subtype ST4 , penicillins (blaTEM-1B), macrolides , chloramphenicol , streptothricin (sat1), sulfonamides (sul3), tetracyclines [tet(A)], and trimethoprim (dfrA14) and shared 99% nucleotide identity with IncHI2 plasmid pSA26-MCR-1 (GenBank accession no. KU743384), which was previously described in a strain of E. coli (ST68) isolated from a patient in Saudi Arabia in June 2012 (mcr-1-positive plasmids pS38 (GenBank accession no. KX129782) (LT599829) (KP347127) (E. coli isolates belonging to ST602 (Switzerland), ST362 (Germany), and an undetermined ST (China), respectively.The genome of SA186 consisted of one 4,828,837-bp chromosome with a GC content of 50.68% and five plasmids designated pSA186-MCR-1 , pSA186-2 , pSA186-3 , pSA186-4 , and pSA186-5 . Plasmid pSA186-MCR-1 carried the prim dfrA and sharfimH22 subclone non-extended-spectrum β-lactamase (non-ESBL)/carbapenemase-producing UPEC strain from Saudi Arabia, which harbors the mcr-1 gene. Its detection highlights a new challenge in the emergence and evolution of antimicrobial resistance plasmids in the Arabian Peninsula.In summary, this is the first report of a complete genome sequence of a multiresistant ST131 CP022730 for the chromosome, CP022731 for pSA186_2, CP022732 for pSA186_3, CP022733 for pSA186_4, CP022734 for pSA186_5, and CP022735 for pSA186_MCR. These sequences are part of BioProject no. PRJNA395653, and the SRA accession number is SRX3097879.The complete genome sequence of UPEC isolate SA186 has been deposited in GenBank under the accession no.


Correction to: J ImmunoTher Cancerhttps://doi.org/10.1186/s40425-019-0689-7Following publication of the original article , the autshould be replaced with“Ipilimumab and AGEN1884 are IgG1 class antibodies. AGEN1884 data show CTLA-4 inhibition and activity against T regulatory cells (reference 8).”


G93A and SOD1G85R, but not wild type SOD1, directly interact with VDAC1 and reduce its channel conductance. No such interaction with N-terminal-truncated VDAC1 occurs. Moreover, a VDAC1-derived N-terminal peptide inhibited mutant SOD1-induced toxicity. Incubation of motor neuron-like NSC-34 cells expressing mutant SOD1 or mouse embryonic stem cell-derived motor neurons with different VDAC1 N-terminal peptides resulted in enhanced cell survival. Taken together, our results establish a direct link between mutant SOD1 toxicity and the VDAC1 N-terminal domain and suggest that VDAC1 N-terminal peptides targeting mutant SOD1 provide potential new therapeutic strategies for ALS.Mutations in superoxide dismutase (SOD1) are the second most common cause of familial amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the death of motor neurons in the brain and spinal cord. SOD1 neurotoxicity has been attributed to aberrant accumulation of misfolded SOD1, which in its soluble form binds to intracellular organelles, such as mitochondria and ER, disrupting their functions. Here, we demonstrate that mutant SOD1 binds specifically to the N-terminal domain of the voltage-dependent anion channel (VDAC1), an outer mitochondrial membrane protein controlling cell energy, metabolic and survival pathways. Mutant SOD1 Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease caused by the death of upper and lower motor neurons in the brain and spinal cord . The age1 including active dismutase mutants, such as SOD1G93A and SOD1G37R, and inactive dismutase mutants in which the mutation affects the metal-binding region, such as SOD1G85R and SOD1H46R (WT) can become misfolded and toxic, thus sharing an aberrant conformation with SOD1 mutants, when oxidatively modified ,SOD1H46R . The latmodified . The examodified . These imodified .2+ dysregulation has been reported in spinal cord and skeletal muscles of familial and sporadic ALS patients , dithiothreitol (DTT), HEPES, leupeptin, phenylmethylsulfonyl fluoride (PMSF), propidium iodide (PI), sucrose and Tris were purchased from Sigma . Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco . The CellTiter 96 AQueous one-solution cell proliferation assay was purchased from Promega .The peptides used in this study , followed by ion exchange chromatography using a HiTrap Q-Sepharose anion exchange column (Amersham Biosciences), as described previously (Recombinant hSOD1eviously .NheI/XhoI sites. Escherichia coli BL21(DE3) cells were transformed with plasmid pET21a harboring the VDAC1 or ΔN-VDAC1 genes. Protein expression was induced for 3 h using 1 mM isopropyl-β-D-thiogalactopyranoside . Proteins were purified on agarose-packed nickel-nitrilotriacetic acid resin  in the presence of 8 M urea. Refolding of the eluted protein was performed essentially as described previously /(Fmax − Fmin).Microscale thermophoresis analysis was performed using a NanoTemper Monolith NT.115 apparatus, as recently described . Brieflywt, hSOD1G93A, or hSOD1G85R to the cis compartment using a Bilayer Clamp BC-535B amplifier . Current amplitude histograms were prepared using AxoGraph X software. Relative conductance was determined as the average steady-state conductance at a given voltage normalized to the conductance at 10 mV, the maximal conductance. Relative conductance-voltage plots were prepared using Microsoft Excel software.The reconstitution of recombinant WT or ΔN-VDAC1 into a planar lipid bilayer (PLB) prepared from soybean asolectin, and subsequent single channel current recordings and data analysis were carried out as described previously . CurrentNSC-34 cells, a mouse motor neuron-like hybrid cell line, the A549 human lung adenocarcinoma and U-87MG human glioblastoma cell lines were incubated with the (1-26)N-Ter-Antp, D-(15-26)N-Ter-Antp, (1-20)N-Ter-Antp, (5-20)N-Ter-Antp, or (10-20)N-Ter-Antp peptides in the appropriate serum-free growth medium for 5 h at 37°C. Cells were harvested, and analyzed for cell death using propidium iodide (PI) staining and flow cytometry. For XTT, the CellTiter 96 AQueous one-solution cell proliferation assay was used to follow cell viability as described previously .Apoptotic cell death was also analyzed using Acridine Orange (AcOr)/ethidium bromide (EtBr) staining . Cells i4) were grown on sterilized coverslips in 24-well plates. 30 h post-transfection, cells were fixed using 4% paraformaldehyde  for 15 min, and then washed 3 times with PBS (5 min each wash). Cells were then permeabilized with 0.3% Triton X-100 in PBS for 5 min followed by washing with PBS. Cells were then blocked for 1 h with blocking buffer (1% BSA free fatty acids diluted in PBS). Anti-VDAC1 polyclonal antibody (ab15895) and mouse anti misfolded SOD1  were incubated at room temperature for 1–2 h in a buffer of 1% BSA free fatty acids and 0.3% Triton-X100 in PBS. Following incubation with primary antibodies, cells were washed with PBS and incubated with fluorescent conjugated secondary Alexa Flour 488 anti-rabbit and Alexa flour 647 anti-mouse antibodies. The coverslips were carefully dried and mounted on slides using Immumount . After overnight drying, images were acquired on an Olympus IX81 confocal microscope.For immunostaining, SH-SY5Y cells (4.5 × 10G93A) were a kind gift from Dr. Kevin Eggan (Harvard Stem Cell Institute). This cell line carries green fluorescent protein (GFP) under the control of the promoter for the motor neuron (MN)-specific transcription factor HB9 (HB9:GFP cells) (G93A mESC line to derive GFP+ MNs.Mouse embryonic stem cell lines harboring human mutant SOD1 (SOD1P cells) . We usedMotor neuron differentiation was induced as described previously with some modifications . Cells win vitro differentiation assay, EBs were enzymatically dissociated after 7 days in culture with TrypLE Express (Gibco) and seeded on 0.01% poly-l-ornithine (Sigma) pre-coated coverslips followed by laminin . Cells (5 × 104) were seeded on coverslip in 24-well plates with ADFNB cell medium supplemented with of Ciliary neurotrophic factor  and Glial cell-derived neurotrophic factor . During plating, 1-20N-Ter-Antp VDAC1-peptide was added to the cultures. Half of the medium was replaced with fresh medium every second day and at the indicated time points. The cultures were fixed in 4% paraformaldehyde in PBS.For an + soma and neurite intersections with test lines of an unbiased counting frame superimposed on images of cell cultures obtained using a 20× objective (NA 0.75) of a Nikon Eclipse E800 epifluorescence microscope equipped with a Nikon DXM1200F CCD camera. The absolute length, L, of neurites per cell was subsequently estimated from the number of neurite intersections, I, per cell by means of the equation L = (πd/2)I describing the relationship between the number of neurite intersections and the vertical distance, d, between the test lines used.Stereological estimation of neurite lengths to evaluate neurite outgrowth in cultured cells was carried out as described previously . The tott-test. The mean ± SEM of results obtained from at least three independent experiments are presented. The significance of differences was calculated by a two-tailed Student’s t-test and is reported as ∗∗p < 0.01. Statistical comparisons between conditions in the mESC MN assays was performed by one-way ANOVA followed by Dunnett’s Multiple Comparison test against the control condition  values being derived from the depletion curves  for the three versions of SOD1 , had no effect on the current  D-(15-26)-N-Ter-Antp, a peptide like (1-26)-N-Ter-Antp but shortened by 13 amino acids from the N-terminus, and presenting all residues in the D configuration; (3) (1-20)-N-Ter-Antp peptide, again a peptide like (1-26)-N-Ter-Antp but shortened by six amino acids at the C-terminus; (4) (5-20)-N-Ter-Antp peptide, a peptide like (1-20)-N-Ter-Antp peptide but shortened by four amino acids at the N-terminus; and (5) (10-20)-N-Ter-Antp peptide, a peptide like (1-20)-N-Ter-Antp peptide but shortened by nine amino acids at the N-terminus of the peptide -N-Ter-Antp and D-(15-26)-N-Ter-Antp peptides induced massive cell death in NSC-34 , U-87MG in vitro whether the different shortened versions of the VDAC1 N-terminal peptide could prevent mutant SOD1 toxicity in neurons, NSC-34 cells were transfected to express SOD1WT or mutant SOD1G93A protein and then treated with or without the above indicated VDAC1 N-terminal peptides. Cell viability was quantified using the XTT assay and apoptosis using acridine orange and ethidium bromide staining. Whereas SOD1WT had not effect on cell viability, expressing SOD1G93A reduced cell viability by 25–30%. The presence of VDAC1 N-terminal peptides reduced the toxic effect of SOD1G93A in a concentration-dependent manner -N-Ter-Antp peptide, decreased SOD1G93A and SOD1G37R-mediated cell death by 68 and 81%, respectively -N-Ter-Antp peptide. Co-localization of misfolded SOD1 with VDAC1 was determined by immunostaining analysis using an anti-VDAC1 antibody that does not target the N-terminus region of the protein, and the B8H10 antibody specifically recognizing misfolded SOD1. In cells transfected with SOD1G93A or SOD1G37R, VDAC1 staining is punctuated as expected for mitochondrial localization, while misfolded SOD1 shows both punctuated staining but mostly diffused, indicating that part of the misfolded SOD1 protein is mitochondria bound -N-Ter-Antp peptide on three characteristics of SOD1eviously . SOD1G931st week . TherefoG93A-expressing MNs (p < 0.05) improved neurite outgrowth of SOD1G93A-expressing MNs  improved MN density (G93A-expressing mature MNs (p < 0.05) improved MN survival when administered at a 10 μM concentration  and ROS  following this domain represent a GXXXG motif that connects the N-terminal domain to β-strand 1, and confer the flexibility required for N-terminal domain translocation out of the channel pore -N-Ter-Antp VDAC1-based peptide significantly improved neurite outgrowth and the survival of SOD1G93A-expressing MNs in a concentration-dependent manner . This not only corroborates the insight gained from studies on NSC-34 cells but also demonstrates that the VDAC1-based peptide acts as a neuroprotectant against misfolded SOD1-mediated MN toxicity. Further, these results underline the importance of a direct misfolded SOD1-VDAC1 interaction for the appearance of misfolded SOD1 toxicity. It remains to be shown whether the VDAC1-based (1-20)-N-Ter-Antp peptide also has the same effect on fully mature human motor neurons. Indeed, the results justify more thorough in vitro and in vivo analysis of the therapeutic potential of the VDAC1-based (1-20)-N-Ter-Antp peptide in treating ALS. These include improving peptide stability, such as by introducing amino acids in the D-conformation and carrying out toxicological studies, as performed with another VDAC1-based peptide specifically inducing cell death of cancer cells (Finally, experiments with mESC-derived SOD1er cells .In summary, we have shown here that SOD1 mutants interact with VDAC1 through the VDAC1 N-terminal domain to exert their inhibitory effect on VDAC1 channel conductance. Moreover, we have shown that VDAC1 N-terminal-derived peptides specifically bind mutant SOD1 and inhibit mutant SOD1-induced toxicity in motor neuron-like NSC-34 cells expressing mutant SOD1 or in mouse embryonic stem cell-derived motor neurons. Thus, we suggest that this VDAC1-based peptide represents a new strategy for interfering with mutant SOD1-mediated cell toxicity.AMe, EK, VS-B, and AI designed the research. SA, RG, AS-K, NS, SA-H, YG, JH, and AMa conducted the experiments. SA, RG, AS-K, SA-H, NS, JH, AMa, AMe, EK, VS-B, and AI analyzed the data. VS-B and AI wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.