Zika virus (ZIKV) is a mosquito-borne flavivirus and can cause neurodevelopmental disorders in fetus. As a neurotropic virus, ZIKV persistently infects neural tissues during pregnancy but the viral pathogenesis remains largely unknown. ZIKV has a positive-sense and single-stranded RNA genome, which encodes 7 non-structural (NS) proteins, participating in viral replication and dysregulation of host immunity. Like those in many other viruses, NS proteins are considered to be products evolutionarily beneficiary to viruses and some are virulence factors. However, we found that some NS proteins encoded by ZIKV genome appeared to function against the viral replication. In this report we showed that exogenously expressed ZIKV NS2A and NS4A inhibited ZIKV infection by inhibiting viral RNA replication in microglial cells and astrocytes. To understand how viral NS proteins suppressed viral replication, we analyzed the transcriptome of the microglial cells and astrocytes and found that expression of NS4A induced the upregulation of ISGs, including MX1/2, OAS1/2/3, IFITM1, IFIT1, IFI6, IFI27, ISG15 or BST2 through activating the ISGF3 signaling pathway. Upregulation of these ISGs seemed to be related to the inhibition of ZIKV replication, since the anti-ZIKV function of NS4A was partially attenuated when the cells were treated with Abrocitinib, an inhibitor of the ISGF3 signaling pathway, or were knocked down with STAT2. Aborting the protein expression of NS4A, but not its nucleic acid, eliminated the antiviral activity of NS4A effectively. Dynamic expression of viral NS proteins was examined in ZIKV-infected microglial cells and astrocytes, which showed comparatively NS4A occurred later than other NS proteins during the infection. We hypothesize that NS4A may possess intrinsic features to serve as a unique type of pathogen associated molecular pattern (PAMP), detectable by the cells to induce an innate immune response, or function with other mechanisms, to restrict the viral replication to a certain level as a negative feedback, which may help ZIKV maintain its persistent infection in fetal neural tissues. The birth of microcephaly infants due to ZIKV infection in pregnant women is related to ZIKV persistent infection. However, it is unclear how ZIKV maintains its persistent infection. In this work, we observed the delayed appearance of ZIKV NS4A protein in neuroglia including microglia and astrocytes compared with other non-structural proteins. Subsequently, we revealed that ZIKV NS4A inhibited viral RNA replication by activating the ISGF3 signaling pathway and inducing the production of ISGs. Aborting NS4A protein expression totally rescued ZIKV viral replication. Our study, combined with the previous findings, suggests that viral non-structural proteins may regulate viral replication, thus perpetuating ZIKV infection. Our hypothesis provides a mechanism for ZIKV to maintain its status of a persistent infection during viral infection in fetus, which can shed lights on our further understanding of viral neuropathogenesis in ZIKV infection. The virus (WNV) . The strus (WNV) . In geneus (WNV)  or mild us (WNV) .When ZIKus (WNV) –8. ZIKV us (WNV) –11. The In vitro culture and histology of fetal brain tissues from the brain with microcephaly have shown that ZIKV activates microglia and induces high levels of pro-inflammatory cytokines [Microglia, the resident macrophages of the central nervous system (CNS), have a perivascular localization which allows them to monitor the influx of blood-borne components into the CNS . Our andytokines ,15. The ytokines . In addiytokines . Becauseytokines .Astrocytes, the most abundant cells of the CNS, have a number of neuroprotective functions, including maintaining the integrity of the blood-brain barrier (BBB), regulating synapses function and promoting neuronal repair . Viral iProgress has been made in the study on ZIKV NS proteins in viral pathogenesis. ZIKV NS1, NS3, NS4B and NS5 all inhibit the interferon signaling pathway –26 and NIn this study, we confirmed that ZIKV could infect human microglial cells and astrocytes by examining the replication of viral genome and the expression of viral proteins. We observed a late appearance of NS4A during viral infection in human microglia and astrocytes and wondered whether this delayed appearance of NS4A had a unique role in viral neuropathogenesis. To our surprise, expression of NS4A exhibited an activity against ZIKV replication, which prompted us to analyze the underlying mechanism and possible ramification of this intrinsic “viral protein against itself” feature. We propose that ZIKV utilizes its NS4A to suppress its replication via a unique mechanism, so that viral persistent infection could be maintained and ZIKV stay in fetal neural tissues for extended presence.2. U251, HeLa, HEK293, Vero and BHK21 cells were maintained in Dulbecco’s modified Eagle medium (DMEM)  with 10% FBS. ZIKV strain SZ01/2016 (GenBank number: KU866423) was isolated from a patient who returned from Samoa [HMC3 cells were maintained in Minimum Essential Medium (MEM)  supplemented with 10% fetal bovine serum  at 37°C in 5% COom Samoa . ZIKV stom Samoa . EV-A71 om Samoa . All virTo construct microglial and astrocytes cells expressing ZIKV NS proteins, the cDNA of NS1, NS2A, NS2B, NS3, NS4A and NS4B were amplified from ZIKV SZ01/2016 and cloned into pLVML-3×HA-MCS-IRES-Puro  or pRK5-HA vector. The empty vector pLVML-3×HA-MCS-IRES-Puro or pLVML-3×HA-MCS-IRES-Puro-NS, psPAX2 and PMD-2.G were co-transfected into HEK293 cells in a 1:1:1 ratio by Vigofect  and the lentiviruses in culture supernatant were collected at 48 h post transfection. Microglial and astrocytes, HMC3 and U251 cells, respectively, were infected by the lentiviruses and the infected cells were screened with 2 or 8 μg/ml puromycin  for 48 h post infection (p.i.). Two weeks later, HMC3 and U251 cells stably expressing ZIKV NS proteins were validated by immunofluorescence assay (IFA) and western blot analysis (WB). Cell lines, which were generated using lentiviruses with empty vector, served as a control for cells expressing NS proteins. We uniformly labeled these cells as “Vector” in all figures.To obtain cell lines expressing NS4A-X, nucleotides TAA were inserted before the start codon ATG of NS4A sequence on the pLVML-3×HA-MCS-IRES-Puro-NS4A plasmid, allowing the retention of the NS4A nucleic acid and the termination of protein expression (NS4A-X). Subsequently, cell lines stably expressing NS4A-X were obtained according to the method described above.To achieve U251 cells knocking down STAT2, p-GIPZ-shSTAT2 and p-GIPZ were purchased from Shanghai Jiao Tong University. Subsequently, together with psPAX2 and PMD-2.G, lentiviruses were constructed and used to infect U251 cells with or without NS4A expression. The lentiviruses packaged by empty plasmid p-GIPZ were set as the negative control for knockdown of STAT2.5 per well. 24 h later, cells were fixed with 4% paraformaldehyde  for 15 min, then perforated by 0.1% Triton X-100 for 15 min, blocked by 3% BSA for 30 min. Then the cells were incubated with anti-HA rabbit antibodies  for 1 h at room temperature. After three PBS washes, the cells were incubated with AlexaFluor 488-labelled donkey anti-rabbit IgG  for 1 h at room temperature. Following five washes by PBS, the coverslips were sealed with Prolong Gold Antifade reagent with DAPI  and observed under the Olympus FLUOVIEW FV3000 confocal microscope. To detect the expression of viral dsRNA in HMC3 cells expressing NS2A or NS4A, the HMC3 cells were infected by 0.1MOI of ZIKV SZ01/2016 for 24 or 48 h. The J2 mouse monoclonal anti-dsRNA antibody  was used as a primary antibody and Alexa Fluor 488-labelled donkey anti-mouse IgG  was used as a secondary antibody.To verify whether HMC3 and U251 cells stably expressed ZIKV NS protein, cells were seeded on sterile coverslips in 24-well plates at 1x10To test the expression of viral proteins in HMC3 or U251 cells, cells were lysed at various time points after ZIKV infection using cell lysis buffer for Western and IP  with protease inhibitors . The polyclonal anti-ZIKV E rabbit antibodies , anti-ZIKV NS2B, NS3, NS4A, or NS5 rabbit antibodies , or the monoclonal anti-GAPDH antibodies  were used as the primary antibodies. The horseradish peroxidase (HRP)-conjugated antibodies (1:3000) against mouse IgG or rabbit IgG obtained from Proteintech  were used as the secondary antibodies. To verify whether the expression of NS2A and NS4A activates the ISGF3 signaling pathway, HMC3 cells expressing NS2A or NS4A were collected for detection of STAT1, phospho-STAT1 (Tyr701), STAT2, phospho-STAT2 (Tyr690) and IRF9. Antibodies for the above proteins were purchased from ABclonal .To examine the response of HMC3 or U251 cells stably expressing ZIKV NS proteins to ZIKV infection, the above cells were infected with 0.1 MOI of ZIKV SZ01/2016. 72 or 48 h p.i. later, the cells were prepared according to the instructions of the apoptosis kit  as follows. Briefly, the cells were washed with PBS, then digested with 0.25% EDTA-free trypsin. After 2 washes with PBS, the cells were suspended with 100 μl binding buffer with 5 μl Annexin V and 5 μl propidium iodide (PI). After 15 min incubation at room temperature, 400 μl binding buffer was added to each sample. Finally the cells were filtered through 200 mesh gauze and immediately analyzed on a BD FACS Calibur. The collected data were further analyzed by FlowJo V10.5 per well the day before infection. Ten-fold serially diluted viruses were inoculated to BHK21 cells in triplicates. After 2 h of infection, the virus inoculum was discarded and replaced by DMEM containing 2% FBS in 1% low melting point agarose. After the agarose was solidified at room temperature, the plates were transferred to an incubator. 4–5 days later, when viral plaques were visible, the cells were stained with crystalline violet dye in 4% paraformaldehyde (PFA) for 4 h at room temperature. The plates were washed and viral plaques were counted.BHK21 cells were seeded on a 12-well plate at 5x10To quantify viral RNA copies in cell culture medium after virus infection in HMC3 cells expressing NS2A or NS4A, the culture medium was collected at 24, 48, 72 and 96 h p.i. Viral RNA was extracted using the TIANamp Virus RNA Kit , and viral RNA copies were quantified by One Step PrimeScript RT-PCR Kit  with the QuantStudio 5 PCR instrument (ABI). The following primers were used: ZIKV-E-F-Taq (5’- GGTCAGCGTCCTCTCTAATAAACG -3’), ZIKV-E-R-Taq(5’- GCACCCTAGTGTCCACTTTTTCC-3’), Probe (5’-6-FAM-AGCCATGACCGACACCACACCGT -BHQ1-3’). Standard curves were prepared using a plasmid encoding ZIKV E gene.To determine the expression of viral genes in cells after viral infection or the expression of NS4A-induced ISGs, total RNA was isolated from the cells using RNAiso Plus reagent (Takara Bio) and reversed by HiScript III RT SuperMix for synthesis of cDNA and qPCR analysis (+gDNA wiper) . Then gene expressions were quantified by TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio) with the QuantStudio 5 or ABI Viia 7 PCR instrument. The primers are listed in 6 cells per well. 24 h later, the cells were infected with ZIKV SZ01/2016 (MOI = 2) and incubated at 4°C for 1 h. The unbound viruses were removed and the cells were washed twice with PBS, followed by preparation of total RNA from the cells for measuring viral RNA copies by qRT-PCR. For the entry assay, after the incubation of the cells and virus at 4°C for 1 h, the cells were washed twice with PBS, and subsequently treated with pre-warmed MEM for 10 min at 37°C. After 3 time washes with PBS, the cells were treated with 0.25% trypsin. After another 3 time washes with PBS, the cells were collected for preparing total RNA and measuring viral RNA copies by qRT-PCR.ZIKV attachment and entry experiments were performed as previously described . For thein vitro according to the instructions of mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific). Subsequently, 5x106 HMC3 cells expressing NS2A or NS4A were resuspended in 400 μl electrotransfer solution cytomix buffer. The mixtures of cells and 5 μg Zika RNA were transferred to sterile electroporation cuvette . After electroporation the cells every 3 s at 450V, 25 uF, total 3 times, by Gene Pulser Xcell Electroporation System , the cells were transferred to 3.5 ml MEM containing 2% FBS immediately, and seeded in 6 cm dishes for culture at 37°C, 5% CO2. Finally, the cells were collected at 48 and 96 h for measuring viral RNA copies by qRT-PCR or at 24, 48, 72 and 96 h for determining the expression of NS2B by western blot assay.The replicon assay was referred to previous reports ,35 with The HMC3 cells expressing NS2A or NS4A were plated on 10 cm culture plates and infected with 0.1 MOI of ZIKV/SZ01. Total RNA was prepared from the cells using RNAiso Plus reagent (Takara Bio) at 24 h p.i. and sent to Gene Donovo  for RNA-Seq.Statistical analyses were performed by SPSS 13.0. The differences between groups of ZIKV NS proteins and controls were evaluated by two-tailed Student’s t test and the p-value of each test was chosen according to the result of Levene’s Test for Equality of Variances. *, P < 0.05; **, P < 0.01; ***, P < 0.001.ZIKV could infect human microglial and astrocytic cells and induce cytopathic effect and cell death . We examTo investigate the effect of ZIKV NS proteins on viral infection, we constructed HMC3 and U251 cell lines stably expressing ZIKV NS proteins. As shown in To investigate the effect of NS protein expression on ZIKV infection, HMC3 cells expressing NS proteins were infected by 0.1 MOI of ZIKV, and the viral titers in the culture supernatants were measured by a plaque formation unit (PFU) assay. As shown in in vitro and then the infectious RNA was electrotransfected into HMC3 cells expressing NS2A or NS4A. Total RNA were prepared from the transfected cells for viral RNA detection and cell lysates were collected to determine the expression of NS2B. NS2B is part of the replication complex and is responsible for grouping together the other components of the replication complex [To examine which step of viral life cycle was targeted by NS2A and NS4A, HMC3 cells expressing NS2A or NS4A were infected with ZIKV at 4°C for 1 h, followed by washing the cells to remove unbinding viruses. Total RNA was prepared from the cells to determine the viral attachment by qRT-PCR. The results showed that NS4A expression inhibited 28.9% of ZIKV attachment to HMC3 cells but NS2A expression had no such effect . Subsequ complex ,38. As sbunyaviridae, SFTSV, and another from piconaviridae, EV-A71, to see if NS2A and NS4A could have any impact on them from different families. As shown in The above results showed NS2A and NS4A from ZIKV/SZ01 inhibited ZIKV/SZ01 infection. Here, we detected whether NS2A and NS4A inhibited the infection of ZIKV/MR766, SFTSV and EV-A71. As shown in Mx1/2, Oas1/2/3, Ifitm1, Ifit1, Ifi6, Ifi27, Xaf1, Bst2, Isg15, and Usp18, were up-regulated by NS4A expression  ,40 was uIfi27 and Bst2 or Mx1 and Isg15, a different pattern from observed in HMC3 cells, probably suggesting that NS4A up-regulated some ISGs in a cell type-dependent manner. According to the above, the activation of ISGF3 signaling pathway may play a partially role in the antiviral effect of NS4A.In addition, we observed that NS4A in U251  or HeLa To further explore the inhibitory effect of NS4A on ZIKV replication, we generated the cell lines expressing mutant NS4A (NS4A-X) by transfecting HMC3 or U251 cells with plasmids expressing HA-NS4A, which has a stop codon introduced to abort the translation of NS4A. We specifically detected HA-NS4A RNA replication by designing the upstream primers in the HA sequence and the downstream primers in the NS4A sequence. As shown in In this report, we observed a delayed appearance of ZIKV NS4A in human microglial cells and astrocytes . We consZIKV proteins are produced from one open reading frame into a polyprotein that is subsequently cleaved into different proteins. It is interesting how NS4A is delayed in appearance compared to other NS proteins. Even though the difference could come from various sensitivities of antibodies, we consider that the later appearance of NS4A may be due to other reasons. One would be the half-life of NS4A, which could be shorter than other NS proteins possibly due to its post-translational modifications and proteolytic processes present in the infected cells. The half-lives of NS3, NS4A, and NS5A of hepatitis C virus (HCV) were 12, 11, and 10 h, respectively, when detected by kinetic analysis in human cell lines expressing HCV open reading frame. We haveDuring infection, ZIKV persists in the male reproductive system for as long as six months  likely rExpression of NS2A and NS4A not only inhibited the infection of ZIKV/SZ01, ZIKV/MR766, but also suppressed SFTSV and EV-A71 replication , suggestFlaviviridae. Knockdown of MX1 significantly inhibited anti-ZIKV activity by IFN-λ in a human trophoblast line (JEG3) [Numerous studies have shown that ISGs are effective inhibitors of viral infections. In this report we showed that NS4A significantly induced the expression of 13 ISGs, including MX1/2, OAS1/2/3, IFITM1, IFIT1, IFI6, IFI27, XAF1, BST2, ISG15, and USP18  in microe (JEG3) . Silencie (JEG3) . We specNS4A may up-regulate ISGs in a cell type-dependent manner. Microglial cells have a unique RNA expression profile and even are heterogeneous in different locations in the brain . ZIKV inIn conclusion, we observed that the expression of ZIKV NS2A and NS4A inhibited ZIKV infection in microglia and astrocytes probably through activation of antiviral mechanisms for suppression of viral RNA replication. NS4A enhanced ISGs expression by activating ISGF3 signaling pathway, and blocking the ISGF3 pathway could partially repress the antiviral activity of NS4A. Aborting the expression of NS4A protein eliminates its antiviral activity effectively. NS4A protein occurred later than NS2B, NS3 and NS5 during ZIKV infection. We hypothesize that ZIKV NS4A may regulate viral replication by inducing an innate immune response, working as a PAMP, or functioning with other mechanisms, to help maintain a status of viral persistent infection. Our study will be helpful in further characterizing and understanding viral neuropathogenesis in ZIKV infection.S1 Fig(a & d) ZIKV/SZ01 infection of HMC3 or U251 cells was detected by IFA. ZIKV E was stained by an anti-E mouse antibody 4G2 (green); cell nuclei were stained by 4,6-diamidino-2-phenylindole . Scale bar, 50 μm. (b) Cell death induced by ZIKV infection in HMC3 cells was analyzed by flow cytometry and Annexin V/PI apoptosis Kit. (c & f) ZIKV replication in HMC3 or U251 cells was determined by a viral plaque formation unit assay. (e) Cytopathic effect was observed under a light microscope after ZIKV infection in U251 cells. Scale bar, 50 μm.(TIF)Click here for additional data file.S2 Fig(a) The expression of ZIKV NS proteins in HMC3 cells was analyzed by IFA. HA-tagged ZIKV NS proteins were detected by staining with anti-HA rabbit antibodies (green); cell nuclei were stained by DAPI (blue). Scale bar, 20 μm. (b) The expression of ZIKV NS proteins in HMC3 cells was analyzed by western blot. HA-tagged ZIKV NS proteins were detected by anti-HA rabbit antibodies. The red box indicates the targeted band.(TIF)Click here for additional data file.S3 Figa) The expression of ZIKV NS proteins in U251 cells was analyzed by IFA. HA-tagged ZIKV NS proteins were detected by anti-HA rabbit antibodies (green); Cell nuclei were stained by DAPI (blue). Scale bar, 20 μm. (b) The expression of ZIKV NS proteins in U251 cells was analyzed by western blot. HA-tagged ZIKV NS proteins were detected by anti-HA rabbit antibodies. The red box indicates the targeted band.((TIF)Click here for additional data file.S4 Fig(a-b) HMC3 or U251 cell death was determined by flow cytometry and Annexin V/PI apoptosis kit at 48 (U251) or 72 h (HMC3) post ZIKV/SZ01 (0.1 MOI) infection.(TIF)Click here for additional data file.S5 FigDsRNA production was analyzed by IFA in HMC3 cells infected with 0.1 MOI of ZIKV for 48 h. DsRNA was probed by the J2 mouse monoclonal anti-dsRNA antibody (green) with cell nuclei stained by 4,6-diamidino-2-phenylindole . Magnification: 60X.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Interestingly, in melanoma clinical datasets, PAK4 expression levels negatively correlate with the presence of CCL21, the ligand for CCR7 expressed in CD103+ DCs. Furthermore, we extensively characterized the transcriptome of PAK4 knockout (KO) tumors, in vitro and in vivo, and established the importance of PAK4 expression in the regulation of the extracellular matrix, which can facilitate immune cell infiltration. Comparison between PAK4 wild type and KO anti-PD-1 treated tumors revealed how PAK4 deletion sensitizes tumors to ICB from a transcriptomic perspective. In addition, we validated genetically and pharmacologically that inhibition of PAK4 kinase activity is sufficient to improve antitumor efficacy of anti-PD-1 blockade in multiple melanoma mouse models. Therefore, this study provides novel insights into the mechanism of action of PAK4 inhibition and provides the foundation for a new treatment strategy that aims to overcome resistance to PD-1 blockade by combining anti-PD-1 with a small-molecule PAK4 kinase inhibitor.PAK4 inhibition can sensitize tumors to immune checkpoint blockade (ICB) therapy; however, the underlying mechanisms remain unclear. We report that PAK4 inhibition reverses immune cell exclusion by increasing the infiltration of CD8 T cells and CD103Our findings provide new insights into PAK4 inhibition mechanism of action as well as the scientific foundation for specifically blocking PAK4 kinase activity using a novel and specific PAK4 kinase inhibitor to overcome resistance to PD-1 blockade. Cancer immunotherapy has changed the treatment landscape of multiple tumor types, including advanced melanoma . PD-1/PDPAK4 overexpression is associated with tumorigenesis in several tumor types including breast, pancreatic, bladder, ovarian cancer, and melanoma -T immunotherapy in glioblastoma (PAK4 is a member of the group II p21-activated kinases (PAK) family and functions as a central player in the reorganization of the cytoskeleton . PAK4 ismelanoma , and con therapy . In addiblastoma . However+ dendritic cell (DC) infiltration, an important subset of dendritic cells that excel at cross-presenting tumor antigens and priming T cells. We also describe how lack of PAK4 expression alters the tumor microenvironment and sensitizes murine melanoma to anti-PD-1 therapy from a transcriptomic perspective. Finally, we demonstrate that the kinase activity of PAK4 is responsible for the improved response and show that pharmacologic inhibition of PAK4 activity with a specific PAK4 kinase inhibitor improves the efficacy of anti-PD-1 immunotherapy. Taken together, these findings provide the rationale for a novel treatment strategy to overcome PD-1 blockade resistance by administering a combination of anti-PD-1 in combination with a specific PAK4 kinase inhibitor.Here, we show how PAK4 inhibition increases not only T-cell infiltration, but also CD1032. All cells were maintained and confirmed Mycoplasma negative using MycoAlert Mycoplasma Detection Kit (Lonza). An early passage stock for each cell line was thawed before experiments and only used for in vitro and in vivo assays within passages 3 and 13 after thawing. To generate a B16 PAK4 KD cell line, we took advantage of the previously generated lentiviral vector with the PAK4 open reading frame (ORF) and performed site directed mutagenesis with the following primers containing the mutation to change AAG codon to ATG (K352M): 5-GGCAAACTGGTGGCCGTCATGAAGATGGACTTGCGCAAGC-3 and 5-GCTTGCGCAAGTCCATCTTCATGACGGCCACCAGTTTGCC-3. After cloning and vector amplification on Stbl3, 293T cells were used for lentiviral particle generation and B16 PAK4 KO cells were transduced at 20% confluency. A total of 24 hours after transduction, media was changed and cells were expanded and sorted on the basis of Thy1.1 expression. PAK4 expression and loss of kinase activity was validated by Western blot and Topflash assay.Murine B16 cells were obtained from the ATCC and YUMM2.1 cells from Bosenberg's lab. Cell lines were authenticated with IDEXX BioAnalytics. YUMM2.1, B16 cells and cells derived from this parental cell line [B16 PAK4 knockout (KO) and B16 PAK4 kinase dead (KD)] were maintained in DMEM, supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 5% COin vivo effects of genetically blocking the kinase activity of PAK4 effect on PD-1 blockade efficacy, we subcutaneously injected 0.3 × 106 B16 PAK4 KD and B16 PAK4 rescue into the flanks of C57BL/6 syngeneic mice. A total of 96 hours after tumor injection, mice were randomly assigned into the different groups. Anti PD-1  treatment was injected intraperitoneally three times per week at 300 μg per dose. We followed the same protocol to test the impact of pharmacologic inhibition of PAK4 kinase activity. For these experiments, we also used the YUMM2.1 melanoma cell line, which was injected subcutaneously (1 × 106 cells) into the flanks of C57BL/6 syngeneic mice. We established four treatment groups of B16 wild type (WT) CRISPR control (CC), or YUMM2.1 melanoma cells: (i) vehicle, (ii) anti-PD-1, (iii) PAK4 inhibitor (A0317859), and (iv) combination. The PAK4 inhibitor was administered by oral gavage at 300 mg/kg daily and anti-PD-1 was injected intraperitoneally three times per week at 300 μg per dose. In all in vivo studies, tumor progression was monitored three times per week by measuring two perpendicular dimensions with a caliper.All mouse studies were approved by the University of California, Los Angeles (UCLA) Animal Research Committee (protocol #2004-159-23). C57BL/6 mice were bred and kept under defined-flora pathogen-free conditions at the Association for Assessment and Accreditation of Laboratory Animal Care–approved animal facility of the Division of Experimental Radiation Oncology, UCLA. To study the To characterize and quantify the DC population, we collected mouse tumor samples from B16 WT CC or PAK4 KO melanoma cells treated with anti-PD-1 or vehicle at two different timepoints, day 6 and day 10. As described previously , tumor sin vitro samples (12 KO and 6 WT), using the RNeasy mini kit (Qiagen). These samples also included some that have been previously stimulated with either TNF at 100 ng/mL for 6 hours (Peprotech), IFNγ at 100 UI/mL for 6 hours (Peprotech) or Wnt-3a at 200 ng/mL for 8 hours (R&D Systems). We also isolated RNA from in vivo B16 WT CC and KO tumors as described previously . Antigen retrieval was performed in a high pH buffer using PT Link  at 95°C for 20 minutes. IHC was carried out using a DAKO Autostainer Link 48 platform . Briefly, slides were blocked for endogenous peroxidases and subsequently stained using the following primary antibodies: rabbit anti-mouse CD8 (D4W27 at 1:200), Rabbit anti-mouse CD45 , and Rabbit anti-mouse CD31 antibodies (Abcam EPR17259 at 1:500) in Da Vinci diluent . MACH2 Rabbit AP polymer  was used to detect primary antibodies, followed by detection using Enzo Red chromogen (Enzo Life Sciences). Slides were then counterstained with Tacha's Hematoxylin , dehydrated, and coverslipped using the DAKO Coverstainer. Once dried, slides were scanned using a 3D Histech Pannoramic MIDI II Scanner (3DHISTECH), then image/spatial analyses were performed using HALO software (Indica Labs).Protein levels and S675-phosphorylation of β-catenin were evaluated by Western blot analysis as described previously  using thCXCL10 expression was measured by RT-PCR following the manufacturer's protocol for the Power SYBR Green RNA-to-CT 1-Step Kit (Applied Biosystems) and using the following FW and RV primers: 5-AATCATCCCTGCGAGCCTAT-3 and 5-TTTTTGGCTAAACGCTTTCAT-3.CCL21 protein expression was measured with the mouse 6-Ckine (CCL21A) ELISA kit (Thermo Fisher Scientific) following the manufacturer's protocol.Mouse MHC class I and II were analyzed by flow cytometry with the following antibodies: MHC class I (H-2Kb) mAb (AF6-88.5.5.3), APC, eBioscience and MCH Class II (I-A/I-E) mAb (M5/114.15.2), APC, eBioscience.+ DC were performed using an unpaired t test with Welch correction. As described previously  were obtained by applying the Benjamini–Hochberg method. For in vivo studies, statistical significance and correction for multiple comparisons was calculated using the Holm-Sidak method. For in vivo studies with YUMM2.1 cells, we also performed a linear mixed-effects model analysis for tumor volume ran with terms for group, time and group*time interaction with a random mouse effect. Slopes were estimated for each group and pairwise contrasts were estimated using the model. Models were run using SAS V9.4 (SAS Institute) and P values <0.05/6, or 0.008 Bonferroni-adjusted) were considered statistically significant. When comparing the expression of a gene or signature between only two groups, we used a two-tailed unpaired t test. If multiple groups were tested, then we used a one-way ANOVA and corrected for multiple comparisons using statistical hypothesis testing with the Tukey test. Differences were considered statistically significant if P < 0.05.GraphPad Prism7 , R software (v3.5.1), and FlowJo Version 10.7.1 was used for graphic representation and statistical analysis. Comparisons of CD103eviously , differehttps://www.ncbi.nlm.nih.gov/bioproject/).The data generated in this study are available within the article and its Supplementary Data. The sequence data generated in this study has been submitted to the NCBI BioProject database PRJNA876309  had less than 5% CD103+ DC infiltration. In line with the increase in DC infiltration, we also observed a significant increase in the percentage of CD45+ CD8+ cells in the PAK4 KO group compared with WT tumors . Of note, we did not find any differences in CCL21 secretion between B16 WT CC and PAK4 KO cell lines in vitro , which included cultures that were treated with either IFNγ, TNF, or Wnt3a. PCA showed that the main source of variance is due to lost PAK4 expression , and antigen presentation  on a total of 18 in vitro analysis  and day 10  so we could investigate the progression of transcriptomic changes over time. DGE analysis of day 6 tumors showed that anti-PD-1 treatment had no impact on either WT nor PAK4 KO tumors yet, as the mice had only have received one dose at this timepoint . Therefoanalysis . B16 PAKg others . In addianalysis .2FC > 2 or < −2 and FDR < 0.05; PAK4 expression facilitates changes in the tumor microenvironment, which become more evident when given anti-PD-1 therapy, to sensitize melanoma B16 tumors to anti-PD-1 treatment , the cadherin/protocadherin gene family (n = 11), the integrin gene family (n = 8), and the adam gene family (n = 9) among others  on day 10, to evaluate the protein expression of CD8 and CD31, which is required for leukocyte transendothelial migration. We observed that PAK4 KO tumors treated with anti-PD-1 presented higher levels of both, CD31 (WT: 4% and KO: 10%) and CD8 expression  at position 352 changed to a methionine  than anti-PD-1 alone (18.3) but it was not statistically significant -mediated immunosuppression . Ideallye models . However+ DCs. We focused on this particular subset of DCs because they have been previously associated with antitumor immunity in melanoma  and with the PAK4 kinase inhibitor, and could potentially affect the tumor architecture and impact its immunogenicity, as it has been observed in other targets , combining an anti-PD-1 antibody with a dual PAK4 and NAMPT inhibitor, KPT-9274, which decreases whole PAK4 protein expression. Our work lays the foundation for the translation of a novel, unique and specific PAK4 kinase inhibitor that could be used in combination with PD-1 blockade immunotherapy.In summary, in this study we showed how PAK4 inhibition remodels the tumor microenvironment, enabling the infiltration of key immune cell subtypes and changing the expression of genes involved in the tumor architecture. In addition, we established that blocking PAK4 kinase function is sufficient to overcome PD-1 blockade resistance Supplementary Figure 1Supplementary Figure 1: Impact of PAK4 deletion on the expression of chemokines.Click here for additional data file.Supplementary Figure 2Supplementary Figure 2: PAK4 deletion does not affect CCL21 secretion.Click here for additional data file.Supplementary Figure 3Supplementary Figure 3: No difference in signalling through different stimuli between PAK4 KO and WT cells.Click here for additional data file.Supplementary Figure 4Supplementary Figure 4: PAK4 deletion does not affect MHC-I and II surface expression.Click here for additional data file.Supplementary Figure 5Supplementary Figure 5: PAK4 deletion increases CXCL10 expression in vivo.Click here for additional data file.Supplementary Figure 6Supplementary Figure 6: Spatial colocalization of CD8 and CD31 positive cells.Click here for additional data file.Supplementary Figure 7Supplementary Figure 7: PAK4 protein expression levels in the different cell lines.Click here for additional data file.Supplementary Figure 8Supplementary Figure 8: PAK4 KO in human melanoma cells impairs B-catenin/WNT signalling.Click here for additional data file.Supplementary Figure 9Supplementary Figure 9: PAK4 inhibitor, A0317859, also synergizes with PD-1 blockade in YUMM2.1 melanoma cells.Click here for additional data file.Supplementary Figure 10Supplementary Figure 10: PAK4 kinase inhibition modulates WNT signalling in vivo.Click here for additional data file.Supplementary Table 1Supplementary Table 1: Panel of immune markers for dendritic cell characterization.Click here for additional data file.Supplementary Table 2Supplemental Table 2: Differential gene expression analysis between B16 KO and WT cells in vitro. Showing genes with q < 0.05 and log2FC > 1 or < -1.Click here for additional data file.Supplementary Table 3Supplemental Table 3: Differential gene expression analysis between B16 KO and WT tumors in vivo at day 6. Showing genes with q < 0.05 and log2FC > 1 or < -1.Click here for additional data file.Supplementary Table 4Supplemental Table 4: Differential gene expression analysis between B16 PAK4 KO anti-PD-1 vs PAK4 KO isotype tumors in vivo at day 10. Showing genes with q < 0.05 and log2FC > 2 or < -2.Click here for additional data file.Supplementary Table 5Supplemental Table 5: Differential gene expression analysis between B16 PAK4 KO anti-PD-1 vs WT anti-PD-1 tumors in vivo at day 10. Showing genes with q < 0.05 and log2FC > 1 or < -1.Click here for additional data file.Supplementary Table 6Supplemental Table 6: List of extracellular matrix genes enriched in PAK4 KO anti-PD-1 treated samples.Click here for additional data file.


S. cerevisiae model to decipher the cytotoxic effect of fludioxonil. Through genome wide transposon mutagenesis, we have identified Bem2, a Rho GTPase activating protein, which is involved in this process. The deletion of BEM2 resulted in fludioxonil resistance. Our results showed that both the GAP and morphogenesis checkpoint activities of Bem2 were important for this. We also provided the genetic evidence that the role of Bem2 in the cell wall integrity (CWI) pathway and cell cycle regulation could contribute to the fludioxonil resistance phenotype.Fludioxonil belongs to the phenylpyrrole group of fungicides with a broad antifungal spectrum that has been widely used in agricultural practices for the past thirty years. Although fludioxonil is known to exert its fungicidal action through group III hybrid histidine kinases, the downstream effector of its cytotoxicity is poorly understood. In this study, we utilized a  S. cerevisiae exhibits a natural resistance towards phenylpyrrole classes of fungicides. However, the heterologous expression of HHK3 orthologs from Magnaporthe grisea, Alternaria brassicicola, Candida albicans, Candida lusitaniae, or Debaryomyces hansenii confers a sensitivity towards these drugs to S. cerevisiae cells that indicates that the fungicidal effect of these compounds is mediated through HHK3 orthologs [DRK1, a HHK3 ortholog from Blastomyces dermatitidis, indicate that fludioxonil does not bind to Drk1 [Fludioxonil belongs to the phenylpyrrole group of fungicides with a broad antifungal spectrum. It is used to control a variety of plant-pathogenic fungi ,2,3,4. Prthologs ,12,13,14S. cerevisiae model and found Bem2, a known Rho GAP (GTPase activating proteins), to be involved in the antifungal action of fludioxonil. The deletion of BEM2 resulted in fludioxonil resistance. We provide the genetic evidence that the role of Bem2 in the cell wall integrity (CWI) pathway and cell cycle regulation could contribute to the fludioxonil resistance phenotype.In this study, we, therefore, carried out a transposon-based approach to identify the signaling pathways or components controlling the fludioxonil sensitivity using S. cerevisiae strains BY4742 or BY4741 background. YPD  and SD minimal medium (2% glucose and 0.67% yeast nitrogen base without amino acids) (Difco) with only required supplements were used for growing S. cerevisiae strains. Strains used in this study are listed in S. cerevisiae strains with different plasmids was done by lithium acetate method [Different deletion mutants used in this study were either procured from EUROSCARF or made in e method . Plasmidhttp://ygac.med.yale.edu/mtn/reagent/avail_reagents/lacZLEU2_lib_p.stm accessed on 20 August 2010). Plasmid DNA from the pools of the library was digested with NotI and the linear DNA corresponding to the 8 kb region was purified and transformed into S. cerevisiae strain BY4742 harbouring pClNik1 plasmid using lithium acetate protocol. Transformants were selected on SD-Leu medium. After 24 h of incubation, the plates were replica plated on SD media containing fludioxonil (50 µg/mL) and incubated further for 2–3 days at 30 °C. Colonies that appeared on these plates were designated as fludioxonil resistant mutants. To identify the dominant resistant mutant in this pool, each resistant mutant was crossed with S. cerevisiae strain BY4741/pClNik1. The resulting diploids that grew on SD media containing fludioxonil (50 µg/mL) were considered as dominant resistant mutant. To rule out whether the appearance of fludioxonil resistance was due to the transposon insertion in HOG1 or PBS2, osmo-sensitivity of all the resistant mutants was checked on plates containing 0.7 M NaCl. For transposon insertion in SSK1 gene each recessive fludioxonil-resistant mutant was crossed with Δssk1 (YPDahl143) harbouring pClNik1 and fludioxonil sensitivity of the resulting diploids was checked on SD media containing fludioxonil (50 µg/mL). Transposon mutants corresponding to those diploids still resistant to fludioxonil were discarded. To identify the transposon insertion site, genomic DNA immediately adjacent to the inserted lacZ sequences was obtained by a plasmid rescue method using pRSQ–URA3 plasmid (GenBank U64694). Positive plasmids were sequenced using primer LacZ R having the binding site within the lacZ sequences of the terminal repeats. The nucleotide sequences were compared with the GenBank database using the BLASTN program.We obtained mTn3-LacZ/LEU2 mutagenized library  from theBEM2 along with the upstream promoter region (7.1 kb) was amplified in two parts using BY4742 genomic DNA as a template. The first PCR fragment (~4.3 kb) was amplified by primers Bem2 PF and Bem2 Kas R, and the second fragment (~2.8 kb) was amplified by primers Bem2 Kas F and Bem2 ORF R  or LEU2 marker (2.5 kb) genes was amplified from plasmid pUG72 or pUG73 [To construct double deletion mutants, or pUG73 , respectMPK1 ORF (1.4 kb) along with its native promoter (555 bp) and terminator region (130 bp) using MPK1 F and MPK1 R as forward and reverse primers from S. cerevisiae genomic DNA. PCR product and vector pRS313 digested with BamH1 and Xho1 were ligated and transformed in E. coli strain DH10B2. Transformants were screened by restriction digestion and positive clone pMPK1-313 was confirmed by automated DNA sequencing.We amplified full-length Three mutants of Mpk1, ATP binding site mutant (K54R), substrate binding (SB) site mutant (R196A) and D-motif binding (DB) site mutant (K83A) were made by site-directed mutagenesis using overlap extension PCR. First PCR was done to amplify 717 bp for K54R, 1.1 kb for R196A, 805 bp for K83A using MPK1 F/K54R R, MPK1 F/R196A R, and MPK1 F/K83A R primer sets, respectively. Second PCR was done to amplify 1.4 kb for K54R, 997 bp for R196A, 1.3 kb for K83A using K54R F/MPK1 R, R196A F/MPK1 R, and K83A F/MPK1 R primer sets, respectively. All the fragments were gel purified and used as the template for final overlap extension PCR to amplify 2.1 kb PCR fragments of all mutants using forward MPK1 F and reverse MPK1 R primers. All mutant constructs were then cloned in pRS313 at BamH1 and Xho1 site as described above to get pMPK1-83, pMPK1-54, and pMPK1-196. Positive clones were confirmed by automated DNA sequencing.Δmpk1) strain along with p426ClNik1. The growth pattern of these strains was checked on SD plate (-His-Ura) containing 25 µg/mL fludioxonil. For liquid media growth assay, Y00993 (Δmpk1) strain expressing MPK1, and its mutant constructs were grown overnight in SD media (-His-Ura) and then re-inoculated in the fresh media at an initial OD600 of 0.1. Cells were grown for four hours at 28 °C with shaking to attain log phase. The cultures were then inoculated in the 96 well plates (98 µL/well). All wells were then dispensed with serial dilutions of fludioxonil (2 µL/well). The final concentration of fludioxonil in the well was from 0–24 µg/mL. OD600 of the culture was measured at 0 h and after 24 h of incubation at 28 °C.pMPK1-313 (MPK1) and its mutant constructs  were transformed in Y00993 , Y04225 (Δsac7), and Y03937 (Δlrg1). The expression of ClNik1 in these mutant strains conferred sensitivity to fludioxonil that was similar to that of the wild type strain  that acts as GAP for various GTPases Rho1, Rho2, Rho4, and Cdc42 ,24,25,26e strain A. In S. il plate B. Thus, BEM2, R2003A and Δ2-1749. The R2003A mutant has a point mutation in the GAP domain, which completely abolishes the GAP activity of Bem2. In contrast, the Δ2-1749 mutant has a deletion in the N terminal region of Bem2, which is essential for its role in the morphogenesis checkpoint [BEM2 in the Δbem2/p426ClNik1 strain completely reversed its resistant phenotype. However, R2003A and Δ2-1749 mutants also remained resistant to fludioxonil  observed in the absence of methylglyoxal. At a lower concentration (5 mM) of methylglyoxal the relative growth of Δbem2 and parental BY4741 was quite similar. However, at a higher concentration of methylglyoxal (25 mM), Δbem2 showed significantly better growth than the parental strain BY4741  and Rlm1 ,32,33,34gth MPK1 B. To betf action C. Thus, BEM2 as a potential regulator of fludioxonil toxicity. Bem2 encodes a Rho GTPase activating protein that regulates GTPases to maintain morphogenesis and cell polarity in S. cerevisiae [BEM2 deletion leads to various cellular defects, which includes an actin polarity defect, a septin assembly defect, a cytokinesis defect, hyper-activation of CWI pathway, enlarged vacuoles, and a morphogenesis checkpoint defect [S. cerevisiae. Similarly, Cdc42 is also regulated by other GAP proteins, e.g., Rga1, Rga2, and Bem3. Our results showed that the fludioxonil sensitivity of the cell was not affected by the deletion of anyone of these GAPs  assay of fludioxonil indicate that the CWI pathway contributes to the resistance to the drug [C. neoformans revealed that the deletion of MPK1 leads to fludioxonil hypersensitivity [S. cerevisiae strain expressing ClNik1 [Δbem2 strains (BEM2 deletion strain. The role of Mpk1 in fludioxonil sensitivity was further explored by creating a set of mutations: K83A, R196A, and K54R, which abolished the binding of proteins at the DB site, the SB site, and ATP binding, respectively. The SB site mutant R196A, which was defective in transcription through Rlm1, showed the two-fold decrease in growth compared to MPK1, suggesting the role of Rlm1-driven transcription of cell wall genes in providing resistance to the cell on fludioxonil treatment (The hypersensitivity of the drug . The CWIsitivity , and simg ClNik1 ,42. We h strains A. Bem2 ireatment C. All th


G93A) mouse model of ALS, the double transgenic mouse (SynCav1 TG/hSOD1G93A) exhibited better motor function and longer survival. The objective of the current study was to test whether neuron-targeted Cav-1 upregulation in the spinal cord using AAV9-SynCav1 could improve motor function and extend longevity in mutant humanized mouse and rat (hSOD1G93A) models of familial (F)ALS.Elevating neuroprotective proteins using adeno-associated virus (AAV)-mediated gene delivery shows great promise in combating devastating neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is one such disease resulting from loss of upper and lower motor neurons (MNs) with 90-95% of cases sporadic (SALS) in nature. Due to the unknown etiology of SALS, interventions that afford neuronal protection and preservation are urgently needed. Caveolin-1 (Cav-1), a membrane/lipid rafts (MLRs) scaffolding and neuroprotective protein, and MLR-associated signaling components are decreased in degenerating neurons in postmortem human brains. We previously showed that, when crossing our SynCav1 transgenic mouse (TG) with the mutant human superoxide dismutase 1 (hSOD1Methods: Motor function was assessed by voluntary running wheel (RW) in mice and forelimb grip strength (GS) and motor evoked potentials (MEP) in rats. Immunofluorescence (IF) microscopy for choline acetyltransferase (ChAT) was used to assess MN morphology. Neuromuscular junctions (NMJs) were measured by bungarotoxin-a (Btx-a) and synaptophysin IF. Body weight (BW) was measured weekly, and the survival curve was determined by Kaplan-Meier analysis.Results: Following subpial gene delivery to the lumbar spinal cord, male and female hSOD1G93A mice treated with SynCav1 exhibited delayed disease onset, greater running-wheel performance, preserved spinal alpha-motor neuron morphology and NMJ integrity, and 10% increased longevity, independent of affecting expression of the mutant hSOD1G93A protein. Cervical subpial SynCav1 delivery to hSOD1G93A rats preserved forelimb GS and MEPs in the brachial and gastrocnemius muscles.Conclusion: In summary, subpial delivery of SynCav1 protects and preserves spinal motor neurons, and extends longevity in a familial mouse model of ALS without reducing the toxic monogenic component. Furthermore, subpial SynCav1 delivery preserved neuromuscular function in a rat model of FALS. The latter findings strongly indicate the therapeutic applicability of SynCav1 to treat ALS attributed to monogenic (FALS) and potentially in sporadic cases . ALS is a progressive, paralytic disease due to loss of motor neurons (MN) in the brain and spinal cord. ALS signs include diffuse muscle weakness, fasciculations (spontaneous muscle twitching), hyperreflexia (upper), and paralysis (lower), and ultimately reducing life expectancy to 2-5 years Amyotrophic lateral sclerosis (ALS) derives its name from denervational atrophy of the muscles in the tongue, oropharynx, and limbs (SOD1) For FALS, genetic interventions focused on knocking down key genes such as mutant form of super oxide dismutase 1 (SynCav1) promotes hippocampal synaptic and neuroplasticity G93A mouse model of ALS, we observed preserved body weight (BW), longer survival, better running wheel (RW) performance, preserved spinal cord α  motor neurons, and more MLR-associated neurotrophin receptors (NTRs) in the spinal cord. In terms of human clinical relevance, a seminal finding from the United Kingdom (University of Sheffield) revealed decreased Cav-1 protein expression and disrupted MLRs in samples from sporadic human ALS cases due to mutations in the Cav-1 DNA enhancer region, suggestive of a novel ALS-associated risk variant Previously we showed that neuron-targeting of the membrane/lipid raft (MLR) protein caveolin-1 , and extend survival, independent of targeting mutant hSOD1G93A protein. We now show for the first time that a one-time subpial delivery of AAV9-SynCav1 to hSOD1G93A mice at lumbar section significantly extends longevity and preserves BW in both male and female mice, preserves lumbar motor neuron morphology and NMJ morphology in the lower limb muscle, and maintains better RW performance independent of affecting expression of mutant hSODG93Aprotein. This study further extends the neuroprotective and therapeutic potential of SynCav1 gene therapy to another distinct neurodegenerative condition , irrespective of disease etiology, and thus SynCav1 has the potential to treat a variety of neurodegenerative conditions attributed to either familial or sporadic forms. Furthermore, SynCav1 gene therapy may work synergistically in combination with other interventions  that specifically target monogenic familial forms of ALS and other neurodegenerative conditions.Over the past few years, the predominant approach to treat ALS is to target known monogenic causes of FALS G93A)-transgenic (Tg) and C57BL/6 mice were purchased from Jackson Laboratory  and bred in-house. Wild type (WT) littermates were used as control. All animal protocols were approved by the Veterans Administration San Diego Healthcare System Institutional Animal Care and Use Committee (#A20-030). Mice were reared (3-5/cage) with free access to food and water. WT, and hSOD1G93A mice were allocated to 4 groups randomly: WT, hSOD1G93A naïve (no surgery), hSOD1G93A + control , and hSOD1G93A + SynCav1 to receive subpial lumbar vector injections. Spinal cord tissue was processed for biochemistry, histopathology, and immunofluorescence at the indicated time points.Human mutant superoxide dismutase-1 (hSOD1G93A) exhibit an ALS-like neurodegenerative phenotype characterized by diminished body weight from muscle atrophy, weakness/paralysis, fasciculations, and early death, along with reductions in functional cortical alpha motor neurons and ventral horn alpha motor neurons that can be observed histologically G93A transgenic male rats  were bred with WT female rats to produce WT and hSOD1G93A positive male rats.Sprague-Dawley rats were treated in compliance with the Guide for the Care and Use of Laboratory Animals  in the Marsala lab and approved by UCSD. Transgenic rats expressing ALS-linked human SOD1 mutant protein . PCR was performed for the hSOD1 genes by using the following protocol: denaturation at 94 °C for 2 min, followed by 10 cycles at 94 °C for 20 s, 65 °C for 15 s (- 0.5 per cycle decrease), and 68 °C for 10 s, then followed by 28 cycles at 94 °C for 15 s, 60 °C for 15 s, 72 °C for 10 s, then 72 °C for 2 min and hold at 10 °C. All primers were purchased from Integrated DNA Technologies . hSOD1G93A-positive mice were confirmed using the following primers: primer 1, hSOD1 Forward [CAT CAG CCC TAA TCC ATC TGA (oIMR0113)]; primer 2, hSOD1 Reverse [CGC GAC TAA CAA TCA AAG TGA (oIMR0114)]; primer 3, Internal control Forward [CTA GGC CAC AGA ATT GAA AGA TCT (oIMR7338)]; and primer 4, Internal control Forward [GTA GGT GGA AAT TCT AGC ATC ATC C (oIMR7339)]. These primers were used to detect a product size of 800 for mouse endogenous SOD1 and 600 base pair (bp) for human SOD1 as previously described G93A. PCR products were separated on a 1% agarose gel (35 min at 135 volts).hSOD1scAAV9-SynCav1, scAAV9-SynRFP, or scAAV9-SynNull vectors were produced by transient transfection of HEK293T cells with each vector plasmid, pRep2/Cap9 and pAd-Helper plasmid. Helper virus-free AAV vectors were produced by Polyethylenimine (PEI)-mediated transient co-transfection of HEK293T cells with three plasmids . Cells were collected at 72 hrs post-transfection and cell lysates were made by 3x freeze/thaw cycle. AAV vectors in the cell lysates were purified by combination of sucrose-cushion ultracentrifugation and anion-exchange column chromatography. Virus titers were measured by real-time Q-PCR to determine genome copy (g.c.) number in the vector preparations (g.c./ml) as a measure of AAV particles with full genome content. Final virus titers for all three vectors are ~2.0 x1013 g.c./ml.A self-complementary AAV (scAAV) construct expressing neuron specific synapsin (Syn) promoter with the Cav-1 cDNA was generated at the UCSD Viral Vector core as previously described Mouse subpial procedure: Lumbar (L1-L3) subpial vector injection in mice was performed as previously described Rat subpial procedure: Wild type (WT) or hSOD1G93A Sprague Dawley rats were anesthetized by isoflurane with 2% in room air and placed into a Lab Standard Stereotaxic frame . Rats were elevated 3 cm by placing on a plastic box. A homeothermic heating blanket which was set at 37 °C with feedback from a rectal thermometer  was used to maintain body temperature of rats during the procedure. Rats were then placed in Spine Adaptors  and a C4-5 laminectomy was performed to expose the dorsal surface of spinal segment C4-5 using an air-powered dental drill and binocular microscope. The dura and pia mater were carefully incised using a 27- G needle and a polyethylene tubing catheter, of which its size was 0.008 ID x 0.02 OD,  was inserted into the subpia-space. Injections were made using 100-μL Hamilton Gas Tight syringe . Mice received a total of 10 μl to the lumbar spinal cord and rats received a total of 20 μl of AAV9-SynCav1 (~2.0 x 1013 genome copies (g.c.)/ml) vector to the cervical spinal cord by subpial delivery  using a 50 μl Hamilton syringe and digital infusion pump . After vector delivery, the muscle and skin were closed using 7/0 Prolene and suture clips. Before recovery from anesthesia, the animals received subcutaneous fluids, antibiotics (cefazolin sodium 50 mg/kg) and the initial dose of pain medication (slow release Buprenex 0.05 mg/kg). Control group received laminectomy and subpial injection of or equal volume of either AAV9-SynNull or sterilized saline.All surgical procedures were done under sterile conditions and approved by the local IACUCs. Disease onset and late disease were retrospectively defined as the first sign of weight loss from peak body weight and 10% weight decline from peak body weight, respectively At 8 and 16-week-age, mice were housed individually in plastic cages measuring 40 x 40 x 35 cm (length x width x height) with a 11.5 cm diameter running wheel mounted on the cage side . The mice had free access to food and water ad libitum and were checked daily for health and wellness. The mice had open access to running wheels under conditions of a 12 h light/dark cycle (lights off from 6 AM to 6 PM). The duration of each experiment was 36 h including 2 dark cycles. The mice will be placed in the cages at least 24h prior to data collection. A sensory beam, connected to a desktop computer via a software , was used to record wheel rotations. Running velocity (m/min), and total distance (m) were used for analysis.Rats were anesthetized by intramuscular injection of ketamine (100 mg/kg) for motor-evoked potentials (MEP) recordings in the branchial or gastrocnemius muscles. Two 30-G stimulating electrodes were placed subcutaneously overlying the left and right motor cortex. In all rats, MEP of upper forelimb  and lower limb (gastrocnemius) were recorded from the muscles elicited by transcranial electrical stimulation with a pulse duration of 200 μs at 15 mA. Recording electrodes were connected to an active head stage , and the recorded signal is amplified using DP-311 differential amplifier . The amplified signal was acquired by the PowerLab 8/30 data acquisition system  at sampling frequency of 20 kHz, digitized and stored using LabChart 7  in PC for analysis.Cohorts of age-matched transgenic rats and littermate controls were tested for loss of forelimb grip strength by using a grip strength meter  at 8 wk  and weekly from 10 to 20 wk. Rats were held in front of a horizontal bar, such that only the forelimb paws were able to grasp the bar. The rats were gently pulled back with steady force until both paws released the bar. Each rat was given 6 attempts and the mean grip strength was recorded for analysis.Motor neuron morphology and Cav-1 target validation was assessed by IF. Specifically, mice were perfused with saline and the spinal cord was dissected followed by 48 hrs post-fix in z-fix  and dehydration in 30% sucrose. 30 µm thickness coronal spinal cord section was then incubated with Alexa Fluor 488 conjugated anti-choline acetyltransferase (ChAT)  and Alexa Fluor 594 conjugated caveolin-1 antibody (7C8)  for overnight at 4 °C. Nuclei were counterstained with antifade DAPI mounting medium . A minimum of three coronal spinal cord sections per mouse were used for quantitation of motor neuron.For NMJ analysis, 30 µm thickness horizontal gastrocnemius muscle section was stained with 488-conjugated bungarotoxin-a (Btx-a)  to label post-synaptic structure and synaptophysin  to label pre-synaptic vesicles followed by 1 h incubation of secondary  at room temperature. Occupancy of synaptophysin labeled presynaptic structure in Btx-a labeled postsynaptic was analyzed to assess NMJ morphology Thoracic (T12) to lumbar (L2) spinal cord tissue were homogenized at 4 ºC in 500 mM sodium carbonate (pH 11.0) (containing protease and phosphatase inhibitors) and then sonicated 3x for 15 s. Samples (0.5 mg/ml) were subjected to sucrose density gradient fractionation as previously described Sample size is indicated under each section of the Methods and in the figure legends. Before determining statistical significance, data were checked for normal distribution. Data were analyzed by Student's t test, one-way analysis of variance (ANOVA) or 2-way ANOVA followed by Fisher's LSD, Tukey's, Šídák's, or Bonferroni's multiple comparisons post hoc analyses as appropriate using Prism 7 software . The Kaplan-Meier survival curve was analyzed using a log-rank test. Mean and median survival were analyzed by an unpaired Student's t-test. Significance was assumed when P < 0.05. All experimental groups were blinded, and code was broken only for analysis. For correlation analysis, a simple linear regression with 95% confidence intervals was performed. G93A mice using different methods and parameters G93A mice, several weeks prior to the standard measurable onset of overt clinical symptoms in this mouse model AAV9-SynCav1 at week 8 via subpial delivery, a highly effective route of administration used to accomplish effective strong transgene expression in the mammalian spinal cord AAV9-SynRFP resulted in a strong expression of RFP in the lower thoracic up to lumbar (T11 to L5), with minimal expression detected in upper thoracic and cervical regions. Our previous study demonstrated that SynCav1+/ hSOD1G93A+ transgenic mice exhibit slowing of disease progression, we therefore initially performed a dose-dependent study for subpial AAV9-SynCav1 delivery to determine the optimal dose necessary to achieve Cav-1 protein expression in the spinal cord similar to that measured in SynCav1+ transgenic mice AAV9-SynCav1 to achieve 4-fold Cav-1 overexpression in the thoracic and lumbar spinal cord was 2 x 1011 total g.c. (10 μl of 2 x 1013 g.c./ml). This dose was used in all the following studies.Previous work from other groups have reported that the occurrence of symptomatic onset varies from 8 to 12 weeks (wk) AAV9-SynCav1 could provide therapeutic benefit to the hSOD1G93A mouse model of ALS. As shown in Figure G93A + control, hSOD1G93A + AAV9-SynCav1 mice showed significantly delayed BW loss in both sexes. As shown in Figure G93A + AAV9-SynCav1 mice was 18 wk for male  and female , an approximately 15% delay compared to the control group. In terms of disease progression, late disease onset  G93A + control mice, while AAV9-SynCav1-treated hSOD1G93A female mice exhibited delayed late disease onset until 23 wk (~11% BW decline); AAV9-SynCav1-treated hSOD1G93A male mice only exhibited ~4% BW decline at 22 wk (end of body weight record due to loss of multiple objects in the hSOD1G93A + control groups).We next assessed whether subpial delivery of G93A + control versus hSOD1G93A naïve, we observed a greater decline in BW in the sham surgery control group compared to naïve hSOD1G93A mice between 18-22 wk  and the surgery group  to remove the confounding effects from subpial surgery on any measurable efficacy afforded by SynCav1 gene delivery. As shown in Figure  AAV9-SynCav1 gene delivery significantly extended survival in male and female (10%) hSOD1G93A mice when compared to hSOD1G93A + control mice  vs hSOD1G93A + control (162 d), ***p = 0.0046, Kaplan-Meier; n = 11-18; Figure G93A + SynCav1 (181 d) vs hSOD1G93A + control (165 d); ****p < 0.0001, Kaplan-Meier; n = 12-15). These findings are direct evidence that SynCav1 significantly delays disease onset and extends longevity  in hSOD1G93A mice.In terms of survival, although subpial surgery had no effect on survival rates for hSOD1G93A mice  showed elevated B8H10 (mutant and WT human SOD protein) and decreased Cav-1 expression. IB of MLR fractions revealed that Cav-1 was predominantly localized to MLR fractions 4-5 . One limitation to IB assays is that when performed on whole spinal cord (or brain) tissue, one is incapable of distinguishing cell-specific protein changes . To better elucidate the decreased Cav-1 expression as measured by IB, we additionally performed IF on lumbar spinal cord tissue from 10 wk (early symptomatic) and 23 wk (end stage) using neuronal marker NeuN and Cav-1 , mice from all groups were trained for voluntary RW test. hSOD1SynCav1 delivery . Together, these results indicate that SynCav1 gene delivery affords significant neuroprotection in hSOD1G93A spinal cord MNs in both Sexes.We next assayed for lower motor neuron (MN) cell counts and morphology in the lumbar ventral horn region L3-L5) after subpial  after suSynCav1 delivery, we analyzed NMJ morphology in the gastrocnemius muscle, one of the larger lower limb muscles receiving synaptic innervation from L5-S1 spinal cord MN axons  compared to hSOD1G93A + controls, results that in line with the greater RW performance observed in the hSOD1G93A+ SynCav1 group.Finally, because we observed better motor function and preservation of MNs at lower lumbar level with s Figure A. At 19 AAV9-SynCav1 delivery in a larger animal model  could mitigate ALS-like disease onset. Transgenic negative or positive rats (G93A + control) or AAV9-SynCav1 hSOD1G93A + SynCav1) by cervical (C4/C5) subpial delivery at 2 m. At 4 m (2 m post vector delivery), IB and IF of spinal cord tissue confirmed strong Cav-1 expression in MN from SynCav1 administered rats (G93A+ SynCav1 rats exhibited significantly greater peak force (g) grip strength at 13, 14, and 15 wk compared to hSOD1G93A+ control rats . These data from hSOD1G93A rats treated with SynCav1 builds upon the findings already demonstrated in hSOD1G93A mice and provides further direct evidence of the therapeutic potential properties afforded by SynCav1 in a larger ALS animal model.We next tested whether subpial ive rats A receivered rats B-S2C. Werol rats B and shorol rats C in the rol rats D and lowrol rats E in the G93A mouse model of ALS. These results show that subpial delivery of AAV9-SynCav1 delays disease onset and early disease progression, preserves ventral horn spinal motor neurons in a familial mouse model of ALS, independent of reducing the toxic monogenic protein engineered to cause the ALS-like neuropathology. Delivering genes that augment neuroprotective proteins may mitigate spinal motor neurodegeneration, prolong survival in individuals afflicted with ALS. The present study showed AAV9-SynCav1 subpial delivery significantly delays disease onset and extends survival in both male and female hSOD1G93A mice, results that are comparable to previous studies that used ASOs to target mutant monogenic hSOD1 in hSOD1G93A rodents SynCav1 gene therapy proved efficacious in both sexes, independent of affecting expression of mutant hSOD1 protein. This latter finding suggests that SynCav1 possesses the therapeutic potential to combat other forms of FALS  SynCav1 can work synergistically with other biologics or small molecules designed to knockdown known monogenic links to FALS.The present study tested a neuroprotective, non-monogenic targeting gene delivery in the hSOD1G93A mice  AAV9-SynCav1 delivery in the small rodent limits broad CNS tissue AAV9 distribution and transduction and subsequent Cav-1 transgene expression. As shown in Figure SynCav1 predominantly distributed in the lumbar cord (~ 4-fold) and to a less degree in the thoracic cord (~ 2-fold), with no over-expression in the cervical cord after subpial delivery. Failure to target the cervical spinal cord G93A mice, the most common cause of death from ALS. However, even with this limitation of achieving cervical AAV9 spinal cord distribution/transduction and subsequent Cav-1 transgene expression, the therapeutic efficacy afforded by subpial SynCav1 gene delivery is comparable to approaches targeting the known monogenic cause of the mutant SOD1 neuropathology G93A mice, which demonstrated a similar extension of survival. A combined therapy consisting of SynCav1 with approaches targeting knockdown of monogenic causes of ALS may work in concert to treat ALS more effectively.A limitation of the current study is that the extended survival achieved by exogenous vector delivery is slightly lower  than that observed when crossing SynCav1 transgenic mice with hSOD1G93A) mice FUS) C9orf72) SynCav1 may in part be through the maintenance/restoration of functional axonal transport of cellular cargoes such as NTR signaling endosomes in vitro model systems consisting of human motor neurons co-cultured with human myotubes in specialized microfluidic chambers SynCav1.On a cellular level, Cav-1 regulates cellular endocytosis and vesicular trafficking AAV9-SynCav1 preserved NMJ integrity and improved running wheel performance in bothG93A hSOD1 male and female mice. Could SynCav1 be applied at this MN 'compensatory stage' to prevent or overcome the “dying back” mechanism and maintain functional NMJ synapses? As noted above, the use of specialized in vitro models consisting of human motor neurons forming functional NMJs with human myotubes could optimize the temporal window at which to deliver SynCav1 in the presence of otherwise toxic dipeptide species to overcome this “dying back” cellular event. Another limitation to the current study is the lack of more sensitive neuromuscular measurements of functional NMJ physiology in the upper or lower limbs  performed specifically in the hSOD1G93A mouse ALS model. However, using the larger hSOD1G93A rat ALS model, subpial cervical spinal cord SynCav1 delivery resulted in significantly better forelimb grip strength and MEPs (greater amplitude and decreased latency) in both upper limb brachial and lower limb gastrocnemius muscles, further evidence of SynCav1's efficacy using two different ALS model species.Preservation of NMJ morphology is essential for preserving motor function and life quality. NMJs are unique synapses that facilitate communication between MNs and the effector muscles. Due to their long axons, MNs are extremely susceptible to retraction during pathological conditions such as ALS, thus resulting in disrupted communication between the MN and effector muscles. Initially, the MNs try to compensate for the retraction by inducing axonal sprouting and collateral innervation. However, with disease progression, this compensation fails and results in final MN death, a neuropathological event known as the “dying back” mechanism SynCav1 at week 8 (pre-symptomatic stage) to the lumbar cord which exhibited similar folds increase as observed in the healthy wild type mice. However, vector dosing needed to achieve efficacious transgene expression levels may depend upon disease stages G93A mice, with no effect on disease onset or survival. A possible explanation is that the surgery may have exacerbated back muscle weakness, which in turn could have affected the ability to retrieve food located on top of the cage for late stage hSOD1G93A mice. This inability to retrieve food would result in a more rapid BW decline compared to naïve hSOD1G93A mice. The fact that we did not measure any difference in survival, MN morphology or MN count between hSOD1G93 control and hSOD1G93 naïve mice, indicate that the adverse effect from subpial surgery on BW is not through affecting MN survival directly. Nevertheless, since the lumbar subpial surgery involves an invasive laminectomy, which is difficult to perform successfully on older, severely symptomatic hSOD1G93A mice. Exploration of alternative routes of SynCav1 administration  are worthy of investigation.The current study delivered SynCav1 gene delivery to the lumbar spinal cord of hSOD1G93A mice prolongs survival and maintains body weight in both males and females, preserves alpha motor neurons in the ventral horn with a direct correlation to soma size and Cav-1 expression, and preserves NMJs in the lower limbs, all of which occurred independent of affecting expression of mutant hSOD1. These data suggest that SynCav1 might serve as a novel gene therapy for neurodegenerative conditions in ALS and other forms of CNS disease of unknown etiology. Further studies are needed to determine the effect of SynCav1 on disease progression when delivered at later stages of the disease.In summary, the present study demonstrates that subpial Supplementary figures.Click here for additional data file.S.W. guided the overall study design, writing the manuscript, IB and IF microscopy; T.I. performed subpial gene delivery, behavioral tests and IF microscopy; P.S. and D.W. assisted with animal breeding and behavior tests; A.S. performed all rat subpial vector delivery and motor tests; X.L, T.D., and W.L assisted with data analysis; J.A.B. and E.K. assisted with editing of the manuscript; A.M. generated AAV vectors ; D.M.R., H.H.P. and P.M.P. assisted with manuscript editing; T.T and M.M. provided subpial surgical guidance; B.P.H. guided the overall study design, scientific concept, edited the manuscript, and funded the project (VA SPiRE award 5I21RX003324).All animal protocols were approved by the Veterans Administration San Diego Healthcare System Institutional Animal Care and Use Committee (#A20-030).


In this study, we investigated the mechanistic interaction of PNPLA3 with lipid droplet (LD)-associated proteins of the perilipin family, which serve as gatekeepers for LD degradation. In a collective of 106 NASH, ASH and control liver samples, immunohistochemical analyses revealed increased ballooning, inflammation and fibrosis, as well as an accumulation of PNPLA3–perilipin 5 complexes on larger LDs in patients homo- and heterozygous for PNPLA3(I148M). Co-immunoprecipitation demonstrated an interaction of PNPLA3 with perilipin 5 and the key enzyme of lipolysis, adipose triglyceride lipase (ATGL). Localization studies in cell cultures and human liver showed colocalization of perilipin 5, ATGL and PNPLA3. Moreover, the lipolytic activity of ATGL was negatively regulated by PNPLA3 and perilipin 5, whereas perilipin 1 displaced PNPLA3 from the ATGL complex. Furthermore, ballooned hepatocytes, the hallmark of steatohepatitis, were positive for PNPLA3 and perilipins 2 and 5, but showed decreased perilipin 1 expression with respect to neighboured hepatocytes. In summary, PNPLA3- and ATGL-driven lipolysis is significantly regulated by perilipin 1 and 5 in steatohepatitis.The single nucleotide polymorphism I148M of the lipase  Fatty liver disease , the accumulation of lipid droplets (LDs) in hepatocytes, is a steadily increasing health risk affecting more than one-third of the population in the western world. Chronic hepatitis C, alcohol abuse and metabolic syndrome are the main causes of hepatocyte steatosis  . NAFLD iPNPLA3 , PNPLA3(I148M) [Pnpla3 knockout mice do not develop steatosis [The most common and strongest genetic risk factor for steatotic liver diseases is a common missense variant of 3I148M) ,7,8. To M ,7,8. T3(I148M) . Interesteatosis ,12; therteatosis . PNPLA3 aggravates SLD.2. Cell lines were tested for mycoplasma contamination on regular basis.HEK293T, Huh7, and HepG2 cells, obtained from ATCC, were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum  and 1% of 10,000 U/mL penicillin, and 10 mg/mL streptomycin (Sigma) at 37 °C and 5% COCryopreserved (snap-frozen) as well as formalin-fixed paraffin-embedded human tissue samples were obtained from the tissue biobank of the University Medical Center Mainz in accordance with the regulations of the tissue biobank and the ethics vote of Rhineland-Palatinate to BKS and HRW . The collective of cryopreserved tissues included 15 liver specimens from patients with colorectal liver metastases taken from a location distant from the metastasis.PNPLA3 genotypes 23× C/C, 17× C/G, and 7× G/G, respectively (study in accordance with the ethics committee of the University of Heidelberg (no: S280-2011).For immunohistochemistry, 59 formalin-fixed, paraffin-embedded liver biopsies of 23 patients with NASH, 19 patients with ASH, including 4 with alcoholic and metabolic (BASH) as well as 21 liver biopsies from control patients  triton X-100, pH = 8.0) supplemented with 1:100 Halt™ protease and phosphatase inhibitor , and lysed with the tip of a sonicator for 10 s. The homogenate was then centrifuged at 12,000× g for 5 min. Protein concentration was determined by the Bradford protein assay (Bio-Rad).After washing cultured cells (10 cm plates) with PBS, cells were pelleted at 1000 rpm, resuspended in 600 µL RIPA buffer  and then lysed with a Precellys 24 homogeniser 2 times at 6500 rpm for 20 s.The antibodies and conditions are described in the Transfection of HEK293T, Huh7, and HepG2 cells was performed as previously described ,33.Co-immunoprecipitation was performed as previously described .® 546 goat anti-mouse IgG (H + L), Alexa Fluor® 635 goat anti-rabbit IgG (H + L), Alexa Fluor® 635 goat anti-mouse IgG (H + L) or without (EGFP- and mRFP fusion proteins), the nuclear dye DAPI and the LD-dye BODIPYTM 493/503 in 1% FBS, 0.1% Triton X-100 in PBS at RT for 1 h. After a final washing step with 0.1% Triton X-100 in PBS, cells were mounted with MOWIOL and images were taken with a Leica SP8 confocal microscope using either a 40× 1.30 NA Oil CS2 HC Plan Apo or a 63× 1.40 NA Oil CS2 HC Plan Apo objective operating at 25 °C. Both objectives were operated with Type F immersion liquid (Leica Microsystems). Leica Application Suite X was used to acquire the images. LD-size was then analysed with ImageJ . For detailed instructions, please see the HEK293T, Huh7, and HepG2 cells were seeded on 13 mm (⌀) coverslips in 12-well plates, transfected the next day and fixed after an additional incubation period of 24 h or 48 h. After washing with PBS, cells were fixed for 10 min with 3.7% formaldehyde in PBS. Subsequently, cells were washed with PBS, permeabilized, and blocked with 10% FBS, 0.1% Triton X-100 in PBS at 37 °C. Cells were then incubated with the primary antibody in blocking solution at RT for 1 h. After a brief wash with 0.1% Triton X-100 in PBS, cells were either incubated with the secondary antibodies (Alexa FluorStained sections were digitised with a NanoZoomer 2.0 HT (Hamamatsu) at a magnification of 40× and the intensity of the staining was quantified using the digital image analysis software QuPath (v.0.1.2) . To deteImmunohistochemistry of formalin-fixed and paraffin-embedded liver biopsies was performed as previously described ,29. StaiTransmission electron microscopy of glutaraldehyde fixed liver biopsies was performed as previously described ,29. ImagHindIII test digest when a 6-well plate size was reached and finally genotyped by sequencing.HEK293T cells did not harbour the PNPLA3(I148M) missense variant on either of the two alleles. Therefore, a previously described protocol  was usedPNPLA3_gtp_for 5′-AGGCCTTGGTATGTTCCTGC-3′, PNPLA3_gtp_rev 5′-TCCACCTTCCAGGGGTAACA-3′ were used as primers for genotyping. The sequence of the ssODN was PNPLA3_ssODN_HindIII 5′-CTATAACTTCTCTCTCCTTTGCTTTCACAGGCCTTGGTATGTTCCTGCTTCATGCCTTTTTATAGCGGACTTATCCCTCCAAGCTTCAGAGGCGTGGTAAGTCGGCTTTCTCTGCTAGCG-3′Lipolysis was induced by a combination of 10 µM forskolin with 500 µM IBMX for 1–3 h and compared with the vehicle (DMSO). Steatosis was induced with 240 µM oleic acid BSA complex for up to one week and compared with BSA treatment alone.TM Solution (Invitrogen) was added, and lysis was performed with a Precellys 24 homogeniser twice for 20 sec at 6500 rpm. RNA was isolated following the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems™), diluted, and used for real-time PCR . For sequences of real-time primers, please see the Cryopreserved samples were collected in tissue homogenizing kit CKMix tubes (Precellys). A total of 1 mL TRI Reagentwww.graphpad.com. First, the data were tested for normal distribution using the Shapiro–Wilk normality test. If the data were normally distributed, a t-test was performed. If the standard deviations of the mean values of two groups differed significantly, Welch’s correction was also applied. If, on the other hand, the data were not normally distributed, the non-parametric Mann–Whitney test was used. p-values < 0.05 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****) were considered significant, whereas p-values > 0.05 not significant (n.s.).Data were visualized as bar diagrams or scatter diagrams, each bar representing mean ± standard deviation. The statistical analysis was performed with GraphPad Prism v.9.4.0  for Windows, GraphPad Software, San Diego, California USA, Since the PNPLA3(I148M) polymorphism is associated with steatohepatitis and increased fibrogenesis in ASH and NASH patients, we analysed the histology as well as the expression pattern of the LD-associated proteins of the perilipin-family in liver biopsies of patients with steatotic liver disease with respect to PNPLA3 status. Immunohistochemistry against perilipins and in part together with PNPLA3 was performed with a total of 106 liver biopsies. In total, 23 NASH, 19 ASH (including 4 BASH-cases), 9 HCV and 8 control patients  were anaPNPLA3 genotypes (We could thereby recapitulate a positive correlation of PNPLA3(I148M) with steatosis, inflammation, the amount of microgranulomas, ballooning and fibrogenesis . GeneralPNPLA3 genotype. These hepatocytes were also positive for PNPLA3 and perilipin 5; however, negative or only mildly positive for perilipin 1 -carriers showed a weak, and homozygous carriers an even stronger localization of PNPLA3 at LDs in zones 1 and 2 of the liver which were negative or only weakly positive for perilipin 1 A,C, wherilipin 1 A–C. In cilipin 1 A,D,E.PNPLA3-genotype and the amount of PNPLA3-protein  without reaching significance, PLIN2 and PLIN 3 mRNA levels were not significantly altered [In order to investigate the underlying molecular mechanisms of PNPLA3 action, we examined the expression of PNPLA3 and perilipins at the protein and transcript level. Western blot analysis of 15 non-neoplastic livers of patients resected for colorectal metastases showed no obvious association between -protein F. Perili-protein F. Real-tmorphism G,H. Inte altered . SimilarOur findings confirm our histological data and suggest a regulatory link between PNPLA3 and perilipin 1 and 5 on the protein and mRNA level, respectively, in human livers in situ.Since perilipin 5 physically interacts with ATGL, the protein sharing the highest similarity with PNPLA3 , we hypoRepetition of the in vitro studies with ATGL revealed interactions of ATGL with perilipin 5 and ABHD5 B as alreAs shown for constitutive perilipins, perilipin 1 localized to LDs when overexpressed in cells D. SurpriSince PNPLA3 and ATGL both localized to LDs , we carrAs perilipin 5 was the only perilipin shown to co-precipitate with PNPLA3 and ATGL A, the imATGL catalyzes the first step in the hydrolysis of TAGs ,39,40. OWhen compared to wild-type PNPLA3, PNPLA3(I148M) did not show an altered interaction with perilipin 5 and ATGL or an altered localization in vitro . PNPLA3. In contCo-expression of ATGL and PNPLA3 led to a significant increase in LD-size compared to cells expressing ATGL alone, which was even higher in the presence of PNPLA3(I148M) suggesting a negative effect on the lipolytic activity of ATGL.PNPLA3 allele, LDs were significantly larger (To further investigate the negative effect of PNPLA3(I148M) on lipolysis, PNPLA3(I148M) knockin cells were generated C. Unmodiy larger E. Followy larger . Lipolysy larger D,E.To investigate the impact of perilipin 1 on the stability of the PNPLA3-ATGL-complex, co-IP and subsequent immunoblot were carried out. Increasing amounts of perilipin 1 led to a dose-dependent release of up to 80% of PNPLA3 from the PNPLA3–ATGL complex A.Cells co-expressing perilipin 1 and PNPLA3 exhibited granular, cytoplasmic localization for PNPLA3, which was negative for ATGL, whereas LDs were positive for perilipin 1 but not PNPLA3 B. In addFinally, the overexpression of PNPLA3 or PNPLA3(I148M) was examined to determine the effects on the expression of perilipin 1–5, the lipases ATGL, HSL, and PNPLA3, as well as the co-activator of ATGL, ABHD5. Overexpression of either PNPLA3 or PNPLA3(I148M) strongly induced perilipin 1 irrespective of cell type C, pointiOur comprehensive in situ and in vitro data provide evidence for the pivotal role of deregulated basal lipolysis in the progression of steatosis to steatohepatitis. In this process, we could demonstrate that PNPLA3 competes with ATGL for perilipin 5 binding within lipolytic perilipin 5-ATGL complexes. We are the first to show that PNPLA3 physically interacts with perilipin 5 and ATGL at larger LDs found in carriers of the PNPLA3(I148M) polymorphism. In addition, we are the first to be able to localize PNPLA3 in a large collective of patients with steatohepatitis in human liver biopsies in situ with respect to different PNPLA3-polymorphisms. We demonstrated that PNPLA3 competitively displaces ATGL from LDs and thereby strongly reduced the lipolytic activity, thereby favouring LD-accumulation. Both perilipins 1 and 5 have been shown to be key regulators of lipolysis in different tissues. In adipocytes, perilipin 1 regulates lipolysis via HSL and ABHD5, whereas in cell types using oxidative energy supply, perilipin 5  controlsWe have demonstrated a possible pathophysiologic mechanism of how the polymorphism I148M of the lipase PNPLA3  mayOur findings demonstrate important implications for the mechanism by which PNPLA3(I148M) promotes progression to steatohepatitis in FLD, irrespective of the causative agent. We hypothesize that cell stress caused by supernutrition, alcohol or HCV together with a block of basal lipolysis caused by PNPLA3(148M) leads to hepatocyte ballooning and inflammation. In ballooned hepatocytes, the hallmark of steatohepatitis, perilipin 1 was markedly reduced, whereas perilipin 2 was strongly expressed but localized diffusely in the cytoplasm. Cytoplasmic localization of perilipin 2 in ballooned hepatocytes is a phenomenon that has not been observed in any other cell type under physiological conditions and can therefore be interpreted as a sign of a severely damaged/dying cell.Our study identified several key players that are dysregulated in steatohepatitis and therefore may have both a diagnostic and a therapeutic potential. Generally, the presence of ballooned hepatocytes in non-heavy drinkers indicates a heterozygous or homozygous PNPLA3(I148M)-mutation . The antWith this study involving human, mouse and cell culture, we could unravel the LD-associated protein perilipin 5 as a binding partner of PNPLA3 and ATGL in vitro and in situ, and the functional role of perilipin 5 ATGL/PNPLA3 complex formation during steatohepatitis, especially in the frequent PNPLA3 polymorphism I148M. Our data point towards an important role of deregulated lipolysis during the formation of ballooned cells in steatohepatitis, triggering the progression of inflammation and fibrosis.


The 313 bp paired-end sequence reads were classified into operational taxonomic units (OTUs), their species boundaries were delineated using the Generalised Mixed Yule Coalescent (GMYC) method, and taxonomic names of the GMYC species were assigned using basic local alignment search tool (BLAST) against International DNA Databases .Riverine metabarcoding data were obtained from the Takamigawa River, a tributary of the Kinokawa River, in Nara Prefecture . We extracted DNA from bulk community samples of aquatic organisms, most of which could not be morphologically identified at species level due to their small body size (0.12 - 2 mm length). A partial coding region of the mitochondrial cytochrome  Specifications Table•The metabarcoding result enables us to evaluate the efficacy of DNA-based biodiversity assessment in comparison with conventional morphology-based species identification data collected from the same localities.•The new OTU clustering algorism that compare the read abundances between neighbouring OTUs is available as an R script, which could be used widely to process amplicon sequence data.•The results are beneficial for researchers working on both DNA metabarcoding and conventional morphology-based faunal surveys.•The data will serve as a reference barcode sequence data of benthic fauna in mountain streams in the Central Honshu, Japan.1cox1 in riverine metagenomes are deposited in DNA Data Bank of Japan (DDBJ). DDBJ accession numbers DRR311861 and DRR311862 were obtained from the run environment (slow riffle) of Aridoshi (ARD), DRR311863-DRR311865 from the rapid environment of Shisetsu (SST), and DDR311866-DRR311868 from the run environment of Shisetsu (SST). There were 472,697 reads in total of DRR311861- DRR311868, and they were classified into 888 OTUs.Raw sequence reads of partial coding regions of Phylogenetic tree of the 888 OTUs was reconstructed by MrBayes 3.2.7a based on the GTR+I+G molecular clock model, and the species boundaries were delineated using the Generalised Mixed Yule Coalescent (GMYC) method . A totalRepresentative sequences of the GMYC species  in a perturbed riverbed by kicking at two sampling stations: ARD (on 17 March 2017) and SST (on 29 June 2017). Sampling was performed in the run environment (slow riffle) of both stations ARD and SST, and in rapid environment of SST . The specimens were immersed in 70% ethanol until DNA was isolated. DNA of the three bulk community samples (one from ARD and two from SST) were separately extracted using conventional proteinase K treatment/phenol extraction/ethanol precipitation, as previously described 2.2cox1) were performed using PrimeSTAR® HS DNA Polymerase  for 35 cycles of 98°C-10 s / 40°C-5 s / 72°C-60 s with an initial incubation at 95°C for 1 min and a final elongation at 72°C for 7 min, using a set of primers IntF and HCOm IntF, ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGWACWGGWTGAACWGTWTAYCCYCC;HCOm, GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTAHACTTCNGGGTGKCCRAARAATCA.The DNA libraries for sequencing were constructed by 2-step tailed PCR. The first PCR amplifications of the target gene . The second PCR was performed using ExTaq® DNA Polymerase (Takara Bio Co. Ltd.) for 12 cycles of 94°C-30 s / 60°C-30 s / 72°C-30 s with an initial incubation at 94°C for 2 min and a final elongation at 72°C for 5 min, with a set of DNA primers containing octanucleotide tag for indexing. The nucleotide sequences of primers were as follows:https://gikenbio.com/).Two technical replicates for ARD and three technical replicates for each of two SST samples were prepared with different tags. The tag sequences for DRR311861, DRR311862, DRR311863, DRR311864, DRR311865, DRR311866, DRR311867, and DRR311868 were TCGACTAG, TTCTAGCT, CCTAGAGT, GCGTAAGA, CTATTAAG, AAGGCTAT, GAGCCTTA, and TTATGCGA, respectively. Quality of the DNA libraries was assessed by Fragment Analyzer with dsDNA 915 Reagent Kit . High-throughput parallel sequencing was performed by Miseq  capable of generating 2 × 300 base paired-end reads. The library construction (after the first PCR) and sequencing were outsourced to Seibutsu Giken Co. Ltd.  (2.310.17632/pfhf4kt37s.1).Among the raw sequence reads , reads with quality scores less than 20 were discarded, and those of the 313 bp net length of paired sequences were sorted out. Valid sequences  were collapsed into 41,010 groups with 100% identity, and the groups consisting of less than three reads were discarded. Neighbouring groups with a single nucleotide difference were combined each other to form an OTU, and a sequence with the largest number of reads within the OTU was defined as the OTU representative sequence. A total of 888 OTUs were obtained and subjected to GMYC analysis. The R script used to produce OTUs and OTU representatives in FASTA format are deposited in Mendeley Data (DOI:2.4A phylogenetic tree of 888 OTUs was reconstructed using Markov chain Monte Carlo (MCMC) methods by MrBayes 3.2.7a with the GTR+I+G molecular clock model. GMYC species boundaries were estimated using SPLITS package in R.Kei Wakimura: Investigation, Writing – original draft. Koji Inai: Software, Investigation. Kazumi Tanida: Resources, Writing – review & editing. Kozo Watanabe: Conceptualization, Writing – review & editing, Funding acquisition. Mikio Kato: Project administration, Conceptualization, Resources, Writing – review & editing, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.