Porcine reproductive and respiratory syndrome virus triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replication.

Macroautophagy/autophagy is a cellular degradation and recycling process that maintains the homeostasis of organisms. A growing number of studies have reported that autophagy participates in infection by a variety of viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe financial losses to the global swine industry. Although much research has shown that PRRSV triggers autophagy for its own benefits, the exact molecular mechanisms involved in PRRSV-triggered autophagy remain to be fully elucidated. In the current study, we demonstrated that PRRSV infection significantly induced Golgi apparatus (GA) fragmentation, which promoted autophagy to facilitate viral self-replication. Mechanistically, PRRSV nonstructural protein 2 was identified to interact with and degrade the Golgi reassembly and stacking protein 65 dependent on its papain-like cysteine protease 2 activity, resulting in GA fragmentation. Upon GA fragmentation, GA-resident Ras-like protein in brain 2 was disassociated from Golgi matrix protein 130 and subsequently bound to unc-51 like autophagy activating kinase 1 (ULK1), which enhanced phosphorylation of ULK1 and promoted autophagy. Taken together, all these results expand the knowledge of PRRSV-triggered autophagy as well as PRRSV pathogenesis to support novel potential avenues for prevention and control of the virus. More importantly, these results provide the detailed mechanism of GA fragmentation-mediated autophagy, deepening the understanding of autophagic processes.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) infection results in a serious swine disease affecting pig farming worldwide. Despite that numerous studies have shown that PRRSV triggers autophagy for its self-replication, how PRRSV induces autophagy is incompletely understood. Here, we identify that PRRSV Nsp2 degrades GRASP65 to induce GA fragmentation, which dissociates RAB2 from GM130 and activates RAB2-ULK1-mediated autophagy to enhance viral replication. This work expands our understanding of PRRSV-induced autophagy and PRRSV replication, which is beneficial for anti-viral drug development.

Split but merge: Golgi fragmentation in physiological and pathological conditions.

The Golgi complex is a highly dynamic and tightly regulated cellular organelle with essential roles in the processing as well as the sorting of proteins and lipids. Its structure undergoes rapid disassembly and reassembly during normal physiological processes, including cell division, migration, polarization, differentiation, and cell death. Golgi dispersal or fragmentation also occurs in pathological conditions, such as neurodegenerative diseases, infectious diseases, congenital disorders of glycosylation diseases, and cancer. In this review, current knowledge about both structural organization and morphological alterations in the Golgi in physiological and pathological conditions is summarized together with the methodologies that help to reveal its structure.

The Golgi complex of dopaminergic enteric neurons is fragmented in a hemiparkinsonian rat model.

Since gastrointestinal disorders are early consequences of Parkinson's disease (PD), this disease is clearly not restricted to the central nervous system (CNS), but also significantly affects the enteric nervous system (ENS). Large aggregates of the protein α-synuclein forming Lewy bodies, the prototypical cytopathological marker of this disease, have been observed in enteric nervous plexuses. However, their value in early prognosis is controversial. The Golgi complex (GC) of nigral neurons appears fragmented in Parkinson's disease, a characteristic common in most neurodegenerative diseases. In addition, the distribution and levels of regulatory proteins such as Rabs and SNAREs are altered, suggesting that PD is a membrane traffic-related pathology. Whether the GC of enteric dopaminergic neurons is affected by the disease has not yet been analyzed. In the present study, dopaminergic neurons in colon nervous plexuses behave as nigral neurons in a hemiparkinsonian rat model based on the injection of the toxin 6-OHDA. Their GCs are fragmented, and some regulatory proteins' distribution and expression levels are altered. The putative mechanisms of the transmission of the neurotoxin to the ENS are discussed. Our results support the possibility that GC structure and the level of some proteins, especially syntaxin 5, could be helpful as early indicators of the disease. RESEARCH HIGHLIGHTS: The Golgi complexes of enteric dopaminergic neurons appear fragmented in a Parkinson's disease rat model. Our results support the hypothesis that the Golgi complex structure and levels of Rab1 and syntaxin 5 could be helpful as early indicators of the disease.

Morphological alterations of the neuronal Golgi apparatus upon seizures.

AIMS: Epilepsy is one of the most common chronic neurological disorders, affecting around 50 million people worldwide, but its underlying cellular and molecular events are not fully understood. The Golgi is a highly dynamic cellular organelle and can be fragmented into ministacks under both physiological and pathological conditions. This phenomenon has also been observed in several neurodegenerative disorders; however, the structure of the Golgi apparatus (GA) in human patients suffering from epilepsy has not been described so far. The aim of this study was to assess the changes in GA architecture in epilepsy. METHODS: Golgi visualisation with immunohistochemical staining in the neocortex of adult patients who underwent epilepsy surgery; 3D reconstruction and quantitative morphometric analysis of GA structure in the rat hippocampi upon kainic acid (KA) induced seizures, as well as in vitro studies with the use of Ca2+ chelator BAPTA-AM in primary hippocampal neurons upon activation were performed. RESULTS: We observed GA dispersion in neurons of the human neocortex of patients with epilepsy and hippocampal neurons in rats upon KA-induced seizures. The structural changes of GA were reversible, as GA morphology returned to normal within 24 h of KA treatment. KA-induced Golgi fragmentation observed in primary hippocampal neurons cultured in vitro was largely abolished by the addition of BAPTA-AM. CONCLUSIONS: In our study, we have shown for the first time that the neuronal GA is fragmented in the human brain of patients with epilepsy and rat brain upon seizures. We have shown that seizure-induced GA dispersion can be reversible, suggesting that enhanced neuronal activity induces Golgi reorganisation that is involved in aberrant neuronal plasticity processes that underlie epilepsy. Moreover, our results revealed that elevated cytosolic Ca2+ is indispensable for these KA-induced morphological alterations of GA in vitro.

Tetramethylpyrazine ameliorates endotoxin-induced acute lung injury by relieving Golgi stress via the Nrf2/HO-1 signaling pathway.

PURPOSE: Endotoxin-induced acute lung injury (ALI) is a severe disease caused by an imbalanced host response to infection. It is necessary to explore novel mechanisms for the treatment of endotoxin-induced ALI. In endotoxin-induced ALI, tetramethylpyrazine (TMP) provides protection through anti-inflammatory, anti-apoptosis, and anti-pyroptosis effects. However, the mechanism of action of TMP in endotoxin-induced ALI remains unclear. Here, we aimed to determine whether TMP can protect the lungs by inhibiting Golgi stress via the Nrf2/HO-1 pathway. METHODS AND RESULTS: Using lipopolysaccharide (LPS)-stimulated C57BL/6J mice and MLE12 alveolar epithelial cells, we observed that TMP pretreatment attenuated endotoxin-induced ALI. LPS + TMP group showed lesser lung pathological damage and a lower rate of apoptotic lung cells than LPS group. Moreover, LPS + TMP group also showed decreased levels of inflammatory factors and oxidative stress damage than LPS group (P < 0.05). Additionally, LPS + TMP group presented reduced Golgi stress by increasing the Golgi matrix protein 130 (GM130), Golgi apparatus Ca2+/Mn2+ ATPases (ATP2C1), and Golgin97 expression while decreasing the Golgi phosphoprotein 3 (GOLPH3) expression than LPS group (P < 0.05). Furthermore, TMP pretreatment promoted Nrf2 and HO-1 expression (P < 0.05). Nrf2-knockout mice or Nrf2 siRNA-transfected MLE12 cells were pretreated with TMP to explore how the Nrf2/HO-1 pathway affected TMP-mediated Golgi stress in endotoxin-induced ALI models. We observed that Nrf2 gene silencing partially reversed the alleviating effect of Golgi stress and the pulmonary protective effect of TMP. CONCLUSION: Our findings showed that TMP therapy reduced endotoxin-induced ALI by suppressing Golgi stress via the Nrf2/HO-1 signaling pathway in vivo and in vitro.

A combined experimental-computational approach uncovers a role for the Golgi matrix protein Giantin in breast cancer progression.

Our understanding of how speed and persistence of cell migration affects the growth rate and size of tumors remains incomplete. To address this, we developed a mathematical model wherein cells migrate in two-dimensional space, divide, die or intravasate into the vasculature. Exploring a wide range of speed and persistence combinations, we find that tumor growth positively correlates with increasing speed and higher persistence. As a biologically relevant example, we focused on Golgi fragmentation, a phenomenon often linked to alterations of cell migration. Golgi fragmentation was induced by depletion of Giantin, a Golgi matrix protein, the downregulation of which correlates with poor patient survival. Applying the experimentally obtained migration and invasion traits of Giantin depleted breast cancer cells to our mathematical model, we predict that loss of Giantin increases the number of intravasating cells. This prediction was validated, by showing that circulating tumor cells express significantly less Giantin than primary tumor cells. Altogether, our computational model identifies cell migration traits that regulate tumor progression and uncovers a role of Giantin in breast cancer progression.

NKX3.1 Expression and Molecular Characterization of Secretory Myoepithelial Carcinoma (SMCA): Advancing the Case for a Salivary Mucous Acinar Phenotype.

BACKGROUND: Secretory myoepithelial carcinomas (SMCA) are rare, mucinous, signet ring predominant tumors with primitive myoepithelial features. While many mucinous salivary gland tumors have now been molecularly characterized, key drivers in SMCA have yet to be elucidated. Recently, NKX3.1, a homeodomain transcription factor implicated in salivary mucous acinar development was also shown in a subset of salivary mucinous neoplasms, salivary intraductal papillary mucinous neoplasms (SG-IPMN). To date, NKX3.1 expression has not been characterized in other mucinous salivary lesions. Here, we report molecular and extended immunophenotypic findings in SMCA and NKX3.1 expression in the context of other head and neck lesions. METHODS: We retrieved 4 previously reported SMCA, performed additional immunohistochemical and targeted next-generation sequencing (NGS). We also investigated the use of NKX3.1 as a marker for SMCA in the context of its prevalence and extent (using H-score) in a mixed cohort of retrospectively and prospectively tested head and neck lesions (n = 223) and non-neoplastic tissues (n = 66). RESULTS: NKX3.1 positivity was confirmed in normal mucous acini as well as in mucous acinar class of lesions (5/6, mean H-score: 136.7), including mucinous adenocarcinomas (3/4), SG-IPMN (1/1), and microsecretory adenocarcinoma (MSA) (1/1). All SMCA were positive. Fluorescence in situ hybridization for SS18 rearrangements were negative in all successfully tested cases (0/3). NGS was successful in two cases (cases 3 and 4). Case 3 demonstrated a PTEN c.655C>T p.Q219* mutation and a SEC16A::NOTCH1 fusion while case 4 (clinically aggressive) showed a PTEN c.1026+1G>A p.K342 splice site variant, aTP53 c.524G>A p.R175H mutation and a higher tumor mutation burden (29 per Mb). PTEN immunohistochemical loss was confirmed in both cases and a subset of tumor cells showed strong (extreme) staining for P53 in Case 4. CONCLUSION: Despite a partial myoepithelial phenotype, SMCA, along with mucinous adenocarcinomas/SG-IPMN and MSA, provisionally constitute a mucous acinar class of tumors based on morphology and NKX3.1 expression. Like salivary mucinous adenocarcinomas/SG-IPMN, SMCA also show alterations of the PTEN/PI3K/AKT pathway and may show progressive molecular alterations. We document the first extramammary tumor with a SEC16A::NOTCH1 fusion.

A neurodevelopmental disorder associated with an activating de novo missense variant in ARF1.

ADP-ribosylation factor 1 (ARF1) is a small GTPase that regulates membrane traffic at the Golgi apparatus and endosomes through recruitment of several coat proteins and lipid-modifying enzymes. Here, we report a pediatric patient with an ARF1-related disorder because of a monoallelic de novo missense variant (c.296 G > A; p.R99H) in the ARF1 gene, associated with developmental delay, hypotonia, intellectual disability and motor stereotypies. Neuroimaging revealed a hypoplastic corpus callosum and subcortical white matter abnormalities. Notably, this patient did not exhibit periventricular heterotopias previously observed in other patients with ARF1 variants (including p.R99H). Functional analysis of the R99H-ARF1 variant protein revealed that it was expressed at normal levels and properly localized to the Golgi apparatus; however, the expression of this variant caused swelling of the Golgi apparatus, increased the recruitment of coat proteins such as coat protein complex I, adaptor protein complex 1 and GGA3 and altered the morphology of recycling endosomes. In addition, we observed that the expression of R99H-ARF1 prevented dispersal of the Golgi apparatus by the ARF1-inhibitor brefeldin A. Finally, protein interaction analyses showed that R99H-ARF1 bound more tightly to the ARF1-effector GGA3 relative to wild-type ARF1. These properties were similar to those of the well-characterized constitutively active Q71L-ARF1 mutant, indicating that the pathogenetic mechanism of the R99H-ARF1 variant involves constitutive activation with resultant Golgi and endosomal alterations. The absence of periventricular nodular heterotopias in this R99H-ARF1 subject also indicates that this finding may not be a consistent phenotypic expression of all ARF1-related disorders.

Engineered Platelet Microparticle-Membrane Camouflaged Nanoparticles for Targeting the Golgi Apparatus of Synovial Fibroblasts to Attenuate Rheumatoid Arthritis.

Synovial fibroblasts in rheumatoid arthritis (RA) joints mediate synovial hyperplasia, progressive joint destruction, and the potential spread of disease between joints by producing multiple pathogenic proteins. Here, we deliver all-trans retinoic acid (ATRA) to selectively down-regulate these pathogenic factors, with a Golgi-targeting platelet microparticle-mimetic nanoplatform (termed Gol-PMMNP) which comprises a nanoparticle core and a platelet microparticle membrane coating labeled with a Golgi apparatus-targeting peptide. Gol-PMMNPs are shown to target synovial fibroblasts derived from RA patients via integrin α2ß1-mediated endocytosis and accumulate in the Golgi apparatus by retrograde transport. ATRA-loaded Gol-PMMNPs (ATRA-Gol-PMMNPs) cause structural disruption of the Golgi apparatus, leading to an efficient reduction of pathogenic protein secretion in RA synovial fibroblasts. In rats with collagen-induced arthritis, Gol-PMMNPs display an arthritic joint-specific distribution, and ATRA-Gol-PMMNPs effectively reduce concentrations of pathogenic factors therein, including inflammatory cytokines, chemokines, and matrix-degrading enzymes within these joints. Additionally, ATRA-Gol-PMMNP treatment results in inflammatory remission and decreased bone erosion in both arthritic and proximal joints. Furthermore, ATRA-Gol-PMMNPs induce negligible toxicity to major organs. Taken together, ATRA-Gol-PMMNPs inhibit the progression of RA through reducing the production of multiple pathogenic mediators by synovial fibroblasts.

[Preliminary Study on Drug-Loaded Chondroitin Sulfate-Modified Micelles Targeting Golgi Apparatus in Tumor Cells for the Treatment of Tumor Metastasis].

Objective: To make preliminary exploration into the Golgi apparatus targeting of chondroitin sulfate-modified micelles (CSmicelles) co-loaded with pirarubicin (THP) and vinorelbine (VRL) in tumor cells, as well as their in vitro anti-tumor metastasis effect. Methods: The cellular uptake efficiency and internalization mechanism of CSmicelles in 4T1 mouse breast cancer cell line were investigated by flow cytometry. Preliminary study of the Golgi apparatus targeting CSmicelles in tumor cells was conducted by co-localization experiment. Then, the effect of CSmicelles co-loaded with THP and VRL (THP+VTL-CSmicelles) on the structure of Golgi apparatus was investigated by GM130 immunofluorescence experiment. Finally, the i n vitro anti-tumor metastasis ability of THP+VTL-CSmicelles was evaluated by wound healing assay and Transwell migration/invasion assay. Results: It was found that CSmicelles could significantly increase cellular uptake of drugs. CSmicelles were internalized into cells through clathrin-mediated and caveolin-mediated endocytosis, which was energy-dependent active transport and exhibited substantial ability of targeting Golgi apparatus in tumor cells. THP+VTL-CSmicelles could break down the structure of Golgi apparatus and significantly inhibit the migration and invasion of tumor cells. Conclusion: THP+VTL-CSmicelles demonstrate high affinity towards Golgi apparatus in tumor cells, exert targeted effects and inhibit tumor cell metastasis, which provides a novel idea and method for the treatment of cancer metastasis.

ZIF-based carbon dots with lysosome-Golgi transport property as visualization platform for deep tumour therapy via hierarchical size/charge dual-transform and transcytosis.

The poor penetration of nanomaterials in solid tumours and difficulty in monitoring their penetration depth are major obstacles in their application for the treatment of solid tumours. Herein, pH-responsive carbon dots (ZCD) based on a zeolitic imidazolate framework (ZIF-8) were fabricated to achieve the deep delivery of the chemotherapeutic doxorubicin (DOX) via a hierarchical size/charge dual-transformation and transcytosis. The as-prepared ZCD accumulated in the solid tumour and the acidic tumour microenvironment further triggered its decomposition. Firstly, ZCD was decomposed by the weakly acidic extracellular microenvironment of the solid tumour, enabling it to transform into small and neutrally charged particles. Subsequently, these particles were endocytosed by lysosomes, and further disintegrated into smaller and positively charged particles, which could target the Golgi apparatus. Consequently, ZCD delivered DOX deep into the solid tumour via a size-shrinking strategy and Golgi-mediated transcytosis, thus significantly improving its antitumour efficacy. In addition, carbonization endowed ZCD with superior fluorescence property, which was enhanced in the acidic microenvironment, thus improving the sensitivity and accuracy of ex vivo monitoring of the penetration depth of the nanomedicine in real time. Collectively, our results confirmed that the carbon dots obtained via the direct carbonization of ZIF-8 simultaneously exhibited enhanced deep penetration into solid tumours and fluorescence, which could be monitored, and that the carbonization of functional materials is effective to enhance their fluorescence, and further broaden their applications.

GRASP depletion-mediated Golgi fragmentation impairs glycosaminoglycan synthesis, sulfation, and secretion.

Synthesis of glycosaminoglycans, such as heparan sulfate (HS) and chondroitin sulfate (CS), occurs in the lumen of the Golgi, but the relationship between Golgi structural integrity and glycosaminoglycan synthesis is not clear. In this study, we disrupted the Golgi structure by knocking out GRASP55 and GRASP65 and determined its effect on the synthesis, sulfation, and secretion of HS and CS. We found that GRASP depletion increased HS synthesis while decreasing CS synthesis in cells, altered HS and CS sulfation, and reduced both HS and CS secretion. Using proteomics, RNA-seq and biochemical approaches, we identified EXTL3, a key enzyme in the HS synthesis pathway, whose level is upregulated in GRASP knockout cells; while GalNAcT1, an essential CS synthesis enzyme, is robustly reduced. In addition, we found that GRASP depletion decreased HS sulfation via the reduction of PAPSS2, a bifunctional enzyme in HS sulfation. Our study provides the first evidence that Golgi structural defect may significantly alter the synthesis and secretion of glycosaminoglycans.

GM130 regulates pulmonary surfactant protein secretion in alveolar type II cells.

Pulmonary surfactant is a lipid-protein complex secreted by alveolar type II epithelial cells and is essential for the maintenance of the delicate structure of mammalian alveoli to promote efficient gas exchange across the air-liquid barrier. The Golgi apparatus plays an important role in pulmonary surfactant modification and secretory trafficking. However, the physiological function of the Golgi apparatus in the transport of pulmonary surfactants is unclear. In the present study, deletion of GM130, which encodes for a matrix protein of the cis-Golgi cisternae, was shown to induce the disruption of the Golgi structure leading to impaired secretion of lung surfactant proteins and lipids. Specifically, the results of in vitro and in vivo analysis indicated that the loss of GM130 resulted in trapping of Sftpa in the endoplasmic reticulum, Sftpb and Sftpc accumulation in the Golgi apparatus, and an increase in the compensatory secretion of Sftpd. Moreover, global and epithelial-specific GM130 knockout in mice resulted in an enlargement of alveolar airspace and an increase in alveolar epithelial autophagy; however, surfactant repletion partially rescued the enlarged airspace defects in GM130-deficient mice. Therefore, our results demonstrate that GM130 and the mammalian Golgi apparatus play a critical role in the control of surfactant protein secretion in pulmonary epithelial cells.

Alcohol-Induced Liver Injury: Down-regulation and Redistribution of Rab3D Results in Atypical Protein Trafficking.

Previous work from our laboratories has identified multiple defects in endocytosis, protein trafficking, and secretion, along with altered Golgi function after alcohol administration. Manifestation of alcohol-associated liver disease (ALD) is associated with an aberrant function of several hepatic proteins, including asialoglycoprotein receptor (ASGP-R), their atypical distribution at the plasma membrane (PM), and secretion of their abnormally glycosylated forms into the bloodstream, but trafficking mechanism is unknown. Here we report that a small GTPase, Rab3D, known to be involved in exocytosis, secretion, and vesicle trafficking, shows ethanol (EtOH)-impaired function, which plays an important role in Golgi disorganization. We used multiple approaches and cellular/animal models of ALD, along with Rab3D knockout (KO) mice and human tissue from patients with ALD. We found that Rab3D resides primarily in trans- and cis-faces of Golgi; however, EtOH treatment results in Rab3D redistribution from trans-Golgi to cis-medial-Golgi. Cells lacking Rab3D demonstrate enlargement of Golgi, especially its distal compartments. We identified that Rab3D is required for coat protein I (COPI) vesiculation in Golgi, and conversely, COPI is critical for intra-Golgi distribution of Rab3D. Rab3D/COPI association was altered not only in the liver of patients with ALD but also in the donors consuming alcohol without steatosis. In Rab3D KO mice, hepatocytes experience endoplasmic reticulum (ER) stress, and EtOH administration activates apoptosis. Notably, in these cells, ASGP-R, despite incomplete glycosylation, can still reach cell surface through ER-PM junctions. This mimics the effects seen with EtOH-induced liver injury. Conclusion: We revealed that down-regulation of Rab3D contributes significantly to EtOH-induced Golgi disorganization, and abnormally glycosylated ASGP-R is excreted through ER-PM connections, bypassing canonical (ER→Golgi→PM) anterograde transportation. This suggests that ER-PM sites may be a therapeutic target for ALD.

CRABP2 involvement in a mechanism of Golgi stress and tumor dry matter in non-small cell lung cancer cells via ER dependent Hippo pathway.

OBJECTIVE: The paper aimed to explore the mechanism of cellular retinoic acid binding protein 2 (CRABP2) involvement in Golgi stress and tumor dryness in non-small cell lung cancer (NSCLC) cells through the estrogen receptor (ER) dependent Hippo pathway. METHODS: Human NSCLC cell line A549 was purchased from ATCC andcultured in RPMI-1640 with 10% FBS. Attractene reagent was used for plasmid transfection. ER (sh) RNA was designed using RNAi Designer. Seventy-six hours after infection, stable cells were obtained after treated with puromycin for 3 weeks. ER silencing cells (with inhibited ER expression) were compared to the control cells (normal cultured NSCLC cell line A549, CRABP2 normal expression). CRABP2 and ER expression levels were detected by RT-PCR. MTT assay was used to detect cell proliferation, and the cell localization of ER and Golgi was observed by confocal microscopy. The invasion and metastasis of cells were analyzed by Boden chamber invasion and migration assays. Western blotting assays was used for detecting the protein expression of E-cadherin, vimentin, ZO-1 protein and epithelial-mesenchymal transition (EMT) related factors. RESULTS: The lower expression level of mRNA was detected in the ER-silencing group compared to the control group (P<0.05). We also found a higher proliferation level of cells, the number of invading and metastatic cells, the expression of vimentin, p-Lats1T1079, Lats1 and p-YAPS127 mRNA in the control group compared to the ER silencing group (P<0.05). And the expression level of protein kinase RNA-like endoplasmic reticulum kinase (PERK), phosphorylate eukaryotic initiation factor 2 (p-eIF2 alpha), activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP) in the control group was higher than that in the ER silencing group (P<0.05). Adversely, a lower expression level of E-cadherin and ZO-1 protein was found in the control group compared to the ER silencing group (P<0.05). CONCLUSION: The expression of CRABP2 in NSCLC cells was regulated by ER, and cell proliferation and invasion were regulated by the Hippo pathway. At the same time, it was found that decreased expression of CRABP2 enhanced endoplasmic reticulum/Golgi stress response.

OM-MSCs Alleviate the Golgi Apparatus Stress Response following Cerebral Ischemia/Reperfusion Injury via the PEDF-PI3K/Akt/mTOR Signaling Pathway.

The mechanism of Golgi apparatus (GA) stress responses mediated by GOLPH3 has been widely studied in ischemic stroke, and the neuroprotection effect of olfactory mucosa mesenchymal stem cells (OM-MSCs) against cerebral ischemia/reperfusion injury (IRI) has been preliminarily presented. However, the exact role of OM-MSCs in the GA stress response following cerebral IRI remains to be elucidated. In the present study, we used an oxygen-glucose deprivation/reoxygenation (OGD/R) model and reversible middle cerebral artery occlusion (MCAO) model to simulate cerebral IRI in vitro and in vivo. Our results showed that the level of GOLPH3 protein, reactive oxygen species (ROS), and Ca2+ was upregulated, SPCA1 level was downregulated, and GA fragmentation was increased in ischemic stroke models, and OM-MSC treatment clearly ameliorated these GA stress responses in vitro and in vivo. Subsequently, the knockdown of PEDF in OM-MSCs using PEDF-specific siRNA further demonstrated that secretion of PEDF in OM-MSCs protected OGD/R-treated N2a cells and MCAO rats from GA stress response. Additionally, rescue experiment using specific pathway inhibitors suggested that OM-MSCs could promote the phosphorylation of the PI3K/Akt/mTOR pathway, thereby mitigating OGD/R-induced GA stress response and excessive autophagy. In conclusion, OM-MSCs minimized the GA stress response following cerebral IRI, at least partially, through the PEDF-PI3K/Akt/mTOR pathway.

Single-cell analysis reveals androgen receptor regulates the ER-to-Golgi trafficking pathway with CREB3L2 to drive prostate cancer progression.

Androgen receptor (AR) plays a central role in driving prostate cancer (PCa) progression. How AR promotes this process is still not completely clear. Herein, we used single-cell transcriptome analysis to reconstruct the transcriptional network of AR in PCa. Our work shows AR directly regulates a set of signature genes in the ER-to-Golgi protein vesicle-mediated transport pathway. The expression of these genes is required for maximum androgen-dependent ER-to-Golgi trafficking, cell growth, and survival. Our analyses also reveal the signature genes are associated with PCa progression and prognosis. Moreover, we find inhibition of the ER-to-Golgi transport process with a small molecule enhanced antiandrogen-mediated tumor suppression of hormone-sensitive and insensitive PCa. Finally, we demonstrate AR collaborates with CREB3L2 in mediating ER-to-Golgi trafficking in PCa. In summary, our findings uncover a critical role for dysregulation of ER-to-Golgi trafficking expression and function in PCa progression, provide detailed mechanistic insights for how AR tightly controls this process, and highlight the prospect of targeting the ER-to-Golgi pathway as a therapeutic strategy for advanced PCa.

Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection.

SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.

Dysregulation of the Acrosome Formation Network by 8-oxoguanine (8-oxoG) in Infertile Sperm: A Case Report with Advanced Techniques.

8-Hydroxyguanine (8-oxoG) is the most common oxidative DNA lesion and unrepaired 8-oxoG is associated with DNA fragmentation in sperm. However, the molecular effects of 8-oxoG on spermatogenesis are not entirely understood. Here, we identified one infertile bull (C14) due to asthenoteratozoospermia. We compared the global concentration of 8-oxoG by reverse-phase liquid chromatography/mass spectrometry (RP-LC/MS), the genomic distribution of 8-oxoG by next-generation sequencing (OG-seq), and the expression of sperm proteins by 2-dimensional polyacrylamide gel electrophoresis followed by peptide mass fingerprinting (2D-PAGE/PMF) in the sperm of C14 with those of a fertile bull (C13). We found that the average levels of 8-oxoG in C13 and C14 sperm were 0.027% and 0.044% of the total dG and it was significantly greater in infertile sperm DNA (p = 0.0028). Over 81% of the 8-oxoG loci were distributed around the transcription start site (TSS) and 165 genes harboring 8-oxoG were exclusive to infertile sperm. Functional enrichment and network analysis revealed that the Golgi apparatus was significantly enriched with the products from 8-oxoG genes of infertile sperm (q = 2.2 × 10-7). Proteomic analysis verified that acrosome-related proteins, including acrosin-binding protein (ACRBP), were downregulated in infertile sperm. These preliminary results suggest that 8-oxoG formation during spermatogenesis dysregulated the acrosome-related gene network, causing structural and functional defects of sperm and leading to infertility.