Mesencephalic Astrocyte-Derived Neurotrophic Factor Inhibits Liver Cancer Through Small Ubiquitin-Related Modifier (SUMO)ylation-Related Suppression of NF-κB/Snail Signaling Pathway and Epithelial-Mesenchymal Transition.

BACKGROUND AND AIMS: Endoplasmic reticulum (ER) stress is associated with liver inflammation and hepatocellular carcinoma (HCC). However, how ER stress links inflammation and HCC remains obscure. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress-inducible secretion protein that inhibits inflammation by interacting with the key subunit of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) p65. We hypothesized that MANF may play a key role in linking ER stress and inflammation in HCC. APPROACH AND RESULTS: Here, we found that MANF mRNA and protein levels were lower in HCC tissues versus adjacent noncancer tissues. Patients with high levels of MANF had better relapse-free survival and overall survival rates than those with low levels. MANF levels were also associated with the status of liver cirrhosis, advanced tumor-node-metastasis (TNM) stage, and tumor size. In vitro experiments revealed that MANF suppressed the migration and invasion of hepatoma cells. Hepatocyte-specific deletion of MANF accelerated N-nitrosodiethylamine (DEN)-induced HCC by up-regulating Snail1+2 levels and promoting epithelial-mesenchymal transition (EMT). MANF appeared in the nuclei and was colocalized with p65 in HCC tissues and in tumor necrosis factor alpha (TNF-α)-treated hepatoma cells. The interaction of p65 and MANF was also confirmed by coimmunoprecipitation experiments. Consistently, knockdown of MANF up-regulated NF-κB downstream target genes TNF-α, interleukin (IL)-6 and IL-1α expression in vitro and in vivo. Finally, small ubiquitin-related modifier 1 (SUMO1) promoted MANF nuclear translocation and enhanced the interaction of MANF and p65. Mutation of p65 motifs for SUMOylation abolished the interaction of p65 and MANF. CONCLUSIONS: MANF plays an important role in linking ER stress and liver inflammation by inhibiting the NF-κB/Snail signal pathway in EMT and HCC progression. Therefore, MANF may be a cancer suppressor and a potential therapeutic target for HCC.

Hepatocyte-derived MANF alleviates hepatic ischaemia-reperfusion injury via regulating endoplasmic reticulum stress-induced apoptosis in mice.

BACKGROUND: Endoplasmic reticulum (ER) perturbations are novel subcellular effectors involved in the ischaemia-reperfusion injury. As an ER stress-inducible protein, mesencephalic astrocyte-derived neurotrophic factor (MANF) has been proven to be increased during ischaemic brain injury. However, the role of MANF in liver ischaemia reperfusion (I/R) injury has not yet been studied. METHODS: To investigate the role of MANF in the process of liver ischaemia-reperfusion, Hepatocyte-specific MANF knockout (MANFhep-/- ) mice and their wild-type (WT) littermates were used in our research. Mice partial (70%) warm hepatic I/R model was established by vascular occlusion. We detected the serum levels of MANF in both liver transplant patients and WT mice before and after liver I/R injury. Recombinant human MANF (rhMANF) was injected into the tail vein before 1 hour occlusion. AST, ALT and Suzuki score were used to evaluate the extent of I/R injury. OGD/R test was performed on primary hepatocytes to simulate IRI in vitro. RNA sequence and RT-PCR were used to detect the cellular signal pathway activation while MANF knockout. RESULTS: We found that MANF expression and secretion are dramatically up-regulated during hepatic I/R. Hepatocyte-specific MANF knockout aggravates the I/R injury through the over-activated ER stress. The systemic administration of rhMANF before ischaemia has the potential to ameliorate I/R-triggered UPR and liver injury. Further study showed that MANF deficiency activated ATF4/CHOP and JNK/c-JUN/CHOP pathways, and rhMANF inhibited the activation of the two proapoptotic pathways caused by MANF deletion. CONCLUSION: Collectively, our study unravels a previously unknown relationship among MANF, UPR and hepatic I/R injury.

Synapse Dysfunction of Layer V Pyramidal Neurons Precedes Neurodegeneration in a Mouse Model of TDP-43 Proteinopathies.

TDP-43 is a major protein component of pathological neuronal inclusions that are present in frontotemporal dementia and amyotrophic lateral sclerosis. We report that TDP-43 plays an important role in dendritic spine formation in the cortex. The density of spines on YFP+ pyramidal neurons in both the motor and somatosensory cortex of Thy1-YFP mice, increased significantly from postnatal day 30 (P30), to peak at P60, before being pruned by P90. By comparison, dendritic spine density was significantly reduced in the motor cortex of Thy1-YFP::TDP-43A315T transgenic mice prior to symptom onset (P60), and in the motor and somatosensory cortex at symptom onset (P90). Morphological spine-type analysis revealed that there was a significant impairment in the development of basal mushroom spines in the motor cortex of Thy1-YFP::TDP-43A315T mice compared to Thy1-YFP control. Furthermore, reductions in spine density corresponded to mislocalisation of TDP-43 immunoreactivity and lowered efficacy of synaptic transmission as determined by electrophysiology at P60. We conclude that mutated TDP-43 has a significant pathological effect at the dendritic spine that is associated with attenuated neural transmission.

Inhibition of Hepatitis B virus replication by SAMHD1.

Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is a newly identified intracellular antiviral factor. By depleting the dNTPs pool of host cells to a low level that cannot support the efficient synthesis of viral cDNA, it restricts replication of some retroviruses. As a DNA virus, Hepatitis B virus (HBV) experiences a process of reverse transcription in its life cycle akin to that of retroviruses. However, whether SAMHD1 can restrict HBV replication in liver cells is unknown. Here, we reported that SAMHD1 expression was detectable in four liver cell lines. Exogenous expression of SAMHD1 in SMMC-7721 cells restrained HBV replication. Similarly, SAMHD1 impeded HBV replication in another liver cell line, BEL-7402. Remarkably, the catalytically inactive mutant, SAMHD1 HD/AA also hampered HBV replication. Additionally, HBV replication reduced SAMHD1 expression in HepG2 cells. Moreover, it was found that IFN-α induced expression of SAMHD1 in liver cells. Together, these findings suggested that IFN-α-inducible SAMHD1 inhibited HBV replication in liver cells.

Endoplasmic reticulum stress is involved in the neuroprotective effect of propofol.

Propofol is a common clinically used intravenous anaesthetic agent with antioxidative property. It has been thought to have neuroprotection in vitro and in vivo. However, the underlying mechanisms remain unclear. Endoplasmic reticulum (ER) stress plays an important role in regulating the signaling pathways concerning cell death and survival. Therefore, we wondered whether the neuroprotective effects of propofol are associated with its regulation on ER stress. In this study, we found that propofol up-regulated BiP and attenuated tunicamycin-induced neural cell death. Propofol pretreatment also inhibited tunicamycin-induced up-regulation of C/EBP homologous protein (CHOP). We also found that propofol or tunicamycin alone increased the levels of spliced XBP1 (XBP1s) and cleaved activating transcription factor 6 (ATF6), an active form of ATF6. However, pretreatment with propofol attenuated the levels of phosphorylated protein kinase receptor-like ER kinase, phosphorylated elF2α, ATF4, and caspase-3, but failed to affect the increase of cleaved ATF6 and XBP1s, induced by tunicamycin. Knockdown endogenous BiP with siRNA abolished the suppression of propofol on tunicamycin-mediated activation of CHOP and caspase-3. Meanwhile, knockdown BiP attenuated the protective effects of propofol on the neural cells exposed to tunicamycin. These data suggest that ER stress is involved in the neuroprotection of propofol via differentially regulating the unfolded protein response pathway, in which BiP plays an important role in initiating the adaptive ER stress and inhibiting the apoptotic ER stress.

Activated macrophage-like synoviocytes are resistant to endoplasmic reticulum stress-induced apoptosis in antigen-induced arthritis.

OBJECTIVE: To explore the characteristic expression of endoplasmic reticulum (ER) stress protein in antigen-induced arthritis models and the role of ER stress in arthritis. METHODS: Effective animal models of rheumatoid arthritis in rabbits and rats were induced by methylated bovine serum albumin and Freund's complete adjuvant. Pathological changes were assessed by magnetic resonance imaging and histological analysis. The expression and localization of ER stress proteins in synovium and peritoneal macrophages (PMΦ) were analyzed by double immunofluorescence staining. RT-PCR was performed to detect mRNA expression of ER stress-related genes. Tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) levels in synoviocytes were measured by RT-PCR and radioimmunoassay. RESULTS: We found that the ER stress marker BiP was highly up-regulated in arthritis synovium and extensively expressed in fibroblast-like synoviocytes (FLS) and macrophage-like synoviocytes (MLS). The expression of the pro-apoptotic factor CHOP/GADD153 was slightly elevated in inflammatory synovium and mainly localized in FLS, but insignificant in MLS. Unexpectedly, increased expression of CHOP was observed in PMΦ in arthritis rats. Likewise, cleaved caspase-3 was rarely expressed in MLS. In addition, induction of ER stress by tunicamycin resulted in significantly increased expression of pro-inflammatory molecules such as IL-1ß and TNF-α in cultured inflammatory FLS. CONCLUSION: Differential activation of the ER stress proteins in synovium MLS may contribute to the resistance of synoviocytes to ER stress-induced apoptosis. Furthermore, ER stress is a potential mediator of arthritis inflammation.

Desmoglein-2 Affects Vascular Function in Moyamoya Disease by Interacting with MMP-9 and Influencing PI3K Signaling.

The pathogenesis and development of Moyamoya disease are still unclear. This study aimed to investigate the effect of desmoglein-2 (DSG2) on Moyamoya disease and determine the inhibitory effect of DSG2 in vascular remodeling in Moyamoya disease.RNA sequencing, immunohistochemistry (IHC), and western blotting were used to detect the expression of DSG2 in the superficial temporal artery (STA) tissues of Moyamoya disease. The association between DSG2 and endothelial cells' biological activities was investigated by cell counting kit-8 (CCK-8), migration assay, tube formation assay, flow cytometry with Annexin V-FITC/PI staining, and TUNEL apoptotic cell detection kit. Pathways affected by overexpression or knockdown of DSG2 were identified in endothelial cells.The expression of DSG2 in the STA tissues of Moyamoya disease was lower than that in normal controls. Overexpression of DSG2 inhibits the proliferation and migration but promotes apoptosis in endothelial cells, and low DSG2 levels result in impaired angiogenesis. In addition, there was an interaction between DSG2 and MMP-9, and DSG2 acted through the PI3K signaling in endothelial cells.Our results indicate that DSG2 affects PI3K signaling in vascular endothelial cells, and MMP-9 is involved in DSG2-mediated vascular changes in Moyamoya disease.

Metastasis pattern and prognosis of large cell neuroendocrine carcinoma: a population-based study.

PURPOSE: As a rare type of tumor, the metastasis pattern of large cell neuroendocrine carcinoma (LCNEC) is still unclear. Our aim was to investigate metastatic patterns and develop a predictive model of prognosis in patients with advanced LCNEC. METHODS: Patients of LCNEC diagnosed between 2010-2015 from the Surveillance, Epidemiology and End Results (SEER) database were retrospectively included. Chi-square test was used for baseline characteristics analysis. Survival differences were assessed using Kaplan-Meier curves. Independent prognostic factors identified by multivariate Cox proportional risk model were used for the construction of nomogram. RESULTS: 557 eligible patients with metastasis LCNEC (median (IQR), 64 (56 to 72) years; 323 males) were included in this research. Among patients with isolated metastases, brain metastases had the highest incidence (29.4%), and multisite metastases had worse OS (HR: 2.020: 95% CI 1.413-2.888; P < 0.001) and LCSS (HR: 2.144, 95% CI 1.480-3.104; P < 0.001) in all age groups. Independent prognostic indicators including age, race, T stage, N stage, chemotherapy, radiotherapy and metastatic site were used for the construction of nomogram. Concordance index (C-index) and decision-curve analyses (DCAs) showed higher accuracy and net clinical benefit of nomogram compared to the 7th TNM staging system (OS: 0.692 vs 0.555; P < 0.001; LCSS: 0.693 vs 0.555; P < 0.001). CONCLUSIONS: We firstly established a novel comprehensive nomogram to predict the prognosis of metastasis LCNEC. The prognostic model demonstrated excellent accuracy and predictive performance. Chemotherapy and metastasis pattern were the two strongest predictive variables. Close follow-up of patients with LCNEC is necessary to make individualized treatment decisions according to different metastasis patterns.

Role of Mesencephalic Astrocyte-Derived Neurotrophic Factor in Alcohol-Induced Liver Injury.

Consumption of alcohol in immoderate quantity induces endoplasmic reticulum (ER) stress response (alcohol-induced ER stress). Mesencephalic astrocyte-derived neurotrophic factor (MANF), an ER stress-inducible protein, works as an evolutionarily conserved regulator of systemic and liver metabolic homeostasis. In this study, the effects of MANF on alcohol-induced liver injury were explored by using hepatocyte-specific MANF-knockout mice (MANF ΔHep) in a chronic-plus-binge alcohol feeding model. We found that alcohol feeding upregulated MANF expression and MANF ΔHep mice exhibited more severe liver injury with extra activated ER stress after alcohol feeding. In addition, we found that MANF deficiency activated iNOS and p65 and increased the production of NO and anti-inflammatory cytokines, which was further enhanced after alcohol treatment. Meanwhile, MANF deletion upregulated the levels of CYP2E1, 4-HNE, and MDA and downregulated the levels of GSH and SOD. These results indicate that MANF has potential protection on alcohol-induced liver injury, and the underlying mechanisms may be associated with meliorating the overactivated ER stress triggered by inflammation and oxidative stress via inhibiting and reducing NO/NF-κB and CYP2E1/ROS, respectively. Therefore, MANF might be a negative regulator in alcohol-induced ER stress and participate in the crosstalk between the NF-κB pathway and oxidative stress in the liver. Conclusions. This study identifies a specific role of MANF in alcohol-induced liver injury, which may provide a new approach for the treatment of ALI.

Amyotrophic lateral sclerosis mutant TDP-43 may cause synaptic dysfunction through altered dendritic spine function.

Altered cortical excitability and synapse dysfunction are early pathogenic events in amyotrophic lateral sclerosis (ALS) patients and animal models. Recent studies propose an important role for TAR DNA-binding protein 43 (TDP-43), the mislocalization and aggregation of which are key pathological features of ALS. However, the relationship between ALS-linked TDP-43 mutations, excitability and synaptic function is not fully understood. Here, we investigate the role of ALS-linked mutant TDP-43 in synapse formation by examining the morphological, immunocytochemical and excitability profile of transgenic mouse primary cortical pyramidal neurons that over-express human TDP-43A315T In TDP-43A315T cortical neurons, dendritic spine density was significantly reduced compared to wild-type controls. TDP-43A315T over-expression increased the total levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropinionic acid (AMPA) glutamate receptor subunit GluR1, yet the localization of GluR1 to the dendritic spine was reduced. These postsynaptic changes were coupled with a decrease in the amount of the presynaptic marker synaptophysin that colocalized with dendritic spines. Interestingly, action potential generation was reduced in TDP-43A315T pyramidal neurons. This work reveals a crucial effect of the over-expression mutation TDP-43A315T on the formation of synaptic structures and the recruitment of GluR1 to the synaptic membrane. This pathogenic effect may be mediated by cytoplasmic mislocalization of TDP-43A315T Loss of synaptic GluR1, and reduced excitability within pyramidal neurons, implicates hypoexcitability and attenuated synaptic function in the pathogenic decline of neuronal function in TDP-43-associated ALS. Further studies into the mechanisms underlying AMPA receptor-mediated excitability changes within the ALS cortical circuitry may yield novel therapeutic targets for treatment of this devastating disease.

Activator protein­1 is a novel regulator of mesencephalic astrocyte­derived neurotrophic factor transcription.

Mesencephalic astrocyte­derived neurotrophic factor (MANF) is an endoplasmic reticulum stress­inducible protein, which has been suggested to be upregulated in inflammatory diseases; however, how inflammation regulates its transcription remains unclear. Activator protein­1 (AP­1), which is a transcription factor complex composed of c­Fos and c­Jun, is activated during the inflammatory process. The present study aimed to investigate whether the AP­1 complex regulates MANF transcription. The results of a luciferase reporter assay revealed that one of three putative AP­1 binding sites in the MANF promoter region is essential for enhancement of MANF transcription. Mechanistically, AP­1 was revealed to directly bind to the promoter region of the MANF gene by chromatin immunoprecipitation assay. Furthermore, MANF was strongly expressed in the liver tissues of patients with hepatitis B virus (HBV) infection, compared with in normal liver tissues from patients with hepatic hemangioma. Furthermore, c­Fos and c­Jun were also upregulated in the nuclei of hepatocytes from patients with HBV infection. In mice treated with carbon tetrachloride, the expression patterns of MANF, c­Fos and c­Jun were similar to those in patients with HBV. These results suggested that the AP­1 complex may be a novel regulator of MANF transcription, which may be involved in liver inflammation and fibrosis.

Facile fabrication of water-dispersible nanocomposites based on hexa-peri-hexabenzocoronene and Fe3O4 for dual mode imaging (fluorescent/MR) and drug delivery.

The facile fabrication of multifunctional nanocomposites (Fe3O4/HBC@F127) consisting of superparamagnetic Fe3O4 nanoparticles and fluorescent organic hexa-peri-hexabenzocoronene (HBC) molecules incorporated in block copolymer diacylphospholipid-polyethyleneglycol F127 have been demonstrated for dual mode imaging (fluorescent/MR) and drug delivery. The obtained nanocomposites were water-dispersible, stable and biocompatible, as confirmed by dynamic light scattering (DLS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Relativity measurements showed a T 2 relaxivity (r 2) of 214.61 mM-1 s-1, which may be used as T 2-weighted MR imaging agents. In vitro imaging studies indicated that the nanocomposites had good MR and fluorescence imaging effects with low cytotoxicity. Besides, the developed nanocomposites could also be applied as drug delivery vehicles. Doxorubicin (DOX) loaded Fe3O4/HBC@F127 nanocomposites significantly inhibited the growth of human hepatoma cells (HepG2). These findings suggested that the facile synthesized multifunctional nanocomposites may be used as a platform for dual mode imaging (both MR and fluorescence) and drug delivery.

Mild and repetitive very mild axonal stretch injury triggers cystoskeletal mislocalization and growth cone collapse.

Diffuse axonal injury is a hallmark pathological consequence of non-penetrative traumatic brain injury (TBI) and yet the axonal responses to stretch injury are not fully understood at the cellular level. Here, we investigated the effects of mild (5%), very mild (0.5%) and repetitive very mild (2×0.5%) axonal stretch injury on primary cortical neurons using a recently developed compartmentalized in vitro model. We found that very mild and mild levels of stretch injury resulted in the formation of smaller growth cones at the tips of axons and a significantly higher number of collapsed structures compared to those present in uninjured cultures, when measured at both 24 h and 72 h post injury. Immunocytochemistry studies revealed that at 72 h following mild injury the axonal growth cones had a significantly higher colocalization of ßIII tubulin and F-actin and higher percentage of collapsed morphology than those present following a very mild injury. Interestingly, cultures that received a second very mild stretch injury, 24 h after the first insult, had a further increased proportion of growth cone collapse and increased ßIII tubulin and F-actin colocalization, compared with a single very mild injury at 72 h PI. In addition, our results demonstrated that microtubule stabilization of axons using brain penetrant Epothilone D (EpoD) (100 nM) resulted in a significant reduction in the number of fragmented axons following mild injury. Collectively, these results suggest that mild and very mild stretch injury to a very localized region of the cortical axon is able to trigger a degenerative response characterized by growth cone collapse and significant abnormal cytoskeletal rearrangement. Furthermore, repetitive very mild stretch injury significantly exacerbated this response. Results suggest that axonal degeneration following stretch injury involves destabilization of the microtubule cytoskeleton and hence treatment with EpoD reduced fragmentation. Together, these results contribute a better understanding of the pathogenesis of mild and repetitive TBI and highlight the therapeutic effect of microtubule targeted drugs on distal part of neurons using a compartmentalized culturing model.