IRGM promotes the PINK1-mediated mitophagy through the degradation of Mitofilin in SH-SY5Y cells.

Mitochondria is a double membrane-bound cellular organelle that generates energy to maintain the homeostasis of cells. Immunity-related GTPase M (IRGM) in human locates at the inner membrane of mitochondria and is best known for its role in regulating autophagy against intracellular pathogens. Previous studies have shown that IRGM is crucial for the normal function of mitochondria, yet, the molecular mechanisms underlying IRGM-mediated quality control of mitochondria are still not fully understood. In this study, we showed that knocking-down IRGM inhibits CCCP induced mitophagy in SH-SY5Y cells. Furthermore, we reported that IRGM decreases the stability of Mitofilin (IMMT, MIC60) in the damaged mitochondria. Knocking down Mitofilin rescues the loss of mitophagy that is observed in the IRGM KD cells, suggesting that IRGM regulates mitophagy through the inhibition of Mitofilin. These data together provide molecular insight regarding how IRGM regulates mitophagy to control the quality of mitochondria.

Up-regulation of MSH6 is associated with temozolomide resistance in human glioblastoma.

The impact of DNA mismatch repair (MMR) on resistance to temozolomide (TMZ) therapy in patients with glioblastoma (GBM) is recently reported but the mechanisms are not understood. We aim to analyze the correlation between MMR function and the acquired TMZ resistance in GBM using both relevant clinical samples and TMZ resistant cells. First we found increased expression of MSH6, one of key components of MMR, in recurrent GBM patients' samples who underwent TMZ chemotherapy, comparing with those matched samples collected at the time of diagnosis. Using the cellular models of acquired resistance to TMZ, we further confirmed the up-regulation of MSH6 in TMZ resistant cells. Moreover, a TCGA dataset contains a large cohort of GBM clinical samples with or without TMZ treatment reinforced the increased expression of MSH6 and other MMR genes after long-term TMZ chemotherapy, which may resulted in MMR dysfunction and acquired TMZ resistance. Our results suggest that increased expression of MSH6, or other MMR, may be a new mechanism contributing to the acquired resistance during TMZ therapy; and may serve as an indicator to the resistance in GBM.

A high frequency of MSH6 G268A polymorphism and survival association in glioblastoma.

MSH6 (mutS homolog 6), one of the five key mismatch repair (MMR) genes, was found to play an important role in conferring resistance to alkylating agents-temozolomide (TMZ) in malignant glioma. This study aims to investigate whether genetic variations in MSH6 gene are associated with the survival outcomes in patients with malignant glioma. Each exon of the MSH6 gene was sequenced, and single nucleotide polymorphism (SNP) analysis was performed using 74 tumor tissues from glioblastoma multiforme (GBM) patients. Among these patients, 54 patients received radiotherapy plus TMZ treatment; 20 patients had radiotherapy only. The promoter methylation of O6-methylguanine methyltransferase (MGMT) was measured by methylation-specific polymerase chain reaction. Literature mining and related data collection were done with NCBI and PubMed databases. Of the 74 GBM patients, 50% (n = 37) harbored MSH6 G268A polymorphism, and no significant rates of other SNP or gene mutation across MSH6 exons were detected. The median overall survival (OS) was 15.6 months for who harbored the SNP and 12.6 months for SNP-negative patients (log-rank test: p = .324). The median OS for the MGMT promoter methylation group (n = 25) and nonmethylation group (n = 29) of the 54 GBM patients treated with TMZ was 21.3 and 8.9 months, respectively, (p = .002). In conclusion, we identified a high frequency of MSH6 G268A polymorphism in MSH6 gene, which did not have a notable influence on survival for the malignant glioma patients with/without TMZ treatment.

IRGM promotes melanoma cell survival through autophagy and is a promising prognostic biomarker for clinical application.

Previously, we showed that mouse immunity-related guanosine triphosphatase (GTPase) family M protein 1 (Irgm1) promotes malignant melanoma progression by inducing cellular autophagy flux and metastasis. Human IRGM, a truncated protein functionally distinct from its mouse counterpart, has several splice isoforms. In this study, we analyzed the association of IRGM and human melanoma clinical prognosis and investigated the function of IRGM in human melanoma cells. Data from the training cohort (n = 144) showed that overexpression of IRGM is proportional to melanoma genesis and clinical stages in human tissue chips. A validation cohort (n = 78) further confirmed that IRGM is an independent risk factor promoting melanoma progression and is associated with poor survival of patients. Among IRGM isoforms, we found that IRGMb is responsible for such correlation. In addition, IRGM promoted melanoma cell survival through autophagy, both in vitro and in vivo. We further showed that the blockade of translocation of high-mobility group box 1 (HMGB1) from the nucleus to cytoplasm inhibits IRGM1-mediated cellular autophagy and reduces cell survival. IRGM functions as a positive regulator of melanoma progression through autophagy and may serve as a promising prognostic marker and therapeutic target.

Combination of mild therapeutic hypothermia and adipose-derived stem cells for ischemic brain injury.

Mild therapeutic hypothermia has been shown to mitigate cerebral ischemia, reduce cerebral edema, and improve the prognosis of patients with cerebral ischemia. Adipose-derived stem cell-based therapy can decrease neuronal death and infiltration of inflammatory cells, exerting a neuroprotective effect. We hypothesized that the combination of mild therapeutic hypothermia and adipose-derived stem cells would be neuroprotective for treatment of stroke. A rat model of transient middle cerebral artery occlusion was established using the nylon monofilament method. Mild therapeutic hypothermia (33°C) was induced after 2 hours of ischemia. Adipose-derived stem cells were administered through the femoral vein during reperfusion. The severity of neurological dysfunction was measured by a modified Neurological Severity Score Scaling System. The area of the infarct lesion was determined by 2,3,5-triphenyltetrazolium chloride staining. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The regeneration of microvessels and changes in the glial scar were detected by immunofluorescence staining. The inflammatory responses after ischemic brain injury were evaluated by in situ staining using markers of inflammatory cells. The expression of inflammatory cytokines was measured by reverse transcription-polymerase chain reaction. Compared with mild therapeutic hypothermia or adipose-derived stem cell treatment alone, their combination substantially improved neurological deficits and decreased infarct size. They synergistically reduced the number of TUNEL-positive cells and glial fibrillary acidic protein expression, increased vascular endothelial growth factor levels, effectively reduced inflammatory cell infiltration and down-regulated the mRNA expression of the proinflammatory cytokines interleukin-1ß, tumor necrosis factor-α and interleukin-6. Our findings indicate that combined treatment is a better approach for treating stroke compared with mild therapeutic hypothermia or adipose-derived stem cells alone.

WITHDRAWN: Correlations of CTLA-4 exon-1 rs231775A>G and promoter region rs5742909C>T polymorphisms with the therapeutic efficacy of 131I radionuclide in Graves' disease.

The Accepted Manuscript version of this article (published on 6 June 2017) was withdrawn on 16 November 2017 at the request of the authors.

Intravenous Administration of Adipose-Derived Stem Cell Protein Extracts Improves Neurological Deficits in a Rat Model of Stroke.

Treatment of adipose-derived stem cell (ADSC) substantially improves the neurological deficits during stroke by reducing neuronal injury, limiting proinflammatory immune responses, and promoting neuronal repair, which makes ADSC-based therapy an attractive approach for treating stroke. However, the potential risk of tumorigenicity and low survival rate of the implanted cells limit the clinical use of ADSC. Cell-free extracts from ADSC (ADSC-E) may be a feasible approach that could overcome these limitations. Here, we aim to explore the potential usage of ADSC-E in treating rat transient middle cerebral artery occlusion (tMCAO). We demonstrated that intravenous (IV) injection of ADSC-E remarkably reduces the ischemic lesion and number of apoptotic neurons as compared to other control groups. Although ADSC and ADSC-E treatment results in a similar degree of a long-term clinical beneficial outcome, the dynamics between two ADSC-based therapies are different. While the injection of ADSC leads to a relatively mild but prolonged therapeutic effect, the administration of ADSC-E results in a fast and pronounced clinical improvement which was associated with a unique change in the molecular signature suggesting that potential mechanisms underlying different therapeutic approach may be different. Together these data provide translational evidence for using protein extracts from ADSC for treating stroke.

Irgm1 promotes M1 but not M2 macrophage polarization in atherosclerosis pathogenesis and development.

BACKGROUND AND AIMS: Atherosclerosis is a chronic inflammatory vascular disease related to macrophages uptake of low-density lipoprotein and their subsequent transformation into foam cells. M1 (inflammatory)/M2 (anti-inflammatory) balance was suggested to impact disease progression. In this study, we investigated whether the immunity related GTPase (Irgm1) regulates macrophage polarization during atherosclerosis development. METHODS: We used apolipoprotein E (ApoE) knockout and Irgm1 haplodeficient mice and induced atherosclerosis with high-cholesterol diet for the indicated months. Atherosclerotic arteries were collected from patients undergoing vascular surgery, to determine the lesional expression of Irgm1 and distribution of M1/M2 populations. RESULTS: Our results showed that IRGM/Irgm1 expression was increased in atherosclerotic artery samples (1.7-fold, p=0.0045) compared with non-atherosclerotic arteries, which was consistent with findings in the murine experimental atherosclerosis model (1.9-fold, p=0.0002). IRGM/Irgm1 expression was mostly found in lesional M1 macrophages. Haplodeficiency of Irgm1 in ApoE(-/-) mice resulted in reduced infiltrating M1 macrophages in atheroma (94%, p=0.0002) and delayed development of atherosclerotic plaques. In vitro experiments also confirmed that Irgm1 haplodeficiency reduced iNOS expression of polarized M1 macrophages (81%, p=0.0034), with negligible impact on the M2 phenotype. Moreover, we found that Irgm1 haplodeficiency in mice significantly reduced expression level of M1 function-related transcription factors, interferon regulatory factor (Irf) 5 and Irf8, but not Irf4, an M2-related transcription factor. CONCLUSIONS: This study shows that Irgm1/IRGM participates in the polarization of M1 macrophage and promotes development of atheroma in murine experimental atherosclerosis.

IRGM1 enhances B16 melanoma cell metastasis through PI3K-Rac1 mediated epithelial mesenchymal transition.

Melanoma is one of the most aggressive skin cancers and is well known for its high metastatic rate. Studies have shown that epithelial mesenchymal transition (EMT) is essential for melanoma cell metastasis. However, the molecular mechanisms underlying EMT are still not fully understood. We have shown that IRGM1, a member of immunity-related GTPase family that regulates immune cell motility, is highly expressed by melanoma cells. The current study aimed to explore whether and how IRGM1 may regulate melanoma cell metastasis. To test this, we modified IRGM1 expression in B16 melanoma cells. We found that over-expression of IRGM1 substantially enhanced pulmonary metastasis in vivo. In keeping with that, knocking-in IRGM1 strongly enhanced while knocking-down IRGM1 impaired B16 cell migration and invasion ability in vitro. Interestingly, we observed that IRGM1 enhanced F-actin polymerization and triggers epithelial mesenchymal transition (EMT) through a mechanism involved in PIK3CA mediated Rac1 activation. Together, these data reveals a novel molecular mechanism that involved in melanoma metastasis.

IRGM1 regulates oxidized LDL uptake by macrophage via actin-dependent receptor internalization during atherosclerosis.

Macrophage derived foam cells are actively involved in the initial phase of atherosclerosis. Uptake of modified lipoprotein such as oxidized LDL (oxLDL) is a critical step for foam cell formation. CD36 is the major receptor mediating oxLDL uptake by macrophage. However, the molecular mechanism underlying CD36 mediated oxLDL uptake remains unclear. Here we reported that IRGM1 (IRGM in human), a member of immunity-related small GTPase family, is essential for the actin-dependent CD36 mediated oxLDL uptake by macrophage. IRGM/IRGM1 was highly expressed by macrophage around the atherosclerotic plaque and was up-regulated by oxLDL both in vitro and in vivo. Moreover loss of IRGM/IRMG1 significantly decreased oxLDL uptake in both mouse and human. Furthermore, the IRGM1 knock-out mice displayed impaired CD36 internalization in macrophage, which was associated with the deficiency of F-actin polymerization. These results revealed a novel function of IRGM1 in regulating oxLDL uptake by macrophage during atherosclerosis.

TP53 codon 72 polymorphism and glioma risk: A meta-analysis.

TP53 codon 72 polymorphism has been reported to affect regulatory networks central to glioma development. Although a number of published studies noted the association between TP53 codon 72 polymorphism and glioma risk, their conclusions were inconsistent. A meta-analysis was used to assess the possible association between TP53 codon 72 polymorphism and glioma risk. The PubMed databases were searched, relevant articles were identified and data were retrieved based on the inclusion criteria. The odds ratio (OR) and 95% confidence interval (95% CI) were determined on the pooled dataset. We retrieved eight different studies including 2,260 glioma cases and 3,506 controls. However, no association was found between the TP53 codon 72 polymorphism and glioma risk regarding the comparison between glioma cases and the controls. By further stratification based on criteria such as tumor grade, and the geographical location of the patients and the relevant controls, we found a significant association in the subgroup of patients with high-grade glioma in Europeans compared to controls in two models of TP53 codon 72 polymorphism, which include the dominant model [C/C + G/C vs. G/G: OR=1.35, 95% CI (1.14, 1.59), P=0.0005, P(h)=0.13] and the additive model [C allele vs. G allele: OR=1.16, 95% CI (1.02, 1.33), P=0.03, P(h)=0.37]. Our analysis suggests that TP53 codon 72 polymorphism is associated with an increased risk of high-grade glioma development in Europeans.

Immune-related GTPase M (IRGM1) regulates neuronal autophagy in a mouse model of stroke.

Autophagy is an important cellular recycling mechanism through self-digestion in responses to cellular stress such as starvation. Studies have shown that autophagy is involved in maintaining the homeostasis of the neural system during stroke. However, molecular mechanisms underlying neuronal autophagy in ischemic stroke remain poorly understood. Previously, we and others have shown that immune-related GTPase M (IRGM; termed IRGM1 in the mouse nomenclature) can regulate the survival of immune cells through autophagy in response to infections and autoimmune conditions. Here, using a permanent middle cerebral artery occlusion (pMCAO) mouse model, we found that IRGM1 was upregulated in the ischemic side of the brain, which was accompanied by a significant autophagic response. In contrast, neuronal autophagy was almost complete lost in Irgm1 knockout (KO) mice after pMCAO induction. In addition, the infarct volume in the Irgm1-KO pMCAO mice was significantly increased as compared to wild-type mice. Histological studies suggested that, at the early stage (within 24 h) of ischemia, the IRGM1-dependent autophagic response is associated with a protection of neurons from necrosis in the ischemic core but a promotion of neuronal apoptosis in the penumbra area. These data demonstrate a novel role of IRGM1 in regulating neuronal autophagy and survival during ischemic stroke.