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Transcriptional regulation of mouse mesencephalic astrocyte-derived neurotrophic factor in Neuro2a cells.

ABSTRACT: Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel type of trophic factor. Recent studies indicate that the MANF gene is induced in response to endoplasmic reticulum (ER) stress through ER stress response element II (ERSE-II) in its 5'-flanking region. In this study, we evaluated the roles of six ER stress response transcription factors in the regulation of the promoter activities of the mouse MANF gene via ERSE-II using various types of mutant MANF luciferase reporter constructs. Treatment with thapsigargin (Tg) induced MANF mRNA generation in parallel with the elevation of ATF6?, sXBP and Luman mRNA levels in Neuro2a cells. Of the six transcription factors, ATF6? most strongly increased the MANF promoter activity via ERSE-II, while the effects of ATF6? and sXBP1 were moderate. However, overexpression of Luman or OASIS did not enhance ERSE-II-dependent MANF promoter activity in Neuro2a cells. To evaluate the relationships between transcription factors in the regulation of ERSE-II-dependent MANF promoter activity, we transfected two effective transcription factor constructs chosen from ATF6?, ATF6?, uXBP1 and sXBP1 into Neuro2a cells with the MANF reporter construct. The MANF promoter activity induced by co-transfection of ATF6? with ATF6? was significantly lower than that induced by ATF6? alone, while other combinations did not show any effect on the ERSE-II-dependent MANF promoter activity in Neuro2a cells. Our study is the first to show the efficiency of ER stress-related transcription factors for ERSE-II in activating the transcription of the mouse MANF gene in Neuro2a cells.

SUBMITTER: Oh-Hashi K

PROVIDER: S-EPMC6275609 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Transcriptional regulation of mouse mesencephalic astrocyte-derived neurotrophic factor in Neuro2a cells.

Oh-Hashi Kentaro K Hirata Yoko Y Kiuchi Kazutoshi K

Cellular & molecular biology letters 20130717 3

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel type of trophic factor. Recent studies indicate that the MANF gene is induced in response to endoplasmic reticulum (ER) stress through ER stress response element II (ERSE-II) in its 5'-flanking region. In this study, we evaluated the roles of six ER stress response transcription factors in the regulation of the promoter activities of the mouse MANF gene via ERSE-II using various types of mutant MANF luciferase reporter constru ...[more]

PMID: 23864333

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Project description:Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) form a family of atypical growth factors discovered for their neuroprotective properties in the central nervous system (CNS) in animal models of neurodegenerative diseases. Although their mechanism of protective action still remains unclear, it has been suggested that both MANF and CDNF promote cell survival through regulating the unfolded protein response (UPR), thereby relieving endoplasmic reticulum (ER) stress. Recent studies identified MANF for its emerging roles in metabolic function, inflammation and pancreatic β-cells. We have found that MANF deletion from the pancreas and β-cells leads to postnatal depletion of β-cells and diabetes. Moreover, global MANF-deficiency in mice results in severe diabetes-independent growth retardation. As the expression pattern of MANF in mouse tissues has not been extensively studied, we set out to thoroughly investigate MANF expression in embryonic and adult mice using immunohistochemistry, histochemical X-gal staining, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription PCR (RT-qPCR). We found that MANF is highly expressed in brain neurons regulating energy homeostasis and appetite, as well as in hypothalamic nuclei producing hormones and neuropeptides important for different body functions. Strong expression of MANF was also observed in peripheral mouse tissues and cells with high secretory and metabolic function. These include pituitary gland and interestingly we found that the anterior pituitary gland is smaller in MANF-deficient mice compared to wild-type mice. Consequently, we found reduction in the number of growth hormone- and prolactin-producing cells. This combined with increased expression of UPR genes, reduced number of proliferating cells in the anterior pituitary and dysregulated expression of pituitary hormones might contribute to the severe growth defect seen in the MANF knockout mice. Moreover, in this study we compared MANF and CDNF levels in mouse tissues. Unlike MANF, CDNF protein levels are generally lower in mouse tissues, and the highest levels of CDNF was observed in the tissues with high-energy demands and oxidative roles, including heart, muscle, testis, and brown adipose tissue.

Project description:Alcohol exposure has detrimental effects on both the developing and mature brain. Endoplasmic reticulum (ER) stress is one of the mechanisms that contributes to alcohol-induced neuronal damages. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress-responsive protein and is neuroprotective in multiple neuronal injury and neurodegenerative disease models. MANF deficiency has been shown to exacerbate alcohol-induced ER stress and neurodegeneration. However, it is unknown whether MANF supplement is sufficient to protect against alcohol neurotoxicity. Alcohol alters MANF expression in the brain, but the mechanisms underlying alcohol modulation of MANF expression remain unclear. This study was designed to determine how alcohol alters MANF expression in neuronal cells and whether exogeneous MANF can alleviate alcohol neurotoxicity. We showed that alcohol increased MANF transcription and secretion without affecting MANF mRNA stability and protein degradation. ER stress was necessary for alcohol-induced MANF upregulation, as pharmacological inhibition of ER stress by 4-PBA diminished alcohol-induced MANF expression. In addition, the presence of ER stress response element II (ERSE-II) was required for alcohol-stimulated MANF transcription. Mutations or deletion of this sequence abolished alcohol-regulated transcriptional activity. We generated MANF knockout (KO) neuronal cells using CRISPR/Cas9. MANF KO cells exhibited increased unfolded protein response (UPR) and were more susceptible to alcohol-induced cell death. On the other hand, MANF upregulation by the addition of recombinant MANF protein or adenovirus gene transduction protected neuronal cells against alcohol-induced cell death. Further studies using early postnatal mouse pups demonstrated that enhanced MANF expression in the brain by intracerebroventricular (ICV) injection of MANF adeno-associated viruses ameliorated alcohol-induced cell death. Thus, alcohol increased MANF expression through inducing ER stress, which could be a protective response. Exogenous MANF was able to protect against alcohol-induced neurodegeneration.

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Transcriptional regulation of mouse mesencephalic astrocyte-derived neurotrophic factor in Neuro2a cells.

Publications

Transcriptional regulation of mouse mesencephalic astrocyte-derived neurotrophic factor in Neuro2a cells.

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