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Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors.

ABSTRACT: Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress-responsive protein with neuroprotective effects in animal models of neurodegeneration, but the underlying mechanism is not understood. We constructed a set of lentiviral vectors that contain or lack the highly conserved final four amino acids of MANF ("RTDL"), which resemble the canonical ER retention signal ("KDEL"), to study MANF regulation in neuroblastoma cells and rat primary cortical neurons. The RTDL sequence was required for both ER retention and secretory response to ER stress. Overexpression of KDEL receptor paralogs (KDELRs) differentially reduced MANF secretion but had no effect on MANF lacking RTDL. MANF binding to the plasma membrane also required the RTDL sequence and was inhibited with a peptide known to interact with KDELRs, suggesting MANF binds KDELRs at the surface. We detected surface localization of FLAG-tagged KDELRs, with levels increasing following ER stress. Our study provides new insight into the regulation of MANF trafficking and has implications for other secreted proteins containing a KDEL-like retention signal.

SUBMITTER: Henderson MJ

PROVIDER: S-EPMC3567670 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors.

Henderson Mark J MJ Richie Christopher T CT Airavaara Mikko M Wang Yun Y Harvey Brandon K BK

The Journal of biological chemistry 20121219 6

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress-responsive protein with neuroprotective effects in animal models of neurodegeneration, but the underlying mechanism is not understood. We constructed a set of lentiviral vectors that contain or lack the highly conserved final four amino acids of MANF ("RTDL"), which resemble the canonical ER retention signal ("KDEL"), to study MANF regulation in neuroblastoma cells and rat primary cortical neurons. ...[more]

PMID: 23255601

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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:BackgroundExtracellular accumulation of amyloid ?-peptide (A?) is one of pathological hallmarks of Alzheimer's disease (AD) and contributes to the neuronal loss. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress-inducible neurotrophic factor. Many groups, including ours, have proved that MANF rescues neuronal loss in several neurological disorders, such as Parkinson's disease and cerebral ischemia. However, whether MANF exerts its protective effect against A? neurotoxicity in AD remains unknown.MethodsIn the present study, the characteristic expressions of MANF in A?1-42-treated neuronal cells as well as in the brains of APP/PS1 transgenic mice were analyzed by immunofluorescence staining, qPCR, and Western blot. The effects of MANF overexpression, MANF knockdown, or recombination human MANF protein (rhMANF) on neuron viability, apoptosis, and the expression of ER stress-related proteins following A?1-42 exposure were also investigated.ResultsThe results showed the increased expressions of MANF, as well as ER stress markers immunoglobulin-binding protein (BiP) and C/EBP homologous protein (CHOP), in the brains of the APP/PS1 transgenic mice and A?1-42-treated neuronal cells. MANF overexpression or rhMANF treatment partially protected against A?1-42-induced neuronal cell death, associated with marked decrease of cleaved caspase-3, whereas MANF knockdown with siRNA aggravated A?1-42 cytotoxicity including caspase-3 activation. Further study demonstrated that the expressions of BiP, ATF6, phosphorylated-IRE1, XBP1s, phosphorylated-eIF2?, ATF4, and CHOP were significantly downregulated by MANF overexpression or rhMANF treatment in neuronal cells following A?1-42 exposure, whereas knockdown of MANF has the opposite effect.ConclusionsThese findings demonstrate that MANF may exert neuroprotective effects against A?-induced neurotoxicity through attenuating ER stress, suggesting that an applicability of MANF as a therapeutic candidate for AD.

Dataset Information

Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors.

Publications

Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors.

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