Abstract
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress–regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone glucose-regulated protein 78 (GRP78). We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time that MANF promotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting several UPR pathways simultaneously. Interestingly, MANF does not affect naïve neurons. We hypothesize that MANF regulates UPR signaling toward a mode more compatible with neuronal survival. Screening of MANF interacting proteins from two mammalian cell lines revealed a conserved interactome of 15 proteins including several ER chaperones such as GRP78, GRP170, protein disulfide isomerase family A member 1, and protein disulfide isomerase family A member 6. Further characterization confirmed previously published finding that MANF is a cofactor of GRP78 interacting with its nucleotide binding domain. Using microscale thermophoresis and nuclear magnetic resonance spectroscopy, we discovered that MANF is an ATP binding protein and that ATP blocks the MANF–GRP78 interaction. Interestingly, functional analysis of the antiapoptotic properties of MANF mutants in cultured neurons revealed divergent roles of MANF as a GRP78 cofactor and as an antiapoptotic regulator of UPR. We conclude that the co-factor type interaction with GRP78 is dispensable for the survival-promoting activity of MANF in neurons.
Highlights
Title: The cytoprotective protein Mesencephalic astrocyte-derived neurotrophic factor (MANF) promotes neuronal survival independently from its role as a glucose-regulated protein 78 (GRP78) cofactor
Overexpression of MANF by plasmid or protein microinjection into superior cervical ganglion (SCG) neurons has been shown to promote their survival against serum deprivation, topoisomerase II inhibitor etoposide, and protein kinase inhibitor staurosporine, whereas MANF added to the culture medium has no effect on the survival of SCG neurons [15, 47]
The survival of Tm-treated SCG neurons injected with MANF plasmid (Fig. 1, A and B) or MANF protein (Fig. 1C) was significantly increased as compared with neurons injected with pCR3.1 control plasmid or PBS, respectively
Summary
Title: The cytoprotective protein MANF promotes neuronal survival independently from its role as a GRP78 cofactor. Initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone glucose-regulated protein 78 (GRP78). We show for the first time that MANF promotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting several UPR pathways simultaneously. Mesencephalic astrocyte-derived neurotrophic factor (MANF, known as arginine-rich, mutated in early stage tumors—ARMET) was originally characterized as a protein secreted from the rat type-1 astrocyte ventral mesencephalic cell line as a growth factor able to promote the survival of cultured midbrain dopamine (DA) neurons [1].
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