Role of two sequence motifs of mesencephalic astrocyte-derived neurotrophic factor in its survival-promoting activity.

K Mätlik,M Hellman,A Eesmaa,P Permi,J Peränen,M Airavaara,Li-Ying Yu,J Anttila,M Saarma,P Lindholm,E Galli,U Arumäe

doi:10.1038/cddis.2015.371

Abstract

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a prosurvival protein that protects the cells when applied intracellularly in vitro or extracellularly in vivo. Its protective mechanisms are poorly known. Here we studied the role of two short sequence motifs within the carboxy-(C) terminal domain of MANF in its neuroprotective activity: the CKGC sequence (a CXXC motif) that could be involved in redox reactions, and the C-terminal RTDL sequence, an endoplasmic reticulum (ER) retention signal. We mutated these motifs and analyzed the antiapoptotic effect and intracellular localization of these mutants of MANF when overexpressed in cultured sympathetic or sensory neurons. As an in vivo model for studying the effect of these mutants after their extracellular application, we used the rat model of cerebral ischemia. Even though we found no evidence for oxidoreductase activity of MANF, the mutation of CXXC motif completely abolished its protective effect, showing that this motif is crucial for both MANF's intracellular and extracellular activity. The RTDL motif was not needed for the neuroprotective activity of MANF after its extracellular application in the stroke model in vivo. However, in vitro the deletion of RTDL motif inactivated MANF in the sympathetic neurons where the mutant protein localized to Golgi, but not in the sensory neurons where the mutant localized to the ER, showing that intracellular MANF protects these peripheral neurons in vitro only when localized to the ER.

Highlights

In line with its role in counteracting cell death, mesencephalic astrocyte-derived neurotrophic factor (MANF) promotes pancreatic β-cell proliferation and survival in vivo, and lack of MANF leads to chronic unfolded protein response (UPR) activation in pancreatic islets.[18]
We have shown that microinjected intracellular MANF protects cultured sympathetic neurons of the superior cervical ganglion (SCG) against apoptosis-inducing toxins, whereas it does not protect or bind these neurons when applied to the culture medium.[19]
In its C-terminal domain, MANF has a conserved sequence 148CKGC151.19,20. To test whether this motif is essential for the survival-promoting function of MANF, we mutated cysteine 151 into serine (CKGC to CKGS) and overexpressed this construct (MANF-C151S) in the SCG neurons treated with etoposide, a cytotoxic drug that causes cell death by inhibition of topoisomerase II

Summary

Introduction

In line with its role in counteracting cell death, MANF promotes pancreatic β-cell proliferation and survival in vivo, and lack of MANF leads to chronic unfolded protein response (UPR) activation in pancreatic islets.[18]. We mutated the CXXC and RTDL motifs and tested the activity of the mutants by microinjecting plasmid DNAs encoding the mutant proteins into apoptotic sympathetic SCG neurons, our validated model for testing the neuroprotective bioactivity of MANF,[19,21] and sensory dorsal root ganglion (DRG) neurons. We show that the CXXC motif is critically required for the survival-promoting activity of MANF, as mutation of this motif prevents MANF from being neuroprotective in both types of neurons in vitro and in the in vivo rat model of cerebral ischemia.

Methods

Results

Conclusion