Characterization of the structural and functional determinants of MANF/CDNF in Drosophila in vivo model

PLoS One. 2013 Sep 3;8(9):e73928. doi: 10.1371/journal.pone.0073928. eCollection 2013.

Abstract

Mammalian MANF and CDNF proteins are evolutionarily conserved neurotrophic factors that can protect and repair mammalian dopaminergic neurons in vivo. In Drosophila, the sole MANF protein (DmManf) is needed for the maintenance of dopaminergic neurites and dopamine levels. Although both secreted and intracellular roles for MANF and CDNF have been demonstrated, very little is known about the molecular mechanism of their action. Here, by using a transgenic rescue approach in the DmManf mutant background we show that only full-length MANF containing both the amino-terminal saposin-like and carboxy-terminal SAP-domains can rescue the larval lethality of the DmManf mutant. Independent N- or C-terminal domains of MANF, even when co-expressed together, fail to rescue. Deleting the signal peptide or mutating the CXXC motif in the C-terminal domain destroys the activity of full-length DmManf. Positively charged surface amino acids and the C-terminal endoplasmic reticulum retention signal are necessary for rescue of DmManf mutant lethality when DmManf is expressed in a restricted pattern. Furthermore, rescue experiments with non-ubiquitous expression reveals functional differences between the C-terminal domain of human MANF and CDNF. Finally, DmManf and its C-terminal domain rescue mammalian sympathetic neurons from toxin-induced apoptosis in vitro demonstrating functional similarity of the mammalian and fly proteins. Our study offers further insights into the functional conservation between invertebrate and mammalian MANF/CDNF proteins and reveals the importance of the C-terminal domain for MANF activity in vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Animals, Genetically Modified
  • Apoptosis / physiology
  • Dopamine / metabolism
  • Drosophila Proteins / chemistry*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / physiology*
  • Drosophila melanogaster / growth & development
  • Drosophila melanogaster / physiology*
  • Genes, Lethal
  • Humans
  • Larva / metabolism
  • Molecular Sequence Data
  • Nerve Growth Factors / chemistry*
  • Nerve Growth Factors / genetics
  • Nerve Growth Factors / physiology*
  • Protein Conformation
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid

Substances

  • CDNF protein, Drosophila
  • Drosophila Proteins
  • MANF protein, Drosophila
  • Nerve Growth Factors
  • Dopamine

Grant support

RL was supported by Viikki Doctoral Programme in Molecular Biosciences (http://www.helsinki.fi/vgsb/), The Finnish Parkinson Foundation (http://www.parkinsonsaatio.fi/), The Ella and Georg Ehrnrooth Foundation (http://www.ellageorg.fi/), The University of Helsinki Funds, and Alfred Kordelin Foundation (http://www.kordelin.fi/). PL was supported by The Academy of Finland (grant No. 139910; http://www.aka.fi/en-GB/A/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.