MANF is widely expressed in mammalian tissues and differently regulated after ischemic and epileptic insults in rodent brain

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Abstract

The mesencephalic astrocyte-derived neurotrophic factor (MANF) has been described as a survival factor for dopaminergic neurons in vitro, but its expression in mammalian tissues is poorly known. MANF and a homologous protein, the conserved dopamine neurotrophic factor (CDNF), form a novel evolutionary conserved family of neurotrophic factors. Here we used in situ hybridization and immunohistochemistry to characterize MANF expression in developing and adult mouse. MANF expression was widespread in the nervous system and non-neuronal tissues. In the brain, relatively high MANF levels were detected in the cerebral cortex, hippocampus and cerebellar Purkinje cells. After status epilepticus, Manf mRNA expression was transiently increased in the dentate granule cell layer of hippocampus, thalamic reticular nucleus and in several cortical areas. In contrast, following global forebrain ischemia changes in Manf expression were widespread in the hippocampal formation and more restricted in cerebral cortex. The widespread expression of MANF together with its evolutionary conserved nature and regulation by brain insults suggest that it has important functions both under normal and pathological conditions in many tissue types.

Introduction

Neurotrophic factors are secreted proteins that promote neuronal survival and enhance formation and maintenance of neuronal connections in the vertebrate nervous system (Huang and Reichardt, 2001, Airaksinen and Saarma, 2002). The role of neurotrophic factors is well characterized mainly in the peripheral nervous system (PNS). In recent years, substantial knowledge has accumulated also about the involvement of growth factors in regulating the development and maintenance of neuronal populations in the central nervous system (CNS).

The characteristic motor symptoms of Parkinson's disease (PD) result from gradual degeneration of midbrain dopaminergic neurons. Several growth factors can act as survival factors for dopaminergic neurons (Krieglstein, 2004, Bespalov and Saarma, 2007), but their roles in the development and maintenance of these neurons in vivo are still unknown. Glial cell line-derived neurotrophic factor (GDNF; Lin et al., 1993) protects and even repairs the dopaminergic system in rodent and primate models of PD (Hoffer et al., 1994, Sauer et al., 1995, Kearns and Gash, 1995, Tomac et al., 1995, Gash et al., 1996, Grondin et al., 2002). However, results from clinical trials with GDNF on Parkinsonian patients have been controversial, since either clinical improvement (Gill et al., 2003, Patel et al., 2005) or low clinical efficacy (Lang et al., 2006) has been reported. Therefore, identification and characterization of new factors which could be neuroprotective and support functional neurorestoration is highly warranted.

Ischemic and epileptic insults, which are associated with neuronal death, trigger changes in the expression of several neurotrophic factors in the brain such as the GDNF family ligands (Kokaia et al., 1999, Arvidsson et al., 2001) and the neurotrophins, e.g., BDNF (Ernfors et al., 1991, Lindvall et al., 1992). Changes in neurotrophic factor levels following brain insults may regulate cell survival, synaptic plasticity, epileptogenesis, and neurogenesis from endogenous neural stem cells (Binder et al., 2001, Gustafsson et al., 2003, Jin et al., 2003, Kuipers and Bramham, 2006, Schäbitz et al., 2007). Therefore, modulation of neurotrophic factor levels could become of therapeutic value in ischemic and epileptic conditions.

A novel neurotrophic factor mesencephalic astrocyte-derived neurotrophic factor (MANF; also known as arginine-rich, mutated in early stage tumors; ARMET) was described few years ago as a survival-promoting factor for embryonic midbrain dopaminergic neurons in vitro (Petrova et al., 2003). The observed survival promoting effect of MANF was specific for dopaminergic neurons, and no effects on serotonergic or GABAergic neurons were detected (Petrova et al., 2003). Our group has recently characterized a vertebrate-specific paralog of MANF, named the conserved dopamine neurotophic factor (CDNF). CDNF (ARMET-like1) protects and rescues midbrain dopaminergic neurons in vivo in a rat 6-OHDA model of Parkinson's disease (Lindholm et al., 2007). MANF and CDNF proteins form a new family of evolutionarily conserved factors (Lindholm et al., 2007). Importantly, the effects of MANF and CDNF on dopaminergic neurons suggest that these factors could be used in treatment of neurodegenerative disorders (Petrova et al., 2003, Lindholm et al., 2007). However, in vivo data of MANF are still lacking.

To reveal tissues in which MANF may have function, and since the tissue expression of mammalian MANF is poorly known, we characterized the distribution of Manf mRNA and protein in developing and adult brain and in non-neuronal tissues. We also analyzed the regulation of Manf mRNA after two pathological conditions, status epilepticus and global forebrain ischemia.

Section snippets

MANF is a secreted protein with intramolecular disulfide bridges

We cloned full-length cDNA encoding human and mouse MANF from liver and brain, respectively, by RT-PCR. Based on sequence analysis, human MANF amino acid sequence (SwissProt Acc. No. P55145) was 98% homologous with mouse (GenBank Acc. No. NP_083379), 82% with Xenopus laevis (Genbank Acc. No. AAH82888) 72% with zebrafish Danio rerio (GenBank Acc. No. AAI24317) and 50% with Caenorhabditis elegans MANF protein (GenBank Acc. No. NP_500273), respectively (Fig. 1A, Table 1). It is important to notice

Discussion

Neurotrophic factors, including GDNF family ligands and neurotrophins, have essential roles in the development, maintenance and function of the vertebrate nervous system (Huang and Reichardt, 2001, Airaksinen and Saarma, 2002). Neurotrophic factors promote the survival of neurons during developmental periods of programmed cell death (PCD) and regulate the density of target innervation. However, it is still poorly understood how trophic factors regulate the development and maintenance of the

Cloning and expression analysis by RT-PCR

Full-length MANF cDNA from human and mouse was cloned by RT-PCR (reverse transcription polymerase chain reaction). Human RNAs of peripheral and total brain tissues were obtained from Clontech (Mountain View, CA). RNAs from post-mortem human brain regions and mouse total RNAs were isolated using Ambion's RNAwiz reagent (Applied Biosystems, Austin, TX). Experiments with human tissues were approved by the local ethical committee. First strand cDNAs were synthesized with SuperscriptII reverse

Acknowledgments

This study was supported by the Sigrid Jusélius Foundation (M.S. and T.T.), the Academy of Finland Neuroscience Program Grant 1111926 (M.S.), the Estonian Ministry of Education and Research Grant 0222602 (T.T.), and by the Swedish Research Council. T. T. is a Wellcome Trust International Senior Research Fellow. We thank Dr. Veli-Matti Leppänen for help in protein purification, Dr. Avtandil Nanobashvili and Dr. Elin Gustafsson for help with epilepsy and ischemia experiments, and Dr. Enn Jõeste

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