Mesencephalic astrocyte-derived neurotrophic factor prevents neuron loss via inhibiting ischemia-induced apoptosis

https://doi.org/10.1016/j.jns.2014.06.042Get rights and content

Highlights

  • MANF prevented neuronal loss induced by ischemia in hippocampus and cortex.

  • MANF rescued neural cells from apoptosis induced by focal cerebral ischemia

  • MANF repressed the cleavage of caspase-3 triggered by focal cerebral ischemia.

  • MANF reduced the elevation of BIP/Grp78, p-IRE1, and XBP1s induced by MACO.

  • MANF did not affect the expression of XBP1u and XBP1s mRNA up-regulated by MACO.

Abstract

Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been shown to be up-regulated under the focal cerebral ischemia and protected against ischemic injury in rats. However, the underlying mechanisms are unclear. The aim of this study was to verify the protection of MANF on the cerebral ischemic injury and further investigate the possible mechanisms. Rat focal ischemic model was established by middle cerebral artery occlusion (MCAO). The recombinant human MANF was therapeutically administrated to the ipsilateral ventricle at 2 h after MCAO. MANF decreased the number of the propidium iodide (PI)- and TUNEL-positive neural cells. Contrarily, MANF protected the NeuN-positive cells in hippocampus and cortex from death induced by ischemia. The more interesting results in this study were that MANF repressed the cleavage of caspase-3 triggered by focal cerebral ischemia. MANF also reduced the elevated levels of BIP/Grp78, phosphorylated IRE1, and splicing XBP1 induced by focal cerebral ischemia, but not affect CHOP expression. Meanwhile, focal cerebral ischemia elevated the levels of XBP1 mRNA, including unspliced XBP1 (XBP1u) and spliced XBP1 (XBP1s). However, MANF did not affect the expression of XBP1 mRNA, neither XBP1u nor XBP1s. These results suggest that MANF can prevent the neuron loss via inhibiting ischemia-induced apoptosis and regulating unfolded protein response-related genes.

Introduction

Mesencephalic astrocyte-derived neurotrophic factor (MANF), also known ARMET, is a secreted protein [1], [2]. The gene encoding MANF is mapped to human chromosomal band 3p21 [3]. Belonging to the family of evolutionarily conserved dopamine neurotrophic factors, MANF has been found to have trophic effects on dopaminergic neurons and non-dopaminergic cells [4], [5]. Drosophila MANF is essential for maintenance of dopamine positive neurites and dopamine levels in the fly [6]. MANF is involved in the regulation of dopaminergic neuron development in zebrafish [7] and modulation of GABAergic transmission to the DA neurons of the substantia nigra [8].

MANF mRNA and protein were widely expressed in the brain [9]. The expression and secretion of MANF were enhanced after endoplasmic reticulum (ER) stress in cell lines [2], [10], [11], [12] and mouse models [13]. MANF was increased after epileptic insults and ischemia [2], [9], [14], [15]. Recent study showed that MANF was up-regulated in the activated glial cells under focal cerebral ischemia and in vitro ER stress or nutrient deprivation [16]. MANF was selectively secreted upon sarco ER calcium deletion [12]. Pretreatment with the recombinant human MANF significantly reduced the volume of infarction as well as improved the behavior in stroke rats [2], [17], [18]. However, the mechanisms are still unknown. In this study, we found that administration of recombinant human MANF protein after ischemic injury reduced the cerebral ischemia-induced neuron loss and promoted behavioral recovery in rats. Further, we found that MANF inhibited the cleavage of caspase-3, but not affected the expressions of ER stress-related proteins in the stroke rats. These results suggest that MANF rescues the neuron loss via inhibiting ischemia-induced apoptosis, which may shed light on the potential therapy of ischemic brain diseases.

Section snippets

Animals

Male Sprague Dawley rats (6 to 8 weeks old, weighing 150–200 g, license number is SCXK 2011–002) were obtained from the Anhui Experimental Animal Center (Hefei, China) and housed in SPF conditions for 1 week prior to experimentation. The animals had access to food and water ad libitum. All the experimental procedures for animal surgery were approved by the Ethics Committee of Anhui Medical University for Animal Experimentation and performed in accordance with the Guideline of the Declaration of

MANF promotes behavioral recovery and reduces the size of infarction induced by MCAO

To verify the protective effects of MANF on neurons during ischemia/reperfusion, MANF, diluted in PBS, was administrated to rat ipsilateral ventricle. PBS was injected as the vehicle control. Bederson's score, generally showing a negative relationship to the neurological deficit, was used to evaluate behavioral recovery before and after treatment. We found that Bederson's score was lower in the rats treated with MANF (1, 5, 10, and 20 μg) than that in the control rats from day 2 to day 14 after

Discussion

In this study, we found a protective effect of MANF on transient cerebral ischemia, in which MANF significantly reduced the size of cerebral infarction as well as improved the behavior in the stroke rats. There is an inverted U-shaped relationship between the doses and the effects of MANF. The maximal protective dose of MANF was 5 μg each rat. The underlying mechanisms are still unknown. It was probably associated with the saturability of MANF receptor or cofactor. This notion needs to be

Conflict of interest

The authors declare no conflict of interests.

Acknowledgment

This work was supported by grants (81372576, 81301060, 81173074 and 91129729) from the National Natural Science Foundation of China.

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    These authors contributed equally to this work.

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