Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Regulates Neurite Outgrowth Through the Activation of Akt/mTOR and Erk/mTOR Signaling Pathways.

Wen Wen,Mei Xu,Jacqueline A Frank,Yongchao Wang,Jia Luo,Hong Xu,Murong Ma,Hui Li

doi:10.3389/fnmol.2020.560020

Abstract

Neurite outgrowth is essential for brain development and the recovery of brain injury and neurodegenerative diseases. In this study, we examined the role of the neurotrophic factor MANF in regulating neurite outgrowth. We generated MANF knockout (KO) neuro2a (N2a) cell lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and demonstrated that MANF KO N2a cells failed to grow neurites in response to RA stimulation. Using MANF siRNA, this finding was confirmed in human SH-SY5Y neuronal cell line. Nevertheless, MANF overexpression by adenovirus transduction or addition of MANF into culture media facilitated the growth of longer neurites in RA-treated N2a cells. MANF deficiency resulted in inhibition of Akt, Erk, mTOR, and P70S6, and impaired protein synthesis. MANF overexpression on the other hand facilitated the growth of longer neurites by activating Akt, Erk, mTOR, and P70S6. Pharmacological blockade of Akt, Erk or mTOR eliminated the promoting effect of MANF on neurite outgrowth. These findings suggest that MANF positively regulated neurite outgrowth by activating Akt/mTOR and Erk/mTOR signaling pathways.

Highlights

Neurite outgrowth is the first step for the formation of axons and dendrites, which are necessary components for the development of a functional neuronal network
Mesencephalic astrocyte-derived neurotrophic factor knockout (KO) N2a cells were generated by transfection of MANF clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9
To examine if MANF is required for neurite outgrowth in N2a cells, control and KO cells were treated with 10 μM retinoic acid (RA) or DMSO control in media with 2% FBS for 3 days

Summary

Introduction

Neurite outgrowth is the first step for the formation of axons and dendrites, which are necessary components for the development of a functional neuronal network. Neurite outgrowth is regulated by neurotrophic factors (NTF) that activate cell signaling pathways for neuron differentiation and migration, and support neuron survival and synaptic function (da Silva and Dotti, 2002; Cui, 2006). Increasing evidence have shown that MANF promotes the development and survival of neurons in both normal and pathological conditions (Petrova et al, 2003; Palgi et al, 2009; Voutilainen et al, 2009; Airavaara et al, 2010; Chen et al, 2012). MANF is required for dopaminergic neuron survival in Drosophila and zebrafish (Palgi et al, 2009; Chen et al, 2012), and selectively protects dopaminergic neuron in rat medial ventral mesencephalon cell cultures in vitro (Petrova et al, 2003). CDNF has been reported to be neuroprotective in animal models of PD, AD, and periphery nerve injury (Lindholm et al, 2007; Cheng et al, 2013; Kemppainen et al, 2015)

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