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.
Document Type
Article
Publication Date
9-24-2020
Digital Object Identifier (DOI)
https://doi.org/10.3389/fnmol.2020.560020
Funding Information
This work was supported by the National Institutes of Health (NIH) grants AA017226 and AA015407. And also supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development [Biomedical Laboratory Research and Development: Merit Review (BX001721)].
Related Content
Descriptions of the supplementary materials:
Supplementary Figure S1 | Expression of exogenous MANF in N2a cells after adenovirus transduction. (A) Protein was extracted from cells transduced with AD-vector and AD-MANF at 24, 48, 72, 96, and 120 h after transduction, then subjected to immunoblot. The expression of HA tag and MANF was examined. β-actin was used as a loading control. The size of the proteins (kDa) was labeled next to each band. (B) Exogenous MANF protein levels were quantified as relative levels normalized to β-actin. Two-way ANOVA followed with the Bonferroni’s post hoc test, ∗∗P < 0.01, ∗∗∗P < 0.0001. The data were expressed as the mean ± SEM of three independent experiments.
Supplementary Figure S2 | MANF cannot be reintroduced into MANF KO cells. (A) Immunofluorescent images showing control and MANF KO cells were transduced with AD-vector and AD-MANF and then immunolabeled with HA tag (green) and MANF (red). Cell nuclei were stained by DAPI. (B) The percentage of cells with HA tag expression was quantified. Student’s t-test, ∗∗∗P < 0.0001. The data were expressed as the mean ± SEM of three independent experiments. (C) Protein was extracted from cells 36 h after being incubated with AD-vector and AD-MANF for 1–4 h, and then subjected to immunoblot with HA tag and MANF antibodies. β-actin was used as a loading control. (D) Protein was extracted from control and MANF KO cells, and then immunoblot with Cas9 antibody. β-actin was used as a loading control. The size of the proteins (kDa) was labeled next to each band. The experiment was replicated three times.
Supplementary Figure S3 | The effect of pharmacological inhibition or activation of Akt, Erk and mTOR on the expression of p-Akt, p-Erk, and p-mTOR in response to RA treatment. (A) Quantification of p-Akt, p-Erk, p-mTOR, and p-P70S6 protein expression in control cells treated with DMSO, RA or RA+inhibitors. β-actin was used as a loading control. One-way ANOVA followed with the Tukey’s post hoc test, ∗P < 0.05, ∗∗P < 0.01 compared to DMSO treated group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to RA treated group. The data were expressed as the mean ± SEM of three independent experiments. (B) Quantification of p-Akt, p-Erk, p-mTOR, and p-P70S6 protein expression in MANF KO cells treated with DMSO, RA or RA+activators. β-actin was used as a loading control. One-way ANOVA followed with the Tukey’s post hoc test, n.s. not statistically significant, ∗P < 0.05 compared to DMSO treated group; #P < 0.05 compared to RA treated group. The data were expressed as the mean ± SEM of three independent experiments.
Repository Citation
Wen, Wen; Wang, Yongchao; Li, Hui; Xu, Hong; Xu, Mei; Frank, Jacqueline A.; Ma, Murong; and Luo, Jia, "Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Regulates Neurite Outgrowth through the Activation of Akt/mTOR and Erk/mTOR Signaling Pathways" (2020). Pharmacology and Nutritional Sciences Faculty Publications. 102.
https://uknowledge.uky.edu/pharmacol_facpub/102
Supplementary Figure S1
Image_2_Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Regulates Neurite Outgrowth Through the Activation of Akt_mTOR and Erk_mTOR Signali.TIF (3182 kB)
Supplementary Figure S2
Image_3_Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Regulates Neurite Outgrowth Through the Activation of Akt_mTOR and Erk_mTOR Signali.TIF (2055 kB)
Supplementary Figure S3
Notes/Citation Information
Published in Frontiers in Molecular Neuroscience, v. 13, article 560020.
© 2020 Wen, Wang, Li, Xu, Xu, Frank, Ma and Luo.
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