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Fndc5 knockdown significantly decreased the expression of neurotrophins and their respective receptors during neural differentiation of mouse embryonic stem cells

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Abstract

Fibronectin type III domain-containing-5 (Fndc5) is a trans-membrane protein which is involved in a variety of cellular events including neural differentiation of mouse embryonic stem cells (mESCs) as its knockdown and overexpression diminishes and facilitates this process, respectively. However, downstream targets of Fndc5 in neurogenesis are still unclear. Neurotrophins including NGF, BDNF, NT-3, and NT-4 are the primary regulators of neuronal survival, growth, differentiation, and repair. These biomolecules exert their actions through binding to two different receptor families, Trk and p75NTR. In this study, considering the fact that neurotrophins and their receptors play crucial roles in neural differentiation of ESCs, we sought to evaluate whether knockdown of Fndc5 decreased neural differentiation of mESCs by affecting the neurotrophins and their receptors expression. Results showed that at neural progenitor stage, the mRNA and protein levels of BDNF, Trk, and p75NTR receptors decreased following the Fndc5 knockdown. In mature neural cells, still, the expression of Trk and p75NTR receptors at mRNA and protein levels and BDNF and NGF expression only at protein levels showed a significant decrease in Fndc5 knockdown cells compared to control groups. Taken together, our results suggest that decreased efficiency of neural differentiation following the reduction of Fndc5 expression could be attributed to decreased levels of NGF and BDNF proteins in addition to their cognate receptors.

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Abbreviations

ANOVA:

Two-way analysis of variance

ATP:

Adenosine triphosphate

BDNF:

Brain-derived neurotrophic factor

bHLH:

Basic helix–loop–helix

BSA:

Bovine serum albumin

cAMP:

Cyclic adenosine monophosphate

CNS:

Central nervous system

CREB:

CAMP-response element binding protein

DAPI:

4, 6‐Diamidino‐2‐phenylindole

DMEM/F12:

Dulbecco’s modified Eagle’s medium/Hams F12 medium

Dox:

Doxycycline

EB:

Embryoid body

ECL:

Enhanced chemiluminescence

ERK:

Extracellular signal-regulated kinase

ESC:

Embryonic stem cell

ES-FBS:

Embryonic stem cell qualified fetal bovine serum

FITC:

Fluorescein isothiocyanate

Fndc5:

Fibronectin type III domain-containing-5

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

HRP:

Horse radish peroxidase

JNK:

Jun N-terminal kinase

Ko-DMEM:

Knock-out DMEM

LIF:

Leukemia inhibitory factor

MAP2:

Microtubule-associated protein 2

MAPK:

Mitogen-activated protein kinases

mESC:

Mouse embryonic stem cell

miRNA:

MicroRNAs

mTOR:

Mammalian target of rapamycin

NB:

Neurobasal

NC:

Neural cell

NGF:

Nerve growth factor

NP:

Neural progenitor

NT:

Neurotrophin

NT-3:

Neurotrophin-3

NT-4:

Neurotrophin-4

p75NTR:

P75 neurotrophin receptor

PBS:

Phosphate-buffered saline

PeP:

Proxisomal protein

PI3:

Phosphatidylinositol-3-kinase

PLC:

Phospholipase C

PVDF:

Polyvinylidenedifluoride

RA:

Retinoic acid

RNAi:

RNA interference

ROS:

Reactive oxygen species

RT:

Room temperature

RT-qPCR:

Real-time quantitative polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SEM:

Standard error of the mean

shRNA:

Small hairpin RNA

TRI:

Total RNA isolation

Trk:

Tropomyosin-related kinase

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Acknowledgements

Authors would like to thank University of Sistan and Baluchestan and Royan Institute for the financial support of this project. The authors are thankful to other members of Royan Institute for their excellent technical assistance and advice.

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RE: experimental design, data collection, data analysis, data interpretation, and manuscript writing. FR: data analysis, data interpretation. DMKT: experimental design, financial support, data analysis, data interpretation, and final approval of the manuscript. MHNE: experimental design, financial support, data analysis, data interpretation, and final approval of manuscript. KG: experimental design, data analysis, data interpretation, manuscript writing, and final approval of the manuscript.

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Correspondence to Mohammad Hossein Nasr-Esfahani or Kamran Ghaedi.

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Approval for this study was obtained from the Institutional Review Board of Royan Institute (Tehran, Iran).

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Ebadi, R., Rabiee, F., Kordi-Tamandani, D. et al. Fndc5 knockdown significantly decreased the expression of neurotrophins and their respective receptors during neural differentiation of mouse embryonic stem cells. Human Cell 34, 847–861 (2021). https://doi.org/10.1007/s13577-021-00517-z

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