Experimental Procedures for Demonstration of MicroRNA Mediated Enhancement of Functional Neuroprotective Effects of Estrogen Receptor Agonists

Methods Mol Biol. 2016:1366:359-372. doi: 10.1007/978-1-4939-3127-9_29.


Protection of motoneurons is an important therapeutic goal in the treatment of neurological disorders. Recent reports have suggested that specific microRNAs (miRs) could modulate the expression of particular proteins for significant alterations in the pathogenesis of different neurological disorders. Thus, combination of overexpression of a specific neuroprotective miR and treatment with a neuroprotective agent could be a novel strategy for functional protection of motoneurons. The protocols described herein demonstrate that miR-7-1, a neuroprotective miR, can enhance the functional neuroprotective effects of estrogen receptor agonists such as 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), Way 200070 (WAY), and estrogen (E2) in preventing apoptosis in A23187 calcium ionophore (CI) exposed VSC4.1 motoneurons. This article describes the protocols for the cell viability assay, transfection of VSC4.1 motoneurons with miRs, Annexin V/propidium iodide staining for apoptosis, Western blotting, patch-clamp recording of whole-cell membrane potential, and JC-1 staining for detection of mitochondrial membrane potential. Taken together, these protocols are used to demonstrate that miR-7-1 caused significant enhancement of the efficacy of estrogen receptor agonists for functional neuroprotection in VSC4.1 motoneurons.

Keywords: Apoptosis; Estrogen; PPT; VSC4.1 motoneurons; WAY; miR-7-1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Blotting, Western
  • Calcium Ionophores / toxicity
  • Cell Fusion
  • Cell Line
  • Cell Survival / drug effects
  • Combined Modality Therapy
  • Cytoprotection
  • Dose-Response Relationship, Drug
  • Estrogens / pharmacology*
  • Flow Cytometry
  • Genetic Therapy / methods*
  • Membrane Potential, Mitochondrial / drug effects
  • Membrane Potentials / drug effects
  • Mice
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Motor Neurons / drug effects*
  • Motor Neurons / metabolism
  • Motor Neurons / pathology
  • Neuroprotective Agents / pharmacology*
  • Patch-Clamp Techniques
  • Rats
  • Receptors, Estrogen / drug effects*
  • Receptors, Estrogen / genetics
  • Receptors, Estrogen / metabolism
  • Transfection
  • Workflow


  • Calcium Ionophores
  • Estrogens
  • MicroRNAs
  • Neuroprotective Agents
  • Receptors, Estrogen