Transfection and Activation of CofActor, a Light and Stress Gated Optogenetic Tool, in Primary Hippocampal Neuron Cultures

Abstract

Proteins involved in neurodegeneration can be coupled with optogenetic reagents to create rapid and sensitive reporters to provide insight into the biochemical processes that mediate the progression of neurodegenerative disorders, including Alzheimer's Disease (AD). We have recently developed a novel optically-responsive tool (the 'CofActor' system) that couples cof ilin and act in (key players in early stage cytoskeletal abnormalities associated with neurodegenerative disorders) with light-gated optogenetic proteins to provide spatial and temporal resolution of oxidative and energetic stress-dependent biochemical events. In contrast to currently available small-molecule based biosensors for monitoring changes in the redox environment of the cell, CofActor is a light-activated, genetically encoded redox sensor that can be activated with precise spatial and temporal control. Here we describe a protocol for the expression and activation of the CofActor system in dissociated hippocampal neuron cultures prepared from newborn mice. Cultures were transfected with Lipofectamine on the fifth day in vitro (DIV5), then exposed to cellular stress inducing stimuli, leading to the formation of actin-cofilin rods that can be observed using live cell imaging techniques. The protocol described here allows for studies of stress-related cytoskeletal dysregulation in live neurons exposed to neurodegenerative stimuli, such as toxic Aβ42 oligomers. Moreover, expression of the sensor in neurons isolated from transgenic mouse models of AD and/or mice KO for proteins involved in AD can advance our understanding of the molecular basis of early cytoskeletal dysfunctions associated with neurodegeneration.

Keywords: Cellular stress; Cofilin-actin rods; Cytoskeletal dysregulation; Hippocampal neurons; Neurodegeneration; Optogenetics.