RESUMEN
The presence of atypical cytoskeletal dynamics, structures, and associated morphologies is a common theme uniting numerous diseases and developmental disorders. In particular, cytoskeletal dysregulation is a common cellular feature of Alzheimer's disease, Parkinson's disease, and Huntington's disease. While the numerous activators and inhibitors of dysregulation present complexities for characterizing these elements as byproducts or initiators of the disease state, it is increasingly clear that a better understanding of these anomalies is critical for advancing the state of knowledge and plan of therapeutic attack. In this review, we focus on the hallmarks of cytoskeletal dysregulation that are associated with cofilin-linked actin regulation, with a particular emphasis on the formation, monitoring, and inhibition of cofilin-actin rods. We also review actin-associated proteins other than cofilin with links to cytoskeleton-associated neurodegenerative processes, recognizing that cofilin-actin rods comprise one strand of a vast web of interactions that occur as a result of cytoskeletal dysregulation. Our aim is to present a current perspective on cytoskeletal dysregulation, connecting recent developments in our understanding with emerging strategies for biosensing and biomimicry that will help shape future directions of the field.
RESUMEN
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 Aß42 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.
RESUMEN
The hallmarks of neurodegenerative diseases, including neural fibrils, reactive oxygen species, and cofilin-actin rods, present numerous challenges in the development of in vivo diagnostic tools. Biomarkers such as ß-amyloid (Aß) fibrils and Tau tangles in Alzheimer's disease are accessible only via invasive cerebrospinal fluid assays, and reactive oxygen species can be fleeting and challenging to monitor in vivo Although remaining a challenge for in vivo detection, the protein-protein interactions underlying these disease-specific biomarkers present opportunities for the engineering of in vitro pathology-sensitive biosensors. These tools can be useful for investigating early stage events in neurodegenerative diseases in both cellular and animal models and may lead to clinically useful reagents. Here, we report a light- and cellular stress-gated protein switch based on cofilin-actin rod formation, occurring in stressed neurons in the Alzheimer's disease brain and following ischemia. By coupling the stress-sensitive cofilin-actin interaction with the light-responsive Cry2-CIB blue-light switch, referred to hereafter as the CofActor, we accomplished both light- and energetic/oxidative stress-gated control of this interaction. Site-directed mutagenesis of both cofilin and actin revealed residues critical for sustaining or abrogating the light- and stress-gated response. Of note, the switch response varied depending on whether cellular stress was generated via glycolytic inhibition or by both glycolytic inhibition and azide-induced ATP depletion. We also demonstrate light- and cellular stress-gated switch function in cultured hippocampal neurons. CofActor holds promise for the tracking of early stage events in neurodegeneration and for investigating actin's interactions with other proteins during cellular stress.