CofActor: Investigation of the Role of Cytoskeletal Dysregulation in Neurodegenerative Disease - Cytoskeletal dysregulation is a prominent hallmark of neurodegenerative disease progression. Its role in conditions like Alzheimer's Disease (AD) and Huntington's Disease (HD), however, remains poorly understood. New tools are therefore required to investigate the connections between cytoskeletal dysregulation and the disease state. In response to this need, we have created a genetically encoded, light and stress-gated switch inspired by the biochemical phenomenon of cofilin-actin rods (“CofActor”), which form in response to high levels of oxidative and energetic stress associated with neurodegenerative disorders, including AD, HD and Parkinson's Disease (PD). CofActor can be a useful tool for rapid and reversible readout of oxidative and energetic stress levels in cells and to assess the cellular stress-dependent changes in actin-dependent processes. Furthermore, CofActor can be activated with both spatial and temporal control, a major advance over currently available methods of studying cofilin-actin rods. We created CofActor by coupling the proteins cofilin and actin with the well-characterized optogenetic switch Cryptochrome 2 (Cry2) – CIB, and in a recent publication, characterized the activity of this sensor in immortalized cell lines and in dissociated hippocampal neuron cultures. In this proposal, we will investigate the utility of CofActor for dissecting the upstream signaling pathways leading to cofilin-actin rod formation by determining its impacts on synaptic function and dendritic spine remodeling in cellular models of AD using dissociated neuron cultures treated with toxic amyloid oligomers and neuron cultures derived from AD model mice (3xTg-AD). This proposal will use CofActor to test the hypothesis that cofilin-actin rod formation contributes to AD pathology by disrupting neuronal function, resulting in synapse loss and impaired structural plasticity of dendritic spines. The specific aims of this proposal are: (i) Investigate how various triggers of oxidative, energetic and excitotoxic stress (including H2O2, ATP depletion, Aβ42 fibrils and oligomers and excessive glutamate receptor stimulation) impact CofActor-mediated cofilin-actin cluster formation and investigate small molecules as inhibitors and activators of the CofActor system in immortalized cells and in cellular models of AD. (ii) Investigate novel mutations of the ATP-binding pocket of actin for modulation of the stress response of the CofActor system, followed by investigating the mutants' ability to induce stress-independent neuronal dysfunction in a mouse model of AD (3xTg-AD); and (iii) Investigate the effect of CofActor-mediated cofilin-actin cluster formation on dendritic spine remodeling associated with dendritic spine structural plasticity induced by 2-photon glutamate uncaging in organotypic hippocampal slice cultures. This proposal will shed light on cofilin-actin rod formation, an important biochemical and structural phenomenon underlying the progression of neurodegenerative diseases (specifically AD), which could lead to the development of targeted therapeutics to slow or stop disease progression.
