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, Aß42 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.
