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Proteins of the actin depolymerizing factor (ADF)/cofilin family are ubiquitous among eukaryotes and are essential regulators of actin dynamics and function. Mammalian neurons express cofilin-1 as the major isoform, but ADF and cofilin-2 are also expressed. All isoforms bind preferentially and cooperatively along ADP-subunits in F-actin, affecting the filament helical rotation, and when either alone or when enhanced by other proteins, promotes filament severing and subunit turnover. Although self-regulating cofilin-mediated actin dynamics can drive motility without post-translational regulation, cells utilize many mechanisms to locally control cofilin, including cooperation/competition with other proteins. Newly identified post-translational modifications function with or are independent from the well-established phosphorylation of serine 3 and provide unexplored avenues for isoform specific regulation. Cofilin modulates actin transport and function in the nucleus as well as actin organization associated with mitochondrial fission and mitophagy. Under neuronal stress conditions, cofilin-saturated F-actin fragments can undergo oxidative cross-linking and bundle together to form cofilin-actin rods. Rods form in abundance within neurons around brain ischemic lesions and can be rapidly induced in neurites of most hippocampal and cortical neurons through energy depletion or glutamate-induced excitotoxicity. In ~20% of rodent hippocampal neurons, rods form more slowly in a receptor-mediated process triggered by factors intimately connected to disease-related dementias, e.g., amyloid-ß in Alzheimer's disease. This rod-inducing pathway requires a cellular prion protein, NADPH oxidase, and G-protein coupled receptors, e.g., CXCR4 and CCR5. Here, we will review many aspects of cofilin regulation and its contribution to synaptic loss and pathology of neurodegenerative diseases.

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Cofilin is an actin-binding protein that plays a major role in the regulation of actin dynamics, an essential cellular process. This protein has emerged as a crucial molecule for functions of the nervous system including motility and guidance of the neuronal growth cone, dendritic spine organization, axonal branching, and synaptic signalling. Recently, other important functions in cell biology such as apoptosis or the control of mitochondrial function have been attributed to cofilin. Moreover, novel mechanisms of cofilin function regulation have also been described. The activity of cofilin is controlled by complex regulatory mechanisms, with phosphorylation being the most important, since the addition of a phosphate group to cofilin renders it inactive. Due to its participation in a wide variety of key processes in the cell, cofilin has been related to a great variety of pathologies, among which neurodegenerative diseases have attracted great interest. In this review, we summarized the functions of cofilin and its regulation, emphasizing how defects in these processes have been related to different neurodegenerative diseases.

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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.

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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.

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Aging brain becomes susceptible to neurodegenerative diseases due to the shifting of microglia and astrocyte phenotypes to an active "pro-inflammatory" state, causing chronic low-grade neuroinflammation. Despite the fact that the role of neuroinflammation during aging has been extensively studied in recent years, the underlying causes remain unclear. The identification of relevant proteins and understanding their potential roles in neuroinflammation can help explain their potential of becoming biomarkers in the aging brain and as drug targets for prevention and treatment. This will eventually reduce the chances of developing neurodegenerative diseases and promote healthier lives in the elderly. In this review, we have summarized the morphological and cellular changes in the aging brain, the effects of age-related neuroinflammation, and the potential role of cofilin-1 during neuroinflammation. We also discuss other factors contributing to brain aging and neuroinflammation.

RESUMEN

Accumulating evidence indicates that cucurbitacin B (CuB), as well as other cucurbitacins, damages the actin cytoskeleton in a variety of cell types. However, the underlying mechanism of such an effect is not well understood. In this study, we showed that CuB rapidly induced actin aggregation followed by actin rod formation in melanoma cells. Cofilin, a critical regulator of actin dynamics, was dramatically dephosphorylated (i.e., activated) upon CuB treatment. Notably, the activated cofilin subsequently formed rod-like aggregates, which were highly colocalized with actin rods, indicating the formation of cofilin-actin rods. Cofilin knockdown significantly suppressed rod formation but did not prevent actin aggregation. Furthermore, knockdown of the cofilin phosphatase Slingshot homolog 1 (SSH1), but not chronophin (CIN), alleviated CuB-induced cofilin hyperactivation and cofilin-actin rod formation. The activity of Rho kinase and LIM kinase, two upstream regulators of cofilin activation, was downregulated after cofilin hyperactivation. Pretreatment with a thiol-containing reactive oxygen species (ROS) scavenger N-acetyl cysteine, but not other ROS inhibitors without thiol groups, suppressed CuB-induced actin aggregation, cofilin hyperactivation and cofilin-actin rod formation, suggesting that thiol oxidation might be involved in these processes. Taken together, our results demonstrated that CuB-induced formation of cofilin-actin rods was mediated by SSH1-dependent but CIN-independent cofilin hyperactivation.

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