Browsing by Author "Bamburg, James, advisor"
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Item Open Access A cellular prion protein-dependent signaling pathway for proinflammatory cytokine- and β-amyloid-induced cofilin-actin rod formation(Colorado State University. Libraries, 2014) Walsh, Keifer P., author; Bamburg, James, advisor; Zabel, Mark, committee member; Tjalkens, Ron, committee memberStimulus of oxidative stress in neurodegeneration leads to synaptic dysfunction and the eventual loss of neurons in the central nervous system. The actin cytoskeleton of neurons under acute or chronic stress experiences dynamic remodeling due to functional alterations in the actin depolymerizing factor (ADF)/cofilin family of actin-binding proteins. Once oxidized, disulfide cross-linked cofilin incorporates into the formation of tandem arrays of 1:1 cofilin:actin rod-like bundles (rods). Rods sequester cofilin, which is required for synaptic remodeling associated with learning and memory, and interrupt vesicular transport by occluding the neurite within which they form. Different rod-inducing stimuli target distinct neuronal populations within the hippocampus. Rods form rapidly (5-30 min) in >80% of cultured hippocampal neurons which are treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% maximum response in ~6 h) occurs in ~20% of neurons upon exposure to soluble beta-amyoid dimer/trimer (Aβd/t), a physiologically relevant species in Alzheimer disease (AD). Here we show that proinflammatory cytokines (TNFα, IL-1β, IL-6) induce rods at the same rate and in the same subpopulation of hippocampal neurons that respond to Aβd/t. Rod formation by proinflammatory cytokines may link the neuroinflammatory hypothesis for AD with the Aβ hypothesis by providing a common target. Neurons from PrPC-null mice form rods in response to glutamate or antimycin A, but not in response to Aβd/t or proinflammatory cytokines. Prion-dependent rod inducers require the activation of NADPH oxidase (NOX) to generate reactive oxygen species (ROS), but NOX activity is not required for rods induced by glutamate or energy depletion. Aβd/t and TNFα stimulate cofilin dephosphorylation and increased ROS production in a subpopulation of neurites at levels that exceed a minimum threshold to maintain stable rods. Removing inducers or inhibiting NOX activity in cells containing prion-dependent rods causes rod disappearance with a half-life of ~36 minutes. Interestingly, the overexpression of PrPC alone is sufficient to induce rods in >40% of hippocampal neurons, nearly twice the number that respond to Aβd/t or TNFα. This suggests that membrane microdomains containing PrPC recruit the oxidizing machinery necessary to initiate and sustain rod formation. Our hypothesis is supported by the inhibition and reversal of prion-dependent rods by the naturally occurring plant triterpene, ursolic acid (UA), and the pharmacological peptide RAP310. UA and related compounds to RAP310 have been proposed to inhibit changes in the membrane lipid profile that permit LR coalescence. The vast majority of neurodegenerative disorders are considered sporadic in incidence and multifactorial in cause, making treatment at an early stage a significant challenge. If cofilin-actin rods indeed bridge multiple disease initiating mechanisms into a common pathway leading to synapse loss, they provide a valuable target for therapeutic intervention.Item Open Access Actin dynamics in silico, in waves, and in rods(Colorado State University. Libraries, 2009) Pak, Chi W., author; Bamburg, James, advisorThe current paradigm of actin dynamics and superorganization has advanced in the past decade from emerging technologies and perspectives, which include the discovery of actin nucleators, real time imaging of the dynamics of single filaments in vitro, and single molecule imaging of actin superstructures in vivo. These advances have influenced each of our studies on multiple levels, sometimes directly. A novel analysis of single actin filament dynamics revealed faster than expected dynamics during treadmilling but not during bulk polymerization. Using an exact stochastic simulation, we investigated whether filament-annealing and -fragmentation might account for faster than expected dynamics; their influence on actin dynamics had not been investigated before in a comprehensive model. Results from our work demonstrated that filament-annealing and -fragmentation alone cannot account for faster than expected dynamics during treadmilling. Thus, strictly through computational modeling, we are able to investigate various hypothetical models and offer insights into a process that cannot be achieved by experimentation. A concept that has also gained support during the past decade has been the self-organizing nature of actin, which was demonstrated by the Listeria actin-comet-tail reconstitution assay. We have proposed that this is a fundamental property of all actin superstructures, whether they are assembled in vitro or in vivo or whether they are involved in development or disease. The concept of actin's self-organization has influenced our study of neuronal waves, which are growth cone-like structures that travel along neurites and which were hypothesized to transport actin to growth cones and support neuritogenesis. Using diffusional analysis, we were able to demonstrate that neuronal waves transport actin. Neuronal waves provide a unique mechanism for transporting actin in that the delivery of actin is dependent upon actin itself and its dynamics. In disease states, the self-organization of actin is often changed but not disrupted, sometimes resulting in the formation of orderly-structured aggregates of cofilin and actin known as cofilin-actin rods (or rods). Using glutamate excitotoxicity as a model system for the cofilin pathology observed in Alzheimer disease (AD), we have determined signaling mechanisms for cofilin-actin rod induction, which in young rat hippocampal neurons require AMPA receptors and are calcium-independent. In addition, cofilin-actin rod interactions with microtubule associated proteins, and associated changes to the microtubule cytoskeleton were studied for its potential relevance to the pathology of AD. Our results suggest that disruptions to the normal organization of actin and microtubules might underlie several pathological hallmarks of early AD.Item Open Access Interruption of neuron-microglia bidirectional communication to modulate cofilin:actin rod formation(Colorado State University. Libraries, 2022) Zoller, Maia, author; Bamburg, James, advisor; Chanda, Soham, committee member; Zabel, Mark, committee memberImmune responses in the central nervous system are mediated by microglia, whose responses to CNS threats can be replicated in vitro to study the role of microglia in the onset, progression, and treatment of neurodegenerative diseases. Previous work has identified a pathway common to neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, and HIV-Associated Neurocognitive Dementia in which the actin-severing protein, cofilin, forms a 1:1 bundle with actin making rod-shaped inclusions (rods) that can be found in the dendrites and axons of neuronal cells. This thesis focuses on developing methods for examining the role of primary microglia, activated by different factors, to secrete rod-inducing chemokines/cytokines or directly attacking neurons leading to neuronal death. Understanding both of these mechanisms is important in study of neuroinflammation and disease progression. Hemin, a hemoglobin metabolite, and alarmin, S100B, a astrocyte secreted, calcium binding protein, protein, were used to model the environment of intracerebral hemorrhage and general neuroinflammation respectively. Preliminary experimental results suggest blockage of actin-rod inducing signaling pathways via CXCR4/CCR5 receptor antagonist improves neuronal survival to both microglia conditioned medium and direct exposure the microglia-activating hemin or S100B. Further studies are in progress to obtain sufficient statistical data to verify these results.