Theses and Dissertations

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Open Access
A surface protease of Lyme disease bacteria degrades host extracellular matrix components and induces inflammatory cytokines in vitro
(Colorado State University. Libraries, 2012) Russell, Theresa Michelle Tidd, author; Bamburg, James R., advisor; Johnson, Barbara J. B., advisor; Luger, Karolin, committee member; Cohen, Robert E., committee member; Gentry-Weeks, Claudia, committee member
For nearly two decades, the paradigm in Lyme disease research has been that Borrelia burgdorferi does not produce proteases capable of damaging host molecules. Lyme disease has been considered, therefore, to be the consequence of an exuberant inflammatory response to infecting bacteria. This prevailing concept, however, has created a conundrum for the field. The bacterial burden in infected tissue is low, but the degree of inflammation is remarkable and seemingly out of proportion to this burden. The studies described in this dissertation provide evidence that, contrary to current thinking, B. burgdorferi does possess a protease that degrades numerous molecules of the host extracellular matrix (ECM). In addition to destabilization of the ECM which would be expected to benefit the organism, characterization of this proteolytic activity demonstrates that ECM fragments are produced that are known to be pro-inflammatory. These bioactive fragments may amplify the inflammatory processes triggered by the presence of the bacteria itself. When this hypothesis was tested directly by exposing chondrocytes to the borrelial protease in vitro, inflammatory cytokines and chemokines that are hallmarks of Lyme disease were induced. The studies herein suggest a new model for the pathogenesis of Lyme disease and offer an explanation for the paradox of debilitating inflammatory disease in the presence of few infecting organisms. Lastly, in contrast to current serology-based Lyme disease diagnostic tests, the activity of this protease in vitro may generate diagnostic biomarkers enabling detection of active B. burgdorferi infection.
Open Access
Cofilin-actin rods, a hippocampal pathology in mouse models of human dementias, form from different inducers through a common molecular pathway
(Colorado State University. Libraries, 2020) Babcock, Isaac W., author; Bamburg, James R., advisor; Di Pietro, Santiago M., committee member; Zabel, Mark D., committee member
To view the abstract, please see the full text of the document.
Open Access
Cofilin-actin rods: quantification and comparison to tau pathology in a human longitudinal aging study and developing probes to measure localization and activity of NADPH oxidase 2, a component of the prion-dependent rod inducing pathway
(Colorado State University. Libraries, 2015) Carlson, Adlei B., author; Bamburg, James R., advisor; Di Pietro, Santiago, committee member; Amberg, Gregory C., committee member
The presence of extracellular amyloid plaques composed mainly of fibrils of the β-amyloid peptide (Aβ) as well as intracellular neurofibrillary tangles composed mainly of hyperphosphorylated tau protein, are used for post-mortem confirmation of the diagnosis of Alzheimer's disease (AD). However, a shift in disease hypothesis has changed over the years. It is now generally accepted that soluble forms of Aβ oligomers and not fibrils, which are deposited in plaques, are most responsible for the synaptic loss and eventual neuronal death that accompanies AD progression. In cultured mammalian neurons, treatment with this more relevant, soluble form of Aβ induces the formation of cofilin-actin rods within neurons. Rods may grow to occlude the neurite and block transport, leading to loss of microtubules and synapses. Tau is a microtubule binding protein whose hyperphosphorylation depends upon its release from microtubules. Thus, rod formation might play a role in the loss of synapses and the development of tau pathology in AD. To determine if cofilin-actin rods might play a role in AD progression, we obtained samples of frontal cortex and the hippocampal formation from nearly identical regions of multiple subjects who were part of a longitudinal study and thus could be grouped as non-cognitively impaired (NCI), early AD (eAD), or mid to late AD. All samples were obtained with a short postmortem interval and the average age of subjects in each group was between 86 and 91 years. We prepared 30 μm sections of cortical and hippocampal tissue, and following immunofluorescence staining for cofilin and phosphorylated tau protein, quantified rod and neuropil thread areas in brain sections from each subject. Rods in the hippocampal formation were most prevalent in the entorhinal cortex, the first brain region to show pathology during development of AD. Comparison of rod and neuropil thread pathology in the frontal cortex revealed a correlation of neuropil thread pathology with disease transition. However, there was no correlation between rod density and disease transition, while the cortical sections revealed a surprisingly high deposition of cofilin rod pathology across all subject cohorts. This may suggest that rods play a different role within brain cortical regions than what was observed in the hippocampus. Additionally, recent work has revealed the implication of a prion-dependent rod inducing pathway dependent on the activation of the reactive oxygen producing NADPH oxidase 2 (NOX). If prion-protein density is responsible for whether a rod forms, can we investigate the NOX intensity and duration of activity in relation to where rods form in a neurite? For future study we sought to develop the sensitive NOX probes, p47-roGFP and NOX-2-redtrack. These probes will give us new tools to analyze the effects that NOX activity and expression have on rod formation. The adenoviral constructs for expression of these two probes have been made and characterized within mammalian cell lines. Evidence presented here provides the basis for the use of these probes to analyze NOX activity as it relates to the generation of rods within neurites.
Open Access
Lipid raft signaling in cofilin-actin rod formation induced by amyloid-β and TNFα
(Colorado State University. Libraries, 2012) Mi, Jianjie, author; Bamburg, James R., advisor; Chen, Chaoping, committee member; Partin, Kathryn M., committee member
Rod-like inclusions (rods), composed of actin saturated with cofilin, are induced in neurons by energetic and oxidative stresses, excitotoxic levels of glutamate, and amyloid beta treatment. Cofilin is an F-actin assembly regulatory protein critical to various actin-dependent processes, such as cytokinesis, cell migration, and neurite formation. Overexpression or hyperactivation (excessive dephosphorylation) of cofilin coupled with its oxidation can lead to formation of rods. Rods represent a likely mechanism to explain the synaptic loss associated with early stages of Alzheimer's disease (AD) and thus represent a novel target for therapeutic intervention. In live neurons, the study of cofilin-actin rod formation induced by specific mediators of stress has been limited because overexpression of fluorescent protein-tagged wild type (WT) cofilin results in formation of considerable numbers of spontaneous rods. A fluorescent cofilin mutant that could incorporate into induced rods but form no spontaneous rods even when overexpressed would offer a useful alternative for live-cell imaging. The R21Q mutant cofilin-RFP has been reported to not induce rods when overexpressed but incorporates into rods containing endogenous cofilin, thus serving as a rod marker in live cells. Here we show that expression of WT cofilin driven by promoters that result in a high or moderate steady-state level of exogenous protein produces a significant number of spontaneous rods, three to four fold over controls. However, R21Q cofilin-RFP expressed behind these same promoters will only incorporate into rods formed from endogenous protein, but not enhance spontaneous rod, even when accompanied by the photo stress induced by microscopic observation. Using the R21Q cofilin- RFP to measure rod formation, we then showed that the proinflammatory cytokine (TNFα) induced about a 3 fold increase in rod formation over untreated controls quantified either as the percent of neurons with rods (percent rod index) or as the number of rods per field (number rod index). Amyloid beta dimer/trimer (Aβd/t) induced about a 2.5 fold increase over controls in the percent of neurons with rods, and close to a 2 fold increase in the number of rods per field. To determine the fidelity of the R21Q cofilin-RFP in labeling all of the rods, we induced rods in control infected or R21Q cofilin-RFP expressing neurons with ATP depletion for 30 min, or with either Aβd/t (250 pM) or TNFα (50 ng/ml; 2.9 nM) for 24 h. Neurons were fixed and immunostained with a primary antibody for cofilin and an Alexa 647 nm-labeled secondary antibody. The percent of rods in RFP expressing cells that co-labeled with mRFP and Alexa 647 were then quantified. Although 100% of rods induced by ATP depletion co-labeled, surprisingly only 48% of the rods induced by TNFα co-labeled, similar to Aβd/t treatment. The reasons for this are not clear but taken together, our results demonstrate that R21Q cofilin-RFP can be used for a live cell marker for following induced rod formation but not as a quantitative measure of the total rod response. Induction of cofilin-actin rods by amyloid beta and TNFα is mediated by the cellular prion protein, a component of lipid raft domains which can signal to activate NADPH oxidase. Lipid rafts are cholesterol/sphingolipid enriched detergent resistant membrane domains in which many membrane receptors associate. Rafts can be visualized with an Alexa labeled cholera toxin B subunit which binds to GM1 ganglioside. Here we used neurons expressing R21Q cofilin-RFP to determine if rod formation is associated with coalesced lipid raft domains and if the coalesced lipid rafts form before or after rods are visible. In the three rods we visualized forming during the period in which lipid rafts were labeled we saw no lipid raft coalescence at sites of the newly formed rods. If we looked at the total R21Q cofilin-RFP labeled rods, about 45% of them co-localize with enlarged lipid raft domains. Thus results suggest that rods may bring about the reorganization of the membrane raft domains, although more data are required to make a definitive conclusion.
Open Access
The amyloid beta dimer/trimer: a potent stimulator of neuronal amyloid beta secretion and cofilin-actin rod formation
(Colorado State University. Libraries, 2010) Marsden, Ian Thomas, author; Bamburg, James R., advisor; Di Pietro, Santiago M., committee member; Partin, Kathryn M., committee member
Amyloid beta (Aβ) peptides, a heterogeneous mixture of 39-43 amino acid peptides produced from β- and γ-secretase cleavage of the amyloid precursor protein (APP), are one of the causative agents of Alzheimer disease (AD). Although sensitive enzyme-linked immunosorbent assays (ELISAs) for specific rodent Aβ peptides and for total and specific human Aβ peptides have been commercially available, no commercial assay for total rodent Aβ was available when we began these studies. Such an assay is desirable to determine the effects of the human Aβ peptides on production of Aβ from cultured rodent neurons, the major model system used in AD research. Here we report an ELISA for total rodent Aβ and show that it can be used without interference from physiologically relevant concentrations of human Aβ. We then apply the assay to measure the production of Aβ in cultured dissociated rat cortical neurons and rat and mouse hippocampal organotypic slices in response to oxidative stress or treatment with human Aβ dimer/trimer (Aβd/t) obtained from culture medium of Chinese hamster ovary cell line 7PA2 expressing a mutant form of human amyloid precursor protein. Neither of the treatments leads to accumulation of intracellular Aβ peptides. Peroxide increases Aβ secretion by about 2 fold, similar to results from previous reports that used an immunoprecipitation and western blot assay. Of greater significance is that physiologically relevant concentrations (250 pM) of human Aβd/t increase rodent Aβ secretion by >3 fold over 4 days, providing support for an Aβ-mediated feed-forward model of AD progression. The over two fold increase in rodent Aβ secreted in response to human Aβd/t was nearly identical between organotypic hippocampal slices of TAU knock-out mice and TAU knock-out mice expressing the human tau transgene, demonstrating that tau plays no role in the enhanced production of Aβ. Previous studies showed oligomers of synthetic amyloid beta (Aβ1-42) induced cofilin activation and formation of cofilin-actin rods in a neuronal subpopulation of rat hippocampus primarily localized within the dentate gyrus. Here we demonstrate that Aβd/t at ~250 pM is more potent in rod induction in both dissociated hippocampal neuronal cultures and organotypic slices than is 1 μM synthetic Aβ as typically prepared oligomers, about a 4000 fold difference. Treatment of the Aβd/t fraction with an Aβ-neutralizing antibody eliminates its rod inducing activity. Traditionally prepared synthetic Aβ oligomers contain SDS-stable trimers and tetramers, but are devoid of dimers. When synthetic human Aβ was incubated under conditions that generate a tyrosine oxidized dimer, the concentration that was required to induce rods decreased dramatically. The oxidized dimer had a maximum rod-inducing activity at ~2 nM (10 ng/mL), suggesting it is the presence of the SDS-stable tyrosine oxidized Aβ dimer in a low-n state that is largely responsible for the potency of the secreted Aβd/t. Aβd/t-induced rods are highly localized to the dentate gyrus and mossy fiber pathway and form more rapidly (significant over controls by 2 h compared to 8 h for those induced by synthetic Aβ-oligomers). Aβd/t-induced rods are reversible, disappearing by 24 h after washout. Cofilin dephosphorylation in response to Aβd/t is greatest within the hippocampal regions of rod formation. Overexpression of cofilin phosphatases slingshot and chronophin increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t treatment both in dissociated neurons and organotypic slice cultures. Overexpression of the cofilin kinase, LIM kinase 1, inhibits Aβd/t-induced rod formation. Together these data support a mechanism through which Aβd/t produces selective synaptic dysfunction affecting learning and memory at least in part via primary effects on cofilin regulation and rod formation in sensitive hippocampal regions.

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