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Department of Biochemistry & Molecular Biology

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These digital collections include theses, dissertations, and datasets from the Department of Biochemistry & Molecular Biology.

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Browsing Department of Biochemistry & Molecular Biology by Subject "Amyloid beta protein"

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    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.