Mark S. Kindy - US grants

Amyloid deposits result in organ dysfunction and death. Amyloidogenesis can be influenced at the level of precursor synthesis, precursor catabolism or fibrillogenesis. The least studied of these processes is fibrillogenesis, yet it may be the most feasible target for therapeutic assault. This is due to the extracellular nature of the deposit, and the commonality in all amyloid processes. All amyloid deposits consist of characteristic antiparallel cross Beta-pleated sheets, with proteoglycans, serum amyloid P component and ApoE as significant subcomponents. Although a diverse, yet select, group of protein precursors have been shown to be incorporated into amyloid, the precise molecular motif(s) that is an essential prerequisite for these unique fibrillar deposits has not been identified. We believe that the "structural motif" is the presence of a cross beta-pleated sheet structure or the ability of the protein to form a cross beta-pleated sheet structure in the presence of heparan sulfate proteoglycan(s) and calcium. The mouse amyloid model is ideal to initiate our understanding of fibrillogenesis. Other systems present major problems of precursor diversity or paucity. In the mouse, serum amyloid A proteins (SAA) are the most dramatic acute phase reactants with levels increasing up to 1000-fold following inflammation. Of the two major SAA isotypes in the mouse (SAA1 and SAA2), only one (SAA2) is selectively cleared and deposited in amyloid. Our laboratory has identified that the CE/J mouse is totally amyloid resistant and that it has a single SAA1 1/2 hybrid isotype. Analyses of this isotype have allowed us to deduce that one or more of 5 amino acids hold the key to the amyloidogenicity of SAA2. To test our hypothesis. the structural and pathophysiological effects of specific alterations in the SAA1\2 cDNA with subsequent mutant protein expression will be examined. Specifically, the CE/J SAA1/2 cDNA has been cloned into an expression vector and synthesized in vitro to generate protein for analysis. Site-directed mutagenesis will be performed to identify those amino acids essential for fibrillogenesis. The structural folding of these mutants will be determined by circular dichroism, tryptophan fluorescence and correlated with their biological ability to be deposited in amyloid. Finally, transgenic mice overexpressing S A A 2 in response to administration of zinc have been generated. This would allow fibrillogenesis to be studied separate from complicating systemic inflammation.

In order to facilitate Projects 2,3,4, and 5, and animal core laboratory has been established to do the following: provide a well-defined animal model of brain injury which produces nonfatal, focal and reproducible injury resulting in a histopathological picture similar to the clinical condition: handle and process all experimental and control animals for the experimenters; perform all surgeries on the animals under the most careful of conditions; perform tests of sensory-perceptual and motor functioning on the animals; perform behavior assessment of cognitive functions on the animals; and process tissue for histological tests. Non-penetrating, localized deformation of the cortex has been the mouse successful and extensively used rodent model. We will employ an electronically-controlled pneumatic impactor to achieve a closely controlled strike of the brain's cortical surface, in a fully anesthetized animal. This device produces precisely controlled brain contusions which are reproducible and which resemble many of the aspects of closed head injury seen clinically.

DESCRIPTION (provided by applicant): This proposal tests the hypothesis that oxidized lipoproteins induce neurodegeneration directly by acting on neurons and indirectly by activating microglia through a mechanism involving scavenger receptors. Oxidative stress mediated neuronal cell loss has been demonstrated in neurodegenerative disorders including Alzheimer's disease (AD) and stroke. Reactive oxygen species (ROS) can increase the rapid oxidation of lipids and proteins generating lipid peroxidation and oxidized protein products. Once formed, these oxidatively modified lipids and proteins may be the primary means by which ROS toxicity is elicited. High-density lipoproteins (HDLs) in the central nervous system are vulnerable to oxidative modification by trace metals, ROS, and enzymatic pathways. Preliminary data demonstrate the detrimental effects of oxidized HDL (oxHDL) on neuronal cells and the activation of microglial response in vitro. The specific aims of this proposal are: 1) To test the hypothesis that HDL induces neurodegeneration both in vitro and in vivo by activating ROS. We will characterize the neuronal and microglial response to oxHDL by activating oxidative stress, calcium and apoptotic pathways. 2) To test the hypothesis that oxHDL functions through interaction with scavenger receptors on neuronal and microglial cells. We will examine cell lines expressing scavenger receptors (SR) and cells isolated from SRgene-inactivation mice for altered response to oxHDL. 3) To test the hypothesis that the apolipoprotein E (apoE) genotype may affect the level of oxidation and the neuronal and microglial response to oxHDL. We will isolate apoE-specific HDL particles and determine their susceptibility to oxidation and their effects on neurodegeneration. 4) To test the hypothesis that oxidized HDL and scavenger receptors are present in AD brain in a regional pattern related to selective vulnerability. We will examine HDL isolated from control and AD brain for oxidative status and the relationship to apoE genotype. We will also examine the expression of SR and other molecules potentially relevant to the effects of oxHDL in the AD brain. These studies should provide insights into the normal function of HDL and SR in the CNS and in the pathogenesis of AD.

The goal of this Training Program is to prepare promising graduate students and postdoctoral fellows for successful careers in the neurobiology of aging. We will provide broad-based training in modern concepts in the neurobiology of aging with specific emphasis on changing cellular and molecular interactions that occur in the aging brain. The unifying focus of the training faculty is our interest in understanding the mechanisms by which the nervous system responds to changes that occur with age under normal and pathological conditions. This Training Program provides the formal framework for faculty in different departments, who share a common interest in the molecular and cellular basis of brain aging, to provide in depth training in aging. The emphasis of the Training Faculty complement each other: some focus on processes that occur in the brain during normal aging, others emphasize the neuropathological diseases that predominate in the aging brain, and still others use in vivo and in vitro systems to model the aging brain. Thus, trainees will learn from faculty who utilize a broad spectrum of state-of-the-art methodological approaches to probe critical questions in aging research. During the previous granting period, our record of recruiting promising pre- and post-doctoral fellows, providing state-of- the-art training, and placing them in excellent positions is outstanding. Our success attests to our ability to identify, attract, train and place promising young investigators in the area of aging is clear due to our changing demography and the increasing average lifespan. Normal aging or pathology of the nervous system account for a large percent of deaths in the elderly. Only if we better understand the basic mechanisms that regulate the aging of the brain and apply this to the treatment of the elderly, can we hope to improve the quality of life during the latter half of the lifespan. Our ability to attract and train graduate students and postdoctoral fellows to meet the challenges in the area of neurobiology of aging will be greatly enhanced by this Training Program.

The central hypothesis for this application is that Alzheimer's disease (AD) is a multi-factorial disorder in which a number of agents, including environmental determinants, contribute to a disparity between amyloid beta (Abeta) - production and Abeta clearance. This disparity leads to the accumulation and subsequent deposition of Abeta peptides that eventuates in the neuropathological presentation of AD. Based on this hypothesis, numerous laboratories have emphasized the biological aspects of amyloid precursor protein (APP) synthesis and metabolism and the mechanisms of Abeta production in AD. However, the molecular mechanisms of Abeta overproduction appear to contribute to only a portion of the total AD cases. The nature of Abeta degradation and clearance is the least studied mechanism in the process of AD progression and it is a likely target for therapeutic intervention. Recent studies indicate that the neutral endopeptidase neprilysin plays an important role in the degradation of Abeta(1-42) in the brain. The Specific Aims of this proposal are: 1) to provide evidence that NEP can regulate Abeta levels and deposition in vivo, which will be done by crossing transgenic mice overexpressing NEP or NEP-deficient mice with APP transgenic mice and assessing progeny biochemically and pathologically; 2) to use a cell-based system to study the susceptibility of NEP to oxidative damage; and 3) to compare NEP levels/activity in human AD brain and age-matched controls and to determine whether oxidative damage contributes to altered NEP activities. These studies will help determine how Abeta is degraded extracellularly, whether NEP plays a key role in this process, and how Abeta levels are regulated by the activity of NEP. The results generated from this study will help us to understand how Abeta peptides are degraded and may provide a unique therapeutic window for the treatment of AD.

It is generally postulated that the progressive neurodegeneration occurring in sporadic Alzheimer's disease (AD) is a result, at least in part, of the neurotoxic properties of the amyloid beta protein (Abeta). Deposits of this protein are widespread within the brains of individuals affected with AD. The neurotoxic effects of Abeta are mediated by free-radicals and depend on the predominant secondary structure of the protein and/or in its polymerization into fibrils. In addition, the mentioned structural characteristics of Abeta determine the rate of amyloid formation, resistance to proteolysis and tissue clearance. Since these features are also considered key to amyloid accumulation, the mechanisms and factors implicated in fibrillogenesis and neurotoxicity are among the most important issues in AD. We recently found that melatonin, a pineal hormone with a proposed role in the aging process, has remarkable cytoprotective properties against Abeta-induced neurotoxicity. While investigating the mechanisms of action of melatonin, we found that in addition to protecting cells against oxidative stress and death, this substance markedly inhibited the progressive formation of beta-sheet structures of Abeta as well as its polymerization into fibrils. Thus, the hormone exhibits anti- amyloidogenic properties that may be synergistic with its powerful antioxidant activity. In addition, melatonin shows many advantages over conventional antioxidants. The lack of toxicity and the ease and rapidity with which this molecule enters the central nervous system makes it an ideal candidate for experimental testing. In contract with conventional antioxidants, melatonin has a proposed role in the aging process. Its rhythmic secretion is known to be altered in aging and more profoundly in populations with dementia. This grant proposes to use a transgenic mouse model to demonstrate whether the novel in vitro effects can be corroborated in vivo. Moreover, the neuroprotective effects of melatonin will be compared with another conventional antioxidant (PBN), which is structurally unrelated to melatonin and is known to protect neurons against Abeta toxicity but exhibits no anti-amyloidogenic properties in vitro. These studies are necessary for the design of human trials and to further characterize mechanistic aspects (i.e., oxidative stress) of the disease pathogenesis.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall objective of the Animal Pathobiology Core is to provide the SC COBRE in Lipidomics and Pathobiology investigators with the ability to utilize animal models in the execution of their proposed research projects as well as establish new animal models for the investigation of the mechanisms involved in the pathogenesis of disease. The Animal Pathobiology Core will provide 1) gene-altered animals including transgenic and knockout mice;2) the necessary facilities, faculty and staff expertise for animal models including neurodegeneration, cardiovascular disease, cancer and inflammation;3) expertise in animal pathology;and 4) a database of all project animals.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is most commonly associated with deposition of amyloid-beta (Abeta) peptide in the brain. The presence of Abeta peptide in the brain plays an important role in the development of aggregates that may result in neuronal damage, dysfunction, microglial activation and neuropathological features of AD. Abeta [with or without tau/neurofibrillary tangles (NFTs)] perturbs cellular properties mainly by oxidant stress, which overwhelms the cellular antioxidant defense mechanisms. Recent studies suggest that environmental toxins (ETs) may contribute to the pathogenic process of AD and neurodegenerative diseases, disrupting neuronal function resulting in oxidative stress (OS), inflammation and cell death. We and others have shown that paraoxonase-1 (PON-1) activity is reduced in the plasma of individuals with AD, and that ETs (organophosphates) exacerbate AD pathogenesis in APP transgenic mice. We hypothesize that AD partially results from early (embryonic/neonatal) or chronic low level exposure to ETs or a defect in paraoxonase activity that gives rise to increased ET levels in plasma and brain triggering oxidative stress, inflammation and Abeta production. This paradigm facilitates Abeta aggregation and exacerbates cellular perturbation and AD pathogenesis. The focus of this proposal is to understand the function of organophosphates (OPs as symbolic ETs) and interrelationship between ETs and paraoxonase (PON-1) in oxidative stress and inflammation associated with AD. The long-term goal of this proposal is to understand the role of ETs and PON-1 in oxidative stress in order to determine if alteration in the level of toxins is a potential therapeutic strategy for individuals with AD. To this end, the following specific aims will be tested: Specific Aim 1: To determine the effect of organophosphates (OPs) on AD pathogenesis in APP transgenic mice. Specific Aim 2: To determine the effect of paraoxonase 1 deficiency on organophosphate induced AD pathogenesis in APP transgenic mice. Specific Aim 3: To study the therapeutic effects of galantamine and adenoviral delivered PON1 on organophosphate provoked AD. Specific Aim 4: To determine if PON1 allotype or functional deficits in PON1 activity predict the development and progression of AD that may be stimulated by environmental toxins. These studies will help to define the role of environmental factors in the pathogenesis of AD. PUBLIC HEALTH RELEVANCE: The overall goal of this project is to determine the role of environmental toxins (ETs) in their contribution to the pathogenic process of AD and neurodegenerative diseases. We hypothesize that AD partially results from early (embryonic/neonatal) or chronic low level exposure to ETs or a defect in paraoxonase activity that gives rise to increased ET levels in plasma and brain triggering oxidative stress, inflammation and Abeta production. The focus of this proposal is to understand the function of organophosphates (OPs as symbolic ETs) and interrelationship between ETs and paraoxonase (PON- 1) in oxidative stress and inflammation associated with AD.

DESCRIPTION (provided by applicant): Many current pharmacological approaches to combating Alzheimer's disease (AD) seek to block Abeta production through inhibition of the amyloidogenic enzymes known as beta- and gamma-secretases. An alternative approach is to activate the alpha-secretase processing of amyloid precursor protein (APP), which is mediated by several members of the disintegrin family of metalloproteases, ADAM9, ADAM10 and ADAM17. Processing of APP by these alpha-secretases is thought to be beneficial with respect to AD since it limits production of Abeta and generates the neuroprotective soluble APPalpha (sAPPalpha) product. Fibulin-1 (Fbln1) is an extracellular matrix protein, expressed in the brain by neurons, that binds the amino terminus of APP and sAPPalpha. The significance of this interaction is not yet established however, we have found that Fbln1 also binds to other membrane anchored alpha-secretase substrates, heparin binding-epidermal growth factor (HB-EGF) and neuregulin-1 (NRG1). We also show that Fbln1 acts to inhibit the proteolytic release of soluble forms of HB-EGF and NRG1. Furthermore, we have found increased levels of sAPPalpha in the conditioned culture medium of Fbln1 null mouse embryo fibroblasts (MEFs) as compared to wildtype MEFs. Based on these findings it is hypothesized that Fbln1 serves as an inhibitor of alpha-secretase processing of APP and therefore may represent a therapeutic target that if inhibited might lead to augmented alpha-secretase processing of APP and reduced pathological APP cleavage. To address this hypothesis there are three specific aims: 1) Determine whether brain APP proteolytic cleavage is altered in Fbln1-deficient mice, 2) determine whether transgenic overexpression of Fbln1 accelerates Abeta production and exacerbates AD pathogenesis, and 3) determine whether Fbln1 inhibits alpha-secretase processing of APP in cultured neuronal cells.