Steven W. Barger - US grants

The shared pathological processes that initiate Alzheimer's disease likely reflect aberrancies in basic aspects of cell biology and thus are amenable to study through cell culture approaches, which offer a degree (and rate) of manipulation unavailable in other models. In this project, tissue culture models will be used to test various aspects of glial and neuronal interactions theoretically operative in Alzheimer's. Preliminary astroglial and microglial culture, neuronal cultures, and relevant cell lines will be utilized to assay the effects of cytokines on parameters being explored in other projects because of their potential importance in the initiation and/or progression of Alzheimer's and related conditions. This project is focused on the hypothesis that S100beta and other glial cytokines modulate neuritic and synaptic integrity, and that this modulation can involve pathogenic over- expression of betaAPP. These interactions appear to be at the root of Alzheimer pathology and clinical dementia; they will be explored through three specific aims. Aim 1: Determine the effects of S100beta on expression and processing of betaAPP. Initial studies have shown that S100beta can elevate betaAPP, which has implications for both amyloid deposition and aberrant neuritogenesis. Proposed studies will determine mechanisms involved in this induction and its relevance to neurite growth and amyloid production. Aim 2: Determine the potential influence of cytokines on the interaction between calcium homeostasis, neuritic outgrowth, and synapses. While S100beta elevates betaAPP, it also could have unrelated effects on neuronal processes and connections through its impact on calcium levels. A more general influence on these parameters is indicated for other cytokines, as well. Determine the effectiveness of anti-inflammatory cytokines and rugs against glial and neuronal activation. The primary endpoint in aim 3 will be microglial activation, with the goal of reducing production of cytokines and neurotoxins which negatively impact neuronal health and function. However, it is also plausible that direct effects of pro-inflammatory cytokines on neurons may be susceptible to manipulation by drugs that target basic cytokine signaling events. Accomplishment of these goals will be of direct benefit to the identification of basic elements of Alzheimer's pathology and potential sites for therapeutic intervention.

DESCRIPTION (Abstract): Increasing evidence points to a role for inflammation in Alzheimer's disease. Data also implicate metabolites of the beta-amyloid precursor protein (beta-APP) in the etiology of Alzheimer's disease. We previously have demonstrated that secreted forms of beta-APP (sAPP) protect neurons against several toxic insults. However, we recently determined that sAPP can stimulate proinflammatory processes in microglia, a monocytic cell in the brain. Interestingly, these two distinct bioactivities of sAPP can be differentially modulated by genetic and biochemical determinants, including a physical interaction with apolipoprotein E (ApoE). These data suggest the hypothesis that the ultimate effect of sAPP on neuronal viability and function results from the integration of its neuroprotective and proinflammatory activities, and that the balance of these activates depends on sAPP structural variations and interactions with ApoE. The hypothesis will be tested through the following objectives: 1) Characterize the interaction between ApoE and sAPP; 2) Determine the signal transduction mechanisms through which sAPP activates inflammatory events in microglia 3) Determine the structural elements responsible for APP's proinflammatory activity; 4F) Determine how various activities of sAPP are ultimately integrated with respect to neuronal function and survival. A diversity of methods will be applied. Solution binding assays will be used to measure the affinity of sAPP for various ApoE isoforms. Microglial activation will be measured through assays of nitrite production, cytokine expression, and neurotoxicity. These endpoints will be applied to pharmacological tests of sAPP signal transduction mechanisms, which will be complemented by biochemical tests of the activation of these signal transduction pathways. Deletional and site-specific mutagenesis will be used to delineate the structural determinants of sAPP proinflammatory activity so that they can be compared to those required for ApoE binding and relevant signaling events. Neuron-microglia co-cultures and other unique culture systems will be utilized to determine the ultimate interaction of sAPP activities at the level of neuronal survival and synaptic integrity, and its modulation by molecular-structure issues. These studies may reveal a key component of Alzheimer's pathogenesis, explain existing implications of inflammatory involvement in Alzheimer's, and suggest therapeutic strategies directed at modifying the actions of sAPP. Specifically, elucidation of the cellular mechanisms through which sAPP activates microglia may provide targets for therapeutic intervention in Alzheimer's disease.

DESCRIPTION (provided by applicant): Infusion of the biological sciences with genetic sequencing of entire genomes has sped discovery, but complete utilization of this information awaits detailed information about gene regulation. Nowhere is this more apparent than in the nervous system, where environmental interactions with genetic information take the form of learning and memory. Glutamate is a paramount neurotransmitter in these processes, but also becomes a neurotoxin in several neurodegenerative conditions. Published literature suggests that activation of glutamate receptors can induce the transcription factor NF-kappa-B in CNS neurons; glutamatergic input has also been implicated in an apparent constitutive basal activity of NF-kappa-B in these cells. However, more stringent tests performed in support of this proposal indicate that glutamate does not activate NF-kappa-B DNA-binding in neurons cultured from the cerebral cortex. Furthermore, the only factors constitutively binding NF-kappa-B target sites are Sp1 and related proteins, and glutamate decreases this activity. Together, these results indicate that NF-kappa-B is not a glutamate-responsive transcription factor as previously thought. But considerable evidence still suggests that kappa-B elements control genes important for neuronal viability. The following hypothesis will be tested: Sp1-related proteins influence survival-promoting genes through binding to kappa-B elements, and glutamate toxicity involves a reduced binding of kappa-B elements by both Sp1 and NF-kappa-B. First, glutamate-evoked events that squelch NF-kappa-B activity in cortical neurons will be elucidated through tests of post-translational modifications and interactions with other proteins. Second, the role of NF-kappa-B in protection of neurons against glutamate toxicity will be examined. Third, mechanisms by which glutamate suppresses the activity of Sp1-related factors will be explored. Finally, the ability of Sp1-related proteins to control neuronal transcription through kappa-B cis elements, with particular regard to survival-promoting genes, will be explored. These studies should shed light on a contentious issue, namely, the role of NF-kappa-B and its DNA target sites in cerebral neurons. As such, this body of work could have direct impact on the mechanisms by which a major neurotransmitter impacts on the genetic correlates of memory, learning, and excitotoxic cell death, paradigms of considerable significance to human mental health.

Alzheimer's disease involves inflammatory sequalae, as supposed by the overall hypothesis guiding this Program, and microglia make a substantial contribution to neuroinflammatory molecules and events. Although much has been learned about the initial stages of microglial activation by amyloid (3-peptide and other Alzheimer-related stimuli, little is known about the subsequent stages that are likely to make a larger contribution to brain biology in a slow, chronic disease like Alzheimer's. The relationship between feedback signals, a refractory state ("innate tolerance"), and alternative activation are not clear. Our ongoing studies conducted under this Program have pointed to a role for genetics in the resolution of microglial activation. The human interleukin-1a (IL-1a) gene contains single-nucleotide polymorphism (SNP) at position +4845, creating an amino acid coding change one residue from the site of proteolytic maturation of the cytokine; possession of the rarer allele 2 at this SNP is correlated with increased risk for Alzheimer's disease. We have found that microglia expressing pro-Illa of the allele 1 sequence become desensitized to activation by other stimuli (a phenomenon known as "innate cross-tolerance")whereas those expressing from allele 2 do not. We have also found roles for calcium regulation and cellular oxidation in critical aspects of microglial activation, including their release of excitotoxins. We intend to examine these events in three main areas of investigation. The role of IL-1a (or pro-IL1a) in tolerance will be studied in a mechanistic and genotype- specific manner. Genotypic differences in hydrolysis by the calcium-dependent protease calpain will be examined along with other impacts of calcium regulation on microglial activation. The interplay between calcium and cellular oxidation will be elucidated, taking particular advantage of a novel, targeted mutation in mice that accumulates lipid peroxidation products. Finally, the impact of AD pathology and oxidative stress on the memory-related behavior of these mice will be assessed. Together, these studies will enhance our understanding of novel aspects of microglial behavior, specifically, the meaningful interactions between proinflammatory cytokines, oxidation, calcium, and excitotoxicity. Elucidation of these issues will provide insight into events that might be amenable to therapeutic intervention, as well as explaining phenotypic differences resulting from genetic variability in the IL1A gene.

The purpose of Core C is to provide, on request, molecular analysis services to the investigators in this Program Project. The services will include Western immunoblot; in situ hybridization; quantitative (real-time) reverse-transcriptase polymerase chain reaction (qRT-PCR) analyses; electrophoretic mobility shift assays; and cytokine bioactivity analyses. In addition, analyses of other proteins and their encoding mRNAs that may be of interest (e.g., other cytokines, growth factors, or structural proteins) will be available upon request. We will have A(3, S100B, and GFAP ELISAs available for rapid screening of large numbers of samples. As a rapid screen for specific mRNAs in cell cultures, micro-//? situ hybridization will also be available. The tissue to be analyzed will be from human and rodent brain (Projects 1 and 2) and from cell cultures (Projects 1 and 3). A key aspect of the Core will be provision of expertise in time-consuming steps, such as antibody selection and optimization of PCR primer design. In addition, standardization across projects will help to achieve valid comparisons and improve efficiency. Results from analyses performed in this Core will be provided to Project Leaders primarily as raw data, including appropriate controls to document sample integrity and equipment function. Data for some analyses will be supplemented by routine extrapolation, and Core personnel will consult with Project Leaders in developing sophisticated parsing of data and experimental parameters. Residual samples and processed specimens, such as tissue sections, will be returned to the respective Project Leader for archiving. Records of services performed and quantitative data from the Core will be archived in the Core's computer database, under protection of state-of-the-art firewalls and data backup. Achieving the goals of this Core will allow standardization of methods for data acquisition and analyses across projects and will help ensure efficient collection and dissemination of data for publication.

DESCRIPTION (provided by applicant): The [unreadable]-amyloid precursor protein ([unreadable]APP) is connected to Alzheimer's disease by genetics, pathology, and biochemistry. A secreted form of this protein, sAPPa, can inhibit the activity of a class of neurotransmitter receptor called the NMDA receptor. This "neuromodulation" requires the unique C-terminus of sAPPa;the secreted form of [unreadable]APP produced by [unreadable]-secretase (sAPP[unreadable]) differs at the C-terminus and is much diminished in neuromodulatory activity. These differences in bioactivity suggest differences in binding to cell-surface receptor(s). Preliminary results suggests that the bioactivity of sAPPa is mediated by binding to the ApoE receptor 2 (apoE-R2;a.k.a. LRP8), known to be a receptor for another neuromodulatory protein, Reelin. Reelin facilitates NMDA-R activity critical for memory by eliciting homodimerization of LRP8 or a closely related receptor termed VLDL-R. Existing as a monomer at low concentrations, sAPPa could be envisioned to bind one or both of these receptors without effecting their dimerization. It is hypothesized here that the neuromodulatory activity of sAPPa is mediated by monovalently binding a LRP8 and/or VLDL-R molecule, inhibiting the receptor(s)'s dimerization and signaling. This effect may be lost as sAPPa homodimerizes at higher concentrations. Key components of this hypothesis will be tested by accomplishment of two main objectives. First, the binding of sAPPa to LRP8 and VLDL-R will be tested by forced expression of these receptors in an ectopic cell type. Second, the role of LRP8 and VLDL-R in the neuromodulatory activity of sAPPa will be examined by reducing their expression in mammalian neurons. In addition to providing key mechanistic insights into the basic biology of sAPPa, this project will facilitate future studies exploring a broader hypothesis that focuses on the dimerization potential of other LRP ligands such as apolipoprotein E. PUBLIC HEALTH RELEVANCE: Most immediately, the results of this study will influence hypotheses about the function of the [unreadable]-amyloid precursor protein ([unreadable]APP), which may play a role in Alzheimer's disease as well as aspects of normal brain development that are compromised in human conditions such as cerebral palsy and Down's syndrome. The findings from this project will also be expanded into a larger hypothesis concerning the interactions of [unreadable]APP with apolipoprotein E (ApoE), with greater emphasis on Alzheimer's disease, where genetic variations in both [unreadable]APP and ApoE contribute to disease etiology.

DESCRIPTION (provided by applicant): The LDL receptor-related protein (LRP) family is of interest in Alzheimer disease (AD) research because it includes several receptors for apolipoprotein E (ApoE), and polymorphisms in the gene for the latter (APOE) create the most demographically significant genetic risk factor for developing AD. Another genetic contribution is made by the genes for presenilins (PS)-1 and - 2; mutations in these genes are responsible for the largest fraction of early-onset AD inherited in an autosomal-dominant manner. Preliminary data indicate that such PS1 mutants reduce the steady-state levels of LRPs below what is seen with wild-type PS1. The proposed studies will characterize this relationship further and test the mechanisms involved and the consequences for LRP function. Successful completion of these studies will inform a novel hypothesis that places LRP dysfunction at a central point in the mechanisms common to AD pathogenesis.

Summary The purpose of Core C is to provide, on request, molecular analysis services to the investigators in this Program Project. The services will include western immunoblot, quantitative (real-time) reverse-transcriptase polymerase chain reaction (qRT-PCR) analyses, ELISAs, and 2D-gel proteomic preparation. In addition, other forms of macromolecular analysis will be developed as the need arises. The tissue to be analyzed will be from human and rodent brain and from nematode and cell cultures. All three Projects will utilize the services of Core C. A key aspect of the Core will be provision of expertise in time-consuming steps, such as antibody selection and optimization of PCR parameters. In addition, standardization across projects will help to achieve valid comparisons and improve efficiency. Results from analyses performed in this Core will be provided to Project Leaders primarily as raw data, including appropriate controls to document sample integrity and equipment function. Data for some analyses will be supplemented by routine extrapolation, and Core personnel will consult with Project Leaders in developing sophisticated interpretation of data and experimental parameters. Residual samples and processed specimens, such as tissue sections, will be returned to the respective Project Leader for archiving. Records of services performed and quantitative data from the Core will be archived in the Core's computer database, under protection of state-of-the-art firewalls and data backup. Achieving the goals of this Core will provide efficiency and standardization of methods for data acquisition and analyses across projects and will help ensure effective collection and dissemination of data for publication.

Summary ? Project 2 Type-2 diabetes mellitus (T2DM) is a growing health-related concern, in part because of increases in its incidence. However, T2DM is also significant because of emerging evidence regarding the breadth of its effects on various body systems. It is now recognized that T2DM is associated with CNS deficits such as dementia, including Alzheimer's disease. We have found several ways in which a gene related to Alzheimer's influences diabetic tendencies in mice. The amyloid precursor protein (APP) serves as the source of amyloid ?-peptide (A?), and its mutation is capable of causing Alzheimer's. Our preliminary studies indicate that APP exerts a powerful impact on insulin/glucose metabolism, so to test the role of A? itself, we have turned to mice that over-produce this peptide alone in the CNS. These mice develop a prediabetic state (insulin resistance) by default. We propose to determine the effects of A? on CNS elements that control peripheral energy metabolism, placing particular emphasis on inflammatory processes. We also will test whether any effects of A? on insulin/glucose regulation can be modulated by expression of human apolipoprotein E (ApoE). This protein's gene (APOE) contributes the largest demographic risk to Alzheimer's disease, and ApoE both impacts inflammation and binds A? in genotype-specific manners. Thus, we expect ApoE to modify the effects that A? has on insulin/glucose metabolism. Finally, we will test whether the effects of A? on insulin/glucose metabolism can be similarly modified by interrupting inflammation or protein aggregation; this will be accomplished in part through cooperative strategies for drug development throughout the parent Program. Together, these experiments will characterize the interactions between T2DM-related syndromes and proteins of critical importance to Alzheimer's disease. The project will ultimately test pharmacological agents that directly impact these interactions and may thus provide novel therapeutic opportunities for Alzheimer's and T2DM.

SUMMARY Aging is the most powerful risk factor for Alzheimer?s disease (AD), and it contributes to the odds of type-2 diabetes mellitus (T2D) as well. Aging is also associated with a decline in the brain?s use of glucose, its most important fuel. Astrocytes play a key role in shuttling glucose from the bloodstream to where it is needed by the neuronal units of activity deeper in the brain tissue. We find evidence of a defect in a key glucose transport molecule of astrocytes in AD and in a mouse line genetically modified to reproduce some aspects of AD. This mouse line, overproducing the ?-amyloid peptide (A?), exhibits dysregulation of circulating glucose, as well as a decline in brain glucose use. These effects are correlated with poor performance in a test of spatial memory. Further mimicking human AD, the mice show these problems in the absence of obesity, hyperglycemia, disruption of appetite, changes in physical activity, pancreatic abnormality, or insulin resistance. Together, these findings inspire the hypothesis that A?, the levels of which begin to rise in the aging brain even without frank AD, perturbs the ability of astrocytes to bring peripheral glucose to neurons, where it is needed for the increased neurological activity associated with memory and other functions. This idea will be tested through studies of the status and function of glucose transport proteins in aging mouse and human brains, along with comparisons between normal aging, AD, and T2D. Through genetic modification of mice, we will modulate the levels of the most important astrocytic glucose transporter to determine if it is i) sufficient to bring about interrupted glucose delivery and memory deficits, and ii) necessary for the presentation of these problems in an AD model. These studies thus explore a novel hypothesis about a specific element of energy utilization in the aging brain and its connection to age-related cognitive impairment. As such, the project may provide innovative strategies for therapeutic intervention.