Walter J. Lukiw - US grants

DESCRIPTION (Provided by applicant): Alzheimer's disease (AD) represents an insidious, progressive, neurodestructive process of the human brain clinically characterized by the deterioration of memory and higher cognitive function. Familial AD and the more prevalent sporadic AD share final common neuropathological features that include a presenilin-1 (PS1) mediated catabolism of b-amyloid precursor protein (BAPP) generating neurotoxic amyloid-beta (AB) peptides that are deposited as insoluble senile plaque (SP). AB peptides and SPs increase reactive oxygen species (ROS) production which, in turn, fuel the expression of brain genes that promote proinflammatory (PI) episodes and brain cell death. The appearance in both familial and sporadic AD brain of reactive astrocytes, activated microglia and PI cytokines such as interleukin-1 beta (IL-1 beta) associated with AB and SP suggests that AD brain may be in a chronic state of inflammation. Abundant data that NSAIDs may be effective in ameliorating AD progression further supports a PI component to AD etiopathology. The goal of this project is to clarify the contributions of two key PI gene signaling pathways in AD brain which culminate in excessive stimulation of the neuroinflammatory response: (a) the inducible oxidoreductase cyclooxygenase-2 (COX-2) gene, which encodes the prostaglandin synthase responsible for prostanoid and other PI mediator production and (b) the presenilin-1 (PS1) gene, which drives aberrant processing of BAPP to accelerate AB production, Inducible COX-2 and PS1 gene over expression can both be regarded as upstream PI signaling events centrally involved in AD pathophysiology. The generation of ROS by AB also activates the binding to promoter DNA of NF-kB, a potent PI transcription factor (TF). Thus, NF-kB-DNA binding, driving COX-2 and PS1 gene transcription represent pivotal activating forces for PI signaling. The COX-2 and PS1 genes (a) exhibit down-regulation during human brain development, (b) are both sharply up-regulated in AD hippocampus, (c) are co-induced by AB42+ IL-I beta] in cultured human brain cells and (d) share at least 9 DNA regulatory motifs in their immediate promoters, including multiple AP1-, HIF1-, NF-Kb and STAT1-DNA binding sites, suggesting correlated gene activation. Using human control and AD brain analysis, human neural cells in primary culture and COX-2- and PS1-promoter, luciferase-reporter transfection models, we propose to test the hypothesis that signaling factors including PI TFs are key in the control of COX-2-and PS1-transcription-mediated over-stimulation of PI pathways that fuel neuronal cell degeneration in AD brain.

DESCRIPTION (provided by applicant): Abundant evidence supports the idea that progressive up-regulation of complex pro-inflammatory signaling, A[unreadable]42 peptide evolution, and loss of neurotrophic support underlie the initiation and propagation of Alzheimer's disease (AD). Micro RNA (miRNA)-mediated messenger RNA (mRNA) interference is a newly discovered genetic mechanism involved in the regulation of gene expression at the post-transcriptional level. The major mode of miRNA action is to target and bind to the 3'un-translated region of specific mRNAs and in doing so, quench expression of that mRNA, thereby acting as a negative regulator of gene expression. AD affects only specific areas of the brain, and mis-regulated miRNA expression is strongly linked to altered gene expression within these regions. In AD models, these same AD-enriched miRNAs induce specific pathological changes that are strikingly similar to those observed in AD brain. Accumulating evidence strongly suggests that altered miRNA-mediated processing of specific mRNA populations triggers the pathogenic expression of genes that drive the AD process. The overall goal of this project is to significantly move the miRNAin- AD field forward, and impact the scientific understanding of AD, by defining mechanistically how specific miRNAs induce molecular-genetic mechanisms that result in AD-type change. Human brains utilize only a fraction of all known miRNA species;and only a subset of these are altered in AD brain. Increases in the expression of a specific NF-?B-sensitive miRNA-146a down-regulates the abundance of at least three major mRNA targets, complement factor H (CFH), interleukin-1 receptor associated kinase (IRAK) and tetraspanin 12 (TSPAN12), that encode key modulators of the brain's innate immune system, inflammatory response and the generation of neurotrophic or neurotoxic amyloid peptides. Down-regulated TSPAN12 restricts ADAM10 (a-secretase) dependent cleavage of beta-amyloid precursor protein ([unreadable]APP). Stressed human brain cells up-regulate miRNA-146a in parallel with increased amyloidogenesis and shedding of A[unreadable]42 peptides. Hypothesis: up-regulated miRNA-146a contributes to altered innate immune and inflammatory responses, increased A[unreadable]42 peptide generation and decreased neurotrophic support. Three Specific Aims test predicted outcomes based on this hypothesis in AD, in stressed human brain primary cells, and in specific transgenic models of AD. These Specific Aims include (1) identification of alterations in miRNA-146a abundance in AD brain;(2) testing whether stressed human neuronal, astrocyte or microglial cells exhibit miRNA changes similar to those conducive to inflammatory neurodegeneration and A[unreadable]42 peptide generation in AD tissues, and testing if added exogenous miRNA-146a or anti-miRNA-146a oligonucleotides will promote or neutralize these changes;and (3) testing whether miRNA-146a, anti-miRNA-146a and modulation of CFH, IRAK or TSPAN12 expression are key regulators of the inflammatory response and amyloidogenesis in amyloid over-expressing Tg2576 mice. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is a progressive, fatal, neurodegenerative disorder of the human brain and is a major and expanding health care concern in this country, currently affecting an estimated 5 million Americans. Recent evidence indicates that progressive changes in the brain's inflammatory signaling systems, involving complement factor H (CFH), the interleukin-1 receptor associated kinase-1 and -2 (IRAK-1, IRAK-2), tetraspanin12 (TSPAN12) and A[unreadable]42 peptide evolution, against a background of brain aging, underlie the initiation and progression of the AD process. Our proposed molecular-genetic approach will define the role and mechanism of action of the recently discovered human brain-enriched micro RNA 146a (miRNA-146a) in CFH, IRAK-1, IRAK-2 and TSPAN12 signaling, and A[unreadable]42 peptide generation, that regulate key components of pathogenic signaling in the AD-affected human brain.

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) is the leading cause of infectious blindness in the industrialized nations. In the USA, 300,000 cases are treated yearly. Ocular HSV causes a self-limiting epithelial keratitis, blepharitis, con- junctivitis, stromal keratitis, and the most damaging disease, necrotizing herpetic stromal keratitis (HSK). HSK is the most difficult to manage; about half of the patients do not respond to combination chemotherapy and will have significant loss of vision or blindness. HSV is a neurotrophic virus characterized by its ability to establish lifelong latency in neurons and to reactivate at peripheral sites, causing recurrent disease. The ocular recur- rences cause significant corneal scarring, leading to HSK. Our proposal will examine the relationship between low and high HSV-1 phenotypic reactivators in a mouse model highly susceptible to ocular pathogenesis. The e4 allele of Apolipoprotein E (ApoE) is a risk factor for ocular herpes. We will demonstrate that different phenotypes (strains) of HSV have different ocular pathogenesis in e4 knock-in mice, a transgenic mouse strain created by insertion of the human ApoE gene into a trans- genic mouse from which the mouse ApoE gene was removed. We will evaluate two ApoE mimetic receptor- based peptides that separately exhibit anti-viral and anti-herpetic activity in a combination therapy to treat the severe ocular pathogenesis resulting from HSV-1 infection in e4 mice. We hypothesize that our investigation using the two ApoE peptides will document a reduction in ocular pathogenesis and oxidative damage in neurons and epigenetic control that is affected by this therapy. To this end, we plan to follow these Specific Aims: Specific Aim 1: To test the hypothesis that significant differences in ocular pathogenesis will occur in e4 mice when infected with high and low phenotypic reactivating strains of HSV-1. Specific Aim 2: To test the hypothesis that more pathogenesis will occur when ApoE e4 mice latently infected with the high phenotypic reactivator are induced to reactivate than in those latently infected with the low phenotypic reactivator. Specific Aim 3: To test the hypothesis that therapy with two unique ApoE peptides will successfully inhibit the induction of HSV-1 reactivation of the high phenotypic reactivator in ApoE e4 mice. These studies, if successful, will demonstrate 1) the role of contrasting HSV-1 phenotypic strains in ocular herpes, 2) the susceptibility and increased pathogenesis of the e4 allele carrier using mice latent with a high phenotypic reactivator and given an HSV-1 induction stimulus, and 3) that two ApoE peptides can inhibit HSV- 1 reactivation and ocular pathogenesis, even in highly susceptible e4 mice that are given an HSV-1 inducer. These pharmacological studies will offer the possibility of a new combination therapy for patients with ocular herpes and possibly for other HSV diseases such as encephalitis and genital herpes. These studies involving translational research, epigenetics, and ocular pathogenesis are in accordance with the research goals of the December 2006 National Plan for Eye and Vision Research. PUBLIC HEALTH RELEVANCE: Herpes Simplex Virus (HSV) is the leading cause of infectious, non-traumatic blindness in all industrialized nations, with over 300,000 cases treated in the United States per year. While therapies are available, some patients remain strongly resistant to current treatments, thus our research targets a specific human gene, the e4 allele of apolipoprotein E (ApoE), which has been implicated in the pathogenesis of ocular HSV- 1. The use of two receptor-based ApoE mimetic peptides that exhibit potent anti-viral and anti-inflammatory activity, and which act to suppress this gene, holds great promise as a new therapy for ocular and other HSV-related diseases.

microRNA (miRNA) signaling in Alzheimer's disease (AD) Through extensive miRNA- and DNA-based expression array-, LED-Northern-, ELISA, Western-, immunological and bioinformatics-based analysis we have discovered a highly interactive network of NF-kB sensitive, up-regulated pro-inflammatory microRNAs (miRNAs) and their down-regulated messenger RNA (mRNA) targets and the proteins these mRNAs encode in sporadic Alzheimer's disease (AD) brain. The up-regulation of these pathogenic miRNAs and their down-regulated mRNA targets has been further analyzed in stressed human brain cells in primary culture and in 5xFAD amyloid over-expressing transgenic mouse lines. Through miRNA and mRNA abundance analysis, miRNA-mRNA complementarity mapping, association energy (EA) indexing, ELISA and Western analysis we can explain much of the observed neuropathology characteristic of the AD process by analyzing significant disruptions in selective miRNA-mRNA signaling. Our hypothesis is that there exists a small family of at least 6 critical pro-inflammatory miRNAs in AD brains responsible for targeting and down-regulating a group of pathogenic messenger RNA (mRNA) targets responsible for amyloidogenesis, tau pathology and neuroinflammation with accompanying deficits in synaptogenesis, innate- immunity, phagocytosis and a progressive impairment in A?42 peptide clearance. This renewal of our previous 5 year NIA R01 will investigate the integrated actions of this group of 6 pro-inflammatory, pathogenic miRNAs up-regulated in sporadic AD brain, in stressed human brain cells in primary culture in the brains of 5xFAD mice. The 6 up-regulated pro- inflammatory microRNAs to be studied in detail are miRNA-7, miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a and miRNA-155. Stressors will be those encountered as are found in aging AD brain ? these include reactive oxygen species (ROS), the pro-inflammatory cytokines IL-1? and TNF? and A?42 peptides. Specific Aim 1 will analyze the contribution of these factors in moderate-to- advanced sporadic AD and Down's syndrome (DS) brain; Specific Aim 2 will analyze the contribution of these factors in stressed human brain cells, i.e. in neuronal-glial primary cell co- cultures; Specific Aim 3 will analyze the contribution of these factors in 5xFAD amyloid over- expressing transgenic mouse lines. We will specifically accentuate the study of the most up- regulated miRNAs: miRNA-7, miRNA-9, miRNA 34a and miRNA-146a and their targeted disruption of UBE2A (ubiquitin conjugase protein) and TREM2 (triggering receptor expressed in microglial cells) signaling in AD brain, and in in vitro and in vivo AD models. We will also analyze the applicability of selective NF-kB inhibitors and anti-miRNA (AM) strategies in restoring homeostasis in this system. Our long term goal is the therapeutic manipulation of these miRNA-regulated epigenetic pathways to provide an efficacious treatment for the clinical management of AD at an early stage.