Raymond Scott Turner, MD, PhD - US grants

The major components of amyloid in Alzheimer's disease (AD) brain are Abeta peptides that are derived by proteolytic cleavage of amyloid precursor protein (APP). Our preliminary data demonstrates that the X11alpha protein strongly influences APP metabolism in transfected HEK 293 cells. Specifically, X11alpha prolongs the half-life of cellular APP and retards recovery of its metabolic fragments, including the secreted amino-terminal fragments APPs, as well as Abeta40 and Abeta42 in conditioned medium. These effects are mediated by direct binding of the PTB-PI domain of Xllalpha to the YENPTY motif in the intracellular carboxy-terminus of APP. In addition, to a PTB-PI domain, X11alpha also contains two PDZ domains as well as an extended amino-terminus that may modulate Xllalpha effects on APP processing. We have recently found that Xllalpha exists as a heterotrimeric complex in mouse brain complexed with the mammalian homologues of the C. elegans Lin-2 and Lin-7 proteins. We propose to determine the potential modulatory influence of mammalian Lin-2 and Lin-7 on the inhibitory effects of X11alpha on cellular APP metabolism. We will study these effects by over-expression of wild type and dominant negative constructs of X11alpha, mLin-2 and mLin-7. Initial studies will be performed with transfected Hek 293 cells. Since APP metabolism is to some degree cell-type specific and Xllalpha is primarily a neuronal protein, we will also analyze the effects of Xllalpha, mLin-2 and mLin-7 on APP processing in neurons. We will perform these experiments in NT2 neurons infected with Semliki Forest Virus expressing the wild type and dominant negative constructs. We hypothesize that Xllalpha influences PP metabolism by altering its cellular trafficking. Thus, we will examine the cellular localization of PP, Xllalpha, mLin-2, and mLin-7 in wild type and infected cells. Finally, we hypothesize that the regional expression of APP, X11alpha, mLin-2, and mLin-7 in brain may inversely correlate with the regional neuropathology of AD, particular amyloid plaques. We will determine the regional expression and localization of these genes/proteins in normal and AD brain sections by immunohistochemistry and in situ hybridization. Collectively, this data will provide insights into the regulation of cellular APP trafficking and metabolism via these specific protein-protein interactions, and their potential roles in the development of AD. This information may lead to novel therapeutic strategies to delay the onset or slow the progression of amyloid deposition, dementia, and AD.

The neuronal adaptor protein X11alpha/mint-1 interacts with amyloid precursor protein (APP) to regulate its trafficking and processing in vitro. In transfected non-neuronal cells, X11alpha inhibits alpha- and gamma- but not beta- cleavage of APP. Because the phosphotyrosine binding (PTB) domain of X11alpha interacts specifically with the APP family, X11alpha appears to inhibit gamma-cleavage of APP specifically while sparing gamma-cleavage of Notch and other substrates of regulated intramembranous proteolysis. To determine the in vivo significance of these in vitro data, we will generate and characterize novel hX11alpha transgenic mice and X11alpha knockout mice and examine murine APP metabolism. Crosses of these mice to Tg2576 mice (transgenic for the Swedish mutation of hAPP, or hAPPswe) will probe the effects of XI la on hAPPswe metabolism and on the development of partial AD-like phenotypes in aging brain. A recent human genome-wide analysis revealed significant linkage of sporadic AD to single nucleotide polymorphisms (SNPs) on chromosome 9, perhaps including the X11alpha region. We will probe genetic linkage of SNPs in the X11alpha region to sporadic AD by using genomic DNA extracted from samples from AD subjects versus carefully-matched control subjects. The specific aims are to: 1) generate X11alpha knockout and hX11alpha transgenic mice and determine effects on murine APP metabolism in brain and in primary neuronal cultures, 2) cross X11alpha knockout and hX11alpha transgenic mice with Tg2576 mice to determine a) modulatory effects on hAPPswe metabolism in brain and in neuronal cultures, and b) development of AD-like phenotypes with aging, and 3) elucidate the SNPs and haplotypes within or adjacent to the X11alpha gene of AD cases and age-, gender-, and ApoE-matched controls to determine if there is a statistically significant link to sporadic AD. Specific aim 3 will study DNA samples obtained from the Pathology Core and from other ADRCs. These results will 1) inform the normal functions of X11alpha as well as APP and its derivatives in CNS neurons, 2) lay the groundwork for viral-vector based gene therapy of AD using either X11alpha or its PTB domain in hAPP transgenic mouse models, and 3) probe a potential genetic risk factor of sporadic AD.

The neuronal adaptor protein X11alpha/mint-1 interacts with amyloid precursor protein (APP) to regulate its trafficking and processing in vitro. In transfected non-neuronal cells, X11alpha inhibits alpha- and gamma- but not beta- cleavage of APP. Because the phosphotyrosine binding (PTB) domain of X11alpha interacts specifically with the APP family, X11alpha appears to inhibit gamma-cleavage of APP specifically while sparing gamma-cleavage of Notch and other substrates of regulated intramembranous proteolysis. To determine the in vivo significance of these in vitro data, we will generate and characterize novel hX11alpha transgenic mice and X11alpha knockout mice and examine murine APP metabolism. Crosses of these mice to Tg2576 mice (transgenic for the Swedish mutation of hAPP, or hAPPswe) will probe the effects of XI la on hAPPswe metabolism and on the development of partial AD-like phenotypes in aging brain. A recent human genome-wide analysis revealed significant linkage of sporadic AD to single nucleotide polymorphisms (SNPs) on chromosome 9, perhaps including the X11alpha region. We will probe genetic linkage of SNPs in the X11alpha region to sporadic AD by using genomic DNA extracted from samples from AD subjects versus carefully-matched control subjects. The specific aims are to: 1) generate X11alpha knockout and hX11alpha transgenic mice and determine effects on murine APP metabolism in brain and in primary neuronal cultures, 2) cross X11alpha knockout and hX11alpha transgenic mice with Tg2576 mice to determine a) modulatory effects on hAPPswe metabolism in brain and in neuronal cultures, and b) development of AD-like phenotypes with aging, and 3) elucidate the SNPs and haplotypes within or adjacent to the X11alpha gene of AD cases and age-, gender-, and ApoE-matched controls to determine if there is a statistically significant link to sporadic AD. Specific aim 3 will study DNA samples obtained from the Pathology Core and from other ADRCs. These results will 1) inform the normal functions of X11alpha as well as APP and its derivatives in CNS neurons, 2) lay the groundwork for viral-vector based gene therapy of AD using either X11alpha or its PTB domain in hAPP transgenic mouse models, and 3) probe a potential genetic risk factor of sporadic AD.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative affliction associated with memory dysfunction. Pathological mutations in familial AD patients have been identified in several genes, and transgenic mice carrying pathological genes have been generated. The generation of transgenic models of amyloidosis has significantly aided progress in the development of therapeutic approaches designed to lower brain amyloid beta (Abeta) load. One of these approaches is called "immunization". Immunologically provoked (or passively administered) antibodies against Abeta have been shown to enhance microglial phagocytosis and reduce Abeta load in the brains of transgenic mice. Significantly, immunization also reversed Abeta associated memory dysfunction. Concomitant with reduced CNS Abeta is the simultaneous observation by us and others that plasma Abeta levels are significantly elevated following immunization. As a result of this observation, we have devised a new methodology to alter brain Abeta load, which has proved to be effective in preliminary studies. In the human active immunization clinical trial, Abeta vaccine approach was terminated after severe brain inflammation was found approximately in 6% of patients. The cause of brain inflammatory side effects is not clear yet, but it is most likely due to immune modulation. Although the first clinical trial of vaccine therapy was terminated, follow up reports are encouraging. Sequestration approach does not modulate immune reaction;therefore, it therapy has higher flexibility in drug development, and drugs based on sequestration approaches have less side effects. In addition, plasma Abeta elevation is a possible biomarker when this approach translates to clinical use. In this study, we will investigate the mechanism of Abeta sequestration mechanism and identify optimal molecular property and drugable target for future development of pharmacological therapy.

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. This is a (Category A) NIA-ACDS/Baxter Healthcare-sponsored 20 month (76 wk) trial comparing two doses of IVIG (200 or 400 mg/kg, q 2wk x 36 infusions) with placebo (0.25% albumin) to decrease the progression of dementia due to mild-moderate Alzheimer's disease already on routine therapy (cholinesterase inhibitors or memantine). Primary endpoints are based on ADAS-Cog and ADAS-CGIC instruments. Secondary measures are other neuropsychiatric measures, resource utilization, safety and tolerability, plasma biomarkers, and changes in (noncontrast) MRI. There are optional sub-studies which would allow 10y banking of DNA, repeated LP for CSF biomarkers (at baseline, 9 mo, and 18 mo), and/or FDG-PET (at baseline and 9 mo).

DESCRIPTION (provided by applicant): Druggable regulators of nitric oxide production as new Alzheimer's disease therapeutics Summary: Nitrosative stress is a critical mediator of the onset and progression of Alzheimer's disease (AD): it precedes and is associated with neuritic dystrophy and dendritic spine loss, A¿/amyloid accumulation and deposition, cholinergic denervation and a memory loss phenotype in animal models of disease. Normally, nitric oxide (NO) is an important signaling molecule and the enzyme that produces it, nitric oxide synthase (NOS), regulates ApoE and its other protein partners via nitrosylation. Under pro-inflammatory conditions (e.g. AD), oxidative stress upregulates NOS. Excess NO combines with oxygen radicals forming the reactive nitrosylating species peroxynitrite, which in turn causes promiscuous dysregulation and indiscriminate damage. Because direct inhibition of NOS results in systemic toxicity, our unique strategy is to selectively reduce NO activity at sites of inflammation. This will be achieved by targeting Sigma- 1 receptors (S1R), because they become important regulators of NOS activity only under conditions of oxidative stress. Our hypothesis is that elevated brain NO levels can be lowered at inflammatory sites by drugs that promote S1R-mediated reductions in NOS activity. The path for discovery and proof-of-concept phases includes: A) synthesis of additional novel candidate molecules to impart the appropriate selectivity and drug-like characteristics to compounds that reduce NO levels and promote neuronal and/or glial cell survival in vitro under conditions of nitrosative stress; B) evaluate 1- of these leads in a transgenic mouse model of AD by measuring reductions in CNS A¿/amyloid burden, 3-nitrotyrosine levels and improvements in cognition. Novel high affinity S1R candidates (hits) have been identified in our preliminary work and now we seek to create leads with refined drug-like properties for testing our target and mechanism- based hypothesis for therapeutics designed to slow the progression of AD. PUBLIC HEALTH RELEVANCE: The objective is to discover and develop news drugs for the treatment of Alzheimer's disease, a leading cause of morbidity and mortality in the US.