Steven Estus - US grants

Amyloid beta (Abeta) is a 4 kDa peptide that spontaneously aggregates and that deposits in the Alzheimer's Disease (AD) brain. Multiple studies have suggested that Abeta accumulation and deposition may be critical to AD (reviewed in (1,2)). Agents that block Abeta aggregation have been proposed as potential preventative and therapeutic treatments for AD (reviewed in (3)). For example, fragments of Abeta block Abeta aggregation as well as deleterious actions resulting from Abeta aggregation, i.e., in vitro neural toxicity (4-8) Moreover, such peptides prevent Abeta deposition when injected with Abeta into rat brains (6). Hence, the benefits that may result from peptides that block Abeta aggregation include amelioration of harmful actions of aggregated Abeta and facilitated Abeta peptides that block Abeta aggregation as well as deleterious actions resulting from Abeta aggregation, i.e., in vitro neural toxicity (4-8). Moreover, such peptides prevent Abeta deposition when injected with Abeta into rat brains (6). Hence, the benefits that may result from peptides that block Abeta aggregation include amelioration of harmful actions of aggregated Abeta and facilitated Abeta clearance. A primary concern about the use of fragments is the relatively low affinity of Abeta fragments of Abeta. To identify peptides that bind to Abeta with a high affinity, we, we propose to use phage display to screen phage peptide libraries. Peptides identified by this approach, or their derivatives, will then be evaluated and optimized for their ability (i) to block Abeta aggregation, (ii) to block Abeta actions, as modeled by Abeta in vitro toxicity, and (iii) to facilitate Abeta clearance. To this end, we propose the following Specific Aims: (i) identify peptides that bind Abeta with high affinity, we propose to use phage display to screen phage peptide libraries. Peptides identified by this approach, or their derivatives, will then be evaluated and optimized for their ability (i) to block Abeta aggregation, (ii) to block Abeta actions, as modeled by Abeta in vitro toxicity, and (iii) to facilitate Abeta clearance. To this end, we propose the following Specific Aims: (i) identify peptides that bind Abeta with high affinity, (ii) determine Abeta region and conformation responsible for peptide: Abeta interactions, (iii) evaluate and optimize peptide ability to inhibit Abeta aggregation and/or enhance Abeta disaggregation, (iv) evaluate peptide ability to modulate Abeta toxicity in vitro, and (v) evaluate and optimize peptide ability to modulate Abeta peptide ability to modulate Abeta toxicity in vitro and (v) evaluate and optimize peptide ability to modulate Abeta clearance. Overall, these studies will evaluate the hypothesis that Abeta binding peptides identified through phage display are capable of inhibiting Abeta aggregation in vitro, toxicity in vitro, and accumulation in vivo. These studies range from using a relatively novel technique for identifying Abeta-binding peptides to evaluation of the ability of these peptides to inhibit Abeta aggregation, toxicity, and clearance. Our studies of the structure of the minimal peptide necessary to modulate Abeta could, in work beyond the scope of this proposal, lead to the development of organic molecules manifesting similar properties. For individuals at risk for AD, such agents could potentially inhibit Abeta accumulation and thereby lead to a reduction in Abeta burden, providing a novel AD therapy.

DESCRIPTION (provided by the applicant): Identifying genetic-risk factors associated with Alzheimers disease (AD) is critical to progress against the disease. Recently, several groups identified a region of chromosome-10 as containing at least one susceptibility locus for late-onset AD (1-3), with one group additionally associating this region with enhanced plasma levels of amyloid-Beta (AB) (1). The gene encoding urokinase-type plasminogen activator (uPA) maps within this implicated region. Previously, we reported that uPA is induced by AB-treated neurons in vitro and in the Hsiao mouse model of AB burden in vivo (4). Moreover, uPA converts plasminogen to the active protease plasmin, which degrades both non-aggregated and aggregated AB with physiologic efficiency (Preliminary Results and [4, 5]). AB accumulation is a hallmark of AD, and may be causal to this disease. Considering these data overall, we hypothesize that one of the chromosome-10 loci is an uPA polymorphism that modulates uPA's ability to contribute, to AB clearance. To assess this hypothesis, we propose to: 1) evaluate the frequency of uPA polymorphisms in AD and control patients to identify polymorphism(s) segregating with AD. In preliminary work, we have identified an uPA polymorphism that significantly segregates with AD susceptibility. This polymorphism causes a leu for pro-change at position 141 within uPA, and alters binding of the uPA zymogen to aggregated fibrin; 2) gain insight into the possible roles of leu-uPA versus pro-uPA by comparing individuals homozygous, for leu-uPA versus pro-uPA for relevant clinical and neuropathologic markers of AD, including uPA localization in AD brain; 3) evaluate the ability of leu- uPA versus pro-uPA to bind AB, activate plasminogen, and inhibit AB neurotoxicity in vitro; and 4) evaluate the role of uPA in AB clearance in vivo by quantifying AB accumulation in mice that are wild-type or genetically deficient for uPA. Overall, the focused approach proposed here will: 1) directly evaluate the possible role of uPA polymorphisms as a risk factor(s) for AD; and 2) provide insights into possible mechanisms underlying the differential uPA actions. These studies are significant, in that the identification of additional genetic risk factors for AD will aide in early AD diagnosis, and thereby facilitate drug discovery by identifying patients at high-risk for AD prior to symptomology. Moreover, by evaluating possible mechanisms underlying the enhanced susceptibility to AD, these studies may lead to the discovery of novel insights into the molecular mechanisms underlying AD, and thereby suggest new therapeutic approaches.

DESCRIPTION (provided by applicant): Genetic factors that contribute to Alzheimer's disease (AD) susceptibility are critical to our understanding and early diagnosis of the disease. A chromosome 10 region contains at least one susceptibility locus for late onset Alzheimer's disease, and is associated with increased amyloid-B (ADi) plasma levels. The gene encoding urokinase-type plasminogen activator (uPA) is within this implicated region. UPA is induced by AB-treated neurons in vitro and in the Hsiao mouse model of AB burden in vivo. Moreover, uPA converts plasminogen to the active protease plasmin, which degrades both nonaggregated and aggregated AB with physiologic efficiency. In summation, ADi induces uPA, which can in turn lead to AB degradation, suggesting a self-regulated system for clearance of AB aggregates. Considering these data overall we hypothesize that the chromosome 10 loci includes a uPA polymorphism(s) that modulates uPA's ability to contribute to AD clearance. To evaluate this hypothesis, we propose to (i) identify uPA polymorphisms that segregate with Alzheimer's disease. In preliminary work we have identified two uPA polymorphisms that significantly segregate with AD susceptibility and are in strong linkage disequilibrium, including (i) a substitution of leu for pro at position 141 within uPA, which alters binding of the uPA zymogen to aggregated fibrin and (ii) a SNP two basepairs 3' to an AP-I site that is known to be critical for uPA induction. We also propose to (ii) Gain insight into the possible role of the at-risk uPA haplotype by comparing individuals homozygous for each genotype for relevant clinical and neuropathologic markers of Alzheimer's disease, (iii) Evaluate the effect of the uPA polymorphisms associated with AD risk on uPA expression and function, and (iv) Evaluate the role of uPA in AB clearance in vivo by quantifying AB accumulation in mice that are wildtype or genetically deficient for uPA. Overall, the focused approach proposed here will (i) directly evaluate the possible role of uPA polymorphisms as a risk factor(s) for Alzheimer's disease, and (ii) provide insights into possible mechanisms underlying differential uPA actions. These studies are significant in that the identification of additional genetic risk factors for Alzheimer's disease will aid in early AD diagnosis, and thereby facilitate drug discovery by identifying patients at high risk for AD prior to symptomology. Moreover, by evaluating possible mechanisms underlying the enhanced susceptibility to Alzheimer's disease, these studies may lead to the discovery of novel insights into the molecular mechanisms underlying Alzheimer's disease, and thereby suggest new therapeutic approaches.

DESCRIPTION (provided by applicant): Genetic factors that contribute to Alzheimer's disease (AD) susceptibility are critical to our understanding and early diagnosis of the disease. A chromosome 10 region contains at least one susceptibility locus for late onset Alzheimer's disease, and is associated with increased amyloid-B (ADi) plasma levels. The gene encoding urokinase-type plasminogen activator (uPA) is within this implicated region. UPA is induced by AB-treated neurons in vitro and in the Hsiao mouse model of AB burden in vivo. Moreover, uPA converts plasminogen to the active protease plasmin, which degrades both nonaggregated and aggregated AB with physiologic efficiency. In summation, ADi induces uPA, which can in turn lead to AB degradation, suggesting a self-regulated system for clearance of AB aggregates. Considering these data overall we hypothesize that the chromosome 10 loci includes a uPA polymorphism(s) that modulates uPA's ability to contribute to AD clearance. To evaluate this hypothesis, we propose to (i) identify uPA polymorphisms that segregate with Alzheimer's disease. In preliminary work we have identified two uPA polymorphisms that significantly segregate with AD susceptibility and are in strong linkage disequilibrium, including (i) a substitution of leu for pro at position 141 within uPA, which alters binding of the uPA zymogen to aggregated fibrin and (ii) a SNP two basepairs 3' to an AP-I site that is known to be critical for uPA induction. We also propose to (ii) Gain insight into the possible role of the at-risk uPA haplotype by comparing individuals homozygous for each genotype for relevant clinical and neuropathologic markers of Alzheimer's disease, (iii) Evaluate the effect of the uPA polymorphisms associated with AD risk on uPA expression and function, and (iv) Evaluate the role of uPA in AB clearance in vivo by quantifying AB accumulation in mice that are wildtype or genetically deficient for uPA. Overall, the focused approach proposed here will (i) directly evaluate the possible role of uPA polymorphisms as a risk factor(s) for Alzheimer's disease, and (ii) provide insights into possible mechanisms underlying differential uPA actions. These studies are significant in that the identification of additional genetic risk factors for Alzheimer's disease will aid in early AD diagnosis, and thereby facilitate drug discovery by identifying patients at high risk for AD prior to symptomology. Moreover, by evaluating possible mechanisms underlying the enhanced susceptibility to Alzheimer's disease, these studies may lead to the discovery of novel insights into the molecular mechanisms underlying Alzheimer's disease, and thereby suggest new therapeutic approaches.

[unreadable] DESCRIPTION (provided by applicant): Identifying genetic variants that significantly alter the function of critical proteins provides insights into related disease processes and possible therapies. The low density lipoprotein receptor (LDLR) is remarkable in this regard because LDLR mutations are a primary cause of familial hypercholesterolemia, i.e., loss of a single LDLR allele causes an approximately 100% increase in LDL-cholesterol levels. Although LDLR SNPs and their haplotypes have been associated with altered cholesterol levels, specific SNPs that alter LDLR function have not been identified. Functional LDLR variants may provide insights into diseases that may be associated with increased cholesterol, e.g., Alzheimer's Disease (AD). Indeed, a robust genetic factor for both cholesterol and AD is the apoE4 allele of the apoE gene. Although apoE is implicated in amyloid-beta (Abeta) deposition in Alzheimer's disease, apoE is known more widely for its role in cholesterol delivery and homeostasis; apoE4 is associated with increased LDL-cholesterol via its role as a ligand for the LDL family of cell surface receptors that mediate lipoprotein uptake. This proposal focuses on the global hypothesis that SNPs that modulate LDLR splicing or expression, and the haplotypes defined by these functional SNPs, are associated with altered cholesterol homeostasis and Alzheimer's disease. Hence, we propose the following Specific Aims: 1. Evaluate LDLR SNPs to identify those that alter LDLR splicing and /or expression, 2. Evaluate the expression and function of LDLR proteins resulting from alternative LDLR splicing. 3. Evaluate LDLR genotypes and haplotypes for their association with cholesterol homeostasis and Alzheimer's disease. Overall, this focused approach will directly evaluate the hypothesis that LDLR polymorphisms modulate LDLR splicing and expression to increase LDL-cholesterol and Alzheimer's disease odds. [unreadable] [unreadable] [unreadable]

these studies are significant and innovative because they have strong relevance to our understanding of the possible mechanistic etiology of AD, potentially facilitating therapy, and to AD genetics, facilitating early diagnosis and treatment.

DESCRIPTION (provided by applicant): The overarching theme of this proposal is that polymorphisms identified by recent Alzheimer's disease (AD) genome wide association studies biologically define rate-limiting steps in AD pathways. Hence, elucidating their mechanism of action will identify robust pharmacologic targets. Notably, a SNP with modest molecular actions may reduce AD risk by 10% but a drug that acts strongly at the same target may have a large effect on AD risk. This proposal will elucidate the mechanism of action of rs3865444 (rs444), an AD-associated SNP in CD33, and translate this mechanism into a proof of concept AD treatment. In our highly compelling preliminary results, we associate the AD-protective minor allele of rs444 with (i) a robust increase in the proportion of CD33 lacking exon 2 (D2-CD33) which appears critical to CD33 function. Large pharma have developed humanized monoclonal antibodies against CD33 for acute myeloid leukemia (AML); these antibodies and their derivatives have potential AD relevance as CD33 antagonists. This leads to our global hypothesis: Reduced CD33 function decreases AD risk whether CD33 inhibition is due to genetics or pharmacologic agents. To test our hypothesis, we will (i) Elucidate the mechanism underlying the CD33 AD SNP, (ii) Compare CD33 and D2- CD33 function, especially relative to AD pathogenic mechanisms, and (iii) Translate CD33 genetics into a novel AD therapeutic mimic. Overall, this focused proposal will develop our compelling mechanistic genetic results, elucidate the differences in D2-CD33 and CD33 function, and begin to translate these changes into an AD-preventive agent. In work beyond the scope of this focused proposal, we anticipate that these therapeutic agents will be tested in AD murine models that are transgenic for human CD33 and, eventually, humans.

Genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNP)s associated with Alzheimer disease (AD) risk. We propose that these SNPs point to mechanisms that affect AD development and thus represent targets for AD drugs. More specifically, we propose that drugs that mimic the actions of the protective SNP allele represent potential ?genetically validated? pharmacologic agents. Notably, a SNP with modest molecular actions may impact disease modestly but a drug that acts strongly at the same target may impact disease robustly. While this has been accomplished in other diseases, the lack of GWAS translation to pharmacologic agents represents a key knowledge gap in AD. In this proposal, we will focus on rs35349669 (rs669), a common SNP within INPP5D that is strongly associated with AD. Collectively, our work on INPP5D, encoding the lipid phosphatase SHIP1, over the last 17 years has provided the framework for its role in neuro-immune modulation through a physical interaction with TREM2, another critical factor recently implicated in AD. Building on these discoveries, we have now developed SHIP1 small molecule inhibitors that are safe and effective in murine models, as well as SHIP1 conditionally deficient mice. New data from our group establish a direct mechanistic connection between SHIP1 and AD pathophysiology, by showing that INPP5D expression is increased with the rs669 allele associated with AD risk and with AD pathology. Overall, this study brings together an interdisciplinary team to critically test our global hypothesis: Pharmacologic or genetic inhibition of SHIP1 decreases AD risk or progression. For this effort, we propose the following Specific Aims: (i) Elucidate the mechanism underlying rs669, (ii) Elucidate SHIP1 molecular actions relative to AD pathogenic mechanisms, (iii) Define the optimal SHIP inhibition strategy to impact macrophage and microglial function in the CNS and (iv) Translate INPP5D genetics into a novel therapeutic agent. Overall, this focused proposal will develop our compelling molecular genetic results, elucidate the actions of SHIP1 in an AD context, and translate these changes into a possible AD-preventive agent.

Genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNP)s associated with Alzheimer disease (AD) risk. We propose that these SNPs point to mechanisms that affect AD development and thus represent targets for AD drugs. More specifically, we propose that drugs that mimic the actions of the protective SNP allele represent potential ?genetically validated? pharmacologic agents. Notably, a SNP with modest molecular actions may impact disease modestly but a drug that acts strongly at the same target may impact disease robustly. While this has been accomplished in other diseases, the lack of GWAS translation to pharmacologic agents represents a key knowledge gap in AD. In this proposal, we will focus on rs35349669 (rs669), a common SNP within INPP5D that is strongly associated with AD. Collectively, our work on INPP5D, encoding the lipid phosphatase SHIP1, over the last 17 years has provided the framework for its role in neuro-immune modulation through a physical interaction with TREM2, another critical factor recently implicated in AD. Building on these discoveries, we have now developed SHIP1 small molecule inhibitors that are safe and effective in murine models, as well as SHIP1 conditionally deficient mice. New data from our group establish a direct mechanistic connection between SHIP1 and AD pathophysiology, by showing that INPP5D expression is increased with the rs669 allele associated with AD risk and with AD pathology. Overall, this study brings together an interdisciplinary team to critically test our global hypothesis: Pharmacologic or genetic inhibition of SHIP1 decreases AD risk or progression. For this effort, we propose the following Specific Aims: (i) Elucidate the mechanism underlying rs669, (ii) Elucidate SHIP1 molecular actions relative to AD pathogenic mechanisms, (iii) Define the optimal SHIP inhibition strategy to impact macrophage and microglial function in the CNS and (iv) Translate INPP5D genetics into a novel therapeutic agent. Overall, this focused proposal will develop our compelling molecular genetic results, elucidate the actions of SHIP1 in an AD context, and translate these changes into a possible AD-preventive agent.