Huntington Potter, PhD - US grants

Specialized nucleic acid elements are involved in the expression of antibody genes. These elements are 'enhancers' which act positively to promote transcription. The DNA rearrangements that occur during the construction of an antibody gene bring the enhancer near an otherwise weak promoter, thus activating the promoter. Specially significant is the fact that unlike other enhancers, which are carried on viruses, the immunoglobulin enhancers are tissue specific - they only function in B lymphocytes. This suggests that B cells contain as a result of their differentiation, proteins which are designed to function as specific transcriptional factors at this site. This application proposes two general approaches for identifying and isolating the transcriptional factors involved in the expression of immunoglobulin genes. These factors and the control of their synthesis during differentiation may serve as prototypes for intracellular regulatory factors involved in other aspects of development. The first proposed approach is biochemical and involves trying to isolate the enhancer recognition protein directly, by assuming that it will specifically bind to enhancer DNA. In addition, an in vitro transcription system will be developed in which messenger RNA synthesis from DNA fragments - containing immunoglobulin genes with or without the enhancer - is asked to be stimulated by the addition of a protein fraction derived from B lymphocytes. The second general strategy is to seek mutants in the gene which codes for the enhancer recognition protein, and then use the mutants to isolate the gene itself. The fact that the immunoglobulin enhancer works only in B cells has allowed the design of three specific experimental strategies. Each is based on being able to construct and introduce into cells a drug resistance gene whose expression is dependent on an active immunoglobulin promoter and enhancer. With both the protein and nucleic acid components involved in immunoglobulin gene expression in hand, it should be possible to study this example of a regulated eukaryotic gene with the same depth that has been possible with, for example, the lactose and tryptophan operons in bacteria. A deeper goal is to understand how the irreversible regulatory switch is thrown at the start of the differentiation of B lymphocytes, and how this switch is related to the initiation of transformation in B cell myelomas. Ultimately, purification of the enhancer recognition protein and analysis of mutations in the enhancer site that affect the proteins' ability to bind to the DNA will begin to provide such a basis molecular description of immunoglobulin regulation and provide key insights into the processes of development and differentiation in general.

We are currently involved in three lines of experimentation that deal with DNA replication and recombination. 1. The first line of experimentation deals with the isolation and characterization of the DNA recognition elements(s) which serve as the origin for negative strand synthesis in the viruses G4, St-1, alpha 3, and 0K. 2. A second line of experimentation involves the isolation of the rolling circle/phage-capsid complex, which is the actively replicating DNA molecule in the 0X-174 late life cycle. 3. A third project involves the use of single-stranded DNA phages to study SOS repair. 4. Fourth, we are using the electron microscope and in vivo and in vitro studies to explore the process of DNA recombination. We can observe and analyze intermediates in the recombination process. Current experiments deal mainly with the development of an in vitro system for studying the enzymology of generalized recombination.

Transcription from Recombinationally Activated Immunoglobulin Genes. Specialized nucleic acid elements are involved in the expression of immunoglobulin genes. These elements are 'enhancers' which act positively to promote transcription. The DNA rearrangements that occur during the construction of an antibody gene bring the enhancer near an otherwise weak promoter, thus activating the promoter. Particularly significant is the fact that unlike other enhancers, which are carried on viruses, the immunoglobulin enhancers are tissue specific--they only function in B lymphocytes. This suggests that B cells contain, as a result of their differentiation, proteins which are designed to function as specific transcriptional factors at this site. This application proposes two general lines of experimentation aimed at identifying and isolating the transcriptional factors involved in the expression of immunoglobulin genes. The first proposed approach is biochemical and involves trying to isolate the enhancer recognition protein directly, for example by assuming that it will specifically bind to enhancer DNA. In addition, an in vitro transcription system will be developed in which messenger RNA synthesis from DNA fragments--containing immunoglobulin genes with or without the enhancer--is tested for stimulation by protein fractions derived from B lymphocytes. The second general strategy is to seek mutants in the gene which codes for the enhancer recognition protein, and then use the mutants to isolate the gene itself. The fact that the immunoglobulin enhancer works only in B cells has allowed the design of three specific experimental strategies. Each is based on the idea of placing a drug resistance gene (neo) under immunoglobulin enhancer control and introducing the construction into various cells. For example, in B cells which are also caused to contain extra copies of the cloned enhancer DNA sequence, only a mutation to overproduction of the enhancer recognition protein will allow the neo gene to be expressed. This mutation would aid the biochemical studies described above, and potentially also allow the regulatory gene which produces the enhancer recognition protein to be cloned. With both the protein and nucleic acid components involved in immunoglobulin gene expression in hand, it should be possible to study this example of a regulated eukaryotic gene to the same depth that has been possible with, for example, the lactose and tryptophan operons in bacteria. Ultimately, purification of the enhancer recognition protein and analysis of mutations in the enhancer site that affect the proteins' ability to bind to the DNA will begin to allow a basic molecular description of immunoglobulin regulation and may provide key insights into the processes of development and differentiation in general. Malaria, DNA Rearrangements and Burkitt Lymphoma. Another experiment related to the recombinational activation of antibody genes attempts to probe the relationship between malaria infection and Burkitt lymphoma, which show a geographic coincidence. By co-cultivation of malaria-infected erythrocytes in vitro with tester mammalian cells, we will test the idea that infected erythrocytes release a clastagenic (chromosome-breaking) compound which could trigger the chromosome translocation associated with Burkitt lymphoma.

Transcription from Recombinationally Activated Immunoglobulin Genes. Specialized nucleic acid elements are involved in the expression of immunoglobulin genes. These elements are 'enhancers' which act positively to promote transcription. The DNA rearrangements that occur during the construction of an antibody gene bring the enhancer near an otherwise weak promoter, thus activating the promoter. Particularly significant is the fact that unlike other enhancers, which are carried on viruses, the immunoglobulin enhancers are tissue specific--they only function in B lymphocytes. This suggests that B cells contain, as a result of their differentiation, proteins which are designed to function as specific transcriptional factors at this site. This application proposes two general lines of experimentation aimed at identifying and isolating the transcriptional factors involved in the expression of immunoglobulin genes. The first proposed approach is biochemical and involves trying to isolate the enhancer recognition protein directly, for example by assuming that it will specifically bind to enhancer DNA. In addition, an in vitro transcription system will be developed in which messenger RNA synthesis from DNA fragments--containing immunoglobulin genes with or without the enhancer--is tested for stimulation by protein fractions derived from B lymphocytes. The second general strategy is to seek mutants in the gene which codes for the enhancer recognition protein, and then use the mutants to isolate the gene itself. The fact that the immunoglobulin enhancer works only in B cells has allowed the design of three specific experimental strategies. Each is based on the idea of placing a drug resistance gene (neo) under immunoglobulin enhancer control and introducing the construction into various cells. For example, in B cells which are also caused to contain extra copies of the cloned enhancer DNA sequence, only a mutation to overproduction of the enhancer recognition protein will allow the neo gene to be expressed. This mutation would aid the biochemical studies described above, and potentially also allow the regulatory gene which produces the enhancer recognition protein to be cloned. With both the protein and nucleic acid components involved in immunoglobulin gene expression in hand, it should be possible to study this example of a regulated eukaryotic gene to the same depth that has been possible with, for example, the lactose and tryptophan operons in bacteria. Ultimately, purification of the enhancer recognition protein and analysis of mutations in the enhancer site that affect the proteins' ability to bind to the DNA will begin to allow a basic molecular description of immunoglobulin regulation and may provide key insights into the processes of development and differentiation in general. Malaria, DNA Rearrangements and Burkitt Lymphoma. Another experiment related to the recombinational activation of antibody genes attempts to probe the relationship between malaria infection and Burkitt lymphoma, which show a geographic coincidence. By co-cultivation of malaria-infected erythrocytes in vitro with tester mammalian cells, we will test the idea that infected erythrocytes release a clastagenic (chromosome-breaking) compound which could trigger the chromosome translocation associated with Burkitt lymphoma.

Certain neuropathological lesions characterize the brains of normal aged humans and monkeys, and particularly Alzheimer's disease patients, where the lesions occur much earlier and in far greater numbers. The substantial minority (between 15 and 50%) of Alzheimer cases which appear to be the result of the inheritance of an autosomal dominant mutation indicate that a single genetic defect can cause the neuropathology. Using an immunochemical/ molecular genetic approach, the gene coding for one component of the extracellular protein deposits (termed "amyloid") of normal aged and Alzheimer's disease brain was cloned, and found to code for a protease inhibitor, alpha 1-antichymotrypsin. Further experiments confirmed the intimate association of this protease inhibitor with the proteinaceous amyloid filaments of normal aged and Alzheimer's disease brain. Most of the next five years will be devoted to determining how alpha 1-antichymotrypsin contributes to amyloid deposition, either directly as a structural amyloid component, or indirectly as a protease inhibitor. For example, the fact that alpha 1- antichymotrypsin is overexpressed in Alzheimer brain suggests that it may prevent the normal clearing of amyloid deposits. Studies will determine the protease targets of alpha 1-antichymotrypsin inhibition in the brain, the cells expressing alpha 1- antichymotrypsin, and any alterations which may arise in the alpha 1-antichymotrypsin gene, its expression, or its encoded protein, during normal aging or Alzheimer's disease. Transgenic mice will be made in which alpha 1-antichymotrypsin will be overexpressed in a regulated manner to determine whether such over-expression leads to neuropathology, and to provide an animal model for testing potential therapeutic approaches to Alzheimer's disease and 'normal' senile neuropathy.

The overall goal of this project is to understand how the expression of proteins important in Alzheimer's disease is controlled in the brain and how that control is altered in the disease state. The identification of the two protein components (alpha-1-antichymotrypsin and the Beta-protein) of the neuropathological amyloid deposits in Alzheimer's disease (AD) brain has opened the way of how these molecules come to be expressed. We have found that alpha-1-antichymotrypsin (ACT) is over expressed in those regions of Alzheimer brain affected by the disease, specifically in astrocytes. There are also differences between Alzheimer's disease patients and controls in the levels of the different beta-protein precursor mRNAs, and beta-protein precursor can be shown to be over- expressed in astrocytes in models of brain damage. A protease, Clipsin, that we have purified, is a strong candidate for a cleavage enzyme that can release the beta-protein from its precursor, and is also expressed in brain, probably in mast cells. Thus changes in the expression of two and possibly three proteins characterize AD and may underlie the mechanism of the disease. We wish to discover how the changes in gene expression observed in AD come about and whether they are a direct result of mutation. (25-50 percent of AD cases arise in certain families and appear to be inherited as an autosomal dominant trait.) Specifically, we propose a series of experiments designed to determine (1) the signaling molecules responsible for inducing ACT, beta-protein precursor, and potentially Clipsin expression in cultured cells-astrocytes and mast cells, (2) the DNA sequences in the regulatory region of these genes which respond to the inducing signals, (3) whether over-expression of ACT in transgenic mice can mimic the over-expression in AD and lead to neuropathology, and (4) whether, and how, the gene coding ACT may be mutant in AD, resulting in aberrant expression.

Alzheimer's disease is a neurodegenerative disorder of the central nervous system resulting in a progressive loss of memory and other intellectual functions beginning in middle to late life. The disease is characterized by the accumulation of certain neuropathological lesions in the brains of affected individuals. The identification of alpha1-antichymotrypsin (ACT) as a component of Alzheimer amyloid filaments and the ? that the beta- protein-the major amyloid protein-is a proteolytic fragment of a larger precursor protein, ? the subject of antiproteases and proteases into the biochemical discussion of Alzheimer's disease. The discovery of a Kunitz inhibitor domain in the beta-protein precursor, and our finding that the beta-protein itself ? a striking resemblance to the active site of serine proteases and can stably bind to ACT via a protease-antiprotease interaction further underscores the need to understand the biochemistry and physiology of protease and antiprotease function in Alzheimer brain. This application proposes to continue our study of two ? of the role of proteases and their inhibitors in Alzheimer's disease-in the structure of the amyloid ? and in the proteolytic processing of the beta-protein from its precursor. With respect to the structure of Alzheimer amyloid filaments, we will determine the specific amino acids in the beta-protein and in the active ? of ACT necessary for their interaction in vitro. We will also design peptides that should bind ACT better in the beta-protein and might serve to inhibit ACT-beta-protein binding in vitro and potentially in vivo. We have discovered two chymotrypsin-like proteases (CLIPs) with the substrate specificity to cleave the terminus of the beta- protein from its precursor. One (clipsin) has been purified to homogeneity. The enzyme ? been partially sequenced and is likely identical to rat mast cell protease I. It preferentially degrades the protein precursor in brain membrane extracts, is inhibited by ACT, and can make the correct cleavage in ? peptide substrates. We will establish whether clipsin can cleave the beta-protein from its native precursor and are in the process of cloning the clipsin gene from rat and human brain cDNA. While clipsin/RMCP I is the best beta-protein-generating candidate protease now known, we are using PCR identify and clone other brain CLIPs. These will be expressed in a baculovirus system and their encoded ? tested for their ability to cleave the beta-protein N-terminus. We will also test the level and distribution their expression in normal, Alzheimer, and Down syndrome brain, to determine why the beta-protein is differentially generated in the disease process.

DESCRIPTION: Thus far, five genes and their protein products have been implicated in the pathogenesis of Alzheimer's disease--APP (AD1), apoE (AD2), antichymotrypsin (ACT; putatively AD5), and the recently identified genes S182 (AD3) and its relative STM2 (AD4). The localization of the products of three of these genes A-beta( (from APP), ACT (by the applicant), and apoE (by others)--in the Alzheimer amyloid plaques provided the first evidence that their interaction might be essential for the development of these neuropathological lesions. The applicants have confirmed this prediction by showing that ACT and apoE4 strongly promote the polymerization of A-beta into amyloid filaments in vitro, which are toxic to human cortical neurons in culture. Furthermore, they have shown that activated microglial cells in the affected area of the Alzheimer brain express IL-1, that IL-1 is produced in vitro by cultured microglia from only these regions of normal brain, and that this IL-1 binds to the IL-1 receptor on astrocytes to induce them to synthesize the amyloid promoting factor ACT. These results reinforce our growing conviction that a glial-based inflammation cascade plays an important role in Alzheimer's disease pathogenesis. In the coming years, the applicants plan to use two experimental systems to further delineate the pathogenic pathway in Alzheimer's disease and to understand the role that changes in gene expression and protein localization play in this pathway. Specifically, they will use cultures of human neurons, astrocytes, and microglial cells to study the expression of the five known Alzheimer-related genes. They will focus on understanding how lymphokines, particularly IL-1, control the expression of these proteins, and the identification the basis of the region-specific differences in IL-1 production that appears to underlie the region-specific production of amyloid in Alzheimer's disease. They also plan to use these culture systems to investigate the effect of A-beta peptides and their macromolecular conformers on IL-1, apoE, and ACT expression in target glial cells. Finally, they plan to determine whether, as predicted, the novel ACT-A polymorphism associated with Alzheimer's disease increases the production of this amyloid-promoting factor. The second general approach is to use immunocytochemistry at the light and electron microscope levels to localize the protein products of the five Alzheimer's disease-related genes. They plan to focus particularly on the synapse, where three of these proteins (APP, ACT, and apoE) have already been localized, and which ours and others work indicates may be the birthplace of the amyloid deposits in the Alzheimer brain.

Biochemical, genetic, and epidemiological evidence indicates that inflammation is an essential part of the pathogenesis of Alzheimer's disease. Over the last decade of this grant, we have focused on the role that specific inflammatory molecules play in the Alzheimer's pathogenic pathway. We have learned, from both in vivo and in vitro experiments in our and other labs, that several acute phase/inflammatory molecules in the brain, specifically anti-chymotrypsin (ACT) (ACT) and apolipoprotein E (apoE) can promote the formation of the neurotoxic amyloid deposits that are the main pathological hallmark of the disease. They do this by binding directing to the Abeta peptide and promoting its polymerization into amyloid filaments. Furthermore, ACT is greatly overproduced in affected areas of the AD brain, providing a potential mechanism by which the regional specificity of amyloid deposition can be explained. ACT over- expression is evidently caused by activation of ACT mRNA synthesis in astrocytes by the inflammatory cytokine IL-1 released from activated microglia. ApoE was recently found to also be over expressed in affected areas of AD brain, but the mechanism is not yet known. We have extended this inflammatory cascade to include the amyloid precursor protein (APP) itself, by finding that IL-1 up-regulates the production of APP in astrocytes, but at the translational rather than the transcriptional level. We propose to extend this findings in order to solidify and clarify the role of ACT and apoE in AD. Specifically, we will test the hypothesis that ACT and apoE directly affect amyloid formation in vivo. TO this end, we have created a transgenic mouse line that expresses human ACT in astrocytes and will use it alone, or together with apoE and over- expression of APP. We will identify aspects of the interaction between Abeta and the "pathological chaperones" ACT and apoE, and use this information to develop peptide inhibitors of the interactions that may form the basis for the development of therapeutic compounds to slow or prevent neurotoxic amyloid formation. Such blocking peptides will be introduced by several means into the transgenic mice to determine their ability to reduce the level of amyloid formation.

[unreadable] DESCRIPTION (provided by applicant): Transgenic h-APP mice models of Alzheimer's disease indicate that the production of the Abeta 1-42 peptide, or its polymerization into amyloid filaments, or both, are part of the pathogenic pathway leading to Alzheimer's disease. In the parent grant to this Supplemental Application, we proposed to determine whether two inflammatory proteins, apolipoprotein E and antichymotrypsin, played a role in amyloid formation in APP transgenic mice, as previous in vitro and epidermiological evidence had predicted. We also proposed to introduce peptide blockers of the ACT-ABeta and apo E-ABeta interactions into the transgenic mice and to determine if they prevent apoE- and ACT-dependent amyloid formation. Recent results have clearly demonstrated that ACT is an amyloid promoter in vivo and have confirmed earlier work showing that, in mice, no filamentous amyloid forms in the absence of apoE (mice lack ACT already). The conclusion is that ABeta cannot form Alzheimer amyloid in mice without help from a "pathological chaperone". The critical question that remains is whether amyloid formation causes the cognitive and memory deficits associated with Alzheimer's disease or whether it is merely a marker of the increased production of ABeta 1-42 induced, for example, by APP mutations. Unfortunately, it has not been easy to measure age- and amyloid associated memory decline in the transgenic APP mice. This problem has recently been solved by the Co-PI of this Supplement, Dr. Gary Arendash, through the use of a new test, the Radial Arm Water Maze (RAWM). The test has successfully correlated working memory deficits with amyloid formation in Tg2576 and Tg2576-PS1 mice and shown that ABeta vaccination prevents the development of memory loss. In preliminary results presented here, aged PD-APP mice were also found to develop memory loss detected by the RAWM. More important, we have found that apoE is essential for both the amyloid formation and the cognitive decline. We now propose to solidify and extend this result by a thorough behavioral analysis of the transgenic mice that are the subject of the parent grant. If the preliminary results are confirmed, we are likely to discover that it is either the process or product of ABeta polymerization that is neurotoxic and leads to cognitive decline in mice and that Abeta 1-42 and diffuse amyloid is by itself fairly harmless. Such a conclusion will be important for the design of Alzheimer therapies. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): There are an estimated 430,000 Alzheimer's disease patients in Florida, with the Tampa Bay and Miami/Dade County areas having a large, ethnically diverse and growing population with this illness. Indeed, Florida is the fourth largest state in the country in terms of population, and the second largest in total number of Alzheimer's disease patients. Clearly Florida would be ideal place to site an NIA-funded Alzheimer's disease Research Center (ADRC). Yet no such Florida-based ADRC exists. Therefore, a group of researcher s and physicians from across the state have joined together to design and seek funding for the Florida ADRC (FADRC) described in this application. The general goals of the proposed FADRC are represented by the three Projects which aim: 1. To better understand the process of transition from normal to MCI to AD by determining which combinations of clinical, epidemiologic, imaging, neuropsychological, and biological markers best identify individuals who will experience a rapid rate of cognitive decline toward AD or other dementing illnesses. 2. To investigate the ability of cognitive rehabilitation to intervene in and slow disease progression in MCI and early AD patients. 3. To use mouse models of AD to determine which aspects of environmental enrichment (including cognitive rehabilitation) best slow or reverse cognitive impairment and might be similarly applied to human patients. These projects are mutually supportive and synergistic because markers found in Project 1 can be used to measure success and/or to distinguish populations in Project 2, and model results obtained in Project 3 can be used to help decide which type of intervention may be most successful in humans. The three projects are supported by 5 Cores- Clinical, Data Management, Neuropathology, Education, and Mouse Behavior and Neuropathology. The end result of the synergy inherent in the design of the FADRC may be the development a novel therapeutic intervention that can slow the clinical course of the disease. To aid in the development, administration and financial support of the planned FADRC, in 2002, the Florida Legislature established and funded the Johnnie B. Byrd Sr. Alzheimer's Center and Research Institute on the campus of the University of South Florida. Byrd Institute funds and personnel will be used to supplement the NIA grant in a State-Federal collaboration that will help assure the success of the proposed FADRC.

DESCRIPTION (provided by applicant): Microtubule (MT) dysfunction, neurotoxicity, chromosome mis-segregation, and defective neuronal plasticity are all induced by A¿ peptide and implicated in Alzheimer's disease (AD) pathogenesis. Our recent data support the unifying hypothesis that A¿-induced pathologies are caused in part by A¿ inhibiting specific MT motors, and we propose to test this hypothesis and its implications. Microtubules serve as the highways upon which ATP driven motor proteins move key cellular components such as proteins, vesicles, chromosomes and large macromolecules, including microtubules themselves, from one part of the cell to another. Many neurodegenerative diseases show defects in the microtubule transport system, underlining its importance in normal cellular physiology. Previously, we found that in AD patients, tg mice, and cultured cells, mutant amyloid precursor protein and presenilin genes that cause familial AD induce chromosome mis-segregation and aneuploidy, processes that are intimately involved with microtubule function. Confirmatory results from other labs showed that 30% of neurons in early AD cortex are aneuploid/ hyperdiploid. Recently, we found that after addition to human cells or Xenopus egg extracts, A¿ impairs the formation and stability of mitotic spindles and directly inhibits three microtubule motor kinesins, Eg5, KIF4A and MCAK, which are essential for the normal structure and function of the mitotic spindle, and, remarkably, are also present in neurons. In particular, Eg5 has severely reduced activity in extracts from brains of the APP/PS transgenic mice, a model of Alzheimer's disease, is inhibited in neurons treated with A¿, and harbors polymorphisms that increase AD risk. Chemical inhibition of Eg5 results in mitotic defects, mis-localization of the NMDA receptor away from the plasma membrane, and inhibition of LTP. A¿snegative impacton LTP, together with our new data regarding its influence on microtubule function, suggests that A¿ inhibition of memory processes in AD may derive in part from its inhibition of specific kinesins, which can disrupt both neurogenesis and neuroplasticity. By determining the effects of exposing cells, mouse brain slice cultures, and adult mice to chemical inhibitors of Eg5 and/or to A¿ on 1. Neurotoxicity, 2. LTP in slice cultures, and 3. learning and memory and AD-like neuropathology in adult mice, the proposed experiments will allow us to conclude whether or not the ability of the Alzheimer A¿ peptide to inhibit certain microtubule motors contributes importantly to its disruption of neurogenesis and neuronal function in Alzheimer's disease and whether such motor inhibition constitutes a novel target for AD therapy.

DESCRIPTION (provided by applicant): We seek to elucidate the interactive roles that atherosclerosis and Abeta play in the pathogenesis of co-morbid Alzheimer's disease (AD) and vascular dementia. Although, most of the field's efforts have been focused on the role of LDL, ApoE and their receptors play in AD pathogenesis, we have obtained evidence that APP/Abeta alters the expression and localization of the LDLR, such that it becomes concentrated in single perinuclear aggregate, rather than sorting to the cell surface. Other experiments suggest that Abeta induces a general defect in the MT network that likely leads to the mis-localization of some, but not all membrane proteins, including the LDLR, potentially rendering it (them) less active. We therefore propose to Test the Hypotheses that 1. APP/Abeta affects LDLR expression and intracellular localization. 2. Abeta-induced LDLR mis-localization leads to LDLR functional deficits, hyperlipidemia and atherosclerosis. The proposed experiments are significant because they may allow us to conclude that one reason why atherosclerosis, especially in the brain so often accompanies AD is because it is promoted by LDLR functional deficits induced by Abeta. Furthermore, the differences in affinity/activation of the different isoforms of ApoE for LDLR may be made more acute when the LDLR is less active, thus accounting in part for the increased risk of AD imparted by ApoE4. These findings and conclusions form the foundation for a comprehensive series of experiments to test the above hypotheses and determine whether Abeta induces the mis-sorting and functional inactivity of other important neuronal and non-neuronal proteins in the AD, particularly, growth factor receptors and the insulin receptor.

PROJECT ABSTRACT Alzheimer?s disease (AD) treatments designed to target the amyloid-beta peptide have shown encouraging results in transgenic animal models but less encouraging results in human trials, which have also been plagued with serious adverse events (SAEs), including amyloid-related imaging abnormalities (ARIAs). Our proposed innovative therapeutic approach is based on epidemiological evidence that patients with the inflammatory disease rheumatoid arthritis (RA) have a reduced risk of developing AD, unrelated to their use of non-steroidal anti-inflammatory drugs (NSAIDs). We identified the innate immune system stimulant Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) as a hematopoietic factor upregulated in RA, which we found reduced brain amyloidosis and reversed cognitive impairment in transgenic AD mice. Other studies have shown GM-CSF to be neuroprotective, anti-apoptotic, and neurogenic in several models of neurological diseases and injuries. We also found that recombinant human GM-CSF(sargramostim/Leukine) treatment is associated with cognitive improvements in leukemia patients after bone marrow chemo-ablation and hematopoietic cell transplant therapy. Notably, sargramostim is an FDA-approved drug for increasing the production and differentiation of white blood cells with an excellent safety record over 30 years. Most importantly, we recently completed a Phase I/II safety and efficacy trial (NCT01409915) in which mild-to-moderate AD participants were treated with sargramostim (250 mcg/m2/day SC) or placebo five days/week for three weeks (20:20 participants per group) with neurological, neuropsychological, neuroimaging, and blood biomarker assessments. Sargramostim treatment was safe (Primary Endpoint) with no drug-related SAEs and no ARIAs. Furthermore, the Mini-Mental State Exam (MMSE) showed cognitive improvement in the sargramostim group at the end of treatment (EOT) compared to baseline (p=0.0074) and in the sargramostim group compared to the placebo group at the EOT (p=0.037) and at 45 days after the EOT (p=0.0281). Other assessments showed no treatment benefits, but there was a trend negative correlation between changes in MMSE versus amyloid-PET. We now propose to carry out a randomized, double- blind, placebo-controlled trial in 42 mild-to-moderate AD participants, 28 of whom will receive sargramostim (250 mcg/m2/day SC) and 14 of whom will receive placebo, five days/week for 24 weeks with a 45-day follow-up visit. We have received both an IND exemption (134291) and IRB approval (17-0215) but will submit improved versions in the coming months. Our Specific Aims are: 1) Assess the long-term safety and tolerability of sargramostim in mild-to-moderate AD participants (Primary Endpoint). 2) Assess the effects of sargramostim treatment on cognition and activities of daily living in mild-to-moderate AD participants (Secondary and Exploratory Endpoints). 3) Assess changes in biomarkers associated with sargramostim treatment in mild-to- moderate AD participants (Exploratory Endpoints).

PROJECT SUMMARY/ABSTRACT People with Down syndrome (DS) exhibit significant hypoplasia of the frontal lobe, hippocampus, and cerebellum and mild to severe intellectual disability, which challenges their ability to function independently. Any enhancement of their cognitive ability would greatly improve their quality of life and activities of daily living, but currently there are no therapeutics available for enhancing cognitive function in people with DS. This proposal aims to design and complete a clinical trial in adults with DS using recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF/sargramostim), an FDA-approved drug for increasing the production and differentiation of various white blood cells with almost 30 years of excellent safety history in numerous patient populations. In a previous retrospective study, we found that sargramostim treatment is associated with cognitive improvements in leukemia patients after bone marrow chemoablation and hematopoietic cell transplantation. In a recently concluded Phase II clinical trial (NCT01409915), we found that three weeks of sargramostim treatment was safe and well tolerated in mild-to-moderate Alzheimer?s disease (AD) participants and was associated with improvement in the MMSE, reduced biomarkers of neurodegeneration (Tau and UCH-L1), but no evidence of reduced amyloid. Furthermore, we have found that treatment with murine GM-CSF improves cognition and ameliorates astrogliosis in a mouse model of DS (which has no AD pathology), that it rapidly reverses cognitive impairment and removes some cerebral amyloid pathology in mouse models of AD, and that it improves age- related cognitive decline in aged wild-type mice. Numerous other studies have shown that GM-CSF is neuroprotective, anti-apoptotic, neurogenic, and beneficial in several neurological diseases and injuries, for example, in a clinical trial with Parkinson?s disease subjects and in animal models of stroke, spinal cord injury, and traumatic brain injury. Specifically, this proposal is designed to investigate whether treatment with sargramostim at the FDA-recommended dose is safe and tolerable in adults with DS, whether it can improve measures of cognitive function, quality of life, and activities of daily living, and whether it can reduce the Tau and UCH-L1 biomarkers in the blood that we have shown to evidence neuroinflammation and neurodegeneration in people with DS and to be reduced in AD patients by GM-CSF treatment.