Debomoy K. Lahiri - US grants

Beta-amyloid is the major proteinaceous component of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer's disease (AD). Beta-protein is derived from a larger amyloid protein precursor (APP) which is encoded by a gene on human chromosome 21. The APP gene encodes multiple transcripts for a family of secreted proteins that are generated by alternate splicing. The APP gene is differentially expressed in all major tissues. The hypothesis is that the upregulation of the promoter activity of the APP gene resulting in its enhanced transcription can set in motion the series of events culminating in amyloid deposits. The long term goal is to study the transcriptional control of APP gene. The specific aims are: 1) to examine the transcription at the APP promoter in different cell types and study the effect of different factors on the promoter; 2) to characterize the regulatory regions (cis-acting) of the APP promoter and identify the cell type-specific (trans-acting) factor(s) that might modulate the transcription of APP; 3) to study the effect of homeobox gene products on the promoter and 4) to determine whether there is a mutation in the APP gene promoter that is isolated from persons with familial Alzheimer's disease (FAD). Expression studies of the APP promoter will be done by constructing chloramphenicol acetyltransferase (CAT) based promoter plasmids with a 0.6-3.2kb fragment of the APP promoter. The strength of the promoter is measured by assaying CAT activity and determining the size of transcripts by ribonuclease protection assay in different cell extracts. Cell lines are of neuronal, glial, and chromaffin origin. The effect of interleukins, phorbol esters and heat shock treatment on the promoter will be studied in different stably transfected cells. To characterize the regulatory domains on the APP promoter, a progressive deletion from the 5' -end is made by limited nuclease digestion and using polymerase chain reaction (PCR). These deleted fragments are ligated to the reporter gene and the promoter activity is measured in different cell-types. Such experiments could detect the presence of an enhancer/silencer sequence in the promoter. The site-directed mutagenesis will be employed to determine the specificity of DNA regulatory elements of the APP promoter. The identification of any cell-specific factor(s) that might affect the promoter activity will be performed by gel shift assay using the appropriate promoter fragment and proteins from cell extracts. The promoter region of the APP gene contains 5 potential binding sites for a homeobox gene (Hox-1.3). Homeobox gene products act as transcriptional factors. We will examine the role of homeobox gene products with a cotransfection model using plasmid constructs with activator gene (Hox-3.l) and target gene (APP). To determine a mutation within the promoter region isolated from persons with presenile cases of FAD, two flanking oligonucleotide primers that span the O.8kb promoter will be synthesized. The flanked promoter from different sources will be amplified by PCR technique and then sequenced to locate a mutation.

Beta-amyloid is the major proteinaceous component of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer's disease (AD). Beta-protein is derived from a larger amyloid protein precursor (APP) which is encoded by a gene on human chromosome 21. The APP gene encodes multiple transcripts for a family of secreted proteins that are generated by alternate splicing. The APP gene is differentially expressed in all major tissues. The hypothesis is that the upregulation of the promoter activity of the APP gene resulting in its enhanced transcription can set in motion the series of events culminating in amyloid deposits. The long term goal is to study the transcriptional control of APP gene. The specific aims are: 1) to examine the transcription at the APP promoter in different cell types and study the effect of different factors on the promoter; 2) to characterize the regulatory regions (cis-acting) of the APP promoter and identify the cell type-specific (trans-acting) factor(s) that might modulate the transcription of APP; 3) to study the effect of homeobox gene products on the promoter and 4) to determine whether there is a mutation in the APP gene promoter that is isolated from persons with familial Alzheimer's disease (FAD). Expression studies of the APP promoter will be done by constructing chloramphenicol acetyltransferase (CAT) based promoter plasmids with a 0.6-3.2kb fragment of the APP promoter. The strength of the promoter is measured by assaying CAT activity and determining the size of transcripts by ribonuclease protection assay in different cell extracts. Cell lines are of neuronal, glial, and chromaffin origin. The effect of interleukins, phorbol esters and heat shock treatment on the promoter will be studied in different stably transfected cells. To characterize the regulatory domains on the APP promoter, a progressive deletion from the 5' -end is made by limited nuclease digestion and using polymerase chain reaction (PCR). These deleted fragments are ligated to the reporter gene and the promoter activity is measured in different cell-types. Such experiments could detect the presence of an enhancer/silencer sequence in the promoter. The site-directed mutagenesis will be employed to determine the specificity of DNA regulatory elements of the APP promoter. The identification of any cell-specific factor(s) that might affect the promoter activity will be performed by gel shift assay using the appropriate promoter fragment and proteins from cell extracts. The promoter region of the APP gene contains 5 potential binding sites for a homeobox gene (Hox-1.3). Homeobox gene products act as transcriptional factors. We will examine the role of homeobox gene products with a cotransfection model using plasmid constructs with activator gene (Hox-3.l) and target gene (APP). To determine a mutation within the promoter region isolated from persons with presenile cases of FAD, two flanking oligonucleotide primers that span the O.8kb promoter will be synthesized. The flanked promoter from different sources will be amplified by PCR technique and then sequenced to locate a mutation.

One of the major hallmarks of Alzheimer's disease (AD) is cerebral depositions of the beta-amyloid protein (AB) which is derived from a larger beta-amyloid precursor proteins (APP). Neurochemically, AD is characterized by a reduction in the presynaptic markers of the cholinergic system particularly in the areas of the brain related to memory and learning. This proposal addresses the processing of APP and how it is affected by a selected group of cholinesterase inhibitors (ChE-Is), specially the chemical tacrine. The primary clinical effect of tacrine is by its inhibition of the enzyme cholinesterase (ChE), thus resulting in a general increased level of acetylcholine whose levels appear to be reduced in A.D. However, this treatment has only moderate benefits in a limited number of patients. The cholinergic effect of tacrine may improve the synapses, while another action of the drug (e.g., lysosomotropic property) could be different. Our proposal will attempt to demonstrate non-cholinergic functions of tacrine using a cell culture model. It is based on the finding that treatment with tacrine markedly suppressed secretion of APP in cultured cells. Hypotheses are: i) noncholinergic functions of tacrine are independent of its well known anti-ChE activity, and ii) the lysosomotropic function of tacrine can be used to study the processing of APP. The specific aims are: l) to investigate the effect of ChE-Is on the secretion of APP and 2) to determine the effect of the selected ChE-Is (from S.A.1) on the carboxyl-truncated fragments (CTFs) of APP. Experiments will be performed in neuronal and astrocytic cell lines in the presence of tacrine and a selected group of ChE-Is. APP will be analyzed by immunochemicaI and molecular biological techniques. Effects of ChE-Is will also be studied in neuroblastoma cells transfected with either wild type APP751 or mutants of APP751 identified in familial AD (FAD). As control, we will study the effects of ChE-Is on other proteins such as heat-shock proteins, protease nexin- l and synaptophysin. Possible interpretations of the outcomes of this proposal are as follows: if the effect of tacrine is solely on ChE then one would expect the same effect of APP secretion to be seen with other ChE-Is. If the effect of tacrine is on the intracellular processing of APP, for instance due to the drug's lysosomotropic property then it would suggest future potential therapies should be focused on that feature of tacrine rather than on its ChE activity only. If the drug has unique effects on processing of APP, that would suggest tacrine or one of its derivatives might be further investigated and or developed to maximize potential effects in decreasing amyloid depositions.

DESCRIPTION: (Verbatim from the Applicant's Abstract) Misregulation of transcription of theB-amyloid precursor protein (APP) gene is implicated in the Pathogenesis of Alzheimer's disease (AD). Several early studies indicated that the APP gene expression might be increased in AD brains. That increases in the APP expression can lead to AD is strongly suggested by its high incidence in Down's syndrome patients with three copies of the gene. AD is a complex disorder with potentially multiple triggers. It is quite possible that disturbance in the transcriptional regulation of APP affects a subset of AD patients. Alternatively, failure to observe consistently a detectable increase in APP mRNA in post-mortem brains may be due to a transient over expression of APP, which may lead to nucleation of amyloid plaques. Our hypothesis is that the APP regulatory pathways play a critical role in AD pathogenesis. Our long-term goal is to study the transcriptional control of the APP gene. We have recently cloned and characterized a 7.9 kb APP promoter region. Here we will examine the role of the upstream regulatory elements (URE) on APP promoter activity and in late-onset AD. This proposal is based on our novel finding that a -75 to +104 bp region regulates APP promoter activity in a cell-type specific manner. The specific aims are: 1) To determine the coredomain of APP promoter that is essential for its activity and indelibility. Certain upstream regulatory regions that confer basic promoter activity in neurons and astrocytes may respond to activation by growth factors and cytokines, which are produced by activated macrophages during the inflammatory response in AD. Functional characterization of the regulatory regions will be done by a serial deletion strategy and DNA transfection in cell lines and primary cultures. 2) To identify whether URE interacts with a cell type specific nuclear protein. We will study how the specific upstream region interacts with a cell type-specific factor in neurons and astrocytes and how this interaction will be affected in the presence of cytokines and growth factors. 3) To determine the role of URE in the developmental and disease state. Differential effects of the promoter region in cell types might suggest a role for URE during development and differentiation. We plan to examine the levels of URE-binding factor in developmental rat brains, normal and AD brains. 4) To determine the genetic variability of the regulatory region in AD. The genetic variability of the regulatory region may be important for late-onset AD sporadic subjects. We propose to screen genomic DNA for 'promoter elements' and to compare the 'promoter binding factors' in control and AD subjects. 5) To manipulate promoter activity in cell culture. We plan to modify the specific regulatory effect of the APP promoter by in vivo competition experiment and using the antisense oligodeoxynucleotide strategy. Finally, we will correlate promoter studies with levels of APP and AB in control and AD subjects. Understanding the complex interplay between the promoter domains and the transcription factors may suggest novel methods for therapeutic intervention.

Alzheimer's disease (AD) is characterized by the severe loss of cholinergic neurons and depositions of amyloid beta peptide (Abeta). Three FDA- approved drugs (tacrine, donepezil and rivastigmine) for treating AD subjects belong to the category of cholinesterase (ChE) inhibitor (ChEI), which works by increasing the brain's supply of acetylcholine, a nerve communication chemical that is deficient in AD. These drugs are approved for treatment of mild to moderate AD and may not be as useful in more advanced stages. Our goal is to study the mechanism of ChEI drugs on amyloidogenic pathways that process beta-amyloid precursor protein (APP) to potentially neurotoxic Abeta. Such study is significant as there is increasing evidence that Abeta plays an important role in AD pathogenesis. This proposal is based on our discovery that treating cultured cells with certain ChEIs, such as tacrine and phenserine, significantly reduced levels of secreted APP (sAPP) and Abeta and may serve to slow the progression of AD as well as improve cognition. Notably, the mechanism of reduction of Abeta did not increase known alternative processing pathways and may therefore be less damaging. We are interested in identification of the mechanisms by which ChEIs block Abeta secretion to take advantage of the Abeta lowering property in developing novel therapeutic agents. SPEC. AIMS: The specific aims are: 1. To study the effects of acetyl- ChEI (AChEI) and butyrl-ChEI (BchEI) on sAPP and Abeta levels. To examine the specificity of their actions, effects of i) AchEI (e.g., pheneserine) ii)BChEI (e.g. cymserine), and iii) compounds that are tacrine-derivatives (e.g. velnacrine) will be tested to identify structural aspects that lower Abeta. 2. To investigate the role of ChEIs on APP metabolism. Effects of ChEIs on i)APP processing in FAD-APP mutant cell lines and ii) the fate of APP carboxyl-truncated fragments will be tested. 3. To determine the possible targets of the drugs. Effects of ChEIs on the i) APP-cleaving enzyme (BACE), ii) 5' -untranslated region and iii) inhibition of Abeta levels in transgenic mice model of AD. We will mechanically select ChEIs that interact with the peripheral allosteric binding domain of ChEenzyme, or with the esteractic and anionic binding domains (phenserine and cymserine) and test it in APP/PS1 double transgenic mice. These results will indicate a unique effect of ChEIs on APP processing, which is independent of their selectivity for the enzyme. This property will be further investigated to maximize their potential effects in decreasing amyloid depositions, and which can be utilized to design better drugs for the treatment of AD.

DESCRIPTION (provided by applicant): Our goal is to study gene regulation in Alzheimer's disease (AD), based on the "amyloid hypothesis" of Alzheimer's disease. Overproduction of the amyloid beta-peptide (Abeta) causes a cascade of neurodegenerative steps resulting in plaque formation and neuronal loss that characterize Alzheimer's disease. The unresolved key question in the field is what factors cause overproduction of Abeta and its large Abeta precursor protein (APP). Increased Abeta production may result from an increase in APP expression, or in its proteolytic processing by the limiting beta-APP cleaving enzyme (BACE). The goals of this proposal are to investigate the transcriptional regulation of i) APP, because APP (and, hence, AP) biogenesis begins at the level of transcription, and ii) BACE gene, as Abeta overproduction may be due to increased BACE level as a result of upregulation in this gene. Specific Aims are: 1) To study the functional domains of the APP promoter and effects of different agents on its activity. We will functionally characterize the 7.9 kb APP promoter and study how intrinsic (cytokines) and extrinsic (metals) factors regulate promoter activity. Promoter will be studied by serial deletions, mutagenesis and transfection experiments in different cell types and primary neuronal cultures. 2) To identify the effects of specific factors and cvtokines common to both APP and BACE gene regulation. We will characterize the role of IL-1alpha, TNF-alpha and CREB transcription factor (TF) on 4.1kb BACE promoter activity. 3) To identify cell type-specific nuclear factors. A 30 bp novel region (-76-47) of the APP promoter contains a regulatory domain that interacts with at least two proteins, PuF and SkiP. We will test i) the candidate TFs that control APP promoter activity and ii) the status of such TF in normal and AD brain tissues using gel shift assay and DNA-affinity chromatography. 4) To characterize APP gene polymorphisms that influences the risk of late-onset Alzheimer's disease. We discovered two polymorphisms at -3829 and -1023 that may be associated with Alzheimer's disease. We will i) do functional and DNA-protein binding studies with promoter variants and ii) correlate promoter studies with levels of APP and Abeta. 5) To study the APP-5'-UTR region. APP expression is also regulated via the 5'-untranslated region (UTR). We will test a dual role for the APP5'-UTR at both transcriptional and post-transcriptional levels, and study its interaction with cytokines. Cell lines from families with characterized FAD will be analyzed for differential expression of the APP and BACE genes. Studying APP and BACE gene regulation is crucial to understand APP production leading to Aa generation. These studies should help developing suitable drug targets for the treatment of Alzheimer's disease.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is characterized by cholinergic dysfunction and depositions of the amyloid beta-peptide (AB) derived from AB precursor protein (APP). Four current FDA-approved drugs for AD belong to the category of cholinesterase inhibitor (ChEI). These drugs are useful for treatment of mild to moderate AD but limited use in advanced stages. Our goal is to test novel acetylcholinesterase inhibitors (AChEIs) or butyrylcholinesterase inhibitors (BChEIs) against pivotal steps in the pathophysiology of AD to better understand the drugs'effects. Our focus is to test new therapeutic strategies to both validated and novel disease targets. Our hypothesis is that certain ChEIs have neuroprotective activity independent of their cholinergic activity due to their AB-lowering property. Our proposal to study the mechanism of ChEI drugs on amyloidogenic pathways is based on our results that certain ChEIs significantly reduced AB levels in cultured cells and animals. The specific aims are: 1] To study the effect of novel AChEIs and BChEIs on APP pathway steps. We will examine i) the effect of novel ChEIs on APP pathway: AB and BACE levels, ii) the effect of substitution of functional groups, iii) APPmRNA 5'-UTR, and iv) synaptic proteins. 2] To test the effect of a novel group of ChEIs on AB deposition in vivo. We will study the effects of different doses of selective AChEIs and BChEIs on APP and AB in double transgenic APPSWE-Tau amyloid plaque plus tangle producing mice. 3] To examine the role of ChE enzyme in AChE knockout mice. We will study the role of the AChE enzyme on APP, AB peptides and synaptic protein markers in the brain tissue samples from novel AChE knockout mice. 4] To investigate the effect of ChEIs on molecular markers in human (archived) samples. To validate the animal studies, we will test the effect of treatment of selected ChEIs on APP and AB levels in human plasma and/or CSF samples. The primary end-points are quantitative and functional: Cell survival, enzyme assay and levels of APP pathway protein/peptides and synaptic protein markers. We will mechanistically select ChEIs that interact with the peripheral allosteric binding domain of ChE and with the esteratic and anionic binding domains and test them in cell culture and animal models. This work will indicate unique effect of ChEIs on AB and synaptic proteins, independent of their selectivity for the enzyme. This property will help maximize their beneficial effects on amyloid and synaptic proteins, which can be utilized to design better therapeutic agents for AD. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is characterized by a reduction in the presynaptic markers of the cholinergic system, particularly in areas of the brain related to memory and learning, and by depositions of the amyloid beta peptide (AB), which is derived from the AB precursor protein (APP). Four of five current FDA-approved drugs for AD are cholinesterase inhibitors (ChEI), which increase the brain's supply of acetylcholine (ACh) by inhibiting cholinesterases (ChE) enzyme and thus preserve cholinergic circuits, which are believed to mediate memory pathways. Rather surprisingly, these drugs did not have a substantial effect on memory, but had an unexpected and welcome outcome of preserving cognition for a slightly extended period of time. The present proposal attempts to characterize the mechanisms of neuropreservation and protection by the ChEIs and fine tune this pathway to improve this beneficial property of ChEIs. Our goal is to test novel acetylcholinesterase inhibitors (AChEIs) or butyrylcholinesterase inhibitors (BChEIs) against pivotal steps in the pathophysiology of AD to better understand the effects of the drugs. We propose to study the mechanism of ChEI drugs on amyloidogenic pathways that process APP to potentially toxic AB. Our studies initially focused on the effects of these drugs on AB production and have recently been extended to synaptic protein markers and other neuroprotective effects. The outcome of the proposal is to identify mechanisms by which ChEIs block potentially toxic AB levels and to utilize this property in developing novel therapeutic agents.

DESCRIPTION (provided by applicant): Human MicroRNA as a potential therapeutic target in Alzheimer's disease Alzheimer's disease (AD) is the most common cause of dementia in the elderly. Current treatments provide only modest symptomatic relief and do not slow disease progression. Thus, new therapeutic strategies are needed. We propose to identify and validate members of a new class of drug targets. Major hallmarks of AD include amyloid plaques, neurofibrillary tangles, synaptic dysfunction and cognitive decline. These aberrations are believed to result, in part, from the overproduction of amyloid-beta peptide (A¿), a proteolytic product of the A¿ precursor protein (APP). Dysregulation of proteins involved in A¿ production may contribute to excess A¿ deposition. Here, we propose the novel approach of targeting microRNA (miRNA) for therapeutic intervention. These are short, non-coding RNAs that act to inhibit protein expression by interacting with specific recognition elements in the 3'-UTR of targe transcripts. We hypothesize that specific miRNA species regulate endogenous levels of APP gene products and that disruption of miRNA-mediated regulation will modulate A¿ levels. Specific Aim (SA) 1a will test the endogenous role of specific miRNAs in governing expression of APP. Rationale: We have recently discovered that specific miRNA (miR-101 and miR-153) regulate APP expression when delivered exogenously. To demonstrate the physiological relevance of this pathway, we will show that such interactions occur endogenously by using miRNA inhibitors and 'Target protector' experiments. Impact: Existence of such a regulatory pathway will stimulate research on its disease relevance. It will also open up therapeutic opportunities to limit APP/ A¿ levels and prevent neurodegeneration in AD and Down syndrome. SA1b will assess how disruption of miRNA regulatory interactions with APP affects A¿ homeostasis. Rationale: Since our focus is on APP and its role in A¿, we will examine how manipulation of endogenous miRNA-3'-UTR regulatory interactions affects downstream molecular pathways implicated in AD. We will assay the products of APP processing: APP and A¿ levels. Impact: Modulation of these pathways would indicate that these miRNAs likely have endogenous regulatory roles that would make them attractive therapeutic targets. SA 2 will examine the effects of in vivo overexpression of miR-101 and miR-153 in an AD animal model. Rationale: Experiments outlined above will thoroughly assess the therapeutic potential of these miRNAs. The experiments outlined in this aim will directly test the preclinical suitability of thes miRNA for therapeutic modulation in relevant AD in vivo models. A suitable miRNA target would be expected to modulate A¿ levels in a salutary fashion. Impact: Testing this will lend further validity to their status as therapeutic targets. Significance: The proposed experiments should provide strong evidence as to whether miRNA regulate APP expression at the exogenous and endogenous level, as well as in vitro and in vivo. These experiments will address whether manipulating the interactions of miRNA with the APP transcript produce salutary effects on downstream molecular pathways implicated in AD, and a potential use of these new drug targets for better therapeutic agents. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is the most common cause of dementia in the elderly; however, current treatments provide only modest symptomatic relief and do not slow disease progression. Here, we propose to study novel mechanisms to regulate amyloid-¿ (A¿) precursor protein (APP) mediated by microRNA (miRNA), which are short, non-coding RNAs that typically regulate protein levels by inhibiting translation of message RNA. The significance of this proposal is that miRNA regulation of APP represents a novel strategy to reduce the toxic A¿ peptide levels in the AD brain, and the proposed work will identify and validate members of a new class of drug targets, and the impact of this work will be in the eventual use of these new drug targets to produce better therapeutic agents to slow or reverse disease progression in AD.

? DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common cause of dementia in the elderly. Because current treatments provide modest symptomatic relief and do not slow AD progression, a better understanding of molecular bases of AD pathology is needed. This proposal will identify and validate microRNAs (miRNAs) as a new class of drug targets. miRNAs are endogenous, short, non-coding RNAs that typically inhibit protein expression by interacting with specific recognition elements of target transcripts. AD is believed to result from overproduction of amyloid-ß peptide (Aß), derived from Aß precursor protein (APP), and dysregulation of proteins involved in Aß production (e.g. APP, ß-secretase/BACE1) contributes to excess Aß deposition. We have also recently found that miRNA can stimulate APP expression in interaction with iron homeostasis. We propose to study APP and BACE1 regulation by miRNA. We hypothesize specific miRNAs regulate endogenous levels of APP and BACE1, are dysfunctional in AD, and manipulation will reduce Aß. Specific Aim 1(SA1) will identify functional miRNA target sites in APP and BACE1 and validate miRNA post-transcriptional regulation of native APP and BACE1 expression. Rationale: Discover functional miRNA targets in UTRs of APP and BACE1 transcripts using. Endpoints are APP and BACE1 mRNA & proteins, and Aß peptides, which we predict to change with miRNA. Impact: Manipulation of miRNA regulation is a novel therapeutic approach and may be feasible for correcting gene dysregulation in AD. SA2 will test physiological interactions between miRNA validated in SA1 and their regulatory networks over APP and BACE1 expression. Rationale: Test other mechanisms of regulation likely linked to maintain homeostasis of APP, BACE1 and Aß. Identifying roles of miRNA in this network is vital to pharmacologically target miRNA-transcript interactions. Impact: Reveal novel mechanisms for miRNA function in AD. SA3 will assess effects of in vivo manipulation of validated miRNA in AD animal models. Rationale: Test our validated miRNAs as therapeutic targets in AD transgenic animals, including interaction with iron homeostasis, by inducing miRNA-dependent changes in translation. Impact: Validate specific miRNAs as drug targets in vivo and identify novel AD-related regulatory networks. SA4 will examine whether miRNAs implicated in regulatory control of gene products involved in Aß homeostasis are dysregulated in AD patients. Rationale: In SA1-3, we will identify pertinent miRNAs that modulate expression of gene products implicated in Aß production. If these miRNAs are also involved in AD pathology, we expect their regulation to vary in anatomical- and pathology-dependent patterns. Impact: Further demonstrate validity of miRNAs as therapeutic targets to treat AD-related dysregulation. Research dictated by our central hypothesis could cause a significant paradigm shift on the field by elucidating novel regulatory mechanisms and identifying specific miRNAs that regulate important gene products implicated in AD. Final impact will be in eventual use of these new drug targets to produce therapeutic agents to slow or halt progression in AD.

? DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common cause of dementia in the elderly. Because current treatments provide modest symptomatic relief and do not slow AD progression, a better understanding of molecular bases of AD pathology is needed. This proposal will identify and validate microRNAs (miRNAs) as a new class of drug targets. miRNAs are endogenous, short, non-coding RNAs that typically inhibit protein expression by interacting with specific recognition elements of target transcripts. AD is believed to result from overproduction of amyloid-ß peptide (Aß), derived from Aß precursor protein (APP), and dysregulation of proteins involved in Aß production (e.g. APP, ß-secretase/BACE1) contributes to excess Aß deposition. We have also recently found that miRNA can stimulate APP expression in interaction with iron homeostasis. We propose to study APP and BACE1 regulation by miRNA. We hypothesize specific miRNAs regulate endogenous levels of APP and BACE1, are dysfunctional in AD, and manipulation will reduce Aß. Specific Aim 1(SA1) will identify functional miRNA target sites in APP and BACE1 and validate miRNA post-transcriptional regulation of native APP and BACE1 expression. Rationale: Discover functional miRNA targets in UTRs of APP and BACE1 transcripts using. Endpoints are APP and BACE1 mRNA & proteins, and Aß peptides, which we predict to change with miRNA. Impact: Manipulation of miRNA regulation is a novel therapeutic approach and may be feasible for correcting gene dysregulation in AD. SA2 will test physiological interactions between miRNA validated in SA1 and their regulatory networks over APP and BACE1 expression. Rationale: Test other mechanisms of regulation likely linked to maintain homeostasis of APP, BACE1 and Aß. Identifying roles of miRNA in this network is vital to pharmacologically target miRNA-transcript interactions. Impact: Reveal novel mechanisms for miRNA function in AD. SA3 will assess effects of in vivo manipulation of validated miRNA in AD animal models. Rationale: Test our validated miRNAs as therapeutic targets in AD transgenic animals, including interaction with iron homeostasis, by inducing miRNA-dependent changes in translation. Impact: Validate specific miRNAs as drug targets in vivo and identify novel AD-related regulatory networks. SA4 will examine whether miRNAs implicated in regulatory control of gene products involved in Aß homeostasis are dysregulated in AD patients. Rationale: In SA1-3, we will identify pertinent miRNAs that modulate expression of gene products implicated in Aß production. If these miRNAs are also involved in AD pathology, we expect their regulation to vary in anatomical- and pathology-dependent patterns. Impact: Further demonstrate validity of miRNAs as therapeutic targets to treat AD-related dysregulation. Research dictated by our central hypothesis could cause a significant paradigm shift on the field by elucidating novel regulatory mechanisms and identifying specific miRNAs that regulate important gene products implicated in AD. Final impact will be in eventual use of these new drug targets to produce therapeutic agents to slow or halt progression in AD.

Project Summary ? Research Education Component (REC) Alzheimer's disease (AD) research has been at the forefront of neuroscience due to its substantive innovations in molecular biology, genetics, neuropathology, and imaging technologies. Nevertheless, a critical need exists for professionals with specialized training to bring basic and clinical neuroscience of AD into the next generation. Therefore, it is vital for the IADC to develop and implement a new Research Education Core (REC). The REC's goal is to develop new researchers specifically trained to discover innovative approaches to improve understanding, diagnosis, and treatment of AD and other dementias. REC's significance is fostering the mission of the IADC to stimulate education, information, and training; coordinate across laboratories; and evaluate success in research education. REC's resources and expertise will stimulate synergy among IADC components and improve faculty development and research in neurodegeneration. REC's objective is to promote and disseminate concepts and functions of IADC cores to students and trainees, facilitate coordination among established faculty, and guide junior faculty in obtaining funding and facilitate their career stability. REC's impact is to develop translational skills to move basic findings to clinical interventions and clinical findings to mechanistic studies. REC's critical innovation is to improve upon current practices by adding rational, methodical training, and mentoring specifically devoted to critical skills underlying publication and successful grant preparation. REC's five Specific Aims are: (1) Provide organizational infrastructure within IADC for research education. REC leadership will define and coordinate training logistics and assemble a team of senior NIH-sponsored mentors for students/trainees. (2) Instill integrated fundamental knowledge of brain health and dementing illnesses via a) didactic courses related to AD, brain aging, neuropathology, and neurodegeneration linked with behavioral, cellular, genetic, imaging, and biostatistical approaches, b) biweekly seminar series and annual Research Symposium for AD and dementia; c) a Behavioral Neurology and Neuropsychiatry Fellowship program; and, d) Continuing the Neurobehavioral Rotation for psychiatry and neurology residents. (3) Recruit, retain, and assist students and faculty at multiple career levels, including historically underrepresented groups. Trainees will get structured guidance in writing, reviewing, and submitting scientific communication and successful grant applications. (4) Define specific metrics to assess REC's service to students, trainees, fellows, including rates and quality of conference participation, peer-reviewed publication, and grants submitted, funded, and rate of success. REC will monitor career progression, post-training employment, promotion in employment, and satisfaction from participants. (5) Foster active cross-disciplinary interaction and collaboration within IADC and beyond. REC will establish a multidisciplinary team of AD researchers, available to students/trainees. The REC will provide a strong framework and resources to help develop the next generation of researchers working to enhance brain health and quality of life.

Alzheimer's disease (AD) affects over 5.4 million Americans. AD is believed to result in whole or in part by accumulation of the toxic amyloid-? peptide (A?), making its generation a candidate for treatment. A? is generated from the A?-precursor protein (APP) via cleavage with ?-site APP cleaving enzyme 1 (BACE1), followed by ?-secretase complex cleavage. BACE1 activity is the rate-limiting step in production of A?. Recent human trials of BACE1-targeting drugs were discontinued due to liver toxicity. This has been explained by invoking ?off-target effects?. We disagree: Such ?failed? drugs may have been molecularly ?on-target? but acting ?off-site? (OnTOS). Specifically for BACE1: In brain, the primary substrate is APP, but in liver, the primary substrate is ?-2,6-sialyltransferase (ST6Gal1). ST6Gal1 is an important enzyme in the body's defense against radiation and oxidative damage. Interference in its processing may have produced hepatotoxicity similar to that of the ?failed? anti-BACE1 drug trials. Animal model-based tests may miss such risks because they often use transgenic knockout (KO) animals, which may have compensatory mechanisms to lacking BACE1 and potentially important uncharacterized artifacts. These animals also have such short lifespans under sheltered conditions. Radiation and oxidative load will not approach that faced by ?wildype (WT) and in the field? humans. Aim 1: Measure levels of BACE1 and orthologs and of APP and ST6Gal1 processing in brain, kidney, and liver samples from BACE1 KO and WT mice treated with vehicle or BACE1 inhibitors (LY2811376 and MK8931) for potential compensating effects in KO vs. drugs. We will use Westerns for total APP and processing of ST6Gal1 and four other BACE1 substrates (distinguished by band migration) and ELISA for soluble APP?. Impact: Establish empirical multi-organ comparisons to demonstrate OnTOS as an ?induced lack? (drug) effect vs. ?congenital? (KO) effect. Aim 2: Determine parallels between mouse models and human samples to lay groundwork for human medical studies. We will obtain at least 15 donor-matched samples of human brain, liver and kidney from AD and non-AD subjects. Proteins and miRNA will be measured as in SA1. We will employ western or quantitative ELISA as in 1-1. RNA-enriched extracts to be used for RT-qPCR of different miRNAs. Impact: Any medical research requires translation to humans. The proposed experiments will lay groundwork for such translation. Aim 3: Measure levels of different miRNAs implicated in regulating BACE1 levels. This will provide a frame of reference for future work to take advantage of different endogenous levels of miRNAs to sidestep OnTOS. We will then validate potentially useful miRNA species in primary mouse brain and liver cultures and in iPSC-derived human brain and liver cultures. Impact: The knowledge generated will provide concrete, mechanistic explanations of a pervasive problem in drug design?ignoring OnTOS. It may also suggest a viable alternative to drugs that may target a molecule specifically but offer no organ specificity: Use of miRNAs with levels that innately insulate a ?non-target? organ against excess.

Alzheimer?s disease (AD) is a devastating neurodegenerative disorder; available therapies only modestly improve symptoms. Our goal is to identify neurobiological mechanisms leading to neurodegeneration in AD. MicroRNAs (miRNA) are endogenous noncoding RNA molecules that typically silence translation. Little research exists on roles of miRNAs that target gene products implicated in AD as participants in neurobiology. We recently discovered a miRNA (miR346) that stimulates translation of amyloid-? precursor protein (APP) as part of iron (Fe) homeostasis. This miRNA binds a site that overlaps an iron responsive element (IRE) in APP. miR346 also has a predicted site in the tau 3?-UTR, and tau facilitates APP activity in Fe homeostasis. Also, we have identified miR298 reducing levels of APP and a specific tau protein moiety. The proposal objective is to test ?balancing feat? among miR346, miR298, Fe, and cellular inflammation networks via APP and tau (FeAT). Our central hy- pothesis is 1) miR346 plays a vital role in maintaining Fe homeostasis; 2) Manipulating miRNA could redirect Fe metabolism to prevent or treat AD and other neurodegenerative disorders. SA1: Test hypothesis?miR346 and 298 interact with APP and tau mRNA untranslated region (UTR) se- quences. Rationale: Fe dyshomeostasis is implicated in AD. APP and tau play roles in Fe metabolism. A co- regulator of both that also takes part in Fe metabolism could be a vital tool for preventing AD-related neurotoxi- city. Impact: Studies reveal miR346 and miR298 coregulate APP and tau. SA2: miR346 and 298 regulation of APP and tau both leads to and responds to Fe dyshomeostasis. Rationale: We intend to establish specific mechanisms of miR346 and miR298 activity within Fe metabolism. miR298 will be indirectly involved in response to Fe levels. Identifying the role of miRNA-mediated regulation within this network should elucidate miRNA-dependent mechanisms for pharmacological manipulation. Impact: These studies are likely to reveal novel biological mechanisms for miRNA function and FeAT trio regulation. SA3: Test hypothesis?miR346 and 298 are dysregulated in AD in stage and brain region specific man- ners. Rationale: We will identify partners of FeAT with miR346 and miR298 in well-characterized clinical sam- ples. We expect regulation to vary in anatomical- and pathology-dependent patterns. Impact: These studies in AD patients will further establish the miRNAs as therapeutic targets. SA4: Translate miR346 and miR298 manipulation on Fe homeostasis, APP and tau, and critical pro- cessing enzymes in transgenic AD model mice. Rationale: We intend to ascertain how or if AD pathology affects the interaction and how the interaction affects AD pathology. We will pay particular attention to differences in response to miRNAs or blockade in wild type vs. transgenic mice. Impact: Determining how manipulation of miR346 in both normal and induced pathology systems will alter pertinent protein levels with an eye toward reducing pathology via miR346 control.