Ottavio Arancio, M.D., Ph.D. - US grants

DESCRIPTION (provided by applicant): The overall purpose of this project is to explore the molecular basis for amyloid-induced changes in basal neurotransmission and synaptic plasticity at synapses between pyramidal neurons in the hippocampus, a structure within the temporal lobe particularly critical for memory storage and with remarkable plastic characteristics of the kind that are required for learning and memory. Our approach is to combine the use of in vitro hippocampal slices and neuronal cultures with in vivo animals. This strategy offers the advantage of identifying changes of synaptic transmission in a preparation with intact neuronal circuits (slice), of giving depth to the knowledge of these changes in a more simplified system with the unique possibility of having direct access to both the pre- and the post-synaptic site (cell culture), and finally of determining whether it is possible to re-establish normal learning and memory by counteracting the effects of these changes in an in vivo complex neuronal system (the whole animal). The following aims will be addressed: a) to determine changes of synaptic transmission induced by amyloid-beta, b) to search for potential mechanisms that might alter neurotransmission following amyloid-beta increase, c) to determine if changes of synaptic transmission induced by amyloid-beta involve the nitric oxide (NO)/soluble guanylyl-cyclase (sGC)/cGMP/cGMPdependent- protein kinase (cGK)/CREB pathway, d) to determine if the disruption of learning and memory occurring in APP/PS1 mice is rescued by drugs acting through the NO/sGC/cGMP/cGK/CREB pathway. On the completion of these studies we will clarify whether amyloid-beta causes synaptic dysfunction through pre- and/or post-synaptic mechanisms. We will also identify a new signaling pathway inactivated by amyloid peptides during synaptic plasticity and learning. Findings derived from these studies will contribute to the design of researchers and therapies for Alzheimer's Disease and a host of other neurodegenerative disorders with staggering social, economic and personal costs to the sufferers, their families and all of society.

laboratory mouse; tissue /cell preparation; tissue resource /registry

The principal component of the extracellular deposits in Alzheimer's disease is the amyloid-beta peptide (Ap). However, much remains to be learned about mechanisms through which Ap disturbs the properties of neurons, ultimately leading to cell dysfunction and death. The discovery and understanding of Amyloid-beta peptide Binding Alcohol Dehydrogenase (ABAD) may clarify the pathogenesis of this elusive disease. Our approach is to combine the use of in vitro hippocampal slices and neuronal cultures with in vivo animals. This strategy offers the advantage of identifying changes in synaptic transmission in a preparation with intact neuronal circuits (slice), of giving depth to the knowledge of these changes in a more simplified system with the unique possibility of having direct access to both the pre-and the post-synapticsite (cell culture), andfinally of determining whether it is possible to re-establish normal learning and memory by counteracting the effects of these changes in an in vivo complex neuronal system (the whole animal). The following aims will be addressed: a) to identify changes of synaptic transmission induced by ABAD/Ap interaction, b) to characterize mechanisms through which ABAD-Ap interaction leads to disruption of synaptic function, c) to identify whether ABAD/Ap interaction interferes with the adenylyl- cyclase(AC)/cAMP/cAMP-dependent-protein-kinase (PKA)/cAMP-regulatory-element-binding (CREB) pathway, a nd d) t o determine w hether a t reatment w ith peptides antagonizing A BAD-Ali interaction is capable of protecting mAPP mice against cognitive and synaptic impairments. Upon the completion of these studies, we will clarify whether and how ABAD/Ap interaction damages synaptic and cognitive function. The importance of analyzing the contributions of ABAD to Ap- induced cell stress is that inhibition of their interaction with Ap might provide a novel means for neuroprotective therapy at a time when cellular damage is still reversible. Project 3 will work closely with Projects 1-2 & 4, and will obtain technical assistance from Core B and Core C. Collaborative interactions will include: exchange of reagents/techniques related to ABAD biology (Project 4), evaluation of cellular stress (Projects 1-2),generation of Tg mice (Core B), and behavioral and pathologic analysis (Core C).

[unreadable] DESCRIPTION (provided by applicant): One of the important targets for developing a causal therapy for Alzheimer's disease (AD) is represented by synapses. The nitric oxide/soluble guanylyl-cyclase/cGMP/cGMP-dependent-protein kinase/cAMP-responsive binding element (NO/sGC/cGMP/PKG/CREB) signaling pathway is thought to play an important role in the synapse during plasticity and memory. Preliminary data from my laboratory have demonstrated the involvement of the pathway in amyloid-beta-induced synaptic dysfunction. Most importantly, sildenafil, an inhibitor the cGMP-degrading enzyme phosphodiesterase V (PDE5), produces an immediate and long lasting amelioration of synaptic and memory abnormalities in an amyloid-depositing mouse model, the APP/PS1 animal. In addition, sildenafil re-establishes normal number of active boutons and glutamate-induced increase in active boutons in cultures from APP/PS1 mice. Thus, the overall purpose of this project is to identify molecules that, by enhancing the NO/sGC/cGMP/PKG/CREB pathway, re-establish normal cognition in the APP/PS1 mouse model. Our approach is to combine expertise in organic chemistry to generate new molecules acting on the signaling pathway with experience in neurobiology to test these compounds at multiple levels including hippocampal slices, neuronal cultures and in vivo animals. This strategy offers the advantage of screening novel drugs in a low cost and fast system (cell cultures), of validating their efficacy in a preparation with intact neuronal circuits (slice from APP/PS1 mice), and finally of determining whether they re-establish normal learning and memory in an in vivo complex neuronal system that is used as an AD model (the whole animal). The following aims will be addressed: a) to identify new enhancers of the NO/sGC/cGMP/PKG/CREB pathway that might be used in CNS diseases; b) to screen the new NO/sGC/cGMP/PKG/CREB pathway enhancers by selecting compounds that rescue amyloid-beta-induced synaptic dysfunction, c) to determine if treatment with novel enhancers of the NO/sGC/cGMP/PKG/CREB pathway have a beneficial effect on cognitive abnormalities in APP/PS1 mice. On the completion of these studies we will identify new therapeutic targets (PDE5, PKG1) and strategies (PDE5 inhibitors, PKG1 agonists) for the treatment of cognitive loss in AD. The whole project will be organized in clear milestones with objective success/failure criteria and GO/NOGO decision points. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Activation of the calpain system might contribute to the impairment of synaptic transmission in Alzheimer's Disease (AD). Calpains regulate the function of many proteins by limited proteolysis and initiate the complete degradation of other proteins. In particular, they modulate processes that govern the function and metabolism of proteins key to the pathogenesis of AD, including tau and amyloid precursor protein. Based on the observation that the protease inhibitors, E64 and BDA-410, are capable of blocking the impairment of synaptic function and cognition in an amyloid-depositing mouse model of AD, the mAPP/mPS1 mouse, we will identify molecules that re-establish normal cognition in mAPP/mPS1 animals by inhibiting calpains. Our approach is to combine expertise in organic chemistry to generate new molecules acting on the signaling pathway with experience in neurobiology to test these compounds at multiple levels including neuronal cultures and in vivo animals. This strategy offers the advantage of screening novel drugs in a less costly system (cell cultures), and of determining whether they re-establish normal learning and memory in an in vivo complex neuronal system that is used as an AD model (the whole animal). The following aims will be addressed: a) to identify new calpain inhibitors and optimize their efficacy for synaptic rescue;b) to screen new calpain inhibitors by selecting compounds that rescue synaptic dysfunction in mAPP/mPS1 mice;c) to further screen calpain inhibitors selected through tests on synaptic function to examine whether they prevent cognitive abnormalities in adult mAPP/mPS1 mice. The whole project will be organized in clear milestones with objective success/failure criteria and GO/NOGO decision points within a collaborative effort with the NIA staff, clinicians and clinical trials experts participating to the annual meeting set by the NIA. The final phase of this work will go beyond the support requested in this application, but will include the crafting of a partnership with industry to bring drugs to patients. On the completion of these studies we will identify new therapeutic agents ameliorating memory loss in AD and other neurodegenerative diseases characterized by elevated levels of amyloid through inhibition of calpain enzymatic activity.

DESCRIPTION (provided by applicant): Amyloid-beta peptides of various lengths (i.e. amyloid-beta 42 and 40) and their accumulation in amyloid-beta plaques are known to play a central role in the pathogenesis of Alzheimer's disease. However, amyloid-beta is normally produced in the brain of healthy individuals throughout life. This may present important issues when designing effective and safe therapeutic approaches against Alzheimer's disease (AD). My laboratory has obtained compelling data showing that administration of amyloid-beta 42, when used at low concentrations, presumably neighboring those found in the normal brain, leads to enhancement of long-term potentiation (LTP), a widely studied cellular model of learning and memory, and post-tetanic potentiation, a type of short- term plasticity that is believed to be an indication of presynaptic function. Picomolar concentrations of amyloid- beta 42 were also able to enhance contextual fear memory and reference memory. Finally, preliminary experiments have shown that depletion of endogenously produced amyloid-beta through antibodies against murine amyloid-beta or through siRNA against murine APP dramatically reduced LTP and hippocampal- dependent memory. Thus, the overall hypothesis of this proposal is that amyloid-beta itself is a critical positive-modulator of synaptic plasticity and memory within the normal CNS. The following three specific aims will be investigated: a) to determine if amyloid-beta is a critical positive modulator of LTP and hippocampal-dependent memory;b) to determine if APP metabolism is altered following tetanic stimulation and memory training, promoting the transient generation of increased amounts of amyloid-beta;c) to determine the mechanisms on how amyloid-beta enhances hippocampal synaptic plasticity and memory. The consequences of our findings go beyond their therapeutic implications, having also relevance for studying the AD pathogenesis and normal learning. PUBLIC HEALTH RELEVANCE: Despite amyloid peptides of various lengths are produced in the brain throughout life in normal individuals, it is not known whether they play a physiological role in the normal brain. This may present important issues when designing effective and safe therapeutic approaches against Alzheimer's disease. We will now explore the possibility that amyloid itself is a critical positive-modulator of synaptic plasticity and memory within the normal brain. The consequences of our findings go beyond their therapeutic implications, having also relevance for studying the pathogenesis of Alzheimer's disease. Indeed, the amyloid hypothesis might take advantage of our findings, as understanding the normal function of a molecule is likely to be relevant to pin point how it gains a new and negative function. Finally, our discoveries will contribute to the understanding of normal mechanisms of learning.

DESCRIPTION (provided by applicant): One of the important targets for developing a causal therapy for Alzheimer's disease (AD) is represented by synapses. The nitric oxide signaling pathway is thought to play an important role in the synapse during plasticity and memory. Published data from Dr. Arancio's laboratory have demonstrated the involvement of the pathway in amyloid-beta-induced synaptic dysfunction. Most importantly, sildenafil, an inhibitor of the cGMP-degrading enzyme phosphodiesterase V (PDE5), one of the components of the pathway, ameliorates synaptic and memory abnormalities in an amyloid-depositing mouse model, the APP/PS1 animal. Thus, the overall purpose of this project is to identify molecules that, by enhancing cGMP levels, re-establish normal cognition in the APP/PS1 mouse model. None of the existing PDE5 inhibitors has been developed to counteract diseases of the CNS and at the same time possesses the selectivity required for chronic administration to an elderly population with comorbid conditions such as AD patients. To that end, a new compound has been synthesized in our laboratories that will serve for lead optimization. YF012403 has high potency and excellent selectivity for PDE5 over other PDE isoforms. Moreover, it crosses the blood brain barrier, and has not shown acute toxicity signs. Most importantly, it ameliorates beta-amyloid induced synaptic and memory dysfunction. The following aims will be addressed by combining med/chem expertise with expertise on the biology of AD and mouse models of the disease: a) to design and synthesize novel PDE5 inhibitors which are optimized for AD;b) to identify compounds with high affinity and good selectivity for PDE5;c) to determine whether new PDE5 inhibitors have good pharmacokinetic and are safe;d) to screen the new PDE5 inhibitors by selecting compounds that rescue synaptic dysfunction in APP/PS1 mice;e) to further screen PDE5 inhibitors selected through tests on synaptic function to examine if they prevent cognitive abnormalities in APP/PS1 mice. On the completion of these studies we will identify a new drug for the treatment of cognitive loss in AD. The whole project will be organized in clear milestones with objective success/failure criteria and GO/NO GO decision points. PUBLIC HEALTH RELEVANCE STATEMENT: Currently used therapies against Alzheimer's disease have limited efficacy. We have found that phosphodiesterase 5 inhibitors might counteract memory deficits in animal of the disease. However, none of the existing inhibitors has been developed to counteract CNS diseases. We now propose to find phosphodiesterase 5 inhibitors that might be used in chronic CNS diseases such as Alzheimer's disease.

DESCRIPTION (provided by applicant): There is intense interest in understanding cellular and molecular mechanisms affected by amyloid-beta elevation, a condition that occurs in patients affected by Alzheimer's Disease (AD). Amyloid-beta is known to impair hippocampal long-term potentiation (LTP) and memory. LTP and memory are elaborate processes that require activation of a number of signaling molecules, pathways and post-translational modifications. This grant addresses the role of SUMOylation in the impairment of LTP and memory following amyloid-beta elevation. SUMOylation is a post-translational modification during which small peptides called small ubiquitin-like modifiers (SUMOs) covalently attach to lysine residues on target substrates. SUMOylation is a reversible process with various effects on protein function, including regulation of localization, stability and activity of many cellular proteins, as well as nuclear integrity, chromosomal segregation and gene expression. Moreover, SUMOylation has been implicated in the pathogenesis of several neurodegenerative disorders, including Parkinson disease, Huntington disease, and AD, but its role in AD remains to be clarified. Our approach is to combine the use of in vitro hippocampal slices and neuronal cultures with in vivo animals. This strategy offers the advantage of identifying changes of synaptic transmission in a preparation with intact neuronal circuits (slice), of giving depth to the knowledge of these changes in a more simplified system with the unique possibility of having direct access to both the pre- and the post-synaptic site (cell culture), and finally of determining whether it is possible to re-establish normal learning and memory by counteracting the effects of these changes in an in vivo complex neuronal system (the whole animal). The following aims will be addressed: 1) to determine whether upregulation of SUMOylation rescues the defect in synaptic function due to amyloid-beta elevation; 2) to determine whether upregulation of SUMOylation ameliorates pre- and post- synaptic mechanisms underlying amyloid-beta-induced synaptic dysfunction; 3) to determine whether upregulation of SUMOylation rescues the memory defect due to amyloid-beta elevation; and 4) to determine whether reduction of CREB SUMOylation is involved in amyloid-beta-induced synaptic and memory dysfunction. Upon the completion of these studies, we will define whether and how SUMOylation is altered in AD. Findings derived from these studies will contribute to identifying the SUMO cascade as a possible target for therapies against AD and a host of other neurodegenerative disorders characterized by amyloid-beta elevation with staggering social, economic and personal costs to the sufferers, their families and all of society.

? DESCRIPTION (provided by applicant): Our poor understanding of the genetic and environmental factors and the mechanisms by which they produce the cognitive and behavioral impairments that characterize Alzheimer's disease stands as a critical barrier to identifying effective preventative measures and treatments for Alzheimer's disease. This project seeks to address this gap in our understanding by examining the ability of the serine/threonine protein phosphatase, PP2A, to control sensitivity to the pathological actions of beta-amyloid, a protein that accumulates in the brain of Alzheimer's disease patients. PP2A is regulated by multiple mechanisms including post-translational methylation of the C-terminus of the catalytic subunit. This methylation is controlled by a dedicated methylesterase, PME-1, and a dedicated methyltransferase, LCMT-1. To perform these studies, we will alter PP2A activity in vivo by manipulating PME-1 and LCMT-1 expression using multiple lines of genetically modified mice. By altering PP2A methylation, the subunit composition and substrate specificity of the mature enzyme will be altered, thereby increasing or decreasing its ability to dephosphorylate Alzheimer's disease relevant substrates. The following specific aims will be pursued: 1) Test the hypothesis that reduced PP2A methylation promotes the development of Alzheimer's disease related impairments by increasing sensitivity to beta- amyloid. 2) Test the hypothesis that over expressing LCMT-1 or reducing PME-1 expression protects against Alzheimer's disease related impairments by decreasing sensitivity to beta- amyloid. 3) Test the hypothesis that PP2A controls beta-amyloid sensitivity by regulating APP phosphorylation at Thr668. These aims will be addressed through a combination of behavioral, electrophysiological, and biochemical techniques. In summary, findings derived from these studies will identify the mechanisms whereby PP2A and its downstream targets may affect the development of Alzheimer's disease by controlling sensitivity to beta-amyloid. Furthermore, they will suggest developing interventions that target this pathway as an effective new therapeutic approach for the disease.

DESCRIPTION (provided by applicant): Aggregation of tau protein to form neurofibrillary tangles together with accumulation of beta-amyloid peptides in amyloid plaques, and neuronal loss are major histopathological hallmarks of Alzheimer's disease. Impairment of processes involved in synaptic strengthening is likely to constitute an early event in the disease that eventually leads to severe cognitive deficits. Recent evidence suggests that extracellular oligomeric tau protein impairs synaptic function and memory. However, synaptic mechanisms affected by tau oligomers have been very poorly explored. With this proposal, the molecular basis of tau oligomer-induced changes in basal neurotransmission and plasticity will be explored. The following specific aims will be tackled: 1) to identify changes of synaptic transmission induced by tau oligomers; 2) to search for potential mechanisms of synaptic dysfunction by tau oligomers; 3) to determine if up-regulation of CREB phosphorylation counteracts tau-induced synaptic dysfunction and memory loss. These aims will be addressed through a combination of electrophysiological, biochemical, imaging and behavioral techniques. Findings derived from these studies will unravel new mechanisms and molecular targets affected by tau protein that might be exploited for developing a treatment against Alzheimer's disease and other disorders characterized by cognitive impairment and abnormal tau pathology.

Our poor understanding of the etiopathogenic mechanisms by which Alzheimer?s disease (AD) starts and propagates throughout the brain stands as a critical barrier to identifying effective treatments for the disease. Extensive literature implicates synaptic dysfunction as an early mechanism affected in AD that involves progressively larger areas of the brain over time, with destabilization of neuronal network activity. This project seeks to address this gap in our understanding by examining the ability of extracellular vesicles (EVs) derived from microglia exposed to amyloid-beta (Abeta-EVs) to dysregulate synaptic function and network activity. EVs are membrane vesicles of endosomal (exosomes) or plasma membrane (ectosomes/microvesicles) origin, which are released extracellularly by most cell types. By exposing cell-type-specific adhesion receptors, EVs can interact with specific cells and deliver complex signals, including proteins, lipids and RNA between cells. Most importantly, several lines of evidence have recently suggested a role for EVs in AD and other neurodegenerative diseases. Abeta-EVs are therefore a good candidate for mediating spreading of synaptic dysfunction with destabilization of neuronal network activity in the AD brain. This will be firmly established by addressing the following specific aims: 1) to test the hypothesis that Abeta-EVs induce alterations of synaptic function and neuronal network activity; 2) to search for a molecular mechanism that is responsible for propagation of Abeta-EV induced synaptic dysfunction across neurons and abnormal neuronal connectivity; 3) to test the hypothesis that transfer of miRNA-146a, a glia-enriched microRNA involved in synaptic function, into the neuron is necessary for impairment of synaptic function and network activity caused by Abeta-EVs. These studies will be performed by combining the expertise of Dr. Verderio, one the pioneers in the EV field, with that of Dr. Arancio on synaptic dysfunction in AD, and Dr. Origlia on recordings from entorhinal cortex slices and EEG in animal models of AD. Moreover, Dr. Verderio has just developed the new elegant approach employed in the research design following a single extracellular vesicle?cell interaction using optical tweezer in primary cultures, and Dr. Origlia is capable of recording long-term potentiation from entorhinal cortex, a technique that has been rarely used (with the last publication in the US in 2004 and none of them in the AD field). Dr. Arancio was a pioneer in the technique of patch clamp recording from pairs of monosynaptically connected neurons. On the completion of these studies we will clarify whether Abeta-EVs spread synaptic dysfunction and destabilize neuronal network activity. We will also identify the molecular mechanisms underlying such phenomena. Findings derived from these studies will contribute to the design of researches and therapies for AD and a host of other neurodegenerative disorders with staggering social, economic and personal costs to the sufferers, their families and all of society.

Our poor understanding of the molecular mechanisms that underlie the cognitive and behavioral impairments that characterize Alzheimer?s disease stands as a critical barrier to identifying effective preventative measures and treatments for Alzheimer?s disease. This project seeks to address this gap in our understanding by examining the ability of enzymes that control the methylation of the serine/threonine protein phosphatase, PP2A, to control the pathological actions of tau ? a central component in the molecular etiology of the disease. PP2A is a heterotrimeric enzyme that exists in multiple isoforms with different expression patterns and substrate specificities, and the activity and composition of these isoforms is regulated by multiple mechanisms including protein methylation. PP2A methylation is controlled by an evolutionarily conserved mechanism involving the competing actions of a dedicated methylesterase and a dedicated methyltransferase ? PME-1 and LCMT-1 in mice respectively. In preliminary studies, we found that transgenic over expression of LCMT-1 protected mice from cognitive impairments caused by acute exposure to soluble tau aggregates, while PME-1 over expression increased sensitivity to these impairments. In this proposal, we will build on these observations by pursuing the following specific aims: 1) Determine which aspects of tau pathology are affected by increased PME and LCMT expression levels. 2) Test the hypothesis that inhibiting PME-1 protects against tau related impairments. 3) Test the hypothesis that PME and LCMT expression levels affect sensitivity to tau by altering the proportion of Ppp2r2a-containing PP2A enzymes. These aims will be addressed through a combination of behavioral, electrophysiological, and biochemical techniques in genetically modified mice. The data obtained from these experiments will identify the mechanisms whereby methylation of the catalytic subunit of PP2A controls the development of tau-related impairments in Alzheimer?s disease, and test the possibility that interventions that target this pathway could constitute an effective therapeutic approach for their prevention or treatment.

Our poor understanding of the molecular mechanisms that underlie the cognitive and behavioral impairments that characterize Alzheimer?s disease stands as a critical barrier to identifying effective treatments for Alzheimer?s disease. This project seeks to address this gap in our understanding by examining the ability of SUMOylation, a post-translational modification during which small peptides called small ubiquitin-like modifiers (SUMOs) covalently attach to lysine residues on target substrates, to control the pathological actions of tau ? a central component in the molecular etiology of the disease. SUMOylation is a reversible process with various effects on protein function, including regulation of localization, stability and activity of many cellular proteins, as well as nuclear integrity, chromosomal segregation and gene expression. There are three known SUMO paralogs in vertebrate brains: SUMO1-3, with SUMO2 and 3 sharing ~95% sequence homology (and not functionally differentiated) often collectively referred to as SUMO2/3. Interestingly, SUMOylation is dysregulated in the hippocampus of Alzheimer?s Disease patients. In preliminary studies we have been able to link tau SUMOylation to tau aggregation and toxicity with SUMO2 conjugation (but not SUMO1) protecting against tau aggregation and toxicity. SUMO2 was also found to decrease aggregated tau release. Moreover, mimicking SUMO2 covalent binding to tau rescued the oligomeric tau-induced impairment of long-term potentiation (LTP), a type of synaptic plasticity thought to underlie memory formation, and memory loss in a mouse model of fronto-temporal dementia. In this proposal, we will build on these observations by pursuing the following specific aims: 1) determine if upregulating SUMO2 conjugation rescues tau-induced defects in synaptic function; 2) determine if upregulating SUMO2 conjugation rescues tau-induced memory defect in mice; 3) test whether the rescuing effects of upregulating SUMO2 conjugation depend upon modulation of tau oligomer formation. These aims will be addressed through a combination of electrophysiological, behavioral, biophysical, and biochemical techniques in wild-type and genetically modified mice. Upon the completion of these experiments, we will identify the mechanisms whereby SUMOylation controls the development of tau-related impairments in Alzheimer?s disease and in fronto-temporal dementia, and test the possibility that interventions that target SUMO2 conjugation could constitute an effective therapeutic approach for their treatment.

ABSTRACT Alzheimer's disease and related dementias (AD/ADRD) have a significant societal impact, yet there are no disease modifying interventions. Root causes of prior clinical trial failures provide instruction for plans to reinvigorate the AD/ADRD therapeutic discovery and development process. Specifically, a diversified portfolio of candidate therapeutic approaches is available based on clinical observations, genetic associations, pathology outcomes and biochemical mechanisms. However, many are neglected in terms of funding and technical pursuit. The prior emphasis on a pathology-based pathway can be avoided by retaining a therapeutic emphasis on discrete but complementary aspects of pathophysiology progression mechanisms. Synaptic dysfunction is one example with diverse potential targets. Synaptic dysfunction underlies subtle amnesic changes occurring prior to the development of the classical histopathologic hallmarks. Deteriorated synaptic strengthening is associated with remodeling of various neurotransmitter systems, including cholinergic, noradrenergic, dopaminergic and serotonergic systems. The serotonergic system is both an underexplored therapeutic mechanism and is especially attractive considering that serotonin is more than a neurotransmitter. Further, clinical findings that 5-hydroxytryptamine receptor 2b (5-HT2bR) expression is increased in AD patient brains and that AD patients respond to a non-selective 5-HT2bR antagonist suggest the potential utility of optimized 5-HT2bR antagonists in AD. We developed a small molecule, MW01-8-071HAB (=MW071), that suppresses LTP defects as well as associative and spatial memory in models of amyloid-beta (A?) and tau elevation. Functional screens for off-target agonist and antagonist activity with 158 known GPCRs demonstrated that MW071 is a selective 5-HT2bR antagonist. Importantly, MW071 lacks 5-HT2BR agonist activity. Avoiding agonist activity is landmark. Approved drugs with 5-HT2bR agonist activity have high risk for cardiac valve toxicity, resulting in withdrawal or black box warnings. Therefore, the promising efficacy in AD relevant models, a pharmacological profile that includes highly selective antagonist activity in the absence of agonist activity, and the availability of a back-up candidate (MW109) adds to the overall appeal of MW071 as a starting point. Our proposed early-stage studies will further de-risk MW071 and MW109 in order to generate and qualify a candidate for a future IND-enabling late stage U01 application: Aim 1. Perform secondary pharmacology analyses following FDA guidance, as a necessary prelude and a firm foundation for future GxP IND-enabling preclinical safety and toxicology research. Aim 2. Validate the efficacy of MW071 and MW109 in prevention/reversal of synaptic and memory impairments in AD-relevant animal models. Quantitative milestones will determine progression through Go/No Go decision points. Successful outcomes and deliverables will allow for future support of GxP IND-enabling evaluation and first-in-human assessment.

PROJECT SUMMARY Alzheimer?s disease (AD) is the most common form of dementia in humans. Despite intense research there is as yet no cure for AD and the increasing incidence of AD in developed countries poses a tremendous cost to society as lifespans increase. There are two forms of AD, those that have genetic determinants and comprise approximately 5% of cases, and those that arise sporadically, particularly upon aging, and comprise the vast majority (~95%) of new AD cases diagnosed. The underlying triggers for sporadic AD are diverse and not well understood. Current therapeutic strategies are limited to those that attenuate AD symptomology without affecting the progression of the disease itself. Thus understanding the etiology of the disease is necessary to generate better therapeutics. A widely accepted hypothesis, known as the ?mitochondrial cascade hypothesis?, posits that aging leads to accumulation of damaged mitochondria that produce mitochondrial reactive oxygen species (mROS), triggering progressive oxidative damage that ultimately results in development of AD. However, despite decades of study, definitive evidence for mROS or aberrant accumulation of damaged mitochondria as a key trigger have not emerged. Our preliminary studies establish a critical role for the mitochondrial complex I assembly factor ECSIT in the regulation of mitochondrial function, mROS production, and mitochondrial quality control. Moreover, we have obtained evidence implicating dysregulation of ECSIT expression/function in AD. Therefore, we propose a series of experiments that leverage the unique expertise of the two principal investigators, and institutional capabilities, to fully characterize the role of ECSIT in neurodegeneration and AD. The proposed experiments will allow us to directly test the mitochondrial cascade hypothesis in murine models of AD and also probe the relationship between ECSIT dysregulation and the development of AD.