Frank Longo - US grants

Research is proposed to test the hypothesis that sperm chromatin dispersion is coordinate with the activity of the maternal chromatin, i.e. changes in nucleocytoplasmic interactions effecting the status/function of the maternal genome also influence the pattern of sperm chromatin dispersion at fertilization. Recent investigations demonstrate that sperm nuclear dispersion does not proceed at a uniform rate but consists of four phases coordinate with major changes in the activity of the maternal chromosones. The presence of distinct phases in the rate of dispersion indicates the existence of major differences in the regulation of chromatin dispersion. What mechanisms comprise each of these phases and how they are related to one another and conditions regulating the status/function of the maternal chromatin are aspects to be explored during the course of this study. Experiments will (1) determine and compare the rate of sperm nuclear dispersion in ova fertilized at different stages of meiotic maturation and (2) determine the kinetics of chromatin dispersion in eggs that, normally fertilized at one stage of meiotic maturation, are inseminated at a different period. If the kinetics of sperm chromatin dispersion is related to the activity of the maternal chromatin, disruption of the normal functioning of the maternal chromatin or factors regulating the status of the maternally derived genome may be reflected in alterations of sperm nuclear transformation. Stages of meiotic maturation of the maternal chromatin will be perturbed and possible effects on the rate of sperm chromatin dispersion will be analyzed. Experiments will be carried out in which the rate of sperm chromatin dispersion will be compared in fertilized nucleate and enucleate eggs and in specimens treated with agents to disrupt the meiotic apparatus. If the kinetics of sperm chromatin dispersion is related to factors regulating the activity of the maternal chromatin, this may be demonstrated by experiments, where extracts from eggs at one stage of fertilization are microinjected into zygotes at a different stage. Correlation of alterations in sperm chromatin dispersion with stages of fertilization from which microinjected extracts were obtained would support the proposed hypothesis. It is anticipated that results of these experiments will lead to further studies, injecting specific components into zygotes, and eventually characterizing regulators of sperm nuclear transformations at fertilization.

The proposal is a correlative study of ultrastructural and electrophysiological events during sperm-egg (Lytechinus) interaction which centers on identifying the primary events involved with egg activation. Sperm attachment initiates one or more signals from the egg surface which results in activation. One possibility is that an excitatory process is initiated immediately after sperm attachment as a result of a sperm ligand-egg receptor type of interaction. Alternatively, the excitatory phase may be induced by fusion between the sperm and egg plasma membranes. It is not known when the gamete plasma membrane fuse, nor is it known whether fusion of the sperm and egg plasma membranes is necessary to induce egg activation. These gaps in our knowledge are addressed by this proposal. The specific aims of this proposal include: (1) to establish by electron microscopic observations how the interacting sperm and eggs are structurally associated at different intervals at and following the onset of the activation current; (2) the ultrastructural relation of interacting gamete plasma membrane in voltage-clamped eggs with changes in the activation current; (3) to correlate the ultrastructural relation of interacting gamete plasma membranes in voltage-clamped oocytes; and (4) to correlate the ultrastructural relation of the plasma membranes of interacting gametes in which the egg has been treated with an agent (cytochalasin B) that blocks sperm incorporation but not egg activation with changes in the activation current. These proposed experiments will establish when fusion of the gamete plasma membranes occurs relative to the onset of the activation current and whether fusion of the sperm and egg plasma membranes is requisite for egg activation.

One of the primary pathological processses occurring in the aging nervous system is neuronal death. For example, in Alzheimer's disease (AD) cholinergic neurons in the basal forebrain, which are thought to function in memory and learning, are lost. Neurotrophic factors are proteins which interact with cell surface receptors to promote the survival and growth of neurons. There is considerable evidence that neurotrophic factors such as nerve growth factor (NGF) can prevent neuronal death or damage and that they may be able to enhance the function of compromised neurons. Basal forebrain cholinergic neurons have NGF receptors and respond to NGF; therefore, NGF may be relevant to understanding and perhaps minimizing the loss of neurons in AD. A significant limitation to both understanding the physiological role of endogenous NGF in vivo and the potential therapeutic application of NGF is the lack of stable, low molecular weight agonists and antagonists of NGF which can effectively reach the central nervous system and penetrate the brain parenchyma. The long term objectives of this work are: i) to begin to define structural features of NGF (NGF's active site) which will allow it to interact with its receptor and ii) to develop a first generation of low molecular weight synthetic NGF peptide analogs which can mimic or inhibit the neurotrophic effect of NGF in vitro and eventually in vivo. The general strategy of this proposal is to map the active site(s) of NGF by synthesizing short peptides corresponding to potential active sites. These peptides will be assayed for their ability to block or mimic several measures of NGF bioactivity in vitro. By systematically varying which residues of NGF are includes in each peptide and by making peptides of different lengths, one can begin to map an active site. Active peptides will then be modified by conventional techniques to form a first generation of NGF analogs. This synthetic peptide strategy has been used many times to define active sites and produce active analogs of many proteins, most of which are considerably more complex than NGF. Indeed, our initial studies with NGF peptides suggest that this method will be successful with NGF as well. The specific aims during the proposed grant period will be: 1) To synthesize and purify NGF peptides and analogs derived from peptides which are active. 2) To test these peptides and analogs for their ability to mimic or inhibit three well-characterized activities of NGF in vitro. 3) To test the most potent peptides for their ability to compete with NGF's binding at its receptor.

DESCRIPTION: (From the applicant's abstract): Long-term objective: Identify novel small molecule targets for preventing neuronal death in neurodegenerative disease. Hypothesis: The LAR tyrosine phosphatase regulates neuronal death and differentiation by modulating signal transduction mechanisms relevant to NGF function and apoptosis. Potential signaling components targeted by LAR include the MEK/ERK, PI-3/AKT, TrkA and JNK kinases. Elucidation of cause-and-effect mechanisms by which LAR counter-balances NGF signaling and promotes apoptosis will lead to novel approaches for inhibiting neuronal death and augmenting neurotrophin effects. Specific aim 1. Establish functional interactions between LAR and TrkA signaling in PC12 cells. Establish the time course for increased NGF-induced TrkA autophosphorylation in LAR-deficient cells. Determine if decreased TrkA dephosphorylation is a candidate mechanism preventing apoptosis in serum-deprived LAR-deficient cells. Determine if co-immunoprecipitation of LAR and TrkA is regulated by NGF or induction of apoptosis. Use confocal microscopy to confirm LAR and TrkA co-localization. Use in vitro enzymatic assays to determine if LAR dephosphorylates TrkA. Specific aim 2. Determine if LAR modulates activation of NGF signaling intermediates regulating PC12 neurite outgrowth. Determine whether baseline and NGF-induced activation states of key NGF signaling intermediates triggering neurite outgrowth are altered in LAR-deficient cells. Establish time courses for NGF-induced activation and deactivation of MEK and its substrate, ERK. Specific aim 3. Determine if LAR modulates activation of signaling intermediates regulating PC12 survival. Determine if activation states of key signaling intermediates known to regulate PCl2 cell survival are altered following serum withdrawal. Measure phosphorylation and dephosphorylation of the following signaling intermediates at multiple time points following withdrawal: MEK, ERK, AKT and JNK. Specific aim 4. Establish cause-and-effect relationships between LAR deficiency, changes in signaling intermediate activation and apoptosis. Measure the effects of the Trk inhibitors K252a and AG879, the MEK inhibitors PD098059 and U0126 and the PI-3/AKT inhibitor LY294002 on serum withdrawal-induced cell death in control and LAR-deficient cells. Further assess LAR-TrkA interactions by determining if LAR deficiency prevents apoptosis in PC12nnr5 cells lacking functional TrkA receptors. Specific aim 5. Determine whether LAR deficiency inhibits neuronal apoptosis in vivo. Determine if the previously established increase in the number of DRG neurons in adult LAR -/- mice is due to decreased developmental cell death. Measure neuronal precursor proliferation rates and developmental death rates by quantitating BrdU- and TUNEL-positive cells in LAR +/+ and -/- DRG.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative disorder that leads to the progressive loss of memory and other cognitive functions. At this time, there are no approved treatments that are capable of delaying its onset or slowing its progression. We have developed a novel category of drug-like, small molecule compounds that specifically target a cell surface receptor that is expressed by neurons affected in AD, known as the p75 receptor. These p75 receptor ligands activate survival-promoting signaling and inhibit degenerative-promoting signaling of the p75 receptor. In tissue culture studies, these ligands are capable of blocking the ability of amyloid beta (A[unreadable]) to activate degenerative signaling within neurons affected in AD. The protective effects of these compounds occur at low nanomolar concentrations and have been verified to occur through their action at p75. Moreover, our ligands block the toxicity of A[unreadable] oligomers, the A[unreadable] species thought to be most toxic to neurons and most relevant to AD. In studies of normal middle aged mice, our lead compound has been demonstrated to reach the brain following daily oral administration and has been found to have no toxic effects including studies of hepatic, cardiac and DNA toxicity. In these mice, our lead compound demonstrates a significant neurotrophic effect of reversing or preventing basal forebrain cholinergic atrophy of the type that occurs during aging and AD in both human and rodent systems. In pilot trials in a well characterized AD mouse model, our lead compound appears to be improving memory function and to be reducing pathological features typical of AD. In this application we will complete the following three milestone-driven projects: i) verification of efficacy in Alzheimer's mice and assessment of potential mechanism-based side effects;ii) cGMP scaled up synthesis and purity necessary for an Investigational New Drug (IND) application to the FDA;and iii)toxicology and pharmacology studies designed to complete an IND application. Completion of these three projects will allow an IND application with the overall goal of obtaining IND approval for conducting the first Phase I trials humans. Completion of the proposed project will also establish a new chemical entity (NCE) and a novel, first in class, drug compound for development in AD therapeutics.

DESCRIPTION (provided by applicant): This application titled "Small Molecule Neurotrophin Mimetics to Treat Huntington's Disease" is in response to NINDS Exploratory/Developmental Projects in Translational Research (R21;program announcement: PAR- 08-232). The current proposal will test whether small molecule agonists for the TrkB neurotrophin receptor will be effective treatments for neurodegeneration associated with Huntington's Disease (HD). The TrkB receptor binds brain derived neurotrophic factor (BDNF), which is a growth factor that is critically involved in neuronal survival and synapse function. Reduced levels of BDNF contribute importantly to pathology in numerous neurodegenerative disorders, including Alzheimer's, Parkinson's and Huntington's (HD). Given the pervasive neuroprotective role of BDNF/TrkB, the demand for small molecule TrkB ligands that lack undesired side- effects is large and long standing. Such compounds have been developed for the first time by the applicant and his colleague. These novel TrkB agonists mimic many of BDNF's effects on TrkB signaling and cell survival in in vitro and in vivo studies. Thus, they are ready to be tested for their effects on preventing pathology in neurodegenerative disease. This proposal will focus on HD since the loss of BDNF in HD brains is a fundamental contributor to the neuropathologies associated with the disorder. HD is a fatal neurodegenerative disease characterized by progressive motor, psychological, and cognitive deficits that emerge in adulthood. It is caused by a mutation in the gene that encodes the huntingtin protein. The proposed studies will use a transgenic mouse model of HD to execute two aims: (i) determine if 2 different BDNF mimetics will slow the emergence of, or prevent, motor, memory and psychiatric deficits associated with HD using a battery of behavioral tests;and (ii) test whether BDNF mimetics ameliorate HD-related neuropathology. Positive results in these studies will identify a novel and feasible therapeutic strategy for reducing numerous HD phenotypes (motor, psychological and cognitive impairments as well as neuropathology), which are currently untreatable, as well as other age-related neurodegenerative diseases. The current R21 proposal is designed to provide proof-of-concept and target validation data that will support a subsequent U01 application to determine the effectiveness of these novel small molecule TrkB ligands against neurodegeneration. PUBLIC HEALTH RELEVANCE: The current proposal will test the efficacy of novel, small molecule agonists for the TrkB neurotrophin receptor to treat neurodegenerative disease, specifically Huntington's Disease (HD). The TrkB receptor binds brain derived neurotrophic factor (BDNF), the loss of which in HD brains is a fundamental contributor to the neuropathologies associated with the disorder. Thus, these first-in-class TrkB agonists are likely candidates to prevent neurodegeneration associated with a disease that currently has no cure and success in the proposed HD studies will point to TrkB agonist applications in other CNS disorders including Alzheimer's disease and depression.

Longo, Frank M. PROJECT ABSTRACT The goal of the Stanford Neurology Resident Research Education Program is to provide residents with training and direct experience in clinical or laboratory neuroscience research with a focus on successful transition to independent funding. A group of Stanford faculty have been selected as mentors that have outstanding records of training young investigators. The ultimate aim is to foster the participants' career development as clinician scientists devoted to investigation of neurological diseases.

DESCRIPTION (provided by applicant): This application titled P75NTR Small Molecule Ligands for Down Syndrome Therapy is in response to NINDS Exploratory/Developmental Projects in Translational Reseach (R21; program announcement: PAR-11-293). The current proposal will test whether small molecule ligands for the p75 neurotrophin receptor (p75NTR) will be effective treatments for neurodegeneration associated with Down syndrome (DS). An alteration most notably associated with DS is reduction of nerve growth factor, which binds to p75NTR expressed by basal forebrain cholinergic neurons (BFCN), the degeneration of which is a pathological hallmark of the disorder. The Longo laboratory previously found that p75NTR ligands, developed by the applicant and his colleague, prevent atrophy of BFCN and cognitive deficits in a mouse model of Alzheimer's disease (AD). To date, there is no known small molecule capable of restoring trophic support to BFCN, functioning at a specific target receptor, penetrating the blood brain barrier via oral administration, and lacking undesirable side-effects. Thus, these novel p75NTR ligands are now poised for efficacy testing in DS. In addition to deleterious effect on neurodevelopment, DS has neurodegenerative consequences that include pathology resembling AD and a significant prevalence of AD-type dementia in the fourth decade of life. The proposed studies will use a transgenic mouse model of DS to execute two aims: (i) determine if two (lead and backup) p75NTR ligands will slow the emergence of, or prevent, memory deficits associated with DS using a battery of behavioral tests; and (ii) test whether p75NTR ligands ameliorate classic DS-related cholinergic neuropathology. Positive results in these studies will identify a novel and feasible therapeutic strategy for reducing hallmark DS phenotypes, which are currently untreatable. The current R21 proposal is designed to provide proof-of-concept and target validation data that will support a subsequent U01 application to determine the effectiveness of these novel small molecule p75NTR ligands against DS.

DESCRIPTION (provided by applicant): This application titled Small Molecule p75 Neurotrophin Receptor Ligand to Treat Huntington's Disease is in response to NINDS Exploratory/Developmental Projects in Translational Research (R21; program announcement: PAR-11-293). The current proposal will test whether a small molecule, non-peptide ligand for the p75 neurotrophic receptor (p75NTR) will be an effective treatment for Huntington's disease (HD). HD is a fatal neurodegenerative disease characterized by progressive motor and cognitive deficits. It is caused by a mutation in the gene that encodes the Huntington (htt) protein, which causes medium spiny neurons in the striatum to die. How mutant htt causes this effect is unclear; however, loss of neurotrophic support is purported to play a key and causal role and p75NTR has been implicated. P75NTR is up-regulated in the striatum of HD patients and mouse models and many central intermediate proteins in p75NTR signaling pathways are shared by pathways affected by mutant htt. Our laboratory has developed first-in-class small molecule, orally bioavailable p75NTR ligands that inhibit degenerative signaling, and prevent neurodegeneration. One lead ligand, LM11A-31, is well into development via our current NIA U01 program. It has completed multiple successful efficacy trials in Alzheimer disease (AD) mouse models and pre-IND studies in rats and dogs. Recently, the ligand received FDA approval for phase I clinical testing. Intellectual property for the compound is intact and owned by UNC and UCSF. These proposed studies will examine whether treatment with LM11A-31 is applicable to HD. The aims of the proposal are to: (i) determine whether LM11A-31 will reduce cognitive and motor deficits as well as neuropathology in a mouse model (R6/2) that develops the HD phenotype rapidly and is therefore cost effective and ideal for PK/PD studies; and ii) determine if LM11A-31 will slow or prevent the development of HD-related behavior deficits and neuropathological features in another mouse model, BACHD. This mouse develop an HD phenotype much slower and less severely but is a better genetic replicate of the disease, thus positive results may be easier to discern and be more readily translatable to the clinic. Thus, targeting p75NTR could offset HD-related deleterious signaling, an entirely new hypothesis for HD treatment that our laboratory is uniquely able to investigate. Positive results in these studies will identify an entirely novel and feasible therapeutic strategy for reducing numerous HD phenotypes (motor and cognitive impairments as well as neuropathology), which are currently untreatable. This R21 proposal is designed to provide target validation data. Positive results obtained here would be the first validation that p75NTR is an effective therapeutic target for an HD model and could fast track LM11A-31 into HD clinical testing.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is growing global health issue. At present, there are no drugs available to halt or reverse disease progression, and all efforts to create such therapies have failed. One reason for these failures is the lack of translatable biomarkers useful in both mouse models and humans, and the relatively small role that appropriate biomarkers have played in the drug discovery and development process. Identification of translatable biomarkers for non-invasive assessment of therapeutic outcomes is imperative to improving the treatment of AD. Here we propose the use of a new imaging strategy for monitoring treatment response in Alzheimer's disease. The strategy involves using a specific positron emission tomography (PET) radioligand ([18F]GE-180) for the translocator protein 18 kDa (TSPO). TSPO PET radioligands are used to non-invasively detect and track microglial activation (a marker of neuroinflammation) in a living subject. Evidence suggests that neuroinflammation takes place very early in the AD, even before the formation of amyloid plaques. In addition, early anti-inflammatory intervention appears to profoundly impact the onset and progression of AD. The early involvement of neuroinflammation in AD, and the fact that it persists throughout disease and contributes to neurodegeneration, make it an ideal candidate biomarker for tracking pathology and monitoring response to early therapeutic interventions. Although there are a number of available TSPO radioligands, [18F]GE-180 has superior binding affinity and in vivo properties for imaging neuroinflammation processes in vivo. And while this tracer has undergone first in-man evaluation with favorable results, it is yet to be studied for it ability to monitor treatment response in AD. Our immediate goal is to evaluate [18F]GE-180-PET for its ability to track AD progression and monitor response to drug therapies in AD mouse models. Following this, our long- term goal is to assess the utility of [18F]-GE180-PET as a biomarker of AD treatment response in clinical trials of novel disease-modifying drugs. Our preliminary studies show that [18F]GE-180-PET can detect microglial activation in a mouse model of AD (APPL/S) at early stages of disease. In this proposal we aim to further evaluate [18F]GE-180 in AD mouse models for its use as a surrogate marker of AD neuroinflammation, and for monitoring response to novel drugs currently under evaluation for AD treatment. We will achieve our goals through the following specific aims: 1) determine whether [18F]GE-180-PET signal correlates with the extent of microglial activation and disease progression in two mouse models of AD, and 2) assess the sensitivity and accuracy of [18F]GE-180-PET for imaging response to two AD therapeutics currently in clinical trials (i.e., LM11A-31 and minocycline). The use of [18F]GE-180 could be a `game-changing' approach with potential far- reaching advantages in the whole arena of biomarker driven diagnostics and therapeutics for AD.

Project Summary This application titled ?Small Molecule Neurotrophin Receptor Ligands to Treat Alzheimer?s Disease? is in response to NIH Drug Discovery for Nervous System Disorders (R21; PAR-16-042). We will determine if small molecule ligands targeted to the TrkB and TrkC neurotrophin (NT) receptors will inhibit fundamental pathophysiological mechanisms underlying AD. In AD, multiple processes including aging, accumulation of amyloid and pathological forms of tau, and inflammation lead to synaptic dysfunction, decreased dendritic spines, and the eventual loss of synapses and neurons. NTs, including brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), are protein ligands essential for the maintenance and function of neuronal synapses. BDNF and NT3 bind to TrkB and TrkC, respectively, to trigger intracellular signaling pathways that are highly integrated with, and hence situated to oppose, the degenerative signaling in AD. Furthermore, disrupted NT signaling contributes to the development of AD neuropathology and memory deficits. We hypothesize that therapeutically restoring or augmenting TrkB and/or TrkC signaling will counteract neurodegenerative signaling in AD thereby inhibiting hallmark AD pathologies. Our laboratory developed small molecule NT receptor ligands that bind to and activate TrkB or TrkB and TrkC. Previous published studies with the TrkB ligand showed that it entered the brain and was neuroprotective in numerous mouse models of neurodegenerative disorders. Our preliminary results with the TrkB/TrkC ligand showed that it reduced tau pathology, decreased dendritic spine loss, and improved cognition in the A?PPL/S mouse model of AD. These results established small molecule TrkB/C ligands as candidate AD therapeutics. While brain levels of the ligands were sufficient to produce neuroprotective effects when given systemically, favorable brain levels after oral delivery are necessary for drug development. To this end, we developed novel derivatives of these ligands that enter the brain more readily producing higher brain concentrations than the original ligands and have neurotrophic effects similar to that of BDNF. Thus, the proposed research aims to determine if these newly derived small molecule TrkB and TrkB/TrkC ligands will activate their intended receptors and initiate downstream signaling in a dose-dependent manner and prevent memory deficits and psychological disturbances as well as neuropathology in the A?PPL/S mouse model of AD. Targeting TrkB together with TrkC is a novel therapeutic strategy for AD, and, to our knowledge, our laboratory is the only source of a small molecule TrkB/TrkC ligand capable of testing the efficacy of simultaneously targeting these two NT receptors. This approach has the potential for synergistic intracellular signaling, as can occur with Trk receptors, and combatting multi-faceted pathological mechanisms seen in AD.

There is an urgent need for a diverse portfolio of new therapeutic and diagnostic targets for Alzheimer?s disease (AD). To hasten progress towards the national goal of developing an effective treatment for AD by 2025, the NIA has created several initiatives designed to identify new AD drug targets, including the Accelerating Medicine Partnership for AD (AMP-AD). Although promising, our understanding of most of the emerging therapeutic hypotheses and nominated targets is not yet sufficient to support their integration into drug discovery programs. Understanding that the amount of evidence required to support the integration of a target into a drug discovery pipeline would far surpass the expertise and capabilities of any one group, here we introduce the Open Drug Discovery Center for Alzheimer?s Disease (Open-AD). The past decade of activity has conclusively shown that the open science approach can be used to de-risk new therapeutic modalities, such as the ones emerging from AMP-AD, to catalyze biological validation of drug targets and to launch new commercial drug discovery programs. To reinvigorate the AD drug discovery pipeline with a diverse portfolio of well-supported next generation AD targets supported by evidence from across the research community, Open-AD will develop and openly disseminate resources (experimental tools, reagents, probes, knowledge, data) that can support target validation and drug discovery across a wide variety of independent evaluations. In Aim 1, we will develop a prioritized set of community-nominated therapeutic hypotheses and targets. In Aim 2, we will develop target enabling packages that include high-quality, well-validated reagents for use in target validation and drug discovery. In Aim 3, we will develop chemical and biological probes. In Aim 4, we will rapidly and openly distribute all Open-AD assets ? including probes ? to enable characterization and experimental validation of candidate drug targets by any interested academic and/or commercial investigator.