Donald C. Lo - US grants

DESCRIPTION: (Applicant's Abstract) Tremendous progress has been made in identifying new neurotrophic factors in their receptors, and in characterizing their roles in neuronal survival and differentiation. Not surprisingly, abnormalities in neurotrophic interactions have been implicated in several neurological disorders including epilepsy and Parkinson's disease. However, current understanding of the neurotrophic regulation of the basic neuronal functions that underlie these processes and disorders, such as neuronal signaling, remains quite limited. The goal of this research proposal is to understand how neurotrophic factors regulate neuronal excitability and synaptic transmission. The experimental approach will be to study how the expression and function of individual ion channels and neurotransmitter receptors are regulated by defined neurotrophic factors. The specific aims are: 1) To test the hypothesis that trk receptors determine the specificity of action of the neurotrophins. This hypothesis will be tested in the context of the regulation of ion channel and neurotransmitter receptor expression by different neurotrophins and trk receptors; 2) To determine if neurotrophic factors that use completely unrelated signal transduction mechanisms have common regulatory actions. The hypothesis of this specific aim is that unrelated neurotrophic factors will have overlapping effects on excitable properties common to all neurons, but distinct effects on excitable properties unique to particular neuronal types. This hypothesis will be tested for the unrelated factors NGF and ciliary neurotrophic factor; and 3) To determine if neurotrophic factors modulate excitability in the short-term. Activation of neurotrophic factor receptors such as the trk receptors initiates multiple signaling cascades within minutes of neurotrophic factor binding. Do these signaling events, many of which are known to affect excitability in other contexts, cause short term modulation of ion channel function? The proposed studies will use electrophysiological, molecular biological, and optical imaging techniques to study the expression and function of individual ion channel and neurotransmitter receptor species in three experimental settings: neuronal cell lines, cells transfected with specific neurotrophic factor receptors, and primary cultured neurons from hippocampus. The results from these experiments will contribute to the understanding of how neurotrophic factors regulate important neuronal functions such as excitability and synaptic transmission, and how deficiencies in neurotrophic interactions can lead to neuronal dysfunction and neurological disorders.

device, based on particle-mediated gene transfer (or 'biolistics'), for the identification and validation of candidate therapeutic drugs and targets. The tremendous pace at which new genomic and proteomic technologies are advancing and generating new information regarding the normal and pathological brain has created a major bottleneck in translational neuroscience, namely, the ability to physiologically validate new candidate therapeutic targets upon which to base target-driven efforts in medicinal chemistry. In this context, a major technological rate-limiting step has been the difficulty of manipulating the expression of genes and proteins in neural disease-specific experimental systems based on bona fide, postmitotic neurons, ranging from primary cultures to tissue explant models. In preliminary experiments, we have demonstrated that a neural transfection approach based on particle-mediated gene transfer, or biolistics, can be used to screen DMA-based and chemical libraries at medium-throughput levels in disease-specific models created in living brain slices. We propose here to develop a new particle acceleration-based technology to increase transfection efficiency and throughput by 10-fold or more. Such an improved and affordable biolistic device would allow laboratory groups of standard size to embark upon high-content, functional screening and validation experiments in neural cells and tissues using the mass of genomic and proteomic information now available. The Research Plan has 3 specific aims: Specific Aim1: To develop a new and highly efficient particle-acceleration transfection device. Specific Aim 2: To determine workable ranges of shooting parameters for the new device. Specific Aim 3: To evaluate the efficiency and effectiveness of the new biolistic device with respect to existing technology.

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a dominant genetic disorder arising from expansions of the polyglutamine domain in the huntingtin gene (htt), affecting some 35,000 people in the US alone. The normal functions of htt remain largely unknown, with disease mechanism(s) involving presumptive gains-of-function from the mutant protein as well as potential loss of function/interference with the normal htt allele. Critically, the lack of clinically validated targets for HD places an urgent need on identifying and understanding the mechanisms of action of potentially beneficial drug targets, and, importantly, on demonstrating that such targets can be addressed using therapeutic candidate molecules with good, drug-like properties. The present proposal focuses on the potential intersection between two such candidate targets/pathways that have increasingly been implicated in HD: the adenosine 2A receptor (A2AR) and the TrkB receptor. Recent evidence suggests that significant aspects of A2AR downstream signaling may actually be mediated through its cross-activation of the TrkB receptor in a manner that is independent of TrkB ligands such as BDNF, whose normal provision to the striatum by the cortex is compromised during HD pathogenesis. If so, such a mechanism, if operant in the context of HD, could present a therapeutic opportunity to use A2AR ligands to provide trophic support to degenerating striatal neurons via their cross-activation of TrkB. Moreover, if this mechanism is supported, BBB-penetrant A2AR ligands in late-stage clinical testing are already available for evaluation in HD models for potential repurposing for clinical use in treating HD. Thus, the goal of this R21 proposal is to provide proof-of-principle for the core hypothesis that A2AR modulation can provide benefit to striatal neurons undergoing neurodegeneration in the context of HD through cross-activation of the TrkB receptor. For these studies, we will use a brain slice-based assay model for HD that, critically, retains the local tissue environment of the striatum and cortex in order to be maximally predictive for the in vivo setting while providing the experimental access of an in vitro/ex vivo preparation. If supportive, these findings in a brain slice-based HD assay should provide the necessary foundation for a full R01 application to examine this mechanism and therapeutic opportunity in whole-animal models of HD using both neurobehavioral as well as neuropathological outcome measures. PUBLIC HEALTH RELEVANCE: Huntington's disease (HD) is a fatal, dominant genetic disorder arising from expansions of the polyglutamine domain in the huntingtin gene (htt), affecting some 35,000 people in the US alone. Currently, no cures are known for this devastating disease, with palliative treatments available that are only partially effective in treating the neuropsychiatric symptoms and motor disabilities that develop over the course of HD. This lack of clinically validated targets for HD places an urgent need on identifying and understanding the mechanisms of action of potentially beneficial drug targets, and, importantly, on demonstrating that such targets can be addressed using therapeutic candidate molecules with good, drug-like properties. The present proposal will test the proposition that existing small molecule drugs targeting the adenosine 2A receptor could be used to tap into a neurotrophic or health-sustaining pathway, the BDNF-TrkB pathway, that becomes deficient in HD and contributes to the degeneration of vital parts of the brain, notably the striatum. To date, the use of the BDNF protein itself has proven to be highly problematic in clinical trials, so activation of its receptor, TrkB, by alternative means provides a potential end run by which drug candidates with much better pharmaceutical properties could be used to supply the critical neurotrophic support to the striatum that is compromised in HD.

DESCRIPTION (provided by applicant): Down syndrome (DS) results from triplication of human chromosome 21 (chr21) and is the most common genetic cause of mental retardation. It is hypothesized that DS is initiated by increased expression of genes located on the triplicated chr21, causing abnormal brain development in DS, but also resulting in further neurological complications, prominently Alzheimer's disease (AD)-like dementia, as DS patients grow into adulthood. By a number of neurological and neuropathological criteria, the DS population is at substantially elevated risk for AD-like neurodegeneration-and by at least 20-30 years earlier than the general population. In conducting a hypothesis-neutral screen of genes located on the DS trisomy for enhancement of amyloid precursor protein (APP)-induced neurodegeneration, we found that one of these genes, the - secretase enzyme BACE2, appears to enhance the rate/extent of neuronal degeneration in a brain slice-based model of AD. Based on previous reports in the literature and our preliminary studies, we hypothesize that BACE2 cleavage of APP generates a truncated version of ¿-amyloid (A¿) that may not be amyloidogenic but nevertheless drives significant neurodegenerative processes even in the absence of full-length A¿. In order to rigorously test this hypothesis, it will be necessary to directly identify and characterize this presumptive A¿ fragment generated by BACE2. However, currently available immunoreagents are not suitable for this purpose. Thus, the goal of this research proposal is to develop and test novel antibodies directed at the presumptive BACE2 A¿ fragment; to use these reagents to demonstrate that a truncated form of A¿ is produced by BACE2 cleavage of APP; and finally to isolate and directly sequence the BACE2-truncated A¿ fragment. The specific aims are thus to: Specific Aim 1: Develop an antibody to the C-terminal half of the canonical A¿ sequence. Specific Aim 2: Directly MS sequence the A¿ fragment produced by BACE2 cleavage. If successful, this research strategy will provide further evidence and the tools necessary for testing the hypothesis that BACE2 could be a favorable potential drug target for slowing/preventing the progression of AD-like neurodegeneration in DS. Moreover, given that BACE2 is also expressed in the brains of the general population, albeit at lower levels, such BACE2-targeted drugs may also be important to investigate in the context of sporadic and familial AD, as the highly-selective BACE1 inhibitor drugs being developed today could unmask neurodegenerative processes mediated by BACE2 that may be latent in the general population as well. PUBLIC HEALTH RELEVANCE: Down syndrome (DS) results from the triplication of human chromosome 21 (chr21) and, with an incidence of 1 in ~750 births, is the most common genetic cause of mental retardation. Tragically, as DS patients grow into adulthood, they suffer from further neurological complications, most notably Alzheimer's disease (AD)-like dementia - at present, there are no drugs available to patients that can slow or halt the progression of early-onset AD in DS. There is thus an urgent need to understand the underlying molecular genetic mechanisms that lead to AD in DS in order to support the rational design of new therapeutics to combat AD in DS. In this context, the present proposal seeks to develop key immunoreagents to enable testing the hypothesis that one of the DS trisomy genes, BACE2, may be an important drug target candidate for the treatment of AD in DS, and potentially for treating AD in the general population as well.

DESCRIPTION (provided by applicant): For new drug and drug target discovery in neurological and neuropsychiatric disorders, transitioning from efficacy in cell-based assays to benefit in whole-animal models has always been difficult and uncertain. Ideally, drug discovery studies would be conducted, as much as possible, in whole-animal models of disease, but in vivo animal experiments are tremendously costly and time- consuming. Conversely, while cell line and primary culture-based assays are rapid and inexpensive, they are compromised by phenotypic changes when neurons are cultured and/or immortalized, and, importantly, by the normal 3-dimensional milieu and local inter-cellular interactions of brain tissue architecture being unavoidably lost. To help bridge this gap between cell-based and whole-animal efficacy studies, we have developed a series of intact brain tissue-based models for CNS disorders including stroke, Huntington's disease (HD), and Alzheimer's disease (AD). In our published studies, we have shown the utility of such assays in advancing a range of gene target identification and drug development programs. To support the use of these brain slice explant models in the context of larger-scale discovery efforts, we have developed numerous technological and process innovations over the last decade, including high-throughput brain slicing and biolistic gene gun devices for transfection of brain slices with disease-relevant genes and assay reporter constructs. The overarching goal of the present proposal is to solve the final rate-limiting barrier to full scalability of this approach, namely, the automation of brain slice-based assay endpoints. To date, all of the brain slice disease models we have developed have been analyzed using laborious manual endpoint assays; nevertheless, assay throughput has been sufficient to support the screening of hundreds to thousands of compounds or gene targets per year even with a modest-sized scientific team. Implementation of turnkey unbiased, automated microscopy and high-content analysis (HCA) platforms would increase the throughput of these assays by 10-fold or more, and enable full support of large-scale systems biology, bioinformatics, and drug discovery programs. Such a bridging stage of high-throughput biology screening between cell-based and whole-animal models should significantly increase the likelihood of success of drug and drug target discovery and development programs, by providing a preview of both efficacy as well as potential adverse off-target effects in intact neurl tissue assays before substantial time and financial commitments are made to full in vivo efficacy studies.

Aspects of microglial activation in neuroinflammation associated with CNS neurodegeneration have alternately been reported to be further damaging or to be protective against disease progression. Thus, the translational potential of targeting microglia in new drug development for CNS neurodegenerative diseases remains uncertain. A major challenge in this context has been that relevant microglial phenotypes and activation states have been exceedingly difficult to recapitulate in cell line models or even in primary cultures of microglia. Conversely, microglial studies in vivo are time- and cost-intensive, and consequently have limited scalability. To address this need, we propose to develop and provide validation for a novel, brain tissue- based drug discovery model for the identification and mechanistic evaluation of new drug and drug target candidates for modulating microglial activation in CNS neurodegenerative disorders. Brain tissue models capture important aspects of intercellular interactions within the intact, local 3-dimensional structure of native neural tissues, and thereby have increased physiological relevance and can be more predictive of clinical benefit compared to cell-based models. Moreover, we have shown previously that brain slice assays can be scaled to useful throughputs for drug discovery in Huntington's disease (HD), Alzheimer's disease (AD), and stroke. The goal of the present proposal is thus to establish the experimental framework for a brain slice-based screening and mechanistic assay for microglial-neuronal interactions, and to provide initial validation that perturbation of microglial activation and/or numbers leads to clear and reproducible effects on rates and/or extents of neurodegeneration. In addition, we will extend the assay to interrogate potential effects of peripheral monocytes, whose infiltration is associated with later stages of CNS disease. We will initially focus on an HD brain slice model that we have used extensively in both screening as well as mechanistic studies, and then ask if our findings are generalizable to different models of CNS neurodegeneration driven by amyloid precursor protein and tau isoforms relevant to AD and frontotemporal dementias (FTD), respectively. If successful, the proposed studies should provide a new 3-D brain tissue-based model for capturing clinically relevant microglial-neuronal interactions scalable to screening throughputs for the discovery of new candidate drugs and drug targets for CNS neurodegeneration, and for their mechanistic evaluation and validation.

Occlusion of the blood vessels supplying the brain leads to ischemic stroke and infarction?irreversible death of brain tissue. Risk factors causing stroke, especially those involving lipid metabolism, form the basis of current therapies to reduce stroke risk. However, despite decades of research on the molecular events occurring during infarction, the translation of these discoveries to ?druggable? targets to treat stroke outcome (death of brain tissue) has been quite disappointing. Novel approaches will be required to identify new and more physiologically relevant targets. The scientific premise of our proposal is that naturally occurring allelic variation underlies the profound differences in seen in stroke outcomes and that these neuro-protective gene variants would provide a novel path towards new targets for stroke treatment. However, genetic mapping approaches for infarct size in the human (e.g., GWAS of infarct volume among ischemic stroke patients) are intrinsically problematic due to wide variation in the extent and location of the occluded vessel, and especially, variation in the time window between first recognized symptoms and medical intervention. To date, we can find no published GWAS for infarct volume in ischemic stroke. The Marchuk lab has taken an alternative, forward genetic approach to discover novel genes modulating infarction. We have surgically occluded the distal middle cerebral artery in over 35 inbred mouse strains and found that infarct volume differs more than 50-fold. These robust and highly reproducible differences in infarct size are at least 10-fold larger than that seen in any engineered mouse lines but, importantly, are caused by natural allelic variation in the mouse genome. We have mapped several of these genetic loci and the goals of Aims 1 and 2 are to identify these novel genes regulating in infarct size. However, this gene discovery approach has required in vivo surgical assays in thousands of adult animals. We need a more scalable yet physiologically relevant screening platform to transform this approach to full genome-wide scale. The Lo lab has pioneered the development of such a discovery platform for cerebral infarction, simulating stroke by Oxygen/Glucose Deprivation (OGD; a well-characterized model for ischemic injury) in ex vivo brain tissue explants. Unlike isolated neurons in culture, brain slice explants retain the complex multicellular nature of the intact organ, and thus retain and represent the complex intercellular interactions occurring in brain tissue during cerebral infarction. In Aims 1 and 2, this ex vivo OGD platform will be used to identify the causative genes in our previously mapped loci. Our experience gained in these aims will lead to Aim 3, where the ex vivo OGD assay will be used to directly map and identify novel cerebral infarction genes, using the genetic mapping resource population of the Collaborative Cross. Our study takes advantage of innovative approaches developed by the co-PIs to implement a novel strategy for identifying novel drug targets to treat ischemic stroke.