Marc W. Halterman, Ph.D. - US grants

Ischemic damage to the brain results in substantial morbidity during the perinatal period as well as mortality in the later decades of life. Studies in the intact animal as well as in vitro have established that the extreme physiologic perturbations which occur during CNS ischemia trigger a delayed form of neuronal death which is dependent on new gene transcription and that hypoxia triggered gene responses precede activation of delayed apoptotic cell death. One approach to the identification of novel therapeutic strategies which would protect against this form of neuronal cell death is through examination of the mechanisms which direct hypoxia responsive gene expression in the CNS. The phylogenetically conserved hypoxia response is manifest in the mammalian systems through transcriptional activation and post- transcriptional mRNA stabilization. In the periphery, transcriptional events are known to be mediated through the hypoxia inducible transcription factor, HIF-1alpha, which binds cognate cis elements in the promoter region of a restricted set of genes thereby simulating the rate of gene transcription. Information regarding the utilization of this hypoxia responsive mechanism within the various cellular compartments of the CNS (neuronal, astrocytic and microglial) and their relationship to apoptotic neuronal loss is lacking, however. Broadly, we hypothesize that early in the post-ischemic CNS hypoxic-regulated gene expression exhibits heterogeneity within the cellular compartments in the CNS and that this response triggers a sequence which either directly or indirectly elicits delayed neuronal death. We plan to exploit hypoxia responsive HSV viral vectors to map the regional and temporal evolution of hypoxic signaling within the compartments of the ischemic murine CNS. Subsequent studies will utilize HIF neuronal isoform specific antibodies to characterize HIF isoform induction, cellular localization and confirm DNA binding reactivity through EMSA supershift assays all under hypoxic conditions. We hypothesize that these experiments will characterize heterogeneous hypoxic response and will define discrete factors in ischemia induced CNS transcriptional activation. Our long-term goals are to identify early responses in the ischemic brain and subsequently identify regulatory nodes in the hypoxic signal cascade which can selectively modulate hypoxia gene activation. Such findings will highlight novel therapeutic strategies directed against hypoxia induced delayed neuronal death.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The overarching goal of this proposal is to develop therapies that protect against delayed neuron loss following stroke. Dr. Halterman is a neurologist and post-doctoral fellow interested in genetic mechanisms governing neuronal survival in ischemic and neurodegenerative conditions. The candidate's long-term objective is to establish an independent, academic research program that implements methodologies to bridge the gap between preclinical disease modeling and the neurotherapeutic discovery process. During the initial 2-year period, Dr. Halterman will receive mentoring in cell death signaling mechanisms, high-content screening and small molecule discovery, and processes to promote the development of investigator- supported basic research into clinical trials. The proposed career development plan will enable the candidate to define hypoxia-induced transcription-dependent death pathways in neurons using novel approaches that combine small molecule inhibitors or genetically modified cell lines with high-content image analysis. Moreover, the candidate will adopt approaches in systems-based bioinformatics, to construct, test and refine hypotheses regarding disease-relevant signaling networks using an in vitro stroke model. These studies will be carried out primarily at the University of Rochester, with additional resources made available by the Drug Discovery Laboratory at the University of Pittsburgh. Didactic experiences supported by the Rochester Clinical Translational Science Institute and several technique-oriented short courses enrich the training plan. During the subsequent 3-year independent phase, the candidate will: 1) investigate cell-type differences in the network of bZIP transcriptional responses triggered by hypoxia, 2) define the cell signaling and protein- protein interactions that govern CHOP-10's apoptotic potential, and 3) define the mechanism by which the CHOP-10 heterodimeric factor c/EBP-beta protects neurons after hypoxic stress. These studies will test the central hypothesis that CHOP-10 and related heterodimeric bZIP partners are critical determinants of neuron survival following stroke. [unreadable] RELEVANCE TO PUBLIC HEALTH: Therapeutic options for the treatment of stroke are limited, and strategies that support the survival of endangered neurons in the sub-acute period are needed. This project aims to identify genetic signaling pathways activated after stroke, and to develop systems that will highlight new treatments for this devastating condition. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): De novo gene expression induced by the hypoxia inducible factor (HIF-1a) plays a decisive role in determining whether neurons live or die after an ischemic insult. However, the molecular mechanisms regulating the balance between HIF's adaptive and pathological effects remain unsettled. We have discovered that the MAP kinase phosphatase MKP-1 stimulates HIF-1a cleavage near the amino-terminal transactivation domain and triggers both BNIP3 expression and a host of related pro-apoptotic responses. In this application we test the hypothesis that together, MKP-1 and HIF-1a function as a molecular switch during ischemia, promoting the expression of genes involved in autophagy and apoptotic signaling. We will use complimentary genetic approaches applied in culture and animal models of ischemic injury to investigate: 1) the mechanism by which MKP regulates HIF-1a post-translational modification, 2) the discrete modifications and factors required for HIF-1a cleavage, and 3) the effects these changes have on neuron survival. Together, these experiments focus on a novel, physiologically responsive signaling node that modulates HIF-1a's latent apoptotic potential. The identification of suitable targets in this network will enable the discovery of small molecules designed to either inhibit or augment transcription- dependent injury. Progress in this area will have broad implications for both ischemic and malignant brain disorders.

? DESCRIPTION (provided by applicant): Cardiac arrest affects an estimated 500,000 Americans annually, and fewer than 25% patients will survive to discharge due in large part to overwhelming CNS injury. The post-resuscitation syndrome following cardiac arrest (PCAS) encompasses the damaging effects of tissue ischemia and the delayed effects of ischemic reperfusion injury (IRI). Activation of the innate immune response is a prime mediator of the delayed microvascular damage, increased vascular permeability and progressive tissue damage observed after resuscitation. In particular, blocking leukocyte recruitment to the CNS significantly reduces the extent of brain injury observed after ischemic challenge. In this proposal we investigate the pathological consequences of peripheral inflammation on global cerebral ischemia, focusing on the relationship between lung inflammation, neutrophil priming and delayed CNS injury. These studies are based on our observation that expression of extracellular superoxide dismutase (EC-SOD) targeted to type II pneumocytes blunts neutrophil trafficking into the post-ischemic brain and confers marked neuroprotection. In this proposal we test the hypothesis that expression of EC-SOD within the lung inhibits IRI by reducing the production of damage associated molecular patterns (DAMPs) and levels of TLR4 activation required for endothelial and PMN activation. Successful demonstration that lung-brain coupling modulates delayed cerebral injury through effects on neutrophil priming may suggest new treatment strategies for patients presenting after cardiac arrest.

Vascular-immune interactions play a pivotal role in the initiation and propagation of ischemic stroke pathology at the onset of ischemia and after reperfusion. In addition to contributing to underlying stroke risk, systemic inflammation amplifies pathological effects of ischemia-reperfusion injury (IRI). And while antiplatelet agents and statins have modest effects on reducing post-stroke inflammation, targeted therapies are desperately needed. We have discovered a novel mechanism of lung-brain coupling induced following an acute ischemic stroke that regulates systemic inflammation, innate immune priming, neurovascular compromise, and secondary ischemic brain damage. Our long-term goal is to identify the mechanistic basis for this response and test whether approaches targeting post-stroke lung pathology could improve outcomes in patients presenting after acute ischemic stroke (AIS). We find that acute cerebral ischemia induces a range of lung pathologies, including 1) simplification of alveolar structures and airway inflammation, 2) increased endothelial permeability and lipid peroxidation, 3) changes in respiratory mechanics, and 4) selective loss of the endogenous lung antioxidant extracellular superoxide dismutase (SOD3). Notably, targeted expression of SOD3 within the distal airways abrogates stroke-induced lung pathology, inhibits systemic inflammation, and reduce cumulative stroke burden. Collectively these data lead us to hypothesize that stroke-induced changes in pulmonary SOD3 activity are a critical determinant of stroke outcomes via effects on systemic immune priming and cerebrovascular resilience. In this proposal, we investigate the mechanism(s) involved in the stroke-dependent loss of SOD3 expression (SA1), demonstrate the effects of SOD3 exhaustion on systemic immune priming (SA2), and explore the influence of stroke risk modifiers on SOD3 regulation and stroke outcomes. These studies provide a new perspective on potential approaches to reduce brain injury, hasten recovery, and mitigate complications associated with AIS. In addition to expanding our understanding regarding the fundamental underpinnings of lung-brain coupling, this work could ultimately lead to the development of inhaled, immune-based therapies for stroke and other acute neurological conditions in which systemic inflammation is a central component.