2004 — 2005 |
Hetman, Michal |
P20Activity Code Description: To support planning for new programs, expansion or modification of existing resources, and feasibility studies to explore various approaches to the development of interdisciplinary programs that offer potential solutions to problems of special significance to the mission of the NIH. These exploratory studies may lead to specialized or comprehensive centers. |
Signaling Pathways in Neuronal Apoptosis @ University of Louisville
mitogen activated protein kinase; serine threonine protein kinase
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2004 — 2008 |
Hetman, Michal |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Survival Signaling in Cns Neurons Exposed to Dna Damage @ University of Louisville
DESCRIPTION (provided by applicant): Neurons are targets for damaging stimuli, which may trigger cell death and in consequence serious neurological diseases. Interestingly, most neurons survive minor damages which they are challenged with during the life span. Therefore one can propose existence of a mechanism that helps neurons to survive the initial injury and resume proper functions after an insult. Identification of this mechanism may result in development of treatments that would harness the intrinsic neuronal defense machinery to combat against neurological diseases. An important damaging stimulus in the CNS is oxidative stress. It exerts its toxic effects at least in part through injury of DNA whose integrity is critical for proper neuronal survival. Indeed, neurons are very sensitive to DNA damage. Interestingly, DNA damaging agents that are used in cancer therapy frequently produce neurological side effects. Our recently published results indicate that Extracellular Signal Regulated Kinase 1/2 (ERK1/2) is activated in rat cortical neurons by a neurotoxic DNA-damaging drug, cisplatin (CPDD) (J Biol Chem 278:43663-43671). Furthermore, we have found that inhibition of ERK1/2 pathway enhances toxic response to CPDD. Consequently, the general aim of our proposal is testing a hypothesis that neuronal DNA damage activates ERK1/2 to suppress neuronal death and to enhance repair of the cellular damage. We will approach this goal by (i) identification of the mechanism behind ERK1/2 mediated protection against DNA damage, (ii) dissection of the pathway linking DNA damage and survival signaling by ERK1/2, (iii) identification of factors which could enhance defensive signaling by ERK1/2 in neurons and finally by (iv) evaluation of ERK1/2 activation as a general compensatory response to diverse forms of DNA injury. To realize the proposal we will use cultured rat primary neurons. The proposed research may result in identification of new drug targets for treatment of neurological diseases and/or deleterious neurological side effects of cancer therapy.
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2006 — 2009 |
Hetman, Michal |
P20Activity Code Description: To support planning for new programs, expansion or modification of existing resources, and feasibility studies to explore various approaches to the development of interdisciplinary programs that offer potential solutions to problems of special significance to the mission of the NIH. These exploratory studies may lead to specialized or comprehensive centers. |
Gsk3b as a Target For Pro-Neuronal Survival in Cns Neurons @ University of Louisville
Apoplexy; Bruise; Budgets; CRISP; Cell Death; Cerebral Stroke; Cerebrovascular Apoplexy; Cerebrovascular Stroke; Cerebrovascular accident; Cessation of life; Chronic; Computer Retrieval of Information on Scientific Projects Database; Condition; Contusions; Death; Demyelinations; Dysfunction; Endoplasmic Reticulum; Ergastoplasm; Functional disorder; Funding; Grant; Institution; Intervention; Intervention Strategies; Investigators; Lead; Mice, Mutant Strains; Mutant Strains Mice; Myelopathy, Traumatic; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nerve Cells; Nerve Degeneration; Nerve Unit; Nervous System Diseases; Neural Cell; Neurocyte; Neurologic Disorders; Neurological Disorders; Neuron Degeneration; Neurons; Oligodendrocytes; Oligodendrocytus; Oligodendroglia; Oligodendroglia Cell; Outcome; Pathology; Pathway interactions; Pb element; Physiopathology; Proteins; Research; Research Personnel; Research Resources; Researchers; Resources; Source; Spinal Cord Trauma; Spinal Trauma; Spinal cord injured; Spinal cord injuries; Spinal cord injury; Stress; Stroke; Testing; United States National Institutes of Health; Vascular Accident, Brain; aberrant protein folding; abnormal protein folding; biological adaptation to stress; brain attack; cerebral vascular accident; gene product; heavy metal Pb; heavy metal lead; interventional strategy; mouse mutant; necrocytosis; nervous system disorder; neural degeneration; neurodegeneration; neurological disease; neuronal; neuronal degeneration; neuronal survival; oligodendrocyte precursor; pathologic protein folding; pathophysiology; pathway; precursor cell; protein mis-folding; protein misfolding; reaction; crisis; response; stress response; stress; reaction; stroke; tool
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2010 — 2013 |
Hetman, Michal |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Regulation of Neuronal Morphogenesis by the Nucleolar Transcription @ University of Louisville Research Foundation Inc
In developing brain, growing the extensions of the nerve cells (neurons) is critical to establish interneuronal connections underlying brain functions. Dendrites are the branched type of such extensions and form the input connection of a neuron. Dendritic growth is stimulated by extracellular cues including a protein, Brain Derived Neurotrophic Factor, and electrical activity of the developing neurons. In the receptive neurons, such stimulating signals activate a cascade of intracellular events that culminates in the growth response. The general goal of this proposal is to identify critical elements of the molecular network underlying dendritic growth. The nucleolus is a subdomain of the cell nucleus, and genes within the nucleolus encode ribosomal RNA (rRNA), a critical component of the protein synthesis machines known as ribosomes. The hypothesis of this proposal is to test whether expression of nucleolar genes regulates dendritic growth. Using whole rat pups and cultured rat forebrain neurons, this hypothesis will be addressed by (i) defining the intracellular molecular signaling events critical for regulation of nucleolar gene expression by signals that increase dendritic growth, (ii) determining whether nucleolar gene expression is required and sufficient to mediate dendritic growth in response to such signals, and, finally, (iii) testing the importance of making new ribosomes for the dendritic growth. This project may identify a previously unrecognized role of the nucleolus in the development of the nervous system. The project will also actively engage undergraduate students some of whom will be recruited from a geographical region in which college-educated individuals are underrepresented.
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2011 — 2015 |
Hetman, Michal Whittemore, Scott R [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Er Stress and Oligodendrocyte Survival After Spinal Cord Injury @ University of Louisville
DESCRIPTION (provided by applicant): The endoplasmic reticulum (ER) is the intracellular organelle in which secretory and membrane proteins are synthesized and folded by resident chaperone proteins. The ER stress response (ERSR) is an evolutionarily conserved cell defense mechanism that protects against excessive accumulation of malfolded proteins in the ER. These malfolded proteins are translocated to the cytoplasm by the machinery of the ER- associated degredation (ERAD) where they are degraded. The ERSR is initiated after multiple cellular stresses including hypoxia, inflammation, trauma, excitotoxicity, and oxidative damage. The ERSR is initially protective, but if malfolded proteins cannot be cleared, apoptotic cell death initiates. The 3 pathways involved in the ERSR involve PERK, IRE1/XBP-1, and ATF6 signaling. Preliminary data demonstrate upregulation of all 3 ERSR pathways following SCI. Mice null for CHOP, a pro-apoptotic transcription factor that is downstream of PERK and activated during ERSR, showed enhanced functional recovery after SCI and we identified oligodendrocytes as highly vulnerable to ER stress. We hypothesize that enhancing the protective or inhibiting the apoptotic aspects of the ERSR will enhance functional recovery after SCI. In Aim 1, we will potentiate the protective effectors of ERSR and in Aim 2 suppress those that initiate oligodendrocyte apoptosis. We will use a combination of pharmacological agents, constitutive and conditional null mice, as well as cell culture studies using wild type (WT) and available null oligodendrocyte precursor cells (OPCs) and/or siRNAs to address these questions.
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2017 — 2018 |
Hetman, Michal Whittemore, Scott R (co-PI) [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Glial-Specific Gene Expression After Contusive Spinal Cord Injury @ University of Louisville
No clinically applicable treatments exist to improve functional outcome after spinal cord injury (SCI). In part, such a lack of progress is due to poor understanding of the complex pathology of this injury. The SCI response is cell type-specific and evolves with time post-injury. Acutely after injury, oligodendrocytes (OL) display disruption of proteostasis including endoplasmic reticulum stress (ERSR) and integrated stress (ISR) responses. The ISR leads to apoptosis and white matter loss that underlies many deficits of sensation and locomotion. Later, oligodendrocyte precursor cells (OPCs) proliferate in an attempt to repair lost myelin. However, that response is limited by inhibitory signals that block OPC differentiation. While global spinal cord transcriptomics have enabled a systematic knowledge about the SCI response, conclusions from these efforts are limited by technological barriers and the complexity of spinal cord anatomy. Thus, SCI-associated changes in mRNA levels may not translate into parallel effects on protein expression. Moreover, homogenized fragments of whole spinal cord used for traditional transcriptomic experiments make identification of novel components of the cell-specific injury response challenging. These limitations may be overcome by Translating Ribosome Affinity Purification (TRAP) technology which isolates and analyzes only mRNAs that are associated with ribosomes (i.e. being likely translated) from specific cell populations that were marked genetically with a ribosomal tag. Thus, we propose to apply TRAP to test the hypothesis that after SCI, both transcriptional and translational reprogramming regulate the expression of critical components of the injury response in a cell type- specific manner. Furthermore, translational regulation will be of particular significance to launch proteostasis responses in OPCs and OLs. We also propose that in the context of OPC/OLs that respond to SCI, identifying translated mRNAs for transcription factors will uncover new targets that can be targeted for OL protection and/or remyelination. Focusing on the injury epicenter, we will characterize translated transcriptome profiles of OPC/OLs at 2, 10 and 42 days after moderate mouse contusive SCI inOPC/OLs from transgenic mice that express EGFP-L10 ribosome tag in a Cre dependent manner selectively in these cells (specific aim 1). In addition, we will use the resulting data sets to identify transcription factors (TF) that orchestrate OPC/OL responses to SCI (specific aim 2). We expect to characterize SCI-associated gene expression events in OPC/OLs with unprecedented accuracy. By focusing on ribosome-associated transcripts, our data will paint a landscape of complete gene expression events that reach the protein level. Such a landscape is unavailable for SCI-challenged OPC/OLs on a whole genome scale. Therefore, this high risk/high reward proposal may redefine the SCI response of OPC/OLs and identify novel targets for white matter protection and/or remyelination therapies.
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2018 — 2020 |
Hetman, Michal Whittemore, Scott R [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Integrated Stress Response and Oligodendrocyte Survival After Spinal Cord Injury @ University of Louisville
Abstract. We identified the ER stress response (ERSR) as a potential target for therapeutic interventions against white matter loss and locomotor impairment after spinal cord injury (SCI). Specifically, the signaling pathway that involves the ERSR kinase PERK, the PERK target elongation initiation factor 2? (eIF2?), and PERK-activated transcription factor CHOP may be manipulated in a time-dependent manner to promote SCI recovery. PERK signaling has a partial overlap with the integrated stress response (ISR) that, via several stress-activated kinases, leads to increased levels of phospho-eIF2? (peIF2?), transient inhibition of protein synthesis, and activation of the transcription factor ATF4. ATF4 regulates CHOP as well as genes involved in ROS metabolism, translational regulation and amino acid synthesis. In this way, the ISR attempts to restore homeostasis. Excessive and prolonged activation of the ISR results in anabolism-associated oxidative stress, mitochondrial damage, cell death and inflammation. Pro-ISR stimuli such as hypoxia, lack of nutrients, oxidative stress, and ER stress are present after SCI. However, the role of the principal ISR components ? eIF2? kinases other than PERK and their common downstream target ATF4 ? has not been addressed in the context of white matter loss after contusive SCI. Our overarching hypothesis is that the ISR plays a critical role in pathogenesis of contusive SCI by promoting OL/OPC death and white matter loss. Aim 1 will examine the role of the 4 upstream ISR kinases (PERK, PKR, GCN2, HRI, which are activated by different stressors) that phosphorylate eIF2?, which in turn inhibits global translation and enhances stress-induced gene expression. We will use a combination of previously optimized gain and loss of function in vitro OPC/OL and in vivo SCI assays that utilize pharmacological inhibitors as well as Hri-/-, Pkr-/-, and Gcn2-/-mice. Preliminary data from ER stressed OPCs or SCI tissue show activation of these kinases as well as compensatory activation of the ISR pathway when PERK is inhibited. Aim 2 will examine the role of the central ISR effector ATF4 in SCI- associated white matter loss. Our preliminary data show activation of ATF4 after SCI or in ER-stressed OPCs. We will determine whether ATF4 is a mediator of OL/OPC death, white matter damage, and functional decline after contusive SCI. In summary, current experimental design is based on data from our previous characterization of the ERSR after SCI. Here, we propose to delineate novel mechanisms of ISR-mediated cell death after SCI as well as define which of ISR mediators may best be suited for therapeutic translation to acutely treat SCI patients. Such treatments are likely to be applicable to other types of CNS trauma such as TBI and stroke.
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2020 — 2021 |
Hetman, Michal Saraswat, Sujata Ohri Whittemore, Scott R (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Bmal1/Arntl Plays a Critical, Non-Circadian Role in Secondary Tissue Damage After Contusive Sci @ University of Louisville
Circadian rhythms regulate a wide spectrum of biological processes of critical importance for organismal health. Perturbations of those rhythms underlie many pathologies including systemic inflammation, depression, and neurodegeneration. Mechanistically, circadian rhythms are driven by intrinsic oscillatory changes in gene expression that are orchestrated by several regulators of gene transcription and mRNA translation forming the core oscillator circuitry. Those key regulators, most importantly the non-redundant transcription/translation factor BMAL1/ARNTL, are active in most cells throughout the body and undergo circadian entrainment by external time cues such as light or feeding. At the organismal level, the pro-rhythmic role of the oscillator is widely recognized as a critical contributor to homeostasis. BMAL1 effects that are independent of the central rhythm include anti-oxidant protection in the brain, life span regulation, contributions to atherosclerosis and endoplasmic reticulum (ER) stress-sensitization of cancer cells. Those, or similar, tissue-specific functions of BMAL1 may affect the outcome after SCI. However, clock function at the molecular level has never been investigated in the context of SCI. Unexpectedly, we found that: (i) moderate contusive thoracic SCI upregulates BMAL1 in penumbral oligodendrocytes (OLs), coinciding with induction of the ER stress response (ERSR)-activated pro-apoptotic transcription factor CHOP and ER stress-mediated apoptosis of OLs, (ii) after SCI, Bmal1-/- mice show improved locomotor recovery and white matter sparing (WMS), as well as selective downregulation of Chop and its pro-apoptotic target gene death receptor 5 (Dr5) and reduced blood extravasation and inflammation, with extensive changes in microglia/macrophage (MM) and endothelial (EC)- specific gene expression. Also, pharmacological enhancement of the negative feedback inhibition of BMAL1 reduces ER stress toxicity in OPC cultures. These exciting findings suggest a novel role of BMAL1 in the pathogenesis of SCI which may include OL-cell autonomous regulation of CHOP-mediated OL apoptosis and/or EC/MM-cell autonomous modulation of post-SCI hemorrhage/vascular dysfunction/cytotoxic neuro- inflammation. Therefore, we will test the hypothesis that BMAL1 regulates OL, EC, and/or MM gene expression that contributes to SCI-associated white matter loss and impaired locomotor recovery. To test this hypothesis, we will: (i) determine the cell autonomous roles of OL-, MM-, and EC-BMAL1 in SCI-associated white matter damage and locomotor recovery, (ii) identify mechanism(s) that underlie BMAL1-mediated enhancement of SCI-driven white matter loss, and (iii) evaluate mediators of the negative feedback inhibition of BMAL1 as pharmacological targets for interventions to reduce SCI-associated white matter loss and locomotor impairment. We will use a moderate T9 SCI contusion model in wild type or cell type-selective Bmal1-/- mice and non-toxic, CNS-permeable drugs targeting the feedback regulation of BMAL1. This research may uncover novel, previously unrecognized contributions of BMAL1 to `secondary tissue injury after SCI.
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