Scott R. Whittemore - US grants

The goal of this COBRE is to develop a multi-disciplinary, highly interactive and collaborative Research Center focused on developing new strategies to facilitate CNS repair and regeneration. This COBRE brings together 5 presently non-funded Principal Investigators, 4 on the UofL faculty and one to be recruited, whose research focuses on various aspects of CNS injury and repair, from 4 Departments: Neurological Surgery, Anatomical Sciences & Neurobiology, Pediatrics, and Pharmacology & Toxicology. This COBRE represents a true Research Center, not a collection of individual projects, as the expertise of these Investigators is broad based and the success of individual projects will depend on strong collaborations with other Projects and Cores. The Specific Aims of this COBRE are: 1. To develop successful independent research projects for each of the 5 Principal Investigators in the COBRE that will ultimately lead to extramurally funded R01 grants. 2. To establish the University of Louisville Center for CNS Injury and Repair, with multiple Core facilities to support all Investigators. The Center and its Core facilities would not be limited to the Investigators on this COBRE, but would be open to all UofL faculty with similar research interests. 3. Using the "Faculty Expansion" provisions of this COBRE, we will recruit a strong junior investigator in the areas of neuroimmunology, neuroprotection, and/or apoptosis. This will be Project 5. 4. To facilitate collaborative research projects between the COBRE Principal Investigators as well as other UofL faculty that will lead to further extramural support. 5. To become a leading Center, recognized both nationally and internationally, in the field of CNS Injury and Repair, and develop a reputation that will attract graduate students, post-doctoral fellows, and additional faculty. 6. To develop new therapeutic approaches to the treatment of CNS injury that will ultimately be utilized clinically. The projects in this COBRE are: Project 1: Signaling pathways in neuronal susceptibility to hypoxia, Evelyne Gozal, Ph.D., PI, Project 2: Reconstructing locomoter circuitry after spinal cord injury, David S.K., Magnuson, Ph.D., Project 3: Adult human olfactory epithelium as a source of multi-potential stem cells for CNS repair, Fred J. Roisen, Ph.D., PI Project 4: Functional regeneration of sensory pathways after spinal cord injury, Stephen M. Onifer, Ph.D., PI.

The goal of this revised PSO grant is to expand the present COBRE-funded Kentucky Spinal Cord Injury Research Center (KSCIRC) Core facilities at the University of Louisville (UofL). We propose to broaden the scope of research that these Cores support in neural development and CNS injury, and 2) bring additional PIs into the Center both to enhance their research programs and to bring their new techniques and approaches to the COBRE PIs. We will increase the number of involved PIs from 8 to 22. The Cores will facilitate the research programs of both NIH funded and non-funded junior and more senior PIs in directions that would not be possible without the highly trained Core personnel. Each Core will be directed by a funded PI and housed in independent, dedicated laboratory space. All PIs will have access to these Cores. We will give priority access to junior and unfunded PIs. The UofL administration will make significant space and financial commitment to the COBRE/KSCIRC Core Facilities. The Cores are: A) Administration and Biostatistics, B) Surgery and Animal Care, C) Behavioral and Electrophysiological Assessment, D) Cell and Tissue Imaging and Histology, and E) Human Translational Studies. The specific aims are: 1. Support and enhance the scope of the strong ongoing UofL NINDS and non-NINDS funded research in the areas of nervous system injury and repair, including our continuing development of innovative technology. 2. Make our technologies available to additional UofL neuroscientists working in other areas of nervous system development and repair to facilitate their individual research programs with novel approaches. 3. Facilitate the development of the research programs of junior and more senior unfunded Investigators. 4. Enhance collaborative opportunities between PIs that utilize these Core facilities.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Core A will be responsible for the overall administration of this COBRE. Core A will coordinate all meetings between Principal Investigators and the Internal (IAC) and External (EAC) Advisory Committees as well as weekly seminar series and Visiting Lecturer Series. Drs. Whittemore, Hagg, and Xu will serve as mentors for each of the Projects and each individual having primary or secondary oversight for 2-4 Projects. CORE A will provide fiscal oversight of all Projects and CORES and also provide expertise in statistical analysis and experimental design for all Projects.

DESCRIPTION (provided by applicant): Our recent work shows that glial restricted precursor cells (GRPs), grafted into a demyelinating or contusive spinal cord injury (SCI), partially restore electrophysiological conduction and hindlimb locomotor recovery. We will build on those data and determine the potential of human embryonic stem cells (hESC) to restore function after engraftment into the damaged spinal cord. Aim 1. Using the focal VLF? demyelinating lesion and grafting D15A-GRPs (A2B5???), we will determine whether inhibition of BMP and Notch signaling pathways, demonstrated in vitro to inhibit oligodendrocyte differentiation, can enhance the remyelination capacity of the engrafted cells. We will examine a number of genetic and molecular biological approaches to inhibiting BMP and Notch signaling. Aim 2. Determine whether delivery of CNTF or NRG1 types I or III, neurotrophic factors known to potentiate oligodendrocyte proliferation and maturation in vitro and in vivo, will enhance remyelination by engrafted GRPs. Aim 3. Using optimal parameters established in Aims 1 and 2 and the more clinically relevant contusion SCI, we will ask whether remyelination can result in enhanced recovery of function. We hypothesize that both remyelination and enhanced white matter sparing are needed for optimal recovery and will distinguish between those effects in these animals.

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.

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.

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.