Theo Hagg - US grants

DESCRIPTION (provided by applicant) Neurotrophic factors (neurotrophins, GDNF and CNTF) can rescue injured dopaminergic neurons of the substantia nigra in adult rodent models of Parkinson's disease by activating transmembrane tyrosine kinase receptors or associated tyrosine kinases. Signaling is terminated by protein tyrosine phosphatases (PTPs), which have a high degree of specificity and are critical for cellular processes, including neural development. A major problem with large neurotrophic factors proteins is their poor ability to reach the brain regions of interest. We have shown that a small general PTP inhibitor can protect injured dopaminergic neurons in rats and dramatically increase the protective effects of a neurotrophin BDNF, suggesting that TrkB signaling was enhanced. Little is known about the identity of PTPs involved in regulating neuronal survival-related signaling. We will use a functional proteomics approach by co-immunoprecipitation techniques to isolate the specific PTPs that associate with the neurotrophic receptors after neurotrophic factor stimulation in rats, mice and cultured neuronal cells. The identification of such important PTPs will provide a basis for assessing their normal and pathophysiological roles and a platform to develop more selective inhibitors for future clinical use. A second aim is to evaluate whether the PTP inhibitor can also enhance protective effects of GDNF or CNTF in an animal model of Parkinson's disease. This could generalize the idea of PTP inhibition as a therapeutic approach, and will be useful for predicting which of the PTPs, identified in the first aim, are affected by the PTP inhibitor. These studies will increase our understanding of how dopaminergic neurons function, validate our new treatment approach and provide us with research tools to improve it for application to Parkinson's disease.

DESCRIPTION (provided by applicant): The endoplasmic reticulum (ER) stress response (ERSR) is one of the major defense mechanisms that protect against cellular insult but if unchecked leads to apoptotic cell death. The ERSR has three arms initiated by PERK, IRE1, ATF6, respectively. Preliminary data show the acute activation of all three ERSR signaling pathways in endothelial cells (ECs) after SCI. Most importantly, we show that attenuation of PERK signaling in CHOP-/- (the downstream effector of PERK) mice or after i.v. salubrinal (which sustains protein synthesis inhibition) leads to enhanced functional recovery after SCI in WT mice. We found an acute vasoconstrictive phase following SCI and can enhance EC protection by the vasodilator nimodipine plus the vasoprotector glibenclamide in WT mice. Specifically, Aim 1 will delineate the specific effectors that underlie ERSR-mediated EC death by PERK signaling. We will determine if reducing PERK or ATF4 signaling in ECs after SCI will enhance functional recovery after SCI. This will be done using available transgenic mice (Aim 1a) and siRNA methods (Aim 1b). We hypothesize that the earlier in the ERSR pathway that inhibition occurs, the more extensive the vasoprotection and recovery. Aim 2 will characterize the acute activation profile of the ERSR in FACS purified ECs when one signaling pathway is deleted (Aim 2a), their effects on spinal cord microvasculature (Aim 2b) and the functional consequences (Aim 2c). Aim 3 will test whether EC rescue by ER stress inhibitors can be improved when combined with the vasodilators, nimodipine or MgSO4, the mainstay treatments for CNS vasospasm. Aim 3a will optimize vasodilation protocols. Aim 3b will optimize treatment regimens for salubrinal and two chemical chaperones that influence ERSR signaling: TUDCA (in clinical trials for ALS) and PBA (FDA-approved). We will then test whether optimized vasodilation would further improve the efficacy of those drugs using both pharmacological and genetic approaches. Aim 3c will define determine the therapeutic window. Collectively, the experiments outlined in these 3 Aims delineate a strategy to optimally inhibit ER stress in ECs to maximize functional recovery after SCI and determine whether this approach is clinically relevant. PUBLIC HEALTH RELEVANCE: This grant examines the role of the endoplasmic reticulum stress response, a cellular defense mechanism induced in every spinal cord cell after SCI, which, if unchecked, leads to cell loss after SCI causing the loss of neurological functions. Our previous data show the crucial role of endothelial cell death of blood vessels in the spinal cord in the ensuing secondary loss of spinal cord tissue. Using both genetic approaches and current FDA approved and/or in clinical trial drugs to rescue the endothelial cells, we expect to identify new acute therapeutic targets that will hopefully extend beyond SCI to other CNS trauma and neurological disease treatment.

Project Summary. CNTF mediates the increased SVZ neurogenesis that occurs in the mouse brain after ischemic stroke. We will follow up on our finding that JNK in astrocytes represses CNTF expression and neurogenesis in naïve mice, to test whether systemic treatment with JNK inhibitor can increase neurogenesis after stroke. We will also block the highly related pro-inflammatory ligand IL-6, which we found reduces stroke- induced neurogenesis, with a gp130 inhibitor. Secondly, we have discovered that blood levels of vitronectin (VTN), produced by the liver, only increase in females after stroke, and leaks into the SVZ to induce IL-6 and repress the neurogenic response. We will identify the mechanisms that regulate VTN, focusing on vagal nerve stimulated muscarinic receptors and FAK. Thirdly, we discovered in males that castration caused an unexpected and very robust effect by increasing basal levels of pro-neurogenic CNTF and decreasing detrimental IL-6, and that this was retained after MCAO. We will define the differential signaling pathways underlying this male-specific mechanism and test whether pharmacological blocking of testosterone would increase neurogenesis in males. Aim 1 will determine whether a gp130 inhibitor promotes neurogenesis when given at 6 h or 2 months after a stroke in adult and aged mice, and whether it acts by blocking IL-6. We will also determine whether JNK inhibition would increase neurogenesis after stroke by increasing CNTF, and/or whether JNK inhibitor would further enhance the neurogenic effects of SC144 after MCAO. Aim 2 will define potentially female-specific mechanisms that regulate liver VTN, including FAK and muscarinic acetylcholine receptors, testing pharmacological FAK inhibition and the role of the nervous system innervation of the liver after MCAO. Aim 3 will define the signaling pathways that mediate testosterone?s effects on CNTF, LIF and IL- 6 expression in the SVZ after MCAO, and using intracerebral injection of testosterone after castration, with or without FAK, JNK, ERK or p38 inhibitors, and whether a testosterone blocker can promote neurogenesis after MCAO. These studies will build on our previous work to define novel fundamental intracellular signaling mechanisms that repress and enhance the key cytokines CNTF and IL-6 involved in SVZ neurogenesis following stroke.

Project Summary. We found that female, but not male, mice have increased plasma vitronectin (VTN) levels and less tissue loss upon genetic VTN deletion after a stroke by temporary middle cerebral artery occlusion (MCAO). This female-specific detrimental role of VTN may be relevant to women because that have worse functional neurological outcomes after stroke. Therefore, this proposal focuses on female mice. We also find that VTN leaks into the brain after MCAO and induces pro-inflammatory cytokine expression. Our in vitro and in vivo data suggest that VTN acts through specific av integrins and possibly through uPAR. Integrins activate intracellular signaling through focal adhesion kinase (FAK). Female mice injected systemically with an FAK inhibitor 6 h after a stroke had better much better outcomes. These data suggest that VTN-Integrin-FAK signaling contributes to tissue loss after stroke. Aim 1 will determine whether individual differences in increased VTN levels in the blood seen after MCAO predict the inflammatory response and loss of brain tissue. This might provide an additional prognostic stroke marker and open new opportunities to develop treatments. We will also determine whether VTN and FAK inhibition act through astroglial or microglial FAK, two early responder cell types with known significant contributions to inflammation. We will identify the most efficacious post-MCAO time and duration of systemic treatments with the FAK inhibitor to improve outcomes, including long-term locomotor function and also test this in aged ?post-menopausal? female mice because stroke occurs mainly in older people, with worse outcomes after menopause. FAK inhibitors are currently in clinical trials for cancer and seem to be well tolerated. Aim 2 will define the extent to which the specific VTN receptors mediate the VTN effects, using pharmacological and genetic approaches after MCAO in vivo. This aim will help to identify molecular targets and treatments that may be more selective than FAK inhibition. Aim 3 will define whether the higher IL-6 induction in the female brain after MCAO is neuroprotective, as has been reported for males, and whether blood IL-6 levels, which are higher after stroke, contribute to induction of VTN. We will also determine whether female sex hormones regulate VTN and whether VTN counteracts the protective effects of estrogen and progesterone. Together, these studies focus on a novel and unique molecular target that contributes to worse outcomes, and will provide new avenues for developing drug treatments after stroke, perhaps specifically for women.