Shaohua Yang - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The demonstration that recombinant tissue plasminogen activator (rtPA) is useful for acute management is changing the approach to ischemic stroke. However, after more than a decade, the use of tPA therapy remains limited. Several factors could contribute to this limitation: irreversible cell damage induced by cerebral ischemia over time; the profile of rtPA treated patients at high risk of hemorrhagic transformation; and the potential detrimental effects of rtPA. Combination pharmacotherapy strategies to expand the rtPA fibrinolysis time window beyond 3 hours are under active investigation. In principle, it may be possible to extend the therapeutic window for rtPA therapy by using a neuroprotective drug. Estrogen is well known to diminish the neurotoxicity caused by NMDA activation. Preliminary evidence is presented in this proposal showing that 17[unreadable]-estradiol stabilizes blood brain barriers (BBB) against cerebral ischemia reperfusion injury through inhibition of matrix metalloproteinase 2 and 9 (MMP2, MMP9) activation, the major factors related to hemorrhagic transformation. 17[unreadable]-estradiol attenuates rtPA induced MMP2 and MMP9 activation in primary astrocytes. Most importantly, we have demonstrated that 17[unreadable]-estradiol extends the therapeutic window of rtPA upon pretreatment in female rats in an embolic middle cerebral artery occlusion (MCAO) model. Thus, the protective properties of estrogen make it a good candidate for combination therapy with rtPA. The neuroprotective effects of estrogen could delay irreversible cell damage. Further, estrogen may attenuate the side effects of rtPA, thereby prolonging the therapeutic window of rtPA and greatly broaden its clinical application. The present application will test the hypothesis that 17[unreadable]-estradiol extends therapeutic window of rtPA in embolic stroke, and determine the underlain mechanisms. To achieve these objectives, the following specific aims will be addressed. 1) To assess the interaction of estrogen and rtPA on glutamate neurotoxicity and matrix metalloproteinases (MMPs) activation. 2) To determine if 17[unreadable]-estradiol extend the therapeutic window of rtPA in female rats in an embolic stroke model. 3) To determine if 17[unreadable]-estradiol extend the therapeutic window of rtPA in male rats in an embolic stroke model. The application's objectives are to explore the feasibility for further clinical trials of combination therapy of estrogen and rtPA for ischemic stroke.The demonstration that recombinant tissue plasminogen activator (rtPA) is useful for acute management is changing the approach to ischemic stroke. However, after more than a decade, the use of rtPA therapy remains limited and has had only a modest impact in the overall burden of ischemic stroke. Several factors could contribute to this limitation: irreversible cell damage induced by cerebral ischemia over time; the profile of rtPA treated patients at high risk of hemorrhage; and the potential side effects of rtPA. Combination pharmacotherapy strategies to expand the 3 hour therapeutic window rtPA are under active investigation. In principle, it may be possible to extend the therapeutic window for rtPA therapy by using a neuroprotective drug. Preliminary evidence is presented in this proposal showing that estrogen can protect against ischemic stroke, and potentially attenuate side effect of rtPA. The present application will test the hypothesis that 17[unreadable]-estradiol extends therapeutic window of rtPA in embolic stroke, and determine the underlain mechanisms. The application's objectives are to explore the feasibility for further clinical trials of combination therapy of estrogen and rtPA for ischemic stroke. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Ischemic stroke is the major cause of vascular dementia. Epidemiological studies have suggested a progressive course of dementia after ischemia stroke. Consistently, the preliminary study presented in this application indicates that, following a transient focal cerebral ischemia, rats exhibit a delayed significant decline of cognitive function. Furthermore, a delayed synaptic alternation was indicated in the hippocampus remote to the primary ischemic area. The relatively slow onset of cognitive dysfunction and synaptic alternation in the hippocampus suggest that therapeutic interventions applied after ischemic stroke could prevent progression of vascular cognitive impairment. In this application, we propose to further determine the long-term effects of transient focal cerebral ischemia on various sensorimotor and cognitive functions of rats, and to delineate the neuropathological mechanisms underlying the vascular cognitive impairment in this model with a focus on the hippocampus synaptic alternation, using behavioral, molecular biochemical, and electrophysiological approaches. In addition, the effects of therapeutic intervention with 172-estradiol and a non-feminizing estrogen analogue will be tested in this experimental model. To achieve this objective, the following aims will be addressed: Specific Aim 1. To characterize vascular cognitive impairment after transient focal cerebral ischemia. In the proposed studies, an extensive behavioral battery will be employed to determine the impact of transient focal cerebral ischemia on long-term sensorimotor functions, cognitive functions, and anxiety-related behavior in a transient middle cerebral artery occlusion model in rats. Specific Aim 2. To determine the mechanisms contributing to the progressive decline of cognitive function after transient focal cerebral ischemia. We will focus on the neuropathological change in hippocampus, which plays a critical role in cognitive function, since delayed synaptic alternation has been identified in this area remote to the primary infarct area after transient middle cerebral artery occlusion in rats. Specific Aim 3. To determine the effects of therapeutic intervention with 172-estradiol and a non-feminizing estrogen analogue on the progression of vascular cognitive impairment induced by transient focal cerebral ischemia, using behavioral, biochemical, and electrophysiological approaches. Collectively, the studies proposed will provide new insight in the mechanism of vascular dementia and identify potential therapeutic target for the prevention of cognitive decline following cerebral ischemia. PUBLIC HEALTH RELEVANCE: In this application, we propose to determine the long-term effects of transient focal cerebral ischemia on various sensorimotor and cognitive functions of rats, and to determine the neuropathological mechanisms underlying the vascular cognitive impairment in this model with a focus on the hippocampus synaptic alternation, using behavioral, molecular biochemical, and electrophysiological approaches. In addition, the effects of therapeutic intervention with 172-estradiol and a non-feminizing estrogen analogue will be tested in this experimental model. The studies proposed will provide new insight on the mechanism of vascular dementia and identify potential therapeutic target for the prevention of cognitive decline following cerebral ischemia.

DESCRIPTION (provided by applicant): Astrocytes respond to all forms of CNS insult by a process commonly referred as reactive astrogliosis characterized as hypertrophy of cellular processes and upregulation of intermediated filament proteins such as glial fibrillary acidic protein (GFAP). Compelling evidence has indicated that reactive astrogliosis is not simply an all-or-none phenomenon but, rather, a finely tuned continuum of molecular, cellular, and functional changes that range from subtle alternations in gene expression to scar formation. Recent research findings have also suggested that reactive astrogliosis may exert both beneficial and detrimental effects in a temporal dependent manner regulated by specific molecular signaling cascades. Increasing evidence has indicated that p38 plays a role in many other biological functions at the CNS. The activation of p38 signaling releases pro-inflammatory cytokines that are known to be involved in various CNS disorders including ischemic stroke, Alzheimer's disease, Parkinson's disease, multiple sclerosis, neuropathic pain and depression. The role of p38 MAPK in peripheral immune and inflammatory response has been extensive studied. However, the action of p38 in astrogliosis, the major component of neuroinflammatory response in CNS, has largely unknown. Our preliminary study demonstrated an activation of p38 signaling in reactive astrogliosis in the glial scar area in a mouse ischemic stroke model. Our laboratory has established a novel transgenic mouse line (hGFAP Cre/p38 loxP) in which the hGFAP promoter-driven Cre transgene deletes the floxed p38?ene and a downregulation of GFAP expression was found in primary astrocytes derived from the transgenic mice. Consistently, attenuation of reactive astrogliosis was observed upon p38 inhibition in primary astrocyte cultures. Reduction of ischemic stroke induced astrogliosis was observed in the hGFAP cre / p38 loxp mice as compared with wild type littermates. As Cre-mediated recombination is irreversible, embryonic expression in neural precursor cells directed by the GFAP promoter have resulted in substantial Cre-mediated recombination in mature neurons as well as glial cell in all GFAP-Cre mice characterized to date. In addition, the constitutive p38 knockout will not be able to study the role of p38 in astrogliosis in a temporal dependent manner. Therefore, there is a clear need of an inducible astrocyte specific p38 knockout mouse line. In this application, we proposed to establish an inducible astrocyte specific p38 knockout mouse line, characterize the inducible gene recombination and p38 knockout in the established transgenic mouse line, and determine the spatial and temporal dependent action of p38 knockout on glial scar formation and functional recovery after ischemic stroke. Specific Aim 1 is to establish the inducible astrocyte specific p38 knockout, hGFAP Cre-ERT2 / p38 loxP, mouse line. Specific Aim 2 is to determine the spatial and temporal dependent action of p38 knockout on glial scar formation and functional recovery after ischemic stroke using the established hGFAP Cre-ERT2 / p38 loxP mouse line. We predict that, in the established hGFAP Cre-ERT2 / p38 loxP mouse line, tamoxifen inducible gene recombination will be achieved predominantly in astrocyte with a minimal in the neural stem cell at subventricular zone. We expect that induction of p38 MAPK knockout in astrocyte after ischemic stroke attenuates glial scar formation, ameliorates the inhibitory action of glial scar on axonal sprouting and synaptogenesis, and enhances functional recovery.

DESCRIPTION (provided by applicant): Neuroglobin (Ngb) is one of the latest discovered globins that preferentially localize to neurons in vertebrate brain with a well-defined neuroprotective effect against a wide range of neurological disorders. However, the mechanism underlying the neuroprotective action of Ngb is still uncertain. Neurons are characterized by high metabolic activity and execute fast-paced energy-demanding processes. Moreover, neurons have poor nutrient storage and are particularly vulnerable to ischemic insult. Ngb expression is at the highest in neonatal brains which is coincident with the peak of lipogenesis in the brain and with the presence of glycogen in neural tissue. Interestingly, the higher Ngb expression in neonatal brain is associated with the long recognized ischemic tolerance in neonates. We have generated stable Ngb overexpression in a neuronal cell line and demonstrated that Ngb overexpression significantly inhibits AMPK signaling and increases cellular glycogen and lipid contents. Consistently, AMPK inhibition and glycogen synthase activation were found in cortical neurons of Ngb overexpression mice. These data provided the first evidence that Ngb might regulate neuron metabolism through AMPK signaling. Enhancing glycogenesis and lipogenesis in neuron could have distinct advantage for ATP production even under anaerobic condition. Our preliminary study further demonstrated that AMPK activation diminished the neuroprotection of Ngb overexpression. Given the well-established function of AMPK signaling in cellular metabolism, the high affinity of Ngb to oxygen, and that both Ngb and AMPK predominately express in neurons at CNS, it is plausible that Ngb might interact with AMPK signaling and play a critical role in neuron metabolism. Our central hypothesis is that Ngb overexpression increases nutrient storage capacity via inhibition of AMPK pathway and, thus, provides neuroprotective action against ischemic stroke. We will address the following two specific aims to test our central hypothesis. 1. Determine the role of Ngb overexpression in neuron metabolism and underlying mechanisms in physiological condition. 2. Determine the involvement of AMPK-mediated anabolic action in the neuroprotective effect of Ngb overexpression against ischemic stroke. The proposed studies are expected to have an important positive impact because they may identify novel therapeutic target that ultimately lead to the discovery of treatment for ischemic stroke.

Stroke is a leading cause of long-term disability in US and worldwide. Historically, both interventions and research have been focused on motor sensory deficits, while cognitive impairment, a decisive determinant of the quality of life for stroke survivors, has been rather neglected. Cognitive impairment is a major sequela after stroke, termed as post-stroke cognitive impairment (PSCI). Stroke and cognitive impairment are common among older persons with an estimate of 1/3 having different degree of cognitive impairment within several months after stroke. However, the prevalence of PSCI may have been significantly underestimated as the traditional Mini-Mental State Examination (MMSE) is not sensitive enough for cognitive assessment. Using more extensive assessment, recent clinical studies have indicated that PSCI is very common in both young and old stroke patients, even in cases of relative mild stroke and cases with excellent clinical recovery. The neuropathology of PSCI remains poorly defined and no FDA approved treatment is available for PSCI. Stroke is a heterogeneous disorder and the pathophysiology of PSCI should be interpreted with reference to clinical subtypes and brain changes in the sample. Accordingly, the mechanisms underlying PSCI should be explored using different stroke models. Our previous study has demonstrated that rats subjected to experimental ischemic stroke induced by transient middle cerebral artery occlusion (tMCAO), despite motor function recovery, developed progressive cognitive function deficit that correlates with hippocampal LTP suppression. A significant reduction of VEGF expression was found in the hippocampus after tMCAO. microRNA miR-20a/20b and 15a/b have been found to inhibit VEGF expression by targeting VEGF 3'-UTR. Consistently, increased levels of miR-15b and miR-20a were found in cerebral spinal fluid (CSF) and hippocampus at 30 days after tMCAO. Our preliminary study further demonstrated that treatment of miR-15b and miR-20a antagomirs at 1 week after stroke attenuated PSCI induced by tMCAO. Mounting evidence has indicated that VEGF enhance neurogenesis and hippocampus-dependent learning and memory. We hypothesize that focal ischemic stroke induces increase of miR-15a/b and ?miR-20a/b expression hence down-regulates VEGF expression in the hippocampus which contributes to PSCI. In the proposed studies, we will determine the effect of miR-15b and miR-20a antagomirs on PSCI induced by transient MCAO and the underlying mechanisms. Aim 1 is to determine the tempo-spatial regulation of miR-15a/b, miR-20a/b and VEGF expression after ischemic stroke induced by transient MCAO. Aim 2 is to determine the preventive effect of miR-15b and miR-20a antagomirs on PSCI induced by transient MCAO. Aim 3 is to determine the VEGF-dependent effect of miR-15b and miR- 20a antagomirs on PSCI induced by transient MCAO. Aim 4 is to determine the neurogenesis-dependent effect of miR-15b and miR-20a antagomirs on PSCI induced by transient MCAO. Aim 5 is to determine if delayed treatment of miR-15b and miR-20a antagomirs reverses PSCI induced by transient MCAO.