Paco S. Herson, Ph.D. - US grants

DESCRIPTION (provided by applicant): Stroke or Brain Attack is a sexually dimorphic disease. Women enjoy protection from stroke relative to men, in part due to endogenous levels of sex steroids, the estrogens and progesterone. While estrogen has been well studied, little is known about progesterone's neuroprotective properties. The steroid is an important but controversial component of hormone therapy in women. Progesterone reduces ischemic brain injury in vivo, however .the mechanism is not known. We hypothesize that one important mechanism of neuroprotection is via progesterone's enhancement of GABA-A receptor activity, counteracting the high levels of excitatory input to neurons during and immediately following ischemia. This R21 application tests this overarching hypothesis, using whole cell voltage-clamp experiments and single cell PCR in cerebellar Purkinje cell (PC) culture, as a novel and initial step in understanding progesterone's neurophysiological actions in complex animal ischemia models. We focus on PCs because of important early observations that PCs, like the well-studied hippocampal CA1 neuron, are uniquely hyper-vulnerable to ischemia. While data from these GABA sensitive cells and cerebral ischemia are few, our recent studies emphasize that non-ischemic female PCs are selectively sensitive to enhancement of GABA-A receptor activity by progesterone metabolites. Furthermore, our preliminary data indicate that female mice require continued exposure of sex steroids to maintain enhanced sensitivity to progesterone metabolites relative to male mice. Therefore, we will test three specific hypotheses 1) Acute progesterone protects PCs from ischemia through activation of the GABA-A receptor. 2) Chronic progesterone enhances female cells to acute progesterone neuroprotection and 3) that chronic progesterone decreases the expression of the gamma-subumt of the GABA-A receptor resulting in increased sensitivity to acute progesterone. Our findings will begin to elucidate the cellular mechanisms of progesterone neuroprotection and sex differences in Purkinje cell response to ischemia.

[unreadable] DESCRIPTION (provided by applicant): Each year approximately 500,000 people suffer from cardiac arrest in the United States, an event associated with poor neurological outcome. Despite intense research over the past 50 years, there are no pharmacological interventions that have proven successful in improving survival and outcome. A hallmark of ischemia-induced neuronal death is excessive release of glutamate leading to excitotoxicity. Unfortunately, glutamate antagonists have proven unsuccessful in humans, predominantly due to side effects. The logical alternative approach would be to apply compounds that activate GABA-A receptors (GABA-A R) in order to counteract excessive glutamate release and excitotoxicity. Interestingly, GABAergic compounds have yielded disappointingly variable results. Recent data has demonstrated that ischemia results in a rapid loss of GABA-A R protein, indicating that the ischemia-induced decrease in GABA-A R protein may cause a decrease in efficacy of GABA-potentiating compounds. Therefore, a treatment that stabilizes GABA-A R protein and function during an ischemic event is an appealing and exciting new approach to neuroprotection. In order to obtain electrophysiological recordings of neuronal GABA-A R function following ischemia, we have developed a cerebellar neuronal culture model. We will use a combination of methods, most notably whole-cell voltage- clamp recordings of synaptic (mIPSCs) and total GABA-A R activity (Current in response to exogenously applied saturating GABA) to confirm and extend upon our preliminary observation that ischemia causes a reduction in functional GABA-A Rs and importantly that ALLO prevents this ischemia-induced loss of function. This RO1 application will test four specific hypotheses 1) that ALLO prevents ischemia-induced reduction in functional GABA-A R, thereby protecting PCs from ischemia. 2) ALLO prevents ischemia-induced reduction in GABA-A R function by maintaining PKC activity and phosphorylation of GABA-A Rs during ischemia. 3) ALLO stabilizes GABA-A R protein during ischemia by preventing proteosome-dependent degradation of GABA-A receptor protein following ischemia and finally 4) that the ALLO-induced protection of GABA-A R function occurs in intact animals exposed to global ischemia (cardiac arrest). Our findings will begin to elucidate the cellular mechanisms of ALLO neuroprotection of PCs and determine molecular pathways that may represent novel targets for neuroprotection. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cardiac arrest/cardiopulmonary resuscitation (CA/CPR) causes ischemia, neuronal excitotoxicity and cognitive decline. Despite intensive efforts, outcome remains poor. Excitotoxicity results from increased glutamate neurotransmission, and the consequent excessive Ca2+ influx through NMDA-type glutamate receptors (NMDAr). Hippocampal CA1 neurons are important to learning and memory and are acutely sensitive to excitotoxicity. We have shown that small conductance Ca2+-activated K+ channels, type 2 (SK2 channels) are expressed together with NMDAr in the spines on hippocampal CA1 neurons where they act to attenuate Ca2+ influx through NMDAr. In addition, SK2 channels are removed from synapses following patterned activity, either normally as for the induction of long term potentiation (LTP), or abnormally after CA/CPR. The loss of synaptic SK2 channels removes the SK channel 'brake' on Ca2+ influx through NMDAr and is due to protein kinase A phosphorylation of the SK2 channels. Our results further show that increasing SK2 channel activity substantially improves neuronal survival after CA/CPR. Therefore, we will use an integrated technical repertoire to test these specific hypotheses: 1. Genetic or pharmacologic enhancement of SK2 channel activity protects CA1 neurons and improves cognitive outcome. We will use genetic mouse models and SK enhancing drugs to determine the i) survival of CA1 neurons and, ii) cognitive performance. 2. CA/CPR-induced ischemia causes a delayed and prolonged loss of synaptic SK2 channels in CA1 neurons, increasing the NMDAr-dependent Ca2+ transient that causes excitotoxicity. Preserving synaptic SK2 channel activity after CA/CPR protects CA1 neurons. We will measure the time course and effects of ischemia on the SK2 and NMDAr contributions to glutamate transmission (EPSP), and NMDAr-mediated Ca2+ transients. 3. CA/CPR-induced ischemia causes PKA phosphorylation of spine SK2 channels, inducing channel endocytosis. Expression of PKA-immune SK2 channels will normalize the SK2 and NMDAr contributions to the EPSP, the NMDAr-dependent Ca2+ transient, and protect CA1 neurons from excitotoxic cell death. We will use control mice or mice expressing PKA-immune SK2 channels to determine: i) the sub-spine distribution of SK2 channels; ii) the SK2 and NMDAr contributions to the EPSP; iii) the spine Ca2+ transient; iv) CA1 viability. 4. The aberrantly sustained ischemia-induced loss of synaptic SK2 channels results in ischemic LTP (iLTP) that shifts ?m, the modification threshold, to higher stimulus frequencies and impairs further potentiation. Maintained expression of functional synaptic SK2 channels prevents iLTP and normalizes ?m. We will measure the long-term effects of CA/CPR-induced ischemia on synaptic plasticity. PUBLIC HEALTH RELEVANCE: Heart attack and the consequent cerebral ischemia is one of the leading causes of death and disability in the United States and, unfortunately, there are currently no drugs available that improve outcome following severe heart attack requiring cardio-pulmonary resuscitation. SK2 channels, one type of Ca2+- activated K+ channel, are anatomically and functionally poised to ameliorate brain damage following stroke. The proposed studies will demonstrate the neuroprotective role of SK2 channels and suggest novel interventional strategies to protect the brain following heart attack, improving survival, diminishing memory deficits, and improving quality of life.

DESCRIPTION (provided by applicant): Ischemic strokes can be treated with either chemical or mechanical means, each with advantages and disadvantages. Tissue plasminogen activator (tPA), a common clot buster, has been used to treat thrombotic clots but can lead to excessive bleeding and must be used soon after symptoms first occur. Mechanical methods can restore blood flow quickly but are invasive and can leave residual prothrombotic material on vessel walls, increasing risk for secondary stroke. To address these drawbacks, we propose a targeted delivery approach performed through an injectable colloidal solution controlled by an external magnetic field. This non-invasive approach combines pharmacological and mechanical methods for clot removal. Here, individual particles in solution are injected into the blood and, upon application of a magnetic field, self-assemble into small microdevices capable of targeting fibrinolytic agents and mechanically attacking a clot in the absence of catheters. As both microdevice assembly and driving forces are provided by the external field, once the procedure is finished, devices self- disassemble into small building blocks removable by the body via phagocytosis. We note that, as the approach is microscale in nature, it can be tuned to more carefully remove any prothrombotic residual clot that can arise in mechanical thrombectomies. Our aims include: Specific Aim 1: Determine the rate at which colloidal-based devices mechanically remove clots. We will investigate clot removal rate by mechanical disruption as a function of operating parameters such as microdevice size and spin-rate within microfluidic vascular mimics. Specific Aim 2: Determine the effectiveness with which fibrinolytic-modified colloidal microdevices can be used to enhance clot removal. Here, we will synthesize tPA-modified magnetic beads and demonstrate their use as fibrinolytic agents within microfluidic vascular mimics. We expect direct coupling of tPA to enhance dissolution rates over mechanical disruption alone. Aim 3: Demonstrate device assembly and targeting within in vivo environments. With a well-established animal stroke model we will demonstrate the delivery, assembly, and targeting of magnetic assemblies to the site of vascular occlusion. Imaged with available small animal MRI facilities, these studies will provide the necessary proof-of-principle for further investigations.

DESCRIPTION (provided by applicant): Each year in the United States ~600,000 people suffer from cardiac arrest (CA) and receive cardiopulmonary resuscitation (CPR), resulting in hypoxia-ischemia (HI) of the brain and consequently in severe neurological deficits in most of the survivors. No pharmacological treatment is available to improve survival or long-term neurological outcome. Ischemic damage in the brain following cerebral ischemia induced by CA is in large part due to glutamate excitotoxicity triggered by a pathological flood calcium through NMDA-type glutamate receptors (NMDAr), ultimately resulting in neuronal cell death. While the pathology and cascade of events leading to injury following cerebral ischemia is complex, overstimulation of NMDAr is considered the major triggering spark for excitotoxicity and ischemic neuronal damage. Excitotoxic stimulation of NMDAr triggers a series of Ca2+-dependent signaling pathways, including activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Our recent findings, and preliminary data, demonstrate that inhibition of CaMKII is a novel approach to minimizing downstream effects of excessive glutamatergic stimulation (excitotoxicity). CaMKII is well established as a major mediator of physiological glutamate signaling involved in synaptic plasticity, particularly synaptic potentiation in CA1 neurons, likel contributing to hippocampal learning. Two major forms of CaMKII regulation have been described, stimulated and autonomous CaMKII activity. Stimulated activity is induced by Ca2+/CaM to CaMKII, and prolonged autonomous (Ca2+-independent) activity is induced by autophosphorylation of residue T286 and/or direct binding to NMDAr subunit NR2B at the synapse (which also mediates CaMKII accumulation at the synapse). Additionally, our preliminary studies reveal a novel link between CaMKII autonomy and nitric oxide (NO), which is produced during excitotoxicity following cerebral ischemia and contributes to oxidative stress and neuronal damage. Our preliminary data indicate that NO-induced nitrosylation/oxidation of CaMKII promotes autonomous CaMKII activity, directly and by protecting T286 from de-phosphorylation. We recently demonstrated that our new CaMKII inhibitor tatCN21 (which blocks both stimulated and autonomous activity) provides robust neuroprotection both in vitro and following experimental stroke. In contrast, traditional CaMKII inhibitors (which block only stimulated activity) do not provide post-insult neuroprotection. This indicates that inhibition of autonomous, but not stimulated CaMKII activity, is a relevant drug target for post-insult neuroprotection. [Importantly, our preliminary results demonstrate that administration of tatCN21 after cardiac arrest results in significant neuroprotection.] The current proposal will utilize our novel mouse CA/CPR model to take advantage of several mutant mouse strains deficient in each form of autonomous CaMKII activity to unravel the complex interactions between these forms of CaMKII activity and their relative contribution to ischemic neuronal cell death. We hypothesize that (i) each autonomy mechanism contributes to neuronal cell death, and that (ii) T286-autophosphorylation mediated CaMKII autonomy is of most direct importance, that (iii) the novel nitrosylation mechanisms contributes to ischemic damage by prolonging T286 phosphorylation, and that (iv) NR2B-binding enables efficient nitrosylation via localizing CaMKII near nNOS.

? DESCRIPTION (provided by applicant): Cardiac arrest (CA) occurs in approximately 600,000 people each year in the United States alone and is a major cause of mortality and morbidity1. Cardiac arrest results in global cerebral ischemia and hypoxic-ischemic injury, and the consequent neuronal damage results in long-term cognitive impairments. The memory disorders commonly observed following CA are readily explained by the selective vulnerability of CA1 pyramidal cells of the hippocampus. However, recent imaging studies in humans indicate that hippocampal injury per se may not correlate with memory dysfunction9, implicating altered physiology following ischemia. Indeed, our recent study indicates that surviving neurons in the hippocampus exhibit physiological changes that likely contribute to cognitive deficits10. This is significant because several interventional clinical trials aimed at reducing neuronal injury have failed to improve outcome following ischemic insults, making it clear that alternative approaches are needed to improve functional recovery. An optimal therapeutic intervention would have the capacity to reduce neuronal injury, maintain functional neuronal networks, and even recover function if administered in the subacute to late chronic phase. Our preliminary data indicate that inhibition of the ion channel TRPM2 fits these criteria. We show that inhibition of TRPM2 during the acute phase (30 min) after cardiac arrest provides neuroprotection and delayed inhibition (6-7 days) reverses CA-induced impairment of neuronal function and plasticity. Memory deficits and hippocampal dysfunction are well accepted sequelae of global cerebral ischemia. Evidence of hippocampal dysfunction following ischemia is provided using electrophysiological recordings of the remaining synaptic network. Synaptic plasticity, in the form of strengthening following physiological stimuli (long-term potentiation; LTP) is a well-established cellular model of learnin and memory. Our recent studies and preliminary studies indicate that CA/CPR causes prolonged impairment (at least 30 days) of hippocampal LTP10 that is prevented by inhibition of TRPM2 channels during the acute phase (30 min) after CA/CPR. Most surprisingly, inhibition of TRPM2 one week after CA/CPR results in reversal of deficits in LTP and enhance memory function. TRPM2 channels have recently been observed to contribute to synaptic inhibition via activation of glycogen synthase kinase 3? (GSK3?); therefore we hypothesize that prolonged activation of TRPM2 channels within hippocampal synapses activates GSK3?, thereby inhibiting the induction of LTP and thus new memories. Our new preliminary data indicates that inhibition of TRPM2 has the potential reduce cognitive impairments and potentially provide a new restorative therapy. The current proposal is aimed at determining the mechanism underlying the regulation of TRPM2 channels during the subacute and chronic phase following ischemia and most-importantly the mechanism of TRPM2-mediated inhibition of synaptic plasticity. We hypothesize that 1) ischemia causes sustained activation of TRPM2 channels, 2) inhibiting synaptic plasticity and 3) that delayed administration of TRPM2 inhibitors will reverse deficits in synaptic plasticity and recover memory function following cardiac arrest.

PROJECT SUMMARY/ABSTRACT Sepsis is a major cause of death and morbidity in both developing and industrialized societies. While great effort has been dedicated to the study of acute organ injury during sepsis, little is known about the mechanisms underlying the chronic morbidities faced by sepsis survivors. One such morbidity is septic neurocognitive dysfunction (also known as chronic septic encephalopathy), a common, severe illness characterized by the accelerated onset of dementia. Recently, the laboratories of Dr. Eric Schmidt (with extensive expertise in the study of sepsis and glycobiology) and Dr. Paco Herson (with extensive expertise in the study of brain injury) have partnered to establish a mouse model of septic neurocognitive dysfunction. This model has allowed the Schmidt and Herson laboratories to explore a novel hypothesis: that septic degradation of the systemic endothelial glycocalyx (a heparan sulfate (HS)-rich layer lining the vascular lumen) releases biologically-active, circulating HS fragments capable of penetrating the hippocampus and disrupting growth factor signaling pathways implicated in cognition. This newly-created collaboration will explore this hypothesis by performing ex vivo electrophysiological studies of living mouse hippocampi, in vivo mechanistic investigations of neurocognitive dysfunction in mouse survivors of endotoxemia or polymicrobial sepsis, and mass spectrometry studies of human plasma samples collected from a cohort of septic patients who underwent rigorous testing of neurocognitive function after sepsis resolution. This novel proposal, which departs from the ?hyperinflammatory? dogma of septic organ injury, promises to identify a mechanistic pathway that is not only responsible for the development of septic neurocognitive dysfunction, but is amenable to therapeutic targeting even in established sepsis.

From Parent Award Project Summary The following aims are developed as the logical next step based on published and unpublished findings from the parent grant (initiated by the late Dr. Traystman) to assess sex-specific signaling following pediatric (juvenile mice) cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Pediatric cardiac arrest is surprisingly common and remains poorly understood and understudied. We made significant progress on the major aims of the previous grant cycle and obtained important new preliminary data that form the foundation for the current aims. We take advantage of our novel juvenile mouse cardiac arrest and cardiopulmonary resuscitation (CA/CPR) model to assess functional outcomes and recovery following CA/ CPR. Emerging evidence from our laboratory, and others, indicate that alterations in the surviving functional networks contribute to cognitive deficits. Synaptic plasticity, in the form of strengthening following physiological stimuli (long-term potentiation; LTP) is a well-established cellular model of learning and memory. Deficits in hippocampal LTP correlate with memory impairments in adult and juvenile mice and therefore, we focus on therapies that target reversing synaptic plasticity deficit to enhance functional recovery (neuro-restoration). We recently made the remarkable observation that juvenile mice exhibit endogenous neuro-restoration; recovery LTP and memory function 14-30 days after CA/ CPR, which we do not observe in adults exposed to the same injury. Our data indicates that the impairments and endogenous recovery of synaptic plasticity and memory function in juvenile mice correlates with expression of brain derived neurotrophic factor (BDNF). Further, we show that stimulation of BDNF-TrkB signaling facilitates recovery of hippocampal function. The recovery in hippocampal function we observed in juveniles corresponds with hormonal maturation that occurs between PND28-56. Our preliminary data indicates that gonadectomy of juvenile male (CAST) and female (OVX) mice prevents recovery of LTP (and recovery of BDNF levels) following CA/CPR. Further, we observed that replacement of sex steroids (estrogen in females and testosterone in males) restores endogenous neuro-restoration in CAST/OVX juvenile mice. Importantly, we observe that estrogen stimulates BDNF expression in juvenile females but not males and that brain estrogen does not facilitate recovery of LTP in males. Therefore, our overarching hypothesis is that 1) increased steroid levels in the brain during puberty facilitate endogenous neuro-restoration following juvenile CA/CPR through activation of sex- specific signaling (Aim 2 male-specific androgen signaling and aim 3 female-specific estrogen receptor signaling) that converges on BDNF and other plasticity gene expression to enhance synaptic plasticity. The proposed research will contribute to our understanding of the mechanisms of functional impairments and recovery following cardiac arrest in the pediatric age group, an understudied population. In particular, this project extends our long-standing research focus regarding sex-specific signaling and the interaction between age, sex, sex steroids and outcomes following brain injury. Further, our studies will extend our focus on developing therapeutic strategies to restore synaptic function within surviving brain networks, rather than attempting to protect neurons from ischemic injury, which may impact treatments of patients of all ages.

Project Summary/Abstract The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a central mediator of two opposing forms of NMDA- receptor (NMDAR)-dependent synaptic plasticity: long-term potentiation (LTP) and depression (LTD). Pathological overstimulation of NMDARs during cerebral ischemia causes excitotoxic neuronal cell death, and we have recently shown that CaMKII mediates also the neuronal damage after global cerebral ischemia (GCI). Importantly, in vivo injection of our optimized CaMKII inhibitor (tatCN19o) provided significant neuroprotection after GCI models that closely mimic the most relevant human conditions: cardiopulmonary resuscitation (CPR) after cardiac arrest in mice or after ventricular fibrillations in pig (unpublished). CaMKII inhibition (i) was done at a highly clinically relevant timepoint for these conditions (30 min after CPR); (ii) was effective also in conjunction with current standard of care (therapeutic hypothermia); and (iii) protected not only from neuronal cell death but also from the long-lasting functional impairments in LTP that are seen in the surviving neurons. Here, three connected but independent aims will directly promote, our mechanistic understanding of CaMKII- mediated regulation of neuronal cell death and LTP impairment. Specifically, the project will investigate (1) the cross-talk of CaMKII autonomy mechanisms in mediating ischemia-induced neuronal damage, (2) a possible dual role of CaMKII in neuronal cell death versus survival, and (3) mechanisms that underly the CaMKII- dependent long-term LTP impairment of the neurons that survive after ischemia. Together, the results of this study will significantly advance our understanding of the molecular mechanisms underlying ischemic neuronal cell death. Additionally, they will inform future development of a therapy in humans.