Richard P. Kraig - US grants

The pathogenesis of ischemic brain cell injury is poorly understood. Necessarily, the future prevention or amelioration of the consequences of ischemic brain cell injury must come from an improved understanding of the cellular mechanisms that result in irreversible cell damage. A highly reproducible (Cornell) model of severe ischemic brain damage in the rat has been developed in this laboratory and will be used in this study. Work with our ischemic model has shown a selective susceptibility of neurons in space and time. Pyramidal cells of the hippocampus can be grouped into susceptible (CA1 and CA 3-5) and resistant (CA 2) zones. Remarkably, cells in CA 1 show delayed histological deterioration which fails to begin until almost 24 hours after the ischemic period while cells in CA 3-5 progressively deteriorate starting immediately after the ischemic period. It is my hypothesis that ischemic brain injury results from the consequences of a loss of pH and calcium homeostasis in the brain. Two recent lines of work suggest that changes in cell calcium or tissue lactate may be fundamentally responsible for ischemic brain damage. However, data in the current literature as well as preliminary results from this laboratory suggest that the interrelation between pH and calcium homeostasis may be the mechanism which initiates the irreversible cell changes of brain ischemia. The objective of this investigation is to examine the electrical characteristics of and ion metabolite changes associated with hippocampal pyramidal cells after an ischemic insult. Microelectrodes sensitive to particular ions, chemicals, and gases (microsensors) will be used to monitor the cellular and adjacent microenvironment in CA 1 and CA 3-5 hippocampal neurons continuously during and after controlled ischemic episodes of 20 to 30 minutes. Measurements will be carried out for 24 hours or more following the transient ischemic exposure. Existing microsensors for sodium, potassium, calcium, pH, and chloride will be used in this study. In addition we expect to develop new sensors for carbon dioxide, oxygen, glucose, lactate, and glutamate.

The pathogenesis of ischemic brain injury is poorly understood. I plan to undertake detailed analyses of the cascade of aberrant microphysiological activities that occur during ischemia and recirculation, a process that has important clinical ramifications. Results from experimental stroke rsearch suggest that either lactic acidosis or disruption of cell calcium homeostasis may be fundamentally responsible for ischemic brain injury. However, little direct evidence exists to confirm these postulated roles for lactate and calcium. A highly reproducible model of severe forebrain ischemia developed in this laboratory will be used in this study. Work with this model has shown a selective vulnerability of neurons in space and time. Histological evidence of damage progresses for days. Microelectrodes sensitive to particular ions, gases, or organic chemicals (microsensors) will be used to test the hypothesis that temporally important local changes in ion and metabolite homeostasis may be detected and be causally related to selective vulnerability. We propose to measure and manipulate the hydrogen and calcium homeostasis and related variables in forebrain regions with varying sensitivity to ischemia during and after the interruption of blood flow. This information will be correlated to histological and electrophysiological characterization of the same areas. Existing microsensors for H+, K+, Ca++, Na+, and Cl- will be used in this study. In addition we expect to develop sensors for CO2, O2, lactate, and glucose.

The pathogenesis of ischemic brain injury remains poorly understood. Brain lactate content is directly proportional to the severity of ischemic brain damage after complete ischemia and implies that acidosis can irreversibly injure brain. However, the molecular mechanisms of the H+ induced injury remain incompletely understood. Our studies suggest that under ischemic conditions which can evolve to infarction, the generation of excess H+ remains confined to a brain space consistent with glia. Such compartmentalization for H+ may be a result of altered properties of glial cell membranes. The contribution of brain cell membranes to H+ homeostasis during ischemia has not been emphasized. A hypothesis is developed based on in vivo recordings that brain infarction from ischemia occurs because of severe acidosis greater than 5.2 pH in glia. Furthermore, this acidosis is likely to result from continued glial lactic acid production coupled to loss of intracellular bicarbonate stores and failure of plasma membrane antiport systems for H+ transport but retained plasma membrane integrity. We plan to examine H+ homeostasis in mammalian neurons, glia, and their interstitial microenvironment. Pairs of H+ selective microelectrodes will be used to simultaneously monitor interstitial and selected intracellular (H+) as well as cell membrane electrical characteristics under ischemic conditions. H+ in ischemia may, in addition to a direct toxic effect, injure brain indirectly through increased modality, loss of cell volume regulation, and resultant postischemic lethal brain edema. Therefore, we will also correlate changes in brain H+ homeostasis to these latter variables of tissue modality, lactate content, and per cent swelling. Cell injury will be assessed by changes in cell electrical characteristics, trans-membrane ion gradients, and visualized by light microscopic techniques. Cells will be identified by their evoked membrane electrical characteristics and through selected horse radish peroxidase staining. The general objective of this study is to characterize the patterns and mechanisms of H+ regulation in mammalian brain cells and their interstitial microenvironment under normal and ischemic conditions so as to test the hypothesis that inhibition of plasma membrane H+ regulatory mechanisms can lead to irreversible dysfunction of glial cells and subsequent brain infarction.

Astrocytes are highly interactive and reactive brain cells that are essential for brain function. Accordingly, damage to brain will evolve directly from injury to astrocytes or these cells will modify their behavior in response to injury of adjacent cells. Examination of how astrocytes respond to injury from brain ischemia should lead to an improved understanding of the cellular pathogenesis of this malady. This philosophy guides the long term goal of this project: To understand how astrocytes respond to ischemic brain injury and how acid-base fluctuations participate in their response. Astrocytes either degenerate from severe ischemia (i.e. infarction) or they are transformed into reactive species by less lethal reduction in flow. Little physiologic information exists that characterizes these two cell changes or alludes to what causes the alterations. Since H+i and K+i modulate vital activities in eukaryotic cells, are interrelated in astrocytes, and are two principal ionic species regulated by these cells, the interrelation of changes in astrocytic H+i and K+i to ischemic injury will be examined in detail here. Ion-selective microelectrodes for pH, K+, as well as a newly developed triple barrel microelectrode array sensitive to pH & CO32, will be used to correlate the patterns of astrocytic acid- base and K+ change from ischemia that are associated with structural markers for these cells (i.e measurements of the cell body size, degree of DNA replication, intensity and distribution of GFAP staining as well as change in regional brain GFAP content). Degeneration of astrocytes will be studied in vivo using a rodent model of focal infarction since this type of ischemic brain injury is clinically most prevalent. Astrocytes become severely acidotic and lose their processes during infarction from global ischemia, perhaps because of a critical reduction in [HCO3-]i. How astrocytic pHi, [HCO3-]I and morphology change with focal infarction is unknown and will be determined. Astrocytic changes in H+i and K+i associated with reactive astrocytosis will be studied in vivo using global ischemia and correlated to the above structural markers of astrocytosis. Furthermore, the effect of hypothermia, which retards neuronal destruction from global ischemia, on ionic and structural variables associated with reactive astrocytosis will be examined since such destruction is thought to be necessary for astrocytic proliferation. Additionally, since astroglial pHi rises at the onset of reactive astrocytosis, the effect of hypercarbia, which acidifies these cells will be studied. Finally, the in vitro brain slice preparation will be used to determine plasma membrane-based mechanisms which interrelate changes in astrocytic pHi and K+i.

[unreadable] DESCRIPTION (provided by applicant): Brain becomes more resilient to injury through activity, including that from spreading depression (SD). While, mechanisms for this intrinsic process remain undefined, they involve innate cytokines. Importantly, innate cytokines are redundant and pleiotropic so multiple cytokines can have similar effects and a single cytokine can have variable effects. This is due to complex interactive signaling at the level of ligands, receptors, and phosphoproteins. Further complexity stems from the diversity of brain cell types, with each capable of unique expression changes. To date such complexity has precluded their systematic study. We will resolve this void via combination of two strategies to accomplish the general goal of defining the innate cytokine signature of SD-induced neuroprotection. The first uses simultaneous, multiplexed measurement of innate cytokine pathway variables (i.e., cytokines, receptors, phosphoproteins). The second applies classical biochemical pathway dissection strategies to mimic (by application of recombinant cytokine ligands) and modulate (by application of cytokine pathway inhibitors) the biological response to NMDA injury or its alteration by SD. Studies will be performed in hippocampal organotypic cultures (HOTCs), which resemble their in vivo counterparts; lack evidence of confounding inflammatory changes and are isolated, and thus, are not influenced by systemic immune changes. Our data show that HOTCs undergo NMDA-mediated injury, SD, and SD-induced neuroprotection like that seen in vivo. Furthermore, this neuroprotection depends on cytokines, via compensatory, adaptive responses. Coupled with novel tools, HOTCs are well suited to our specific aims of defining innate cytokine: 1) ligand; 2) receptor; and 3) related phosphoprotein changes associated with SD-induced neuroprotection from NMDA injury compared to NMDA injury alone - as a means to establish an encompassing signature of the cellular and tissue innate cytokine pathway changes that evoke SD-induced neuroprotection. These aims will be accomplished using: newly developed microsphere-based flow cytometric assays to simultaneously measure innate cytokines (IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma, IL-6, IL-10), their receptors (IL-1R1, TNFR1, IL-6Ralpha/gp130, IFNgammaR, IL-10R), and related downstream phosphoprotein (kinases (ERK1/2, P38MAPK, JNK) and transcription factors (ATF-2, NFkappaB, STAT3)). Changes will be localized to cells using multiplexed real-time RT-PCR of mRNA derived from laser dissection microscopy acquired specific cell enhanced samples (cytokines) and double label immuno- histochemistry (receptors and phosphoproteins). Innate cytokines trigger altered function via gene activation. We suggest that identification of compensatory changes of the innate cytokine system will predict which genes, and related protein pathways, within the system (and within other signaling systems) will be optimal therapeutic targets to emulate protective capacities of intrinsic neuroprotection. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Brain worsens or lessens the severity of injury to itself by means that include altered synaptic activity (SA) that likely involves inflammatory mediators (IM's) since they have both pro- and anti-inflammatory effects on injury plus equally opposite effects on SA. Such divergent, net effects likely involve differential patterns and stimulus dependency of the production, expression and subsequent effects of IM's in brain tissue which is regionally and cellular heterogeneous. However, confirming this notion has previously been impossible due to the absence of appropriate tools that would allow needed simultaneous measurements of multiple candidate molecules from identified cells and the interstitial space (ISS) within functioning brain tissue. We propose to remove this void by formally coupling 2 new investigative tools for measurement of IM protein changes in identified cells (and ISS) from rodent hippocampal organ cultures (HOTC's) and age-matched whole animal controls. Multiple and simultaneous measurements of targeted IM's will be made using the Bio-Plex Protein Array System and specific cell-type samples derived from a Leica laser dissection microscope. Our general goal is to establish detailed protocols for simultaneous measurement of multiple proteins from identified cells plus the ISS and then illustrate the biologic utility for their use with the exemplary SA change seen with seizures. This project will be focused via 3 important steps to more clearly illustrate its potential impact. First, we will alter SA through the use of seizures, since this stereotypic perturbation of brain induces changes in IM's. Second while many IM's change with seizures, we will examine interleukin-1 (IL-1), a prototypic IV. Third, 6 key IL-1 family IM's will be examined since understanding their simultaneous behavior is essential to eventually deciphering how IL-1 effects SA and brain function. IL-1 family IM's consist of 3 receptor ligands (IL-1alpha, IL-1beta & IL-lreceptor antagonist (Ra)), 2 receptor subtypes (IL-1RI & IL-1RII) and an accessory protein (IL-1AcP). While elevated levels of IL-1alpha and IL-1beta enhance seizures, the soluble form of IL-1 Ra (slL-1Ra) acts as an anticonvulsant. Our specific aims are: 1: Develop and confirm protocols for simultaneously measuring IL-1 family IM in identified cells and the ISS from HOTC's and hippocampus in vivo. 2: Determine the spatiotemporal and cell-specific pattern of IL-1 IM changes induced by seizures in hippocampus in vivo and in HOTC's. 3: Determine how excitability conditioning (i.e., from late-long-term potentiation (L-LTP) and exogenous alteration of IL-1 IM homeostasis) alters IL-1 IM expression, seizure susceptibility & injury in HOTC's and whether these changes are interdependent. Accurately determining the where, when and to what degree IL-1 family IM's simultaneously change in identified cells is consistent with the R21 program and should speed deciphering how IL-1 family IM's cause dual effects on seizures and SA.

PROJECT SUMMARY (See instructions): Prenatal brain ischemia irreversibly destroys developing oligodendrocytes leading to hypomyelination and cognitive dysfunction that can remain through adulthood and so is an immense burden to affected patients, families and society. Enhanced neural activity from environmental enrichment (EE) can mitigate this loss. While the mechanisms for these effects are incompletely defined, recent literature and results from our laboratory indicate that tumor necrosis factor alpha (TNF-a) signaling is involved in both processes. TNF-a and its cell of origin, microglia, can exacerbate oligodendrocyte degeneration. Bacterial endotoxin lipopolysaccharide, which commonly worsens stroke in premature infants is mediated by TNF-a only when astrocytes are present, though again mechanisms for this interactive cellular affect are unknown. In contrast, our data show that low-level TNF-a, which rises with EE, is responsible for the related activitydependent neuroprotection and increased myelin content. This dual pattern of TNF-a behavior is consistent with hormesis (i.e, low-dose stimulation and high-dose inhibition) which takes the form here of acute, high dose toxicity and low-dose adaptive compensatory change via insulin growth factor-1 (IGF-1). Astrocytes are an intrinsic source of IGF-1 that vitalizes oligodendrocytes. Importantly, TNF-a can inhibit IGF-1 signaling. Thus, our general goal is to define the TNF-a/IGF-1 neural signaling involved in oligodendrocyte injury from prenatal brain ischemia and myelin recovery by increased neural activity. The specific aims are to determine the role of TNF-a/IGF-1 signaling in: AIM 1: oligodendrocyte death from prenatal brain ischemia in vitro; AIM 2: activity-dependent myelin recovery after prenatal brain ischemia in vitro; and AIM 3: characterize the relation between TNF-a/IGF-1 signaling cascades to oligodendrocyte loss from prenatal brain ischemia and subsequent activity-dependent myelin recovery in vivo. PI4 rat hippocampal slice cultures and P7 CD-I mice will be used to model human prenatal ischemic brain injury using endpoints of hippocampal myelin content and memory that will be compared to measured, mimicked and modulated TNF-a/IGF-1 signaling cascade variables.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is the most common disabling neurological disease of young adulthood. It is an incurable autoimmune disorder involving oligodendrocyte loss, demyelination, and an inability to remyelinate damaged brain. Remyelination declines with age and MS progression, where oligodendrocyte precursor cells (OPCs) are actively prevented from differentiating into myelinating cells. Current therapies are only directed at reducing demyelination via immune suppression which reduces immune competence. Importantly, no therapies exist to prevent the inevitable decline in remyelination. This project is designed to remove this void. OPC differentiation into myelinating cells is regulated by certain microRNAs (miRNAs) that are reduced in MS lesions. Notably, blood from young rats enhances remyelination of aged brain. The rationale for this work comes from pilot data showing that blood-derived exosomes, small vesicles released from cells, contain miRNAs that trigger remyelination. Non-immunogenic exosomal miRNAs from the serum of young and environmentally enriched (EE) rats enhanced remyelination. The long-term goal of our research is to develop neurotherapeutics based on understanding how EE protects brain. The overall goal of this application is to develop exosomes containing miRNAs as a therapeutic for brain remyelination. The central hypothesis is that these exosomes will enhance remyelination of brain and can be crafted into a therapy for MS. The project has four Aims: AIM 1: Establish interferon-gamma (IFN-?) -stimulated dendritic cells (DCs) as an effective source of exosomes that mimic EE-exosomes to enhance remyelination without injury or immune reaction in slice cultures. AIM 2: Establish an efficient means of loading DC exosomes with fabricated targeted miRNA species and establish their ability to enhance remyelination without injury or immune reaction in slice cultures compared to EE-exosomes and IFN-?-stimulated DC exosomes. AIM 3: Establish the optimal efficacy of intravenously (then nasally) delivered (Aims 1-2) exosomes to promote remyelination without injury or immune reaction in a rat lysolecithin model of MS demyelination. AIM 4: Establish the optimal efficacy of intravenously (then nasally) administered (Aims 1-2) exosomes to promote remyelination in a mouse model of MS [experimental autoimmune encephalomyelitis (EAE)] without injury or enhanced immune reaction. Outcome measures will be: 1) OPC, differentiated oligodendrocyte, and compact myelin levels, targeted miRNAs & target levels; 2) toxicity, measured by cell death, liver and renal function assays, plus oncogenesis; 3) immune activation assayed by serum and brain markers; and 4) clinical measures of disease severity. The work is innovative because it uses naturally occurring signaling molecules and vehicles for delivery of EE-like enhancement of myelination. The proposed research is significant because it will establish the preclinical feasibility and utility of EE-based miRNA-containing exosomes as a novel therapeutic to remyelinate MS- damaged brain without immune compromise.

? DESCRIPTION (provided by applicant): Migraine is an immense healthcare burden with limited therapeutic options. Environmental enrichment (EE) reduces migraine. The long-term goal of this project is to determine how EE mitigates migraine, modeled using spreading depression (SD), in order to identify novel targets for development of therapies. SD, the likely cause of migraine with aura (MwA), transiently disrupts myelin. Microglia can polarize to distinct phenotypes that range from classically (M1) to alternatively (M2a) activated states. Microglia generate the oxidative stress that occurs with SD and lowers SD threshold (SDT), suggesting that a pro-inflammatory M1 phenotype enhances SD by damaging myelin and increasing aberrant cross-talk between nerve fibers. In contrast, EE promotes an anti-inflammatory M2a phenotype and raises SDT, while increasing myelination, and oxidative tolerance (OT). EE increases CNS interleukin-11 (IL-11), and IL-11 alone can recreate these effects. The rationale for this project is based on evidence indicating that microglial M2a polarization increases SDT via secreted factors. Preliminary data show that while exosomes released from M1 microglia reduce SDT, those from M2a microglia increase SDT, perhaps by increasing myelination/OT, which could prevent the aberrant excitability that provokes SD. This study will further explore the effects of EE-induced M2a polarization on increasing SDT through production of exosomes. The overall goal is to define exosomal miRNAs from M2a- polarized microglia that most potently increase SDT plus myelination/OT and test their effectiveness in vivo after nasal administration. The central hypothesis is that M2a-polarized microglia release exosomes containing miRNA that mimic the effects of EE on SDT, myelin levels and OT. The aims are: Aim 1: Establish the impact of M2a versus M1 microglia-derived exosomes on SDT, myelination/OT. SD is used to model MwA in rat brain slice cultures and rats. Exosomes from polarized primary microglia are applied to slice cultures for measurement of SDT, myelination/OT. Aim 2: Identify miRNA species differentially expressed in M2a versus M1 microglia-derived exosomes and validate their role in influencing myelination/OT. miRNAs are identified using arrays and bioinformatics. Targets are verified using qPCR and Western blots. Aim 3: Determine the extent to which unique miRNAs identified in Aim 2 impact SDT, myelination/OT. Exosomes from M2a microglia and dendritic cells (DCs) will be electroporated with specific miRNA inhibitors and mimics respectively, then tested for impact on slice culture SDT, myelination/OT. DC exosomes will be used, as they enter brain nasally to produce functional effects. Aim 4: Establish the impact of M2a exosomes in vivo. M2a exosomes will be nasally delivered to brain for measurement of SDT, myelination/OT, and target mRNA/proteins. Trafficking will be determined via in vivo and confocal imaging of tagged exosomes. This work is innovative because it explores M2a microglial exosomes/miRNAs as a means to increase SDT via myelination/OT. It is significant, because it will identify therapeutic miRNAs to be loaded into exosomes for nasal delivery against SD/MwA.