Neuroprotection & Mechanism of Ethanol Therapy: New Prospects for an Ancient Drug

Balanced energy metabolism of neural cells and associated mitochondrial functions are critically important for neural survival. Ischemia/reperfusion injury in acute stroke disrupts energy balance by impairing the metabolic state of neural cells and by interrupting mitochondrial activity. In addition, reactive oxygen species (ROS) generated by the mitochondria and in the cytosol trigger cell death cascades. Previous studies have demonstrated that ethanol significantly and consistently decreased whole-brain metabolism, raising the possibility that ethanol could be used to ameliorate metabolic dysfunction in stroke and serve as a clinical neuroprotectant. Our goal is to establish a safe, inexpensive, easy-to-use, and powerful therapeutic strategy by testing the hypothesis that ethanol reduces cerebral ROS generation and cell death in ischemia/reperfusion injury after stroke by slowing down and thus normalizing glycolytic and oxidative metabolism.

Our current studies investigate 1) whether ethanol exerts its neuroprotective effect by suppressing brain energy metabolism, decreasing hyperglycolysis via inhibition of glucose transporter activity, and inhibiting nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) through reduction of NOX complex formation and glycolysis-produced NADPH; and 2) whether ethanol prevents ROS generation and cell death by ameliorating oxidative phosphorylation via inhibition of a key mitochondrial enzyme, cytochrome c oxidase (CcO); and 3) to test the translational potential of ethanol therapy in a more clinically relevant stroke model with a prolonged ischemic period.

Investigation of ethanol therapy on neural metabolism and mitochondrial function in stroke is fundamental for establishing its clinical potential as a new neuroprotective strategy for this devastating disease. This work incorporates a highly innovative approach, validating an ancient, approved drug for a new therapeutic application and proposing new mechanisms for its effect on brain metabolism. Our study will enable preclinical and clinical investigations of ethanol therapy for acute ischemic stroke.

Ischemic Area Infusion and Regional Hypothermia: A Potential Therapy in Stroke

Clinically, there are no effective therapeutic tools for amelioration of cerebral ischemia/reperfusion caused by stroke. It has been emphasized that ischemia/reperfusion injury is initiated by a series of events occurring at the blood-vascular-parenchymal interface, leading to inflammatory injury, disruption of endothelial integrity, and neuron death. Brain cooling is a remarkable neuroprotectant in stroke therapy if applied soon after onset of ischemia. Due to management difficulties, hypothermic induction by surface cooling in current clinical settings is vastly limited.

Results from our recent studies indicate that highly localized intra-arterial "flushing" of the ischemic territory prior to reperfusion significantly reduces brain injury in experimental stroke. The mechanisms of neuroprotection conferred by hypothermia or vascular infusion are thought to be multifunctional. This leads to a new hypothesis that local intra-arterial cold hypertonic solution infusion, concurrent with regional cerebral hypothermia in ischemic areas prior to reperfusion, synergistically minimizes brain injury. This may provide the ultimate neuroprotective "cocktail" that limits inflammation and neurovascular disruption during reperfusion. In our laboratory, we define the therapeutic and systematic optimization of a combined infusion and cooling procedure in our stroke model by evaluating long-term motor deficits, brain infarct volume, as well as cerebral and pulmonary edema. We also elucidate protective mechanisms of the novel model that targets the brains vascular-parenchymal interface by reducing inflammatory mediators, endothelial activation of nuclear factor kappa-B, leukocyte infiltration, matrix metalloproteinase expression, and blood-brain barrier disruption.

Results from these studies will provide fundamental information on the establishment of a novel therapeutic procedure in stroke beyond the levels achieved by current therapy. Intravascular cold infusion into the ischemic region, which combines recanalization of the occluded middle cerebral artery (mechanically or thrombolytically) and administration of neuroprotective drugs, may improve outcome in stroke patients.

Exercise-Induced Endogenous Neuroprotection in Stroke

There is increasing evidence from us and other investigators that exercise produces endogenous protection in the brain after transient ischemia. Our goal is to establish an endogenous neuroprotective concept of exercise preconditioning in stroke and identify the cellular and molecular mechanisms by which exercise induces neuroprotection. We elucidate TNF and HSP signaling pathways that mediate differential endothelial activation and downstream inflammatory, neurovascular integrity and apoptotic events. The proposed strategy of exercised-induced endogenous neuroprotection can be translated to other therapeutic approaches, such as pharmacology. This strategy will allow the development of combined approaches to inhibit and stimulate appropriate targets simultaneously, thus reaching the highest therapeutic potential.

Traumatic Brain Injury (TBI)

Memory storage and learning have been found to be associated with synaptic plasticity. During acute closed head injury and its aftermath, rapid acceleration and deceleration of the head causes diffuse axonal injury in the entire brain, leading to severe synapse loss and damage. Recent work suggests that brain extracellular matrix (ECM) proteins and their regulatory matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9, play a role in synaptic plasticity. Our study assessed the role of MMP-2 and -9 in synaptic damage after TBI and the role of hypoxia inducible factor-1α (HIF-1α), a transcription factor upregulated during hypoxia, in the regulation of MMP-2 and -9 expression post TBI.

TBI causes vasogenic brain edema, where the extracellular space is expanded by fluids from abnormally permeabilized blood vessels. The detrimental effect of AQPs in brain edema has been reported. AQPs, such as AQP4 and 9, can cause either cytotoxic or vasogenic edema in TBI. HIF-1α is a key component of the cellular response to pathophysiologic conditions and can be harmful in cerebral ischemia. Our study determines the role of HIF-1α in regulating expression of AQP-4 and -9 and associated brain edema after close head TBI.

The Harris laboratory studies hydrocephalus with a prudent focus on bioengineering strategies that could improve treatment. We integrate experimental bench top data with tranlational studies involving patients to deepen our knowledge of this complex disorder. Discover more.

Dr. Parajuli's Neuro-oncology/Immunology laboratory focuses on developing novel adjuvant therapeutic strategies by combining immunotherapy and complementary/alternative approaches for the treatment of malignant gliomas.

Malignant gliomas are one of the most aggressive and lethal solid tumors with a high rate of invasion into normal brain tissue. Each year, more than 18,000 people are diagnosed with this deadly disease in the USA. The median survival rate remains a dismal 12 months, in spite of aggressive surgical and chemotherapeutic interventions.

Immunotherapy using tumor-antigen loaded dendritic cells (DC) has the potential to specifically target and eradicate the invasive tumor cells without affecting the surrounding normal brain tissue. Studies on immunotherapy mostly focus on activation of cytolytic T lymphocytes (CTL) for targeting tumor cells bearing specific antigens with MHC class I molecules.

However, malignant tumors often circumvent an immune attack by both passive and active mechanisms. One of the ways malignant tumors evade immune attack is by lowering the expression of MHC class I molecules. Novel DC-based vaccine strategies for activation of natural killer-T (NK-T) cells along with CTLs are being developed in order to effectively target all tumor cells irrespective of the level of MHC class I expression. In this context, studies on elucidation of molecular mechanisms of NK-T cell activation and tumor cytolysis are also being pursued. The laboratory is also actively collaborating with Dr. Sandeep Mittal and Dr. Lawrence Lum in developing a Phase I/II clinical immunotherapeutic strategy using bi-specific antibodies.

Most malignant gliomas maintain constitutively high TGF-β activity, which is pivotal in modulating cell-mediated immune response either by directly inhibiting DC and CTL activities, or via generation and/or expansion of regulatory T (Treg) cells. Studies are being conducted to combine immunotherapy with administration of a novel herbal product derived from a traditional medicinal plant Scutellaria sp, which significantly inhibits the secretion of TGF-β in malignant gliomas. The phytochemical composition of the herbal product and their molecular mechanisms of anti-glioma/anti-TGF-β activity are being studied. Potential adjuvant therapy of gliomas with the herbal product in combination with the alkylating agent temozolomide is also being explored.

Mechanisms of immune suppression by malignant tumors

Malignant tumors can directly induce Treg cell or T-DC activity via elaboration of several membrane-bound or secreted cytokines/factors. Treg cells and T-DC can also modulate each other via similar cytokine interactions. These suppressor cells or mediators, in turn, inhibit cytolytic functions of effector T cells (CTLs) or NK cells. TGF-β, which is expressed in both membrane-bound and soluble forms, can be very critical in most of these interactions.

CTL: Cytotoxic T lymphocyte; DC: Dendritic cell; NK: Natural killer; PGE2: Prostaglandin E2; T-DC: Tolerogenic/suppressor DC; TGF: Transforming growth factor; Treg: Regulatory T cells.

Parajuli P, Mathupala SM, Mittal S, Sloan, AE. Dendritic cell-based active specific immunotherapy for malignant glioma. Expert Opinion on Biological Therapy, 7:439-48, 2007.

Read more about resident, research associate, and student participants.

Research Interests

Fetal Alcohol Syndrome

Alcohol consumption during pregnancy can result in intrauterine fetal neurotoxicity, i.e., fetal alcohol syndrome/effects (FAS/E). FAS/E infants develop severe impairments in cognitive functions that are largely attributed to volume reductions in the striatum, a brain region which is consistently and dramatically reduced in volume in children exposed to alcohol in utero. Defining the molecular mechanisms and targets that determine sensitivity or resistance to ethanol neurotoxicity in the striatum and other brain regions is critical to the design of therapies for intervention in the pathological sequelae associated with FAS/E.

Ethanol exposure during brain development (synaptogenesis) results in massive apoptotic neuronal death in the rodent brain, with rapid and synchronous apoptosis occurring in the striatum. Ethanol acts to potentiate GABAA receptor activity and the antagonize NMDA receptor function. Antagonism of NMDA receptor-mediated calcium entry results in impairment of intracellular signaling pathways, such as those involving the calcium-stimulated adenylyl cyclases, AC1 and AC8. AC immunoreactivity is highly represented in the pre- and postsynaptic compartments (adjacent to NMDA receptors) making the ACs likely to be key targets for alterations of cellular responses.

Mice with single deficiency of AC1 (AC1KO) or AC8 (AC8KO), and mice with combined AC1 and AC8 deficiency (DKO) have increased vulnerability to neuronal apoptosis in the striatum following treatment with ethanol, as well as to NMDA receptor antagonists and GABAA agonists in the brain during the synaptogenesis period compared to wild type (WT) controls. However, the mechanisms by which AC1 and AC8 confer resistance to ethanol-induced apoptosis are yet unknown.

Recent evidence from our lab has demonstrated that the enhanced neuroapoptotic response observed in the striatum of DKO mice is accompanied by significant reductions in phosphorylation of known pro-survival proteins, insulin receptor substrate-1 (IRS-1), Akt and extracellular signal-regulated kinases (ERKs). These data suggest that AC1/AC8 are crucial activators of cell survival signaling pathways acutely following ethanol exposure and represent molecular factors that may directly modulate the severity of symptoms associated with Fetal Alcohol Syndrome.

DKO mice demonstrate increased apoptosis following acute ethanol treatment. Representative sagittal sections depicting ethanol-induced apoptosis in the striatum 4 h after 2.5 g/kg or 5.0 g/kg in neonatal WT and DKO mice. DKO mice demonstrated 2.5 fold increases in activated caspase-3 expression compared to WT mice at both doses. (Conti, et al., Neurobiol. Dis. (2008) 33(1):111-8)

Neuronal Reactivation after Ethanol Exposure

Ethanol is a widely used central nervous system depressant that results in sedation. In the rodent model, the duration of sedation is affected by neuroadaptation to acute ethanol doses; however, the neuroadaptive mechanisms resulting from ethanol exposure remain unclear. The cAMP signaling pathway has emerged as an important modulator of ethanol sensitivity. Reductions in cAMP signaling increase behavioral sensitivity to ethanol in the mouse. We have previously demonstrated that in DKO mice, which are lacking the calcium-stimulated adenylyl cyclases 1 and 8 (AC1 and AC8) ethanol-induced sedation is increased compared to wild type (WT) controls.

We have demonstrated previously that the increased sensitivity of DKO mice to ethanol-induced sedation was accompanied by impaired protein kinase A (PKA) phosphorylation of target proteins of unknown identity. We hypothesize that ethanol-mediated induction of PKA phosphorylation is part of a compensatory homeostatic mechanism initiated by AC1 and/or AC8. Our recent use of phosphoproteomic techniques has allowed for identification of several PKA target proteins involved with presynaptic function, including synapsin, vacuolar H+-ATPase, and dynein, that are phosphorylated following acute ethanol exposure in WT mice. Identification of additional proteins phosphorylated after ethanol treatment include dynamin and eukaryotic elongation factor-2 (eEF-2). Of these, we have demonstrated that phosphorylation of synapsin I, II, eEF-2 and dynamin is impaired in the brains of DKO, and in some cases, AC1KO mice following acute ethanol exposure.

Together these data suggest that calcium-stimulated ACs, largely involving AC1, contribute to the presynaptic homeostatic response to ethanol-induced inhibition of neuronal function by facilitating PKA activation of proteins involved in presynaptic vesicle release. Further identification of PKA targets uniquely regulated by AC1 and AC8 will provide additional insight into the mechanisms of the neuronal response to the inhibitory effects of ethanol.

Immunohistochemical detection of phospho-synapsin protein following ethanol treatment in WT and ACKO mice. Representative coronal sections demonstrate robust induction of phospho-synapsin in the cortex and hippocampus of ethanol- treated WT mice compared to saline controls. DKO mice demonstrate no induction of phospho-synapsin in either brain region following ethanol treatment compared to saline controls. Representative coronal sections demonstrate robust induction of phospho-synapsin in the hippocampus of ethanol-treated WT and AC8KO mice. In contrast, AC1KO mice impaired induction of phospho-synapsin in following ethanol treatment compared to WT and AC8KO mice. (Conti, et al., (2009) PLoS ONE4(5):e5697) Find Dr. Conti's lab webpage here.

Education and Training

BSE, Bioengineering, University of Pennsylvania
Ph.D., Neuroscience, University of Pennsylvania (Advisor: Julie A. Blendy, PhD)
Postdoctoral Fellow, Pediatrics, Washington University in St. Louis School of Medicine (Advisor: Louis J. Muglia)
Research Instructor, Washington University in St. Louis School of Medicine (Psychiatry)
 

Active Research Funding

K01- AA017683 NIH/NIAAA; Effects of Adenylyl Cyclases 1 and 8 on Neuronal Sensitivity to Ethanol; AC Conti, PI; DM Kuhn, Mentor (2008-2013)

Publications

Contact Information:

Alana C. Conti, Ph.D. (Principal Investigator)
Director of Research, Pediatric Neurosurgery
Assistant Professor, Department of Neurological Surgery
Wayne State University School of Medicine

Mailing Address
VAMC
4646 John R. St. (11R)
Rm. 4250
Detroit, MI 48201

E-mail: aconti@med.wayne.edu
Phone: 313.576.3311
Fax: 313.576.1112