RESEARCH DIVISIONS

Basic Research

ALS/Neuromuscular

The Department of Neurology is currently conducting various research projects pertaining to ALS/Neuromuscular. These include:

Glutamate in ALS: There is evidence suggesting excess excitotoxicity in ALS, probably from glutamate, a stimulatory chemical released from some nerve endings to carry the nerve signal from one neuron to the next in a chain. It is also released if nervous tissue is damaged. Too much glutamate over stimulates neurons, which can cause them to die.

Strong evidence of excess glutamate in ALS comes from Dr. Jeffrey Rothstein's work showing a partial deficiency of the glial glutamate uptake transporter GLT1. This transporter is present on supporting glial cells around neurons, and is responsible for removing glutamate by absorbing it into the glial cells. If there is insufficient GLT1, there will be excess glutamate to over stimulate neurons, predisposing them to early death.

It is uncertain how this deficiency of GLT1 arises. Studies have predominantly used autopsy tissue. To learn more about glutamate in ALS, we have undertaken studies of ALS patients' brains in life using MRI spectroscopy. Using a regular clinical MRI machine with spectroscopy capacity, we have shown that in the motor cortex and brainstem of the brains of ALS patients there is a progressive loss of a chemical, N-acetyl-aspartate (NAA), that is a marker of the number and health of nerve cells. Evidence of an increased signal from glutamate and glutamine in motor cortex and brainstem of ALS patients was searched for. Accurate estimation of the amount of these chemicals requires a higher field-strength magnet MRI than we have available, but our findings suggest that the total levels of glutamate and glutamine, which includes both intracellular and extracellular, are decreased.

Work from other laboratories indicates that GLT1 is a complex protein that exists in several different forms in ALS and normal nervous system tissue, and that there are no mutations of the gene for GLT1 that have been found to cause the decreased level of GLT1 protein. Clearly, a greater understanding of the role of glutamate excitotoxicity in ALS is needed.

Surrogate Markers for ALS: Currently no laboratory test is available to prove that a patient has ALS, except in the one to two percent of patients with mutations of the gene for superoxide dismutase -1 (SOD1). The only marker of disease progression is the extent of the abnormal neurological signs measured by a neurologist, such as the amount of muscle weakness, muscle wasting, breathing tests, and reflex changes.

Programs to discover drugs to treat ALS use these abnormal neurological signs to measure rates of disease progression. However, because ALS is so variable, drug trials need very large numbers of patients. It may take a trial of 2,000 patients treated for 18 months to prove whether a drug is effective or not.

There would be significant improvement in the diagnosing and monitoring ALS and in new drug development if we had a marker that was relatively specific and sensitive for ALS, and which also changed as the disease progressed. The department, as well as other laboratories, used MR spectroscopy to study NAA, a chemical present in neurons. The motor cortex levels of NAA fall as ALS progresses. Other research teams found that treatment with riluzole (which slows ALS disease progression) raise the levels of NAA slightly. More research is needed to find a reliable surrogate marker for ALS to assist in programs for more rapid screening of drugs that may benefit ALS.

Oxidative Damage in ALS: Though oxygen is essential to advanced life forms, it is also potentially toxic. All cells of humans have a complex system of enzymes and other substances designed to control the effect of oxygen. One of these enzymes is SOD1. Discovery of mutations of SOD1 in about 20 percent of patients with familial ALS has increased the awareness that damage of motor neurons by excess oxygen free radicals may be a cause of ALS. Other research groups have shown that there is oxidative damage of proteins and other macromolecules in the nervous tissue of ALS patients.

Surprisingly, oxidative damage is not the mechanism by which SOD1 mutations produce the motor neuron degeneration in ALS. Research is under way to find exactly how the mutant SOD1 protein damages the cells.

Addiction Research Project

Laboratory Staff

Molecular Biology/Pharmacology	Michael Hurley, Ph.D., Research Scientist
Behavioral Pharmacology	John Haracz, M.D., Ph.D., Research Scientist
Neurochemistry	John Pablo, Ph.D., Research Scientist
Neuropharmacology	Stephanie Collins, Ph.D., Research Scientist
Neuroanatomy/Neurohistology	Margaret Basile, M.S., Technical Specialist
Neurochemistry	Qinjie Ouyang, B.S., Technical Specialist
Neurochemistry	Shane Haines, B.S., Technical Specialist

Clinical Staff

Clinical Assessment Coordinator:  Craig Kovera, M.A.
Social Worker: Judith Hitchman, M.S.W., C.A.A.P. 2 (Certified Addiction Professional)

Brain Bank Staff

Research Program Coordinator     Lilian Dominkovics, B.S.
Patient Records Coordinator    Jocely Gonzalez, M.S.
Postdoctoral Associate    John Pablo, Ph.D.
Technical Specialist    Margaret Basile, M.S.
Brain Bank Technical Specialist   Milvio Gomez, P.A.

Goals

• To develop a therapeutic agent to eliminate withdrawal symptoms and drug craving during the detoxification process.
• To develop a therapeutic as an adjunct to treatment to help prevent relapse during the critical period of early abstinence.
• To elucidate the neurochemical alterations that occur with chronic substance abuse by studying the human brain. Understanding how drug abuse affects the brain will provide new avenues for the development of potential therapeutics for substance abuse disorders.
• To interact with addiction professionals in South Florida to bring advances from the research bench to the client's bedside. By serving as a link to treatment providers, our scientific team can provide information on the latest research developments for substance abuse disorders.

Mitochondrial Research

The mitochondrial research program of the Department of Neurology is studying the role of mitochondrial dysfunction in neurological disorders. Principal investigators working in this area include Carlos T. Moraes, Ph.D., Thomas Sick, Ph.D., Miguel Perez-Pinzon, Ph.D. and Antoni Barrientos.

Mitochondria are small structures inside the cell that are the powerhouse of the cell. Most of the energy necessary for various biochemical reactions is produced in the mitochondria.

A large number of diseases of skeletal muscle, heart muscle, the peripheral and central nervous systems, the eye, and other organs of the body are due to mutations of the mitochondrial genome. These diseases frequently show inheritance through the maternal line, because all the mitochondria are inherited from the mother. These diseases show a great variability in their clinical presentation, because there are many thousands of mitochondria per cell, only a small percentage of which may have the abnormal mutated mitochondrial genome, whereas others will be normal.

Research projects in this area include the role of mitochondrial dysfunction in: ALS, Parkinson’s disease, genetic disorders, stroke, seizures, and diabetes. It also involves more basic research on the function of normal mitochondria.

The mitochondrial research program uses such cutting-edge techniques as the introduction of experimental genes, the knockout of normal genes, the regulation of normal genes, and the creation of transgenic animals. All of these techniques are brought to bear on the basic problem of understanding human neurological disease.

Spinal Cord Pharmacology

Research is currently being conducted to examine the actions of amino acid neurotransmitters in the spinal cord. John C. Hackman, Ph.D., professor of neurology and Alexander Y. Valeyev, Ph.D., research assistant professor of neurology, have been researching these transmitters that include the excitatory amino acid glutamate and the inhibitory amino acid gamma-aminobutyric acid (GABA), which are responsible for the majority of fast neurotransmission in the central nervous system. Each transmitter acts on a variety of receptors and the action of each receptor may be altered by other transmitters/modulators. Researchers have recently been studying the interactions between other transmitters such as serotonin and norepinephrine and glutamate and GABA.

The laboratory uses a systems approach with an intact spinal cord slice preparation to study these interactions. The laboratory also studies the pharmacology and biophysical properties of GABA receptors on cultured dorsal root ganglion cells using whole cell and isolated patch clamp techniques. These cells are the cells that bring sensory information into the spinal cord and are important in the transmission of painful stimuli into the spinal cord.

With the assistance of Charles W. Luetje, Ph.D. in the Department of Molecular and Cellular Pharmacology, the laboratory has begun to characterize the subunits of the GABA receptor present in the human dorsal root ganglion using molecular biological techniques. The laboratory also has begun to culture and transfect cells with GABA subunits to study the effects of different subunit combinations on the pharmacology of the GABA receptor.

Interdepartmental collaborations include Paul Schiller, Ph.D. of the Geriatric Research Program at the Miami Veterans Affairs Medical Center and Patrick M. Wood, Ph.D. of the Miami Project to Cure Paralysis.

Parkinson’s and Drugs of Addiction

Established in 1986, the Brain Endowment Bank has coordinated a nationwide network to accept brain donations for clinical and basic research. A variety of studies are currently being conducted at the Brain Endowment Bank, including Parkinson’s disease and other movement disorders, drug addiction, and aging.

Headed by Deborah Mash, Ph.D., professor, director of research, and Levey Chair in Parkinson’s Disease Research, the bank’s research team educates the public about the importance of brain donation as a permanent and invaluable research resource. The donor base includes normal and diseased brains from individuals with neurodegenerative and neuropsychiatric disorders.

The effects of chronic substance misuse is analyzed on the neurochemistry of the human brain. Through the use of molecular biology and neurochemical approaches, research is able to identify pathways that are affected by drug abuse.

Researchers are actively working to develop a therapeutic agent to eliminate withdrawal symptoms and drug craving during the detoxification process and to help prevent relapse. Interaction with addiction professionals in South Florida help to bring advances from the research bench to the client's bedside. By serving as a link to treatment providers, the bank’s scientific team can provide information on the latest research developments for substance abuse disorders.

Parkinson’s disease is one of the primary research focuses. Studies aim at identifying
risk factors associated with Parkinson’s disease and to identify the neurochemical changes that occur as the disease progresses. Researchers have demonstrated that cocaine abusers have an overexpression of the neuronal protein alpha-synuclein. The increase in alpha-synuclein measured in chronic cocaine abusers may be a toxic gain, predisposing addicts to neurodegenerative changes in DA neurons and the development of Parkinsonism.

Neuronal Aging

Neurovirology

Neurovirology includes the study of viral diseases of the nervous system. The laboratory based Neurovirology Program emphasizes the study of the molecular basis of viral neurocytopathogenicity and neural cell biology as it pertains to virus-cell interactions.

To that end, the Neurovirology research program featured novel human neural cell culture systems to support the in vitro study of viral infection, in particular human immunodeficiency virus (HIV-1) and the beta-herpesviruses. The cell cultures consist of mixed cell types necessary to support viral replication but also facilitate cell-cell interactions that may be important in disease. These cell cultures have been developed with the close collaboration of the neurobiologists working in the Miami Project to Cure Paralysis.

Three specific projects of the program include:

Proximal Mechanisms of Neuronal Injury in HIV-1 Infection: The summary objective of this project is to elucidate the early or proximal events and mechanisms of HIV-1-induced neuronal injury. Our hypothesis is that early events in HIV-associated neuronal injury are manifested by structural changes in neuronal cells, including alterations in neuronal connections. Micheline McCarthy, M.D., Ph.D., associate professor of neurology, along with other researchers, are testing this hypothesis with human cell cultures including neurons, astrocytes, and microglia. Thus far, preliminary data suggest that HIV-1 infection stimulates excess production of one of the neurofilament proteins found in neurons. This response has interesting parallels to experimental models of another neurodegenerative disease, amyotrophic lateral sclerosis (Lou Gehrig’s disease).

Herpesvirus-HIV Interactions and the Neuropathogenesis of HIV-1: The study of molecular interactions between human herpesviruses and HIV-1 has focused attention on the role these viruses may play as co-factors during the course of HIV-1 infection. Of the human beta-herpesviruses, cytomegalovirus (CMV) and human herpesvirus-6 (HHV-6) have similar molecular properties and are capable of replicating or persisting in CD4+ lymphocytes as well as invading the central nervous system. These properties make them particularly well suited to function as co-factors in HIV-1 infection in the brain. Researchers have demonstrated in a laboratory-based study that CMV infection or HHV-6 infection summarily activates the major gene-controlling region of HIV-1 (the long terminal repeat region, or LTR). These viruses enhance HIV-1 replication to a similar extent in human astrocytes.

An interesting by-product of this research project was the observation that HHV-6 can grow in human astrocytes and cause a specific type of cytopathology - syncytia formation or cell fusion into giant, multi-nucleated cells. HHV-6 has been reported to be re-activated in the brains of patients with multiple sclerosis(MS), and this virus’ ability to grow in astrocytes and oligodendrocytes may lead to viral-induced or viral-exacerbated attacks in some MS patients.

HIV-1 Infection in Neuroepithelial Stem cells: In the developing central nervous system, neuroepithelial stem cells proliferate and differentiate sequentially into multiple brain cell types, including neurons and astrocytes. We had expected HIV-1 virus to induce cell death (apoptosis) in stem cells or partially differentiated human neurons in laboratory cultures. This expectation was based on prior scientific journal publications from other labs reporting that HIV-1 induces apoptosis in mature neurons in culture. To our surprise, however, the virus did not induce apoptosis in stem cells or immature neurons. These cells seem relatively resistant to HIV-1. Thus we have an opportunity to study how the state of differentiation of the cell may protect against neuronal destruction. In addition, since stem cells are found in certain locations in adult brain, they may constitute a reservoir of potential neurons that could repair neurological damage by HIV-1.

Neuronal Metabolism

Research efforts in the area of neuronal metabolism include the examination of mitochondrial function and brain injury. The laboratory of Thomas Sick, Ph.D., professor and vice chair for research, is currently investigating changes in mitochondrial function and the consequences of these changes following brain ischemia, trauma, and neurodegeneration following exposure to neurotoxins.

The laboratory employs a variety of techniques for assessing changes in mitochondrial function including fluorescence and absorption spectrophotometry, fluorescence imaging, and immunohistochemistry. Standard electrophysiological methods, including extracellular, intracellular, and patch clamp recordings, are employed to monitor neuronal function.

Experiments are typically conducted using acute brain slice and organotypic slice culture models of neural injury. The laboratory boasts a wide international spectrum of investigators including post-doctoral fellows and research associates from China, India, Ukraine, Japan, and Germany. Also supported are graduate doctoral and master’s degree students from the Neuroscience Graduate Program and Biomedical Engineering.

Neuronal Molecular Biology

Research studies conducted in neuronal molecular biology are headed by Bingren Hu, Ph.D., assistant professor, Cerebral Vascular Disease Research Center. Hu focuses on four major areas concerning molecular mechanisms underlying neuronal damage after brain ischemia and traumatic injuries:

Intracellular signaling pathways: As demonstrated in various publications, Hu and his research team have found several signaling pathways that contribute to neuronal damage/protection after brain ischemia and hypoglycemia. These include MAP kinase pathway, AP1 pathways, CREB/ATF-2 pathways, Akt pathways and caspase-3 pathways. Understanding and manipulating these pathways may lead to new compounds that protect neurons against brain damage after ischemia.

Synaptic ultrastructure, biochemistry and function: Hu’s team has found dramatic modifications of synapses in their ultrastructure and molecular composition after brain ischemia. Synapses, the basic neurotransmission units, control all neuronal functions. Synaptic alteration after brain ischemia is mostly responsible for changes in neurological functions.

Protein damage and aggregation: Proteins are dramatically damaged and aggregated in neurons destined to die after brain ischemia and hypoglycemia. Hu’s study has demonstrated that protein aggregation contributes to delayed neuronal death after transient cerebral ischemia.

Neuronal apoptosis during brain maturation: Research has indicated that molecular mechanisms of neuronal death after ischemia are quite different between mature brains and immature brains. This may indicate that brain injury patients of various ages should be treated differently.

Cerebral Vascular Disease Research Center

Director
	Myron D. Ginsberg, M.D. m.ginsberg@miami.edu

Address
	National Parkinson Foundation Building 1501 N.W. 9th Avenue Miami, FL 33136 Phone: 305-243-6449

The Cerebral Vascular Disease Research Center at the University of Miami is a unique national resource devoted to experimental stroke research. Its uniqueness derives not only from its 34-year history of programmatic support by the National Institutes of Health, but more importantly from the fact that this longevity and continuity of funding has allowed it to evolve to its present status as a highly-integrated collaborative program in which virtually every project represents a multidisciplinary effort and in which complementary investigative strategies are brought to bear on experimental issues having high clinical relevance to the pathophysiology and therapy of human ischemic brain injury.

A major strength of this research group is the unparalleled continuity of the technical research personnel working within it. These highly trained individuals have, in many instances, worked with the Center for 1 or 2 decades. The stability of the technical support staff reflects their genuine commitment to, and pride in, the accomplishments of the Center.

A major organizational strength of this Center consists of its multifaceted core facilities supporting work in physiology, neuropathology, neurochemistry, molecular biology, tracer-kinetic strategies, computer science, image-processing, and behavior. The computer/image-processing core has evolved as a central resource of the Center over the past decade. This is reflected in its having successfully competed on two occasions for major hardware funding through Shared Instrumentation Grants of the National Institutes of Health.

The traditional and present strengths of the Center revolve around its expertise in experimental investigations. In addition, Center personnel collaborate closely with the clinical Stroke Division of the Department of Neurology in holding joint conferences, training postdoctoral fellows, and planning clinical research protocols in stroke.