Linking Innate Immune Responses in the CNS to Glutamate-Mediated Excitotoxicity: Studies in Acute Alphavirus Encephalomyelitis in Mice

David Irani, M.D.

Johns Hopkins University School of Medicine

Funded in June, 2004: $300000 for 3 years


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Exploring How Inflammation May Ultimately Lead to Motor Neuron Disease

These investigators will study a mouse model to determine whether immune microglial cells, which reside in the brain and spinal cord, produce inflammatory signals in response to a virus that ultimately leads to the death of motor neurons controlling movement. A similar disease in humans is the devastating and fatal Amyotrophic Lateral Sclerosis (ALS), often referred to as Lou Gehrig's disease. The research findings may demonstrate how anti-inflammatory agents can block initiation of this chain of events, thus preventing motor neuron disease.

As in the brain, microglial cells are the only resident immune cells in the spinal cord. Scientists have not yet determined how effectively microglial cells attack invaders in the brain or the spinal cord, which together comprise the central nervous system (CNS). Additionally, scientists suspect that microglial cells inflict harm to other CNS cells under certain circumstances. In the case of ALS, the researchers suggest, microglial cells become activated and produce inflammatory mediators. These mediators then precipitate a chain of events which results in destruction of the neurons that control movement (the "motor" neurons).

Many neurons communicate with one another using an excitatory neurotransmitter called glutamate. As glutamate is transmitted at the junction (synapse) from one nerve cell to another, excess glutamate spills out into the space surrounding the synapse. The spilled glutamate is ordinarily picked up by cells called astrocytes. This prevents high concentrations of glutamate from forming around the motor neuron synapse. If astrocytes fail to clear this excess glutamate, however, the neurotransmitter will overexcite motor neurons and cause them figuratively to fire to death. Literally, the excess glutamate causes the cells to degenerate and die.

This is what happens in the motor neuron disease ALS, the researchers hypothesize. Specifically, the researchers suggest, astrocytes fail to pick up the spilled glutamate because their receptors for glutamate become damaged and dysfunctional. These receptors are called "glutamate transporters." How these transporters are damaged has been a mystery.

Recent studies may provide a clue. They suggest that specific types of inflammation that are activated by microglial cells within the CNS precede the loss of glutamate transporters' function and their ability to clear away excess glutamate. Attempts to study this relationship between a preceding immune inflammatory response and subsequent astrocyte receptor failure have been difficult. The difficulty arises because the course of the animal model of ALS is exceedingly slow. The Hopkins researchers have circumvented this problem by developing an animal model disease that is similar to ALS but progresses much more rapidly. The disease is produced by a virus known as Sinabis. It causes severe ALS-like hind limb paralysis and progressive motor neuron destruction in the spinal cord in just eight to ten days.

The investigators will use this viral infection animal model to test their hypothesis that the virus activates immune microglial cells in the spinal cord to produce an inflammatory response. This inflammation interferes with glutamate transporters on astrocytes, preventing the astrocytes from taking up the excess glutamate that accumulates in the synaptic junction between two motor neurons. The resultant excessive concentrations of glutamate cause motor neurons to fire endlessly and eventually die.

If the researchers' hypothesis is correct, the results could spur the development of experimental anti-inflammatory therapies to stop this chain of events at its source: the initiation of inflammation by microglial cells.


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Linking Innate Immune Responses in the CNS to Glutamate-Mediated Excitotoxicity: Studies in Acute Alphavirus Encephalomyelitis in Mice

Glutamate-mediated excitotoxic injury of neurons has been implicated in the pathogenesis of many neurological disorders. Impaired reuptake of extracellular glutamate via down-regulation or dysfunction of astrocytic glutamate transporters is a common feature in many of these conditions. Although astrocytes express at least two distinct glutamate transporters, functional studies suggest that up to 95% glutamate reuptake is mediated by the protein, GLT-1/EAAT2. Loss of EAAT2 is particularly evident in motor neuron disorders such as Amyotrophic Lateral Sclerosis (ALS), although the molecular mechanisms involved in these events remain poorly understood. However, recent tissue gene expression studies in an animal model of ALS identify innate immune responses arising from activated microglial cells in the spinal cord as being likely proximal events. Not only does this suggest that anti-inflammatory strategies targeting these innate immune processes could be relevant in the treatment of these diseases, but it also highlights the connection between inflammation and neurodegeneration. Clarifying the molecular underpinning of this connection could have broad eventual clinical relevance.

We study neuroadapted Sindbis virus (NSV) encephalomyelitis in mice, a well-established CNS alphavirus infection model. This disease causes severe hindlimb paralysis and progressive lumbar motor neuron destruction in the spinal cords of infected animals over a period of 8-10 days. Similarities to the poliomyelitis-like illness that develops in humans infected with West Nile virus (WNV) are notable, and the disease evolves with much faster kinetics than animal models of ALS. Pharmacological blockade of AMPA glutamate receptors and inhibition of virus-induced microglial activation both independently prevent NSV-induced paralysis and lumbar motor neuron destruction without altering CNS virus replication or spread, suggesting that they each exert neuroprotective functions. Taken together, we hypothesize that NSV induces local microglial activation in the spinal cord leading to disrupted astrocytic glutamate reuptake, rising extracellular glutamate concentrations, and ultimately AMPA receptor-mediated destruction of infected and non-infected motor neurons alike.

To test this model, we propose the following three Specific Aims:

1. To correlate altered GLT-1 expression and/or function with the degree of hindlimb paralysis and motor neuron injury in the spinal cords of NSV-infected mice;

2. To clarify the role that IL-1ß plays in NSV-induced down-regulation of GLT-1 expression and/or function;

3. To search for other innate immune mediators arising from microglia that contribute to NSV-induced down-regulation of GLT-1 expression and/or function.


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David Irani, M.D.

David Irani, M.D., is an Assistant Professor in the Department of Neurology at the Johns Hopkins University School of Medicine, and is an Assistant Professor in the Department of Molecular Microbiology and Immunology at the Johns Hopkins University Bloomberg School of Public Health. He completed his medical training at the University of Michigan in 1987 and finished his residency training in neurology in 1993. Since that time, his laboratory has been situated within the Neurovirology Research Group now centered in the Johns Hopkins University Bloomberg School of Public Health. The main focus of his research interests are in viral pathogenesis in the nervous system, and, in particular, the role that host responses play in the pathogenesis of viral infections of the brain and spinal cord.


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Prow N.A. and Irani D.N.  The opioid receptor antagonist, naloxone, protects spinal motor neurons in a murine model of alphavirus encephalomyelitis.  Exp Neurol. 2007 Jun;205(2):461-70.