Imaging Neuro-Immune Interactions in a Model of Multiple Sclerosis

Thomas Misgeld, M.D

Harvard University

Funded in September, 2003: $100000 for 4 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . FINDINGS . SELECTED PUBLICATIONS .

ABSTRACT

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Imaging Neuro-Immune Interactions in a Model of Multiple Sclerosis

Multiple sclerosis (MS) is the most common neurological cause of disability in young adults. In MS, immune cells invade the central nervous system and damage nerve and glial cells. Loss of myelin is a cardinal feature of MS, but axon damage determines the long-term outcome of the disease. One prime cellular suspect for causing axon damage in MS and its animal model, experimental autoimmune encephalomyelitis (EAE), is the macrophage. Post-mortem studies show a strong correlation between macrophage infiltration and axon damage. Further, macrophages are in close contact with damaged axons, suggesting that they can directly attack axons. This concept, however, has so far not been tested directly and the mechanisms of macrophage-induced axon damage are not understood.

One reason why we know so little about the mechanism of axon damage in neuro-inflammatory diseases is that it has never been observed at a cellular level in a living animal. We have developed an in vivo microscopy technique that allows us to monitor the interactions between fluorescently labeled macrophages and axons in spinal EAE lesions. This technique will enable us to visualize whether, when, and where macrophages inflict damage on axons in EAE. We hope to elucidate the mechanisms of macrophage-mediated axonal damage and thus provide novel targets for therapeutic intervention.

 

HYPOTHESIS

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Hypothesis:
In multiple sclerosis, immune cells damage axons and myelin in the central nervous system. The extent of axonal damage determines the degree of persistent neurological deficits of multiple sclerosis patients. Macrophages have emerged as prime suspects in mediating this damage.

We plan to monitor the interactions between macrophages and axons by in vivo microscopy in an animal model of multiple sclerosis to test the hypothesis that macrophages directly damage axons, and to pinpoint where and when this damage occurs. Our approach will provide insight into the mechanisms of inflammatory axonal damage. It will allow direct assessment of therapeutic interventions on macrophage-axon interactions and help to suggest new targets and time-windows for therapy.

Goals:
1. Visualize axons and macrophages in the spinal cord of living mice in an animal model of multiple sclerosis using intravital microscopy.
2. Directly image how macrophages induce axonal damage to test whether:

     a. macrophages selectively interact with axons,
     b. there is axonal damage specifically at the interaction site, and whether
     c. axon damage precedes macrophage contact or vice versa.3. Study the mechanisms of macrophage-mediated axonal damage and document the effects of therapeutic interventions.

Methods:
To visualize axons and macrophages in living animals, we use transgenic mice in which axons and macrophages are labeled with fluorescent proteins. By using local injections of immune mediators, we can specifically target inflammatory lesions to parts of the spinal cord, which we can image using in vivo microscopy.

 

FINDINGS

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Lay summary:
When the immune system erroneously turns against our own body, an “autoimmune disease” occurs. In the case of the central nervous system (the brain and the spinal cord), the resulting disease is known as multiple sclerosis. Multiple sclerosis is a relatively common cause of paralysis in young adults. Recently it has become clear that the nerve cell processes themselves, the axons, are damaged in multiple sclerosis and that this axon damage determines how severely a patient is incapacitated by the disease. To understand how nerve processes are damaged will be an important step towards finding ways to prevent or at least reduce such damage. As nerve processes in the inflamed brain interact with many different cells, one big step forward would be to observe which kind of interactions can lead to damage of nerve processes. This is currently not possible in humans, but seems feasible in small animals such as mice, where a disease state similar to multiple sclerosis can be induced.

Supported by the Dana Foundation, we have developed a method that allows us to do just that: watch the interaction of immune cells and nerve processes in the inflamed nervous system. We found that for a nerve process to be damaged it has to be in contact with an immune cell for an extended period of time. Such an interaction can then either result in complete destruction of the axon or its recovery. Locally within the attacked nerve processes, important structures known as mitochondria appear to be damaged early. Mitochondria are intracellular structures that produce energy, but can also be the source of toxic substances such as reactive derivates of oxygen. Currently we are exploring the possibility that the local changes in mitochondria determine whether an axon is doomed or can survive.

Scientific Summary:
Despite being a demyelinating disease, multiple sclerosis (MS) is characterized by substantial axon damage, which critically determines to a patient’s permanent disability. Underlying neuroinflammatory axon damage is a complicated and poorly understood mechanism, which likely involves the dynamic interaction of multiple immune and neural cell types. In order to elucidate the events that elicit axon damage in a mouse model of MS (experimental autoimmune encephalomyelitis, EAE), we have developed an in vivo imaging technique that allows direct visualization of interactions between axons and immune cells. We have focused our investigations on the actively inflamed areas of EAE lesions and found that microphages and axons engage in long-lasting contacts. Axons undergo reversible changes that manifest as local beading, which can either reverse or progress to overt fragmentation. Exploring the subcellular changes at sites of neuro-immune contacts, we found that neuronal mitochondria are disrupted locally even before any changes in axonal morphology become apparent. Currently we are exploring how these mitochondrial changes are induced and whether therapeutic strategies that protect mitochondria can prevent subsequent axon damage.

SELECTED PUBLICATIONS

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Misgeld T.*, Kerschensteiner M.*, Bareyre F.M., Burgess, R.W. and Lichtman J.W. Imaging axonal transport of mitochondria in vivo. Nat Methods. 2007 Jul;4(7):559-61. 
(*equal contribution)

Misgeld T., Nikic I., and Kerschensteiner M. In vivo imaging of single axons in the mouse spinal cord. Nat Protoc. 2007;2(2):263-8.

Misgeld T. and Kerschensteiner M. In vivo imaging of the diseased nervous system. Nat Rev Neurosci. 2006 Jun;7(6):449-63.

Kerschensteiner M., Schwab M., Lichtman J.W., and Misgeld T. In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat Med. 2005 May;11(5):572-7.