University of Washington
School of Medicine

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My laboratory studies the molecular mechanisms that control the amplitude of neuroinflammatory responses, which differ between neuropathologies. Some neuropathologies induce exacerbated neuroinflammatory responses, while others inhibit them. We are particularly interested in determining the signaling molecules produced by microglia and astrocytes that lead to either exacerbated or inhibited neuroinflammatory responses, with the aim of identifying therapeutics that keep these responses in check and reduce the cell damage that uncontrolled neuroinflammatory responses induce.

Microglial cells, the resident macrophages of the brain, are highly plastic cells. Upon "activation", they undergo a rapid, multi-step change in phenotype. For example, activated microglia retract their processes and extend lamellipodia, allowing them to migrate towards injured tissue. They proliferate and can, under certain conditions, release signaling molecules, including cytokines, that orchestrate neuroinflammation and control its expansion. Activated microglia also release cytotoxins and engulf the cellular debris.

Astrocytes, the most abundant glial cells of the CNS, are closely juxtaposed to virtually all neurons. By removing neurotransmitters from the synaptic cleft and buffering potassium ions released by depolarized neurons, astrocytes assist neurons in their ability to communicate between each other. Under neuropathological conditions, astrocytes also release cytokines that control the extent of neuroinflammatory responses mediated by microglial cells and invading immune cells.

Several IN VITRO and IN VIVO experiments suggest that cannabinoids, i.e., molecules that have a similar chemical structure to the marijuana component delta⁹-tetrahydrocannabinol (THC), affect neuroinflammation.

To date, we know that:

1. Plant derived and synthetic cannabinoids reduce inflammation.
2. Activated microglial cells express cannabinoid receptors, the molecular target for cannabinoids. (Figure 1, below.)
3. Resting microglial cells do not express cannabinoid receptors.
4. Cannabinoids stimulate activated microglial cells to migrate and inhibit their ability to release cytokines.
5. Upon acute stimulation, microglial cells increase their production of endocannabinoids, the endogenous ligands for the cannabinoid receptors.
6. Upon prolonged activation, microglial cells reduce their ability to produce endocannabinoids.
7. Healthy, mature astrocytes express cannabinoid receptors.
8. Upon acute stimulation, astrocytes produce endocannabinoids, although to a lesser extent than microglial cells.

To study the cannabinoid signaling system in microglia and astrocytes, we use mouse cells in primary culture and measure the activity of each of the components that constitute the cannabinoid signaling system. We routinely use the following techniques:

1. Chemical-ionization gas chromatography/mass spectrometry (CI-GC/MS) to determine endocannabinoid amounts. (Figure 2, below.)
2. The presence and functionality of cannabinoid receptors is measured by classic pharmacological approaches (binding, signal transduction assay, western blots, immunocytochemistry and cell migration). (Figure 3, below.)
3. Endocannabinoid biosynthesis and inactivation by uptake and hydrolysis is determined using radioactive substrates.
4. Cell transfection is used to assess the diverse molecular components that constitute the cannabinoid signaling system.
5. As part of several collaborative efforts, we have access to different wild type and transgenic mice models of neuroinflammation.

Some of the questions that we are currently addressing are: "Do endocannabinoids orchestrate neuroinflammation?" "Do cannabinoids affect the interactions between neurons, astrocytes and microglia that occur during uncontrolled neuroinflammatory responses?" Our working hypothesis is that uncontrolled neuroinflammatory responses, such as occurring in Alzheimer's disease, AIDS dementia and multiple sclerosis, are associated with - or are due to - malfunction of the cannabinoid signaling system. This malfunction likely disables the negative feedback mechanism that normally keeps neuroinflammatory responses in check.

Another set of questions that we are currently addressing are: "What enables astrocytomas to escape immune surveillance?" "Do the microglial cells that are located within astrocytomas have a compromised phenotype?" Recent data show that astrocytomas likely express a specific subtype of cannabinoid receptors and have an altered endocannabinoid production mechanism. Our aim is to determine if impairment of the cannabinoid signaling system in astrocytomas enables them to escape immune surveillance. This research should identify molecular targets that could enhance the CNS immune response towards astrocytomas, which will hopefully reduce, stop or even reverse tumor growth.

Our broad, long-term goal is to understand the molecular mechanisms underlying immune surveillance in the brain and the basic biology of microglial cell and astrocyte function, which could provide novel therapeutic avenues to treat Alzheimer's Disease, AIDS dementia, multiple sclerosis and brain tumors.