Glial Function in the Nervous System
The role of glia in nervous system development and homeostasis is an important but neglected problem in neurobiology, with important implication for human medicine. Seeking a principled approach to identify glial genes in Drosophila, we combined several techniques into a reverse genetic screen, with genome-wide expression profiling of FAC-sorted glial cells followed by injection-based RNA interference for initial functional analysis. This approach resulted in the discovery of many novel glial genes, including a GPCR pathway involved in blood-brain barrier regulation and a family of phagocytic receptors required for the clearance of apoptotic neurons. In subsequent work, we further characterized the molecular and cellular mechanisms underlying these processes and developed a large arsenal of tools and assays for the study of glia in vivo.
We continue to study the glial blood-brain barrier and phagocytosis, but have also become interested in a new problem - the interaction between glia and neurons in nervous system homeostasis in the mature animal, a question that has much bearing on the causation and development of neuro-degenerative diseases. In our original expression profiling, we had found many genes that might play a role in homeostasis to be upregulated in glia.
In order to identify relevant factors, we are now taking more direct functional genomic approaches using unbiased genome-wide RNAi. In a first screen, we identified genes required in glia for the survival of the adult animal by expressing transgenic RNAi (VDRC) with a pan-glial driver and scoring for lethality/subviability in the F1 progeny. This screen was highly successful and yielded previously known glial genes as well as a large number of new, uncharacterized genes. We are now using different strategies for selecting candidates out of this pool, tailored to each problem under investigation. To discover genes specifically required for blood-brain barrier formation and maintenance, we express RNAi in the relevant glial subpopulation and again score for subviability/lethality. To identify factors involved in phagocytosis, we initially use a cell-based RNAi assay in conjunction with FACS and microscopic analysis, since many phagocytosis genes do not cause lethality in the organism due to molecular redundancy. Upon completion of these large-scale functional screens, we will select small subsets of genes that will be studied in greater molecular and cellular detail, using techniques well established in the lab.
Investigating the contribution of glia to nervous system homeostasis requires the development of new genetic tools and functional, imaging-based assays. In a collaboration with the Janelia Farm Research Campus of HHMI, we are characterizing all glia sub-types in the adult brain and identifying specific drivers for each population. In addition, we are adapting and developing genetically encoded biosensors to assess the health status of neurons. The next crucial step will be to integrate these different tools into an assay system that allows us to measure the effects of perturbations in glia on the health of neurons, by tracking biosensor readouts over time in the living animal.