Glia in Brain Development and Function
Neurons are not the only cells in the nervous system. Glial cells constitute ~60 percent of the cells in the human brain and are emerging as major regulators of nervous system development, function, and health. Despite their abundance in the nervous system, we know surprisingly little about any aspect of glial biology. How are glia made? How diverse are they at the molecular level? How do they take on their elaborate morphologies during development? How do they interact with neurons during neural circuit assembly or plasticity? What is their function in the mature brain? We have been pioneering the use of Drosophila as a model system to delve deeply into the biology of this amazing group of cells.
Development and Function of Astrocytes
Shortly after a mammal is born, astrocytes (star-shaped cells that are the most predominant glial subtype in the brain and spinal cord) invade synapse-rich regions of the brain and become intimately associated with nearly all synapses, the fundamental unit of neuron-neuron signaling. Why are astrocytes so closely coupled with synapses? Recent work points to important roles for astrocytes in synapse formation, maturation, and efficacy, and it is likely that glia are also active participants in synaptic signaling and plasticity. We wish to understand the molecular bases of these interactions and the precise roles of glia in central nervous system (CNS) information processing.
Are there astrocytes in Drosophila? Using a variety of molecular-genetic approaches, we recently identified a cell type in the Drosophila brain that bears a striking resemblance to mammalian astrocytes at the morphological and molecular levels. For example, the fly astrocyte takes on a tortuous morphology, extending profuse membrane specializations throughout the brain, which associate closely with CNS synapses (Figure 1). These cells, like mammalian astrocytes, express transporters essential for the clearance of major neurotransmitters from the synaptic cleft (e.g., glutamate and GABA), arguing for an important role for fly astrocytes in modulating neurotransmitter tone in the brain. Using a collection of tools we recently developed, we have embarked on the first molecular-genetic analysis of astrocyte development and function in an organism amenable to rapid genetic analysis. We are exploring a number of questions: How do astrocytes take on their tortuous morphologies? Do astrocytes occupy unique spatial domains in the CNS? Is astrocyte morphology or synapse association responsive to neural activity? Are astrocytes actively remodeled during development? How do astrocytes modulate synaptic signaling?
Neuron-Glia Signaling Sculpts Axonal and Synaptic Connectivity
It is widely believed that glial cells play an important role in wiring the nervous system, but compelling evidence supporting a requirement for glia in the establishment of neural connectivity has remained scarce. We are exploring the mechanisms by which glia sculpt neural connectivity during development in two contexts: the adult Drosophila antennal lobe and the larval neuromuscular junction (NMJ). Through glial-specific knockdown of a number of signaling molecules we have found that developing adult brain glia are critical players in the wiring of the adult olfactory system (Figure 2). This observation implicates glia in establishment of the precise spatial map of olfactory receptor neuron (ORN) axon connectivity in the antennal lobe. We are now seeking to determine how neuron-glia interactions sculpt axonal connections in this tissue.
To explore how glia sculpt synaptic fields, we turned to the Drosophila larval NMJ (in collaboration with Vivian Budnik, University of Massachusetts Medical School). During larval life, the NMJ increases ~100-fold in size, and thereby acts as a developmental model for synaptic plasticity. We find that suppressing glial phagocytic activity leads to the accumulation of massive amounts of presynaptically derived debris at the NMJ (Figure 3) and a dramatic decrease in the formation of new boutons. We believe that normal synapse expansion entails large-scale shedding of presynaptic membranes, that glia transiently invade the NMJ to engulf this material, and that constitutive clearance of presynaptic material is essential for normal synaptic expansion. We hope to use the Drosophila NMJ to probe additional fundamental questions in glial biology by exploring, in live preparations, the effects of neural activity on glial motility and Ca2+ signaling, and determining additional ways in which glia can modulate synaptic growth and connectivity.
Molecular Pathways Mediating Glial Response to Neurodegeneration
Glia have the ability to sense any neural injury (e.g., spinal cord injury, ischemia, or neurodegenerative disease) and respond by undergoing reactive gliosis, a process whereby glia exhibit dramatic changes in morphology and gene expression patterns, migrate to the injury site, and manage brain responses to trauma. Reactive gliosis has been a major topic of study in the field of glial cell biology for more than a decade, but molecular pathways mediating neuron-glia signaling after injury have remained largely undefined.
We developed a simple assay for acute nerve injury in Drosophila and showed that severed Drosophila axons degenerate after a defined latent phase and elicit potent morphological and molecular responses from glia: within hours after injury, glia up-regulated the expression of Draper (the Drosophila ortholog of the Caenorhabditis elegans cell corpse engulfment receptor CED-1; Figure 3), extended membranes toward severed axons, and phagocytosed degenerating axonal debris. In draper mutants, glia failed to respond to axon injury or clear axonal debris from the CNS. These observations demonstrate that Drosophila glia can respond to brain trauma and phagocytose degenerating axonal debris, and identify Draper as a central mediator of these events. In addition, based on the requirement for the cell corpse engulfment receptor Draper for clearance, this work implies that cell corpses and degenerating axons may present similar engulfment cues to local phagocytes. We are now defining the Draper signaling pathway and identifying additional signaling molecules that mediate the neuron→glia signaling events after axon injury.
Molecular Mechanisms of Axon Autodestruction
We are interested in understanding how axons undergo autodestruction and then autonomously tag themselves for engulfment by glia. Severing axons offers a simple model in which to study axon degeneration: when an axon is cut, the distal portion undergoes catastrophic fragmentation after a defined latent phase. This process, termed Wallerian degeneration, was long thought to represent a passive wasting away of the axon due to a lack of nutrient supplies from the cell body. However, the identification of the slow Wallerian degeneration (Wlds) molecule, whose overexpression can suppress axon degeneration in mice for weeks after axotomy, forced us to reconsider this notion. It is now thought that Wallerian degeneration may be an active program of axon autodestruction, akin to apoptotic death, that is somehow suppressed by Wlds. We recently discovered that severed Drosophila axons undergo Wallerian degeneration that can be strongly suppressed by mouse Wlds (Figure 4); thus the molecular mechanisms driving Wallerian degeneration and Wlds function are conserved in Drosophila and mammals. This observation opened the door to a detailed molecular-genetic analysis of Wallerian degeneration in Drosophila. We are now attempting to dissect the in vivo action of Wlds and are exploring more basic aspects of Wallerian degeneration; for example, is Wallerian degeneration an active process? To answer this question we performed a forward genetic screen in the fly and identified dSarm/Sarm1—the first known molecule that, when knocked out, potently blocks Wallerian degeneration for weeks in both flies and mice. We are now deeply immersed in trying to (1) understand how dSarm/Sarm1 promotes axonal degeneration, (2) identify additional "axon death" signaling pathways, and (3) determine the identity of the long-elusive neuron→glia "injury" signals.
Partial support for these projects was provided by grants from the National Institutes of Health, the Christopher and Dana Reeve Foundation, the Smith Family Foundation, the Worcester Foundation for Biomedical Research, the ALS Therapy Alliance, the Gaffin Family, and the Alfred P. Sloan Foundation.
As of August 12, 2013