Synapses are highly organized subcellular structures that transmit information within the nervous system and to other parts of our body. A typical chemical synapse is composed of three compartments: a presynaptic terminal for rapid neurotransmitter release, a postsynaptic site that is rich in neurotransmitter receptors to receive the signal, and a synaptic cleft that bridges the pre- and postsynaptic partners and maintains them in precise registration. How synapses are formed and maintained throughout the life of an organism are topics under intense investigation. We use the nematode C. elegans as an experimental system to examine the molecular genetic mechanisms regulating synapse formation, maintenance and function. The worm nervous system is composed of 302 neurons, with its entire connectivity known at the ultrastructural level. Synaptic connections can be observed in living animals using transgenic fluorescent protein markers. Our past studies elucidated conserved signaling pathways instructing synaptogenesis and axon guidance in the developing nervous system. In our recent studies, we have focused on the maintenance and function of synapses and mechanisms underlying neuronal response to injury.
Mechanisms of Synapse Maintenance
C. elegans moves in a sinusoidal pattern, which is achieved through dynamic balance of cholinergic excitation and GABAergic inhibition. In the locomotor circuit, the premotor interneurons signal through cholinergic (ACh) motor neurons to control the direction of movement. The ACh motor neurons form synapses to both body muscles and to GABAergic motor neurons, which in turn cross-inhibit contraction of muscles on the side opposite of the body. To understand the signaling network ensuring proper synaptic connection and transmission in the motor circuit, we used forward genetic screening to isolate mutants with altered locomotion. Our studies of these mutants have led to discovery of new regulators of the molecular machinery in synaptic vesicle release and inter-tissue crosstalk in controlling synapse density and transmission. An acetylcholine-gated ionotropic receptor, ACR-2, is localized to the somatodendrites of the ACh motor neurons and regulates locomotion speed. A gain-of-function mutation of acr-2 causes cholinergic over-excitation accompanied by reduction in GABAergic inhibition. The acr-2(gf) mutant animals exhibit spontaneous contraction, mimicking the physiological state of seizures. Interestingly, the molecular nature of the acr-2(gf) mutation is nearly identical to those associated with a form of frontal lobe epilepsy. Using genetics and electrophysiology, we have elucidated two mechanisms involving ion homeostasis and neuropeptide modulation to regulate excitation and inhibition balance of the motor circuit. We have also discovered a role for the epidermis, which encompasses the motor circuit, in neuronal excitability. An immunoglobulin superfamily (IgSF) transmembrane protein ZIG-10 is required in both ACh neurons and in the epidermis to restrict the density of cholinergic synapses. Loss of zig-10 function or misexpression of zig-10 alters the ratio of excitatory to inhibitory synapses. We find that in the epidermis ZIG-10 promotes phagocytosis through a Src kinase and the CED-1 phagocytotic receptor. ZIG-10-related IgSF proteins are broadly expressed in the nervous system of fly and mammals. We are further addressing the cell biology underlying phagocytosis in synapse refinement and maintenance.
Regulation of Synaptic Remodeling
The C. elegans adult motor circuit matures through a precisely timed synaptic remodeling in which the juvenile GABAergic motor neurons undergo selective synapse elimination and reformation. Strikingly, this synapse remodeling occurs without overt changes in neuronal morphology. Our early studies addressed the timing of this remodeling. We recently uncovered a mechanism involving the regulation of microtubule (MT) cytoskeleton in synapse remodeling. We find that increasing MT dynamics promotes synaptic remodeling, partly through modifications of motor proteins in directional trafficking of synaptic components. Neural circuits are frequently reorganized to produce and maintain a functional nervous system. Our studies establish parallels between C. elegans developmental circuit remodeling and synapse refinement in mammals.
Nerve Regeneration Mechanisms
The limited ability of mature neurons to regenerate upon injury is an enigma. Such limitations severely impede our ability to repair damaged nerves and to recover function after injury. We have developed laser-assisted axotomy to sever axons of C. elegans neurons. After femtosecond laser axotomy, the injured axons exhibit neuron-type, neurite-type, and stage-dependent regenerative responses, and can restore partial circuit function. The regrowth from severed axons is correlated with calcium transients that are triggered by axotomy. As C. elegans lacks myelin and has a generally permissive environment for regrowth, it offers an opportunity to investigate intrinsic growth pathways with high cellular specificity. Exploiting the rich genetic resources of C. elegans, we have undertaken a systematic screen for conserved genes on adult axon regeneration. A key pathway involves the conserved MAPKKK DLK-1, which we had previously shown to regulate synapse formation in the developing nervous system. DLK-1 is essential for regenerative responses, and increasing dlk-1 activation enhances growth extent of injured axons. The activation of DLK-1 is dependent on the injury-induced calcium transient. Recent studies indicate the function of the DLK pathway in axon regeneration is conserved in Drosophila and mammals. We have defined a novel mechanism that controls DLK-1 activation in vivo, which is likely conserved in mammalian homologs.
Cytoskeleton remodeling is a major event underlying regrowth of injured axons. Taking advantage of single-axon imaging, we have characterized an intricate sequence of changes in MT cytoskeleton in the seconds, minutes, and hours after injury. We identified a surprisingly rapid and dynamic re-localization of the conserved protein EFA-6 (Exchange-Factor-for-Arf6/EFA6) after injury.
Furthermore, we demonstrated that the relocalization of EFA-6 is concurrent with its inhibitory effects on axon regrowth. We defined a mechanism by which EFA-6 influences MT dynamics and axon regeneration through binding to doublecortin-like kinase (DCLK) and proteins interacting with MT minus ends in axons. Increasing evidence has shown that manipulating MT dynamics is beneficial for recovery from spinal cord injury. Our studies in C. elegans provide deep insights to the understanding of the key steps that an injured neuron must overcome for functional recovery.
Development of a New Optogenetic Method for Manipulation of Molecules and Cells
Optogenetic manipulation of cells and molecules using genetically encoded tags is revolutionizing neurobiology. Mini Singlet Oxygen Generator (miniSOG) is a genetic encoded photosensitizer, created by Roger Tsien's lab (HHMI, University of California, San Diego). Under blue light excitation, miniSOG produces green fluorescence and generates reactive oxygen species (ROS) that allow visualization of molecules by electron microscopy. In collaborative studies with the Tsien lab and the lab of Mark Ellisman (UCSD), we demonstrated that miniSOG could be used for in vivo labeling of organelles and molecules. Taking advantage of the light-inducible ROS-producing ability of miniSOG, we have also developed several applications to ablate cells, proteins, and induce heritable DNA modifications. In combination with other technologies, miniSOG has become a versatile agent to aid functional studies of many cellular processes.
Grants from the National Institutes of Health provided partial support for portions of this work.