Research Projects
Formation and wiring of the brain requires the intricate coordination and regulation of a series of events. Neurons must polarize, and extend axons and dendrites. Axon extension must be terminated in the appropriate location. Finally, synapses must be formed at the correct time, and in the correct location. All of these events must be regulated, coordinated and integrated. While we understand much about the regulation of individual developmental events, we know very little about how different events are coordinated on a molecular level.
The Grill lab studies candidate intracellular coordinators of neuronal development. By unraveling the mechanisms by which these molecules function, we will gain insight into how neurons integrate and manage numerous signals from their environment to form a neural network. We also hope to identify key molecular functions that can be stimulated to trigger new synapse and axonal growth. Such knowledge has tremendous potential to aid in generating new therapies to treat neurodegenerative diseases, and injury to the central nervous system from stroke.
Importantly, the major events in the life of a developing neuron are conserved from the simple microscopic nematode, C. elegans, through the mammalian brain. The Grill lab uses C. elegans as a model system to study the molecular mechanisms of neuronal development for a number of important reasons.
1) Axon and synapse morphology and patterning can be easily visualized in living C. elegans. 2) Powerful genetic and transgenic approaches are well developed in C. elegans, and enable us to obtain important functional information about the molecules we study. 3) The molecular mechanisms that govern synapse formation and axon development in C. elegans are conserved in the mammalian nervous system. 4) The Grill lab uses innovative and powerful proteomic screens that are ideally suited to decipher the composition and role of protein complexes that regulate neuronal development and function.
Several areas of active research are discussed below.
1) How does the Regulator of Presynaptic Morphology (RPM)-1 function in axon termination and synapse formation?
C. elegans RPM-1 has two known functions. It acts as an ubiquitin ligase to negatively regulate the Dual Leucine Zipper-bearing Kinase (DLK)-1, and positively regulates a Rab GTPase pathway. Importantly, studies in flies, fish and mice have established that RPM-1 is part of a larger protein family that functions in axon extension, termination and guidance, as well as synapse formation. RPM-1′s functional promiscuity, gigantic size, and evolutionary conservation make it a compelling candidate as a coordinator of different events in neuronal development. To further understand the mechanism of how RPM-1 functions, we performed a proteomic screen in which RPM-1 was purified from the neurons of C. elegans, and its binding proteins were identified by mass spectrometry. Using this approach, we have identified numerous, conserved RPM-1 binding proteins that, like RPM-1, regulate both axon termination and synapse formation. We are now actively engaged in deciphering the molecular relationship between RPM-1 and its binding proteins, and unraveling the mechanism of how RPM-1 binding proteins function.
Given our initial success with RPM-1 as a target in proteomic screens, we have moved to a second-generation screen that involves sophisticated protein purification techniques, such as Tandem Affinity Purification (TAP), and more sensitive mass spectrometry to obtain a comprehensive profile of RPM-1 binding proteins.
Another major area of focus is the Rab GTPase, Gut Granule Loss (GLO)-1, that mediates a portion of RPM-1′s function. GLO-1 can be locked in active and inactive conformations, which allows us to perform proteomic screens to identify both activating and effector proteins. Given GLO-1′s function in mediating RPM-1 activity, we hope to better understand how RPM-1 functions by expanding our knowledge of GLO-1′s mechanism of action.
2) What are the mechanisms that control the function of the DLK-1 MAPK cascade?
Like RPM-1, DLK-1 functions in synapse formation, axon termination and axon extension, and is highly conserved in mammals. DLK-1 is also essential for axon regeneration in C. elegans, which further heightens our interest in this molecule, and its downstream kinases MKK-4 and PMK-3 (a p38 MAP kinase). While RPM-1′s role as a negative regulator of the DLK-1 pathway is well studied, we have recently found that a phosphatase of the PP2C family, PPM-1, also negatively regulates the DLK-1 pathway. We are actively investigating if other members of the PP2C family also function in neuronal development, and regulation of the DLK-1 pathway. Understanding how the activity of DLK-1 is controlled is essential for the development of novel pharmaceuticals that affect DLK-1 activity.
3) How do we identify novel coordinators of neuronal development and synaptic activity?
We have identified several molecules, aside from RPM-1, that function in behavior or synaptic function, and that also function in synapse formation and axon termination. The functional promiscuity, molecular composition, and evolutionary conservation of these molecules suggest that they may coordinate different events in neuronal development with synaptic activity. To test this hypothesis, we have initiated proteomic screens with candidates in which TAP purification and mass spectrometry are used to identify their binding proteins. By obtaining information about protein interaction networks, we hope to begin assembling the signaling interactome that relays information in a neuron to coordinate different events in development with synaptic function.