Laura Bianchi - US grants

[unreadable] DESCRIPTION (provided by applicant): In nature, mechanical signaling plays fundamental roles in processes as diverse as cell volume regulation and the senses of touch and hearing. Recent work in nematodes, flies and mammals has implicated DEG/ENaC and TRP ion channels in osmo- and touch- sensation. Still, little is understood of how these channels function in vivo and how their activities are coordinated to maximize perception. In this proposal I combine experimental approaches uniquely applicable in C. elegans to characterize specific DEG/ENaC and TRP channels that act in well-characterized neurons to mediate mechanosensory perception. Aim I. I have identified a novel TRP-like stretch-sensitive ion channel in body touch neurons that is independent of the MEC-4 DEG/ENaC ion channel that senses gentle touch. Using in vivo calcium imaging, we have also found that touch receptors respond to harsher touch via a mechanism independent of MEC-4. My hypothesis is that the novel stretch-sensitive ion channel mediates calcium transients elicited by harsh touch and is required for normal responses to harsh touch, I will further characterize the stretch-sensitive channel, identify its gene and generate a knockout to test for its role in harsh touch behavioral responses. Aim II. In independent experiments I found that two novel DEG/ENaCs are expressed in the polymodal sensory neurons ASH (known to also require TRP channels OSM-9 and OCR-2 for function). DEG/ENaC deg-1 is co-expressed in these neurons. I have null mutants for all three DEG/ENaCs and I found that at least one of them (others are untested) shows defects in ASH-mediated nose touch response and osmolarity avoidance behavior. I will combine genetic, electrophysiological, behavioral, and imaging approaches to fully characterize the roles of DEG/ENaCs in ASH neuronal function. At completion of this work, I will be well positioned to address whether TRPs and DEG/ENaCs are functionally redundant or whether they "sense" distinct stimuli [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We depend on the sense of touch for most individual and social activities. Many forms of injuries and inflammation are accompanied by allodynia and mechanical hyperalgesia, conditions in which innocuous touch stimuli now cause severe pain. Despite its fundamental importance, mechanotransduction remains one of the least understood signaling process, both at the cellular and molecular levels. Touch is transduced by specialized mechanosensors embedded in the skin, most of which are composed of nerve endings and associated glial and epithelial cells. The role of these non-neuronal cells in touch sensation is poorly understood. Our goal is to understand touch sensation by dissecting functional interactions between mechanosensory neurons and glia using genetic, molecular and physiological strategies in C. elegans. Specifically, I hypothesize that such interactions employ the ion channel DELM-1, a member of the DEG/ENaC family of cationic channels, expressed in mechanosensory glia. Based on our preliminary results and what is known about the role of DEG/ENaC channels in epithelia, I hypothesize that glial DELM-1 is needed to control the concentration of K+ in the microenvironment between the neuronal ending and glia. Pilot experiments suggest that DELM-1 is inhibited by mechanical forces. Thus, I also hypothesize that the control of extracellular K+ by DELM-1 is highly dynamic and contributes to adaptation of the mechanosensors. The aims of this proposal are: 1) Does the glial DELM-1 channel regulate the activity of mechanosensory neurons? 2) Does DELM-1 elevate [K+] in the microenvironment between glia and touch neurons? 3) Is the glial DELM-1 channel mechanosensitive? Beautiful work in mammalian tissues using skin-nerve preparation and Von-Frey hair has allowed major progress in our understanding of touch sensation. I have now the unprecedented opportunity to capitalize on this previous work to explore a new area in the field. My work is likely to be relevant to our understanding of how associated cells, including glia, control the function of neurons throughout the nervous system. PUBLIC HEALTH RELEVANCE: Without the sense of touch we could not feed ourselves, hold objects, move around and care for our offspring. In many forms of injury and inflammation touch stimuli become painful. Touch is mediated by activation of receptors in the skin, most of which are composed of nerve cells and associated cells. The role of these associated cells in touch sensation is largely unknown. I propose here to advance our understanding of the functional interaction between nerve and associated cells in touch sensation using the tractable model system C. elegans. My work will ultimately help elucidate how cells associated with nerve cells contribute to sensory neuropathy and other human neurological disorders.

DESCRIPTION (provided by applicant): Developing neural circuits are actively remodeled as synapses are created in new locations and dismantled in others. These dynamic events are regulated by neuronal activity to produce mature circuits with specific physiological functions. This phenomenon has been observed throughout animal phylogeny which suggests that the underlying pathways are conserved. However, the molecular mechanisms that drive synaptic remodeling are largely unknown. Here we propose a strategy that exploits the simple model organism, C. elegans, to define a development program that remodels the synaptic architecture of a GABAergic circuit. Ventral synapses for DD class GABA neurons are relocated to new sites on the dorsal side during larval development. This synaptic remodeling program is blocked by the UNC-55/COUP-TF transcription factor in VD motor neurons which normally synapse with ventral muscles. We exploited this UNC- 55 function in a powerful cell-specific profiling strategy that identified 19 conserved genes with roles in synaptic remodeling. We have now shown that one of these UNC-55 targets, the DEG/ENaC cation channel, UNC-8, promotes synaptic remodeling in a mechanism that is activated by GABAergic signaling. This finding is important because DEG/ENaC proteins have been implicated in learning and memory but the mechanism that links DEG/ENaC function to synaptic plasticity is poorly understood. Specific Aim 1 tests the key prediction that UNC-8 is closely associated with GABAergic synapses that are remodeled by UNC-8 activity. Specific Aim 2 is designed to test the novel hypothesis that a Ca2+-dependent mechanism links neural activity to UNC-8 cation transport in a feedback loop that dismantles the presynaptic machinery. Specific Aim 3 defines the cellular origin and molecular components of the proposed activity-dependent pathway that regulates UNC-8 and promotes GABAergic synaptic remodeling. Together, these approaches offer a powerful opportunity to delineate an intricate molecular pathway that controls synaptic plasticity. Moreover, the conservation of these remodeling components in mammals argues that the results of this work are likely to reveal fundamental mechanisms that regulate synaptic plasticity in the human brain.

Isolated microenvironments like the synapse exist throughout the nervous system where the concentration of ions is regulated by accessory cells, including glia, quite independently from the surrounding tissues. The ionic composition of these microenvironments is key for neuronal function. Despite the fact that glial regulation of ion concentration in microenvironments is a main mode for regulating neuronal activity, our understanding of this type of regulation by glia is limited, especially for ions like Cl- and HCO3-. Furthermore, models are lacking where a comprehensive analysis can be performed on the glial ion channels and transporters involved in regulating ion concentrations, and how these proteins impact neuronal output, from molecule to animal behavior. In our over 10 years of effort aimed at advancing understanding of glia-neurons interaction and its impact on animal behavior using the model C. elegans, we have recently taken the unbiased approach of sequencing the mRNA of Amphid sheath glia. In this application, we propose to establish the mechanism by which one of the identified enriched genes, the glial Cl-/HCO3- permeable channel CLH-1, regulates neuronal output and animal behavior. We previously published that CLH-1 mediates pH regulation in the worm nervous system. Our preliminary results now show that CLH-1 is needed for normal nose-touch behavior. We hypothesize that glial CLH-1 regulates the activity of touch neurons via a direct effect of the permeating ions Cl- and HCO3- on neuronal DEG-1 channels. Thus, the specific aims of this application are: 1) In neurons, to establish the mechanism of neuronal dysfunction when clh-1 is knocked-out, 2) In glia, to determine whether it is the loss of permeation of Cl-, HCO3-, or both that produces the phenotype of clh-1 knock-out worms. Furthermore, in aim 3 we will exploit our proven approach to identify additional glial ion channels and transporter genes that are critical for the glial control of neuronal function and animal behavior: 3) To identify novel glial ion channels and transporters involved in glia-neurons interaction. The importance of regulating ion concentrations in neuronal microenvironments is underscored by the fact that several neurological diseases such as deafness, epilepsy, Alzheimer's, and even demyelinating diseases like multiple sclerosis are characterized by loss of ionic homeostasis. We propose here to use methodologies we have developed and proven to be effective to test mechanisms by which dysregulation of Cl- and HCO3- homeostasis in C. elegans leads to severe neuronal pathology. In addition, we will test the involvement of newly identified genes encoding glial channels and transporters in glia-neurons interaction.

Developing neural circuits are actively remodeled as synapses are created in new locations and dismantled in others. These dynamic changes are driven by the combined effects of genetic programs and neural activity that together shape the architecture and function of mature circuits. Synaptic plasticity has been observed throughout animal phylogeny which suggests that the underlying pathways are conserved and thus can be investigated in simple model organisms that are amenable to experimental analysis. Here we propose to use the nematode, C. elegans, to define a development program that remodels the synaptic architecture of a GABAergic circuit. During early larval development, DD-class GABAergic neurons undergo a dramatic remodeling program in which the presynaptic apparatus exchanges locations with postsynaptic components within the DD neuronal process. To reveal the mechanism of this effect, we are investigating the functional roles of ~20 conserved genes that we have determined are transcriptionally regulated to drive GABA neuron remodeling. Our work has shown that two of these targets, the DEG/ENaC cation channel protein, UNC-8, and ARX-5/p21, a conserved component of the Arp2/3 complex, function together in an activity-dependent mechanism that dismantles the presynaptic domain. Aim 1 tests the hypothesis that UNC-8 cation transport elevates intracellular calcium to drive presynaptic disassembly and that this effect is regulated by calcium- dependent phosphorylation. This goal is important because members of the DEG/ENaC protein family have been implicated in learning and memory but the mechanism that links DEG/ENaC function to synaptic plasticity is poorly understood. Aim 2 tests the hypothesis that the UNC-8 function triggers an actin-dependent endocytic mechanism that recycles presynaptic components for reassembly at new locations. These experiments derive from our surprising discovery that a key functional protein of the Arp2/3 actin-branching complex is transcriptionally regulated to effect synapse removal and that newly identified components of an endocytic recycling pathway are involved. Together, these approaches offer a powerful opportunity to delineate intricate molecular pathways that link neural activity to genetic programming in the execution of a synaptic remodeling mechanism. Moreover, the conservation of C. elegans remodeling components in mammals argues that this work is likely to reveal fundamental mechanisms that regulate synaptic plasticity in the human brain.