Felix E. Schweizer - US grants

DESCRIPTION (provided by applicant): The release of neurotransmitter at synapses is controlled by the number of synaptic vesicles that are ready for fusion with the plasma membrane in response to calcium influx. The aim of this proposal is to determine how the number of such readily-releasable vesicles is regulated and what effect this and other pools of vesicles can have on release. At synapses throughout the nervous system, ultrastructural information shows distinct pools of vesicles. A subset of vesicles is docked to the plasma membrane at the active zone, primed for fusion. Following exocytosis, this readily-releasable pool of vesicles must be replenished from a distinct reserve pool of vesicles clustered in the vicinity of the active zone. Slow replenishment will cause synaptic depression, while over-replenishment will lead to augmentation of transmitter release. Changing the size and rate of replenishment of vesicle pools thus offers a powerful mechanism for modulation of synaptic strength. While we understand in considerable detail what governs the fusion of docked vesicles, much less is known about what controls the dynamics of these distinct vesicle pools. Acutely isolated sensory hair cells are an excellent system for these studies since their vesicle pools are well characterized anatomically. Also, release occurs at the basal end of the cell body, offering easy access to the sites of transmitter release and exocytosis can be monitored directly by measuring the increase in cell membrane capacitance that occurs when vesicles fuse. Specific Aim 1 establishes the basic release properties of hair cells and tests the hypothesis that transmitter release is most efficient at a certain stimulation frequency, i.e., that transmitter release is tuned. Specific Aim 2 tests the hypothesis that the size of pools and the interaction between them control exocytosis. It tests whether calcium and/or protein kinases regulate traffic between vesicle pools. Specific Aim 3 tests the hypothesis that glutamate enhances its own release by modulating pool dynamics via presynaptic metabotropic receptors. The findings will significantly advance our understanding of synaptic transmission. By revealing novel mechanisms for the modulation of neurotransmitter release they may identify targets for therapeutic intervention in neurological diseases.

DESCRIPTION (provided by applicant): Mechanosensory hair cells in the vestibular system transduce information about head position and movement into neuronal signals. The aim of this application is to elucidate the molecular mechanisms underlying synaptic transmission from vestibular hair cells to afferent fibers. The hair cells afferent synapse exhibits anatomical (synaptic bodies or ribbons) and functional (responding to graded depolarizations) specializations that distinguish them from other synapses in the nervous system. We here propose to investigate which proteins and protein-interactions important at conventional synapses are important for hair cell synaptic transmission. Furthermore, vestibular physiology lacks an understanding of the functional difference between type I and type II hair cells. Type I hair cells, found only in amniotes, are engulfed by the afferent nerve, suggesting differences in synaptic physiology. The proposed experiments are therefore further aimed at understanding the molecular and functional difference in neurotransmitter release between vestibular type I and type II hair cells. These experiments will further our molecular and biophysical understanding of the first synapse in the vestibular and auditory system. Hair cells release neurotransmitter from the basolateral end of the cell body, offering relatively easy access to synaptic areas for the introduction of toxins and peptides to acutely perturb protein function. Exocytosis will be monitored directly by measuring the increase in cell membrane area that occurs when vesicles fuse with the plasma membrane. Specific Aim 1 tests the hypothesis that exocytosis is regulated by a specific subset of SNARE proteins. Specific Aim 2 addresses whether vesicle recycling is limiting for exocytosis. In Specific Aim 3 the hypothesis is tested that glutamate acts in a positive feedback loop to enhance release especially in type I hair cells. A detailed understanding of the proteins and mechanisms of release at this initial sensory synapse aid in identifying novel therapeutic approaches targeting vestibular dysfunction.

DESCRIPTION (provided by applicant): Environmental risk factors as well as genetic predisposition underlie the development and progression of most neurodegenerative diseases. Although neuronal death characterizes the clinical stages of many of these diseases, dysfunction in the cell periphery, especially at the synapse, likely occur earlier. We find that environmental toxins such as pesticides that are implicated in Parkinson's disease (PD) rapidly affect synaptic transmission and synaptic plasticity. Many of these same toxins also affect the ubiquitin proteasome system (UPS), a major intracellular pathway for degradation of misfolded, oxidized or no-longer needed proteins. Further, several of the genes that have been linked to PD code for proteins involved in the UPS. In PD, the accumulation of protein aggregates in Lewy bodies points towards a deficit in protein processing and degradation. We therefore propose that proteasomal dysfunction in neurons compromises synaptic function and serves as an early indicator of neuronal degeneration. In addition to protein degradation, mitochondrial function appears compromised in PD. Interestingly, some PD-linked genes code for mitochondrial proteins and some pesticides linked to PD inhibit the mitochondrial electron transport chain. Compromised mitochondrial function will decrease cellular ATP levels and increases reactive oxygen species (ROS) and thus oxidized proteins. The UPS, which also degrades oxidized proteins, requires ATP for proper function. Thus, the UPS is a potential central target in the etiology of PD even for genes and toxins that directly affect mitochondrial function. We propose that changes in synaptic transmission triggered by UPS dysfunction represent an early indicator for neuronal vulnerability. Synaptic function thus offers a useful assay to test for the interaction between genetic and environmental factors underlying neurodegenerative diseases such as PD. PD is a progressive disease. Physiological alterations must precede clinical symptoms. Genetic animal models for PD similarly show progressive behavioral and physiological deficits and - like human disease - no deficits are evident early in life. Our studies will determine the impact of environmental toxins on synaptic transmission in neurons derived from PD animal models versus those from controls, and discern whether effects are more severe, occur at different concentrations or simply act in a different way. This work directly tests the interaction of genes and environmental factors in the etiology of PD. Our experiments are based on the hypothesis that genetic mutations found in neurodegenerative diseases interact with environmental toxins to affect synaptic function. We further propose synaptic dysfunction as an early sign of PD. Our experiments will lay the groundwork for identifying potential environmental and genetic risk factors for PD and other neurodegenerative diseases. The experimental paradigms developed will be useful in establishing a screening tool for the toxicity of pesticides and therapeutic interventions in relationship to neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: Genetic predisposition and environmental factors underlie the development and progression of most neurodegenerative diseases such as Parkinson's disease (PD). This grant application proposes to test the hypothesis that communication between neurons at synapses is compromised by environmental toxins such as pesticides and that mutations in specific genes associated with PD interact with these very same pathways to exacerbate the synaptic dysfunction. The experiments are designed to elucidate this interaction between genes and the environment and to find support for the idea that alterations of synaptic function could be used in future studies as predictors of pesticide toxicity with respect to neurodegenerative diseases.

DESCRIPTION (provided by applicant): Synaptic transmission is a critical feature of the nervous system and is as such tightly regulated on many different time-scales. The ubiquitin-proteasome system (UPS) plays an important role in synaptic transmission by maintaining appropriate protein homeostasis. The UPS regulates synapse formation during development, by shaping long-term plasticity, by altering the number of active synapses and by changing the size of vesicle pools. All of these regulatory functions occur on a time scale of hours to days and are consistent with the central role of the UPS in protein degradation: covalent attachment of ubiquitin tags proteins for degradation by the proteasome. We have recently discovered that the UPS also regulates neurotransmitter release on a time-scale of a few minutes or less. This led us to the hypothesis that ubiquitination at the synapse rapidly regulates release by changing the activity level of target proteins rather than changing their half-life. Thus, protein ubiquitination can serve as an acute dynamic regulator of synaptic function. This represents a novel regulatory pathway that is of clear basic science interest given the central role of synaptic transmission in nervous system function. However, the pathway is also of clear clinical interest as it might offer insight into the earliest steps of dysfunction associated with neurodegenerative diseases. In many neurodegenerative diseases signs of synaptic dysfunction precede clinical symptoms. Neurodegenerative diseases are also characterized by a UPS that is impaired either through mutations in the pathway, by misfolded proteins or via environmental toxins. The recognition of the rapid regulatory role of the UPS in shaping synaptic transmission might offer an early causal link between UPS impairment and synaptic dysfunction. In this grant application we will test the physiological mechanisms that mediate the rapid regulation of presynaptic release by ubiquitination (Aim 1). Together with the identification of the synaptic ubiquitome (Aim2) these experiments will enable discovery of pathways and processes that are of basic science interest and that might provide novel druggable targets for neurological diseases. PUBLIC HEALTH RELEVANCE: In this grant application we propose to test the hypothesis that protein ubiquitination at the synapse can serve as a rapid, posttranslational modification that regulates synaptic transmission, rather than simply representing a signal for protein degradation by the proteasome. Early stages of many neurodegenerative diseases, such as Parkinson's, Alzheimer's and Huntington's Disease, are characterized by changes in synaptic function and protein ubiquitination is compromised either by mutations in the pathway or by accumulation of misfolded proteins. Our experiments on the role of rapid protein ubiquitination in the regulation of synaptic transmission suggest a causal link between these observations and thus indicate novel druggable targets and pathways.

DESCRIPTION (provided by applicant): This proposal requests partial support for an international meeting on Synaptic Transmission as part of the Gordon Research Conference series to be held at the Waterville Valley Resort in the mountains of New Hampshire during the week of July 29-August 3, 2012. The broad and long term goal of the conference is to increase our understanding of the fundamental molecular and cellular mechanisms of synaptic transmission. The synapse serves as the basic signaling unit of the nervous system. Synaptic transmission underlies every aspect of brain function and is relevant to most neurological diseases, as well as mental illness and drug addiction. The specific aims of this meeting will be to convene 34 speakers that represent critical areas of synaptic transmission research with a total of 150 participants for a five day conference in a relatively isolated setting. The program will have two keynote lectures and eight sessions that broadly address current issues in the nano-scale structure and molecular machinery of synapses, the regulation of synaptic transmission by calcium, trans-synaptic signaling and postsynaptic modifications. A session devoted to ribbon-type synapses in the eye and ear will highlight structural and functional diversity of synapses. Another session is devoted to the synaptic basis of brain disorders and a meeting will conclude with an integrative session on the role of synapses in circuits. In addition, two evening poster sessions will permit all participants to contribute to these topics. The significance of this application is that the Gordon Research Conference on Synaptic Transmission is a critical component of the yearly series of conferences that propel research in the international community of synaptologists. The health relatedness of this application is that the discussions will define the questions that require experimental resolution of a wide range of devastating brain disorders, including neurodegenerative diseases, schizophrenia, autism spectrum disorders, drug addiction, mood disorders, loss of peripheral sensory function and many others, which are collectively emerging as synapto-pathies.