Thomas Biederer, PhD - US grants

DESCRIPTION (provided by applicant): Neurons communicate with each other in the brain through specialized junctions, called synapses. During brain development, numerous new synapses are established and new synapses continue to form throughout life. The long-term goal of the research proposed in this application is to determine the molecular basis of synapse formation in the vertebrate brain. The first proteins have now been identified that organize synapse formation and development. One such protein is SynCAM 1, a synaptic cell adhesion molecule that connects pre- and postsynaptic sides. Importantly, SynCAM 1 induces the formation of new, fully functional excitatory synapses between neurons. It is highly expressed in the developing brain during intense synaptogenesis, indicating a broad function for this molecule in synapse formation. Such synaptogenic functions have been validated in cultured neurons and in vivo. The objective of this application is to define the signaling pathways through which SynCAM 1 organizes synapses and determine how other trans-synaptic proteins act in concert with it. The central hypothesis of this application is that SynCAM signaling organizes developing synapses and regulates synaptic function at later stages. To attain the objective of this application, three specific aims will be pursued. The first aim of this application is to determine the intracellular signaling pathways through which SynCAM 1- mediated synaptic adhesion instructs synapse development, focusing on changes in the synaptic cytoskeleton. Second, it will be analyzed how trans-synaptic interactions act in concert to assemble synapses and shape their structure. Third, it will be determined to which extent SynCAM 1 functions in vivo together with other synaptic adhesion molecules to organize synapses. These experiments involve the biochemical characterization of SynCAM binding partners and their activities. Functional analyses of SynCAM interactions will be performed by quantitative immunocytochemistry, imaging of synapses in cultured hippocampal neurons, and electrophysiological recordings. In addition, the in vivo relevance of these interactions will be tested using structural and functional studies of synapses, including ultrastructural analyses, electrophysiological recordings, and behavioral analyses. Achieving these goals is important for human health, as altered synapse organization affects the wiring of neuronal circuits and synaptic plasticity. These changes are associated with alterations in human behavior, the ability to learn and remember, and addiction to drugs of abuse. Furthermore, deficits of synapse formation likely underlie neurodevelopmental disorders such as autism. In summary, this application aims to identify the molecular interactions involved in synapse formation. The progress under this application will allow testing to which extent these synapse-organizing processes are affected in disorders of the human brain and whether they represent novel points of therapeutic intervention.

DESCRIPTION (provided by applicant): Neurons communicate with each other in the brain through specialized junctions, called synapses. Drugs of abuse can change synapse structure and function together with behavioral responses related to addiction. Select molecules induce and organize synapses in the developing brain, but their functional roles during the response to drugs of abuse have not been tested. Such roles can be hypothesized based on the morphological changes of synapses in addiction as well as due to human genetic studies. One class of proteins that organize synapses are SynCAMs (also named nectin-like molecules), a family of adhesion molecules that connect pre- and postsynaptic sites. SynCAM 1 induces new, fully functional excitatory synapses in the developing brain and its expression regulates learning and memory. The objective of this application is to define to which extent the synapse-organizing protein SynCAM 1 controls synapse number and structure in brain regions affected by drugs of abuse, and how its functions impact behavioral responses to the psychostimulant cocaine. The central hypothesis of this application is that differences in synapse organization in brain regions affected by addictive drugs impact drug susceptibility, and that these drugs alter synaptogenic pathways. To attain the objective of this application, two specific aims will be pursued. The first aim of this application is to analyze whether synapse organization by SynCAM 1 impacts synapses in the nucleus accumbens and the addiction-related behaviors to which this region contributes. Second, it will be tested how altered synapse organization in the habenula, a brain region involved in reward learning, affects responses to drugs of abuse. These experiments involve the morphometric analysis of spines, the postsynaptic elements of excitatory synapses, in brain regions affected by drugs of abuse. Biochemical studies will determine effects of addictive drugs on the expression of synapse organizing proteins. Functional analyses will be performed using assays of addiction-related behaviors. In addition, a transgenic mouse model will be developed to target excitatory synaptic connectivity in the habenula. Achieving these goals is important for human health, as this application will test to which extent altered synapse organization affects the re-wiring of neuronal circuits during addiction. In summary, this application aims to identify the molecular interactions involved in synapse organization during the first response to drugs of abuse and the subsequent development of addiction. The progress under this application will allow testing to which extent differences in synapse-organizing processes predict vulnerability to drugs of abuse and whether these pathways represent novel points of therapeutic intervention. PUBLIC HEALTH RELEVANCE: Nerve cells in the brain communicate with each other through specialized junctions, called synapses. Changes in synapse formation and function impair the wiring of the brain and can contribute to neurological and behavioral dysfunction, including addiction to drugs of abuse. This research program is relevant to public health because it will analyze how addictive drugs alter the way nerve cells connect with each other, allowing us to understand what steps go wrong when substances are abused.

The synaptic cleft is a conserved and integral component of central synapses. It is comprised of protein complexes that span across it, and their adhesive interactions and signaling roles guide synapse development. Their relevance is underscored by the mutations in synapse-organizing proteins that are linked to complex brain disorders including autism-spectrum disorders and schizophrenia. Yet, the molecular patterning and dynamics of synaptic cleft components remain largely unknown. This contrasts with our understanding of the nanoscale organization of pre- and post-synaptic compartments, which is elucidating our understanding of synaptic structure and function. Moreover, acute synapse-organizing roles of cleft proteins at mature synapses and the functional interplay of trans-synaptic interaction systems remain to be defined. Our central hypothesis is that the properties and plasticity of mature synapses are dynamically instructed by select cleft components. This proposal builds on preliminary and previously published results by the collaborating groups that synaptic adhesion complexes are differentially localized within the cleft, shape this compartment, can undergo rapid changes in synaptic abundance upon plasticity induction, and alter long-term synaptic plasticity. Three specific aims will be pursued to test our hypothesis. First, we will test roles of trans-synaptic interactions in acutely instructing pre- and postsynaptic function. Second, it is our aim to determine the molecular and organizational dynamics of the cleft during long-term plasticity. Third, we will identify cleft proteins that guide changes during plasticity. Our approaches include tools to acutely perturb trans-synaptic interactions, proximity labeling to identify cleft-resident molecules and monitor them during plasticity, superresolution imaging to map their cleft locations, and cell biological and physiological functional assays. We anticipate to determine the molecular patterning and dynamics of the synaptic cleft and to identify how trans-synaptic interactions actively shape synaptic function. This expected progress is significant because it will define the cleft as a molecularly organized and acutely controlled cellular compartment that instructs synaptic properties. Moreover, this research can determine how a dynamic remodeling of the cleft architecture underlies the activity-dependent plastic changes at synapses. Synaptic organization and function are disrupted in neurodevelopmental and neurological disorders, and this program will provide information for defining how these diseases impact the cleft and may even originate in it to alter synaptic properties.

In neurodegenerative brain disorders, aggregates of misfolded protein oligomers can undergo neuron-to- neuron transfer. This results in the seeded assembly of misfolded host proteins in recipient cells, thus propagating the pathogenic protein species. Recent progress has demonstrated that in Parkinson?s disease (PD), ?-synuclein forms pathogenic fibrils that can spread between interconnected neurons across synapses and brain regions. This may contribute to the dementia that develops in PD patients over time. It is unlikely that pathogenic ?-synuclein assemblies have a single neuronal ?receptor?. Indeed, the first few partners of ?- synuclein fibrils are beginning to be identified. However, the pathophysiological interactions of ?-synuclein fibrils with neuronal and synaptic plasma membrane proteins remain unclear, and how their ?-synuclein fibril retention impacts synaptic properties has not yet been addressed. Our central hypothesis is that synaptic adhesion molecules can contribute to ?-synuclein spread due to their cellular location and the synaptic pathology in PD. Our preliminary data on pathological effects of ?-synuclein fibrils support this hypothesis. In addition, we aim to establish an unbiased proteomic approach to screen for neuronal surface partners of ?- synuclein fibrils. Two specific aims will be pursued to test our central hypothesis. First, we will determine the pathophysiological effects of ?-synuclein fibril binding on the load of aggregated endogenous ?-synuclein in recipient cells and on their synapses. Second, we will establish and employ an innovative proteomic approach to identify the complement of neuronal membrane proteins that are targeted by ?-synuclein fibrils. Our approaches include an assay using preformed ?-synuclein fibrils that we have established for application in cultured primary neurons, including microfluidic chambers and assays of synapse assembly, together with a proteomic labeling approach we have established. We anticipate first, to define how synaptic target interactions with ?-synuclein fibrils impact ?-synuclein transmission and synaptic properties, and second, to perform a proteomic screen to identify the complement of ?-synuclein fibril partners in neuronal membranes. This approach will be applicable to multiple types of neurons and can hence advance the field by identifying cell- autonomous factors that affect neuronal vulnerability across distinct regions. The expected progress is significant because it will define the roles of synaptic targets in PD-relevant synaptic pathophysiology and enable identifying therapeutic targets across neuron types. Together, this project can reveal mechanisms underlying ?-synuclein spread and PD progression.

In neurodegenerative brain disorders, aggregates of misfolded protein oligomers can undergo neuron-to- neuron transfer. This results in the seeded assembly of misfolded host proteins in recipient cells, thus propagating the pathogenic protein species. Recent progress has demonstrated that in Parkinson?s disease (PD), ?-synuclein forms pathogenic fibrils that can spread between interconnected neurons across synapses and brain regions. This may contribute to the dementia that develops in PD patients over time. It is unlikely that pathogenic ?-synuclein assemblies have a single neuronal ?receptor?. Indeed, the first few partners of ?- synuclein fibrils are beginning to be identified. However, the pathophysiological interactions of ?-synuclein fibrils with neuronal and synaptic plasma membrane proteins remain unclear, and how their ?-synuclein fibril retention impacts synaptic properties has not yet been addressed. Our central hypothesis is that synaptic adhesion molecules can contribute to ?-synuclein spread due to their cellular location and the synaptic pathology in PD. Our preliminary data on pathological effects of ?-synuclein fibrils support this hypothesis. In addition, we aim to establish an unbiased proteomic approach to screen for neuronal surface partners of ?- synuclein fibrils. Two specific aims will be pursued to test our central hypothesis. First, we will determine the pathophysiological effects of ?-synuclein fibril binding on the load of aggregated endogenous ?-synuclein in recipient cells and on their synapses. Second, we will establish and employ an innovative proteomic approach to identify the complement of neuronal membrane proteins that are targeted by ?-synuclein fibrils. Our approaches include an assay using preformed ?-synuclein fibrils that we have established for application in cultured primary neurons, including microfluidic chambers and assays of synapse assembly, together with a proteomic labeling approach we have established. We anticipate first, to define how synaptic target interactions with ?-synuclein fibrils impact ?-synuclein transmission and synaptic properties, and second, to perform a proteomic screen to identify the complement of ?-synuclein fibril partners in neuronal membranes. This approach will be applicable to multiple types of neurons and can hence advance the field by identifying cell- autonomous factors that affect neuronal vulnerability across distinct regions. The expected progress is significant because it will define the roles of synaptic targets in PD-relevant synaptic pathophysiology and enable identifying therapeutic targets across neuron types. Together, this project can reveal mechanisms underlying ?-synuclein spread and PD progression.

DESCRIPTION (provided by applicant): Synaptic activity is central to all behavior, learning, and memory; synaptic dysfunction leads to neurological disorders such as epilepsy, autism, Alzheimer's disease, depression, and sleep disorders. Developing new therapeutic approaches for preventing or treating synaptic dysfunction requires a continuing cadre of young investigators well-trained in multidisciplinary techniques to address synaptic function. This application requests five years of continued funding for the Synapse Neurobiology Training Program (SNTP), to support 4 predoctoral trainees per year, selected from a pool of highly qualified applicants in years 3-5 of thesis research relevant to synaptic structure and function. Research on synaptic function and dysfunction are strengths of the 20 SNTP faculty mentors. In the first 4 years of the SNTP, 11 trainees received individualized, in-depth, multidisciplinary training, 3 have completed their PhD degree and are engaged in academic research, biotech, and teaching careers. In addition, 6 new faculty members were recruited to the Department of Neuroscience, expanding and strengthening the multifaceted research approaches being taught to SNTP trainees and providing a rich diversity of thesis research labs investigating synapse neurobiology and synaptic disorders. SNTP trainees have access to cutting-edge tools and training in a wide array of research approaches via the NINDS-funded Center for Neuroscience Research Cores (Imaging, Behavior, Genomics, and Electrophysiology). The SNTP is further strengthened by a new, stand-alone Neuroscience Program and a curriculum that increases the depth and breadth of trainees' exposure to fundamental concepts in neuroscience, with particular emphasis on the synapse. The new curriculum shifts focus from lecture-based teaching to interactive, discussion-oriented small group sessions with faculty and hands-on workshops that hone the trainees' critical thinking and technical skills. SNTP trainees have multiple opportunities to interact with physician scientists through coursework, collaborative research projects, and one-on-one interactions with clinicians. The latter activity represents a new initiative that provides SNTP trainees with an opportunity to learn about diseases most relevant to their thesis work. Trainees also benefit from a Career Paths seminar series that exposes them to the diverse post-PhD career options available and to help them start a personal contact network. Annual research seminars by trainees hone their presentation skills and reinforce the highly interactive environment that characterizes the Tufts neuroscience community. SNTP trainees are capable, proactive, and motivated scientists, as evidenced by awards, presentations at national meetings, and student-led outreach activities that include teaching workshops at high schools and colleges with large populations of students from underserved groups. The SNTP provides trainees with the solid multifaceted foundation they need to build successful research careers and contribute to knowledge of both healthy and diseased nervous systems.