Deanna L. Benson - US grants

9419900 Benson As the nervous system develops, exuberant connections are made between cells, many of which are later eliminated to arrive at the final pattern of connections. A question that has yet to be fully answered is the role that neural activity plays in this process of synapse formation, elimination, and consolidation. This research will investigate the formation of synapses between cultured hippocampal neurons, which have been shown to closely resemble their in vivo counterparts molecularly, structurally, and functionally. A number of experimental manipulations will be performed to elucidate the role of neural activity in the formation and stabilization of synapses and synaptic patterns. This entire process is integral to the development of the nervous system, and is one that is modifiable by experience and learning. The outcome of these experiments may provide fundamental information about how the electrical activity associated with neural activation refines the connections between nerve cells.

Dendritic spines are highly specialized postsynaptic structures which receive the vast majority of excitatory input. Changes in their morphology and density have been associated with learning as well as long-term potentiation of synaptic efficacy which is thought to represent the cellular correlate of learning and memory. Aging in humans, nonhuman primates and rodents is usually accompanied by memory deficits, loss of dendritic spines, and in rodents, age-related memory deficits have been correlated with LTP deficits. Such changes in spine function during aging do not arise from significant cell loss, which is normally minimal during aging, but rather may indicate specific alterations in the mechanisms which underlie spine formation and maintenance. Elucidating fundamental mechanisms contributing to spine induction and maintenance becomes critical not only for understanding the basis for spine-related deficits in aging, but also for designing rational therapies aimed at preserving spine function. Estrogen treatment stimulates production of dendritic spines in rat hippocampus, and a growing body of data shows strong links between estrogen replacement therapy in postmenopausal women and a decreased risk of dementia. Estrogen induction of dendritic spines requires NMDAR activation, and binding studies suggest that changes in NMDAR subunit combination may be involved. The signaling pathway also appears to include activation of a serine-threonine protein kinase. One such kinase, type II calcium-calmodulin-dependent protein kinase (CaMKII) is a likely candidate in that it is concentrated within dendritic spines and its activation is required for the induction long-term potentiation . Thus, changes in NMDAR subunit synthesis, association or localization and activation of CaMKII are likely to participate in dendritic spine induction and will be investigated as part of a long term goal to elucidate the mechanisms of estrogen-mediated dendritic spine regulation.

9728003 BENSON The long term goal of this research is to understand how the axons of neurons are guided to particular parts of their target cells, and once there, how they assemble synaptic contacts. Several classes of adhesion molecules, such as cadherins, integrins and members of the immunoglobulin superfamily are known to generate junctions between cells, and recent research has shown that these same molecules are also localized at synaptic junctions in the central nervous system (CNS). These new data strongly suggest that the mechanisms by which pre- and postsynaptic membranes adhere to one another in the CNS may be substantially similar to those that generate intercellular adhesion in other, non-neural tissues. Consistent with this hypothesis is the following evidence: Immunolocalization of N- and E-cadherin at synaptic junctions; the tight adhesive nature of cadherins and their resultant intermembrane distances correlate well with biochemical properties and calculated intermembrane distances described for synapses; and both synapse formation and targeting to the appropriate cellular domain (somata, dendritic shafts, spines) occur in the absence of neural activity. Synapse development and organization are well-characterized in the rat hippocampus and in hippocampal neurons grown in culture. Together they make an excellent model system in which to examine interneuronal synapse formation and targeting. The first aim of Dr. Benson's research is to test the hypothesis that cadherin incorporation is critical for synapse formation. This will be accomplished by examining the deposition of cadherins at synapses in cultured hippocampal neurons in relation to other well- characterized synaptic markers, in the presence and absence of inhibitors and antisense oligonucleotides that block the production of cadherins. The second aim is to examine the relationship between different cadherins and functionally distinct synapses. Co-localization of cadherins or ca tenins and transmitter-specific synaptic markers will be assessed in vivo and in vitro. Together, data from these aims will be used to describe the dynamics of cadherin localization during synapse development and will have broad implications for the mechanisms underlying synapse assembly in CNS.

blocking antibody; tissue /cell culture

The neuropathology observed in fetal alcohol syndrome (FAS) is strikingly similar to that observed in humans with mutations in the cell adhesion molecule L1. Since these L1 mutations are known to disrupt extracellular adhesion and some are presumed to block intracellular signaling, the effects of FAS may be due in part to disrupted LI adhesive signal transduction during development. During development cell adhesion molecules (CAMS) mediate many of the cell-cell interactions essential for appropriate axon pathfinding and synapse formation. In order for CAMS to do this, they are restricted to particular cell types, targeted to either axons or dendrites, and in many cases, to particular regions within axons and dendrites. Virtually nothing is known about how CAMS become targeted to particular cellular domains, how they are retained or for molecules of the Ig superfamily, how adhesive signals are transduced from the surface to the cytoskeleton. The overall goal of this proposal is to define intracellular sorting strategies and signaling pathways that are employed by the axonal CAM L1 and to determine whether these strategies are undermined by exposure to alcohol. To do this, we will identify the molecular domains required to mediate some of the functions of L1 so that ultimately we may identify signaling pathways that are disrupted by alcohol during critical developmental. periods.

Proteins must be directed to different places within a cell, particularly in nerve cells (neurons), where long processes like the axon can extend far from the cell body. This protein 'traffic' is thought to be done by packaging proteins into distinct vesicles, which then are targeted to the correct destination. Virtually nothing is known about this process. Two principal types of vesicles are the large dense-core vesicles (LDCVs) containing neuropeptides or monoamines, and small clear vesicles (SCVs) that contain neurotransmitters like gultamate and gamma-amino butyric acid (GABA). Neurons and neuroendocrine cells both must correctly sort peptide precursors into LDCVs, away from the neurotransmitter-containing SCVs that release and reload their contents through very different mechansims. This project uses molecular and immunocytochemical approaches to identify polypeptide sequences that target VGF to LDCVs and to characterize the novel sorting mechanism or receptor molecule involved.
Results will be important for defining the fundamental mechanisms that underly the sorting of proteins into distinct vesicle populations, ultimately transporting them to specific regions in highly polarized neurons, and to regions related to regulative or constitutive secretory pathways in these complex cells. Graduate training is included in this project.

DESCRIPTION (provided by applicant): In utero exposure of humans to high levels of ethanol can result in a variety of neurological anomalies collectively termed "alcohol related neurodevelopmental disorders" (ARND). Some of the hallmarks of observed cases of ARND bear a striking similarity to those seen in individuals with mutations in the cell adhesion molecule (CAM) L1. This has given rise to the hypothesis that L1 is a target for ethanol, and consistent with this idea, many L1-dependent functions including neuron migration, axon extension, axon fasciculation and myelination are particularly vulnerable to ethanol exposure. The overall goals of this grant are to determine the nature and mechanism of ethanol actions on L1-dependent axon extension. Specifically, the proposal is to examine the effects of ethanol on the surface expression, axonal polarization and membrane fluidity of L1, and to identify the mechanism by which ethanol decreases L1-dependent axon outgrowth. The data from this work will directly address the effects of fetal alcohol exposure on the development of normal neural connectivity, and will also have relevance for mechanisms of recovery following traumatic brain injury or stroke, where many developmentally regulated molecules become re-expressed and recovery of function is impaired by ethanol.

DESCRIPTION (provided by applicant): In the human brain more than 20 billion neurons become precisely connected to one another during development. How this happens, despite significant advances, remains for the most part, a mystery. Recently, we have found that the cell adhesion molecule L1 can bind directly to ezrin, radixin and moesin, members of the ERM family of molecules that link transmembrane proteins to the actin cytoskeleton. Work from other laboratories has established the importance of L1 in axon fasciculation and guidance, and our preliminary studies indicate that the ERM family plays a critical role in translating L1 binding into outgrowth. Work in non-neuronal cells suggests that ERMs act both upstream and downstream of the Rho family of small GTPases and that ERM binding to the tuberous sclerosis1 gene product, hamartin, is required for Rho mediated regulation of adhesion. This suggests that ERMs may be key regulators of actin dynamics during neural differentiation and pathfinding. The goal of the proposed work is to define the nature of ERM function in neurons, to address how ERMs are dynamically regulated in response to particular phases of neurite outgrowth and to changes in substrate, and to investigate the signaling pathways involved. The initial studies will be carried out in culture where environment can be closely controlled. Results from this work will inform the analysis and interpretation of an in vivo study of ERM function in the regulation of axon outgrowth and branching.

DESCRIPTION (provided by applicant): Synapse pathology is common to a variety of neurodegenerative and neurodevelopmental diseases. For many such disorders, including schizophrenia and autism spectrum disorders, changes in synapse composition are suspected to be causally related to some of the most devastating symptoms. Synapses throughout the brain share key structural proteins evidenced by their similar, readily identified structure as see through an electron microscope, but they are also very heterogeneous: only a handful of synapse components is known to be shared by all synapses. Presynaptic membranes adhere to postsynaptic membranes in an exceptionally strong interaction, resistant to disassembly. This interaction can withstand tissue fractionation and has been exploited over the past several decades to purify synaptosomes: pre- to postsynaptic adhesions and attached membranes that re-seal to make an enclosed synaptic organelle. Current proteomics studies have utilized synaptosomes or subfractions of such preparations to assess synapse composition. While this supports the feasibility of using synaptosomes for proteomics-based comparisons, the resulting large datasets have not been very useful since they reflect a highly complex starting material containing an enormously heterogeneous population of synapses. Synapse heterogeneity is based largely on differences in connectivity, but currently there are no methodologies available that can be used to interrogate and analyze differences between identified synapse populations. Here we propose a novel and straightforward method based on the mammalian GFP reconstitution across synaptic partners technique that will enable proteomic comparisons between identified synapse populations in health and disease. We have assembled a team to test this using well- defined synaptic circuits. This approach will be a major step forward as it wll permit a rapid, large-scale assessment of particular synapse populations that can be isolated from complex circuits.

DESCRIPTION (provided by applicant): Disorders of cognitive, social and perceptual functions are associated with abnormalities of cortical synaptic circuit development and plasticity. There is a strong genetic disposition to such diseases, but the precise causes are unknown. Microdeletions in chromosome 15q24 are associated with a syndrome that features autism. A recent study of 15q24 microdeletion syndrome identified a minimal deletion interval that contains only four genes that are expressed in brain, among which, SEMA7A stands apart as highly relevant to sensory dysfunctions associated with the syndrome. Sema7A is an atypical member of the Semaphorin family of guidance cues: it is membrane-anchored by a GPI-linkage; it is expressed principally postnatally in the nervous system; and it can promote axon extension in a ¿1 integrin-dependent manner. These findings point to the idea that Sema7A has roles in late stages of brain development distinct from the customary Semaphorin-Plexin interactions that generate axon repulsion during embryonic development, but this has not been explored. Our data show that Sema7A is particularly enriched in somatosensory (S1) cortex at a time when synapses develop and sensory experience drives the refinement of connectivity. Accordingly, we hypothesize that Sema7A functions in the maturation and fine-tuning of cortical microcircuitry that occurs during early postnatal development. In mouse S1 barrel cortex our preliminary data show that when Sema7A is genetically ablated thalamocortical axons reach layer IV, but their synapses fail to mature functionally and their postsynaptic dendritic targets fil to orient their arbors appropriately. In contrast, somatosensory maps in subcortical centers are normal. These data outline an entirely novel molecular contribution to the functional and structural development of cortical sensory maps, the absence of which may perturb information processing through cortical microcircuits that in turn, produce symptoms relevant to 15q24 microdeletion syndrome. Our preliminary data serve as the basis for the hypothesis that Sema7A is essential for normal S1 maturation and function.

DESCRIPTION (provided by applicant): It is estimated than more 6% of all Americans (or nearly 19 million individuals) suffer from a serious mental disorder. Many such disorders arise during development or adolescence, and strong evidence supports that they are caused or exacerbated by gene mutations or variations in gene copy number. Treatments exist, but there are few cures. Human genetic studies have identified CYFIP1 as a gene that is dysregulated in a wide variety of developmental brain disorders including certain forms of Angelman and Prader-Willi syndromes, autism spectrum disorders, and schizophrenia. How does a single gene contribute to so many disorders? CYFIP1 produces a cytoplasmic protein, cytoplasmic FMRP interacting protein (Cyfip1), having two independent and highly conserved functions. Cyfip1 represses cap-dependent translation of FMRP target mRNAs as a noncanonical initiation factor 4E binding protein (4E-BP), and it regulates the generation of branched actin filaments at the plasmalemma as an integral component of the WAVE complex. Regulation of both cap-dependent translation and actin cytoskeleton are known to be important for synapse assembly, morphology, and plasticity and are also known to be pathways that are vulnerable to developmental brain disorders. Thus it is easy to appreciate why loss of even a single copy of Cyfip1 could have broad consequences, but the function of Cyfip1 at developing synapses is not well understood. In this proposal we will investigate how Cyfip1 contributes to synapse development, function, and plasticity using a mouse model we developed expressing reduced levels of Cyfip1. Preliminary experiments show that neurons with reduced Cyfip1 levels display a strong, principally presynaptic phenotype during development that is missing in adolescence, and an equally strong, but postsynaptic phenotype in adolescence that is missing in younger animals. Based on these findings, we will test the hypothesis that Cyfip1's actions in cap-dependent protein synthesis and actin polymerization contribute differentially to presynaptic function during development and postsynaptic physiology and plasticity in maturity.

? DESCRIPTION (provided by applicant): Autism Spectrum Disorders (ASDs) comprise a range of neurodevelopmental abnormalities in cognitive abilities and behaviors associated with dysfunctional circuitry between the prefrontal cortex (PFC) and the neostriatum. Behavioral abnormalities emerge early after birth and are thought to reflect defects in the finetuning and plasticity of developing functional synaptic connectivity. We have shown that mRNA encoding cadherin8 (Cdh8) - a type II, synaptically localized classic cadherin - is highly enriched in PFC and dorsal striatum during early postnatal development. Moreover, the timing, anatomical distribution, and axon targeting function of Cdh8 suggest strongly that Cdh8 may be crucial for the development and plasticity of PFC?striatal circuitry. This is significant because several recent studies have linked CDH8 genetically to susceptibility to ASDs. Thus, we hypothesize that cognitive ASD like phenotypes reflect impaired synaptic development of PFC?striatal direct and/ or indirect pathway circuitry due to deficient Cdh8 dependent molecular control over these pathways. We will test this hypothesis by combining mouse genetics, anatomy, electrophysiology and behavioral assessment. The vertical integration across these objectives (spanning molecules, synapses, circuits and behaviors) will provide novel insight into molecular control of brain pathways implicated in cognitive and behavioral deficits associated with ASDs. This is important, because corticostriatal circuit defects are central to a number of aberrant behaviors associated with autism and anxiety disorders, but there is surprisingly little known about the normal development and plasticity of such circuits.

This application requests funds to purchase a Leica SP8 TCS STED 3X microscope to serve investigators at the Icahn School of Medicine at Mount Sinai and in New York City. The instrument will be housed and administered by the institution's Microscopy Core, a facility that has been in continuous operation for more than twenty-five years. The goal is to bring a critical research capability to nine Major Users and one Minor User in seven departments and supported by fifteen NIH grants as well as to make the technology available to other qualified users in the New York City area. The capabilities of the instrument are not duplicated by any on campus. Very few individual labs have the means to purchase, house and maintain their own super-resolution microscope and the Leica SP8 TCS STED 3X is an outstanding, flexible and user friendly instrument that will bring acquisition of high quality, high resolution data within reach of junior and senior investigators who rely on light microscopy to advance their science. The acquisition of the Leica SP8 TCS STED 3X at Mount Sinai will contribute directly to the advancement of several research initiatives that will further current understanding in several major areas relevant to human health including molecular and cell biological mechanisms of viral infection, bacteria-mediated inflammation, neurodegenerative disease, developmental brain disorders, vision, stress and depression. The research that will benefit covers a broad range of significant, unsolved questions central to the overall mission of NIH.

The G2019S mutation is the most common of several mutations in leucine-rich repeat kinase 2 (LRRK2) causing up to 40% of familial Parkinson's disease in certain populations. This pathogenic point mutation is autosomal dominant and increases kinase activity 2-3 fold. Disease progression in both motor and non-motor symptoms of mutant LRRK2 carriers is similar to idiopathic cases suggesting common mechanisms, but progress has been limited because LRRK2 biology is poorly understood and little is known of pathogenic cellular or synaptic actions of G2019S-LRRK2. LRRK2 expression is high in spiny projection neurons (SPNs) of dorsal and ventral striatum, and rises rapidly during axon ingrowth and excitatory synaptogenesis. The timing and location of expression suggests that mutant LRRK2 may be maladaptively influencing development of excitatory circuits that impact striatal function. To begin to test this idea, we probed glutamatergic synaptic function in SPNs in G2019S-LRRK2 knockin mice. We showed that early in postnatal life, G2019S-SPNs in dorsal striatum exhibit a significantly abnormal increase in spontaneous excitatory synaptic currents (sEPSCs) compared to WT mice or mice expressing a LRRK2 kinase-dead knockin mutation (D2017A). Such abnormal excitatory activity was observed in both direct- and indirect-pathway SPNs, was normalized by LRRK2 kinase inhibitors, and was associated with larger SPN dendritic spine-heads and sEPSC amplitudes. Dorsal striatal SPNs receive convergent input from cerebral cortex and control many types of goal- directed behaviors, and the latter are thought to reflect balanced control of bidirectional changes in corticostriatal synaptic strength. The early abnormalities in SPN synaptic function and structure suggest that synaptic plasticity will be altered by G2019S-LRRK2 with consequences for striatally-based behaviors. Preliminary data support both of these ideas. Together, we hypothesize that the normal balance between mechanisms that strengthen or weaken synaptic transmission is altered in SPNs expressing G2019S-LRRK2 in a way that both reveals molecular signaling pathways targeted by mutant LRRK2 and that has predictable consequences for behaviors. The proposed experiments will assess the impact of mutant LRRK2 on synapse strengthening and weakening in subtype-identified SPNs; they will identify the molecular pathways and mechanisms involved; they will determine if mutant LRRK2 alters behaviors associated with SPN synapse plasticity; and they will test whether in vivo LRRK2 inhibition early in life ameliorates maladaptive effects on synaptic and behavioral plasticity documented later in life.

PROJECT SUMMARY/ABSTRACT The Tisch Cancer Institute (TCI) Microscopy Shared Resource Facility (MSRF) provides TCI members access to equipment, training, expert consultation, and collaborative opportunities across a full-range of light and electron microscopy applications, including: confocal, multiphoton, super-resolution, in vivo imaging, light sheet, widefield, and transmission electron microscopy (EM). Three high-end workstations equipped with state-of-the- art software accompanied by a local server and high-speed connectivity permit a wide range of image analysis and quantification strategies. Five full time scientists staff the MSRF during regular hours and provide real-time assistance to both novice and experienced users. All of the MSRF scientists can be tapped for implementing advanced approaches. Each holds a PhD or was otherwise trained intensively in a field having particular relevance to their imaging specialty such as super-resolution microscopy or computer engineering. In particular, the EM specialist is a leader in the field and has published over ninety papers covering everything from correlative light-EM (CLEM) to traditional EM techniques, and the scientific advisor is also a TCI member who is expert in intravital imaging approaches and works collaboratively with novice TCI users needing to apply this strategy to cancer-related questions. The MSRF also sponsors monthly seminars featuring scientists using microscopy or vendors developing relevant new technology and its scientists co-direct an annual hands-on microscopy course for graduate school trainees and host workshops throughout the year. The capabilities of the microscope systems together with the skills and training of the team are crucial for supporting TCI studies that include multichannel immunofluorescence for tumor diagnostics, localization of cell signaling and growth regulatory molecules, tumor cell migration and invasion, and cell biological mechanisms of metastasis and dormancy.

The actin cytoskeleton anchors synapse adhesion molecules and generates the flexible architecture that characterizes dendritic spine shape. It regulates and supports membrane traffic and defines synapse compartments. It also drives the generation of new synapses and lasting changes in synapse size and shape that occur in response to salient stimuli. These are long-standing, widely accepted facts. The broad acceptance of these facts makes it all the more surprising that relatively little is known about how synaptic actin is generated. What drives its assembly when new synapses are forming, and how does this process differ from the reorganization that drives spine expansion or shrinkage? This gap in knowledge limits the understanding of Alzheimer's Disease and related dementias where synapse loss and changes in spine shape are well documented. It also impacts brain disorders and pathologies, including amyotrophic lateral sclerosis, schizophrenia, intellectual disability and autism, that can be seeded in the mutation, loss, or gain of actin regulatory function, and disorders that involve derailed mechanisms of synapse plasticity such as drug addiction. This gap in knowledge is not an oversight or due to lack of interest. It exists because synapses are small and difficult to study, actin filaments are exceptionally thin, fragile and dynamic, and several molecular components important for nucleating actin have only recently been identified. The purpose of this proposal is to identify the principal actin nucleators relevant to the generation of synapses, and to assess the time, place, and context in which they act. Not knowing the relevant players is a rate limiting step in the field and the proposed experiments are a first step toward identifying the nature and location of actin scaffolds relevant to particular stages of synapse formation, biological actions (e.g. adhesion or trafficking) or to changes in state (e.g. potentiation or depression).