Nicole Calakos - US grants

DESCRIPTION (Applicant's Abstract): The candidate proposes to undertake a program of research which will involve extensive training in the fields of electrophysiology and synaptic plasticity. The addition of expertise in these fields will complement the candidate's already extensive background in the molecular biology and biochemistry of synaptic vesicle function. Specifically, the goals of the research are to understand the basis of long-term potentiation (LTP) in the mossy fiber synapses of the hippocampus, an area that relies on presynaptic mechanisms for this plasticity. Two proteins, rab3a and protein kinase A (PKA), whose essential role at mossy fiber LTP (mfLTP) has already been established will be the focus of investigation. To determine the downstream effectors of rab3a which mediate mfLTP, extensive electrophysiologic analysis of a null mouse strain for the candidate rab3a effector protein, rim, will be undertaken. Once the basis of the rim (-/-) mutant phenotype on mfLTP is characterized, rescue experiments in primary cultures of hippocampal granule cells using transient expression systems will be performed. This approach will further be used to identify the specific domains of rim required for mfLTP. A second line of study will be the pursuit of targets of PKA phosphorylation required to establish mfLTP. In addition to standard techniques of studying phosphoproteins, a specific analysis of proteins important for synaptic vesicle docking and fusion will be performed as there is evidence that suggests they may be targets of PKA. The final area of investigation will return to the role of rim in mfLTP and seek to understand the molecular basis of this effect by identifying interacting proteins. In the long term, using such approaches to study synaptic plasticity, the candidate hopes to understand the role of plasticity in memory, recovery from brain injuries such as stroke, and neurodegenerative diseases such as Alzheimer's with the goal of improving therapy in disease states. The candidate's background of graduate study in Neuroscience and residency training in Neurology is particularly suited to make basic science discoveries and apply them to areas of clinical relevance. The mentor, Dr. Robert Malenka is also a physician-scientist and serves as an ideal role model for excellence in research. The research environment at Stanford University School of Medicine is well suited both in its physical plant and intellectual resources to embark upon these studies. Lastly, in addition to acquiring new research skills, the candidate will be active in a variety of relevant scientific meetings (journal clubs, lecture series, national meetings) and the Department of Neurology to prepare for a career in academic medicine with both research and clinical responsibilities.

DESCRIPTION (provided by applicant): Elucidating the molecular basis of synaptic plasticity will lead to a more sophisticated understanding of the neural circuit modifications which underlie experience-dependent plasticity in both health and disease. Much is known about the mechanisms of postsynaptic forms of long lasting plasticity. By comparison, however, relatively little is known about the mechanisms of presynaptic plasticity. This proposal focuses on understanding the synaptic functions of a class of presynaptic, active zone proteins, RIMs, because of their requirement in a prominent form of presynaptic LTP and their additional roles in basal neurotransmitter release and short-term plasticity. RIMs have several protein binding domains that interact with key components of synaptic vesicles and active zones. Because of this, RIMs have been described as presynaptic "scaffold" proteins. As a scaffold, RIM is a powerful tool to gain insight to the coordinate activities of several important presynaptic proteins. A key outstanding question is to understand how RIM integrates the activities of its binding partners to achieve synaptic plasticity. In this proposal, we will perform rescue experiments in the RIM1a knockout background to delineate the functional significance of RIM1a's interactions with other presynaptic proteins. We will also study the functional significance of a key PKA phosphorylation site that is implicated in mediating LTP. Lastly, we will evaluate the functional significance of the RIM2 gene products. Are they functionally redundant or does expression of a particular RIM isoform convey distinct synaptic properties? To perform these experiments we will use electrophysiological recording techniques on both culture and acute slice preparations in combination with molecular techniques to allow gene transfer into the knockout background. A precise understanding of the molecular interactions that underlie presynaptic plasticity as described in this proposal is critical both to enable studies of the behavioral significance of presynaptic plasticity and to enable targeting these proteins for the development of therapies for a wide range of neuropsychiatric diseases that may involve synaptic plasticity such as dementia, dystonia and addiction.

DESCRIPTION (provided by applicant): Animal models are an important tool for studying human disease mechanisms and testing new therapies. We have identified a compulsive grooming disorder in mice following deletion of a key scaffolding component of the post-synaptic density (PSD). The pathogenesis of this behavior in mice may relate to disorders in the Obsessive Compulsive Disorder (OCD)-like spectrum of anxiety disorders in humans. This mouse model now affords us an opportunity to study pathogenesis from gene to synaptic function to circuit to behavior. The experiments proposed will begin to establish these links by delineating how the loss of this PSD component alters post-synaptic composition, synaptic transmission, and cortico-striatal circuitry. We further propose rescue experiments to restore synaptic function at cortico-striatal synapses and eventually to determine the critical circuitry sufficient to restore normal behavior to the animal. In sum, the results of these experiments will advance our understanding of post-synaptic assembly and synaptic transmission at cortico- striatal synapses. These underpinnings are critical to direct future therapies in humans for OCD-like disorders and other entities arising from abnormal basal ganglia synaptic transmission.

DESCRIPTION (provided by applicant): Dystonia is among the top 3 prevalent movement disorders and a cause of unremitting disability from a relatively young onset. As yet, its mechanisms are largely unknown. It is not considered a neurodegenerative disease and abnormalities in brain plasticity are suggested. In a patient with sporadic, late-onset, focal dystonia, we recently identified a novel rare sequence variant of TOR1A (p.F205I). TOR1A mutation is a known cause of familial early-onset, generalized dystonia (DYT1, c.GAG). After revealing in silico and in vitro evidence that the p.F205I variant impairs TorsinA function, we developed a knockin mutation mouse model to test the behavioral significance. In preliminary studies, we have found that F205I mutant mice have robust and replicable behavioral abnormalities in a motor learning task. We propose to further develop this novel mouse model and use it to understand the changes in brain activity and neuropathology due to F205I TorsinA and their relationship to behavior. The F205I TOR1A mouse model provides a useful tool to establish the causal relationship between TorsinA dysfunction, neuronal pathology and altered behavior. By furthering knowledge of TorsinA biology, we hope to accelerate insights for the treatment of dystonia.

DESCRIPTION (provided by applicant): Tourette Syndrome (TS) and its associated conditions (tics, OCD, ADHD) constitute a substantial societal burden, as TS alone is estimated to affect nearly 1% of the population. A critical barrier to progress in understanding the mechanisms for these disorders is the lack of animal models with etiological relevance to the human condition. We propose to take advantage of the recent discovery of a genetic etiology for TS involving a nonsense mutation within the histidine decarboxylase gene to overcome this barrier. In this proposal we will create, validate and behaviorally characterize a novel mouse model for TS. To most faithfully replicate the protein-based pathobiology as it occurs in humans, we will create humanized mouse models that express the human protein in place of the endogenous mouse protein. Moreover, because such a model will express the human TS mutant protein, once validated, this mouse model will be uniquely suited for screening novel therapeutics in a way that is not afforded by conventional mouse knockin approaches.

DESCRIPTION (provided by applicant): Compulsive motor behaviors reflect a loss of normal adaptive behavioral responsiveness and result in rigid behaviors that range in severity from detrimental to disabling. Obsessive compulsive disorder (OCD) is the prototypical form of such behavioral disorders, but maladaptive compulsions emerge in diverse contexts affecting people across the lifespan. Development of effective therapeutics for compulsivity has been hampered by our lack of understanding of the underlying cellular and circuit mechanisms. In order to address this need, we have assembled a unique set of genetic reagents and developed a novel approach to examine the local striatal circuit. We propose to: (1) test our hypothesis that overactivity of striatal group 1 metabotropic glutamate receptors (mGluRs) drives compulsive-like behaviors in mice, (2) build upon our recent progress of defining a role for mGluR5 dysregulation by determining whether mGluR1 signaling is also dysregulated in mice with OCD-like behaviors, (3) evaluate the integrated effects of abnormal striatal group 1 mGluR signaling on striatal output in mice with OCD-like behaviors and (4) determine whether mGluR-dependent reconfiguration of striatal output is a common mechanism associated with animal models for compulsive behaviors. Together these studies have the potential to be paradigm-shifting by providing the first direct experimental evidence to support a role for striatal group 1 mGluRs in causing compulsive motor behaviors. Such preclinical results will newly direct attention to this highly druggable class of receptors as well as illuminate specific cellular and circuit mechanisms to advance the treatment of related disorders in humans.

DESCRIPTION (provided by applicant): The TOR1A gene encodingTorsinA protein is mutated in the most common form of inherited primary dystonia, DYT1. Both our understanding of the cellular biology and efficacy of treatments is very limited for dystonia. The human DYT1 disease-causing mutation, deltaGAG causes major cellular disruption of membrane flow and fluorescent indicators of TorsinA show an irregular punctuate pattern (inclusions). We hypothesize that the identification of modifiers of cellular inclusion pathology caused by mutant TorsinA proteins will provide novel targets to advance both our understanding of dystonia pathogenesis and to provide novel targets for the treatment of dystonia. Using a novel high-throughput assay that our group recently developed, we propose to perform whole genome RNAi screening for modifiers that normalize mutant TorsinA-associated cellular pathology. We expect that this screen because of its comprehensive scope and unbiased nature may identify novel therapeutic candidates and further suggest entire signaling pathways to target for the treatment of dystonia.

ABSTRACT The basal ganglia are critical for the learning and subsequent selection of motor programs. In health, adaptive plasticity in the basal ganglia enables easy execution of complex motor tasks through formation of habits. Conversely, in disease, repetitive behaviors, addictions and compulsions are thought to derive from maladaptive plasticity involving basal ganglia circuitry. The efficacy of medications and deep brain stimulation targeting the basal ganglia to treat Parkinson disease offer examples of the benefits to be gained by understanding the mechanisms by which this circuitry modulates behavior. However, major gaps in our understanding of the functional principles of the basal ganglia limit more widespread and more effective targeting of this circuitry to alleviate other ailments deriving from dysfunction of this circuitry, such as addictions, compulsions, and dystonia. Historically, there have been wide gaps in the levels of analysis of brain plasticity mechanisms, typically involving isolated study of particular candidate synapses ex vivo and regional activity in awake behaving animals. However, emerging technologies to image and selectively manipulate brain circuitry now provide several key opportunities to bridge these levels of analysis. These opportunities can reveal how plasticity is organized across the local microcircuitry and enable finer levels of monitoring and perturbing activity in the awake behaving animal. In this mentored career award, an accomplished investigator in the field of synaptic plasticity and the basal ganglia, Dr. Calakos, proposes to undertake career enhancement activities to acquire new expertise in in vivo physiology and familiarity with the latest cutting-edge opportunities to evaluate and manipulate activity in vivo. In collaboration with key mentors, the research objectives will be to develop an approach to simultaneously image both classes of striatal projection neurons in vivo and to use that approach to evaluate the novel variable of relative latency to fire between the two neuronal classes of striatal projection neurons as a driver of habitual behavior.

ABSTRACT A distinguishing feature of the brain is that its circuitry isn?t computationally static, it adapts to experience. Understanding the circuit mechanisms for adaptive behavior carries two-fold potential benefits - revealing the brain?s learning rules and identifying key behaviorally significant functional ?nodes?. These nodes suggest potent sites to target for therapy development and may also be instructive to suggest more basic circuit principles underlying behavior. Using striatal circuitry and habit learning as a model system, we recently uncovered a set of paradigm- challenging findings in a striatum-dependent habit learning task. In particular, we discovered a new circuit-level signature, termed dviLP (direct vs indirect Latency Plasticity), which distinguishes striatal slices prepared from habitual vs goal-directed animals. The features of dviLP shift long-held attention on rate differences between the two principle projection neuron types, those to the direct and indirect pathways, to consider that behaviorally adaptive signals may be generated by plasticity of their relative timing to fire. Moreover, the origin of this plasticity appears to involve striatal fast-spiking interneurons, a highly non-canonical site for the expression of long-lasting plasticity. Beginning with this highly novel foundation, here we propose to generate a robust predictive computational model for striatal-dependent learning mechanisms by joining multiple disciplines and multiple levels of analysis through an iterative process of circuit modeling and experimentation. In Aim 1, we will comprehensively map functional changes in synaptic and cellular activity that define the behavioral transition from goal-directed to habitual in an operant lever press task. We will use a layered suite of molecular genetic tools to assign coordinates that specify inputs, outputs, compartments (striosome/matrix) and regions (medial, dorsal). In Aim 2, we will measure the activity of genetically specified components of the striatum in behaving mice, identifying the dynamic changes that correlate with and cause the shift from goal- directed to habitual behavior. Our team offers multidisciplinary strengths. Dr. Calakos and Yin have expertise in habit behavior, plasticity mechanisms and in vivo circuit dynamics; ideal for spearheading this effort. The success and impact of this effort will be amplified by tightly incorporating Dr. Brunel?s expertise in computationally modeling brain learning mechanisms and Dr. Tadross?s novel pharmacogenetic reagents that are ideally positioned to test causality of synaptic plasticity events, offering the unique opportunity to manipulate a specific synaptic receptor in a genetically defined cell type. Ultimately, we expect that the knowledge gained through this highly collaborative proposal will provide a foundational resource to accelerate understanding of striatal learning rules for adaptive behavior.