Fernanda Laezza - US grants

DESCRIPTION (provided by applicant): Psychiatric diseases are chronic, devastating disorders thought to arise from maladaptive brain plasticity, and potent and safe pharmacotherapies are in great need. Identifying the mechanistic links that might sustain these aberrant neuroadaptations will advance our understanding of the biology of mental disorders, potentially providing new platforms for medication development. Using an innovative bioluminescence-based molecular screening approach combined with biochemical, electrophysiological, and imaging assays, we provide breakthrough results showing a link between glycogen synthase kinase 3 (GSK3), a critical enzyme found dysfunctional in mood disorders, depression and schizophrenia, and neuronal excitability, which we propose as a potential mechanism underlying dysfunction of neuronal circuitries associated with psychiatric disorders and certain addictive behaviors. Building on previous discoveries demonstrating that fibroblast growth factor 14 (FGF14) is a functionally relevant component of the Nav channelosome that controls neuronal excitability, we present exciting new data showing that the FGF14:Nav channel complex formation is bi-directionally controlled by GSK3 and by the GSK3 constitutive repressor, protein kinase B (Akt), and that GSK3 directly phosphorylates FGF14. Pharmacological inhibition of Akt and GSK increases and prevents, respectively, the FGF14:Nav channel complex formation, whereas inhibition of GSK3 occludes the effect of Akt inhibition. In hippocampal neurons, GSK3 inhibition disperses the FGF14:Nav channel complex from the axonal initial segment (AIS), the site of action potential initiation, impairs intrinsic fring and reduces excitatory synaptic transmission, whereas inhibition of Akt leads to opposite phenotypes. Furthermore, we show that Fpep1, a small interfering peptide modeled upon the FGF14:Nav channel interface, prevents the FGF14:Nav channel complex assembly, providing a tool for minimizing the effect of GSK3 on neuronal excitability in vivo. In this proposal we will employ a combination of bioluminescence-based technology, mass spectrometry, phosphorylation assays, confocal imaging and electrophysiology to determine the molecular mechanism by which GSK3 controls the FGF14:Nav channel complex formation (Aim 1) and promotes targeting of the FGF14:Nav channel complex in neurons (Aim 2) and to evaluate whether GSK3 exerts an effect on excitability and neuroplasticity in cortico-limbic circuits through the FGF14:Nav channel complex that could be reversed by pharmacological or genetic approaches targeting FGF14 (Aim 3). Positive outcomes of this study will provide new insights into the molecular mechanisms of GSK3 in the brain and offer an unprecedented opportunity for new medication development against GSK3-linked psychiatric disorders. PUBLIC HEALTH RELEVANCE: Innovative and integrated approaches are needed to enhance the success of therapeutic interventions against psychiatric disorders. Through a multidisciplinary project including molecular biology, biochemistry mass spectrometry, single cell imaging and electrophysiology in rodent models, we will validate FGF14 as a novel downstream target of GSK3, creating a novel platform for intervention against psychiatric disorders associated with GSK3 dysfunction.

ABSTRACT Psychiatric diseases and addictive behaviors are complex brain disorders associated with maladaptive plasticity of the brain circuitry. The lack of adequate platforms to rapidly screen against novel targets in physiological environments has significantly hampered probe discovery initiatives that could inform the circuitry alterations leading to mental disorders and addiction and enable the design of future therapeutics. Protein- protein interactions (PPI) within ion channel complexes fine-tune neuronal excitability and are emerging as links to the biology of psychiatric disorders. Their highly specific and flexible interfaces could make protein- channel interactions ideal targets for probe development. Such molecular probes would provide the neuropharmacology community with optimal research tools to parse out brain disease complexities and ultimately enable future drug design. We have identified the PPI between the voltage-gated Na+ (Nav) Nav1.6 channel and its accessory regulator protein, fibroblast growth factor 14 (FGF14) as a novel, functionally relevant regulator of neuronal excitability in brain areas such as the cortico-mesolimbic circuit, which is associated with disorders of the affective and cognitive domains. Through a successful bioluminescence-based high-throughput screen (HTS) and subsequent in vivo studies in the nucleus accumbens (NAc), we discovered that the FGF14:Nav1.6 channel complex is part of the glycogen synthase kinase 3 (GSK3) pathway, a signaling cascade found aberrant in bipolar disorder, depression, anxiety and addiction. To explore the druggability of the FGF14:Nav1.6 complex, we employed a minimal functional domain (MFD) approach to design a peptide-derivative mapped to the PPI interface and showed it has in vitro-to-ex vivo activity in the NAc circuit. These discoveries have prompted us to develop a new pipeline to identify chemical probes against the FGF14:Nav1.6 complex to interrogate its function in the cortico-mesolimbic circuit. To advance a probe discovery campaign against this new target, we have designed an integrated multi-modal screening platform, based on the latest MFD principles of pharmacology that includes a newly designed and validated bioluminescence primary screening assay to reconstitute the FGF14:Nav1.6 C-tail complex in cells. This pipeline also includes the necessary counter, toxicity, and cell-free orthogonal assays (Aim 1), automated patch-clamp electrophysiology as a functional screen in combination with structure-activity relationship efforts and in silico analysis (Aim 2), and ex vivo validation of selected probes in the NAc circuitry (Aim 3). The proposed pipeline introduces a new, rapid and integrated platform that will accelerate the discovery of novel chemical probes for neuronal excitability, providing the foundation for pre-therapeutic development of a new class of PPI-based leads for a broad spectrum of psychiatric disorders.

ABSTRACT The development of pharmacotherapeutics for cocaine use disorder has lagged behind treatments for other neuropsychiatric conditions, emphasizing the need for new targets. Preliminary data suggest that rats with a protective phenotype for cocaine taking/seeking produced by environmental enrichment have less retinoic acid signaling in the nucleus accumbens, a brain region highly implicated in addiction. Additional preliminary data show that increasing retinoic acid signaling increases neuronal firing and EPSCs in the nucleus accumbens (NAc) shell and increases cocaine taking/seeking in rats. Inversely, decreasing one aspect of retinoic acid signaling in the NAc in vivo decreases neuronal firing and cocaine taking. The current proposal will generate specific mechanistic evidence regarding how retinoic acid signaling confers susceptibility to cocaine self- administration. Accordingly, the overall hypothesis of this proposal is that specific components of the retinoic acid signaling pathway in the NAc shell control susceptibility/resilience to cocaine self-administration and will represent valuable novel targets for the future treatment of cocaine dependence. Thus, the successful completion of this project will prioritize high-quality ?druggable? targets in the retinoic acid pathway for subsequent pharmacotherapeutic development that can be developed and translated into clinical practice. The first aim will determine if decreasing retinoic acid synthesis in the NAc shell alone confers a protective phenotype for cocaine self-administration. Expression of the retinoic acid synthesis enzyme Aldh1a1 will be knocked down in vivo using a novel adeno-associated viral vector (AAV) prior to behavioral phenotyping and electrophysiological analysis. Next, the acute effects of Aldh1a1 inhibition on behavior, firing, and synaptic transmission will be tested using a small-molecule inhibitor. The second aim will determine the relative influence of retinoic acid receptor (RAR) vs. peroxisome proliferative receptor (PPARbeta/delta) signaling mechanisms. AAV vectors will be used to either knock down PPARbeta/delta or overexpress RARbeta to create a protective behavioral and electrophysiological phenotype in susceptible rats. The results will determine which of the competing mechanisms contains the most promising therapeutic target. The third aim will determine the relative genomic vs. non-genomic influence of retinoic acid signaling in rat NAc. Chromatin immunoprecipitation will be employed with DNA sequencing to assess the genomic aspect. The non-genomic side of the equation will focus on rapid RA-dependent homeostatic synaptic plasticity from neuronal RNA granules. The final result of this project will be a much-needed novel candidate target for therapeutic development for cocaine addiction.

ABSTRACT Neuropsychiatric disorders are thought to arise from complex changes of brain plasticity. Recent evidence points toward ion channel complexes as cellular hubs of plasticity that confer disease vulnerability or protection depending on the channel regulatory state. In medium spiny neurons (MSNs) in the nucleus accumbens (NAc), a subtype of highly vulnerable cells, neuroadaptive changes in intrinsic firing are mediated by neurotrophin brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signaling. Yet, the molecular mechanisms by which these changes occur are still poorly understood. Intrinsic firing in MSN relies on the integrity of the macromolecular complex of the voltage-gated Na+ (Nav) channel Nav1.6 and its accessory regulatory fibroblast growth factor 14 (FGF14) and is subject to regulation by glycogen synthase kinase 3 (GSK3) ?, a downstream effector of BDNF/TrkB signaling. Here, we provide exciting new evidence for the Nav1.6, FGF14 and GSK3? as a macromolecular signaling complex downstream of BDNF/TrkB critical for MSNs neuronal plasticity. Using an array of in vitro and in cell assays, cell imaging, and electrophysiology, we show that stability, phosphorylation and functional activity of the Nav1.6 channel are proportional to the level of BDNF and the kinase activity, whereby low level of BDNF predicts resilience and high level mediates a susceptible phenotype conferred by changes in neuron firing. We will conduct a full range of biophysical, biochemical and electrophysiological studies combined with pharmacological and viral vector-based in vivo gene transfer methods to evaluate the impact of BDNF/TrkB signaling on macromolecular composition (Aim 1), subcellular targeting (Aim 2) and functional properties (Aim 3) of the Nav1.6 channel in the context of neuroadaptive plasticity of MSNs. Outcomes of these studies could potentially lead to the development of biomarkers of susceptibility to neuropsychiatric disorders by investigating molecular pathways in relevant experimental models, an area of great interest for biological psychiatry.

ABSTRACT Epidemiological studies identify early life exposure to pyrethroids as a threatening risk factor for attention- deficit hyperactivity disorder (ADHD). Because the reported risk of exposure is within no-observed-adverse- effect level (NOAEL) guidelines, environmental exposure to pyrethroids could be an underestimated leading cause of ADHD and other neurodevelopmental disorders in the general population. Animal models of early-life exposure to the pyrethroid pesticide deltamethrin (DM), a potent neurotoxin that acts on the insect voltage- gated Na+ (Nav) channel, recapitulates ADHD-like behavior through disruption of dopamine signaling in the nucleus accumbens (NAc), the brain region implicated in the human disease. Yet, the mechanism of toxicity of DM in the developing brain has not yet been determined. Recent studies from our group provide evidence for cross reactivity of DM with the mammalian Nav1.1 channel, an isoform expressed in fast-spiking parvalbumin (PV) inhibitory interneurons during development. These cells exert a powerful inhibitory control over the output of the NAc, which, if disrupted, leads to dopamine dysfunction with effects on locomotor activity, attention, and impulsivity, endophenotypes that characterize ADHD. In supporting studies conducted in an early-life DM exposure animal model we show regional accumulation of DM and loss in GABA in the NAc/striatum and demonstrate disruption of PV interneuron firing in the same brain region accompanied by ADHD-like behaviors. Building on this premise, we propose molecular (Aim 1), functional (Aim 2) and behavioral (Aim 3) studies to test the hypothesis that the primary mechanism of DM toxicity in the developing brain is to disrupt PV interneuron function leading to loss of local inhibitory control in the NAc and behavioral phenotypes common to ADHD. Outcomes of this study will provide new insights into the molecular-based understanding of risk factors for neurodevelopmental disorders providing guidance for therapeutic development against exposure.