James A. Bibb - US grants

In striatal neurons, one important step in dopaminergic signal transduction involves the activation of the protein phosphatase 1 inhibitors, DARPP-32 and inhibitor 1. In response to dopamine, these proteins are phosphorylated by cAMP-dependent protein kinase and block dephosphorylation of downstream effectors which regulate the physiological state of the neurons. An objective of this proposal is to identify and characterize new first messenger pathways occurring in medium spiny neurons of the basal ganglia which regulate the phosphorylation state of DARPP-32 and inhibitor 1 and understand the molecular mechanisms by which they achieve their effects. The specific aims of this proposal are (1) to identify novel protein phosphorylation events in which DARPP-32 and inhibitor 1 serve as substrates and characterize these phosphorylation events in vitro, (2) to demonstrate that the novel phosphorylation events occur in vivo and to study their role in signal transduction pathways in the basal ganglia. Enzymatic phosphorylation of DARPP32 and inhibitor 1 will be studied in vitro with the resulting data being applied to studies of brain tissue. Novel phosphorylation sites will be identified and these sites will be examined for phosphorylation state in neostriatal tissue under basal conditions, during developmental, and after pharmacological treatments. It is possible that new targets for antipsychotic drugs may be developed on the basis of this research.

Drug addiction has a detrimental sociological and economic impact upon the U.S. as well as the entire wond. By exacting its toll on families and communities, it not only affects the present, but also future generations. Addiction to the psychomotor stimulant cocaine is characterized by a progressive escalation and loss of control over drug intake despite the negative consequences associated with continued drug use. This process involves long-term neuroadaptations within the circuitry of the brain that is normally dedicated to reward and incentive learning. Our goal is to provide a clearer understanding of the biochemical basis of the effects that cocaine has on behavior. Reward learning, as well as drug seeking behavior is dependent upon both increased glutamatergic and dopaminergic neurotransmission within the striatum. Glutamate activates Ca2+-dependent signaling cascades, while dopamine invokes G-protein coupled cAMP-dependent signaling pathways. The neuronal protein kinase Cdk5 integrates Ca2+ and cAMP signaling and mediates the effects of cocaine. We have found that Cdk5 is constitutively active under basal conditions and provides a negative tonus toward PKA signaling. while activation of NMDA receptors by glutamate in striatal neurons inhibits Cdk5. Thus Cdk5 is uniquely positioned to mediate the biochemIcal effects of cocaine that are necessary for addiction. We propose to study the role of Cdk5 in addiction by characterIzing the effect of conditional loss of Cdk5 on the behavioral responses of mice to cocaine administration, These stud res wilt utilize conditional knockout transgenic technology to delete the Cdk5 gene in adult mice [and will be complemented by the use of novel systemic Cdk5 inhibiting drugs]. The cellular basis for these effects wlR be pursued by characterizing the regulation of Cdk5 via ionotropic glutamate receptors. and the effects of cocaine upon this regulation will be examined in intact brain tissue using a neuropharmacological approach. To specifically define the molecular mechanisms by which Cdk5 mediates cocaine's effects and contributes to addiction, we will characterize novel interactions between Cdk5 and NMDA receptors, which we have discovered contribute to synaptic plasticity that underlies memory formation. By understanding the biology of drug abuse at the biochemical and molecular level, we strive to contribute to the development better treatmems for addiction. (The use of inhibiting drugs will be eliminated.]

DESCRIPTION (provided by applicant): Dopamine neurotransmission is important for many physiological functions and its dysregulation contributes to numerous neurological disorders. Previously, we showed that the neuronal protein kinase Cdk5 modulates dopamine efficacy by converting the striatal phosphatase inhibitor DARPP-32 into a PKA inhibitor. Further studies of the regulation of dopamine signaling by Cdk5 using pharmacology and transgenic technology have revealed novel mechanisms by which Cdk5 is likely to govern PKA activity in the striatum. Here we provide evidence that Cdk5 regulates PKA through phosphorylation of the PKA holoenzyme RII2 subunit. Furthermore, Cdk5 regulates intracellular cAMP levels through direct phosphorylation the phosphodiesterase PDE4B or controlling its regulation by PKA and MAPK. We made conditional Cdk5 knockout models and found that knockout in adult mice results in severe alterations in dopamine signaling. We propose to further study Cdk5- dependent regulation of PKA signaling pathways in striatal neurons using a conditional knockout approach. We propose to characterize the biochemical mechanisms by which Cdk5 regulates PKA both in vitro and in intact striatal tissue. These studies will identify targets and suggest strategies for the development of new treatments for neurological and neuropsychiatric disorders. PUBLIC HEALTH RELEVANCE: Neuropsychiatric disorders that are caused by disruptions in dopamine neurotransmission include schizophrenia, attention deficit hyperactivity disorder, and numerous other mental illnesses. The goal of this research is to delineate new mechanisms by which dopamine neurotransmission is regulated involving the neuronal protein kinase Cdk5 so that new pharmacotherapeutic treatments for these disorders can be developed.

DESCRIPTION (provided by applicant): Cognition is dependent upon the proper formation of neural circuits and the adaptations of those circuits in response to experience. The cellular and molecular mechanisms underlying declarative learning and memory constitute a predominant area in basic neurobiological research and are of considerable clinical relevance. Dysregulation of the biochemical mechanisms underlying cognition likely contributes to neurodevelopmental and neurodegenerative disorders including autism, Alzheimer's, mental retardation, schizophrenia, and attention deficit and hyperactivity disorder. We have focused on synaptic signal transduction mechanisms involving the neuronal protein kinase Cdk5, with the goal of determining its role in learning and memory. We developed an innovative transgenic approach that allowed the induction of knockout of the neuronal protein throughout the brain of adult mice. This led to the discovery that the neuronal protein kinase Cdk5 governs synaptic plasticity, learning, and memory. We found that this was due to changes in the levels and surface expression of the NR2B subunit of the NMDA receptor. Here we propose to characterize the interactions between Cdk5 and NR2B, evaluate the role of Cdk5-NR2B interactions in synaptic plasticity, and assess the role of Cdk5-NR2B interaction in learning, and memory. The proposed studies are based on our preliminary findings that Cdk5 phosphorylates NR2B at a novel site and this phosphorylation controls the translocation of the receptor to the synaptic cell surface. We will characterize the regulation of this important site with regard to phosphorylation/dephosphorylation and define its physiological function. We hypothesize that the phosphorylation state of NR2B is dynamically regulated during learning and essential for consolidation of memory in the hippocampus. Furthermore, we show that are developing small drug-like interfering peptides based that disrupt Cdk5-NR2B interactions in vitro and in vivo. We will use this selective targeting approach to manipulate Cdk5-NR2B interactions and bidirectionally control the phosphorylation state and surface levels of NR2B. This will allow us to modulate synaptic plasticity, learning and memory in the hippocampus. Thus by combining advanced transgenic, biochemical, neurophysiological and behavioral approaches, we will define an important new mechanism that mediates cognition and demonstrate it as a target for the possible development of innovate disruption strategies to treat cognitive disorders.

DESCRIPTION (provided by applicant): Drug addiction is a widespread and severe neuropsychiatric disorder and a major public health concern. It is characterized by loss of behavioral control as the neurobiological processes of learning and memory of information that motivates actions to acquire rewards are overwhelmed by the pharmacological effects of the drug. Combined with other environmental and emotional factors, motivated drug taking leads to compulsive craving, seeking, and taking that define addiction. Reward and addiction learning are mediated by molecular mechanisms of synaptic remodeling at dopaminergic and glutamatergic synapses. Identifying and validating these mechanisms is key to understanding addiction and developing effective strategies to treat it. We have discovered a new mechanism that mediates cognition through the regulation of NMDA receptors (NMDARs). This mechanism involves the modulation of the phosphorylation state of Ser1116 of the NR2B subunit of these receptors. This site is phosphorylated by the neuronal protein kinase, Cdk5. Cdk5 knockout (KO) or inhibition reduces phospho-Ser1116 NR2B, increases cell surface levels of the receptor, increases NMDAR-mediated current, and enhances cognition. Interestingly acute cocaine exposure, causes dephosphorylation of this site, likely facilitating reward learning. In contrast, chronic cocaine exposure potentiates this site, possibly attenuating further learning, thereby contributing to the perpetuation of the addicted state. We believe this mechanism provides a new avenue to understanding the molecular basis of addiction. We propose to study the regulation of this mechanism by cocaine and dopamine signal transduction. We will characterize its modulation during acquisition of self-administration (SA), chronic SA, and extinction after withdrawal from sucrose and cocaine SA in mice. We will define the effects of loss of Cdk5 and reduced phospho-Ser1116 NR2B on sucrose and cocaine SA by temporally and spatially controlled Cdk5 KO. Finally, we will specifically target and validate the role of this mechanism in acquisition, extinction, and reinstatement of sucrose and cocaine SA by viral gene delivery of novel drug-like small interfering peptides that disrupt NR2B-Cdk5 interactions. This translational research will significantly advance our understanding of the mechanisms of addiction and may contribute to the development of treatments to help addicted individuals recover.

DESCRIPTION (provided by applicant): Stroke is the leading cause of adult disability in the U.S. and the third leading cause of mortality world-wide. The most common form, ischemic stoke, is caused by blood clots occluding arterial supply, resulting in permanent neurological damage or death. For a minority of patients, blood clots can be removed or lysed to prevent or limit stroke injury. However, neuroprotectants that improve survival and recovery are not available. Following arterial occlusion, neuronal death spreads outward from the initial infarct as neurons depolarize causing massive glutamate release and hyperactivation of NMDA receptors (NMDAR). As a result, excitotoxic Ca2+ flows into cells where it activates the protease calpain. This excitotoxic cascade is a major component of stroke pathophysiology. However, efforts to block the cascade with NMDAR antagonists have not proven effective in clinical trials due to unwanted effects. We discovered that the protein kinase Cdk5 physically links calpain to the NR2B subunit of NMDARs. Cdk5 is also bound to its activator p35. Through this NR2B-calpain- Cdk5/p35 signaling complex, excitotoxic activation of NMDARs causes calpain to convert p35 to p25. Cdk5 associated with p25 causes neuronal death and has been suggested to mediate to ischemic injury. However little is yet known of how Cdk5 or p35/p25 contributes to stroke pathophysiology or how it may be effectively targeted to improve outcome. Here we propose to characterize the dysregulation of Cdk5 following middle cerebral artery occlusion (MCAO) and determine the neuroprotective effects of loss of Cdk5 or p35 in mice. As a novel strategy to achieve neuroprotection we will target the NR2B-calpain-Cdk5/p35 cascade by identifying functional protein-protein interaction motifs and developing small interfering peptides (SIPs) that prevent calpain-Cdk5 and NR2B-Cdk5 interactions. These SIPs will be optimized as molecules that potently and specifically dissociate the NR2B-calpain-Cdk5/p35 and prevent Cdk5/p25 generation by NMDAR hyperactivation without otherwise affecting the functions of the receptors, calpain, or Cdk5. We will then assess their ability to neuroprotect and improve behavioral and histological outcome in mice subjected to MCAO. These translational studies will advance our knowledge of the mechanisms that mediate stroke injury and derive novel neuroprotectants that will potentially lead to the development of therapies that improving recovery and reduce stroke-related disability.

STRIATAL EXCITATORY AND METABOTROPIC PKA REGULATION IN STRESS AND RESILIENCE Mental disorders such as anxiety and depression are major health concerns that contribute unabated to a large portion of all morbidity and mortality. These complex disorders may be viewed as mal- adaptations that arise in brain circuitry. In order to achieve more effective treatments, better mechanistic understanding of brain circuitry integration is needed. Normally, motivated behaviors and executive functions require processing of sensory-triggered excitatory neurotransmission and assignment of emotional context. This occurs in the striatum where cortical glutamatergic and midbrain dopaminergic inputs converge to mediate brain functions such as reward and stress responses. Striatal dysfunction is broadly implicated in the etiology of many mental illnesses. For example, stress-induced alterations in the activity of reward-related brain regions, such as the nucleus accumbens (NAc), are linked to the pathophysiology of depression. Insight into the mechanisms by which glutamate and dopamine neurotransmission are integrated within the NAc may shed light on some causes of mental illness, or implicate new drug targets and treatment strategies. Here, we introduce a new signaling mechanism which we hypothesize is controlled by striatal glutamatergic and dopaminergic signaling to allow concerted regulation of PKA activity. Specifically, our preliminary data indicates that glutamate controls constitutive phosphorylation of the RII-beta (RIIb) regulatory subunit by Cdk5, which then directly affects PKA activation by D1-type dopamine receptors via a second PKA-dependent auto-phosphorylation mechanism. We hypothesize that this unique mechanism mediates striatal plasticity and behavioral responses to stress and that chronic stress can cause mal-adaptations in this mechanism so that glutamate and dopamine signaling are uncoupled and PKA signaling is dysregulated. We further hypothesize that this mechanism may be targeted to improve striatal plasticity and behavioral resilience. To pursue this novel premise, we propose to 1) study the regulation of RIIb/PKA and explore downstream effectors in vitro and in vivo; 2) study the role of RIIb/PKA phosphorylation in ventral striatal neuronal excitability and synaptic plasticity and 3) study the regulation of this mechanism by acute and chronic stress, and determine how it contributes to behavioral responses to stress. These studies will yield important information on the mechanisms that integrate brain circuitry and how they are affected by stress. Thus, we will better understand some of the basis by which stress may contribute to complex mental disorders such as anxiety and depression and how they may be more effectively treated.

PERIPHERAL INFLAMMATION AND STRESS DRIVE VENTRAL STRIATAL MALADAPTATIONS PROJECT SUMMARY Mental illnesses such as depression and anxiety are a major disease burden linked to suicide and mortality. It is well known that exposure to stress can precipitate neuropsychiatric complications. The immune system also has a large influence on psychological symptoms and chronic conditions like inflammatory bowel disease dramatically increase risk of depression and anxiety. Surprisingly, little is known of the brain circuitry-specific mechanisms that drive this comorbidity. Our goal is to address this need by studying how systemic inflammation and stress cause maladaptations in reward/aversion circuitry of the ventral striatum (nucleus accumbens, NAc). In preliminary studies, we found that gastrointestinal (GI) inflammation, as a pervasive form of systemic inflammation, modulates stress-response behavior, and dysregulates NAc synaptic plasticity and excitability of D1 dopamine (DA) receptor (D1R) expressing medium spiny neurons (MSNs). Multi- omic exploration of the mechanistic basis for these effects implicated a novel dynorphin (DYN)-kappa opioid receptor (kOR)-Cdk5/p35-b adducin (ADD2) signaling cascade in the NAc which we hypothesize mediates these maladaptations. Based on these findings we propose to study the effects of peripheral inflammation, stress, and their interactions on neurobehavioral functions (Aim 1), and NAc synaptic plasticity, cell type-specific excitability, and DA neurotransmission (Aim 2). The novel kOR-Cdk5/p35-ADD2 pathway we have identified provides a mechanism by which maladaptive changes in DA neurotransmission can actuate alterations in DA-cAMP-PKA signaling and alter structural plasticity. We will study the mechanisms by which this pathway functions and its contribution to the effects of inflammation and stress on structural plasticity (Aim 3). Innovative components of this proposal include the study of inflammation/stress interactions, NAc cell type-specific interrogation of the role of kOR-Cdk5/p35-ADD2 signaling in mediating these effects, in vivo fiber photometry to study DA dynamics, and a novel systemic Cdk5 inhibitor as a targeted therapeutic approach. This research connects a strong field of striatal signal transduction to a major clinical problem. The impact will be to provide a detailed picture of the mechanistic basis for systemic inflammation-mental illness comorbidity and possible new approaches for therapeutic intervention.