Paul J. Lombroso, MD - US grants

The basal ganglia is a brain region rich in dopamine and the principal site where dopamine blocking agents, including neuroleptic drugs, exert their central side effects. Although it has been established that neuroleptics block dopamine receptors in the basal ganglia, their effects on messenger RNA (mRNA) transcription are not known. The availability of a panel of complementary DNA (cDNA) clones for messenger RNAs which are effected by neuroleptic treatment would be an important step toward understanding the action of neuroleptics. I propose to study changes in mRNA levels in basal ganglia from animals treated with neuroleptics and dopamine neurotoxins. The first phase of the proposed project is to isolate and characterize basal ganglia enriched mRNA using subtractive hybridization and cDNA cloning techniques. These studies in normal animals represent a novel approach to studying selective gene transcription in the basal ganglia. I will initially use tissue derived from bovine brains to develop expertise in cDNA subtractive hybridization. I will then use brain tissue from untreated African green monkeys in a series of identical experiments. In both cases, mRNA from cerebellar tissue will be used to subtract common messages. These studies will result in the isolation of a panel of cDNA clones enriched to basal ganglia relative to cerebellum, and will be the basis for subsequent studies on the effects of dopamine blocking agents. In the second phase, I will examine the effect of decreased dopamine (DA) levels on mRNA transcription rates in this region. Two methods will be used to deplete postsynaptic levels of DA. First, DA pathways will be blocked by chronic treatment with the neuroleptic, Haldol. In the second method, a selective neurotoxin called MPTP, will be administered. The use of these agents in conjunction with subtractive hybridization techniques presents a unique opportunity to study the effects of dopamine depletion on mRNA transcription within neurons of the basal ganglia. The third and final phase of this project will be to characterize basal ganglia specific mRNAs, particularly those altered by drug treatment. For these studies, I will use in situ hybridization and immunocytochemistry to compare the anatomical distribution of experimentally isolated cDNAs from basal ganglia. Within the basal ganglia, the distribution of cDNAs will be compared with known neurotransmitter substances and other cell type specific markers. My long range goal is to understand the molecular bases for neuropsychiatric diseases and this proposal represents a first step toward that end.

For the last 30 years, the dopamine hypothesis of schizophrenia has been the dominant theoretical model which has guided clinicians in their treatment strategies and researchers in their experimental paradigms. In brief, it proposes an alteration in central dopaminergic pathways within the brains of schizophrenic patients. Particular attention has been given to dopaminergic and dopaminoceptive neurons of the basal ganglia and related structures as a possible area of pathology and/or site of action for neuroleptic treatment. The long-term objectives of may laboratory has been the isolation and characterization of genes specifically enriched within regions of the basal ganglia. A putamen-enriched cDNA library was constructed using subtractive hybridization techniques. This methodology has led to the isolation of a novel, intracellular protein tyrosine phosphatase (PTP) (Lombroso et al., 1991) which is highly enriched within the CNS and is localized within dopaminoceptive neurons of the basal ganglia and related structures (Lombroso et al., 1992). As the cDNA message was originally found by Northern analysis to be highly enriched within rat striatum, it was termed STEP for STriatal Enriched Phosphatase. Further characterization of this and other related members of this PTP family forms the basis of this FIRST (R29) application. The specific aims of this application include the identification of additional members of the STEP family of PTPs. We have evidence that the originally cloned STEP cDNA is one of several alternatively spliced variants. We now propose to isolate full-length PTPs related to STEP. This will be accomplished by screening brain cDNA libraries with the original STEP cDNA probe, and sequencing the isolated clones and demonstrating tyrosine phosphatase activity. We will generate a panel of monoclonal antibodies which recognize individual family members, which can then be used to further localize STEP isoforms. Potential substrates of STEP will be investigated using a number of strategies. In addition, we propose to investigate the regulation of STEP in vivo after chronic administration of dopamine receptor antagonists and to demonstrate a change in STEP immunoreactivity within the striatum and nucleus accumbens. STEP expression will also be investigated in PC12 cells. STEP message and protein are not present until these cells are exposed to nerve growth factor (NGF). We now propose to identify the STEP-related PTP in PC12 cells that is induced after NGF treatment. We have recently cloned the mouse genomic clone. We now propose to sequence and characterize the promoter region. This work will lead to the identification of regions within the promoter region that controls the high degree of tissue specificity that is found with expression of STEP. This work will lead directly to the generation of transgenic mice in which STEP protein has been knocked out, and these mice will be then investigated for neurological or behavioral abnormalities.

DESCRIPTION: The central goal of the proposed investigation is the study the structure and function of a family of protein tyrosine phosphatases termed STEP ("striatal-enriched" phosphatases). STEP members are brain-specific and highly enriched within neurons of the basal ganglia. A striking feature is that some members contain transmembrane domains, PEST sequences and polyproline-rich sequences. The hypotheses to be tested is that some of these domains provide a mechanism for subcellular targeting of these different isoforms, determine substrate specificity for the different STEP isoforms and regulate their activity. Preliminary data suggest that STEP participates in signal transduction pathways through the formation of protein-protein complexes. The proposed experiments would use biochemical, electron microscopic, and molecular techniques to address these hypotheses. Abnormalities in basal ganglia underlie the pathogenesis of many neurological disorders including Huntington's and Parkinson's disease and are also involved in neuropsychiatric disorders such as Tourrette's Syndrome and obsessive-compulsive disorder. This collection of nuclei is also the site of action of many of the unwanted side effects of neuroleptic drugs. Disruption of the neural signaling mechanisms within the basal ganglia is a common component of all of these disorders and further knowledge is needed of the normal signaling pathways that occur in this region of the CNS.

DESCRIPTION (Adapted from applicant's abstract): My application for a K02 award reflects a natural progression of my scientific development, first as a child psychiatrist and then as a molecular biologist. From the beginning of my career as a research fellow in psychiatry and in molecular biology, and more recently as a faculty member in the Yale Child Study Center, the focus of my research has been on understanding underlying mechanisms in signaling pathways within the basal ganglia. Disruption of the normal signal transduction pathways within this region occurs in a number of neuropsychiatric disorders, including Tourette's syndrome and obsessive compulsive disorder, as well as being the site of action of the unwanted side effects of the major neuroleptics. The central goal of the proposed investigation is to study the structure and function of a family of protein tyrosine phosphatases, termed STEP. The family consists of both cytosolic and membrane-associated isoforms, as well as truncated variants that lack an active catalytic site. A striking feature is that some STEP isoforms contain transmembrane domains, PEST sequences, and polyproline-rich sequences. We hypothesize that these domains provide a mechanism for the subcellular targeting, as well as determining their substrate specificity and enzymatic activity. These hypotheses require additional investigations beyond my expertise in molecular biology. Specifically, there are four areas of career development that I have arranged over the course of the next five years. These include the identification of interacting proteins that form complexes with STEP members using both the yeast 2 hybrid system, as well as affinity chromatography to biochemically purify associated proteins. STEP knockout mice will also be characterized. The proposed investigations will be done in the supportive environment of the Child Study Center with its commitment to clinically informed basic science research.

DESCRIPTION (from applicant's abstract): This R01 application focuses on the role of protein tyrosine phosphatases (PTPs) in signal transduction pathways in the basal ganglia. Compeling evidence suggests that the basal ganglia are involved in a number of neuropsychiatric disorders, including schizophrenia, Tourette's syndrome and obsessive compulsive disorder. In Huntington and Parkinson's disorders, abnormalities of the basal ganglia, or in their efferent and afferent pathways have been found. This collection of nuclei are also the site of action for many of the unwanted side effects of neuroleptic drugs. Disruption of the neural signaling mechanisms within the basal ganglia is a common component of all of these disorders, and further knowledge is needed of the normal signaling pathways that occur in this region of the CNS. The long-term goal of my research are to characterize genes that contribute to neuropsychiatric disorders. Under previous funding mechanisams (a K20 and then a FIRST award from NIMH), my laboratory has isolated a novel family or protein tyrosine phosphatases (PTPs) uniquely present within dopaminoceptive neurons of the basal ganglia and related structures. As the protein is highly enriched within rat striatum, it is termed STEP for Striatal Enriched Phosphatase. The present application is a natural continuation of my FIRST Award in which we isolated four isoforms of the STEP family. The first specific aim proposes to continue to isolate the characterize members of this large and important group of PTPs. The family consists of cytosolic and membrane-associated isoforms, as well as truncated forms that lack on active catalytic siste. One of the most striking features is that some STEPs contain transmembrane domains, PEST sequences, and polyproline-rich sequences. We hypothesize that these provide a mechanism for the subcellular targeting of STEP members, for the regulation of their enzymatic activity, and for their substrate specificity. In addition, we propose that phosphorylation asnd proteolysis of STEP occurs after dopamine D1 signaling. We will identify the physiological substrates of STEP, one of which is proposed to be the NMDA receptor. We will these these hypotheses using biochemical, molecular, and electrophysiological techniques.

DESCRIPTION (provided by applicant): Chronic intermittent exposure to stimulant drugs produces persistent and progressive changes in behavior including responses to subsequent drug challenges. This phenomenon is termed sensitization. Although animal models have provided information with regard to the initial biochemical changes involved in sensitazation, less is known about subsequent steps. The present RO1 will investigate this question. Our laboratory has discovered a family of protein tyrosine phosphatases (PTPs) that are expressed within neurons of the CNS. This PTP, termed STEP, is highly enriched in the striatum, nucleus accumbens, amygdala, and the frontal cortices. We recently determined that STEP regulates the activity of the MAP kinase members, ERKI/2. STEP is itself regulated by dopamine and glutamate signaling. Dopamine stimulation leads to the phosphorylation of key regulatory residues and inactivates STEP. Glutamate signaling dephosphorylates and activates STEP. STEP thus acts as a switch that regulates the activity ERK1/2 and limits its ability to activate downstream substrates. We propose that STEP regulates the effects of amphetamine. It does so by controlling the activity of ERKI/2. Consistent with this hypothesis, preliminary data in rats demonstrate that STEP is phosphorylated after amphetamine treatment. The sites phosphorylated are key regulatory residues in a domain required for binding to the ERKs. When STEP is phosphorylated at that site, the ERK signaling cascade is sustained. We will conduct time-course and dose-response analyses with psychostimulants, and perform immunocytochemical and Western blot analyses to determine the patterns of p-ERK, p-STEP, p-CREB, p- Elk-1 and the immediate early genes c-fos, jun and AfosB. We propose that the duration that ERKI/2 remain active determines the pattern of gene expression. Functional studies will disrupt the ability of STEP binding to ERK or disrupt the ability of STEP to let go of ERK. This will be tested by using TAT-peptide technology that introduces protein sequences that either prevent STEP from binding to ERK or never let go of ERK. We show preliminary feasibility data on this approach. A complementary set of experiments will study the STEP knock-out mouse that we predict is hypersensitive to stimulant treatment.

DESCRIPTION (provided by applicant): The goals of the proposed investigation are to study the regulation of the tyrosine phosphatase STEP. Previous studies established that STEP inactivates ERK1/2, p38 and the tyrosine kinase Fyn. Over the last cycle, we have discovered that STEP also induces NR1/NR2B and GluR1/GluR2 internalization. STEP has been implicated in the pathophysiology of Fragile X syndrome (FXS), Alzheimer's disease (AD), and schizophrenia (SZ). In these disorders, there is an increase in STEP levels. The current model suggests that the increase in STEP leads to the inappropriate internalization of NR1/NR2B and GluR1/GluR2 receptors. However, the mechanisms by which STEP levels are increased differ in these disorders. In AD and SZ, there is a disruption in the ubiquitination and degradation STEP. In FXS, there is an increase in the translation of STEP mRNA due to the absence of FMRP. STEP is rapidly degraded after specific forms of synaptic activity. Synaptic stimulation results in the ubiquitination of STEP, which is then degraded through the proteasome system. We hypothesize that STEP is degraded at synaptic sites to promote synaptic strengthening. Extrasynaptic NMDAR stimulation results in a calpain-mediated proteolysis STEP. We propose that this mechanism prolongs p38 signaling and initiates cell death pathways. The molecular mechanisms that regulate these processes are unknown. Aim 1 focuses on STEP ubiquitination. We include functional experiments that mutate STEP so that it can no longer be ubiquitinated and degraded. We predict that this construct will disrupt both LTP, as well as the consolidation of fear conditioning after infusion into the amygdala. We will determine the kinases that promote or prevent STEP ubiquitination. Aim 2 focuses on STEP proteolysis. We include functional studies that disrupt proteolysis of STEP, and will determine the effects this has on cell death using in vivo excitotoxic models. We predict that blocking STEP proteolysis will significantly decrease cell death. Aim 3 focuses on STEP translation. Two cytoplasmic polyadenylation elements (CPEs) are present in STEP mRNA and we will determine whether these elements regulate the translation of STEP mRNA. We include functional experiments that will mutate these CPEs. We predict this will disrupt the normal function of STEP in the regulation of its substrates, including glutamate receptor trafficking. These studies will advance our understanding of how STEP is involved in normal cognitive processes and possible mechanisms by which it is disrupted in several diseases in which there are significant cognitive deficits. PUBLIC HEALTH RELEVANCE: A major discovery over the last cycle is that STriatal-Enriched tyrosine Phosphatase (STEP) regulates glutamate receptor trafficking, and removes both NR1/NR2B and GluR1/GluR2 receptors from synaptic membranes after specific forms of synaptic stimulation. These latter findings have implicated STEP in the pathophysiology of fragile X syndrome, schizophrenia, and Alzheimer's disease. The proposed studies examine the molecular mechanisms that regulate STEP activity and will clarify how this important family of regulatory proteins are involved in synaptic plasticity, cell death, and human disease.

DESCRIPTION (provided by applicant): Striatal-Enriched tyrosine Phosphatase 61 (STEP61) is targeted to synaptic compartments and induces glutamate receptor internalization. We recently discovered that STEP61 is elevated in cingulate cortex of persons with schizophrenia (SZ). Our preliminary findings suggest that the increase of STEP61 results in internalization of glutamate receptors, a model consistent with the glutamate hypothesis of SZ. We also propose that the beneficial effects of antipsychotic medications are mediated through STEP61. The overall goals of this proposal are to define the mechanisms by which STEP61 mediates the loss of glutamate receptors in SZ, as well as its role in the beneficial effects of neuroleptics. In Aim 1, we will increase our samples of SZ cingulate cortex and include dorsolateral PFC. Our prediction is that STEP61 will again be elevated. We will determine the molecular mechanism by which this occurs. STEP61 is normally ubiquitinated and degraded by the proteasome. We propose that this process is disrupted in SZ. PKA phosphorylation of STEP61 is one signal that is necessary for the ubiquitination of STEP61. In the absence of PKA-phosphorylation, STEP61 is no longer ubiquitinated and degraded. We hypothesize, however, that additional kinases are required, and will identify these kinases and their phosphorylation sites in STEP61. Aim 1 will also test the hypothesis that the beneficial effects of neuroleptics are mediated through STEP61. We predict that dopamine D2R antagonists activate PKA, leading to the phosphorylation and inactivation of STEP61. Inactivation of STEP61 promotes trafficking of NMDAR to neuronal membranes. Aim 1 is significant as it explains aspects of the glutamate hypothesis of SZ as well as how neuroleptics function to ameliorate symptoms. Aim 2 will test our hypothesis in an animal model of SZ (NRG1 mice). NRG1 mice have decreased functional NMDARs on neuronal surfaces. Our hypothesis is that the loss of these receptors is mediated by activation of STEP61. We will first test our hypothesis by stimulating neuronal cultures and slices derived from WT and STEP KO mice with NRG1. Our prediction is that the absence of STEP61 will prevent NRG1-induced internalization of NMDARs. We will also test our hypothesis by crossing NRG1 with STEP KO mice. We predict that progeny null for STEP will rescue the loss of NMDARs from neuronal membranes. STEP KO mice have elevated levels of NMDARs and AMPARs on neuronal membranes. Aim 3 will test the hypothesis that these mice are less sensitive to the effects of psychotomimetic agents. Preliminary data indicate that this is the case: STEP KO mice are less sensitive to the acute effects of PCP on locomotor activity, as well as the subchronic effects of PCP on cognition. We have developed a STEP inhibitor called LDN-33960, and have shown that this compound reduces the acute effects of PCP on locomotor activity. We will test if this compound also reduces the cognitive deficits induced by PCP. Ultimately, our work has the potential of discovering a new family of therapeutic agents for SZ.