Frederick C. Nucifora

FKBP12 Binds the Inositol 1,4,5-Trisphosphate Receptor at Leucine-Proline (1400–1401) and Anchors Calcineurin to this FK506-like Domain

The immunophilin FKBP12 is one of the most abundant and conserved proteins in biology. It is the primary receptor for the immunosuppressant actions of the drug FK506 in whose presence FKBP12 binds to and inhibits calcineurin, disrupting interleukin formation in lymphocytes. The physiologic functions of FKBP12 are less clear, although the protein has been demonstrated to physiologically interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the ryanodine receptor, and the type 1 transforming growth factor β receptor. We now report that FKBP12 binds the IP3R at residues 1400–1401, a leucyl-prolyl dipeptide epitope that structurally resembles FK506. We further demonstrate that binding to IP3R at this site enables FKBP12 to interact with calcineurin, presumably to anchor the phosphatase to IP3R and modulate the receptor’s phosphorylation status. We propose that FK506 promotes an FKBP12-calcineurin interaction by mimicking structurally similar dipeptide epitopes present within proteins that use FKBP12 to anchor calcineurin to the appropriate physiologic substrates.

Differential cellular expression of isoforms of inositol 1,4,5-triphosphate receptors in neurons and glia in brain.

Inositol 1,4,5‐trisphosphate receptors (IP3R) are mediators of second messenger‐induced intracellular calcium release. Three isoforms are known to be expressed in brain, but their regional distributions and cellular localizations are little known. In order to better understand the roles of IP3 receptor isoforms in brain function, a first step is to define their distributions. We have used affinity‐purified antibodies directed against peptides unique to each isoform to determine their sites of expression in rat brain. Type 1 IP3R (IP3R1) is dramatically enriched in Purkinje neurons in cerebellum and neurons in other regions, consistent with previous studies. By contrast, IP3R2 is only detected in glia, whereas IP3R3 is predominantly neuronal, with little detected in glia. IP3R3 is enriched in neuropil, especially in neuronal terminals (which often contain large dense core vesicles) in limbic and basal forebrain regions including olfactory tubercle, central nucleus of the amygdala, and bed nucleus of the stria terminalis. In addition, IP3R1 and IP3R3 have clearly distinct time courses of expression in developing brains. These data suggest separate roles for inositol 1,4,5‐trisphosphate receptor isoforms in development, and for glial and neuronal function. The IP3R3 may be involved in regulation of neurotransmitter or neuropeptide release in terminals within specific nuclei of the basal forebrain and limbic system. J. Comp. Neurol. 406:207–220, 1999.

Comparison of type 2 inositol 1,4,5-trisphosphate receptor distribution and subcellular Ca2+ release sites that support Ca2+ waves in cultured astrocytes.

Abstract: We have examined the mechanisms that underlie Ca2+ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca2+ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca2+ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca2+ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5‐trisphosphate receptor (InsP3R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP3Rs with Ca2+ release kinetics in subcellular regions during propagation of norepinephrine‐evoked waves. 3,3′‐Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP3Rs (InsP3R2) detected a 260‐kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca2+ responses and InsP3R2 immunofluorescence were compared in the same cell, domains of elevated Ca2+ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP3R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca2+ release sites that support regenerative wave propagation include increased levels of InsP3R2 expression.

Clozapine as a Model for Antipsychotic Development

Schizophrenia is a devastating illness that affects up to 1% of the population; it is characterized by a combination of positive symptoms, negative symptoms, and cognitive impairment. Currently, treatment consists of one class of medications known as antipsychotics, which include typical (first-generation) and atypical (second-generation) agents. Unfortunately, antipsychotic medications have limited efficacy, with up to a third of patients lacking a full response. Clozapine, the first atypical antipsychotic developed, is the only medication shown to be superior to all other antipsychotics. However, owing to several life-threatening side effects and required enrollment in a registry with routine blood monitoring, clozapine is greatly underutilized in the US. Developing a medication as efficacious as clozapine with limited side effects would likely become the first-line therapy for schizophrenia and related disorders. In this review, we discuss the history of clozapine, landmark studies, and its clinical advantages and disadvantages. We further discuss the hypotheses for clozapine’s superior efficacy based on neuroreceptor binding, and the limitations of a receptor-based approach to antipsychotic development. We highlight some of the advances from pharmacogenetic studies on clozapine and then focus on studies of clozapine using unbiased approaches such as pharmacogenomics and gene expression profiling. Finally, we examine how these approaches could provide insights into clozapine’s mechanism of action and side-effect profile, and lead to novel and improved therapeutics.

Ubiqutination via K27 and K29 chains signals aggregation and neuronal protection of LRRK2 by WSB1.

A common genetic form of Parkinsons disease (PD) is caused by mutations in LRRK2. We identify WSB1 as a LRRK2 interacting protein. WSB1 ubiquitinates LRRK2 through K27 and K29 linkage chains, leading to LRRK2 aggregation and neuronal protection in primary neurons and a Drosophila model of G2019S LRRK2. Knocking down endogenous WSB1 exacerbates mutant LRRK2 neuronal toxicity in neurons and the Drosophila model, indicating a role for endogenous WSB1 in modulating LRRK2 cell toxicity. WSB1 is in Lewy bodies in human PD post-mortem tissue. These data demonstrate a role for WSB1 in mutant LRRK2 pathogenesis, and suggest involvement in Lewy body pathology in sporadic PD. Our data indicate a role in PD for ubiquitin K27 and K29 linkages, and suggest that ubiquitination may be a signal for aggregation and neuronal protection in PD, which may be relevant for other neurodegenerative disorders. Finally, our study identifies a novel therapeutic target for PD.

A Mutation in NPAS3 That Segregates with Schizophrenia in a Small Family Leads to Protein Aggregation.

Schizophrenia and other major mental illnesses result from a complex interplay of genetic and environmental factors. We previously identified a mutation in NPAS3 that results in a valine to isoleucine (V304I) amino acid substitution segregating with schizophrenia in a small family. The amino acid change occurs in a potentially critical region for protein function. Furthermore, the same amino acid substitution in proteins related to familial Alzheimers disease and transthyretin amyloidosis has been associated with protein aggregation. In this study, we demonstrate that NPAS3 is prone to aggregation, and that the V304I mutation in NPAS3 increases this propensity in both bacterial and mammalian expression systems. We also show that NPAS3-V304I reduces soluble endogenous NPAS3, and increases insoluble endogenous NPAS3 and leads to alteration of transcriptional activity. These results suggest that protein aggregation, potentially leading to cell dysfunction via a loss of protein function through sequestration, may contribute to the pathogenesis of schizophrenia and other forms of mental illness. Further exploration of the mechanisms leading to abnormal protein quality control could lead to new therapeutic targets.

Analysis of differential gene expression mediated by clozapine in human postmortem brains.

Clozapine is the only medication indicated for treating refractory schizophrenia, due to its superior efficacy among all antipsychotic agents, but its mechanism of action is poorly understood. To date, no studies of human postmortem brain have characterized the gene expression response to clozapine. Therefore, we addressed this question by analyzing expression data extracted from published microarray studies involving brains of patients on antipsychotic therapy. We first performed a systematic review and identified four microarray studies of postmortem brains from antipsychotic-treated patients, then extracted the expression data. We then performed generalized linear model analysis on each study separately, and identified the genes differentially expressed in response to clozapine compared to other atypical antipsychotic medications, as well as their associated canonical pathways. We also found a number of genes common to all four studies that we analyzed: GCLM, ZNF652, and GYPC. In addition, pathway analysis highlighted the following processes in all four studies: clathrin-mediated endocytosis, SAPK/JNK signaling, 3-phosphoinositide synthesis, and paxillin signaling. Our analysis yielded the first comprehensive compendium of genes and pathways differentially expressed upon clozapine treatment in the human brain, which may provide insight into the mechanism and unique efficacy of clozapine, as well as the pathophysiology of schizophrenia.

Risk of Hospitalization Due to Medication Nonadherence Identified Through EMRs of Patients With Psychosis

OBJECTIVE This study examined whether outpatients with a psychotic disorder who are at risk of hospitalization can be identified by using data from electronic medical records (EMRs). METHODS Data from EMRs of outpatients enrolled in two clinics for treatment of psychotic disorders were abstracted. Monthly data were collected for 75 patients over two years. The study examined the association of medication nonadherence, substance use, participation in psychiatric rehabilitation, and long-acting injectable antipsychotic use in any given month with the risk of hospitalization in the subsequent month by using generalized estimating equations. RESULTS The only variable found to increase the relative risk of future hospitalization was recorded medication nonadherence (adjusted relative risk=7.19, p<.001). CONCLUSIONS Results suggest that recording medication nonadherence in EMRs is feasible and that these data may be used to identify patients at high risk of future hospitalization, who may require more intensive intervention.

Case of Secondary Tics Associated With Olanzapine in an Adult

Atypical antipsychotic medications, such as risperidone, aripiprazole, and olanzapine, have utility in treating motor tics, particularly in Tourette syndrome. In rare cases, atypical antipsychotic medications have been associated with adult-onset motor tics. Such adverse drug reactions have been documented in response to quetiapine, aripiprazole, and amisulpride. Here, we report, to our knowledge, the first case of adult-onset motor tics related to olanzapine administration.

A mouse model of 22q11.2 deletions: Molecular and behavioral signatures of Parkinson’s disease and schizophrenia

22q11.2 deletions, a genetic risk for schizophrenia, could be susceptible to Parkinson’s disease through elevated expression of α-synuclein. Individuals with chromosome 22q11.2 deletions are at increased risk of developing psychiatric conditions, most notably, schizophrenia (SZ). Recently, clinical studies have also implicated these recurrent 22q11.2 deletions with the risk of early-onset Parkinson’s disease (PD). Thus far, the multiple mouse models generated for 22q11.2 deletions have been studied primarily in the context of congenital cardiac, neurodevelopmental, and psychotic disorders. One of these is the Df1/+ model, in which SZ-associated and developmental abnormalities have been reported. We present the first evidence that the mouse model for the 22q11.2 deletion exhibits motor coordination deficits and molecular signatures (that is, elevated α-synuclein expression) relevant to PD. Reducing the α-synuclein gene dosage in Df1/+ mice ameliorated the motor deficits. Thus, this model of the 22q11.2 deletion shows signatures of both SZ and PD at the molecular and behavioral levels. In addition, both SZ-associated and PD-relevant deficits in the model were ameliorated by treatment with a rapamycin analog, CCI-779. We now posit the utility of 22q11.2 deletion mouse models in investigating the mechanisms of SZ- and PD-associated manifestations that could shed light on possible common pathways of these neuropsychiatric disorders.