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1.
Transl Psychiatry ; 6(10): e915, 2016 10 11.
Article in English | MEDLINE | ID: mdl-27727244

ABSTRACT

Cognitive impairment is a key feature of schizophrenia (SZ) and determines functional outcome. Nonetheless, molecular signatures in neuronal tissues that associate with deficits are not well understood. We conducted nasal biopsy to obtain olfactory epithelium from patients with SZ and control subjects. The neural layers from the biopsied epithelium were enriched by laser-captured microdissection. We then performed an unbiased microarray expression study and implemented a systematic neuropsychological assessment on the same participants. The differentially regulated genes in SZ were further filtered based on correlation with neuropsychological traits. This strategy identified the SMAD 5 gene, and real-time quantitative PCR analysis also supports downregulation of the SMAD pathway in SZ. The SMAD pathway has been important in multiple tissues, including the role for neurodevelopment and bone formation. Here the involvement of the pathway in adult brain function is suggested. This exploratory study establishes a strategy to better identify neuronal molecular signatures that are potentially associated with mental illness and cognitive deficits. We propose that the SMAD pathway may be a novel target in addressing cognitive deficit of SZ in future studies.


Subject(s)
Cognitive Dysfunction/genetics , Cognitive Dysfunction/pathology , Olfactory Mucosa/pathology , Schizophrenia/genetics , Schizophrenia/pathology , Smad5 Protein/genetics , Adult , Biopsy , Cognitive Dysfunction/diagnosis , Down-Regulation/genetics , Female , Humans , Male , Middle Aged , Neuropsychological Tests , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Schizophrenia/diagnosis
2.
Transl Psychiatry ; 3: e323, 2013 Nov 05.
Article in English | MEDLINE | ID: mdl-24193728

ABSTRACT

Several psychiatric and neurological diseases are associated with altered hippocampal neurogenesis, suggesting differing neural stem cell (NSC) function may play a critical role in these diseases. To investigate the role of resident NSCs in a murine model of psychiatric disease, we sought to isolate and characterize NSCs from alpha-calcium-/calmodulin-dependent protein kinase II heterozygous knockout (CaMK2α-hKO) mice, a model of schizophrenia/bipolar disorder. These mice display altered neurogenesis, impaired neuronal development and are part of a larger family possessing phenotypic and behavioral correlates of schizophrenia/bipolar disorder and a shared pathology referred to as the immature dentate gyrus (iDG). The extent to which NSCs contribute to iDG pathophysiology remains unclear. To address this, we established heterogeneous cultures of NSCs isolated from the hippocampal neuropoietic niche. When induced to differentiate, CaMK2α-hKO-derived NSCs recapitulate organotypic hippocampal neurogenesis, but generate larger numbers of immature neurons than wild-type (WT) littermates. Furthermore, mutant neurons fail to assume mature phenotypes (including morphology and MAP2/calbindin expression) at the same rate observed in WT counterparts. The increased production of immature neurons which fail to mature indicates that this reductionist model retains key animal- and iDG-specific maturational deficits observed in animal models and human patients. This is doubly significant, as these stem cells lack several developmental inputs present in vivo. Interestingly, NSCs were isolated from animals prior to the emergence of overt iDG pathophysiology, suggesting mutant NSCs may possess lasting intrinsic alterations and that altered NSC function may contribute to iDG pathophysiology in adult animals.


Subject(s)
Bipolar Disorder/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Dentate Gyrus/growth & development , Neural Stem Cells/cytology , Neurogenesis/genetics , Neurons/cytology , Schizophrenia/genetics , Animals , Cells, Cultured , Dentate Gyrus/metabolism , Disease Models, Animal , Hippocampus/growth & development , Hippocampus/metabolism , Mice , Mice, Knockout , Neural Stem Cells/metabolism , Neurogenesis/physiology , Neurons/metabolism , Phenotype
3.
Transl Psychiatry ; 3: e243, 2013 Apr 02.
Article in English | MEDLINE | ID: mdl-23549417

ABSTRACT

Quetiapine is an atypical neuroleptic with a pharmacological profile distinct from classic neuroleptics that function primarily via blockade of dopamine D2 receptors. In the United States, quetiapine is currently approved for treating patients with schizophrenia, major depression and bipolar I disorder. Despite its widespread use, its cellular effects remain elusive. To address possible mechanisms, we chronically treated mice with quetiapine, haloperidol or vehicle and examined quetiapine-specific gene expression change in the frontal cortex. Through microarray analysis, we observed that several groups of genes were differentially expressed upon exposure to quetiapine compared with haloperidol or vehicle; among them, Cdkn1a, the gene encoding p21, exhibited the greatest fold change relative to haloperidol. The quetiapine-induced downregulation of p21/Cdkn1a was confirmed by real-time polymerase chain reaction and in situ hybridization. Consistent with single gene-level analyses, functional group analyses also indicated that gene sets associated with cell cycle/fate were differentially regulated in the quetiapine-treated group. In cortical cell cultures treated with quetiapine, p21/Cdkn1a was significantly downregulated in oligodendrocyte precursor cells and neurons, but not in astrocytes. We propose that cell cycle-associated intervention by quetiapine in the frontal cortex may underlie a unique efficacy of quetiapine compared with typical neuroleptics.


Subject(s)
Antipsychotic Agents/pharmacology , Cell Cycle/drug effects , Dibenzothiazepines/pharmacology , Frontal Lobe/drug effects , Haloperidol/pharmacology , Schizophrenia/metabolism , p21-Activated Kinases/genetics , Analysis of Variance , Animals , Astrocytes/metabolism , Disease Models, Animal , Frontal Lobe/metabolism , Gene Expression , In Situ Hybridization , Male , Methamphetamine/administration & dosage , Mice , Neurons/metabolism , Oligodendroglia/metabolism , Principal Component Analysis , Quetiapine Fumarate , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Schizophrenia/chemically induced , p21-Activated Kinases/metabolism
4.
Transl Psychiatry ; 2: e135, 2012 Jul 10.
Article in English | MEDLINE | ID: mdl-22781168

ABSTRACT

Hippocampus-associated cognitive impairments are a common, highly conserved symptom of both schizophrenia (SCZ) and bipolar disorder (BPD). Although the hippocampus is likely an impacted region in SCZ/BPD patients, the molecular and cellular underpinnings of these impairments are difficult to identify. An emerging class of mouse models for these psychiatric diseases display similar cognitive impairments to those observed in human patients. The hippocampi of these mice possess a conserved pathophysiological alteration; we term the 'immature dentate gyrus' (iDG), characterized by increased numbers of calretinin-positive immature neuronal progenitors, a dearth of calbindin-positive mature neurons and (often) constitutively increased neurogenesis. Although these models provide a link between cellular dysfunction and behavioral alteration, limited translational validity exists linking the iDG to human pathophysiology. In this study, we report the initial identification of an iDG-like phenotype in the hippocampi of human SCZ/BPD patients. These findings suggest a new motif for the etiology of these diseases and link an emerging class of mouse models to the human disease condition.


Subject(s)
Bipolar Disorder/genetics , Dentate Gyrus/growth & development , Nerve Tissue Proteins/genetics , S100 Calcium Binding Protein G/genetics , Schizophrenia/genetics , Adult , Analysis of Variance , Animals , Bipolar Disorder/physiopathology , Calbindin 2 , Calbindins , Dentate Gyrus/physiopathology , Disease Models, Animal , Female , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Middle Aged , Nerve Tissue Proteins/metabolism , Phenotype , S100 Calcium Binding Protein G/metabolism , Schizophrenia/physiopathology
6.
Exp Hematol ; 28(3): 305-10, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10720695

ABSTRACT

OBJECTIVE: To identify tyrosine phosphorylated proteins that interact with CrkL in Bcr-Abl-expressing cells and analyze the function of that association. MATERIALS AND METHODS: Immunoprecipitation of CrkL was performed on lysates from parental cells (Rat-1, MO7e, or 32D) or Bcr-Abl-expressing cells (Rat-1p185, MO7p210, 32Dp210, K562) followed by immunoblotting for pTyr, Stat5, or CrkL. Interactions were confirmed in vitro using GST-CrkL fusion proteins. Electrophoretic mobility shift assays were performed on K562 nuclear extracts using a beta-casein promoter-derived probe. Supershift analysis was performed with CrkL, Stat5, Stat1, Grb2, and peptide-blocked CrkL and Stat5 antibodies. CrkL localization in Rat-1 and Rat-1p185 cells was detected with indirect immunofluorescence. Transcriptional activation was analyzed in COS7 cells transfected with a Stat-responsive luciferase reporter construct and Bcr-Abl, kinase-defective Bcr-Abl, CrkL, or Grb2. RESULTS: We show that, in Bcr-Abl-expressing cells, CrkL+ interacts with tyrosine phosphorylated Stat5. Additionally, in the presence of Bcr-Abl, CrkL is found in the nucleus, can be detected in a Stat5/DNA complex, and increases transcriptional activation from a Stat-responsive reporter construct. CONCLUSION: This suggests a novel role for CrkL, functioning as a nuclear adaptor protein that can associate with and activate Stat proteins in Bcr-Abl-expressing cells.


Subject(s)
Adaptor Proteins, Signal Transducing , Fusion Proteins, bcr-abl/genetics , Milk Proteins , Nuclear Proteins/genetics , Transcriptional Activation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fusion Proteins, bcr-abl/biosynthesis , Gene Expression Regulation, Neoplastic , Humans , Nuclear Proteins/metabolism , STAT5 Transcription Factor , Signal Transduction/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , Tumor Cells, Cultured , src Homology Domains/genetics
7.
Endocrinology ; 140(10): 4713-24, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10499530

ABSTRACT

The insulin-like growth factor I (IGF-I) receptor is a transmembrane tyrosine kinase that mediates the growth-promoting effects of IGF-I and IGF-II. Changes in IGF-I receptor messenger RNA levels are reflected in cell surface receptor number, and modulation of IGF-I receptor levels affects tumorigenicity in numerous cellular models; thus, control of IGF-I receptor gene expression appears to be an important level at which cellular proliferation and tumorigenic potential may be regulated. We have previously shown that the product of the WT1 Wilms' tumor suppressor gene represses IGF-I receptor gene expression both in vitro and in vivo, and that decreased WT1 levels are correlated with up-regulation of IGF-I receptor gene expression in Wilms' tumor, benign prostatic hyperplasia, and breast cancer. Gene regulation by WT1 is complex, in that the WT1 gene encodes a variety of products as a result of alternative splicing and RNA editing, and a number of missense point mutations have been characterized in Wilms' tumor-associated syndromes. Additionally, the WT1 protein has been demonstrated to self-associate through its N-terminal domain, although the role of this intermolecular interaction in transcriptional regulation by WT1 is unclear. In this report, we analyze the relative activity of wild-type and mutant versions of the WT1 protein with respect to IGF-I receptor promoter activity in transient transfection assays and assess the potential contribution of WT1 self-association to IGF-I receptor regulation using the yeast two-hybrid system. Of the naturally occurring variations in WT1 structure, only the presence of a three-amino acid KTS insert in the zinc finger domain introduced by alternative splicing of exon 9 had a significant effect on WT1 repression of IGF-I receptor promoter activity. The N- and C-terminal domains of WT1 also exhibited partial repression, as did the most common mutant version of the WT1 protein associated with Denys-Drash syndrome. Mutations in the WT1 N-terminus attenuated WT1 self-association in the yeast two-hybrid system, but did not impair transcriptional repression. Our results suggest that 1) the DNA-binding capacity of WT1 is critical for maximal repression of the IGF-I receptor promoter, but some effects may be mediated through protein-protein interactions involving the N-terminal domain; 2) WT1 self-association may not be required for repression of the IGF-I receptor promoter; and 3) the Denys-Drash syndrome version of the WT1 protein may exhibit residual or possible gain of function activity in some contexts rather than exerting dominant negative effects, as has been proposed previously.


Subject(s)
DNA-Binding Proteins/genetics , Insulin-Like Growth Factor I/metabolism , Mutation/physiology , Promoter Regions, Genetic/physiology , Receptors, Somatomedin/genetics , Transcription Factors/genetics , Animals , CHO Cells , Cricetinae , DNA, Recombinant , DNA-Binding Proteins/metabolism , Humans , RNA Editing , Rats , Reference Values , Transcription Factors/metabolism , Transfection , WT1 Proteins
8.
J Biochem ; 116(3): 636-42, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7852284

ABSTRACT

Using the cDNA fragment of chicken c-sea receptor tyrosine kinase as a probe, we isolated from a chicken lung cDNA library overlapping cDNA clones encoding a novel protein kinase, which we termed LIM-kinase (LIMK). The predicted polypeptide of 642 amino acid residues contains remarkable structural features, composed of the N-terminal two tandemly arrayed LIM/double zinc finger motifs and the C-terminal unusual protein kinase domain. To our knowledge, a protein kinase containing the LIM motif in the molecule has not heretofore been described. The protein kinase domain of LIMK shares highly conserved residues with the known protein kinases, but LIMK is unique in that it contains the sequence DLNSHN in subdomain VIB and a short, highly basic insert sequence, which may function as a signal for nuclear localization, between subdomain VII and VIII in the protein kinase domain. Northern blot analysis revealed that the single species of LIMK mRNA of 3.8 kb is expressed predominantly in the lung, and faintly in the kidney, liver, brain, spleen, gizzard, and intestine. As the LIM motif is thought to be involved in protein-protein interactions by binding to another LIM motif, and is often present in the homeodomain-containing proteins involved in cell fate determination and in the oncogenic nuclear proteins (rhombotins), it is likely that LIMK is involved in developmental or oncogenic processes through interactions with these LIM-containing proteins.


Subject(s)
DNA, Complementary/genetics , Lung/enzymology , Protein Kinases/genetics , RNA, Messenger/biosynthesis , Zinc Fingers , Amino Acid Sequence , Animals , Base Sequence , Chickens , Cloning, Molecular , Genetic Code , Lim Kinases , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid
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