An immortalized rat ventral mesencephalic cell line, RTC4, is protective in a rodent model of stroke.

One therapeutic approach to stroke is the transplantation of cells capable of trophic support, reinnervation, and/or regeneration. Previously, we have described the use of novel truncated isoforms of SV40 large T antigen to generate unique cell lines from several primary rodent tissue types. Here we describe the generation of two cell lines, RTC3 and RTC4, derived from primary mesencephalic tissue using a fragment of mutant T antigen, T155c (cDNA) expressed from the RSV promoter. Both lines expressed the glial markers vimentin and S100beta, but not the neuronal markers NeuN, MAP2, or beta-III-tubulin. A screen for secreted trophic factors revealed substantially elevated levels of platelet-derived growth factor (PDGF) in RTC4, but not RTC3 cells. When transplanted into rat cortex, RTC4 cells survived for at least 22 days and expressed PDGF. Because PDGF has been reported to reduce ischemic injury, we examined the protective functions of RTC4 cells in an animal model of stroke. RTC4 or RTC3 cells, or vehicle, were injected into rat cortex 15-20 min prior to a 60-min middle cerebral artery ligation. Forty-eight hours later, animals were sacrificed and the stroke volume was assessed by triphenyl-tetrazolium chloride (TTC) staining. Compared to vehicle or RTC3 cells, transplanted RTC4 cells significantly reduced stroke volume. Overall, we generated a cell line with glial properties that produces PDGF and reduces ischemic injury in a rat model of stroke.

The use of microarrays to characterize neuropsychiatric disorders: postmortem studies of substance abuse and schizophrenia.

Neuropsychiatric disorders are generally diagnosed based on a classification of behavioral and, in some cases, specific neurological deficits. The lack of distinct quantitative and qualitative biological descriptors at the anatomical and cellular level complicates the search for and understanding of the neurobiology of these disorders. The advent of microarray technology has enabled large-scale profiling of transcriptional activity, allowing a comprehensive characterization of transcriptional patterns relating to the pathophysiology of neuropsychiatric disorders. We review some of the unique methodological constraints related to the use of human postmortem brain tissue in addition to the generally applicable requirements for microarray experiments. Microarray studies undertaken in neuropsychiatric disorders such as schizophrenia and substance abuse by the use of postmortem brain tissue indicate that transcriptional changes relating to synaptic function and plasticity, cytoskeletal function, energy metabolism, oligodendrocytes, and distinct intracellular signaling pathways are generally present. These have been supported by microarray studies in experimental models, and have produced multiple avenues to be explored at the functional level. The quality and specificity of information obtained from human postmortem tissue is rapidly increasing with the maturation and refinement of array-related methodologies and analysis tools, and with the use of focused cell populations. The development of experimental models of gene regulation in these disorders will serve as the initial step towards a comprehensive genome-linked analysis of the brain and associated disorders, and help characterize the integration and coordinate regulation of complex functions within the CNS.

Transcriptional profiling in the human prefrontal cortex: evidence for two activational states associated with cocaine abuse.

CNS-focused cDNA microarrays were used to examine gene expression profiles in dorsolateral prefrontal cortex (dlPFC, Area 46) from seven individual sets of age- and post-mortem interval-matched male cocaine abusers and controls. The presence of cocaine and related metabolites was confirmed by gas chromatography-mass spectrometry. Sixty-five transcripts were differentially expressed, indicating alterations in energy metabolism, mitochondria and oligodendrocyte function, cytoskeleton and related signaling, and neuronal plasticity. There was evidence for two distinct states of transcriptional regulation, with increases in gene expression predominating in subjects testing positive for a metabolite indicative of recent 'crack' cocaine abuse and decreased expression profiles in the remaining cocaine subjects. This pattern was confirmed by quantitative polymerase chain reaction for select transcripts. These data suggest that cocaine abuse targets a distinct subset of genes in the dlPFC, resulting in either a state of acute activation in which increased gene expression predominates, or a relatively destimulated, refractory phase.

Reduction of synapsin in the hippocampus of patients with bipolar disorder and schizophrenia.

Several studies suggest that decreased expression of presynaptic proteins may be characteristic of schizophrenia. We examined one such protein, synapsin, in schizophrenia and bipolar disorder. Samples of hippocampal tissue from controls (n = 13), patients with schizophrenia (n = 16), or bipolar disorder (n = 6), and suicide victims (n = 7) were used. The membrane and cytosolic fractions were analyzed by Western immunoblotting for synapsin using an antibody that detects synapsin Ia, IIa, and IIIa proteins. Synaptophysin was also measured for comparison. Total synapsin was decreased significantly in patients with schizophrenia (P = 0.034) and in bipolar disorder (P = 0.00008) as compared to controls. The synapsin/synaptophysin ratios were decreased in schizophrenia and bipolar disorder, and additionally in suicide victims (P = 0.014). Age, postmortem interval, percentage of protein extracted, and pH of brain were not different between groups. No changes in total synapsin or synaptophysin in the hippocampus were produced by injecting rats with either lithium or haloperidol for 30 days. Reductions in synapsin in both patients with schizophrenia (synapsin IIa and IIIa) and bipolar disorder (synapsin Ia, IIa and IIIa) imply that altered or reduced synaptic function in the hippocampus may be involved in these disorders.

A murine dopamine neuron-specific cDNA library and microarray: increased COX1 expression during methamphetamine neurotoxicity.

Due to brain tissue heterogeneity, the molecular genetic profile of any neurotransmitter-specific neuronal subtype is unknown. The purpose of this study was to purify a population of dopamine neurons, construct a cDNA library, and generate an initial gene expression profile and a microarray representative of dopamine neuron transcripts. Ventral mesencephalic dopamine neurons were purified by fluorescent-activated cell sorting from embryonic day 13.5 transgenic mice harboring a 4.5-kb rat tyrosine hydroxylase promoter-lacZ fusion. Nine-hundred sixty dopamine neuron cDNA clones were sequenced and arrayed for use in studies of gene expression changes during methamphetamine neurotoxicity. A neurotoxic dose of methamphetamine produced a greater than twofold up-regulation of the mitochondrial cytochrome c oxidase polypeptide I transcript from adult mouse substantia nigra at 12 h posttreatment. This is the first work to describe a gene expression profile for a neuronal subtype and to identify gene expression changes during methamphetamine neurotoxicity.

Characterization of human cleaved N-CAM and association with schizophrenia.

The neural cell adhesion molecule (N-CAM) is a cell recognition molecule involved in cellular migration, synaptic plasticity, and CNS development. A 105- to 115-kDa isoform of N-CAM (cleaved N-CAM or cN-CAM) is increased in schizophrenia in hippocampus, prefrontal cortex, and CSF. We purified and partially characterized cN-CAM, a putative novel isoform, and confirmed that the first 9 amino acids were identical to exon 1 of N-CAM, without the signal sequence. Analysis of trypsin-digested cN-CAM fragments by matrix-assisted laser desorption ionization on a time-of-flight mass spectrometer (MALDI-TOF) yielded peptides that could be identified as being derived from the first 548 amino acid residues of the expected N-CAM amino acid sequence. Immunological identification with four specific N-CAM antisera directed toward cytoplasmic, secreted, variable alternative spliced exon, or GPI epitopes failed to indicate other known splice variants. Neuraminidase treatment of cN-CAM produced a minor alteration resulting in a faster migrating immunoreactive band, indicating partial glycosylation of cN-CAM. Membranous particles from cytosolic brain extract containing cN-CAM were obtained by ultracentrifugation; however, CSF contained few such particles. cN-CAM and synaptophysin were colocalized on these particles. Both cN-CAM and N-CAM 180 were present in synaptosomal preparations of human brain. Following incubation of synaptosomes or brain tissue without protease inhibitors, N-CAM 180 was degraded and cN-CAM was increased. A cN-CAM-like band was present in human fetal neuronal cultures, but not in fetal astrocyte cultures. Thus, cN-CAM represents a protease- and neuraminidase-susceptible fragment possibly derived by proteolytic cleavage of N-CAM 180. An enlargement in ventricular volume in a group of adult patients with schizophrenia over a 2-year interval was found to be correlated with CSF cN-CAM levels as measured at the time of the initial MRI scan (r = 0.53, P = 0.01). cN-CAM is associated with ventricular enlargement; thus, the release of N-CAM fragments may be part of the pathogenic mechanism of schizophrenia in vulnerable brain regions such as the hippocampus and prefrontal cortex. Alternatively, the increases in cN-CAM in schizophrenia may be a reflection of a more general abnormality in the regulation of proteolysis or of extracellular matrix stability.

Application of cDNA microarrays to examine gene expression differences in schizophrenia.

Using cDNA microarrays we have investigated gene expression patterns in brain regions of patients with schizophrenia. A cDNA neuroarray, comprised of genes related to brain function, was used to screen pools of samples from the cerebellum and prefrontal cortex from a matched set of subjects, and middle temporal gyrus, from a separate subject cohort. Samples of cerebellum and prefrontal cortex from neuroleptic naive patients were also included. Genes that passed a 3% reproducibility criterion for differential expression in independent experiments included 21 genes for drug-treated patients and 5 genes for drug-naive patients. Of these 26 genes, 10 genes were increased and 16 were decreased. Many of the differentially expressed genes were related to synaptic signaling and proteolytic functions. A smaller number of these genes were also differentially expressed in the middle temporal gyrus. The five genes that were differentially expressed in two brain regions from separate cohorts are: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide; sialyltransferase; proteasome subunit, alpha type 1; ubiquitin carboxyl-terminal esterase L1; and solute carrier family 10, member 1. Identification of patterns of changes in gene expression may lead to a better understanding of the pathophysiology of schizophrenia disorders.

Trophic factor secreting kidney cell lines: in vitro characterization and functional effects following transplantation in ischemic rats.

Several kidney cell lines were investigated for their ability to produce glial cell line-derived neurotrophic factor (GDNF). Cell line-conditioned medium was analyzed using ELISA and two cell lines were identified which produce GDNF in physiologically active concentrations. ELISA analyses revealed that conditioned medium from these two cell lines also contained PDGF, bFGF, TGFbeta1 and TGFbeta2. Both of these cell lines were then transplanted into the striatal penumbra of rats, 1 h following middle cerebral artery occlusion. Behavioral testing revealed that both cell lines reduced the deficit associated with cerebral ischemia and reduced the infarct volume relative to controls. Reduction of infarct volume was likely achieved by the action of GDNF and/or other growth factors produced by the cells.

Gene expression profile in early stage of retinoic acid-induced differentiation of human SH-SY5Y neuroblastoma cells.

PURPOSE: The human SH-SY5Y cell line is an established model for retinoic acid (RA)-induced neural differentiation. We employed a broad human 15K microarray (15,000 genes) and focused Neuroarray (1152 genes) to examine changes in gene expression early in the process of differentiation (6 hr), before morphology or growth changes are observed. METHODS: 33 P-labeled CDNA probes prepared from RNA extracts of RA-treated and control cultures were hybridized to array membranes, and levels of expression were quantified and compared. RESULTS: In the 15K array, 19 % of the genes were decreased (0.4 % were named genes and the remainder were expressed sequence tags (ESTs) or unknowns), and 9 % were increased (4.2 % named genes). In the Neuroarray, 3 % were decreased and 8 % were increased. CONCLUSIONS: Summary gene profiles are presented, which include transcription factors, genes associated with cell cycle, cell shape, neurotransmission, intermediary filaments, and others. The prevalence of down-regulated genes in the broad 15K array and up-regulated genes in the neuro-focused array suggests a pattern shift in gene expression associated with differentiation. The predominance of ESTs among the down-regulated genes indicates a great number of as-yet-unidentified genes are repressed in early stage neural differentiation.

Assembly and use of a broadly applicable neural cDNA microarray.

cDNA microarrays provide an efficient method to analyze gene expression patterns in thousands of genes in parallel. In some cases, large unfocused collections of cDNAs have been used in hybridization studies, in others small logically defined collections of tissue specific arrays have been used. Here we describe the bioinformatic selection of 1152 named human cDNAs specifically designed for neuroscience applications, arrayed on nylon membranes at high density. cDNAs were chosen which represent all the major cellular types of the brain including; neurons, astrocytes, microglia, and oligodendrocytes. Gene families chosen include cell type specific markers, ion-channels, transporters, receptors, and cell adhesion molecules among many others. These arrays were used with region specific samples from human brain to determine MRNA expression profiles for each region. Used with 33p labeled complex probes, this is a low cost, highly sensitive approach for tbc investigator to focus on tissue specific genes of interest where samples of limiting amounts of RNA are used. This selected set of brain-relevant cDNAs should be widely useful in the analysis of gene expression patterns from brain tissues as well as neural cell lines.

Neuropathology of bipolar disorder.

The literature on the neuropathology of bipolar disorder (BD) is reviewed. Postmortem findings in the areas of pathomorphology, signal transduction, neuropeptides, neurotransmitters, cell adhesion molecules, and synaptic proteins are considered. Decreased glial numbers and density in both BD and major depressive disorder (MDD) have been reported, whereas cortical neuron counts were not different in BD (in Brodmann's areas [BAs] 9 and 24). In contrast, MDD patients showed reductions in neuronal size and density (BA 9, BA 47). There are a number of findings of alterations in neuropeptides and monoamines in BD brains. Norepinephrine turnover was increased in several cortical regions and thalamus, whereas the serotonin metabolite, 5-hydroxyindoleacetic acid, and the serotonin transporter were reduced in the cortex. Several reports further implicated both cyclic adenosine monophosphate and phosphatidylinositol (PI) cascade abnormalities. G protein concentrations and activity increases were found in the occipital, prefrontal, and temporal cortices in BD. In the PI signal cascade, alterations in PKC activity were found in the prefrontal cortex. In the occipital cortex, PI hydrolysis was decreased. Two isoforms of the neural cell adhesion molecules were increased in the hippocampus of BD, whereas the synaptic protein marker, synaptophysin, was not changed. The findings of glial reduction, excess signal activity, neuropeptide abnormalities, and monoamine alterations suggest distinct imbalances in neurochemical regulation. Possible alterations in pathways involving ascending projections from the brain stem are considered. Larger numbers of BD brains are needed to further refine the conceptual models that have been proposed, and to develop coherent models of the pathophysiology of BD.

Elevated concentration of N-CAM VASE isoforms in schizophrenia.

Neural cell adhesion molecule (N-CAM) is a cell recognition molecule, four major isoforms (180, 140, 120, and 105-115 kDa) of which are present in brain. N-CAM has several roles in cellular organization and CNS development. Previously we have found an elevation in CSF N-CAM 120 kDa in the CSF of patients with schizophrenia, bipolar disorder, and depression. We now report an increase in the variable alternative spliced exon (VASE), a 10 amino acid sequence inserted into the fourth N-CAM domain, in the CSF of patients with schizophrenia, but not in bipolar disorder or depression. VASE-immunoreactive (VASE-ir) bands were measured in CSF from patients with schizophrenia (n = 14), bipolar disorder I (n = 7), bipolar disorder II (n = 9), unipolar depression (n = 17) and matched controls (n = 37) by Western immunoblotting. Three VASE-ir bands were distinguished in lumbar CSF corresponding to heavy (165 kDa), medium (155 kDa) and low (140 kDa) MW. A logarithmic transformation was applied to the VASE protein units and analyzed with a MANOVA. There was a 51% and 45% increase in VASE heavy (p = 0.0008) and medium (p = 0.04) MW protein, respectively, in patients with schizophrenia as compared with normal controls. Current neuroleptic treatment in patients with schizophrenia had no effect on CSF VASE concentrations. VASE concentration correlated significantly with behavioral ratings in patients with schizophrenia but not affective disorders. Thus, VASE immunoreactivity is increased in schizophrenia but not in affective disorders. These results provide further evidence of an abnormality of N-CAM protein in chronic schizophrenia and suggest differences between schizophrenia and affective disorders in regulation of N-CAM.

Alterations of hippocampal secreted N-CAM in bipolar disorder and synaptophysin in schizophrenia.

Schizophrenia and bipolar disorder have both been linked to structural abnormalities of the hippocampus, which is consistent with a neurodevelopmental anomaly. One isoform of the neural cell adhesion molecule (N-CAM) protein, cytosolic N-CAM 105-115 kDa, was previously shown to be increased in schizophrenia in the hippocampus and prefrontal cortex. Another isoform of N-CAM, the variable alternative spliced exon of N-CAM, was also increased in the hippocampus and prefrontal cortex of bipolar disorder patients. In the present study, the secreted isoform of N-CAM (SEC N-CAM), synaptophysin, and actin proteins were measured in the hippocampus of controls, suicide victims, and patients with bipolar disorder or schizophrenia by quantitative Western immunoblotting. Previous measurements of cytosolic N-CAM (105-115 kDa) protein, from the same hippocampus samples, were used to calculate the N-CAM (105-115 kDa)/synaptophysin ratio. An affinity purified antibody to SEC N-CAM recognized SEC N-CAM (108 kDa and 115 kDa) in brain but SEC N-CAM was not detectable in CSF. In bipolar disorder, but not in schizophrenia, an increased SEC N-CAM 115 kDa/108 kDa ratio was found as compared to controls (P = 0.03). The synaptophysin/actin ratio was significantly decreased in schizophrenia (P = 0.014) as compared to controls. The cytosolic N-CAM 105-115 kDa/synaptophysin ratio was increased in patients with schizophrenia (P= 0.017), but not in bipolar disorder. Thus, bipolar disorder patients show altered expression of SEC N-CAM in the hippocampus. Patients with schizophrenia show a decrease in synaptophysin and an increase in the cytosolic N-CAM 105-115 kDa/synaptophysin ratio. The results offer further evidence of differences in protein expression between bipolar disorder and schizophrenia in the hippocampus, which is consistent with a distinct neuropathology for each neuropsychiatric disorder.

Bromocriptine protects dopaminergic neurons from levodopa-induced toxicity by stimulating D(2)receptors.

Neuroprotective properties of bromocriptine, a D(2) receptor agonist, were investigated using the in vitro neurotoxicity of levodopa for dopaminergic neurons from rat embryonic ventral mesencephalon. Levodopa, when added to the culture medium, showed toxicity which was specific for dopaminergic neurons. Bromocriptine was found to protect dopaminergic neurons from levodopa toxicity. Another D(2) agonist, 2-(N-phenethyl-N-propyl-amino-5-hydroxytetralin, showed similar protective effects. The neuroprotective effect of bromocriptine was inhibited by supplementation of the culture medium with sulpiride, a D(2) antagonist, or by D(2) receptor knockdown with an antisense oligonucleotide. Dopaminergic neurons treated with levodopa showed an increase in free radicals. These data suggest that neuroprotective properties of bromocriptine seen in this cellular model of neurotoxicity are dependent on dopamine D(2) autoreceptor binding and that levodopa toxicity may be related to increased free radical generation in dopaminergic neurons.

Immunohistochemical localization of the cell adhesion molecules Thy-1 and L1 in the human prefrontal cortex patients with schizophrenia, bipolar disorder, and depression.

L1 and Thy-1 are members of the immunoglobulin (Ig) superfamily of cell adhesion molecules (CAMs) that are vital for normal neural development. Abnormalities in CAM expression could lead to the histological abnormalities that have previously been described in the frontal cortex of patients with schizophrenia. A postmortem immunohistochemical study of L1 and Thy-1 in the normal human prefrontal cortex revealed positive immunostaining of axons in all layers of the cortex. Quantifying the intensity of immunostaining in the prefrontal cortex of patients with schizophrenia, bipolar disorder and depression failed to reveal any significant differences when compared to that of normal controls.

CSF N-CAM in neuroleptic-naïve first-episode patients with schizophrenia.

An increased concentration of neural cell adhesion molecule (N-CAM) 105-115 kDa has been reported in patients with schizophrenia in both CSF and in post-mortem brain samples. To determine whether increased N-CAM is integral to the disease process or, alternatively, results from early treatment, CSF N-CAM was measured in a blind study of first episode (FE) patients, who were either neuroleptic-naïve (NN) or neuroleptic-treated (NT, < 100 mg Haldol equivalents), multi-episode (ME) patients, and controls. Overall, the FE patients displayed lower N-CAM concentrations as compared to controls (p = 0.043). This decrease in N-CAM in FE patients was seen only in the FE-NT group as compared to both controls (p = 0.0006). The FE-NT group also showed a lower CSF N-CAM compared to that in the FE-NN (p = 0.025) group. No difference in CSF N-CAM between the FE-NN and control group was found. ME patients showed an increased N-CAM as compared with FE patients (p = 0.018), but not as compared to controls (p = 0.93). Neuroleptic-naïve first-episode patients do not display a phenotypic increase in N-CAM. Thus, N-CAM is altered in first-episode patients following acute neuroleptic treatment and withdrawal, as compared to neuroleptic-naïve first-episode patients.

Further studies of elevated cerebrospinal fluid neuronal cell adhesion molecule in schizophrenia.

BACKGROUND: The purposes of the present study were to attempt to replicate a previous finding of increased cerebrospinal fluid (CSF) neuronal cell adhesion molecule (N-CAM) in schizophrenia, and to assess whether the increases could be related to medication, clinical state effects, or brain structural measures. METHODS: CSF N-CAM was measured by the Western blot technique in 45 DSM-III-R diagnosed male schizophrenic patients both on and off haloperidol treatment and in 20 healthy male control subjects. RESULTS: CSF N-CAM was significantly increased in schizophrenic patients, with no overlap in the ranges, when compared to controls. There were no significant effects of medication or exacerbation on CSF N-CAM. No associations with measures of brain structure were found. CONCLUSIONS: Because N-CAM levels were not shown to be different on and off treatment or in exacerbated versus nonexacerbated patients, the higher levels seen in schizophrenic patients may be inherent to the disorder and possibly related to neurodevelopment.

Abnormal expression of cell recognition molecules in schizophrenia.

Schizophrenia is a neuropsychiatric disorder of unknown etiology associated with subtle changes in brain morphology. The cell recognition molecules (CRMs) neural cell adhesion molecule (N-CAM) and L1 are involved in morphoregulatory events and numerous neurodevelopmental processes. We found a selective increase of 105- to 115-kDa N-CAM in the hippocampus and prefrontal cortex of patients with schizophrenia while other N-CAM isoforms and L1 proteins were not altered. There was also evidence for an abnormality in CRM expression in schizophrenic patients: concentrations of 200-kDa L1 were strongly correlated with expression of N-CAM isoforms and cleaved L1 proteins in controls, whereas these correlations were absent in patients with schizophrenia. The increase of the 105- to 115-kDa N-CAM isoform in the brains of patients with schizophrenia confirms previous cerebrospinal fluid findings. Increased N-CAM in schizophrenia may result from structural brain abnormalities, from glial processing of N-CAM, or from an aberration in the regulation of N-CAM expression.

A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53-induced growth arrest and immortalizes primary mesencephalic cells.

As an alternative to primary fetal tissue, immortalized central nervous system (CNS)-derived cell lines are useful for in vitro CNS model systems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 large T antigen is commonly used to immortalize mammalian cells, but it interferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentiated phenotypes. In order to increase the phenotypic repertoire of immortalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vector containing a truncated SV40 large T gene that encodes only the amino-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive cell line, T64-7B. Colonies expressing T155 proliferated at the growth-restrictive temperature. T155 was then transfected into primary cultures from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-10, which co-expressed T155 and mature neuronal and astrocytic markers. Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite the absence of a p53-binding region, and (2) immortalize primary CNS cells expressing mature markers while actively dividing. T155 and T155-transfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulation to produce cells with specific properties.