Joseph Gabriele

cDNA array reveals increased expression of glucose-dependent insulinotropic polypeptide following chronic clozapine treatment: role in atypical antipsychotic drug-induced adverse metabolic effects

Clozapine is an atypical antipsychotic drug with unique pharmacological and therapeutic properties. Unlike the typical antipsychotic drug, haloperidol, clozapine does not cause extrapyramidal side effects; however, weight gain, dyslipidemia, and type II diabetes are commonly associated with the use of this drug in subjects with schizophrenia. The aim of this study was to profile gene expression in the rat striatum following clozapine treatment. Chronic treatment with clozapine revealed upregulation of several genes including the glucose-dependent insulinotropic polypeptide (GIP) gene by over 200% in the rat striatum. The cDNA array results for the GIP gene were confirmed by real-time RT-PCR as well as by radioimmunoassay. Expression of the GIP gene in the central nervous system is consistent with the results of retinal GIP gene expression as reported by other investigators. Taken together, these findings implicate the possible role of GIP as a neuromodulator in the central nervous system. GIP is an insulinotropic agent with stimulatory effects on insulin synthesis and release from the pancreas. However, changes in brain GIP levels are most likely unrelated to the metabolic adverse effects (dyslipidemia, type II diabetes, weight gain) associated with clozapine treatment. Therefore, we also measured GIP gene expression in the K-cell-rich regions, duodenum and jejunum (small intestine), and plasma GIP levels using radioimmunoassay following chronic treatment with clozapine. GIP mRNA levels in the small intestine and the plasma GIP at the protein level were significantly elevated in clozapine-treated subjects. Furthermore, as observed in humans, chronic clozapine treatment also caused weight gain, and increased levels of insulin, triglycerides and leptin in the plasma. These results suggest that adverse metabolic effects associated with clozapine treatment may be related to its ability to increase intestinal gene expression for GIP.

Guanosine improves motor behavior, reduces apoptosis, and stimulates neurogenesis in rats with parkinsonism

Parkinsons disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) caused by an abnormal rate of apoptosis. Endogenous stem cells in the adult mammalian brain indicate an innate potential for regeneration and possible resource for neuroregeneration in PD. We previously showed that guanosine prevents apoptosis even when administered 48 hr after the toxin 1‐methyl‐4‐phenylpyridinium (MPP+). Here, we induced parkinsonism in rats with a proteasome inhibitor. Guanosine treatment reduced apoptosis, increased tyrosine hydroxylase–positive dopaminergic neurons and expression of tyrosine hydroxylase in the SNc, increased cellular proliferation in the SNc and subventricular zone, and ameliorated symptoms. Proliferating cells in the subventricular zone were nestin‐positive adult neural progenitor/stem cells. Fibroblast growth factor‐2‐expressing cells were also increased by guanosine. Thus, guanosine protected cells from apoptosis and stimulated “intrinsic” adult progenitor/stem cells to become dopaminergic neurons in rats with proteasome inhibitor–induced PD. The cellular/molecular mechanisms underlying these effects may open new avenues for development of novel therapeutics for PD.

Synapsin II gene expression in the dorsolateral prefrontal cortex of brain specimens from patients with schizophrenia and bipolar disorder: effect of lifetime intake of antipsychotic drugs

Synapsins are neuronal phosphoproteins crucial to regulating the processes required for normal neurotransmitter release. Synapsin II, in particular, has been implied as a candidate gene for schizophrenia. This study investigated synapsin II mRNA expression, using real-time reverse transcriptase–PCR, in coded dorsolateral prefrontal cortical samples provided by the Stanley Foundation Neuropathology Consortium. Synapsin IIa was decreased in patients with schizophrenia when compared with both healthy subjects and patients with bipolar disorder, whereas synapsin IIb was only significantly reduced in patients with schizophrenia when compared with healthy subjects but not in patients with bipolar disorder. Furthermore, lifetime antipsychotic drug use was positively associated with synapsin IIa expression in patients with schizophrenia. Results suggest that impairment of synaptic transmission by synapsin II reduction may contribute to dysregulated convergent molecular mechanisms, which result in aberrant neural circuits that characterize schizophrenia, while implicating involvement of synapsin II in therapeutic mechanisms of currently prescribed antipsychotic drugs.

Translocation of AIF in the human and rat striatum following protracted haloperidol, but not clozapine treatment

Loss of mitochondrial membrane integrity and consequent release of apoptogenic factors may be involved in mediating striatal neurodegeneration after prolonged treatment with the typical antipsychotic drug haloperidol. Apoptosis-inducing factor (AIF), an intramitochondrial protein, may have a large influence on mediating haloperidol-induced striatal neuron destruction. Translocation of this protein from mitochondria to the nucleus promotes cell death independently of the caspase cascade. To examine how AIF may contribute to haloperidol-induced apoptosis, AIF translocation was observed in three haloperidol treatment paradigms. SH-SY5Y cells were treated with both haloperidol and clozapine and examined for AIF immunofluorescence. Immunohistochemistry was also performed on human striatal sections obtained from the Stanley Foundation Neuropathology Consortium and on rat brain sections following 28 days of antipsychotic drug treatment. In the cellular model haloperidol, but not clozapine treatment increased the nuclear AIF immunofluorescent signal and decreased cell viability. Corollary to these findings, striatal sections from patients who had taken haloperidol and rats who were administered haloperidol both had an elevated nuclear AIF signal. The results provide novel evidence implicating the involvement of AIF in haloperidol-associated apoptosis and its relevance to the development of typical antipsychotic drug-related adverse effects such as tardive dyskinesia.

Decreased expression of a 40-kDa catecholamine-regulated protein in the ventral striatum of schizophrenic brain specimens from the Stanley Foundation Neuropathology Consortium

The majority of heat shock proteins (HSP) act as molecular chaperones protecting cells from deleterious stress. These proteins are able to inhibit the aggregation of partially denatured proteins and refold them into the correct conformation. They have also been shown to be involved in the pathogenesis of many neurodegenerative and psychiatric disorders. Previous reports from our laboratory have described a 40-kDa catecholamine-regulated heat-shock-like protein (CRP40). This study investigates CRP40 expression in ventral striatal specimens obtained from the Stanley Foundation Neuropathology Consortium (SFNC). CRP40 levels were significantly reduced in schizophrenic patients relative to the control group. However, ventral striatal samples of individuals diagnosed with major depression or bipolar disorder did not show significant changes in the expression of the protein. No differences in CRP40 levels were observed due to age, sex or postmortem interval (PMI). Further analysis of the schizophrenic group revealed that unmedicated and medicated patients showed decreases in ventral striatal CRP40 levels relative to the control group. However, the largest reduction in these levels was seen in unmedicated schizophrenic patients. In addition, relative to the unmedicated individuals, the clozapine- and haloperidol-treated groups showed elevations in ventral striatal CRP40 expression, although not significant. An increase in sample size may clarify this observation. Taken together, these results suggest a functional role of CRP40 in the pathogenesis of schizophrenia.

Modulation of a 40-kDa catecholamine-regulated protein following D-amphetamine treatment in discrete brain regions.

A 40-kDa catecholamine-regulated protein (CRP40) has been demonstrated to be expressed in the central nervous system, and is known to bind to dopamine and related catecholamines. Recently, it has been shown that dopamine D1 receptor antagonist and dopamine D2 receptor antagonist differentially modulated the CRP40 protein in the striatum. In the present study, we examined the effects of the indirect psychostimulant, D-amphetamine, on (CRP40) expression in discrete brain regions. The technique of Western immunoblotting was utilized for quantitation of CRP40 in different experimental paradigms following D-amphetamine treatment. Acute treatment with D-amphetamine (5.0 mg/kg, i.p.) caused no significant change in CRP40 levels in either of the two brain regions studied: striatum and nucleus accumbens. Chronic D-amphetamine administration (2.5 mg/kg, i.p.) significantly increased CRP40 levels in striatum and nucleus accumbens (37.64 +/- 14.57% and 27.86 +/- 8.40%, respectively, P < or = 0.05). Chronic and possibly sensitized D-amphetamine challenged rats (0.5 mg/kg, i.p.) showed a significant increase in CRP40 levels in the nucleus accumbens only (40.49 +/- 15.91%, P < or = 0.05). Although CRP40 has a consensus motif with the 70-kDa heat shock protein (HSP70), levels of HSP70 remained unchanged under identical experimental conditions. The results of this study demonstrate selective modulation of CRP40 by D-amphetamine treatment, without affecting the 70-kDa heat shock protein.

Knockdown of mortalin within the medial prefrontal cortex impairs normal sensorimotor gating.

The 70‐kDa mitochondrial heat shock protein, mortalin, is a ubiquitously expressed, multifunctional protein that is capable of binding the neurotransmitter, dopamine, within the brain. Dopamine dysregulation has been implicated in many of the abnormal neurological behaviors. Although studies have indicated that mortalin is differentially regulated in response to dopaminergic modulation, research has yet to elucidate the role of mortalin in the regulation of dopaminergic activity. This study seeks to investigate the role of mortalin in the regulation of dopamine‐dependent behavior, specifically as it pertains to schizophrenia (SCZ). Mortalin expression was knocked down through the infusion of antisense oligodeoxynucleotide molecules into the medial prefrontal cortex (mPFC). Rats infused with mortalin antisense oligodeoxynucleotide molecules exhibited significant prepulse inhibition deficits, suggestive of defects in normal sensorimotor gating. Furthermore, mortalin misexpression within the mPFC was coupled to a significant increase in mortalin protein expression within the nucleus accumbens at the molecular level. These findings demonstrate that mortalin plays an essential role in the regulation of dopamine‐dependent behavior and plays an even greater role in the pathogenesis of SCZ. Synapse 64:808–813, 2010.

Cloning, characterization, and functional studies of a human 40-kDa catecholamine-regulated protein: implications in central nervous system disorders

Catecholamine-regulated proteins (CRPs) have been shown to bind dopamine and other structurally related catecholamines; in particular, the 40-kDa CRP (CRP40) protein has been previously cloned and functionally characterized. To determine putative human homologs, BLAST analysis using the bovine CRP40 sequence identified a human established sequence tag (EST) with significant homology (accession #BQ224193). Using this EST, we cloned a recombinant human brain CRP40-like protein, which possessed chaperone activity. Radiolabeled dopamine binding studies with recombinant human CRP40 protein demonstrated the ability of this protein to bind dopamine with low affinity and high capacity. The full-length human CRP40 nucleotide sequence was elucidated (accession #DQ480334) with RNA ligase-mediated rapid amplification of complementary DNA ends polymerase chain reaction, while Northern blot hybridization suggested that human CRP40 is an alternative splice variant of the 70-kDa mitochondrial heat shock protein, mortalin. Human SH-SY5Y neuroblastoma cells treated with the antipsychotic drug, haloperidol, exhibited a significant increase in CRP40 messenger RNA expression compared to untreated control cells, while other dopamine agonists/antagonists also altered CRP40 expression and immunolocalization. In conclusion, these results show that we have cloned a splice variant of mortalin with a novel catecholamine binding function and that this chaperone-like protein may be neuroprotective in dopamine-related central nervous system disorders.

Antipsychotic drug use is correlated with CRP40/mortalin mRNA expression in the dorsolateral prefrontal cortex of human postmortem brain specimens

Heat shock proteins act as intracellular chaperones by assisting with proper protein folding in response to various cellular stresses. In doing so, these proteins protect the cell from unwanted protein aggregation, which in turn, plays an important role in the pathogenesis of numerous disorders. Previous reports from our laboratory have described a 40 kDa catecholamine regulated heat shock-like protein (CRP40), an alternate gene product of the 70 kDa mitochondrial heat shock protein, mortalin. CRP40 shares an intimate association with dopaminergic activity, specifically as it pertains to dopamine dysregulation in schizophrenia. This study investigates human CRP40/mortalin mRNA expression within dorsolateral prefrontal cortex postmortem specimens from normal control, schizophrenic and bipolar patients obtained from the Stanley Medical Research Institute. Real-time polymerase chain reaction was carried out for all patient samples (n=105; n=35 per group) in a blinded manner. No significant alterations in CRP40/mortalin mRNA expression levels were observed between control, bipolar and schizophrenic patients. However, multiple regression demonstrated a distinct positive correlation between CRP40/mortalin mRNA expression and lifetime use of antipsychotic drugs within the schizophrenic patient profile, after controlling for important confounding factors. Thus, the data suggest that human CRP40/mortalin is modulated by dopaminergic activity and may act to protect neurons from excess catecholamine activity in regions of the brain associated with psychosis.

Expression of the 40 kDa catecholamine regulated protein in the normal and injured rat retina.

Catecholamine regulated protein 40 (CRP40) has been shown to be expressed in the central nervous system (CNS) of several mammalian species where it may function in a similar manner to members of the heat shock protein (HSP) family. Immunohistochemical and immunoblotting techniques were utilized to investigate whether CRP40 is expressed in normal rat retinas. In addition, changes in CRP40 expression were studied following optic nerve transection. The immunohistochemical results showed that CRP40 is expressed in the normal rat retina. The protein was found to be highly expressed in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer plexiform layer (OPL). In addition, a low level of CRP40 was found in the inner plexiform layer (IPL), and in the inner segment layer (ISL). No expression was found in the outer nuclear layer (ONL) of normal rat retina. The immunoblotting results show that CRP40 expression decreased in a time-dependent fashion after the optic nerve transection. This decrease indicates that the expression of CRP40 is dependent on the neurons normal physiological state and that it plays an important function in physiological and pathological conditions in the retina.