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.

Dopaminergic properties and experimental anti-parkinsonian effects of IPX750 in rodent models of Parkinson disease.

With a view toward improving the neural bioavailability of administered dopaminergic compounds, including dopamine, synthetic efforts have been directed toward enhancing the brain bioavailability of these compounds by accessing cellular sugar transport systems with stereoselective dopaminergic drugs. While synthesis and chemistry of the resultant class of compounds has recently been described in US Patent No. 6,548,484, the associated biologic properties have not previously been reported. One member of this new class, IPX-750, is a pro-drug dopamine-gluconamine designed to retain stereospecificity of binding at: glucose transporters (GLUT 1/GLUT 3 and intestinal Na/glucose co-transporters SGLT1), dopamine transporter (DAT); and, dopaminergic receptors of the D1/D2 families. Designed to be cleavable by tissue amidases, results reported here show that intact IPX-750 pro-drug retains dopaminergic agonist binding and biologic activities both in vitro and in vivo. IPX-750, like dopamine, exhibited predominant D5/D1 binding specificity with lower binding activity at D2. As expected, binding was highly stereo-specific, ie, IPX-760, a benzamide differing in just a hydrogen atom and keto oxygen from IPX-750, bound with 6-fold lower activity at D5. In cell culture, activation resulted from binding of IPX-750 at D1 or D5 in transfected cells was measured by increased intracellular cAMP. Interestingly, considering prior reported in vitro toxicity of dopamine oxidized and metabolic product dopamine, no evidence of in vitro toxicity was observed at up to 72 hrs in cell cultures at the EC50 of IPX-750 for increasing intracellular cAMP. IPX-750 was evaluated in the Parkinson's disease animal models, including MPTP mouse model, the 6-hydroxydopamine (6-OHDA) rat model and the Nurr1(+/-) knockout mouse model. In MPTP-lesioned and Nurr1+/- knockout mice, IPX-750 significantly increased Rota-rod time. In 6-OHDA-lesioned rats, IPX-750 significantly decreased apomorphine (APO)-induced rotation. Worthy of note, after cessation of IPX-750 treatments the anti-parkinsonian activity in MPTP-lesioned and Nurr1+/- mice required about 2 weeks to washout, suggesting a possible biologic reservoir of drug. In addition, after eight weeks of twice daily administration of 20 mg/kg IPX-750, mice did not show statistical difference in the total number of TH-positive neurons in substantia nigra (SN). These combined results suggest (i) that stereo-specific glycoconjugation may be an effective method to improve penetrability of drugs through the blood brain barrier; (ii) treatment with bioavailable IPX-750 in vitro did not show evidence for neurotoxicity; and, (iii) IPX-750 possesses dopaminergic properties and exerts anti-parkinsonian effects in three different PD rodent models, suggesting therapeutic potential for this new class of drugs in treating dopamine deficiency diseases.

Human blood analysis reveals differences in gene expression of catecholamine-regulated protein 40 (CRP40) in schizophrenia.

Heat shock proteins (HSPs) are important players in neurodegeneration and psychiatric disorders. We previously reported significant reductions of a 40-kDa Catecholamine Regulated Protein (CRP40) in schizophrenia post-mortem brain specimens. This study investigated whether gene expression of CRP40 is altered in living subjects with schizophrenia. CRP40 mRNA was analyzed in white blood cells of first episode and chronic/treated schizophrenia subjects compared to healthy controls. Significant reductions in CRP40 mRNA were found among first episode schizophrenia subjects and chronic schizophrenia subjects compared to healthy controls (p<0.05 for both). These results suggest a possible functional role of CRP40 in the pathogenesis of schizophrenia.

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.

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.

Regulation of dopamine transporter activity by carboxypeptidase E.

BACKGROUND: The dopamine transporter (DAT) plays a critical role in terminating the action of dopamine by rapid reuptake into the presynaptic neuron. Previous studies have revealed that the DAT carboxyl terminus (DAT-CT) can directly interact with other cellular proteins and regulate DAT function and trafficking. RESULTS: Here, we have identified that carboxypeptidase E (CPE), a prohormone processing exopeptidase and sorting receptor for the regulated secretory pathway, interacts with the DAT-CT and affects DAT function. Mammalian cell lines coexpressing CPE and DAT exhibited increased DAT-mediated dopamine uptake activity compared to cells expressing DAT alone. Moreover, coexpression of an interfering DAT-CT minigene inhibited the effects of CPE on DAT. Functional changes caused by CPE could be attributed to enhanced DAT expression and subsequent increase in DAT cell surface localization, due to decreased DAT degradation. In addition, CPE association could reduce the phosphorylation state of DAT on serine residues, potentially leading to reduced internalization, thus stabilizing plasmalemmal DAT localization. CONCLUSION: Taken together, our results reveal a novel role for CPE in the regulation of DAT trafficking and DAT-mediated DA uptake, which may provide a novel target in the treatment of dopamine-governed diseases such as drug addiction and obesity.