Subacute administration of both methcathinone and manganese causes basal ganglia damage in mice resembling that in methcathinone abusers.

An irreversible extrapyramidal syndrome occurs in man after intravenous abuse of "homemade" methcathinone (ephedrone, Mcat) that is contaminated with manganese (Mn) and is accompanied by altered basal ganglia function. Both Mcat and Mn can cause alterations in nigrostriatal function but it remains unknown whether the effects of the 'homemade' drug seen in man are due to Mcat or to Mn or to a combination of both. To determine how toxicity occurs, we have investigated the effects of 4-week intraperitoneal administration of Mn (30 mg/kg t.i.d) and Mcat (100 mg/kg t.i.d.) given alone, on the nigrostriatal function in male C57BL6 mice. The effects were compared to those of the 'homemade' mixture which contained about 7 mg/kg of Mn and 100 mg/kg of Mcat. Motor function, nigral dopaminergic cell number and markers of pre- and postsynaptic dopaminergic neuronal integrity including SPECT analysis were assessed. All three treatments had similar effects on motor behavior and neuronal markers. All decreased motor activity and induced tyrosine hydroxylase positive cell loss in the substantia nigra. All reduced 123I-epidepride binding to D2 receptors in the striatum. Vesicular monoamine transporter 2 (VMAT2) binding was not altered by any drug treatment. However, Mcat treatment alone decreased levels of the dopamine transporter (DAT) and Mn alone reduced GAD immunoreactivity in the striatum. These data suggest that both Mcat and Mn alone could contribute to the neuronal damage caused by the 'homemade' mixture but that both produce additional changes that contribute to the extrapyramidal syndrome seen in man.

Delayed O-methylation of l-DOPA in MB-COMT-deficient mice after oral administration of l-DOPA and carbidopa.

1. Catechol-O-methyltransferase (COMT) is involved in the O-methylation of l-DOPA, dopamine, and other catechols. The enzyme is expressed in two isoforms: soluble (S-COMT), which resides in the cytoplasm, and membrane-bound (MB-COMT), which is anchored to intracellular membranes. 2. To obtain specific information on the functions of COMT isoforms, we studied how a complete MB-COMT deficiency affects the total COMT activity in the body, peripheral l-DOPA levels, and metabolism after l-DOPA (10 mg kg-1) plus carbidopa (30 mg kg-1) administration by gastric tube in wild-type (WT) and MB-COMT-deficient mice. l-DOPA and 3-O-methyl-l-DOPA (3-OMD) levels were assayed in plasma, duodenum, and liver. 3. We showed that the selective lack of MB-COMT did not alter the total COMT activity, COMT enzyme kinetics, l-DOPA levels, or the total O-methylation of l-DOPA but delayed production of 3-OMD in plasma and peripheral tissues.

The beneficial effect of a prolyl oligopeptidase inhibitor, KYP-2047, on alpha-synuclein clearance and autophagy in A30P transgenic mouse.

The misfolding and aggregation of α-synuclein (aSyn) eventually lead to an accumulation of toxic forms that disturb normal neuronal function and result in cell death. aSyn rich inclusions are seen in Parkinson's disease, dementia with Lewy bodies and other synucleinopathies. Prolyl oligopeptidase (PREP) can accelerate the aggregation process of aSyn and the inhibition of PREP leads to a decreased amount of aggregated aSyn in cell models and in aSyn transgenic mice. In this study, we investigated the effect of 5- and 28-day PREP inhibitor (KYP-2047) treatments on a mouse strain carrying a point mutation in the aSyn coding gene. Following PREP inhibition, we found a decrease in high molecular-weight oligomeric aSyn and a concomitant increase in the amount of the autophagosome marker, LC3BII, suggesting enhanced macroautophagy (autophagy) and aSyn clearance by KYP-2047. Moreover, 28-day treatment with KYP-2047 caused significant increases in striatal dopamine levels. In cell culture, overexpression of PREP reduced the autophagy. Furthermore, the inhibition of PREP normalized the changes on autophagy markers (LC3BII and p62) caused by an autophagy inhibition or aSyn overexpression, and induced the expression of beclin 1, a positive regulator of autophagy. Taken together, our results suggest that PREP inhibition accelerates the clearance of protein aggregates via increased autophagy and thus normalizes the cell functions in vivo and in vitro. Therefore, PREP inhibition may have future potential in the treatment of synucleinopathies.

The Catechol O-Methyltransferase Inhibitor Entacapone in the Treatment of Parkinson's Disease: Personal Reflections on a First-in-Class Drug Development Programme 40 Years On.

In the 1980s, Orion Pharma, then a mid-ranking Nordic area pharmaceutical company, established a drug development programme on the inhibition of catechol O-methyltransferase (COMT). This enzyme, which plays an important role in the inactivation of catecholamine neurotransmitters and drugs with a catechol structure, thus came under consideration as a target in the innovative translational and clinical programme we describe in this historical review. The starting point was the conjecture that a peripherally acting COMT inhibitor might improve entry of levodopa into the brain. This had potentially significant implications for the medical treatment of Parkinson's disease (PD). The rationale was that more efficient delivery of levodopa to the brain might allow the high therapeutic doses of levodopa to be reduced and the dose interval to be extended. Elucidation of structure-activity relations paved the way for the discovery and development of entacapone, a 5-nitrocatechol that was a potent and highly specific inhibitor of COMT. Experience in phase III clinical trials established that entacapone, used as an adjunct to regular or controlled-release levodopa preparations (also including a peripherally acting dopa-decarboxylase inhibitor), increased ON-time and reduced OFF-time and improved clinical condition in patients with PD experiencing wearing-off, often with a reduced daily levodopa dose. Several of these studies also identified that entacapone improved patients' quality of life and was cost-effective. Subsequently, entacapone has been amalgamated into a triple-combination preparation (Stalevo®) with levodopa and carbidopa to create a flexible and convenient drug therapy for patients with PD who have end-of-dose motor fluctuations not stabilised on levodopa/dopa-decarboxylase inhibitor treatment. This review offers a historical perspective on a successful programme of drug development by researchers who played central roles in the progress from exploratory hypothesis to registered pharmaceutical product.

Are transglutaminase 2 inhibitors able to reduce gliadin-induced toxicity related to celiac disease? A proof-of-concept study.

PURPOSE: Celiac disease is an autoimmune-mediated enteropathy characterized by adaptive and innate immune responses to dietary gluten in wheat, rye and barley in genetically susceptible individuals. Gluten-derived gliadin peptides are deamidated by transglutaminase 2 (TG2), leading to an immune response in the small-intestinal mucosa. TG2 inhibitors have therefore been suggested as putative drugs for celiac disease. In this proof-of-concept study we investigated whether two TG2 inhibitors, cell-impermeable R281 and cell-permeable R283, can prevent the toxic effects of gliadin in vitro and ex vivo. METHODS: Intestinal epithelial Caco-2 cells were treated with peptic-tryptic-digested gliadin (PT-gliadin) with or without TG2 inhibitors and thereafter direct toxic effects (transepithelial resistance, cytoskeletal rearrangement, junction protein expression and phoshorylation of extracellular-signal-regulated kinase 1/2) were determined. In an organ culture of celiac-patient-derived small-intestinal biopsies we measured secretion of TG2-autoantibodies into the culture medium and the densities of CD25- and interleukin (IL) 15-positive cells, forkhead box P3 (FOXP3)-positive regulatory T cells (Tregs) and Ki-67-positive proliferating crypt cells. RESULTS: Both TG2 inhibitors evinced protective effects against gliadin-induced detrimental effects in Caco-2 cells but the cell-impermeable R281 seemed slightly more potent. In addition, TG2 inhibitor R281 modified the gluten-induced increase in CD25- and IL15-positive cells, Tregs and crypt cell proliferation, but had no effect on antibody secretion in celiac-patient-derived biopsies. CONCLUSIONS: Our results suggest that TG2 inhibitors are able to reduce certain gliadin-induced effects related to responses in vitro and ex vivo.

Strong preferences of dopamine and l-dopa towards lipid head group: importance of lipid composition and implication for neurotransmitter metabolism.

The interactions of the neurotransmitter dopamine, and its precursor l-dopa, with membrane lipids were investigated through a set of molecular dynamic simulations with all atom resolution. The results obtained indicate that both dopamine and l-dopa have a pronounced association with the lipid head groups, predominantly mediated through H-bonds. As a result the molecules are anchored to the interfacial region of the membrane. The strength of this interaction is dependent on lipid composition - the presence of phosphatidylserine leads to an increase in the strength of this interaction, resulting in an H-bond network with a lifetime much longer than the timescale of our simulations. Also, bilayers that include sphingomieline and cholesterol interact strongly with dopamine and l-dopa. We postulate that the high membrane association that we have observed for both dopamine and l-dopa could have the following effects: 1) when on the plasma membrane exterior, favour the availability of these compounds for cell membrane uptake processes and, 2) when on an internal membrane surface, accentuate the importance of membrane-bound metabolizing enzymes over their soluble counterparts.

Catechol-O-methyltransferase gene polymorphism and chronic human pain: a systematic review and meta-analysis.

In human studies, low COMT (catechol-O-methyltransferase) activity has been associated with increased sensitivity to acute clinical preoperative or postoperative pain. We explored the association between the COMT genotype and three chronic pain conditions: migrainous headache, fibromyalgia, or chronic widespread pain and chronic musculoskeletal pain. Furthermore, we evaluated whether COMT genotype affects the efficacy of opioids in chronic pain. After a systematic literature review, we carried out meta-analyses on the three chronic pain conditions. The efficacy of opioids was evaluated using a systematic review only. The meta-analyses showed that fibromyalgia or chronic widespread pain is the only type of chronic pain that could be associated with the COMT single nucleotide polymorphism rs4680 (Val158Met). Met158, which results in the low-activity variant of COMT, is the risk allele. In chronic clinical pain, the effect of the COMT polymorphism depends on the pain condition. Low COMT activity is not associated with migrainous headache or chronic musculoskeletal pain conditions, but it may increase the risk for fibromyalgia or chronic widespread pain. Low COMT activity increases opioid receptors and enhances opioid analgesia and adverse effects in some cancer pains. Findings from animal studies that have utilized COMT inhibitors elucidate the mechanism behind these findings. In rodent pain models, COMT inhibitors are pronociceptive, except for neuropathic pain models, where nitecapone was found to be antiallodynic. The complex interplay between enhanced adrenergic and dopaminergic activity in different parts of the nociceptive system probably explains the complicated actions of low COMT activity.

Minocycline protects SH-SY5Y cells from 6-hydroxydopamine by inhibiting both caspase-dependent and -independent programmed cell death.

Minocycline, a tetracyclic antibiotic, exerts both antiinflammation by acting on microglia and a direct protection on neurons by inhibiting the apoptotic machinery at various levels. However, we are not aware of any study investigating the effects of minocycline on caspase-independent programmed cell death (PCD) pathways. This study investigated these alternative pathways in SH-SY5Y cells, a human dopaminergic cell line, challenged with 6-hydroxydopamine (6-OHDA). Minocycline exhibited neuroprotection and inhibition of the toxin-induced caspase-3-like activity, DNA fragmentation, and chromatin condensation, hallmarks of apoptosis. Moreover, we revealed that 6-OHDA also activated caspase-independent PCDs (such as paraptosis), which required de novo protein synthesis. Additionally, by separately monitoring caspase-dependent and caspase-independent pathways, we showed that inhibition of apoptosis only partially explained the protective effect of minocycline. Moreover, we observed that minocycline reduced the protein content of cells but, unexpectedly, increased the protein synthesis. These findings suggest that minocycline may actually increase protein degradation, so it may also accelerate the clearance of aberrant proteins. In conclusion, we report for the first time evidence indicating that minocycline may inhibit PCD pathways that are additional to conventional apoptosis.

Over-expression of a human chromosome 22q11.2 segment including TXNRD2, COMT and ARVCF developmentally affects incentive learning and working memory in mice.

Duplication of human chromosome 22q11.2 is associated with elevated rates of mental retardation, autism and many other behavioral phenotypes. However, because duplications cover 1.5-6 Mb, the precise manner in which segments of 22q11.2 causally affect behavior is not known in humans. We have now determined the developmental impact of over-expression of an approximately 190 kb segment of human 22q11.2, which includes the genes TXNRD2, COMT and ARVCF, on behaviors in bacterial artificial chromosome (BAC) transgenic (TG) mice. BAC TG mice and wild-type (WT) mice were tested for their cognitive capacities, affect- and stress-related behaviors and motor activity at 1 and 2 months of age. An enzymatic assay determined the impact of BAC over-expression on the activity level of COMT. BAC TG mice approached a rewarded goal faster (i.e. incentive learning), but were impaired in delayed rewarded alternation during development. In contrast, BAC TG and WT mice were indistinguishable in rewarded alternation without delays, spontaneous alternation, prepulse inhibition, social interaction, anxiety-, stress- and fear-related behaviors and motor activity. Compared with WT mice, BAC TG mice had an approximately 2-fold higher level of COMT activity in the prefrontal cortex, striatum and hippocampus. These data suggest that over-expression of this 22q11.2 segment enhances incentive learning and impairs the prolonged maintenance of working memory, but has no apparent effect on working memory per se, affect- and stress-related behaviors or motor capacity. High copy numbers of this 22q11.2 segment might contribute to a highly selective set of phenotypes in learning and cognition during development.

Sequential expression, activity and nuclear localization of prolyl oligopeptidase protein in the developing rat brain.

Prolyl oligopeptidase (POP) is a serine protease that hydrolyzes peptides shorter than 30-mer. Some evidence has recently been obtained that POP can generate protein-protein interactions and therefore participate in various physiological and pathological events. Several studies have reported that POP may be involved in neurogenesis since its activity increases during development and can be found in the nucleus of proliferating tissues. In cell cultures, POP has been shown to be localized in the nucleus, but only early in the development, since during maturation it is moved to the cytosol. We have now studied for the first time the expression of POP protein, its enzymatic activity and nuclear localization in vivo in the developing rat brain. We observed that enzymatic activity of POP is highest on embryonic day 18 while the protein amounts reach their peak at birth. Furthermore, POP is located in the nucleus only early in the development but is transferred to the cytosol already before parturition. Our in vivo results confirm the previous cell culture results supporting the role of POP in neurogenesis. A discordance of antenatal protein amounts and enzymatic activities is suggesting a tight regulation of POP activity and possibly even a nonhydrolytic role at that stage.

Distribution of catechol-O-methyltransferase (COMT) proteins and enzymatic activities in wild-type and soluble COMT deficient mice.

Catechol-O-methyltransferase (COMT) has both soluble (S-COMT) and membrane-bound (MB-COMT) isoforms. A specific COMT antibody was used in immunohistochemical and confocal co-localization studies to explore the distribution of COMT in general in normal mice and MB-COMT in particular, in an S-COMT deficient mouse line. In the peripheral tissues, high COMT protein and activity levels were observed in liver and kidney, whereas in the brain, COMT expression and activity were much lower. MB-COMT was widely distributed throughout all tissues, and overall, the MB-COMT distribution mimicked the distribution of S-COMT. MB-COMT displayed some preference for brain tissue, notably in the hippocampus. MB-COMT related enzymatic activity was also pronounced in the cerebral cortical areas and hypothalamus. MB-COMT, like S-COMT, was found to be an intracellular enzyme but it was not associated with plasma membranes in the brain. Both COMT forms were abundantly found in microglial cells and intestinal macrophages, but also in astroglial cells. COMT was also present in some neuronal cells, like pyramidal neurons, cerebellar Purkinje and granular cells and striatal spiny neurons, but not in major long projection neurons. Finally, it seemed that nuclear COMT is not visible in S-COMT deficient mice.

Quantitative role of COMT in dopamine clearance in the prefrontal cortex of freely moving mice.

Catechol-O-methyltransferase (COMT) plays an active role in the metabolism of dopamine (DA) in the prefrontal cortex (PFC). Because of low levels of dopamine transporter (DAT), it is proposed that the majority of released DA is taken up by either norepinephrine transporter (NET) and subsequently metabolized by monoamine oxidize (MAO) or by uptake(2) (to glial cells and post-synaptic neurons) and metabolized by COMT. However, a comprehensive in vivo study of rating the mechanisms involved in DA clearance in the PFC has not been done. Here, we employ two types of microdialysis to study these pathways using DAT, NET and MAO blockers in conscious mice, with or without Comt gene disruption. In quantitative no-net-flux microdialysis, DA levels were increased by 60% in the PFC of COMT-knockout (ko) mice, but not in the striatum and nucleus accumbens. In conventional microdialysis studies, we showed that selective NET and MAO inhibition increased DA levels in the PFC of wild-type mice by two- to fourfold, an effect that was still doubled in COMT-ko mice. Inhibition of DAT had no effect on DA levels in either genotype. Therefore, we conclude that in the mouse, PFC COMT contributes about one half of the total DA clearance.

Are genetic variants of COMT associated with addiction?

The human catechol-O-methyl transferase (COMT) gene contains multiple single-nucleotide polymorphisms, some of which are postulated to have clinical significance. This article reviews human studies that have explored the association between COMT polymorphisms and addiction to drugs, alcohol or tobacco. Most studies concentrate on the Val108/158Met polymorphism. Although there are reports indicating a positive association with COMT polymorphisms and addiction, the majority of the studies failed to detect such a link between them with one exception, smoking. It is unlikely that there would be any single gene that could be designated as 'the addiction gene'. Rather, there seems to be a great number of genes that are associated with addiction, among which COMT seems to have a minor role. Environmental factors and genetic milieu have a great impact on whether the small effects of COMT polymorphisms on risk of addiction can be detected in a given population. Sex differences complicate the gene-environment interplay even further.

Sample purification improves the analysis of nonviral in vivo gene transfection.

Nonviral gene delivery has gained a lot of interest as a promising approach for gene therapy. Despite intensive studies and much progress the outcome of nonviral vectors has remained significantly weaker than that of viral vectors. A weak transfection efficiency of nonviral gene transfection is still limiting their in vivo use. We have tested the possibility to improve the measurement of transfection efficiency by increasing the sensitivity of analysis with sample purification. The BPVlacZ and CMVlacZ plasmids were transfected by i.v. infusion of the PEI/DNA complexes in the rats. An adenovirus lacZ vector was used as a reference. The transfection efficiency was analysed from the lungs and brain. Tissue samples were minced and homogenized for preparation of crude homogenates and for further purification of crude homogenates with a DEAE anion exchange chromatography. The beta-galactosidase activity was measured using a luminometric assay. The obtained activity of beta-galactosidase was higher in the purified than nonpurified samples and the analysis of transfection efficiency as beta-galactosidase activity was improved more than 1000-fold by the purification of samples from perfused target tissues. An increased sensitivity of analysis by sample preparation may be a useful and inexpensive strategy to detect and estimate a low transfection efficiency or transgene expression often associated with a nonviral in vivo gene delivery.

Issues about the physiological functions of prolyl oligopeptidase based on its discordant spatial association with substrates and inconsistencies among mRNA, protein levels, and enzymatic activity.

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyses proline-containing peptides shorter than 30 amino acids. POP may be associated with cognitive functions, possibly via the cleavage of neuropeptides. Recent studies have also suggested novel non-hydrolytic and non-catalytic functions for POP. Moreover, POP has also been proposed as a regulator of inositol 1,4,5-triphosphate signaling and several other functions such as cell proliferation and differentiation, as well as signal transduction in the central nervous system, and it is suspected to be involved in pathological conditions such as Parkinson's and Alzheimer's diseases and cancer. POP inhibitors have been developed to restore the depleted neuropeptide levels encountered in aging or in neurodegenerative disorders. These compounds have shown some antiamnesic effects in animal models. However, the mechanisms of these hypothesized actions are still far from clear. Moreover, the physiological role of POP has remained unknown, and a lack of basic studies, including its distribution, is obvious. The aim of this review is to gather information about POP and to propose some novel roles for this enzyme based on its distribution and its discordant spatial association with its best known substrates.

The role of PEI structure and size in the PEI/liposome-mediated synergism of gene transfection.

Polyethylenimines (PEIs) and cationic liposomes are widely used for nonviral gene delivery. When PEIs have been used alone, the transfection efficiency has been higher for larger or linear than smaller or branched PEIs. We have reported previously that a combination of small PEIs and liposomes results in a potentiation of transfection efficiency in vitro. Here, the role of PEI size and structure in this synergism has been clarified further. Therefore, two structurally different high MW PEIs, i.e. the linear PEI22K and branched PEI25K, were studied in the SMC cells. We found that both linear PEI22K and branched PEI25K resulted in a similar synergism and comparable transfection efficiencies. However, the potentiation for larger PEIs found in the present study was weaker than that for smaller PEIs obtained in our previous studies. In conclusion, our present and previous results demonstrate that the increment of PEI/liposome-mediated gene transfection by different types of PEIs in vitro is a common attribute that is rather associated with their size than the structure. Interestingly, the effect of PEI size seems to be opposite when combined with liposome or given alone, i.e. the small PEIs are more effective when combined and less effective when alone than the larger ones.

Combination of snap freezing, differential pH two-dimensional reverse-phase high-performance liquid chromatography, and iTRAQ technology for the peptidomic analysis of the effect of prolyl oligopeptidase inhibition in the rat brain.

In vitro, prolyl oligopeptidase (POP) cleaves proline-containing bioactive peptides such as substance P, gonadotropin-releasing hormone, thyrotropin-releasing hormone, arginine-vasopressin, and neurotensin. Based on specific in vivo inhibition, POP has been suggested to be involved in cognitive and psychiatric processes but the identity of its physiological substrates has remained inconclusive. We have combined (a) sample snap-freezing and boiling buffer extraction, to limit protein degradation and reduce sample complexity; (b) pH two-dimensional liquid reverse-phase chromatography to enhance resolution; and (c) iTRAQ isobaric labeling to identify the rat brain peptides whose levels were differentially changed due to in vivo POP inhibition. In the hypothalamus, all the substrates found were part of precursors of secreted peptides such as copeptin, PACAP-related peptide, somatostatin, and proSAAS derived peptides, while in the cerebellum the peptides were derived from carcinoma-amplified sequence 1 homolog and calmodulin. In the striatum, somatostatin precursor derived peptide, fragments from E3-SUMO protein ligase RanBP2, and the subunit 5A of cytochrome c oxidase were increased. When analyzing the peptides that were significantly reduced by POP inhibition we found fragments from large protein complexes but, exclusively in the cerebellum, bioactive peptides such as cerebellin and fibrinopeptides A and B were detected.

Localization of prolyl oligopeptidase in the thalamic and cortical projection neurons: a retrograde neurotracing study in the rat brain.

Prolyl oligopeptidase (POP) is a serine endopeptidase which hydrolyses proline-containing peptides shorter than 30-mer. POP is believed to be associated with cognitive functions via neuropeptide cleavage. POP has been also connected to the inositol 1,4,5-triphosphate (IP(3)) signalling but the effects of POP-inhibition to the IP(3) accumulation in vivo are still unclear. However, little is known about the physiological role of POP in the brain. We have previously found that in the rat brain POP was specifically expressed in the pyramidal neurons of the cerebral cortex, particularly in the primary motor and somatosensory cortices, and corresponding projection areas in thalamus. Using a retrograde neurotracer we have now visualized the localization of POP in thalamocortical and corticothalamic projection neurons in ventrobasal complex and medial geniculate nucleus of thalamus and somatosensory/motor and auditory cortices. We observed that both in thalamus and cortex over 50% of projection neurons contained POP. These results support the hypothesis that POP is involved in thalamocortical and corticothalamic signal processing. We also propose, based on our neuroanatomical findings and literature, that POP may take part in the thalamocortical oscillations by interacting with IP(3) signalling in cells.

Site-specific role of catechol-O-methyltransferase in dopamine overflow within prefrontal cortex and dorsal striatum.

Accumulating evidence from clinical and preclinical studies shows that catechol-O-methyltransferase (COMT) plays a significant role in dopamine metabolism in the prefrontal cortex, but not in the striatum. However, to what extent dopamine overflow in the prefrontal cortex and striatum is controlled by enzymatic degradation versus reuptake is unknown. We used COMT deficient mice to investigate the role of COMT in these two brain regions with in vivo voltammetry. A real-time analysis of evoked dopamine overflow showed that removal of dopamine was twofold slower in the prefrontal cortex of mice lacking COMT than in wild-type mice, indicating that half of the dopamine decline in this brain region results from COMT-mediated enzymatic degradation. Lack of COMT did not influence dopamine overflow/decline in the dorsal striatum. COMT-deficient mice demonstrated a small (20-25%) but consistent increase in evoked dopamine release in the prefrontal cortex, but not in the dorsal striatum. Cocaine affected equally dopaminergic neurotransmission in the prefrontal cortex in both genotypes by prolonging 3-4 times dopamine elimination from extracellular space. Paradoxically, this happened without increase of the peak levels of evoked dopamine release. The present findings represent the first demonstration of the significant contribution of COMT in modulating the dynamics of dopamine overflow in the prefrontal cortex and underscore the therapeutic potential of manipulating COMT activity to alter dopaminergic neurotransmission in the prefrontal cortex.

Characterization of membrane-bound prolyl endopeptidase from brain.

Prolyl oligopeptidase (POP) is a serine protease that cleaves small peptides at the carboxyl side of an internal proline residue. Substance P, arginine-vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. POP has been implicated in a variety of brain processes, including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension, and psychiatric disorders. Although POP has been considered to be a soluble cytoplasmic peptidase, significant levels of activity have been detected in membranes and in extracellular fluids such as serum, cerebrospinal fluid, seminal fluid, and urine, suggesting the existence of noncytoplasmic forms. Furthermore, a closely associated membrane prolyl endopeptidase (PE) activity has been previously detected in synaptosomes and shown to be different from the cytoplasmic POP activity. Here we isolated, purified and characterized this membrane-bound PE, herein referred to as mPOP. Although, when attached to membranes, mPOP presents certain features that distinguish it from the classical POP, our results indicate that this protein has the same amino acid sequence as POP except for the possible addition of a hydrophobic membrane anchor. The kinetic properties of detergent-soluble mPOP are fully comparable to those of POP; however, when attached to the membranes in its natural conformation, mPOP is significantly less active and, moreover, it migrates anomalously in SDS/PAGE. Our results are the first to show that membrane-bound and cytoplasmic POP are encoded by variants of the same gene.