Copy number elevation of 22q11.2 genes arrests the developmental maturation of working memory capacity and adult hippocampal neurogenesis.

Working memory capacity, a critical component of executive function, expands developmentally from childhood through adulthood. Anomalies in this developmental process are seen in individuals with autism spectrum disorder (ASD), schizophrenia and intellectual disabilities (ID), implicating this atypical process in the trajectory of developmental neuropsychiatric disorders. However, the cellular and neuronal substrates underlying this process are not understood. Duplication and triplication of copy number variants of 22q11.2 are consistently and robustly associated with cognitive deficits of ASD and ID in humans, and overexpression of small 22q11.2 segments recapitulates dimensional aspects of developmental neuropsychiatric disorders in mice. We capitalized on these two lines of evidence to delve into the cellular substrates for this atypical development of working memory. Using a region- and cell-type-selective gene expression approach, we demonstrated that copy number elevations of catechol-O-methyl-transferase (COMT) or Tbx1, two genes encoded in the two small 22q11.2 segments, in adult neural stem/progenitor cells in the hippocampus prevents the developmental maturation of working memory capacity in mice. Moreover, copy number elevations of COMT or Tbx1 reduced the proliferation of adult neural stem/progenitor cells in a cell-autonomous manner in vitro and migration of their progenies in the hippocampus granular layer in vivo. Our data provide evidence for the novel hypothesis that copy number elevations of these 22q11.2 genes alter the developmental trajectory of working memory capacity via suboptimal adult neurogenesis in the hippocampus.

Elimination of extracellular dopamine in the medial prefrontal cortex of conscious mice analysed using selective enzyme and uptake inhibitors.

We have shown in a previous study that in the medial prefrontal cortex (mPFC) of Comt knockout animals, uptake1 followed by oxidation accounts for approximately 50% and uptake2 followed by O-methylation for the remaining 50% of dopamine clearance. However, compensatory mechanisms in genetically modified animals may have affected the result. Therefore, in the present study, we gave a high dose (30 mg/kg) of tolcapone in combination with pargyline and reboxetine to C57BL/6J mice to see whether the earlier findings could be confirmed. The three drugs were also given together. We used intracerebral microdialysis to determine the levels of extracellular dopamine and its metabolites in the mPFC. In addition, we analyzed dopamine, 3,4-dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) contents in cortical and striatal synaptosomes to estimate the amount of releasable dopamine and dopamine turnover. In the prefrontal cortex of male C57BL/6J mice, the combination of two drugs (pargyline + tolcapone or reboxetine + tolcapone) generally elevated extracellular dopamine levels more than any single drug. Similar responses, although much weaker, were observed in female mice. Unexpectedly, triple treatment with pargyline, reboxetine and tolcapone did not increase dopamine outflow in the mPFC in either sex, and the treatment actually diminished dopamine outflow in the dorsal striatum. This seems to indicate that such an extensive treatment induces a fast and effective shut-down of dopamine release both in the mPFC and striatum to protect the brain from excess dopaminergic stimulation. The observed decrease in extracellular dopamine levels was not due to the depletion of releasable dopamine because abundant amounts of dopamine were present in synaptosomes. These results imply that the relative proportion of COMT-induced dopamine clearance may be somewhat lower than earlier estimated.

Generation of membrane-bound catechol-O-methyl transferase deficient mice with disctinct sex dependent behavioral phenotype.

Catechol-O-methyltransferase (COMT) has two isoforms: soluble (S-COMT), which resides in the cytoplasm, and membrane-bound (MB-MT), anchored to intracellular membranes. COMT is involved in the O-methylation of L-DOPA, dopamine and other catechols. The exact role of MB-COMT is still mostly unclear. We wanted to create a novel genetically modified mouse model that specifically lacks MB-COMT activity and to study their behavioral phenotype. MB-COMT knock-in mutant mice were generated by introducing two point mutations in exon 2 of the Comt gene (ATGCTG->GAGCTC disabling the function of the P2 promoter and allowing only the P1-regulated S-COMT transcription. The first mutation changes methionine to glutamic acid whereas the second one does not affect coding. The expression of the two COMT isoforms, total COMT activity in several areas of the brain and peripheral tissues and extracellular dopamine concentrations after L-DOPA (10 mg/kg) and carbidopa (30 mg/kg) subcutaneous administration were assessed. A battery of behavioral tests was performed to compare MB-COMT deficient mice and their wild type littermates of both sexes. MB-COMT deficient mice were seemingly normal, bred usually and had unaltered COMT activity in the brain and periphery despite a complete lack of the MB-COMT protein. MB-COMT deficient male mice showed higher extracellular dopamine levels than their wild-type littermates in the striatum, but not in the mPFC. In addition, the MB-COMT deficient male mice exhibited a distinct endophenotype characterized by schizophrenia-related behaviors like aggressive behavior and reduced prepulse inhibition. They also had prolonged immobility in the tail suspension test. Both sexes were sensitized to acute pain and had normal motor activity but disturbed short-term memory. Hence the behavioral phenotype was not limited to schizophrenia-related endophenotype and some behavioural findings were not sex-dependent. Our findings indicate that MB-COMT is critical for behavior, and its function in COMT-dependent brain areas cannot be entirely substituted by the remaining S-COMT.

Prolyl oligopeptidase colocalizes with α-synuclein, ß-amyloid, tau protein and astroglia in the post-mortem brain samples with Parkinson's and Alzheimer's diseases.

Prolyl oligopeptidase (EC 3.4.21.26, PREP) is a serine protease that hydrolyzes proline-containing peptides shorter than 30-mer but it has also nonhydrolytic functions. PREP has been shown to accelerate aggregation of wild-type α-synuclein (α-syn) under cell-free conditions, and PREP inhibitors can block this aggregation both in vitro and in vivo. α-syn is the main component of Lewy bodies in Parkinson's disease (PD) and Lewy body dementia. To clarify the possible interaction of PREP with other markers of neurodegenerative diseases, we studied colocalizations of PREP and (1) α-syn, (2) ß-amyloid, (3) tau protein and (4) astroglial and microglial cells in human post-mortem brain samples from PD, Alzheimer's disease (AD) patients and in healthy control brain samples. In the substantia nigra of PD brains, an intense colocalization with PREP and α-syn was evident. PREP colocalized also with ß-amyloid plaques in AD brains and with tau protein in AD and in healthy brains. PREP was also found in astroglial cells in PD, AD and control brains, but not in the microglia. Our findings are the first ones to demonstrate colocalization of PREP and pathological proteins in the human brain and support the view that, at least in spatial terms, PREP could be associated with pathogenesis of neurodegenerative diseases.

Comparison of motor performance, brain biochemistry and histology of two A30P α-synuclein transgenic mouse strains.

Three point mutations in the SNCA gene encoding α-synuclein (aSyn) have been associated with autosomal dominant forms of Parkinson's disease. To better understand the role of the A30P mutant aSyn, we compared two transgenic mouse strains: a knock-in mouse with an introduced A30P point mutation in the wild-type (WT) gene (Snca(tm(A30P))) and a transgenic (Tg) mouse overexpressing the human A30P aSyn gene under the prion promoter [tg(Prnp-SNCA A30P)]. The brain aSyn load, motor performance, brain dopamine (DA) and sensitivity to 6-hydroxydopamine (6-OHDA) were studied in these mice. aSyn was evidently accumulating with age in all mice, particularly in tg(Prnp-SNCA A30P) Tg mice. There were no robust changes in basal locomotor activities of the mice of either line at 6 months, but after 1 year, tg(Prnp-SNCA A30P) Tg mice developed severe problems with vertical movements. However, the younger Tg mice had a reduced locomotor response to 1mg/kg of d-amphetamine. Snca(tm(A30P)) mice with the targeted mutation (Tm) were slightly hyperactive at all ages. Less 6-OHDA was required in tg(Prnp-SNCA A30P) Tg (1 µg) than in WT (3µg) mice for an ipsilateral rotational bias by d-amphetamine. That was not seen with the Snca(tm(A30P)) strain. A small dose of 6-OHDA (0.33 µg) led to contralateral rotations and elevated striatal DA in Tg/Tm mice of both lines but otherwise 6-OHDA-induced striatal DA depletion was similar in all mice, indicating no A30P-aSyn-related toxin sensitivity. 3,4-Dihydroxyphenylacetic acid/DA-ratio was elevated in tg(Prnp-SNCA A30P) mice, suggesting an enhanced DA turnover. This ratio and homovanillic acid/DA-ratio were declined in Snca(tm(A30P)) mice. Our results demonstrate that the two differently constructed A30P-aSyn mouse strains have distinct behavioral and biochemical characteristics, some of which are opposite. Since the two lines with the same background were not identically produced, the deviations found may be partially caused by factors other than aSyn-related genetic differences.

Different interactions of prolyl oligopeptidase and neurotensin in dopaminergic function of the rat nigrostriatal and mesolimbic pathways.

Prolyl oligopeptidase (PREP) is an intracellular enzyme digesting small proline-containing peptides. Since PREP resides the same brain areas as neurotensin in the nigrostriatal and mesolimbic dopaminergic pathways, we were interested to study if there is an intracellular interaction between them. A colocalization of PREP with neurotensin and neurotensin receptor 1 (NTS1) in the rat striatum, nucleus accumbens (NAcc), substantia nigra (SN) and ventral tegmental area (VTA) was studied with immunofluorescence. From the same brain areas, the levels of dopamine and its metabolites were measured 1 h after the injection of saline, NTS1 ligands (JMV-449; 5 µg) or antagonist (SR142948; 5 µg) to the rat striatum or NAcc. We also studied whether an intraperitoneal injection of a PREP inhibitor (KYP-2047; 5 mg/kg) affects the levels of dopamine and its metabolites alone or modifies the effects of the NTS1 ligands. PREP was highly colocalized with neurotensin and NTS1 in the VTA, and with NTS1 in the SN. Colocalization was moderate or low in other brain areas. When injected to the striatum, JMV-449 had a tendency to increase dopamine (p = 0.052) and metabolite levels in the striatum and SN, whereas SR142948 did not. After the injection to the NAcc, JMV-449 but not SR142948, increased dopamine levels in the VTA and dopamine metabolite levels in the NAcc and VTA. KYP-2047 decreased the dopamine levels in the striatum, but increased dopamine metabolite levels in the NAcc and VTA. Our results suggest a novel role for PREP in the modulation of dopaminergic transmission, which may be different in nigrostriatal and mesolimbic pathways.

A prolyl oligopeptidase inhibitor, KYP-2047, reduces α-synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease.

BACKGROUND AND PURPOSE: The aggregation of α-synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on α-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human α-syn and in the brains of two A30P α-synuclein transgenic mouse strains. EXPERIMENTAL APPROACH: Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild-type or transgenic mice were treated for 5 days with KYP-2047 (2 × 3 mg·kg(-1) a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble α-synuclein protein levels were measured by Western blot. α-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and α-synuclein,and the effect of KYP-2047 on cell viability were also investigated. KEY RESULTS: In cell lines, oxidative stress induced a robust aggregation of α-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with α-synuclein inclusions while abolishing the colocalization of α-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated α-synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5-day treatment with the PREP inhibitor reduced the amount of α-synuclein immunoreactivity and soluble α-synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS: The results suggest that the PREP may play a role in brain accumulation and aggregation of α-synuclein, while KYP-2047 seems to effectively prevent these processes.

Complex estrogenic regulation of catechol-O-methyltransferase (COMT) in rats.

Catechol-O-methyltransferase (COMT) activity depends on gender, age and physiological status suggesting that estrogen may regulate COMT activity. In fact, estrogens down-regulate the function of COMT promoters in cell cultures. On the other hand, COMT may play an important role in estrogen-induced cancers due to its ability to inactivate estrogen metabolites and thereby lowering the levels of these potential carcinogens. In this study, we explored the effect of estrogen on COMT activity in vivo in rats. Male and female Wistar rats received 14-day treatments with either estradiol (100 µg/kg/day; s.c.) or tamoxifen (500 µg/kg/day; s.c.), respectively; in addition ovariectomized rats were studied. COMT activity and COMT protein expression were measured from various brain- and peripheral tissues. Although we found a regulatory function of estrogen, its effects were sex and tissue dependent. Antagonizing the effects of estrogen via tamoxifen increased COMT protein expression in several central and peripheral tissues. However, amounts of COMT protein and COMT activities did not always match. Generally, COMT activities were quite resistant to the effects of tamoxifen and estradiol. Estradiol, unexpectedly, doubled the amount of COMT protein in the prostate but exhibited down-regulatory function in the prefrontal cortex and kidneys. Ovariectomy by itself, however, had only minor effects on COMT activity and expression. It is noteworthy that the estrogen down-regulation and tamoxifen up-regulation of COMT were best substantiated in the prefrontal cortex and kidneys where COMT is physiologically important for dopamine metabolism.

Vascular endothelial growth factor C acts as a neurotrophic factor for dopamine neurons in vitro and in vivo.

Neurotrophic factors regulate the development and maintenance of the nervous system and protect and repair dopaminergic neurons in animal models of Parkinson's disease (PD). Vascular endothelial growth factors A (VEGF-A) and B have also neurotrophic effects on various types of neurons, including dopaminergic neurons. We examined the ability of the key lymphangiogenic factor VEGF-C to protect dopaminergic cells in vitro and in vivo. The study was initiated by a finding from microarray profiling of Neuro2A-20 cells which revealed up-regulation of VEGF-C by glial cell-line-derived neurotrophic factor (GDNF). Next, we observed that VEGF-C can rescue embryonic dopaminergic neurons and activate the mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway in vivo. VEGF receptors 1-2 and co-receptors, neuropilins 1-2, were expressed both in mouse embryonic cultures and adult midbrains. In vivo, VEGF-C had a robust functional effect in the rat unilateral 6-hydroxydopamine (6-OHDA) model of PD and there was a small additive effect on the survival of tyrosine hydroxylase (TH)-positive cells with GDNF. The neuroprotective effect of VEGF-C is most likely due to a combination of direct and indirect neurotrophic effects because, VEGF-C, unlike GDNF, induced also angiogenesis in the striatum following 6-OHDA insult as it did in human umbilical vein endothelial cells (HUVEC). However, we detected activation of astroglia and microglia as well as blood-brain barrier disruption after intracerebral delivery of VEGF-C, raising a concern of its safe usage as a therapeutic molecule. Our results provide evidence of VEGF-C as a neurotrophic factor that influences the dopaminergic system through multiple mechanisms.

Epithelial transport and deamidation of gliadin peptides: a role for coeliac disease patient immunoglobulin A.

In coeliac disease, the intake of dietary gluten induces small-bowel mucosal damage and the production of immunoglobulin (Ig)A class autoantibodies against transglutaminase 2 (TG2). We examined the effect of coeliac patient IgA on the apical-to-basal passage of gluten-derived gliadin peptides p31-43 and p57-68 in intestinal epithelial cells. We demonstrate that coeliac IgA enhances the passage of gliadin peptides, which could be abolished by inhibition of TG2 enzymatic activity. Moreover, we also found that both the apical and the basal cell culture media containing the immunogenic gliadin peptides were able to induce the proliferation of deamidation-dependent coeliac patient-derived T cells even in the absence of exogenous TG2. Our results suggest that coeliac patient IgA could play a role in the transepithelial passage of gliadin peptides, a process during which they might be deamidated.

Prolyl oligopeptidase induces angiogenesis both in vitro and in vivo in a novel regulatory manner.

BACKGROUND AND PURPOSE A serine protease, prolyl oligopeptidase (POP) has been reported to be involved in the release of the pro-angiogenic tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (Ac-SDKP) from its precursor, 43-mer thymosin ß4 (Tß4). Recently, it was shown that both POP activity and the levels of Ac-SDKP are increased in malignant tumours. The aim of this study was to clarify the release of Ac-SDKP, and test if POP and a POP inhibitor, 4-phenyl-butanoyl-L-prolyl-2(S)-cyanopyrrolidine (KYP-2047), can affect angiogenesis. EXPERIMENTAL APPROACH We used HPLC for bioanalytical and an enzyme immunoassay for pharmacological analysis. Angiogenesis of human umbilical vein endothelial cells was assessed in vitro using a 'tube formation' assay and in vivo using a Matrigel plug assay (BD Biosciences, San Jose, CA, USA) in adult male rats. Moreover, co-localization of POP and blood vessels was studied. KEY RESULTS We showed the sequential hydrolysis of Tß4: the first-step hydrolysis by proteases to <30-mer peptides is followed by an action of POP. Unexpectedly, POP inhibited the first hydrolysis step, revealing a novel regulation system. POP with Tß4 significantly induced, while KYP-2047 effectively prevented, angiogenesis in both models compared with Tß4 addition itself. POP and endothelial cells were abundantly co-localized in vivo. CONCLUSIONS AND IMPLICATIONS We have now revealed that POP is a second-step enzyme in the release of Ac-SDKP from Tß4, and it has novel autoregulatory effect in the first step. Our results also advocate a role for Ac-SDKP in angiogenesis, and suggest that POP has a pro-angiogenic role via the release of Ac-SDKP from its precursor Tß4 and POP inhibitors can block this action.

Inhibitors of catechol-O-methyltransferase sensitize mice to pain.

BACKGROUND AND PURPOSE: Catechol-O-methyltransferase (COMT) inhibitors are used in Parkinson's disease in which pain is an important symptom. COMT polymorphisms modulate pain and opioid analgesia in humans. In rats, COMT inhibitors have been shown to be pro-nociceptive in acute pain models, but also to attenuate allodynia and hyperalgesia in a model of diabetic neuropathy. Here, we have assessed the effects of acute and repeated administrations of COMT inhibitors on mechanical, thermal and carrageenan-induced nociception in male mice. EXPERIMENTAL APPROACH: We used single and repeated administration of a peripherally restricted, short-acting (nitecapone) and also a centrally acting (3,5-dinitrocatechol, OR-486) COMT inhibitor. We also tested CGP 28014, an indirect inhibitor of COMT enzyme. Effects of OR-486 on thermal nociception were also studied in COMT deficient mice. Effects on spinal pathways were assessed in rats given intrathecal nitecapone. KEY RESULTS: After single administration, both nitecapone and OR-486 reduced mechanical nociceptive thresholds and thermal nociceptive latencies (hot plate test) at 2 and 3 h, regardless of their brain penetration. These effects were still present after chronic treatment with COMT inhibitors for 5 days. Intraplantar injection of carrageenan reduced nociceptive latencies and both COMT inhibitors potentiated this reduction without modifying inflammation. CGP 28014 shortened paw flick latencies. OR-486 did not modify hot plate times in Comt gene deficient mice. Intrathecal nitecapone modified neither thermal nor mechanical nociception. CONCLUSIONS AND IMPLICATIONS: Pro-nociceptive effects of COMT inhibitors were confirmed. The pro-nociceptive effects were primarily mediated via mechanisms acting outside the brain and spinal cord. COMT protein was required for these actions.

Degradation of coeliac disease-inducing rye secalin by germinating cereal enzymes: diminishing toxic effects in intestinal epithelial cells.

Currently the only treatment for coeliac disease is a lifelong gluten-free diet excluding food products containing wheat, rye and barley. There is, however, only scarce evidence as to harmful effects of rye in coeliac disease. To confirm the assumption that rye should be excluded from the coeliac patient's diet, we now sought to establish whether rye secalin activates toxic reactions in vitro in intestinal epithelial cell models as extensively as wheat gliadin. Further, we investigated the efficacy of germinating cereal enzymes from oat, wheat and barley to hydrolyse secalin into short fragments and whether secalin-induced harmful effects can be reduced by such pretreatment. In the current study, secalin elicited toxic reactions in intestinal Caco-2 epithelial cells similarly to gliadin: it induced epithelial cell layer permeability, tight junctional protein occludin and ZO-1 distortion and actin reorganization. In high-performance liquid chromatography and mass spectroscopy (HPLC-MS), germinating barley enzymes provided the most efficient degradation of secalin and gliadin peptides and was thus selected for further in vitro analysis. After germinating barley enzyme pretreatment, all toxic reactions induced by secalin were ameliorated. We conclude that germinating enzymes from barley are particularly efficient in the degradation of rye secalin. In future, these enzymes might be utilized as a novel medical treatment for coeliac disease or in food processing in order to develop high-quality coeliac-safe food products.

Molecular dynamics study of prolyl oligopeptidase with inhibitor in binding cavity.

We used the crystal structure of prolyl oligopeptidase (POP) with bound Z-pro-prolinal (ZPP) inhibitor (Protein Data Bank (PDB) structure 1QFS) to perform an intensive molecular dynamics study of the POP-ZPP complex. We performed 100 ns of simulation with the hemiacetal bond, through which the ZPP is bound to the POP, removed in order to better investigate the binding cavity environment. From basic analysis, measuring the radius of gyration, root mean square deviation, solvent accessible surface area and definition of the secondary structure of protein, we determined that the protein structure is highly stable and maintains its structure over the entire simulation time. This demonstrates that such long time simulations can be performed without the protein structure losing stability. We found that water bridges and hydrogen bonds play a negligible role in binding the ZPP thus indicating the importance of the hemiacetal bond. The two domains of the protein are bound by a set of approximately 12 hydrogen bonds, specific to the particular POP protein.

Importance of membrane-bound catechol-O-methyltransferase in L-DOPA metabolism: a pharmacokinetic study in two types of Comt gene modified mice.

BACKGROUND AND PURPOSE: Catechol-O-methyltransferase (COMT) metabolizes compounds containing catechol structures and has two forms: soluble (S-COMT) and membrane-bound (MB-COMT). Here we report the generation of a mouse line that expresses MB-COMT but not S-COMT. We compared the effects of deleting S-COMT only or both COMT forms on the pharmacokinetics of oral L-DOPA. EXPERIMENTAL APPROACH: L-DOPA (10 mg kg(-1)) and carbidopa (30 mg kg(-1)) were given to mice by gastric tube, and samples were taken at various times. HPLC was used to measure L-DOPA in plasma and tissue samples, and dopamine and its metabolites in brain. Immunohistochemistry and Western blotting were used to characterize the distribution of COMT protein isoforms. KEY RESULTS: Lack of S-COMT did not affect the levels of L-DOPA in plasma or peripheral tissues, whereas in the full COMT-knock-out mice, these levels were increased. The levels of 3-O-methyldopa were significantly decreased in the S-COMT-deficient mice. In the brain, L-DOPA levels were not significantly increased, and dopamine was increased only in females. The total COMT activity in the S-COMT-deficient mice was 22-47% of that in the wild-type mice. In peripheral tissues, female mice had lower COMT activity than the males. CONCLUSIONS AND IMPLICATIONS: In S-COMT-deficient mice, MB-COMT in the liver and the duodenum is able to O-methylate about one-half of exogenous L-DOPA. Sexual dimorphism and activity of the two COMT isoforms seems to be tissue specific and more prominent in peripheral tissues than in the brain.

Expression and traffic of cellular prolyl oligopeptidase are regulated during cerebellar granule cell differentiation, maturation, and aging.

Prolyl oligopeptidase (POP) is an endopeptidase which cleaves short proline-containing neuropeptides, and it is involved in memory and learning. POP also has an intercellular function mediated through the inositol pathway, and has been involved in cell death. POP has been early considered as a housekeeping enzyme, but the recent research indicates that POP expression is regulated across tissues and intracellularly. In the brain, POP is exclusively expressed in neurons and most abundantly in pyramidal neurons of cerebral cortex, in the CA1 field neurons of hippocampus and in cerebellar Purkinje's cells. Intracellularly, POP is mainly present in the cytoplasm and some in intracellular membranes, like rough endoplasmic reticulum and Golgi apparatus. In this paper, we systematically studied the levels of expression of POP along the life of cerebellar granule cells (CGC) in culture and the distribution of POP within different intracellular compartments. We used the tight-binding inhibitor JTP-4819 covalently coupled with fluorescein (FJTP) as a tool to study the changes on expression and localization of POP protein. Our results indicate that POP activity levels are regulated during the life of the neurons. POP was found mainly in cytoplasm and neuronal projections, but at an early developmental phase significant amounts were found also in nuclei. Along the life of the neurons, POP activity fluctuated in 7-day cycles. In young neurons, the cytosolic POP activity was low but increased by maturation so that the activity peak coincided with full differentiation. Over aging, cytoplasmic POP was concentrated around nucleus, but the activity decreased with time. POP was also present in vesicles across the neuron. No major changes were seen in the nuclear or membrane bound POP over aging until activity disappeared upon neuronal death. This is the first time when POP was found in the nuclei of human neuronal cells.

Spatial association of prolyl oligopeptidase, inositol 1,4,5-triphosphate type 1 receptor, substance P and its neurokinin-1 receptor in the rat brain: an immunohistochemical colocalization study.

Prolyl oligopeptidase (POP) is a serine endopeptidase which hydrolyzes proline-containing peptides shorter than 30 amino acids. It has been suggested that POP is associated with cognitive functions, possibly via the cleavage of neuropeptides such as substance P (SP). Recently, several studies have also linked POP to the inositol 1,4,5-triphosphate (IP(3)) signaling. However, the neuroanatomical interactions between these substances are not known. We used double-labeled immunofluorescence to determine the POP colocalization with SP, SP receptor (neurokinin-1 receptor, NK-1R) and IP(3) type 1 receptor (IP(3)R1) in the rat brain. Furthermore, since striatal and cortical GABAergic neurons are involved in SP neurotransmission, we studied the coexpression of POP, SP and GABA by triple-labeled immunofluorescence. POP was moderately present in IP(3)R1-containing cells in cortex; the colocalization was particularly high in the thalamus, hippocampal CA1 field and cerebellar Purkinje cells. Colocalization of POP with SP and NK1-receptor was infrequent throughout the brain, though some POP and SP coexpression was observed in cerebellar Purkinje cells. We also found that POP partially colocalized with SP-containing GABAergic neurons in striatum and cortex. Our findings support the view that POP is at least spatially associated with the IP(3)-signaling in the thalamus, hippocampus and cerebellar Purkinje cells. This might point to a role for POP in the regulation of long-term potentiation and/or depression. Moreover, the low degree of colocalization of POP, SP and its NK-1R suggests that a transport system is needed either for POP or SP to make hydrolysis possible and that POP may act both intra- and extracellularly.

On the role of prolyl oligopeptidase in health and disease.

Prolyl oligopeptidase (POP) is a serine peptidase which digests small peptide-like hormones, neuroactive peptides, and various cellular factors. Therefore, this peptidase has been implicated in many physiological processes as well as in some psychiatric disorders, most probably through interference in inositol cycle. Intense research has been performed to elucidate, on the one hand, the basic structure, ligand binding, and kinetic properties of POP, and on the other, the pharmacology of its inhibitors. There is fairly strong evidence of in vivo importance of POP on substance P, arginine vasopressin, thyroliberin and gonadoliberin metabolism. However, information about the biological relevance of POP is not yet conclusive. Evidence regarding the physiological role of POP is lacking, which is surprising considering that peptidase inhibitors have been exploited for drug development, some of which are currently in clinical trials as memory enhancers for the aged and in a variety of neurological disorders. Here we review the recent progress on POP research and evaluate the relevance of the peptidase in the metabolism of various neuropeptides. The recognition of novel forms and relatives of POP may improve our understanding of how this family of proteins functions in normal and in neuropathological conditions.

Increased p53 levels without caspase-3 activity and change of cell viability in 6-hydroxydopamine-treated CV1-P cells.

The effect of 6-hydroxydopamine (6-OHDA) on the level of p53 protein, the activity of caspase-3 and the nuclear morphology-based assessment of cell viability were compared in the nonneuronal CV1-P fibroblast and neuronal SH-SY5Y neuroblastoma cells. The level of p53 protein was increased in the low-dose range (<100 micromol/L) in both cell types, particularly in fibroblasts. In the neuroblastoma cells, a moderate p53 increase paralleled the elevated caspase-3 activity and apoptotic cell behavior. Interestingly, in the fibroblasts at the low 6-OHDA concentrations, p53 remained high during the whole experiment, and there was neither significant caspase-3 activity nor cell death. In the high-dose range (>100 micromol/L), the increase of p53 was reduced and the cell death was predominantly necrotic as judged from the nuclear morphology in both fibroblasts and neuroblastoma cells. Also, the caspase-3 activity was reduced in SH-SY5Y cells. In contrast to some earlier reports, we have shown that the actual 6-OHDA sensitivity of nonneuronal cells may be equal or even higher than that in neuronal cells if the enhancement of p53 levels is used as a criterion for the response. However, the 6-OHDA toxicity was clearly higher in the neuronal than in fibroblast cells.

Effects of aqueous extracts of Halimeda incrassata (Ellis) Lamouroux and Bryothamnion triquetrum (S.G.Gmelim) Howe on hydrogen peroxide and methyl mercury-induced oxidative stress in GT1-7 mouse hypothalamic immortalized cells.

The current investigation focuses attention on the neuroprotective and antioxidant properties of aqueous extracts from Halimeda incrassata (Hi) and Bryothamniom triquetrum (Bt) in the mouse immortalized hypothalamic GT1-7 cell line. Under basal oxidative conditions, Hi extract reduces intracellular reactive oxygen species production, as assessed by 2',7'-dichlorofluorescein fluorescence, while Bt extract does not contribute to basal ROS generation. Both extracts, at concentrations higher than 0.20 mg/ml, exert protection against hydrogen peroxide-mediated cell death, although only Hi extract can additionally prevent hydrogen peroxide-induced ROS production. The two seaweed aqueous extracts, at concentrations higher than 0.05 mg/ml, also display protection against neuronal death induced by methyl mercury chloride, as well as against methyl mercury chloride-mediated ROS generation. None of the extracts increase GSH intracellular pools, in basal conditions, after depleting its levels with either hydrogen peroxide or methyl mercury chloride. Some comments on the probable targets of the neuroprotection exerted by these two extracts are included in this paper.