Polymorphisms in the gene encoding 11B-hydroxysteroid dehydrogenase type 1 (HSD11B1) and lifetime cognitive change

A rare polymorphism in the gene encoding 11B-hydroxysteroid dehydrogenase type 1 (HSD11B1: rs846911-C/A) has been associated with an increased risk of Alzheimer's disease. We tested the hypothesis that this and 2 other HSD11B1 polymorphisms (rs12086634-G/T and rs846910-A/G) were associated with lifetime cognitive change in humans. Subjects were 194 participants of the Scottish Mental Survey of 1932 who took the same well-validated mental test at age 11 and age 79. The subjects represented the highest and lowest quintiles with respect to cognitive decline between ages 11 and 79. Despite having non-significantly different IQs at age 11, by age 79 the groups had mean (S.D.) IQs of 80.3 (14.1) and 109.6 (9.1), respectively (p<.001). The polymorphism rs846911-C/A was absent from both groups. There were no significant differences in the frequency of polymorphisms of rs12086634-G/T (p=.91) and rs846910-A/G (p=.90) between the groups. We conclude that these variants in HSD11B1 are not significant contributors to the range of cognitive ageing examined here.     

Increased expression of 11 beta-hydroxysteroid dehydrogenase type 2 in the lungs of patients with acute respiratory distress syndrome

We examined the immunohistochemical distribution of 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2), the enzyme responsible for the conversion of bioactive glucocorticoids to their receptor-inactive forms, in lung tissue obtained at autopsy from 14 patients who had died due to acute respiratory distress syndrome (ARDS). We found positive immunoreactivity for 11 beta-HSD2 in 13 cases. The cells expressing 11 beta-HSD2 in the alveolar wall were positive for surfactant apoprotein-A as well as cytokeratin. Immunoreactivity for 11 beta-HSD2 was also detected in the CD68+ cells, which were found in the alveolar spaces. All patients had been treated with glucocorticoids for ARDS and/or the underlying diseases. There was no statistically significant correlation between the use of glucocorticoids and 11 beta-HSD2 immunoreactivity in the alveolar wall (P = 0.0729). However, expression of grade + + was found in three out of five patients who received dexamethasone pulse therapy at relatively large doses, as well as in three other patients treated with prednisolone for a long period of time for the underlying disease. An increase in the expression of 11 beta-HSD2 may result in faster glucocorticoid breakdown in lung cells in patients with ARDS. Impaired glucocorticoid availability in the lungs of such patients may explain, in part, the fact that glucocorticoid therapy does not always rescue patients with ARDS.     

DJ-1 mRNA anatomical localization and cell type identification in the mouse brain

Mutations in DJ-1 cause familial Parkinson's disease (PD). The expression pattern of DJ-1 in the brain remains controversial. In the present study, we used DJ-1 deficient mice as negative controls and examined DJ-1 mRNA expression in mouse brains. In sequential double labeling on the same sections, in situ hybridization of DJ-1 mRNA was followed by immunofluorescence detection of cell type markers. We found that DJ-1 mRNA was expressed in the majority of neurons in all brain areas examined. In particular, all dopamine neurons in the ventral midbrain expressed DJ-1 mRNA. In contrast, the choroid plexus and ependymal cells lining the ventricles were the only non-neuronal regions strongly expressing DJ-1 mRNA. DJ-1 mRNA was not detected in astrocytes. The fact that DJ-1 mRNA is expressed in all nigra dopamine neurons but not in astrocytes suggests that its potential neuroprotective role could be cell-autonomous. Moreover, that DJ-1 expression is not restricted to substantia nigra dopamine neurons suggests that PD-linked mutant DJ-1 may interact with other predisposing factors to cause the relatively selective dopamine neuron degeneration in Parkinson's disease.     

Seizure activity involved in the up-regulation of BDNF mRNA expression by activation of central Mu opioid receptors

Chemical-induced seizures up-regulated brain-derived neurotrophic factor (BDNF) mRNA expression. Intracerebroventricular (i.c.v.) administration of endogenous opioids preferentially activating μ opioid receptor (MOR) could also increase BDNF mRNA expression. The aim of this study was to determine to what extent i.c.v. administration of synthetic MOR-selective agonists in rats can modulate both seizure activity and up-regulation of BDNF mRNA expression. Effects and potencies of i.c.v. administration of morphine and [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO), were directly investigated by scoring behavioral seizures and measuring BDNF mRNA expression. In addition, effects of the opioid receptor antagonist naloxone and antiepileptic drugs, diazepam, phenobarbital, and valproate, on i.c.v. MOR agonist-induced behavioral seizures and up-regulation of BDNF mRNA expression were determined. A single i.c.v. administration of morphine (10–100 μg) or DAMGO (0.15–1.5 μg) dose-dependently elicited behavioral seizures and increased BDNF mRNA expression in the widespread brain regions. However, s.c. administration of MOR agonists neither produced behavioral seizures nor increased BDNF mRNA expression. Pretreatment with naloxone 1 mg/kg significantly reduced behavioral seizure scores and the up-regulation of BDNF mRNA expression elicited by i.c.v. morphine or DAMGO. Similarly, diazepam 10 mg/kg and phenobarbital 40 mg/kg significantly blocked i.c.v. MOR agonist-induced actions. Pretreatment with valproate 300 mg/kg only attenuated behavioral seizures, but it did not affect morphine-induced increase of BDNF mRNA expression. This study provides supporting evidence that seizure activity plays an important role in the up-regulation of BDNF mRNA expression elicited by central MOR activation and that decreased inhibitory action of GABAergic system through the modulation on GABA receptor synaptic function by central MOR activation is involved in its regulation of BDNF mRNA expression.     

Regional distribution of importin subtype mRNA expression in the nervous system: study of early postnatal and adult mouse

Importin-alpha and beta1 mediate the translocation of macromolecules bearing nuclear localization signals across the nuclear pore complex. Five importin-alpha isoforms have been identified in mice and six in human. Some of these importins play an important role in neural activity such as long term potentiation, but the functional differences of each isoform in the CNS are still unclear. We performed in situ hybridization (ISH) using non-isotopic probes to clarify the expression patterns of importin-alpha subtypes (alpha5, alpha7, alpha1, alpha4, alpha3) and importin-beta1 in the mouse CNS of adult and early postnatal stages. The mRNAs of the importin-alpha subtypes and importin beta1 were expressed throughout the CNS with specific patterns; importin-alpha5, alpha7, alpha3, and beta1 showed moderate to high expression levels throughout the brain and spinal cord; importin-alpha4 showed a lack of expression in limited regions; and importin-alpha1 showed a low expression level throughout the brain and spinal cord but with a moderate expression level in the olfactory bulb and reticular system. We also demonstrated that importin-alphas and beta1 mRNAs were predominantly expressed in neurons in the adult mouse brain by using double-labeling fluorescence ISH and immunohistochemistry. Moreover, importin-alphas and beta1 mRNAs were detected throughout the CNS of postnatal mice and were highly expressed in the external granule layer of the cerebellar cortex on postnatal days 0, 4, and 10. This is the first report of importin-alphas and beta1 expression throughout the CNS of adult mice, as well as in the developing brain, including cell type specific localization.     

Type 2 11beta-hydroxysteroid dehydrogenase activity in human fallopian tube and correlation of enzyme levels with endometrial histopathology

PROBLEM: The inter-conversion of hormonally active cortisol and inactive cortisone is catalyzed by 11beta-hydroxysteroid dehydrogenase (11beta-HSD). This conversion controls the level of active glucocorticoid concentration in tissues. As the fallopian tube plays a major role in the process of fertilization, we wanted to investigate whether 11beta-HSD is present in the human fallopian tube to control the glucocorticoid levels as in other tissues. METHOD OF STUDY: Isthmic, ampullary and fimbrial portions of the fallopian tube are obtained from patients undergoing hysterectomy and salpingo-oopherectomy for symptomatic leomyomata uteri. 11beta-HSD activities were measured in the homogenates of the tube, cortisol as the steroid substrate. The enzyme activity was expressed as nanomolar cortisone formed per minute per gram of tissue (mean +/- S.D.). RESULTS: A significant level of 11beta-HSD activity in oxidation direction was found in all three parts of the tube. There is no significant difference in the distribution of the enzyme activity throughout the tube. When tubal 11beta-HSD activity was compared with endometrial histology, the enzyme activity is significantly lower in proliferative endometrium when compared with secretory endometrium (P = 0.002). The enzyme activity in inactive endometrium is significantly higher than the active endometrium (P = 0.05). CONCLUSION: The presence of 11beta-HSD throughout the fallopian tube and its correlation to endometrial histology is indicative of its probable role in controlling the glucocorticoid levels in the tissue, which in turn may influence the fertilization process.     

Local levonorgestrel regulation of androgen receptor and 17beta-hydroxysteroid dehydrogenase type 2 expression in human endometrium

BACKGROUND: The levonorgestrel-releasing intrauterine system (LNG-IUS) is a highly effective contraceptive. However, unscheduled breakthrough bleeding (BTB), leads to discontinuation in a proportion of users. The LNG-IUS down-regulates endometrial progesterone and estrogen receptors and this may play a role in the mechanism responsible for BTB. LNG is an androgenic progestogen and so we examined the regulation of the androgen receptor (AR) in endometrium exposed to intrauterine LNG. Furthermore, as the enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2) regulates intracellular levels of estrogens, progestins and androgens, we evaluated the changes in expression of 17betaHSD2 in the same tissue endometrial samples. METHODS: Immunohistochemistry and real time quantitative RT-PCR were used to compare protein and mRNA expression of AR and 17betaHSD2 in endometrial biopsies from women with normal menstrual cycles and those using a LNG-IUS. RESULTS: Immunohistochemistry showed that AR and 17betaHSD2, which were immunolocalized to the stroma and glands of endometrium respectively, were both suppressed by LNG-IUS treatment, though moderate staining of 17betaHSD2 was evident 1 month after insertion of the LNG-IUS. AR mRNA expression was down-regulated in LNG-exposed endometrium when compared with the proliferative phase of the menstrual cycle. 17betaHSD2 mRNA was significantly increased 3 months (but not 6-12 months) after LNG-IUS insertion. CONCLUSIONS: Endometrial intracellular estradiol levels would have been suppressed by 17betaHSD2 during the first few, but not the later, months of LNG-IUS action, and the lowered endometrial estradiol level may contribute to the frequent BTB evident in the early months of LNG-IUS use. The subsequent decline in 17betaHSD2 would lead to elevated local intracellular estradiol in the later months, when the BTB tends to subside. The suppression of AR by the LNG-IUS may also play a role in BTB, as elevated AR has been associated with amenorrhoea.     

Effects of age and gender on Sirt 1 mRNA expressions in the hypothalamus of the mouse

Sirt1 is a NAD-dependent deacetylase that has been shown as a link between energy metabolism and aging. Its putative role as a target for neurodegenerative disorders has recently been suggested; yet, little is known about the changes that occur in Sirt1 levels in the aging brain. Here we show by in situ hybridization that Sirt1 expression is modified in specific areas of the brain in mice upon aging, and that gender also impacts on this regulation. Mice aged 12 and 24 months had a lower Sirt1 expression specifically in the antero ventral thalamic nucleus (AV) and in the arcuate nucleus (ARC) than their young (4 mo) counterparts, whereas changes were either not noticeable or not significantly modulated in other parts of the brain. Regulation of Sirt1 mRNA levels in the subfornical organ (SFO) and in the substancia nigra part compacta (SNC) depended on gender. These findings suggest that reduced Sirt1 levels upon aging could contribute to a lower Sirt1 activity, and that specific nuclei might be particularly affected.     

Temporal profile of connexin 43 expression after photothrombotic lesion in rat brain

Following focal ischemic injury, several mechanisms lead to secondary expansion of the affected area and therefore increase the initial damage. We thoroughly investigated the expression of astrocytic connexin 43 (Cx43) after photothrombosis in rat brain. The temporal profile of Cx43 mRNA as well as protein expression was studied in remote, structurally uninjured cortical and hippocampal areas. The hippocampal formation revealed an increased number of Cx43 mRNA positive astrocytes and an up-regulated protein expression exclusively in the ipsilateral stratum oriens. We assume a participation of this region in glia scar formation. While Cx43 mRNA positive cells were transiently increased, immunoreactivity was reduced in the somatosensory cortex of injured hemispheres. The observed decrease of Cx43 protein in the post-ischemic cerebral cortex implies an impairment of gap junctional intercellular communication which might be detrimental to the brain.     

Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain

Protocadherins (PCDHs) consist of the largest subgroup of the cadherin superfamily, and most PCDHs are expressed dominantly in the CNS. Because PCDHs are involved in the homophilic cell-cell adhesion, PCDHs in the nervous system have been suggested to play roles in the formation and maintenance of the synaptic connections. Although many PCDHs (>50) are in tandem arranged as a cluster in a specific chromosome locus, there are also considerable numbers of non-clustered PCDH members (approximately 20). In this study, we examined the spatiotemporal distribution of mRNAs for 12 non-clustered PCDHs in rat brain using in situ hybridization. Some of them (PCDH1, PCDH7, PCDH9, PCDH10, PCDH11, PCDH17, and PCDH20) exhibited region-dependent expression pattern in the cerebral cortex during the early postnatal stage (P3), which is a critical period for the establishment of specific synaptic connections: PCDH7 and PCDH20 mRNAs were predominantly expressed in the somatosensory (parietal) and visual (occipital) cortices, whereas PCDH11 and PCDH17 mRNAs were preferentially expressed in the motor (forelimb and hindlimb areas) and auditory (temporal) cortices, and PCDH9 mRNA was highly expressed in the motor and main somatosensory cortices. These PCDHs were also expressed in the specific regions of the connecting thalamic nuclei. These cortical regionalization and thalamic nuclei-specificity appeared to be most distinct in P3 compared with those of embryonic and adult stages. Taken together, these results suggest that PCDHs may play specific roles in the establishment of selective synaptic connections of specific modality of cerebral cortex with other communicating brain regions such as the thalamus.     

Human serum mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1): determination of levels in body fluids and identification of two forms in serum

We developed an ELISA for human serum MASP-1, a Cls-like serine protease which is known to function in C4 and C2 activation. We then determined MASP-1 levels in 1063 sera from normal Japanese subjects ranging in age from 3 to 100 years, as well as in certain body fluids using this assay. Individual serum MASP-1 levels ranged from 148 to 12–83 μg/ml, with a normal frequency distribution pattern. The arithmetic mean ± s.d. of MASP-1 levels in serum was 6–27 ± 185 μg/ml, whereas levels of MASP-1 in cerebrospinal fluid and in urine were almost undetectable. When the mean ± s.d. of serum MASP-1 was calculated for each age group (10 year range) and values were then compared, the age group consisting of 3–9-year-olds (7–54 ± l-39;μ/ml) was found to have the highest value. When MASP-1 was measured in cord blood, it was shown that levels were already as high as those of 3–9-year-olds. The serum MASP-1 level was found to be as strongly dependent on age as is the serum MBL level. MASP-1 and MBL are thought to play an active part in immunity in younger people. It was found that the serum level of MASP-1 was much higher than that of MBL, and the major portion of human serum MASP-1 appeared to exist in the circulation as a form unbound to MBL.     

11beta-Hydroxysteroid dehydrogenase type 2 protects the neonatal cerebellum from deleterious effects of glucocorticoids

11beta-Hydroxysteroid dehydrogenase type 2 is a glucocorticoid metabolizing enzyme that catalyzes rapid inactivation of corticosterone and cortisol to inert 11-keto derivatives. As 11beta-hydroxysteroid dehydrogenase type 2 is highly expressed in the developing brain, but not in the adult CNS, we hypothesized that it may represent a protective barrier to the deleterious actions of corticosteroids on proliferating cells. To test this hypothesis we have investigated the development and growth of the cerebellum in neonatal C57BL/6 mice and mice lacking 11beta-hydroxysteroid dehydrogenase type 2 (-/-). 11beta-Hydroxysteroid dehydrogenase type 2-/- mice had consistently lower body weight throughout the neonatal period, coupled with a smaller brain size although this was normalized when corrected for body weight. The cerebellar size was smaller in 11beta-hydroxysteroid dehydrogenase type 2-/- mice, due to decreases in size of both the molecular and internal granule layers. When exogenous corticosterone was administered to the pups between postnatal days 4 and 13, 11beta-hydroxysteroid dehydrogenase type 2(-/-) mice were more sensitive, showing further inhibition of cerebellar growth while the wildtype mice were not affected. Upon withdrawal of exogenous steroid, there was a rebound growth spurt so that at day 21 postnatally, the cerebellar size in 11beta-hydroxysteroid dehydrogenase type 2-/- mice was similar to untreated mice of the same genotype. Furthermore, 11beta-hydroxysteroid dehydrogenase type 2-/- mice had a delay in the attainment of neurodevelopmental landmarks such as negative geotaxis and eye opening. We therefore suggest that 11beta-hydroxysteroid dehydrogenase type 2 acts as to protect the developing nervous system from the deleterious consequences of glucocorticoid overexposure.     

3,4-Methylenedioxymethamphetamine induces differential regulation of tryptophan hydroxylase 2 protein and mRNA levels in the rat dorsal raphe nucleus

Previous investigations with 3,4-methylenedioxymethamphetamine (MDMA) have suggested that administration of this drug results in a degeneration of 5-HT nerve terminals and subsequent alterations in 5-HT neurotransmission. However, only limited investigations have examined the effects of MDMA on the dorsal raphe nucleus. The present study was designed to assess the effect of MDMA on the rate-limiting enzyme in 5-HT biosynthesis, tryptophan hydroxylase (TPH), by measuring TPH2 protein and mRNA levels in rat dorsal raphe (DR) nucleus. Rats were administered MDMA (20 mg/kg, s.c.) or saline twice daily for 4 days and killed 14 days later. Tissue sections of the DR were processed for quantitative immunoautoradiography and in situ hybridization histochemistry for measurements of the levels of TPH2-immunoreactivity (IR) and TPH2 mRNA. To assess 5-HT axon terminal integrity after MDMA treatment, the density of 5-HT transporter (SERT) binding sites was measured by quantitative autoradiography using [125I]RTI-55 ((-)-2beta-carbomethoxy-3 beta-(4-iodophenyl) tropane) ((125)I-RTI-55) as a ligand. TPH2-IR levels were significantly decreased by 45% in the mid DR and by 40% in the caudal DR in the MDMA-treated rats compared with saline-injected rats. In contrast, TPH2 mRNA levels were significantly increased by 24% in the mid DR and by 12% in the caudal DR. MDMA treatment significantly decreased (125)I-RTI-55 labeled SERT binding sites in the striatum, nucleus accumbens and cingulate cortex demonstrating a loss of 5-HT terminals. The increase in TPH2 mRNA levels in both the mid DR and caudal DR of MDMA-treated rats may reflect a compensatory mechanism in the injured 5-HT neurons to increase TPH2 protein synthesis. Taken together, our results suggest that a serious defect occurs in the biosynthesis of TPH2 in the DR following MDMA administration.     

Circadian and developmental regulation of N-methyl-d-aspartate-receptor 1 mRNA splice variants and N-methyl-d-aspartate-receptor 3 subunit expression within the rat suprachiasmatic nucleus

The circadian rhythms of mammals are generated by the circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Its intrinsic period is entrained to a 24 h cycle by external cues, mainly by light. Light impinging on the SCN at night causes either advancing or delaying phase shifts of the circadian clock. N-methyl-d-aspartate receptors (NMDAR) are the main glutamate receptors mediating the effect of light on the molecular clockwork in the SCN. They are composed of multiple subunits, each with specific characteristics whose mutual interactions strongly determine properties of the receptor. In the brain, the distribution of NMDAR subunits depends on the region and developmental stage. Here, we report the circadian expression of the NMDAR1 subunit in the adult rat SCN and depict its splice variants that may constitute the functional receptor channel in the SCN. During ontogenesis, expression of two of the NMDAR1 subunit splice variants, as well as the NMDAR3A and 3B subunits, exhibits developmental loss around the time of eye opening. Moreover, we demonstrate the spatial and developmental characteristics of the expression of the truncated splice form of NMDAR1 subunit NR1-E in the brain. Our data suggest that specific properties of the NMDAR subunits we describe within the SCN likely influence the photic transduction pathways mediating the clock entrainment. Furthermore, the developmental changes in NMDAR composition may contribute to the gradual postnatal maturation of the entrainment pathways.