A comparative study of Parkinson's disease and leucine-rich repeat kinase 2 p.G2019S parkinsonism

Parkinson disease is a progressive neurodegenerative disease for which leucine-rich repeat kinase 2 (LRRK2 carriers) p.G2019S confers substantial genotypic and population attributable risk. With informed consent, we have recruited clinical data from 778 patients from Tunisia (of which 266 have LRRK2 parkinsonism) and 580 unaffected subjects. Motor, autonomic, and cognitive assessments in idiopathic Parkinson disease and LRRK2 patients were compared with regression models. The age-associated cumulative incidence of LRRK2 parkinsonism was also estimated using case-control and family-based designs. LRRK2 parkinsonism patients had slightly less gastrointestinal dysfunction and rapid eye movement sleep disorder. Overall, disease penetrance in LRRK2 carriers was 80% by 70 years but women become affected a median 5 years younger than men. Idiopathic Parkinson disease patients with younger age at diagnosis have slower disease progression. However, age at diagnoses does not predict progression in LRRK2 parkinsonism. LRRK2 p.G2019S mutation is a useful aid to diagnosis and modifiers of disease in LRRK2 parkinsonism may aid in developing therapeutic targets.     

Developmental expression and distribution of opioid receptors in zebrafish

Zebrafish is a novel experimental model that has been used in developmental studies as well as in the study of pathological processes involved in human diseases. It has been demonstrated that the endogenous opioid system is involved in developmental mechanisms. We have studied the relationship between the different embryonic stages and opioid receptor expression for the four known opioid receptors in zebrafish (mu, delta 1, delta 2 and kappa). The mu opioid receptor is detected at higher levels than the other opioid receptors before the midblastula transition and during the segmentation period. The delta duplicate 2 exhibits only one peak of expression at 21 h postfertilization (hpf), when the motor nervous system is forming. The kappa receptor is expressed at very low levels. In situ hybridization studies at 24 hpf show that the opioid receptors are widely distributed in zebrafish CNS and at 48 hpf their localization is detected in more defined structures. Our results support specific implications of the opioid receptors in developmental processes such as morphogenesis of the CNS, neurogenesis, neuroprotection and development of neuromuscular and digestive system. Pain-related alterations can be a consequence of changes in the endogenous opioid system during development, hence we provide important information that might help to solve pain-related pathological situations.     

Independent occurrence of I2020T mutation in the kinase domain of the leucine rich repeat kinase 2 gene in Japanese and German Parkinson's disease families

To understand the genetic origin of I2020T mutation in the kinase domain of leucine rich repeat kinase 2 (LRRK2), we investigated the original PARK8 Japanese family (Sagamihara family) and a German family (family 32), both of which were found to harbor I2020T as the causal mutation for autosomal dominant familial Parkinson's disease (PD). Microsatellite-haplotype analysis around the LRRK2 gene indicated that the mutation-carrying haplotypes of the two families were distinct from each other. This indicated that the I2020T mutation, an essential pathogenic mutation of PARK8-related PD, had occurred independently in the two PD families.     

MeCP2 expression and promoter methylation of cyclin D1 gene are associated with cyclin D1 expression in developing rat epididymal duct

Hypermethylation-dependent silencing of the gene is achieved by recruiting methyl-CpG binding proteins (MeCPs). Among the MeCPs, MeCP2 is the most abundantly and ubiquitously expressed in various types of cells. We first screened the distribution and expression pattern of MeCP2 in adult and developing rat tissues and found strong MeCP2 expression, albeit rather ubiquitously among normal tissues, in ganglion cells and intestinal epithelium in the small intestine, in Purkinje cells and neurons in the brain, in spermatogonia and in epithelial cells in the epididymal duct of the testis. We then assessed the expression and the methylation pattern of the promoter region of cyclin D1 by immunohistochemistry and sodium bisulfite mapping, and found that cyclin D1 expression in the epididymal duct decreased rapidly during rat development: strong in newborn rats and very weak or almost negative in 7-day-old rats. Mirroring the decrease of cyclin D1 expression, methylated cytosine at both CpG and non-CpG loci in the cyclin D1 promoter was frequently observed in the epididymal duct of 7-day-old rats but not in that of newborn rats. Interestingly, MeCP2 expression also increased concomitant with the increase of methylation. Cyclin D1 expression in the epididymal duct may be efficiently regulated by the epigenetic mechanism of the cooperative increase of MeCP2 expression and promoter methylation.     

Specific tissue expression and cellular localization of human apolipoprotein M as determined by in situ hybridization

Apolipoprotein M (apoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL), and in minor proportion in triglyceride-rich lipoprotein (TGRLP) and low-density lipoprotein (LDL). The gene coding for apoM has been detected in all mammal genomes. The function of apoM is unknown yet. In the present study, we demonstrated that apoM is exclusively expressed in a strong manner in adult liver and kidney, and is expressed weakly in fetal liver and kidney as detected with human multiple tissue expression array. Both immunohistochemical staining and apoM mRNA in situ hybridization demonstrated that apoM was exclusively expressed in hepatocytes in human liver and in tubular epithelial cells in human kidney. The present study helps to elucidate the pathophysiological functions of apoM in vivo.     

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.     

The influence of group III metabotropic glutamate receptor stimulation by (1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid on the parkinsonian-like akinesia and striatal proenkephalin and prodynorphin mRNA expression in rats

Group III metabotropic glutamate receptors (mGluRs) are widely distributed in the basal ganglia, especially on the terminals of pathways which seem to be overactive in Parkinson's disease. The aim of the present study was to determine whether (1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid (ACPT-1), an agonist of group III mGluRs, injected bilaterally into the globus pallidus (GP), striatum or substantia nigra pars reticulata (SNr), can attenuate the haloperidol-induced catalepsy in rats, and whether that effect was related to modulation of proenkephalin (PENK) or prodynorphin (PDYN) mRNA expression in the striatum. Administration of ACPT-1 (0.05-1.6 microg/0.5 microl/side) caused a dose-and-structure-dependent decrease in the haloperidol (0.5 mg/kg i.p. or 1.5 mg/kg s.c.)-induced catalepsy whose order was as follows: GP>striatum>SNr. ACPT-1, given alone to any of those structures, induced no catalepsy in rats. Haloperidol (3 x 1.5 mg/kg s.c.) significantly increased PENK mRNA expression in the striatum, while PDYN mRNA levels were not affected by that treatment. ACPT-1 (3 x 1.6 microg/0.5 microl/side) injected into the striatum significantly attenuated the haloperidol-increased PENK mRNA expression, whereas administration of that compound into the GP or SNr did not influence the haloperidol-increased striatal PENK mRNA levels. Our results demonstrate that stimulation of group III mGluRs in the striatum, GP or SNr exerts antiparkinsonian-like effects in rats. The anticataleptic effect of intrastriatally injected ACPT-1 seems to correlate with diminished striatal PENK mRNA expression. However, since the anticataleptic effect produced by intrapallidal and intranigral injection of ACPT-1 is not related to a simultaneous decrease in striatal PENK mRNA levels, it is likely that a decrease in enkephalin biosynthesis is not a necessary condition to obtain an antiparkinsonian effect.     

Habituation to repeated restraint stress is associated with lack of stress-induced c-fos expression in primary sensory processing areas of the rat brain

Rats repeatedly exposed to restraint show a reduced hypothalamic–pituitary–adrenal axis response upon restraint re-exposure. This hypothalamic–pituitary–adrenal axis response habituation to restraint does not generalize to other novel stressors and is associated with a decrease in stress-induced c-fos expression in a number of stress-reactive brain regions. We examined whether habituation to repeated restraint is also associated with adaptation of immediate early gene expression in brain regions that process and relay primary sensory information. These brain regions may not be expected to show gene expression adaptation to repeated restraint because of their necessary role in experience discrimination. Rats were divided into a repeated restraint group (five 1-hour daily restraint sessions) and an unstressed group (restraint naïve). On the sixth day rats from each group were either killed with no additional stress experience or at 15, 30 or 60 min during restraint. Immediate early gene expression (corticotrophin-releasing hormone heteronuclear RNA, c-fos mRNA, zif268 mRNA) was determined by in situ hybridization. A reduction in stress-induced hypothalamic–pituitary–adrenal axis hormone secretion (plasma corticosterone and adrenocorticotropic hormone) and immediate early gene expression levels in the paraventricular nucleus of the hypothalamus, the lateral septum and the orbital cortex was observed in repeated restraint as compared with restraint naïve animals. This reduction was already evident at 15 min of restraint. Unexpectedly, we also found in repeated restraint rats a reduction in restraint-induced c-fos expression in primary sensory-processing brain areas (primary somatosensory cortex, and ventroposteriomedial and dorsolateral geniculate nuclei of thalamus). The overall levels of hippocampal mineralocorticoid receptor heteronuclear RNA or glucocorticoid receptor mRNA were not decreased by repeated restraint, as may occur in response to severe chronic stress. We propose that repeated restraint leads to a systems-level adaptation whereby re-exposure to restraint elicits a rapid inhibitory modulation of primary sensory processing (i.e. sensory gating), thereby producing a widespread attenuation of the neural response to restraint.     

Decreased expression of Bmal2 in patients with Parkinson's disease

Bmal1 is one of the central regulators of the clock machinery. Recently, we examined the expression profile of Bmal1 in total leukocytes for a 12h duration during the evening, overnight, and the morning, in subjects with Parkinson's disease (PD) and healthy controls. The results indicate that the expression of Bmal1 is significantly lower in PD patients versus control subjects. However, it is still unclear whether other key regulators of the clock machinery, especially Bmal2, the paralog of Bmal1, are also expressed differently in PD. To address this issue, the expression profiles of Bmal2, Clock, and Dec1 were examined in the same samples using real-time RT-PCR assay. The results show a difference in the expression pattern of Bmal2, but not Clock and Dec1. The expression of Bmal2 is significantly lower in PD at 21:00 h (p=0.005) and 00:00 h (p=0.025). These results together with our previous findings suggest that the molecular clock in total leukocytes is disturbed in PD patients.     

Expression of NGF and trkA mRNA in song control and other regions of the zebra finch brain

We used in situ hybridization to measure the expression of NGF and trkA mRNA in the zebra finch brain at posthatch day 11 (P11), P25, and in adulthood. Expression of NGF and trkA was restricted to specific areas of the telencephalon in the adult zebra finch brain. Interestingly the expression of NGF and trkA overlapped in most brain regions, suggesting that NGF acts at sites close to cells that synthesize it. In song regions of adults, both NGF and trkA were clearly expressed in lMAN, HVC, and RA in males and in lMAN and RA in females. At P11, NGF and trkA mRNA were detected only in RA in both sexes. At P25, when sex differences in lMAN and RA begin to emerge, NGF mRNA was expressed in lMAN and RA in both sexes and trkA was detected at low levels in lMAN in both sexes. Whereas the level of trkA expression in RA of males at P25 was consistently low but detectable, expression in females was not detected. The volume of RA defined by NGF was significantly larger in males than females at P25. We also found a tendency for the intensity of NGF in RA to be higher in males than in females at P25, although the difference was not statistically significant. The presence of NGF and trkA mRNA in RA and lMAN at P25 suggests that they may participate in sexually dimorphic neural development of RA and lMAN, possibly by participating in sex-specific cell survival.     

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.     

Stable expression of the vesicular GABA transporter following photothrombotic infarct in rat brain

Before exocytotic release of the inhibitory neurotransmitter GABA, this amino acid has to be stored in synaptic vesicles. Accumulation of GABA in vesicles is achieved by a specific membrane-integrated transporter termed vesicular GABA transporter. This vesicular protein is mainly located at presynaptic terminals of GABAergic interneurons. In the present study we investigated the effects of focal ischemia on the expression of the vesicular GABA transporter. Vesicular GABA transporter mRNA and protein expression was examined after photothrombosis in different cortical and hippocampal brain regions of Wistar rats. In situ hybridization and quantitative real-time RT-PCR were performed to analyze vesicular GABA transporter mRNA. Both vesicular GABA transporter mRNA-stained perikarya and mRNA expression levels remained unaffected. Vesicular GABA transporter protein-containing synaptic terminals and somata were visualized by immunohistochemistry. The pattern of vesicular GABA transporter immunoreactivity as well as the protein expression level revealed by semiquantitative image analysis and by Western blot remained stable after stroke. The steady expression of vesicular GABA transporter mRNA and protein after photothrombosis indicates that the exocytotic release mechanism of GABA is not affected by ischemia.     

Differential expression of five members of the ADAM family in the developing chicken brain

ADAMs (a disintegrin and metalloprotease) are a family of trans-membrane multi-domain metalloproteases with multiple functions. So far, more than 35 ADAM family members have been identified from mammalian and nonmammalian sources. Although some functions of ADAMs have been elucidated, their expression patterns remain poorly investigated, especially during CNS development. Here, we cloned the open reading frames or full-length cDNAs of ADAM9, ADAM10, ADAM12, ADAM22 and ADAM23 from chicken embryonic brain, analyzed their evolutionary relationship, and mapped their expression in the embryonic chicken brain by in situ hybridization for the first time. In general, each of the five ADAMs shows a spatially restricted and temporally regulated expression profile. However, the types of tissues and cells, which express each of the five ADAMs, differ from each other. ADAM9 is predominantly expressed in the choroid plexus and in the ventricular layer. ADAM10 is expressed by developing blood vessels, oligodendrocytes, and subsets of neurons and brain nuclei. ADAM12 is expressed by very few brain nuclei, cerebellar Purkinje cells, restricted regions of the neuroepithelium, and some neurons in the deep tectal layers. ADAM22 expression is strong in some brain nuclei and in the pineal gland. ADAM23 is expressed by most gray matter regions and the choroid plexus. The differential expression patterns suggest that the five ADAMs play multiple and versatile roles during brain development.     

用RNA-RNA原位杂交技术研究Robo2在早期鸡胚发育中的表达

目的:探讨Robo2(roundabout homolog 2)在早期鸡胚发育中的表达。方法:利用分子生物学手段,构建Robo2/pSPT18重组质粒,并且制备地高辛标记的Robo2 RNA探针,进而通过RNA-RNA原位杂交技术检测Robo2在早期鸡胚发育中的表达。结果:原条发生前Robo2表达较弱,原条发生后主要表达在原条和神经板。体节期Robo2主要表达在脊索、神经管、体节和血岛部位。冰冻切片后观察到Robo2主要表达在外胚层,而在中胚层和内胚层只有部分表达。结论:阐明了Robo2在早期鸡胚发育中的表达,为进一步研究Robo2在正常生理和病理条件下的功能及作用机制奠定基础。     

Expression of reelin, its receptors and its intracellular signaling protein, Disabled1 in the canary brain: relationships with the song control system

Songbirds produce learned vocalizations that are controlled by a specialized network of neural structures, the song control system. Several nuclei in this song control system demonstrate a marked degree of adult seasonal plasticity. Nucleus volume varies seasonally based on changes in cell size or spacing, and in the case of nucleus HVC and area X on the incorporation of new neurons. Reelin, a large glycoprotein defective in reeler mice, is assumed to determine the final location of migrating neurons in the developing brain. In mammals, reelin is also expressed in the adult brain but its functions are less well characterized. We investigated the relationships between the expression of reelin and/or its receptors and the dramatic seasonal plasticity in the canary (Serinus canaria) brain. We detected a broad distribution of the reelin protein, its mRNA and the mRNAs encoding for the reelin receptors (VLDLR and ApoER2) as well as for its intracellular signaling protein, Disabled1. These different mRNAs and proteins did not display the same neuroanatomical distribution and were not clearly associated, in an exclusive manner, with telencephalic brain areas that incorporate new neurons in adulthood. Song control nuclei were associated with a particular specialized expression of reelin and its mRNA, with the reelin signal being either denser or lighter in the song nucleus than in the surrounding tissue. The density of reelin-immunoreactive structures did not seem to be affected by 4 weeks of treatment with exogenous testosterone. These observations do not provide conclusive evidence that reelin plays a prominent role in the positioning of new neurons in the adult canary brain but call for additional work on this protein analyzing its expression comparatively during development and in adulthood with a better temporal resolution at critical points in the reproductive cycle when brain plasticity is known to occur.     

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.     

Localization and glucocorticoid regulation of 11beta-hydroxysteroid dehydrogenase type 1 mRNA in the male mouse forebrain

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts the inactive 11-dehydrocorticosterone into the active glucocorticoid corticosterone. There is accumulating evidence indicating widespread expression of 11beta-HSD1 in the brain. However, there is little information about regulation of 11beta-HSD1 expression in this tissue. Using in situ hybridization involving use of 35S-labeled cRNA probe, we have studied the distribution of cells expressing 11beta-HSD1 mRNA in the male mouse forebrain as well as the effects of adrenalectomy (ADX) and acute administration of corticosterone (3 and 24 h) on 11beta-HSD1 mRNA levels. Cells expressing 11beta-HSD1 mRNA were mostly detected in the cerebral cortex, hippocampus, amygdala and medial preoptic area, with the highest expression in the cerebral cortex (retrosplenial granular area) and hippocampus (CA3 and granular layer of the gyrus dentatus). Seven days following ADX, 11beta-HSD mRNA levels were increased by 50% in the gyrus dentatus, by 100% in the CA3 area, and 105% in the cerebral cortex. Administration of corticosterone to ADX mice induced a significant decrease in mRNA, in both the hippocampus and cerebral cortex so that, at the 24 h time interval, the levels were similar to those observed in intact mice. These results clearly indicate that circulating corticosterone is downregulating the expression of 11beta-HSD1 mRNA in the two forebrain areas studied. This downregulation might contribute to maintain low intracellular corticosterone levels in central regions and then prevent the deleterious effects induced by high glucocorticoid levels.