The loss of function GBA1 c.231C > G mutation associated with Parkinson disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by bradykinesia, rigidity, and tremor. However, familial PD caused by single-gene mutations remain relatively rare. Herein, we described a Chinese family affected by PD, which associated with a missense heterozygous glucocerebrosidase 1 (GBA1) mutation (c.231C > G). Clinical data on the proband and her family members were collected. Brain MRI showed no difference between affected and unaffected family members. Whole-exome sequencing (WES) was performed to identify the pathogenic mutation. WES revealed that the proband carried a missense mutation (c.231C > G) in GBA1 gene, which was considered to be associated with PD in this family. Sanger sequencing and co-segregation analyses were used to validate the mutation. Bioinformatics analysis indicated that the mutation was predicted to be damaging. In vitro functional analyses were performed to investigated the mutant gene. A decrease in mRNA and protein expression was observed in HEK293T cells transfected with mutant plasmids. The GBA1 c.231C > G mutation caused a decreased GBA1 concentration and enzyme activity. In conclusion, a loss of function mutation (c.231C > G) in GBA1 was identified in a Chinese PD family and was confirmed to be pathogenic through functional studies. This study help the family members understand the disease progression and provide a new example for studying the pathogenesis of GBA1-associated Parkinson disease.

Cocaine Directly Inhibits α6-Containing Nicotinic Acetylcholine Receptors in Human SH-EP1 Cells and Mouse VTA DA Neurons.

Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits α6N/α3Cß2ß3-nAChR (α6*-nAChRs) function. Human α6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (VTA) were used as a model of presynaptic α6*-nAChR activation since this method preserves terminal boutons. Patch-clamp recordings in whole-cell configuration were used to measure α6*-nAChR function as well as evaluate the effects of cocaine. In SH-EP1 cells containing heterologously expressed human α6*-nAChRs, cocaine inhibits nicotine-induced inward currents in a concentration-dependent manner with an IC50 value of 30 µM. Interestingly, in the presence of 30 µM cocaine, the maximal current response of the nicotine concentration-response curve is reduced without changing nicotine's EC50 value, suggesting a noncompetitive mechanism. Furthermore, analysis of whole-cell current kinetics demonstrated that cocaine slows nAChR channel activation but accelerates whole-cell current decay time. Our findings demonstrate that cocaine-induced inhibition occurs solely with bath application, but not during intracellular administration, and this inhibition is not use-dependent. Additionally, in Xenopus oocytes, cocaine inhibits both α6N/α3Cß2ß3-nAChRs and α6M211L/α3ICß2ß3-nCAhRs similarly, suggesting that cocaine may not act on the α3 transmembrane domain of chimeric α6N/α3Cß2ß3-nAChR. In mechanically isolated VTA DA neurons, cocaine abolishes α6*-nAChR-mediated enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs). Collectively, these studies provide the first evidence that cocaine directly inhibits the function of both heterologously and naturally expressed α6*-nAChRs. These findings suggest that α6*-nAChRs may provide a novel pharmacological target mediating the effects of cocaine and may underlie a novel mechanism of cocaine reward and dependence.

Different Phenotypes at Onset in Neuromyelitis Optica Spectrum Disorder Patients with Aquaporin-4 Autoimmunity.

BACKGROUND: Although rare, brain abnormalities without optic neuritis (ON) or transverse myelitis (TM) diagnosed with neuromyelitis optica spectrum disorder (NMOSD) have been reported in patients positive for the aquaporin-4 (AQP4) antibody. OBJECTIVE: To analyze demographic and clinical differences among NMOSD patients without ON or TM, those with either ON or TM, and patients with simultaneous ON and TM at disease onset. METHODS: In this retrospective study, patients who were positive for the AQP4 antibody, as detected using a cell-based assay, at the Second Affiliated Hospital of Guangzhou Medical University in China were recruited. Demographic and clinical data were obtained from each patient's medical record. RESULTS: A total of 292 patients were included in this study and were divided into four subgroups based on their initial manifestations: (i) NMOSD without ON or TM (NMOSD-ON-TM-, n = 70); (ii) NMOSD with ON (NMOSD-ON+, n = 95); (iii) NMOSD with TM (NMOSD-TM+, n = 116); and (iv) simultaneous ON and TM [neuromyelitis optica (NMO), n = 11]. We found that age at onset was lower in the NMOSD-ON-TM- group than that in the other groups. The interval from the first episode to relapse was shorter in the NMOSD-ON-TM- group than that in NMOSD-TM+ group. Cerebral spinal fluid white cell counts and protein levels were significantly higher in the NMOSD-ON-TM- group than those in the other groups. Lower Expanded Disability Status Scale scores were observed in the NMOSD-ON-TM- group. Brain abnormalities, including in area postrema and hemisphere lesions, were more frequent in the NMOSD-ON-TM- group. Kaplan-Meier analysis showed that patients in the NMOSD-ON-TM- group experienced earlier relapse than those in other groups. Conversion to NMO in the NMOSD-ON+ group was greater than that in the other groups. Only 14 patients (4.8%, 14/292) had pure brain abnormalities, of which 12 had disease duration of several more years and 8 (57.1%) experienced relapses. CONCLUSION: NMOSD patients with different initial manifestations present with significant differences in clinical features during follow-up. Patients with long-term AQP4 autoimmunity in the brain in the absence of ON or TM are not common.

Brain cannabinoid receptor 2: expression, function and modulation.

Cannabis sativa (marijuana) is a fibrous flowering plant that produces an abundant variety of molecules, some with psychoactive effects. At least 4% of the world's adult population uses cannabis annually, making it one of the most frequently used illicit drugs in the world. The psychoactive effects of cannabis are mediated primarily through cannabinoid receptor (CBR) subtypes. The prevailing view is that CB1Rs are mainly expressed in the central neurons, whereas CB2Rs are predominantly expressed in peripheral immune cells. However, this traditional view has been challenged by emerging strong evidence that shows CB2Rs are moderately expressed and function in specific brain areas. New evidence has demonstrated that brain CB2Rs modulate animal drug-seeking behaviors, suggesting that these receptors may exist in brain regions that regulate drug addiction. Recently, we further confirmed that functional CB2Rs are expressed in mouse ventral tegmental area (VTA) dopamine (DA) neurons and that the activation of VTA CB2Rs reduces neuronal excitability and cocaine-seeking behavior. In addition, CB2R-mediated modulation of hippocampal CA3 neuronal excitability and network synchronization has been reported. Here, we briefly summarize recent lines of evidence showing how CB2Rs modulate function and pathophysiology in the CNS.

Icariin has synergistic effects with methylprednisolone to ameliorate EAE via modulating HPA function, promoting anti-inflammatory and anti-apoptotic effects.

BACKGROUND: High-dose methylprednisolone (MP) is a clinically recommended therapeutic regimen for Multiple Sclerosis (MS), whereas some dreadful complications induced by it remain inevitable. Studies implied that estrogens might play neuroprotective and anti-inflammatory roles in EAE and MS and promote glucocorticoid efficacy. Icariin (ICA), a primary active component of Epimedium extracts, also possesses neuroprotective and estrogen-like effects with less adverse complication than estrogen. However, rare study focuses ICA's effects on MS or EAE. OBJECTIVE: Our purpose is to determine whether ICA has synergistic effects with MP in treating EAE and explore the possible mechanisms. METHODS: C57BL/6 EAE mice were received different dose of ICA combined with MP and single MP treatment. Then, the clinical scores and serum Interleukin-17 (IL-17), Corticosterone (CORT), Adrenocorticotropic Hormone (ACTH) concentrations were analyzed. Western blot and Flow Cytometry were used to investigate the expression of glucocorticoid receptor (GR) and cell apoptosis. RESULTS: ICA has cooperative effects with MP in decreasing serum IL-17 and CORT concentrations, up-regulating the expression of GR in cerebral white matter and attenuating the cell apoptosis in spinal cord, especially high-dose ICA combined with MP. CONCLUSION: ICA has synergistic effects with MP to ameliorate EAE via modulating hypothalamic-pituitary-adrenal (HPA) function, promoting anti-inflammatory and anti-apoptotic effects. ICA could be considered as a promising therapeutic option for MS.

Identification and characterization of a novel splice-site mutation in the Wilson disease gene.

This study aimed to identify aberrant transcripts of the new splice-site mutation c.3244-2A>C in the Wilson disease (WD) gene (ATPase, Cu++ transporting, beta polypeptide, ATP7B) and discuss its genotype and clinical phenotype. DNA and RNA were extracted from peripheral blood lymphocytes, amplified by polymerase chain reaction (PCR) and nested reverse transcription PCR (RT-nested PCR) to characterize the aberrant transcripts. RT-nested PCR product sequencing comparison showed that c.3244-2A>C splice-site mutation caused aberrant transcripts and formatted a new splice acceptor. Patient carrying the splice-site mutation c.3244-2A>C presented early onset age, severe clinical manifestations, and poor prognosis. WD patients with the splice-site mutation show severe clinical manifestations, indicating that aberrant transcripts have important implications for WD phenotype.

Novel loss-of-function PRRT2 mutation causes paroxysmal kinesigenic dyskinesia in a Han Chinese family.

BACKGROUND: Mutations in proline-rich transmembrane protein 2 (PRRT2) are a cause of paroxysmal kinesigenic dyskinesia (PKD). In this study, we investigated the PRRT2 gene mutation in a Chinese Han family with PKD and study the pathogenesis of the mutation with PRRT2 gene. METHODS: Peripheral venous blood was taken from the family members. Sanger sequencing was used for novel mutation sequencing. For the pathogenesis with the novel mutation was analyzed by bioinformatics, real-time PCR, subcellular localization and Western blot. RESULTS: The Sanger sequencing showed a novel mutation, c.186-187delGC, a deletion mutation, in exon 2 of the PRRT2 gene, the frameshift mutation generated a truncated protein that was stably expressed in transfected Human embryonic kidney (HEK) 293 cells. A subcellular localization assay in COS-7 cells with GFP-tagged protein showed nuclear localization for the mutant protein while the wild-type protein was localized in membranes. Co-transfection of HEK293 cells with wild-type and mutant expression plasmids cells did not influence mRNA or protein expression from the wild-type plasmid. CONCLUSIONS: Our findings demonstrated that the c.186-187delGC mutation resulted in a truncated protein from the PRRT2 gene to involve in PKD pathogenesis with haploinsufficiency. The results extend the mutation spectrum of the PRRT2 gene and provide a new example for studying the pathogenesis of the mutated PRRT2 gene.

Mutation analysis of 73 southern Chinese Wilson's disease patients: identification of 10 novel mutations and its clinical correlation.

This study was designed to investigate the molecular basis and the correlation between genotype and phenotype in the southern Chinese patients with Wilson's disease (WD). Genotypes of the ATP7B gene in 73 WD patients were examined by denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. A total of 38 different disease-causing mutations were identified, including 10 novel mutations: missense mutations (p.Gln707Arg, p.Cys1079Phe, p.Gly1149Glu, p.Ser855Tyr, p.Ala874Pro and p.Ser921Arg), nonsense mutation (p.Arg1228Stop), splice-site mutations (2121+3A>T and 3244-2A>G) and frameshift mutation (1875_1876insAATT). We found that a pair of siblings carried the same genotype but different clinical type, and two patients were found to have three mutations. In addition, we compared the clinical data for p.Arg778Leu homozygotes and compound heterozygotes. Our research has enriched the mutation spectrum of the ATP7B gene in the Chinese population and can serve as the basis for genetic counseling and clinical/prenatal diagnosis to prevent WD in China.

Super-high-dose methylprednisolone does not improve efficacy or induce glucocorticoid resistance in experimental allergic encephalomyelitis.

OBJECTIVE: To investigate whether a super-high dose (SHD) of methylprednisolone (MP) improves its efficacy or induces glucocorticoid (GC) resistance, and to explore the potential mechanisms of GC resistance in experimental allergic encephalomyelitis (EAE). METHODS: The therapeutic effects of SHD and low-dose MP were evaluated in EAE by analyzing clinical scores, pathological changes and cytokine production. Immunohistochemistry and RT-PCR were used to investigate the expression of GC receptor (GR) isoforms and splicing factor SRp30c. RESULTS: Both MP doses had similar therapeutic effects. The ratio of GRα to GRß was positively correlated with clinical score changes. However, there was no difference in the GRα/GRß ratio between SHD and low-dose MP groups. SRp30c mRNA was correlated with GRß expression. CONCLUSION: This study indicates that the GRα/GRß ratio is associated with GC sensitivity, and SRp30c may play an important role in promoting alternative splicing of GR pre-mRNA to generate GRß in EAE rats. Compared with low-dose MP, SHD MP does not improve efficacy or induce GC resistance.

[Relationship between angiotensin converting enzyme gene and ischemic stroke].

OBJECTIVE: To study relationship between angiotensin converting enzyme (ACE) gene and ischemic stroke (IS). METHODS: (1)Four hundred and fifty-four patients and 334 controls were recruited in our study, their I/D polymorphisms of ACE gene were detected by polymerase chain reaction (PCR) and denaturing high performance liquid chromatogram, and their risk factors of IS were recorded at the same time. (2)In addition, 29 stroke-prone spontaneously hypertensive rats (SHR-SP) and 40 Sprague-Dawley (SD) rats were enrolled, and hypoxia- apnoea animal models and simple apnoea animal models were used at the same time. Their plasma angiotensin II (Ang II) levels were determined. RESULTS: The frequencies of DD, ID and II genotype in IS patients were 22.5%, 43.4% and 34.1%, respectively, and 17.4%, 45.5% and 37.1%, respectively in controls. DD genotype was associated with large artery arteriosclerosis (LAA). Plasma Ang II level in SHR-SP group was (164.49+/-34.58) ng/L, and it was higher than that in control group [(150.92+/-24.92)ng/L] with no significant difference (P>0.05). Ang II levels in apnoea and hypoxia-apnoea group were (382.84+/-62.75) ng/L and (295.90+/-55.07) ng/L, respectively, and they were significantly higher than that in control group (all P<0.01). The relative risks of DD genotype and D alleles in IS patients with smoking, alcohol abuse, or with diabetes mellitus were higher than those in controls, but II genotype and I alleles were lower than those in controls. CONCLUSION: DD genotype is a risk factor for IS, Ang II takes part in the course of hypoxia-stress, and it is correlated with smoking, alcohol abuse and diabetes mellitus in the pathogenesis of IS.

[Relationship between methylenetrahydrofolate reductase gene and ischemic stroke].

OBJECTIVE: To study the relationship between methylenetrahydrofolate reductase (MTHFR) gene and ischemic stroke. METHODS: Four hundred and fifty four ischemic stroke patients were enrolled in the study. They were divided into large artery atherosclerosis (LAA), cardioembolism (CE), small artery occlusion (SAA), stroke of other determined etiology (SOE) and stroke of undetermined etiology (SUE) according to TOAST (Trail of ORG 10172 in Acute Stroke Treatment) criteria; and they were divided into mild, moderate and severe types ischemic stroke according to their scores of neurologic impairment. Three hundred and thirty four subjects, in whom hypertension, coronary heart disease, cerebral vascular disease, diabetes mellitus, cancer, renal failure etc. were excluded, served as controls in the study. Their C677T polymorphisms of MTHFR gene were determined with polymerase chain reaction (PCR) and denaturing high performance liquid chromatography (DHPLC), and their risk factors of ischemic stroke were recorded at the same time. RESULTS: The frequencies of CC, CT and TT genotype in ischemic stroke were 51.8%, 40.5% and 7.7%, respectively, and they were 56.9%, 38.3% and 4.8% respectively in controls. TT genotype and T allele were associated with LAA and CE, moderate type and severe type of ischemic stroke. The frequencies of TT genotype and T allele in ischemic stroke patients were significantly higher in those with smoking, alcohol abuse or diabetes mellitus than those in controls (all P<0.10), but CC genotype and C allele were significantly lower in them than those in controls (all P<0.05). On the other hand, all of genotypes and alleles in ischemic stroke patients with no history of smoking, alcohol abuse or diabetes mellitus were not significantly different from those in controls. CONCLUSION: TT genotype and T allele are risk factors for ischemic stroke. It exists interactions between smoking, alcohol abuse, diabetes mellitus and MTHFR gene in the pathogenesis of ischemic stroke.

[Detecting the polymorphism of methylenetetrahydrofolate reductase gene by denaturing high performance liquid chromatography].

OBJECTIVE: To establish a method for detecting the polymorphism of methylenetetrahydrofolate reductase gene (MTHFR). METHODS: The MTHFR was amplified, and the amplified products were detected by denaturing high performance liquid chromatography (DHPLC), and the amplified MTHFR was confirmed by sequencing and restriction enzyme digesting. RESULTS: A total of 334 individuals of Han people in southern China were recruited in our study, and their polymorphisms of MTHFR were detected. The accurate rate of the DHPLC method, that was very sensitive with 100% detection rate available, was over 99%. The frequencies of CC, CT and TT genotypes were 56.9%, 38.3% and 4.8% individually, and the frequencies of T and C alleles were 23.95% and 76.05% individually. CONCLUSION: The DHPLC method can detect polymorphism of MTHFR rapidly, effectively and economically. And there is the existence of different MTHFR polymorphisms in area and race.

[Transplantation of 3H-thymidine-labeled human bone marrow-derived mesenchymal stem cells in mdx mice].

OBJECTIVE: To investigate the feasibility of using human bone marrow-derived mesenchymal stem cells (hBM- MSCs) for repairing the skeletal muscle sarcolemma lesions in mdx mice and characterize the distribution of the transplanted hBM-MSCs. METHODS: Eighteen 8- to 10-week-old immunosuppressed mdx mice received transplantation with 1x10(7) of hBM-MSCs (the fifth passage) with 3H-thymidine (3H-TdR) labeling by injection of the cells into the tail vein. The mice were killed at 24 h, 48 h, 2 weeks, and 1, 2 and 4 months after the transplantation, respectively, to measure the radioactivity in the tissues and organs. Dystrophin expression on the sarcolemma was detected by immunofluorescence analysis. RESULTS: One month after transplantation, the mice with cell transplantation showed greater radioactivity in most of the tissues and organs than the control mice, especially in the bone marrow, liver and spleen. The radioactivity was then gradually lowered but in the skeletal muscle, the radioactivity increased progressively since 2 weeks after transplantation, reaching the peak of 27.65+/-3.53 Bq/mg at 1 month. Compared with that in the control mice, the radioactivity in the bone marrow and skeletal muscle was persistently higher in mice with cell transplantation 1 month after transplantation. No dystrophin-positive cells were found in the mdx mice at 2 weeks but detected at 1 month. The percentage of dystrophin-positive fibers in each section ranged from a 6.6% (1 month) to 8.9% (4 months). CONCLUSIONS: hBM-MSCs engrafted in immunosuppressed mdx mice may differentiate into skeletal muscle cells to repair the pathological lesion of the skeletal muscle sarcolemma. The hBM-MSCs reside mainly in the bone marrow, liver and spleen in the early stage following transplantation, homing into the bone marrow and skeletal muscle later.

[Application of the Bgl II-Bln I dosage test to gene diagnosis of facioscapulohumeral muscular dystrophy 1A gene].

OBJECTIVE: To increase the sensitivity and specificity of conventional gene diagnosis of facioscapulohumeral muscular dystrophy 1A(FSHD1A) by analyzing the distribution of translocation between chromosomes 4q35 and 10q26 in suspected FSHD cases. METHODS: The Bgl II- Bln I dosage test was performed to detect translocation between chromosomes 4q35 and 10q26 in 7 cases of presymptomatic FSHD patients showing positive result in gene diagnosis and 5 cases of sporadic FSHD patients showing negative result in gene diagnosis. DNA samples were digested with Bgl II and Bln I, followed by agrose gel electrophoresis. Probe p13E-11 was labeled with alpha-(32) P dCTP, followed by Southern hybridization. Then the ratio between the chromosomes 4 and 10 derived signal intensities was judged and hence was made known whether there was interchromosomal translocation between chromosomes 4 and 10. RESULTS: The Bgl II-Bln I dosage test revealed a translocation from chromosome 4q35 to 10q26 in one presymptomatic FSHD patient, thus indicating the result of gene diagnosis for her might be false positive. There was one translocation from chromosome 10q26 to 4q35 detected in one sporadic FSHD patient, indicating the result of gene diagnosis for her might be false negative. There were no translocations between chromosomes 4 and 10 in the other 10 cases. CONCLUSION: The Bgl II-Bln I dosage test can detect the translocation between chromosomes 4q35 and 10q26. It can improve the accuracy of the conventional method for gene diagnosis of FSHD1A.

[Translocation between chromosomes 4q35 and 10q26 in facioscapulohumeral muscular dystrophy].

OBJECTIVE: To investigate the distribution of translocation between chromosomes 4q35 and 10q26 in facioscapulohumeral muscular dystrophy (FSHD) patients and normal individuals. METHODS: The Bgl II-Bln I dosage test was performed to study the distribution of translocation between chromosomes 4q35 and 10q26 in 70 cases of FSHD patients, 55 cases of kindred with FSHD, and 52 cases of normal controls. RESULTS: (1) In normal individuals, the frequency of translocation between chromosomes 4q35 and 10q26 is 19.23%. The frequency of translocation from chromosome 4q35 to 10q26 and that from chromosome 10q26 to 4q35 are both 9.62%. (2) In the FSHD patients, the frequency of translocation between chromosomes 4q35 and 10q26 is 18.57%. The frequency of translocation from chromosome 4q35 to 10q26 and that from chromosome 10q26 to 4q35 are 12.86% and 5.71% respectively. CONCLUSIONS: The translocation between chromosomes 4q35 and 10q26 was frequently observed in both normal Chinese population and FSHD patients. No significant difference was observed between them.