Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-μ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS

CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking

Protein export from the endoplasmic reticulum (ER) is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII)-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking

Gender gap in parental leave intentions: Evidence from 37 countries

This is the final version. Available from Wiley via the DOI in this record. Despite global commitments and efforts, a gender-based division of paid and unpaid work persists. To identify how psychological factors, national policies, and the broader sociocultural context contribute to this inequality, we assessed parental-leave intentions in young adults (18–30years old) planning to have children (N = 13,942; 8,880 identified as women; 5,062 identified as men) across 37 countries that varied in parental-leave policies and societal gender equality. In all countries, women intended to take longer leave than men. National parental-leave policies and women’s political representation partially explained cross-national variations in the gender gap. Gender gaps in leave intentions were paradoxically larger in countries with more gender-egalitarian parental-leave policies (i.e., longer leave available to both fathers and mothers). Interestingly, this cross-national variation in the gender gap was driven by cross-national variations in women’s (rather than men’s) leave intentions. Financially generous leave and gender-egalitarian policies (linked to men’s higher uptake in prior research) were not associated with leave intentions in men. Rather, men’s leave intentions were related to their individual gender attitudes. Leave intentions were inversely related to career ambitions. The potential for existing policies to foster gender equality in paid and unpaid work is discussed.SSHRC Insight Development GrantSSHRC Insight GrantEconomic and Social Research CouncilState Research AgencyGuangdong 13th-five Philosophy and Social Science Planning ProjectNational Natural Science Foundation of ChinaSwiss National Science FoundationSwiss National Science FoundationCenter for Social Conflict and Cohesion StudiesCenter for Intercultural and Indigenous ResearchSSHRC Postdoctoral FellowshipSlovak Research and Development AgencySwiss National Science FoundationCanada Research ChairsSocial Sciences and Humanities Research Council of CanadaOntario Ministry of Research and InnovationHSE University, RFFaculty of Arts, Masaryk Universit

Heart Sound Analysis Using MFCC and Time Frequency Distribution

This paper presents heart sound analysis method based on Time-Frequency Distribution (TFD) analysis and Mel Frequency Cepstrum Coefficient (MFCC). TFD represents the heart sound in term of time and frequency simultaneously which while the MFCC defines a signal in term of frequency coefficient corresponding to the Mel filter scale. There are 100 normal data and 100 data with disease obtained from the hospital which consists of various kinds of problems including mitral regurgitation and stenosis, tricuspid regurgitation and stenosis, ventricular septal defect and other structural related disease. B-Distribution is chosen from a number of time-frequency analysis methods due its capability to represent the signal in the most efficient way in term of noise and cross term reduction. The advantage of MFCC is that it is good in error reduction and able to produce a robust feature when the signal is affected by noise. SVD/PCA technique is used to extract the important features out of the B-Distribution representation. The coefficient obtained from SVD-PCA and MFCC is later used for classification Artificial Neural Network. The results show that the system is able to produce the accuracy up to 90.0% using the TFD and 80.0% using the MFCC

Investigation of the functional roles of the MELAL and GQXXRXG motifs of the human noradrenaline transporter using cysteine mutants

The study examines the roles of the highly conserved MELAL and GQXXRXG motifs, located in the second transmembrane domain and the first intracellular loop of the human noradrenaline transporter (hNET). We have previously shown that this region does not directly participate in the NET substrate translocation pathway [Sucic, S., and Bryan-Lluka, L.J., 2005. Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis. J. Neurochem. 94, 1620-1630.1, while the current report focuses on the importance of this region in determining other functional properties of the hNET. Mutation to cysteine of the wild-type residues was carried out by site-directed mutagenesis of hNET cDNA. The wild-type and mutant hNETs were expressed in transiently transfected COS-7 cells and the effects of these mutations were pharmacologically examined. The results indicate that the GQXXRXG motif is important for the binding of cocaine, but not antidepressants. The hN120C mutant caused an I 1-fold increase in the binding affinity of cocaine, compared to the wild-type hNET, while hQ118C, hY119C, hR121C and hE122C showed smaller increases. Interestingly, the apparent affinities of cocaine for some of these mutants were either decreased or unchanged, contrasting with the effects observed from the binding studies. The hE113C mutant in the MELAL motif caused very marked (over 400-fold) reductions in the binding affinities of substrates, but had no effects on the binding affinities of cocaine or antidepressants. Overall, the MELAL and GQXXRXG motifs are important determinants of NET cell surface expression and substrate and inhibitor binding. The results further suggest that the binding sites for substrates, cocaine and antidepressants on the NET are distinct but overlapping. (c) 2006 Elsevier B.V All rights reserved