The atypical protein kinase C (aPKC) in complex with PAR3 and PAR6 is required for axon-dendrite differentiation, but the upstream factors responsible for regulating its activity are largely unknown. Here, we report that in cultured hippocampal neurons aPKC is directly regulated by Dishevelled (Dvl), an immediate downstream effector of Wnt. We found that downregulation of Dvl abrogated axon differentiation, whereas Dvl overexpression resulted in multiple axon formation. Interestingly, Dvl was associated with aPKC and this interaction resulted in aPKC stabilization and activation. Furthermore, the multiple axon formation resulting from Dvl overexpression was attenuated by expressing a dominant-negative aPKC in these neurons and overexpression of aPKC prevented the loss of axon caused by Dvl downregulation. Finally, Wnt5a, a noncanonical Wnt, activated aPKC and promoted axon differentiation. The Wnt5a effect on axon differentiation was attenuated by downregulating Dvl or inhibiting aPKC. Thus, Dvl-aPKC interaction can promote axon differentiation mediated by the PAR3-PAR6-aPKC complex.

Objective-Investigate L-type calcium channel blockers of the dihydropyridine class for association with Parkinson's disease because these drugs traverse the blood brain barrier, are potentially neuroprotective, and have previously been evaluated for impact on PD risk.Methods-We identified 1,931 patients with a first time diagnosis for PD between 2001 and 2006 as reported in the Danish national hospital/outpatient database and density matched them by birth year and sex to 9,651 controls from the population register. Index date for cases and their corresponding controls was advanced to date of first recorded prescription for anti-Parkinson drugs, if prior to first PD diagnosis in the hospital records. Prescriptions were determined from the national pharmacy database. In our primary analyses, we excluded all calcium channel blockers prescriptions 2-years before index date/PD diagnosis.Results-Employing logistic regression analysis adjusting for age, sex, diagnosis of chronic pulmonary obstructive disorder, and Charlson co-morbidity score we found that subjects prescribed centrally acting calcium channel blockers (excludes amlodipine) between 1995 and two years prior to the index date were less likely to develop Parkinson's disease (Odds Ratio 0.73; 95% Confidence Interval 0.54-0.97); this 27% risk reduction did not differ with length or intensity of use. Risk estimates were close to null for the peripherally acting drug amlodipine and for other antihypertensive medications.Interpretation-Our data suggest a potential neuroprotective role for centrally acting L-type calcium channel blockers of the dihydropyridine class in PD that should be further investigated in studies that can distinguish between types of L-Type channel blockers.

Reprogramming of somatic cells in the enucleated egg made Dolly, the sheep, the first successfully cloned mammal in 1996. However, the mechanism of sheep somatic cell reprogramming has not yet been addressed. Moreover, sheep embryonic stem (ES) cells are still not available, which limits the generation of precise gene-modified sheep. In this study, we report that sheep somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells using defined factors (Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, SV40 large T and hTERT). Our observations indicated that somatic cells from sheep are more difficult to reprogram than somatic cells from other species, in which iPS cells have been reported. We demonstrated that sheep iPS cells express ES cell markers, including alkaline phosphatase, Oct4, Nanog, Sox2, Rex1, stage-specific embryonic antigen-1, TRA-1-60, TRA-1-81 and E-cadherin. Sheep iPS cells exhibited normal karyotypes and were able to differentiate into all three germ layers both in vitro and in teratomas. Our study may help to reveal the mechanism of somatic cell reprogramming in sheep and provide a platform to explore the culture conditions for sheep ES cells. Moreover, sheep iPS cells may be directly used to generate precise gene-modified sheep.

The mammalian nicotinamide-adenine dinucleotide (NAD)-dependent deacetylase Sirt1 impacts different processes involved in the maintenance of brain integrity and in the pathogenic pathways associated with several neurodegenerative disorders, including Alzheimer's disease. Here we used human Sirt1 transgenic mice to demonstrate that neuron-specific Sirt1 overexpression promoted neurite outgrowth and improved cell viability under normal and nutrient-limiting conditions in primary culture systems and that Sirt1-overexpressing neurons exhibited higher tolerance to cell death or degeneration induced by amyloid-β1-42 oligomers. Coincidentally, we found that enhanced Sirt1 expression in neurons downregulated the mammalian target of rapamycin (mTOR) protein levels and its phosphorylation without changes in its mRNA levels, which was accompanied by concomitant inhibition of the mTOR downstream signaling activity as revealed by decreased p70S6 kinase (p70S6K) phosphorylation at Thr389. Consistently with this, using a Sirt1 siRNA transfection approach, we observed that reduction of endogenous mouse Sirt1 led to increased levels of mTOR and phosphorylation of itself and p70S6K as well as impaired cell survival and neurite outgrowth in wild-type mouse primary neurons, corroborating a suppressing effect of mTOR by Sirt1. Correspondingly, the mTOR inhibitor rapamycin markedly improved neuronal cell survival in response to nutrient deprivation and significantly enhanced neurite outgrowth in wild-type mouse neurons. The protective effect of rapamycin was extended to neurons even with Sirt1 siRNA knockdown that displayed developmental abnormalities compared with siRNA control-treated cells. Collectively, our findings suggest that Sirt1 may act to promote growth and survival of neurons in the central nervous system via its negative modulation of mTOR signaling.

Agrin induces, whereas acetylcholine (ACh) disperses, ACh receptor (AChR) clusters during neuromuscular synaptogenesis. Such counteractive interaction leads to eventual dispersal of nonsynaptic AChR-rich sites and formation of receptor clusters at the postjunctional membrane. However, the underlying mechanisms are not well understood. Here we show that calpain, a calcium-dependent protease, is activated by the cholinergic stimulation and is required for induced dispersion of AChR clusters. Interestingly, the AChR-associated protein rapsyn interacted with calpain in an agrin-dependent manner, and this interaction inhibited the protease activity of calpain. Disrupting the endogenous rapsyn/calpain interaction enhanced CCh-induced dispersion of AChR clusters. Moreover, the loss of AChR clusters in agrin mutant mice was partially rescued by the inhibition of calpain via overexpressing calpastatin, an endogenous calpain inhibitor, or injecting calpeptin, a cell-permeable calpain inhibitor. These results demonstrate that calpain participates in ACh-induced dispersion of AChR clusters, and rapsyn stabilizes AChR clusters by suppressing calpain activity.

The dynamic interaction between positive and negative signals is necessary for remodeling of postsynaptic structures at the neuromuscular junction. Here we report that Wnt3a negatively regulates acetylcholine receptor

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Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson's disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopaminelike neurons (pNSC-DAn) in culture. Here, we showed that after the striatal transplantation of pNSC-DAn, (i) pNSC-DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC-DAn (and not from injured original cells). Thus, pNSC-DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proofof-concept for human clinical translation.is a chronic progressive neurodegenerative disorder characterized by the specific loss of dopaminergic neurons in the substantia nigra pars compacta and their projecting axons, resulting in loss of dopamine (DA) release in the striatum (1). During the last two decades, cell-replacement therapy has proven, at least experimentally, to be a potential treatment for PD patients (2-7) and in animal models (8-15). The basic principle of cell therapy is to restore the DA release by transplanting new DA-like cells. Until recently, obtaining enough transplantable cells was a major bottleneck in the practicability of cell therapy for PD. One possible source is embryonic stem cells (ESCs), which can develop infinitely into self-renewable pluripotent cells with the potential to generate any type of cell, including DA neurons (DAns) (16,17).Recently, several groups including us have introduced rapid and efficient ways to generate primitive neural stem cells (pNSCs) from human ESCs using small-molecule inhibitors under chemically defined conditions (12,18,19). These cells are nonpolarized neuroepithelia and retain plasticity upon treatment with neuronal developmental morphogens. Importantly, pNSCs differentiate into DAns (pNSC-DAn) with high efficiency (∼65%) after patterning by sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) in vitro, providing an immediate and renewable source of DAns for PD treatment. Importantly, the striatal transplantation of human ESC-derived DA-like neurons, including pNSC-DAn, are able to relieve the motor defects in a PD rat model (11-13, 15, 19-23) Regarding question 1, a recent study using nafion-coated carbon fiber electrodes (CFEs) reported that the amperometric current is rescued in vivo by ESC (pNSC-DAn-like) therapy (19). Both norepinephrine (NE) and serotonin are present in the striatum (24, 25). However, CFE am...