Notch signaling plays crucial roles for cellular differentiation during development through gamma-secretase-dependent intramembrane proteolysis followed by transcription of target genes. Although recent studies implicate that Notch regulates synaptic plasticity or cognitive performance, the molecular mechanism how Notch works in mature neurons remains uncertain. Here we demonstrate that a novel Notch signaling is involved in expression of synaptic proteins in postmitotic neurons. Levels of several synaptic vesicle proteins including synaptophysin 1 and VGLUT1 were increased when neurons were cocultured with Notch ligands-expressing NIH3T3 cells. Neuron-specific deletion of Notch genes decreased these proteins, suggesting that Notch signaling maintains the expression of synaptic vesicle proteins in a cell-autonomous manner. Unexpectedly, cGMP-dependent protein kinase (PKG) inhibitor, but not gamma-secretase inhibitor, abolished the elevation of synaptic vesicle proteins, suggesting that generation of Notch intracellular domain is dispensable for this function. These data uncover a ligand-dependent, but gamma-secretase-independent, non-canonical Notch signaling involved in presynaptic protein expression in postmitotic neurons.
        
Title: Conclusive identification of the oxybutynin-hydrolyzing enzyme in human liver Sato Y, Miyashita A, Iwatsubo T, Usui T Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 40:902, 2012 : PubMed
The aim of this study was to conclusively determine the enzyme responsible for the hydrolysis of oxybutynin in human liver. Hydrolysis in human liver microsomes (HLMs) and human liver cytosol (HLC) followed Michaelis-Menten kinetics with similar K(m) values. In recombinant human carboxylesterase (CES)-expressing microsomes, CES1 was much more efficient than CES2 and yielded a K(m) value more comparable with that found in HLMs or HLC than did CES2. A correlation analysis using a set of individual HLMs, in which both CESs acted independently showed that the hydrolysis rate of oxybutynin, correlated significantly with a CES1 marker reaction, clopidogrel hydrolysis, but not with a CES2 marker reaction, irinotecan (CPT-11) hydrolysis. Chemical inhibition studies using bis-(p-nitrophenyl) phosphate, clopidogrel, nordihydroguaiaretic acid, procainamide, physostigmine, and loperamide revealed that the effects of these compounds in HLMs, HLC, and recombinant CES1-expressing microsomes were similar, whereas those in CES2-expressing microsomes were clearly different. These results strongly suggest that CES1, rather than CES2, is the principal enzyme responsible for the hydrolysis of oxybutynin in human liver..
Neuroligin NLG a postsynaptic adhesion molecule is involved in the formation of synapses by binding to a cognate presynaptic ligand neurexin Here we report that neuroligin-1 NLG1 undergoes ectodomain shedding at the juxtamembrane stalk region to generate a secreted form of NLG1 and a membrane-tethered C-terminal fragment CTF in adult rat brains in vivo as well as in neuronal cultures Pharmacological and genetic studies identified ADAM10 as the major protease responsible for NLG1 shedding the latter being augmented by synaptic NMDA receptor activation or interaction with soluble neurexin ligands NLG1-CTF was subsequently cleaved by presenilin/gamma-secretase Secretion of soluble NLG1 was significantly upregulated under a prolonged epileptic seizure condition and inhibition of NLG1 shedding led to an increase in numbers of dendritic spines in neuronal cultures Collectively neuronal activity-dependent proteolytic processing of NLG1 may negatively regulate the remodeling of spines at excitatory synapses.
        
Title: Identification of human cytochrome P450 isoforms and esterases involved in the metabolism of mirabegron, a potent and selective beta(3)-adrenoceptor agonist Takusagawa S, Yajima K, Miyashita A, Uehara S, Iwatsubo T, Usui T Ref: Xenobiotica, 42:957, 2012 : PubMed
Human cytochrome P450 CYP enzymes and esterases involved in the metabolism of mirabegron a potent and selective human beta(3)-adrenoceptor agonist intended for the treatment of overactive bladder were identified in in vitro studies Incubations of mirabegron with recombinant human CYP enzymes showed significant metabolism of mirabegron by CYP2D6 and CYP3A4 only Correlation analyses showed a significant correlation between mirabegron metabolism and testosterone 6beta-hydroxylation CYP3A4/5 marker activity In inhibition studies using antiserum against CYP3A4 a strong inhibition at maximum 80 inhibition of the metabolism of mirabegron was observed whereas the inhibitory effects of monoclonal antibodies against CYP2D6 were small at maximum 10 inhibition These findings suggest that CYP3A4 is the primary CYP enzyme responsible for in vitro oxidative metabolism of mirabegron with a minor role of CYP2D6 Mirabegron hydrolysis was catalyzed in human blood plasma and butyrylcholinesterase BChE solution but not in human liver microsomes intestinal microsomes liver S9 intestinal S9 and recombinant acetylcholinesterase solution K(m values of mirabegron hydrolysis in human blood plasma and BChE solution were all similar 13.4-15.2 muM The inhibition profiles in human blood and plasma were also similar to those in BChE solution suggesting that mirabegron hydrolysis is catalyzed by BChE.
Proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases generates beta-amyloid (Abeta) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Abeta production. Previously, we and others reported that the four integral subunits of the gamma-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of gamma-secretase subunits. We report that in cultured cells and in brain the gamma-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys(689), and APH-1 is S-palmitoylated at Cys(182) and Cys(245). S-Palmitoylation-defective nicastrin and APH-1 form stable gamma-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for gamma-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Abeta40, Abeta42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate gamma-secretase processing of APP and other substrates.
Divergent synthesis of multifunctional molecular probes based on caprolactam-derived dipeptidic gamma-secretase inhibitors (GSIs), Compound E (CE) and LY411575 analogue (DBZ), was efficiently accomplished by means of Cu(I)-catalyzed azide/alkyne fusion reaction. Photoaffinity labeling experiments using these derivatives coupled to photoactivatable and biotin moieties provided direct evidence that the molecular targets of CE and DBZ are the N-terminal fragment of presenilin 1 within the gamma-secretase complex. Moreover, these photoprobes directly targeted signal peptide peptidase. These data suggest that the divergent synthesis of molecular probes has been successfully applied to characterize the interaction of GSIs with their molecular targets and define the structural requirements for inhibitor binding to intramembrane-cleaving proteases.
The dementia with Lewy bodies (DLB) Consortium has revised criteria for the clinical and pathologic diagnosis of DLB incorporating new information about the core clinical features and suggesting improved methods to assess them. REM sleep behavior disorder, severe neuroleptic sensitivity, and reduced striatal dopamine transporter activity on functional neuroimaging are given greater diagnostic weighting as features suggestive of a DLB diagnosis. The 1-year rule distinguishing between DLB and Parkinson disease with dementia may be difficult to apply in clinical settings and in such cases the term most appropriate to each individual patient should be used. Generic terms such as Lewy body (LB) disease are often helpful. The authors propose a new scheme for the pathologic assessment of LBs and Lewy neurites (LN) using alpha-synuclein immunohistochemistry and semiquantitative grading of lesion density, with the pattern of regional involvement being more important than total LB count. The new criteria take into account both Lewy-related and Alzheimer disease (AD)-type pathology to allocate a probability that these are associated with the clinical DLB syndrome. Finally, the authors suggest patient management guidelines including the need for accurate diagnosis, a target symptom approach, and use of appropriate outcome measures. There is limited evidence about specific interventions but available data suggest only a partial response of motor symptoms to levodopa: severe sensitivity to typical and atypical antipsychotics in approximately 50%, and improvements in attention, visual hallucinations, and sleep disorders with cholinesterase inhibitors.
Presenilin 1 (PS1) and presenilin 2 play a critical role in the gamma-secretase processing of amyloid precursor protein (APP) and Notch1. Here, we investigate maturation and intracellular trafficking of APP and other membrane proteins in cells expressing an experimental PS1 deletion mutant (deltaM1,2). Stable expression of deltaM1,2 impairs gamma-secretase processing of Notch1 and delays Abeta secretion. Kinetic studies show enhanced O-glycosylation and sialylation of holo-APP and marked accumulation of APP COOH-terminal fragments (CTFs). Surface biotinylation, live staining, and trafficking studies show increased surface accumulation of holo-APP and CTFs in deltaM1,2 cells resulting from enhanced surface delivery of newly synthesized APP. Expression of a loss-of-function PS1 mutant (D385A) or incubation of cells with gamma-secretase inhibitors also increases surface levels of holo-APP and CTFs. In contrast to APP, glycosylation and surface accumulation of another type I membrane protein, nicastrin, are markedly reduced in deltaM1,2 cells. Finally, expression of deltaM1,2 results in the increased assembly and surface expression of nicotinic acetylcholine receptors, illustrating that PS1's influence on protein trafficking extends beyond APP and other type I membrane protein substrates of gamma-secretase. Collectively, our findings provide evidence that PS1 regulates the glycosylation and intracellular trafficking of APP and select membrane proteins.