A Robust End-to-End IoT System for Supporting Workers in Mining Industries.

The adoption of the Internet of Things (IoT) in the mining industry can dramatically enhance the safety of workers while simultaneously decreasing monitoring costs. By implementing an IoT solution consisting of a number of interconnected smart devices and sensors, mining industries can improve response times during emergencies and also reduce the number of accidents, resulting in an overall improvement of the social image of mines. Thus, in this paper, a robust end-to-end IoT system for supporting workers in harsh environments such as in mining industries is presented. The full IoT solution includes both edge devices worn by the workers in the field and a remote cloud IoT platform, which is responsible for storing and efficiently sharing the gathered data in accordance with regulations, ethics, and GDPR rules. Extended experiments conducted to validate the IoT components both in the laboratory and in the field proved the effectiveness of the proposed solution in monitoring the real-time status of workers in mines.

3D AFM Nanomechanical Characterization of Biological Materials.

Atomic Force Microscopy (AFM) is a powerful tool enabling the mechanical characterization of biological materials at the nanoscale. Since biological materials are highly heterogeneous, their mechanical characterization is still considered to be a challenging procedure. In this paper, a new approach that leads to a 3-dimensional (3D) nanomechanical characterization is presented based on the average Young's modulus and the AFM indentation method. The proposed method can contribute to the clarification of the variability of the mechanical properties of biological samples in the 3-dimensional space (variability at the x-y plane and depth-dependent behavior). The method was applied to agarose gels, fibroblasts, and breast cancer cells. Moreover, new mathematical methods towards a quantitative mechanical characterization are also proposed. The presented approach is a step forward to a more accurate and complete characterization of biological materials and could contribute to an accurate user-independent diagnosis of various diseases such as cancer in the future.

Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling.

Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand-receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme-substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.

PS1 FAD mutants decrease ephrinB2-regulated angiogenic functions, ischemia-induced brain neovascularization and neuronal survival.

Microvascular pathology and ischemic lesions contribute substantially to neuronal dysfunction and loss that lead to Alzheimer disease (AD). To facilitate recovery, the brain stimulates neovascularization of damaged tissue via sprouting angiogenesis, a process regulated by endothelial cell (EC) sprouting and the EphB4/ephrinB2 system. Here, we show that in cultures of brain ECs, EphB4 stimulates the VE-cadherin/Rok-α angiogenic complexes known to mediate sprouting angiogenesis. Importantly, brain EC cultures expressing PS1 FAD mutants decrease the EphB4-stimulated γ-secretase cleavage of ephrinB2 and reduce production of the angiogenic peptide ephrinB2/CTF2, the VE-cadherin angiogenic complexes and EC sprouting and tube formation. These data suggest that FAD mutants may attenuate ischemia-induced brain angiogenesis. Supporting this hypothesis, ischemia-induced VE-cadherin angiogenic complexes, levels of neoangiogenesis marker Endoglin, vascular density, and cerebral blood flow recovery, are all decreased in brains of mouse models expressing PS1 FAD mutants. Ischemia-induced brain neuronal death and cognitive deficits also increase in these mice. Furthermore, a small peptide comprising the C-terminal sequence of peptide ephrinB2/CTF2 rescues angiogenic functions of brain ECs expressing PS1 FAD mutants. Together, our data show that PS1 FAD mutations impede the EphB4/ephrinB2-mediated angiogenic functions of ECs and impair brain neovascularization, neuronal survival and cognitive recovery following ischemia. Furthermore, our data reveal a novel brain angiogenic mechanism targeted by PS1 FAD mutants and a potential therapeutic target for ischemia-induced neurodegeneration. Importantly, FAD mutant effects occur in absence of neuropathological hallmarks of AD, supporting that such hallmarks may form downstream of mutant effects on neoangiogenesis and neuronal survival.

Presenilin1 familial Alzheimer disease mutants inactivate EFNB1- and BDNF-dependent neuroprotection against excitotoxicity by affecting neuroprotective complexes of N-methyl-d-aspartate receptor.

Excitotoxicity is thought to play key roles in brain neurodegeneration and stroke. Here we show that neuroprotection against excitotoxicity by trophic factors EFNB1 and brain-derived neurotrophic factor (called here factors) requires de novo formation of 'survival complexes' which are factor-stimulated complexes of N-methyl-d-aspartate receptor with factor receptor and presenilin 1. Absence of presenilin 1 reduces the formation of survival complexes and abolishes neuroprotection. EPH receptor B2- and N-methyl-d-aspartate receptor-derived peptides designed to disrupt formation of survival complexes also decrease the factor-stimulated neuroprotection. Strikingly, factor-dependent neuroprotection and levels of the de novo factor-stimulated survival complexes decrease dramatically in neurons expressing presenilin 1 familial Alzheimer disease mutants. Mouse neurons and brains expressing presenilin 1 familial Alzheimer disease mutants contain increased amounts of constitutive presenilin 1-N-methyl-d-aspartate receptor complexes unresponsive to factors. Interestingly, the stability of the familial Alzheimer disease presenilin 1-N-methyl-d-aspartate receptor complexes differs from that of wild type complexes and neurons of mutant-expressing brains are more vulnerable to cerebral ischaemia than neurons of wild type brains. Furthermore, N-methyl-d-aspartate receptor-mediated excitatory post-synaptic currents at CA1 synapses are altered by presenilin 1 familial Alzheimer disease mutants. Importantly, high levels of presenilin 1-N-methyl-d-aspartate receptor complexes are also found in post-mortem brains of Alzheimer disease patients expressing presenilin 1 familial Alzheimer disease mutants. Together, our data identify a novel presenilin 1-dependent neuroprotective mechanism against excitotoxicity and indicate a pathway by which presenilin 1 familial Alzheimer disease mutants decrease factor-depended neuroprotection against excitotoxicity and ischaemia in the absence of Alzheimer disease neuropathological hallmarks which may form downstream of neuronal damage. These findings have implications for the pathogenic effects of familial Alzheimer disease mutants and therapeutic strategies.

The product of the γ-secretase processing of ephrinB2 regulates VE-cadherin complexes and angiogenesis.

Presenilin-1 (PS1) gene encodes the catalytic component of γ-secretase, which proteolytically processes several type I transmembrane proteins. We here present evidence that the cytosolic peptide efnB2/CTF2 produced by the PS1/γ-secretase cleavage of efnB2 ligand promotes EphB4 receptor-dependent angiogenesis in vitro. EfnB2/CTF2 increases endothelial cell sprouting and tube formation, stimulates the formation of angiogenic complexes that include VE-cadherin, Raf-1 and Rok-α, and increases MLC2 phosphorylation. These functions are mediated by the PDZ-binding domain of efnB2. Acute downregulation of PS1 or inhibition of γ-secretase inhibits the angiogenic functions of EphB4 while absence of PS1 decreases the VE-cadherin angiogenic complexes of mouse brain. Our data reveal a mechanism by which PS1/γ-secretase regulates efnB2/EphB4 mediated angiogenesis.

Presenilin1/γ-secretase protects neurons from glucose deprivation-induced death by regulating miR-212 and PEA15.

Reduced cerebral glucose utilization is found in aged individuals and often is an early sign of neurodegeneration. Here, we show that under glucose deprivation (GD) conditions, decreased expression of presenilin 1 (PS1) results in decreased neuronal survival, whereas increased PS1 increases neuronal survival. Inhibition of γ-secretase also decreases neuronal survival under GD conditions, which suggests the PS1/γ-secretase system protects neurons from GD-induced death. We also show that neuronal levels of the survival protein, phosphoprotein enriched in astrocytes at ∼15 kDa (PEA15), and its mRNA are regulated by PS1/γ-secretase. Furthermore, down-regulation of PEA15 decreases neuronal survival under reduced glucose conditions, whereas exogenous PEA15 increases neuronal survival even in the absence of PS1, which indicates that PEA15 promotes neuronal survival under GD conditions. The absence or reduction of PS1, as well as γ-secretase inhibitors, increases neuronal miR-212, which targets PEA15 mRNA. PS1/γ-secretase activates the transcription factor, cAMP response element-binding protein, regulating miR-212, which targets PEA15 mRNA. Taken together, our data show that under conditions of reduced glucose, the PS1/γ-secretase system decreases neuronal losses by suppressing miR-212 and increasing its target survival factor, PEA15. These observations have implications for mechanisms of neuronal death under conditions of reduced glucose and may provide targets for intervention in neurodegenerative disorders.-Huang, Q., Voloudakis, G., Ren, Y., Yoon, Y., Zhang, E., Kajiwara, Y., Shao, Z., Xuan, Z., Lebedev, D., Georgakopoulos, A., Robakis, N. K. Presenilin1/γ-secretase protects neurons from glucose deprivation-induced death by regulating miR-212 and PEA15.

Presenilin 1 promotes trypsin-induced neuroprotection via the PAR2/ERK signaling pathway. Effects of presenilin 1 FAD mutations.

Mutants of presenilin 1 (PS1) increase neuronal cell death causing autosomal-dominant familial Alzheimer's disease (FAD). Recent literature shows that treatment of neuronal cultures with low concentrations of trypsin, a member of the serine family of proteases, protects neurons from toxic insults by binding to the proteinase-activated receptor 2 and stimulating survival kinase extracellular signal-regulated kinase (ERK 1/2). Other studies show that PS1 is necessary for the neuroprotective activity of specific neurotrophic factors, such as brain-derived neurotrophic factor, against excitotoxicity and oxidative stress. Here, we show that treatment of mouse cortical neuronal cultures with trypsin activates ERK1/2 and protects neurons against glutamate excitoxicity. The trypsin-dependent ERK activation and neuroprotection requires both alleles of PS1 because neither PS1 knockout nor PS1 hemizygous neuronal cultures can use exogenous trypsin to activate ERK1/2 or increase neuronal survival. The protective effect of PS1 does not depend on its γ-secretase activity because inhibitors of γ-secretase have no effect on trypsin-mediated neuroprotection. Importantly, cortical neuronal cultures either heterozygous or homozygous for PS1 FAD mutants are unable to use trypsin to activate ERK1/2 and rescue neurons from excitotoxicity, indicating that FAD mutants inhibit trypsin-dependent neuroprotection in an autosomal-dominant manner. Furthermore, our data support the theory that PS FAD mutants increase neurodegeneration by inhibiting the ability of neurons to use cellular factors as protective agents against toxic insults.

Allosteric signaling through an mGlu2 and 5-HT2A heteromeric receptor complex and its potential contribution to schizophrenia.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) can form multiprotein complexes (heteromers), which can alter the pharmacology and functions of the constituent receptors. Previous findings demonstrated that the Gq/11-coupled serotonin 5-HT2A receptor and the Gi/o-coupled metabotropic glutamate 2 (mGlu2) receptor-GPCRs that are involved in signaling alterations associated with psychosis-assemble into a heteromeric complex in the mammalian brain. In single-cell experiments with various mutant versions of the mGlu2 receptor, we showed that stimulation of cells expressing mGlu2-5-HT2A heteromers with an mGlu2 agonist led to activation of Gq/11 proteins by the 5-HT2A receptors. For this crosstalk to occur, one of the mGlu2 subunits had to couple to Gi/o proteins, and we determined the relative location of the Gi/o-contacting subunit within the mGlu2 homodimer of the heteromeric complex. Additionally, mGlu2-dependent activation of Gq/11, but not Gi/o, was reduced in the frontal cortex of 5-HT2A knockout mice and was reduced in the frontal cortex of postmortem brains from schizophrenic patients. These findings offer structural insights into this important target in molecular psychiatry.

Presenilin 1 is necessary for neuronal, but not glial, EGFR expression and neuroprotection via γ-secretase-independent transcriptional mechanisms.

Epidermal growth factor receptor (EGFR) plays pivotal roles in cell proliferation, differentiation, and tissue development, while EGFs protect neurons from toxic insults by binding EGFR and stimulating survival signaling. Furthermore, recent evidence implicates this receptor in neurometabolic disorders like Alzheimer disease and aging. Here we show that absence of presenilin 1 (PS1) results in dramatic decrease (>95%) of neuronal EGFR and that PS1-null (PS1(-/-)) brains have reduced amounts of this receptor. PS1(-/-) cortical neurons contain little EGFR and show no epidermal growth factor-induced survival signaling or protection against excitotoxicity, but exogenous EGFR rescues both functions even in absence of PS1. EGFR mRNA is greatly reduced (>95%) in PS1(-/-) neurons, and PS1(-/-) brains contain decreased amounts of this mRNA, although PS1 affects the stability of neither EGFR nor its mRNA. Exogenous PS1 increases neuronal EGFR mRNA, while down-regulation of PS1 decreases this mRNA. These effects are neuron specific, as PS1 affects the EGFR of neither glial nor fibroblast cells. In addition, PS1 controls EGFR through novel mechanisms shared with neither γ-secretase nor PS2. Our data reveal that PS1 functions as a positive transcriptional regulator of neuronal EGFR controlling its expression in a cell-specific manner. Severe downregulation of EGFR may contribute to developmental abnormalities and lethal phenotype found in PS1, but not PS2, null mice. Furthermore, PS1 may affect neuroprotection and Alzheimer disease by controlling survival signaling of neuronal EGFR.

A role for noncoding variation in schizophrenia.

A large portion of common variant loci associated with genetic risk for schizophrenia reside within noncoding sequence of unknown function. Here, we demonstrate promoter and enhancer enrichment in schizophrenia variants associated with expression quantitative trait loci (eQTL). The enrichment is greater when functional annotations derived from the human brain are used relative to peripheral tissues. Regulatory trait concordance analysis ranked genes within schizophrenia genome-wide significant loci for a potential functional role, based on colocalization of a risk SNP, eQTL, and regulatory element sequence. We identified potential physical interactions of noncontiguous proximal and distal regulatory elements. This was verified in prefrontal cortex and -induced pluripotent stem cell-derived neurons for the L-type calcium channel (CACNA1C) risk locus. Our findings point to a functional link between schizophrenia-associated noncoding SNPs and 3D genome architecture associated with chromosomal loopings and transcriptional regulation in the brain.

Allelic interference: a mechanism for trans-dominant transmission of loss of function in the neurodegeneration of familial Alzheimer's disease.

Presenilins (PSs) are catalytic components of the γ-secretase complexes that promote the ε-cleavage of cell surface proteins producing cytosolic peptides shown to function in cell signaling and gene expression. In addition, secretase cleavages at γ-sites of amyloid precursor protein substrates produce the amyloid-ß (Aß) peptides found in all people. Aggregation of Aß peptides form the amyloid fibrils found in amyloid plaques of Alzheimer's disease (AD) patients and aged individuals. A common hypothesis suggests that AD is caused by aggregated Aß peptides, but treatments with either inhibitors of Aß production or anti-Aß antibodies showed no therapeutic value. Importantly, recent evidence [Marambaud et al.: Cell 2003;114:635-645] shows that PS familial AD (FAD) mutations cause a loss of γ-secretase cleavage function at the ε-site of substrates manifested by a decreased production of cytosolic peptides and an accumulation of transmembrane γ-secretase substrates. These data support the hypothesis that PS FAD mutations promote neurotoxicity by inhibiting the γ-secretase-catalyzed ε-cleavage of substrates, thus reducing cell signaling while causing accumulation of membrane-bound cytotoxic peptides. Similar mechanisms may be involved in toxicities observed in clinical trials of γ-secretase inhibitors. A model of allelic interference may explain the dominant negative transmission of neurotoxic loss of function in FAD neurodegeneration.

Presenilin-1/γ-secretase controls glutamate release, tyrosine phosphorylation, and surface expression of N-methyl-D-aspartate receptor (NMDAR) subunit GluN2B.

Abnormally high concentrations of extracellular glutamate in the brain may cause neuronal damage via excitotoxicity. Thus, tight regulation of glutamate release is critical to neuronal function and survival. Excitotoxicity is caused mainly by overactivation of the extrasynaptic NMDA receptor (NMDAR) and results in specific cellular changes, including calcium-induced activation of calpain proteases. Here, we report that presenilin-1 (PS1) null mouse cortical neuronal cultures have increased amounts of calpain-dependent spectrin breakdown products (SBDPs) compared with WT cultures. NMDAR antagonists blocked accumulation of SBDPs, suggesting abnormal activation of this receptor in PS1 null cultures. Importantly, an increase in SBDPs was detected in cultures of at least 7 days in vitro but not in younger cultures. Conditioned medium from PS1 null neuronal cultures at 8 days in vitro contained higher levels of glutamate than medium from WT cultures and stimulated production of SBDPs when added to WT cultures. Use of glutamate reuptake inhibitors indicated that accumulation of this neurotransmitter in the media of PS1 null cultures was due to increased rates of release. PS1 null neurons showed decreased cell surface expression and phosphorylation of the GluN2B subunit of NMDAR, indicating decreased amounts of extrasynaptic NMDAR in the absence of PS1. Inhibition of γ-secretase activity in WT neurons caused changes similar to those observed in PS1 null neurons. Together, these data indicate that the PS1/γ-secretase system regulates release of glutamate, tyrosine phosphorylation, and surface expression of GluN2B-containing NMDARs.

Presenilin mediates neuroprotective functions of ephrinB and brain-derived neurotrophic factor and regulates ligand-induced internalization and metabolism of EphB2 and TrkB receptors.

Activation of EphB receptors by ephrinB (efnB) ligands on neuronal cell surface regulates important functions, including neurite outgrowth, axonal guidance, and synaptic plasticity. Here, we show that efnB rescues primary cortical neuronal cultures from necrotic cell death induced by glutamate excitotoxicity and that this function depends on EphB receptors. Importantly, the neuroprotective function of the efnB/EphB system depends on presenilin 1 (PS1), a protein that plays crucial roles in Alzheimer's disease (AD) neurodegeneration. Furthermore, absence of one PS1 allele results in significantly decreased neuroprotection, indicating that both PS1 alleles are necessary for full expression of the neuroprotective activity of the efnB/EphB system. We also show that the ability of brain-derived neurotrophic factor (BDNF) to protect neuronal cultures from glutamate-induced cell death depends on PS1. Neuroprotective functions of both efnB and BDNF, however, were independent of γ-secretase activity. Absence of PS1 decreases cell surface expression of neuronal TrkB and EphB2 without affecting total cellular levels of the receptors. Furthermore, PS1-knockout neurons show defective ligand-dependent internalization and decreased ligand-induced degradation of TrkB and Eph receptors. Our data show that PS1 mediates the neuroprotective activities of efnB and BDNF against excitotoxicity and regulates surface expression and ligand-induced metabolism of their cognate receptors. Together, our observations indicate that PS1 promotes neuronal survival by regulating neuroprotective functions of ligand-receptor systems.

Cellular mechanisms of γ-secretase substrate selection, processing and toxicity.

Presenilins (PSs) are catalytic components of the γ-secretase proteolytic complexes that produce Aß and cell signaling peptides. γ-Secretase substrates are mostly membrane-bound peptides derived following proteolytic cleavage of the extracellular domain of type I transmembrane proteins. Recent work reveals that γ-secretase substrate processing is regulated by proteins termed γ-secretase substrate recruiting factors (γSSRFs) that bridge substrates to γ-secretase complexes. These factors constitute novel targets for pharmacological control of specific γ-secretase products, such as Aß and signaling peptides. PS familial Alzheimer's disease (FAD) mutants cause a loss of γ-secretase cleavage function at epsilon sites of substrates thus inhibiting production of cell signaling peptides while promoting accumulation of uncleaved toxic substrates. Importantly, γ-secretase inhibitors may cause toxicity in vivo by similar mechanisms. Here we review novel mechanisms that control γ-secretase substrate selection and cleavage and examine their relevance to AD.

Presenilin1/gamma-secretase promotes the EphB2-induced phosphorylation of ephrinB2 by regulating phosphoprotein associated with glycosphingolipid-enriched microdomains/Csk binding protein.

Reverse signaling through the ephrinB ligands is important for several morphogenetic events, such as axon guidance, neuronal plasticity, spine maturation, and synaptogenesis. Signaling is initiated by binding of EphB receptors to ephrinB ligands, stimulating their tyrosine phosphorylation via an unclear mechanism. Here we show that this mechanism involves presenilin1 (PS1)/γ-secretase regulation of phosphoprotein associated with glycosphingolipid-enriched microdomains/Csk binding protein (PAG/Cbp), an adaptor protein that controls the activity of Src kinases. Using immunoprecipitation and Western blot of mouse primary neuronal and human embryonic kidney (HEK293) cell extracts overexpressing PAG/Cbp, we show that EphB2 induces tyrosine dephosphorylation of PAG/Cbp in a γ-secretase-dependent manner. In these cells, PAG/Cbp dephosphorylation is promoted by the PS1/γ-secretase-produced fragment of ephrinB2 cleavage (ephrinB2/CTF2), which forms complexes with PAG/Cbp when introduced exogenously. EphB2-induced tyrosine phosphorylation of ephrinB2 depends on PAG/Cbp because EphB2 cannot increase ephrinB2 phosphorylation in cells treated with anti-PAG siRNA or in PAG/Cbp-knockout (KO) cells. Furthermore, in contrast to WT PS1, familial Alzheimer disease (FAD) PS1 mutants expressed in PS1-KO mouse embryonic fibroblasts inhibited both the EphB2-induced dephosphorylation of PAG/Cbp and the phosphorylation of ephrinB2. PS1 FAD mutations may thus inhibit the function of ephrinB in the brain, promoting neurodegeneration in Alzheimer disease.

The CACNA1C and ANK3 risk alleles impact on affective personality traits and startle reactivity but not on cognition or gating in healthy males.

OBJECTIVES: The rs10994336 ANK3 and rs1006737 CACNA1C genetic variants have recently been identified as the most consistent, genome-wide significant risk factors for bipolar disorder, while the CACNA1C variant has also been associated with schizophrenia and major depression. The aim of this study was to examine the phenotypic consequences of the risk CACNA1C and ANK3 alleles in a large homogeneous cohort of healthy young males. METHODS: We recruited 703 randomly selected, healthy army conscripts (mean age 22.1 ± 3.0 years) from the first wave of the Learning on Genetics of Schizophrenia project in Heraklion, Crete. Of those recruited, 530 subjects entered and completed the study. Subjects were assessed for prepulse inhibition (PPI), startle reactivity, neuropsychology, and personality. RESULTS: UNPHASED analysis revealed that the rs1006737 A-allele was associated with lower extraversion and higher harm avoidance, trait anxiety, and paranoid ideation, while the rs10994336 T-allele was associated with lower novelty seeking and behavioral activation scores (p < 0.01). Both alleles were associated with high startle reactivity (p < 0.05). There were no significant associations with any cognitive task performance or PPI. CONCLUSIONS: The CACNA1C genotype was associated with proneness to anxiety and negative mood, while the ANK3 genotype was associated with proneness to anhedonia. Both risk genotypes were associated with high startle reactivity, suggesting a role of these polymorphisms in threat/stress signal processing, probably in the hippocampus and/or amygdala. None of the risk genotypes affected sensorimotor gating or behavioral performance in an extensive battery of executive function tests in this cohort of healthy males.

Inhibitors of γ-secretase stabilize the complex and differentially affect processing of amyloid precursor protein and other substrates.

γ-Secretase inhibitors (GSIs) are drugs used in research to inhibit production of Aß and in clinical trials to treat Alzheimer's disease (AD). They inhibit proteolytic activities of γ-secretase noncompetitively by unknown mechanisms. Here, we used cortical neuronal cultures expressing endogenous levels of enzymes and substrates to study the effects of GSIs on the structure and function of γ-secretase. We show that GSIs stabilize the interactions between the C-terminal fragment of presenilin (PS-CTF), the central component of the γ-secretase complex, and its partners the APH-1/nicastrin and PS1-NTF/PEN-2 subcomplexes. This stabilization dose-dependently correlates with inhibition of N-cadherin cleavage, a process limited by enzyme availability. In contrast, production of amyloid precursor protein (APP) intracellular domain (AICD) is insensitive to low concentrations of GSIs and is limited by substrate availability. Interestingly, APP is processed by both PS1- and PS2-containing γ-secretase complexes, while N-cadherin and ephrinB1 are processed only by PS1-containing complexes. Paradoxically, low concentrations of GSIs specifically increased the levels of Aß without affecting its catabolism, indicating increased Aß production. Our data reveal a mechanism of γ-secretase inhibition by GSIs and provide evidence that distinct γ-secretase complexes process specific substrates. Furthermore, our observations have implications for GSIs as therapeutics because processing of functionally important substrates may be inhibited at lower concentrations than Aß.

The association of schizophrenia risk D-amino acid oxidase polymorphisms with sensorimotor gating, working memory and personality in healthy males.

There is evidence supporting a role for the D-amino acid oxidase (DAO) locus in schizophrenia. This study aimed to determine the relationship of five single-nucleotide polymorphisms (SNPs) within the DAO gene identified as promising schizophrenia risk genes (rs4623951, rs2111902, rs3918346, rs3741775, and rs3825251) to acoustic startle, prepulse inhibition (PPI), working memory, and personality dimensions. A highly homogeneous study entry cohort (n = 530) of healthy, young male army conscripts (n = 703) originating from the Greek LOGOS project (Learning On Genetics Of Schizophrenia Spectrum) underwent PPI of the acoustic startle reflex, working memory, and personality assessment. The QTPHASE from the UNPHASED package was used for the association analysis of each SNP or haplotype data, with p-values corrected for multiple testing by running 10,000 permutations of the data. The rs4623951_T-rs3741775_G and rs4623951_T-rs2111902_T diplotypes were associated with reduced PPI and worse performance in working memory tasks and a personality pattern characterized by attenuated anxiety. Median stratification analysis of the risk diplotype group (ie, those individuals homozygous for the T and G alleles (TG+)) showed reduced PPI and working memory performance only in TG+ individuals with high trait anxiety. The rs4623951_T allele, which is the DAO polymorphism most strongly associated with schizophrenia, might tag a haplotype that affects PPI, cognition, and personality traits in general population. Our findings suggest an influence of the gene in the neural substrate mediating sensorimotor gating and working memory, especially when combined with high anxiety and further validate DAO as a candidate gene for schizophrenia and spectrum disorders.

Phosphatidylinositol-4,5-bisphosphate regulates epidermal growth factor receptor activation.

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2) or PIP(2)] is a direct modulator of a diverse array of proteins in eukaryotic cells. The functional integrity of transmembrane proteins, such as ion channels and transporters, is critically dependent on specific interactions with PIP(2) and other phosphoinositides. Here, we report a novel requirement for PIP(2) in the activation of the epidermal growth factor receptor (EGFR). Down-regulation of PIP(2) levels either via pharmacological inhibition of PI kinase activity, or via manipulation of the levels of the lipid kinase PIP5K1α and the lipid phosphatase synaptojanin, reduced EGFR tyrosine phosphorylation, whereas up-regulation of PIP(2) levels via overexpression of PIP5K1α had the opposite effect. A cluster of positively charged residues in the juxtamembrane domain (basic JD) of EGFR is likely to mediate binding of EGFR to PIP(2) and PIP(2)-dependent regulation of EGFR activation. A peptide mimicking the EGFR juxtamembrane domain that was assayed by surface plasmon resonance displayed strong binding to PIP(2). Neutralization of positively charged amino acids abolished EGFR/PIP(2) interaction in the context of this peptide and down-regulated epidermal growth factor (EGF)-induced EGFR auto-phosphorylation and EGF-induced EGFR signaling to ion channels in the context of the full-length receptor. These results suggest that EGFR activation and downstream signaling depend on interactions of EGFR with PIP(2) and point to the basic JD's critical involvement in these interactions. The addition of this very different class of membrane proteins to ion channels and transporters suggests that PIP(2) may serve as a general modulator of the activity of many diverse eukaryotic transmembrane proteins through their basic JDs.