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Results from recent clinical trials of antibodies that target β-amyloid (Aβ) for Alzheimer’s disease (AD) have created excitement and have been heralded as corroboration of the amyloid cascade hypothesis. However, while Aβ may contribute to disease, genetic, clinical, imaging, and biochemical data suggest a more complex etiology. Here we review the history and weaknesses of the amyloid cascade hypothesis in view of the new evidence obtained from clinical trials of anti-amyloid antibodies. These trials indicate that the treatments have either no or uncertain clinical effect on cognition. Despite the importance of amyloid in the definition of AD, we argue that the data point to Aβ and amyloid playing a minor etiological role. We also discuss data suggesting that the concerted activity of many pathogenic factors contribute to AD and propose that evolving multi-factor disease models will better underpin the search for more effective strategies to treat the disease.

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.

Adherens junctions (AJs) create spatially and mechanically discrete microdomains at the interfaces of cells. Using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering, and mechanical loading, we report 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-µdomain) to initialize receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP-µdomain). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of cholesterol-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP-µdomains, AJs prevents inappropriate enzyme-substrate interactions and suppresses 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 differentiative expansion of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of large cell-surface receptors like 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.

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.

Aberrant cleavage of Notch by γ-secretase is implicated in numerous diseases, but how cleavage is regulated in space and time is unclear. Here, we report that cadherin-based adherens junctions (cadAJs) are sites of high cell-surface γ-secretase activity, as well as sites of constrained physical space that excludes γ-secretase substrates having large extracellular domains (ECDs) like Notch. ECD shedding initiates drastic spatial relocalization of Notch to cadAJs, allowing enzyme-substrate interactions and downstream signaling. Spatial mutations by adjusting the ECD size or the physical constraint alter signaling. Dysregulation of this spatial switch promotes precocious differentiation of ventricular zone neural progenitor cells in vivo. We show the generality of this spatial switch for amyloid precursor protein proteolysis. Thus, cadAJs create spatially distinct biochemical compartments regulating cleavage events involving γ-secretase and preventing aberrant activation of receptors.

Combining NMR, mass spectrometry, AlphaLISA and cell assays, we discovered a compound C1 that binds C-terminal juxtamembrane lysines at the transmembrane domain of the amyloid precursor protein (APPTM) and inhibits γ-secretase production of amyloid-β with μM IC50. Our work suggests that targeting APPTM is a novel and viable strategy in AD drug discovery.

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., We describe a presenilin 1-dependent mechanism by which neurotrophic factors protect neurons rom excitotoxicity, a condition with crucial roles in neurodegeneration and stroke. The mechanism includes presenilin1-dependent N-methyl-d-aspartate receptor complexes with factor receptors. Familial Alzheimer disease mutants alter N-methyl-d-aspartate receptor-presenilin1 associations and block factor-dependent neuroprotection in absence of Alzheimer disease hallmarks., Graphical Abstract Graphical Abstract

Alzheimer’s disease (AD) is the most common type of dementia caused by severe neurodegeneration in the hippocampus and neocortical regions of the brain. In addition to neurodegeneration, AD brains contain high levels of amyloid plaques (APs) and neurofibrillary tangles (NFTs) which are used as neuropathological hallmarks of the disorder. Despite intense research efforts, the mechanism(s) of the AD neurodegeneration are imperfectly understood, hampering efforts for the development of efficient therapeutics. Furthermore, failure of clinical trials to benefit AD patients suggests that AD hallmarks are poor therapeutic targets and supports the suggestion that these hallmarks are sequelae of neurodegeneration. Although genetic evidence seem to support the amyloid theory of AD, additional empirical observations and experimental data are inconsistent with the amyloid/Aβ theories of AD [Robakis and Neve (1998), TINS vol. 21 pp.15–19; Robakis (2011) NBA vol. 32, pp 372–379]. This possibility is further supported by data that amyloid plaques and neurofibrillary tangles are found in a number of distinct neurodegenerative disorders and that animal models expressing high levels of AD pathological structures show little neuronal loss. Furthermore, genetic evidence linking genetic loci to disease reveal little about the molecular mechanisms involved. Mutants of APP, PS1, and PS2 cause familial AD (FAD) suggesting these mutants can be used as models to study mechanisms of neurodegeneration. Recent reports show that the ability of efnB1 and BDNF (factors) to rescue neurons from excitotoxicity depends on PS1 but is independent of γ-secretase. Interestingly, PS1 FAD mutations block the ability of factors to protect neurons from toxicity suggesting that FAD mutants may increase neuronal death by blocking neuroprotective activities of brain neurotrophins. Other reports also suggest that proteins involved in FAD have Aβ-/γ-secretase-independent functions that can play important roles in AD. Furthermore, non-neuronal brain cells like microglia are implicated in AD pathology.

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.

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.

Open chromatin provides access to DNA-binding proteins for the correct spatiotemporal regulation of gene expression. Mapping chromatin accessibility has been widely used to identify the location of cis regulatory elements (CREs) including promoters and enhancers. CREs show tissue- and cell-type specificity and disease-associated variants are often enriched for CREs in the tissues and cells that pertain to a given disease. To better understand the role of CREs in neuropsychiatric disorders we applied the Assay for Transposase Accessible Chromatin followed by sequencing (ATAC-seq) to neuronal and non-neuronal nuclei isolated fromfrozen postmortem human brain by fluorescence-activated nuclear sorting (FANS). Most of the identified open chromatin regions (OCRs) are differentially accessible between neurons and non-neurons, and show enrichment with known cell typemarkers, promoters and enhancers. Relative to those of non-neurons, neuronal OCRs aremore evolutionarily conserved and are enriched in distal regulatory elements. Transcription factor (TF) footprinting analysis identifies differences in the regulome between neuronal and non-neuronal cells and ascribes putative functional roles to a number of non-coding schizophrenia (SCZ) risk variants. Among the identified variants is a Single Nucleotide Polymorphism(SNP) proximal to the gene encoding SNX19. In vitro experiments reveal that this SNP leads to an increase in transcriptional activity. As elevated expression of SNX19 has been associated with SCZ, our data provide evidence that the identified SNP contributes to disease. These results represent the first analysis of OCRs and TF-binding sites in distinct populations of postmortemhuman brain cells and further our understanding of the regulome and the impact of neuropsychiatric disease-associated genetic risk variants.

Recent studies link synaptojanin 1 (synj1), the main phosphoinositol (4,5)-biphosphate phosphatase (PI(4,5)P2-degrading enzyme) in the brain and synapses, to Alzheimer disease. Here we report a novel mechanism by which synj1 reversely regulates cellular clearance of amyloid-β (Aβ). Genetic down-regulation of synj1 reduces both extracellular and intracellular Aβ levels in N2a cells stably expressing the Swedish mutant of amyloid precursor protein (APP). Moreover, synj1 haploinsufficiency in an Alzheimer disease transgenic mouse model expressing the Swedish mutant APP and the presenilin-1 mutant ΔE9 reduces amyloid plaque load, as well as Aβ40 and Aβ42 levels in hippocampus of 9-month-old animals. Reduced expression of synj1 attenuates cognitive deficits in these transgenic mice. However, reduction of synj1 does not affect levels of full-length APP and the C-terminal fragment, suggesting that Aβ generation by β- and γ-secretase cleavage is not affected. Instead, synj1 knockdown increases Aβ uptake and cellular degradation through accelerated delivery to lysosomes. These effects are partially dependent upon elevated PI(4,5)P2 with synj1 down-regulation. In summary, our data suggest a novel mechanism by which reduction of a PI(4,5)P2-degrading enzyme, synj1, improves amyloid-induced neuropathology and behavior deficits through accelerating cellular Aβ clearance. Background: Recent studies have linked synaptojanin 1 (synj1) with Alzheimer disease (AD). Results: We report that synj1 reduction decreases amyloid plaque burden and attenuates cognitive deterioration in an AD mouse model. These effects are mediated through accelerating endosomal/lysosomal degradation of Aβ. Conclusion: Our data suggest a novel mechanism by which synj1 reduction promotes Aβ clearance. Significance: These studies implicate a therapeutic strategy for AD.

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. Background: Presenilin-1/γ-secretase is critical to neuronal function. Results: Inhibition of presenilin-1/γ-secretase increases release of glutamate, stimulating NMDAR and calpain while decreasing phosphorylation and surface expression of neuronal GluN2B. Conclusion: Presenilin-1/γ-secretase regulates glutamate release, NMDAR-dependent calpain activity, and modification of GluN2B. Significance: This study reveals a role for γ-secretase in release of glutamate, a neurotransmitter central to neuronal function, and excitotoxicity, a stress important to neurodegeneration.

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-β (Aβ) peptides found in all people. Aggregation of Aβ 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 Aβ peptides, but treatments with either inhibitors of Aβ production or anti-Aβ 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.

Presenilins (PSs) are catalytic components of the γ-secretase complex that produces Aβ peptides. Substrates of γ-secretase are membrane-bound protein fragments deriving from the cleavage of extracellular sequence of cell surface proteins. APP-derived γ-secretase substrates are cleaved at gamma (γ) sites to produce Aβ while cleavage at the epsilon (ε) site produces AICD proposed to function in transcription. In addition to APP, γ-secretase promotes the ε-cleavage of a large number of cell surface proteins producing cytosolic peptides shown to function in cell signaling. A common hypothesis suggests that Alzheimer's disease (AD) is caused by Aβ peptides or their products. Treatment of patients with inhibitors of Aβ production however, showed no therapeutic benefits while inducing cytotoxicity. Similarly, treatments with anti-Aβ antibodies yielded disappointing results. Importantly, recent evidence shows that PS familial AD (FAD) mutations cause a loss of γ-secretase cleavage activity at ε site of substrates thus inhibiting production of biologically important cell signaling peptides while promoting accumulation of membrane-bound cytotoxic substrates. These data support a hypothesis that FAD mutations may increase neurotoxicity by inhibiting the γ-secretase-catalyzed ε cleavage of substrates thus interfering with cell signaling while also promoting accumulation of cytotoxic peptides. Similar mechanisms may explain γ-secretase inhibitor-associated toxicities observed in clinical trials. Here we discuss evidence that FAD neurodegeneration may be caused by loss of γ-secretase cleavage function at ε sites of substrates.

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.

PMCID: PMC4819166.-- Moreno et al., 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 G-coupled serotonin 5-HTA receptor and the G-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-HT heteromers with an mGlu2 agonist led to activation of G proteins by the 5-HTA receptors. For this crosstalk to occur, one of the mGlu2 subunits had to couple to G proteins, and we determined the relative location of the G-contacting subunit within the mGlu2 homodimer of the heteromeric complex. Additionally, mGlu2-dependent activation of G, but not G, was reduced in the frontal cortex of 5-HT 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., This work was supported, in whole or in part, by the NIH grants R01MH084894 and R56MH084894 (to J.G.-M.), R01HL59949 (to D.E.L.), R37AG017926 and R01AG008200 (to N.K.R.), R01DA025036 and R01DA027460 (to J.A.M.), R01NS047229 and P50AG05138 (to A.G.), and S10RR027411 (to M.C.). This work was also supported by Dainippon Sumitomo Pharma (to J.G.-M.), Spanish MINECO/EDR Funds SAF2009-68460 and SAF2013-48586R (to J.J.M.), the Basque Government (to J.J.M.), the Spanish Government SAF2010-15663 grant (MICINN) (to J.F.L.G.), and Medical Research Council (UK) grants MR/L023806/1 and G0900050 (to G.M.). P.M.-A. and A.G.-B. were recipients of predoctoral fellowships from UPV/EHU and the Basque Government in Spain, respectively.

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.—Georgakopoulos, A., Xu, J., Xu, C., Mauger, G., Barthet, G., Robakis, N. K. Presenilin1/γ-secretase promotes the EphB2-induced phosphorylation of ephrinB2 by regulating phosphoprotein associated with glycosphingolipid-enriched microdomains/Csk binding protein.

Roussos P, Giakoumaki SG, Georgakopoulos A, Robakis NK, Bitsios P. The CACNA1C and ANK3 risk alleles impact on affective personality traits and startle reactivity but not on cognition or gating in healthy males. Bipolar Disord 2011: 13: 250–259. © 2011 The Authors. Journal compilation © 2011 John Wiley & Sons A/S. 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

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.

Despite the progress of the past two decades, the cause of Alzheimer's disease (AD) and effective treatments against it remain elusive. The hypothesis that amyloid-β (Aβ) peptides are the primary causative agents of AD retains significant support among researchers. Nonetheless, a growing body of evidence shows that Aβ peptides are unlikely to be the sole factor in AD etiology. Evidence that Aβ/amyloid-independent factors, including the actions of AD-related genes, also contribute significantly to AD pathogenesis was presented in a symposium at the 2010 Annual Meeting of the Society for Neuroscience. Here we summarize the studies showing how amyloid-independent mechanisms cause defective endo-lysosomal trafficking, altered intracellular signaling cascades, or impaired neurotransmitter release and contribute to synaptic dysfunction and/or neurodegeneration, leading to dementia in AD. A view of AD pathogenesis that encompasses both the amyloid-dependent and -independent mechanisms will help fill the gaps in our knowledge and reconcile the findings that cannot be explained solely by the amyloid hypothesis., National Institutes of Health (U.S.) (Grant P01-AG027916), National Institutes of Health (U.S.) (Grant R01-NS051874)

The presenilin-1 gene is mutated in early-onset familial Alzheimer’s disease. The mutation Pro117Leu is implicated in a very severe form of the disease, with an onset of less than thirty years. The consequences of this mutation on neurogenesis in the hippocampus of adult transgenic mice have already been studied in situ. The survival of neural progenitor cells was impaired resulting in decreased neurogenesis in the dentate gyrus. Our intention was to verify if similar alterations could occur in vitro in progenitor cells from the murine ganglionic eminences isolated from embryos of this same transgenic mouse model. These cells were grown in culture as neurospheres and after differentiation the percentage of neurons generated as well as their morphology were analysed. The mutation results in a significant decrease in neurogenesis compared to the wild type mice and the neurons grow longer and more ramified neurites. A shift of differentiation towards gliogenesis was observed that could explain decreased neurogenesis despite increased proliferation of neural precursors in transgenic neurospheres. A diminished survival of the newly generated mutant neurons is also proposed. Our data raise the possibility that these alterations in embryonic development might contribute to increase the severity of the Alzheimer’s disease phenotype later in adulthood.

Presenilin 1, a protein involved in the development of familial Alzheimer disease, is an important functional component of the gamma-secretase complex that processes many cell surface receptors including the EphB2 tyrosine kinase receptors (Litterst, C., Georgakopoulos, A., Shioi, J., Ghersi, E., Wisniewski, T., Wang, R., Ludwig, A., and Robakis, N. K. (2007) J. Biol. Chem. 282, 16155-16163). Recent evidence reveals that cytosolic peptides produced by the combined metalloproteinase/gamma-secretase processing of cell surface proteins function in signal transduction and protein phosphorylation. Here we show that peptide EphB2/CTF2 released to the cytosol by the gamma-secretase processing of EphB2 receptor, has tyrosine kinase activity, and directly phosphorylates the N-methyl-d-aspartate receptor (NMDAR) subunits in both cell lines and primary neuronal cultures. This phosphorylation occurs in the absence of Src kinases and is resistant to Src inhibitors revealing a novel pathway of NMDAR tyrosine phosphorylation independent of Src activity. EphB2/CTF2, but not a kinase-deficient mutant of EphB2/CTF2, promotes the cell surface expression of NMDAR. Because NMDAR plays central roles in synaptic plasticity and function, our results provide a potential link between the gamma-secretase function of presenilin 1 and learning and memory.

The gamma-secretase complex cleaves many transmembrane proteins, including amyloid precursor protein, EphB and ErbB tyrosine kinase receptors, Notch1 receptors, and adhesion factors. Presenilin 1, the catalytic subunit of gamma-secretase, associates with the cadherin/catenin cell-cell adhesion/communication system and promotes cadherin processing (Georgakopoulos, A., et al. (1999) Mol. Cell 4, 893-902; Marambaud, P., et al. (2002) EMBO J. 21, 1948-1956), but the mechanism by which gamma-secretase and cadherins associate is unclear. Here we report that p120 catenin (p120ctn), a component of the cadherin-catenin complex, recruits gamma-secretase to cadherins, thus stimulating their processing while inhibiting production of Abeta peptide and the amyloid precursor protein intracellular domain. This function of p120ctn depends on both p120ctn-cadherin and p120ctn-presenilin 1 binding, indicating that p120ctn is the central factor that bridges gamma-secretase and cadherin-catenin complexes. Our data show that p120ctn is a unique positive regulator of the gamma-secretase processing of cadherins and a negative regulator of the amyloid precursor protein processing. Furthermore, our data suggest that specific members of the gamma-secretase complex may be used to recruit different substrates and that distinct PS1 sequences are required for processing of APP and cadherins.

In order to study N1 processing, we expressed human N1 (hN1) in HEK293 cells (293-hN1). Following Western blot analysis of 293-hN1 extracts, we detected, in addition to full-length hN1 and the N1 extracellular domain truncated form (N1-TM), a novel extracellular domain truncated form of hN1 with a COOH-terminal deletion, designated hN1-TMdeltaCT. Treatment of cells with the gamma-secretase inhibitor L-685,458 resulted in an accumulation of hN1-TMdeltaCT suggesting that this fragment is a gamma-secretase substrate. To identify the proteolytic activity(ies) that generates hN1-TMdeltaCT, we treated 293-hN1 cells with inhibitors of proteasome, calpains, caspases, serine and cysteine proteases. Despite the presence of a caspase-3 cleavage site within hN1 intracellular domain, none of the caspase inhibitors inhibited hN1-TMdeltaCT production. The proteasomal inhibitors used had also no effect. Incubation of cells with the cysteine protease inhibitor E64d resulted in the accumulation of hN1-TM and the inhibition of hN1-TMdeltaCT production suggesting a precursor-product relationship and that a cysteine protease is involved. Similarly, treatment of cells expressing amyloid precursor protein or E-cadherin with E-64d resulted in the accumulation of COOH-terminal fragments suggesting that these proteins are also processed within their intracellular domain by a cysteine protease. Processing towards hN1-TMdeltaCT requires maturation and transport of hN1 to the cell surface since treatment with brefeldin A inhibited its production and resulted in accumulation of hN1. Processing of hN1 within its intracellular domain could generate fragments that can exert novel functions and/or interfere with the function of hN1 intracellular domain.

The apolipoprotein E4 (ApoE4) allele is the strongest genetic risk factor for developing sporadic Alzheimer's disease (AD). However, the mechanisms underlying the pathogenic nature of ApoE4 are not well understood. In this study, we have found that ApoE proteins are critical determinants of brain phospholipid homeostasis and that the ApoE4 isoform is dysfunctional in this process. We have found that the levels of phosphoinositol biphosphate (PIP2) are reduced in postmortem human brain tissues of ApoE4 carriers, in the brains of ApoE4 knock-in (KI) mice, and in primary neurons expressing ApoE4 alleles compared with those levels in ApoE3 counterparts. These changes are secondary to increased expression of a PIP2-degrading enzyme, the phosphoinositol phosphatase synaptojanin 1 (synj1), in ApoE4 carriers. Genetic reduction of synj1 in ApoE4 KI mouse models restores PIP2 levels and, more important, rescues AD-related cognitive deficits in these mice. Further studies indicate that ApoE4 behaves similar to ApoE null conditions, which fails to degrade synj1 mRNA efficiently, unlike ApoE3 does. These data suggest a loss of function of ApoE4 genotype. Together, our data uncover a previously unidentified mechanism that links ApoE4-induced phospholipid changes to the pathogenic nature of ApoE4 in AD.

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.—Bruban, J., Voloudakis, G., Huang, Q., Kajiwara, Y., Al Rahim, M., Yoon, Y., Shioi, J., Gama Sosa, M. A., Shao, Z., Georgakopoulos, A., Robakis, N. K. Presenilin 1 is necessary for neuronal, but not glial, EGFR expression and neuroprotection via γ-secretase-independent transcriptional mechanisms.

Strong support for a primary causative role of the Abeta peptides in the development of Alzheimer's disease (AD) neurodegeneration derives from reports that presenilin familial AD (FAD) mutants alter amyloid precursor protein processing, thus increasing production of neurotoxic Abeta 1-42 (Abeta 42). This effect of FAD mutants is also reflected in an increased ratio of peptides Abeta 42 over Abeta 1-40 (Abeta 40). In the present study, we show that several presenilin 1 FAD mutants failed to increase production of Abeta 42 or the Abeta 42/40 ratio. Our data suggest that the mechanism by which FAD mutations promote neurodegeneration and AD may be independent of their effects on Abeta production.

Cell culture studies have shown that the Alzheimer amyloid precursor protein (APP) is secreted after full-length APP is cleaved by a putative secretase at the Lys16-Leu17 bond (secretase cleavage I) of the amyloid peptide sequence. Because this cleavage event is incompatible with amyloid production, it has been assumed that secreted APP cannot serve as a precursor of the amyloid depositions observed in Alzheimer's disease. Here we show that in neuronally differentiated PC12 cells and human kidney 293 cell cultures a portion of the secreted extracytoplasmic APP reacted specifically with both a monoclonal antibody recognizing amyloid protein residues Leu17-Val24 and a polyclonal antiserum directed against amyloid protein residues Ala21-Lys28. Furthermore, this APP failed to react with antisera recognizing the cytoplasmic domain of the full-length protein. These data indicate the presence of an alternative APP secretase cleavage site (secretase cleavage II), C-terminal to the predominant secretase cleavage I. Depending on the exact location of cleavage site II, potentially amyloidogenic secreted APP species may be produced.

Neurofibrillary tangles (NFT) are one of the pathologic hallmarks of Alzheimer’s disease (AD). Their major component is tau, a protein that becomes hyperphosphorylated and accumulates into insoluble paired helical filaments. During the course of the disease such filaments aggregate into bulky NFT that get ubiquitinated. What triggers their formation is not known, but neuroinflammation could play a role. Neuroinflammation is an active process detectable in the earliest stages of AD. The neuronal toxicity associated with inflammation makes it a potential risk factor in the pathogenesis of chronic neurodegenerative diseases, such as AD. Determining the sequence of events that lead to this devastating disease has become one of the most important goals for AD prevention and treatment. In this review we focus on three topics relevant to AD pathology and to NFT formation: (1) what triggers CNS inflammation resulting in glia activation and neuronal toxicity; (2) how products of inflammation might change the substrate specificity of kinases/phosphatases leading to tau phosphorylation at pathological sites; (3) the relationship between the ubiquitin/proteasome pathway and tau ubiquitination and accumulation in NFT. The overall aim of this review is to provide a challenging and sometimes provocative survey of important contributions supporting the view that CNS inflammation might be a critical contributor to AD pathology. Neuronal cell death resulting from neuroinflammatory processes may have devastating effects as, in the vast majority of cases, neurons lost to disease cannot be replaced. In order to design therapies that will prevent endangered neurons from dying, it is critical that we learn more about the effects of neuroinflammation and its products.

Alzheimer’s disease (AD) is a neurodegenerative disorder of the central nervous system characterized by progressive loss of neurons in limbic and association cortices affecting almost all cognitive functions and memory. Additional neuropathological hallmarks of AD brains include large numbers of amyloid plaques (APs) containing fibers of Aβ peptides and neurofibrillary tangles (NFTs) consisting of overphosphorylated tau protein. Sporadic AD (SAD) is the most common form of dementia and accounts for more than 90 % of all cases. The etiology of SAD is complex and may be driven by both environmental and genetic factors. Presence of ApoE4 allele is the most important genetic risk factor for SAD although in the last 5 years additional genes were identified with smaller effects on the disorder. Several theories have been developed to explain the molecular basis of AD but none of these seems to offer a satisfactory explanation for the neurodegenerative phenotype of the disorder.

In previous studies we found that overexpression of the inducible form of cyclooxygenase, COX-2, in the brain exacerbated beta-amyloid (A beta) neuropathology in a transgenic mouse model of Alzheimer's disease. To explore the mechanism through which COX may influence A beta amyloidosis, we used an adenoviral gene transfer system to study the effects of human (h)COX-1 and hCOX-2 isoform expression on A beta peptide generation. We found that expression of hCOXs in human amyloid precursor protein (APP)-overexpressing (Chinese hamster ovary (CHO)-APPswe) cells or human neuroglioma (H4-APP751) cells resulting in 10-25 nM prostaglandin (PG)-E2 concentration in the conditioned medium coincided with an approximately 1.8-fold elevation of A beta-(1-40) and A beta-(1-42) peptide generation and an approximately 1.8-fold induction of the C-terminal fragment (CTF)-gamma cleavage product of the APP, an index of gamma-secretase activity. Treatment of APP-overexpressing cells with the non-selective COX inhibitor ibuprofen (1 microM, 48 h) or with the specific gamma-secretase inhibitor L-685,458 significantly attenuated hCOX-1- and hCOX-2-mediated induction of A beta peptide generation and CTF-gamma cleavage product formation. Based on this evidence, we next tested the hypothesis that COX expression might promote A beta peptide generation via a PG-E2-mediated mechanism. We found that exposure of CHO-APPswe or human embryonic kidney (HEK-APPswe) cells to PG-E2 (11-deoxy-PG-E2) at a concentration (10 nM) within the range of PG-E2 found in hCOX-expressing cells similarly promoted (approximately 1.8-fold) the generation of the CTF-gamma cleavage product of APP and commensurate A beta-(1-40) and A beta-(1-42) peptide elevation. The study suggests that expression of COXs may influence A beta peptide generation through mechanisms that involve PG-E2-mediated potentiation of gamma-secretase activity, further supporting a role for COX-2 and COX-1 in Alzheimer's disease neuropathology.

Presenilin1 (PS1), a protein implicated in Alzheimer's disease (AD), forms complexes with N-cadherin, a transmembrane protein with important neuronal and synaptic functions. Here, we show that a PS1-dependent gamma-secretase protease activity promotes an epsilon-like cleavage of N-cadherin to produce its intracellular domain peptide, N-Cad/CTF2. NMDA receptor agonists stimulate N-Cad/CTF2 production suggesting that this receptor regulates the epsilon-cleavage of N-cadherin. N-Cad/CTF2 binds the transcription factor CBP and promotes its proteasomal degradation, inhibiting CRE-dependent transactivation. Thus, the PS1-dependent epsilon-cleavage product N-Cad/CTF2 functions as a potent repressor of CBP/CREB-mediated transcription. Importantly, PS1 mutations associated with familial AD (FAD) and a gamma-secretase dominant-negative mutation inhibit N-Cad/CTF2 production and upregulate CREB-mediated transcription indicating that FAD mutations cause a gain of transcriptional function by inhibiting production of transcriptional repressor N-Cad/CTF2. These data raise the possibility that FAD mutation-induced transcriptional abnormalities maybe causally related to the dementia associated with FAD.

Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system (CNS) characterized by progressive loss of memory and other cognitive skills. Neurons in the limbic and association cortices become progressively dysfunctional affecting almost all cognitive functions and memory. The PSI-regulated epsilon-secretase cleavage of type I transmembrane receptors controls production of transcriptionally active intracellular fragments (ICFs) suggesting that this cleavage is a key factor in surface-to-nucleus signal transduction and gene expression. Signal-induced gene expression mediates neuronal responses to environmental changes and is a key event in neuronal survival and synaptic function. Familial Alzheimer's Disease (FAD) mutations may interfere with nuclear signaling and transcription by interfering with the PS1/epsilon-secretase cleavage and production of transcriptionally active ICFs. This raises the possibility that, similar to polyglutamine induced neurodegeneration like Huntington's chorea, transcriptional abnormalities are involved in the development of FAD.

The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor-like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro-2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope-specific anti-bodies suggested that a CS chain is attached within or proximal to the Aβ sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of ∼100 and 110 kDa were obtained that reacted with an APLP2-specific antiserum, suggesting that non-transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined. The proteoglycan nature of APP and APLP2 suggests that addition of the CS glycosaminoglycan chains is important for the implementation of the biological function of these proteins. However, the differential expression of these two proteoglycans suggests that their physiological roles and their possible involvement in Alzheimer's disease may differ.

Mutations in the presenilin-1 (PS-1) gene are one cause of familial Alzheimer's disease (FAD). However, the functions of the PS-1 protein as well as how PS-1 mutations cause FAD are incompletely understood. Here we investigated if neuronal overexpression of wild-type or FAD mutant PS-1 in transgenic mice affects neurogenesis in the hippocampus of adult animals. We show that either a wild-type or an FAD mutant PS-1 transgene reduces the number of neural progenitors in the dentate gyrus. However, the wild-type, but not the FAD mutant PS-1 promoted the survival and differentiation of progenitors leading to more immature granule cell neurons being generated in PS-1 wild type expressing animals. These studies suggest that PS-1 plays a role in regulating neurogenesis in adult hippocampus and that FAD mutants may have deleterious properties independent of their effects on amyloid deposition.

The functions of the presenilin-1 (PS-1) protein remain largely unknown. In adult brain PS-1 is expressed principally in neurons. However during development PS-1 is expressed more widely including in embryonic neural progenitors. To determine if PS-1 is expressed in neural progenitors in adult hippocampus we used bromodeoxyuridine (BrdU) labeling combined with immunostaining for BrdU, PS-1 and markers of neuronal or glial differentiation. Most BrdU labeled cells also expressed PS-1 at a time when few BrdU labeled cells expressed the early neuronal markers beta-III tubulin or TOAD-64 and none expressed mature neuronal (NeuN or calbindin) or astrocytic (GFAP) markers. Cells expressing PS-1 and the neural progenitor marker nestin were also found. Thus PS-1 is expressed in neural progenitor cells in adult hippocampus implying its possible role in neurogenesis in adult brain.

J. Wang Department of Pathophysiology, Key Laboratory of Neurological Diseases of the Education Committee of China, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China B. Ghetti Department of Pathology and Laboratory Medicine, Indiana Alzheimer Disease Center, Indiana University School of Medicine, 635 Barnhill Drive, MS A138, Indianapolis, IN 46202, USA

Chondroitin sulfate (CS)-D and CS-E, which are characterized by oversulfated disaccharide units, have been shown to regulate neuronal adhesion, cell migration, and neurite outgrowth. CS proteoglycans (CSPGs) consist of a core protein to which one or more CS chains are attached via a serine residue. Although several brain CSPGs, including mouse DSD-1-PG/phosphacan, have been found to contain the oversulfated D disaccharide motif, no brain CSPG has been reported to contain the oversulfated E motif. Here we analyzed the CS chain of appican, the CSPG form of the Alzheimer's amyloid precursor protein. Appican is expressed almost exclusively by astrocytes and has been reported to have brain- and astrocyte-specific functions including stimulation of both neural cell adhesion and neurite outgrowth. The present findings show that the CS chain of appican has a molecular mass of 25-50 kDa. This chain contains a significant fraction (14.3%) of the oversulfated E motif GlcUA beta 1-3GalNAc(4,6-O-disulfate). The rest of the chain consists of GlcUA beta 1-3GalNAc(4-O-sulfate) (81.2%) and minor fractions of GlcUA beta 1-3GalNAc and GlcUA beta 1-3GalNAc(6-O-sulfate). We also show that the CS chain of appican contains in its linkage region the 4-O-sulfated Gal structure. Thus, appican is the first example of a specific brain CSPG that contains the E disaccharide unit in its sugar backbone and the 4-O-sulfated Gal residue in its linkage region. The presence of the E unit is consistent with and may explain the neurotrophic activities of appican.

Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, binds directly to epithelial cadherin (E-cadherin). This binding is mediated by the large cytoplasmic loop of PS1 and requires the membrane-proximal cytoplasmic sequence 604–615 of mature E-cadherin. This sequence is also required for E-cadherin binding of protein p120, a known regulator of cadherin-mediated cell adhesion. Using wild-type and PS1 knockout cells, we found that increasing PS1 levels suppresses p120/E-cadherin binding, and increasing p120 levels suppresses PS1/E-cadherin binding. Thus PS1 and p120 bind to and mutually compete for cellular E-cadherin. Furthermore, PS1 stimulates E-cadherin binding to β- and γ-catenin, promotes cytoskeletal association of the cadherin/catenin complexes, and increases Ca 2+ -dependent cell–cell aggregation. Remarkably, PS1 familial Alzheimer disease mutant ΔE9 increased neither the levels of cadherin/catenin complexes nor cell aggregation, suggesting that this familial Alzheimer disease mutation interferes with cadherin-based cell–cell adhesion. These data identify PS1 as an E-cadherin-binding protein and a regulator of E-cadherin function in vivo .

Amyloid beta peptides (Abetas) of 39-43 amino acids constitute the major protein component of the amyloid plaques found in Alzheimer's disease brain. The generation of Abetas is regulated by the phosphoinositide (PI) pathway, which commonly couples to transmitter receptors. This study reports evidence for the activation of the PI pathway by Abetas in Xenopus oocytes expressing rat brain RNA. The naturally occurring peptides Abeta1-40 and Abeta1-42 were both active, whereas the cytotoxic fragment Abeta25-35 and the reverse peptide Abeta40-1 did not stimulate the PI pathway. Abetas rapidly lost potency in solution, suggesting that they were active only in their non-aggregated form. The Abeta response was saturable and not reduced by a substance P antagonist. This pharmacology excludes the participation of known Abeta binding proteins. The results indicate that a PI coupled receptor for non-aggregated Abeta may be present in brain.

Recent studies have reported that neuronal populations expressing low levels of presenilin-1 (PS-1) display increased vulnerability in late-onset sporadic Alzheimer's disease (AD). To examine whether this phenomenon also occurs in other neurodegenerative diseases, we performed a quantitative immunocytochemical study of PS-1 distribution in the cerebral cortex of Pick's disease (PiD) cases and non-demented individuals. In PiD cases, the percentage of PS-1-containing, Pick body (PB)-free neurons was significantly elevated only in cortical areas showing neuronal loss. In these areas, PS-1 levels, measured by immunoblotting, were often higher in PiD compared to non-demented cases. Moreover, PS-1 immunoreactivity was significantly reduced in PB-containing neurons. These data suggest that as previously shown in AD, low cellular expression of PS-1 may be associated with increased neuronal loss and cellular degeneration.

A growing body of data suggests that free radicals are involved in the pathogenesis of Alzheimer's disease (AD). Increased expression of antioxidant enzymes, such as superoxide dismutase (SOD), and their co-localization to senile plaques and dystrophic neurites have established a firm association between free-radical mediated injury and the disease neuropathology. While several studies have confirmed these findings, there is conflicting information regarding the activity of some of the enzymes. In the current report, we assayed the activity of superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) from the same areas of the tissue showing increased expression of SOD1 and SOD2 (parallel sequential slices). Nine brains with neuropathologically confirmed AD and six neuropathologically normal, age-matched, controls were examined. Despite marked increased expression of SOD1 and SOD2 within senile plaques in all the cases studied, the activities of SOD, GSH-Px and catalase were significantly lower in AD than in control brains. The difference was most profound in the case of catalase followed by GSH-Px and SOD. These data are in qualitative agreement with that of several laboratories, and support a decrease rather than an increase, in antioxidant enzyme activity. The findings suggest two main possibilities. On one hand, the observed reduced activity along with antigenically increased expression may be consistent with inactivation of excess protein that has been synthesized under conditions of high oxidative stress. Increased protein oxidation coupled with enzyme inactivation is a documented, aging-associated phenomenon. Alternatively, the increased immuno-reactivity may reflect a redistribution phenomenon as the enzymes become more concentrated at the sites of increased oxidative stress, despite an over all reduction in their activity.

Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.