Spiros Efthimiopoulos - Academia.edu (original) (raw)
Papers by Spiros Efthimiopoulos
Nature Biomedical Engineering, 2018
In the version of this Article originally published, in Fig. 1c-e, on the x axes, the lines label... more In the version of this Article originally published, in Fig. 1c-e, on the x axes, the lines labelled ' Aβ 42 ' and ' Aβ 42 (F19S;L34P)' grouped the data incorrectly; the line labelled Aβ 42 should have grouped the data for Random 1-2 and Clones 1-10, and the line labelled Aβ 42 (F19S;L34P) should have only grouped the data for Random 1-2 on the right end of the plots and blots. These figures have now been corrected in all versions of the Article.
Nature Biomedical Engineering
Protein misfolding and aggregation are common pathological features of several human diseases, in... more Protein misfolding and aggregation are common pathological features of several human diseases, including Alzheimer’s disease and type 2 diabetes. Here, we report an integrated and generalizable bacterial system for the facile discovery of chemical rescuers of disease-associated protein misfolding. In this system, large combinatorial libraries of macrocyclic molecules are biosynthesized in Escherichia coli cells and simultaneously screened for their ability to rescue pathogenic protein misfolding and aggregation using a flow cytometric assay. We demonstrate the effectiveness of this approach by identifying drug-like, head-to-tail cyclic peptides that modulate the aggregation of the Alzheimer’s disease-associated amyloid β peptide. Biochemical, biophysical and biological assays using isolated amyloid β peptide, primary neurons and various established Alzheimer’s disease nematode models showed that the selected macrocycles potently inhibit the formation of neurotoxic amyloid β peptide aggregates. We also applied the system to the identification of misfolding rescuers of mutant Cu/Zn superoxide dismutase—an enzyme linked with inherited forms of amyotrophic lateral sclerosis. Overall, the system enables the identification of molecules with therapeutic potential for rescuing the misfolding of disease-associated polypeptides.A fluorescence-based assay is used to screen cyclic peptides for their activity in preventing protein misfolding, an event that can generate pathogenic aggregates that lead to diseases such as Alzheimer’s disease or amyotrophic lateral sclerosis..
Journal of Alzheimer's Disease
Alterations in tau synaptic distribution are considered to underlie synaptic dysfunction observed... more Alterations in tau synaptic distribution are considered to underlie synaptic dysfunction observed in Alzheimer's disease (AD). In the present study, brain blood hypoperfusion was simulated in mouse brain slices, and tau levels and phosphorylation were investigated in total extracts, as well as in postsynaptic density fractions (PSDs) and non-PSDs obtained through differential extraction and centrifugation. Oxygen deprivation (OD) resulted in tau dephosphorylation at several AD-related residues and activation of GSK3β and phosphatase PP2A. On the contrary, glucose deprivation (GD) did not affect total levels of cellular tau or its phosphorylation despite inactivation of GSK3β. However, tau distribution in PSD and non-PSD fractions and the pattern of tau phosphorylation in these compartments is highly complex. In PSDs, tau was increased under GD conditions and decreased under OD conditions. GD resulted in tau dephosphorylation at Ser199, Ser262, and Ser396 while OD resulted in tau hyperphosphorylation at Ser199 and Ser404. In the non-PSD fraction, GD or OD resulted in lower levels of tau, but the phosphorylation status of tau was differentially affected. In GD conditions, tau was found dephosphorylated at Ser199, Thr205, and Ser404 and hyperphosphorylated at Ser262. However, in OD conditions tau was found hyperphosphorylated at Thr205, SerSer356, Ser396, and Ser404. Combined OD and GD resulted in degradation of cellular tau and dephosphorylation of PSD tau at Ser396 and Ser404. These results indicate that oxygen deprivation causes dephosphorylation of tau, while GD and OD differentially affect distribution of total tau and tau phosphorylation variants in neuronal compartments by activating different mechanisms.
Eur Neuropsychopharmacol, 1996
Neurobiology of Aging, 2000
Mart cases ofearly onset familial Alzheimer's disease (FAD) are cawed by mutations in presendin 1... more Mart cases ofearly onset familial Alzheimer's disease (FAD) are cawed by mutations in presendin 1 gene. We found that in epithelial cells, presenilin I (PSI) protein localizes at cell-cell contact sites and forms complexes with the cadherin-based adhere"\ junctions. The cytoplarmic domain of cell surface cadherin regulates cell-cell adhesion by interacting with soluble protein factors, including p-and y-catenin. We used E-cadherin deletion mutants which lack the p-, and y-catenin binding wquence to show that the PSl/E-cadherin interaction is Independent of the catenin binding. Cross-linking experiments revealed that the cleaved carboxyterminal fragment of PSI binds directly to E-cadherin and an I I amino acid sequence in the cytoplasmic domain of E-cadherin is necessary for this interaction. Furhtermore, absence of PSI destabilizes both the E-cadherin/P-catenin and E-cadherin/ycatenin complexes. Thus. our data shows that PSI binda directly to the cytopla\mic domam of E-cadherin and atabilizs the cadherin/catemn cell-cell adhesmn complex. Adherens junctions regulate cell-cell adhesion/communication end play important roles not only in organogene\is but also in tissue function of adult organisms.
Neurobiology of Aging, 2000
Journal of Alzheimer's disease : JAD, 2015
Amyloid-β protein precursor (AβPP) metabolism and the accumulation of its derivative amyloid-β (A... more Amyloid-β protein precursor (AβPP) metabolism and the accumulation of its derivative amyloid-β (Aβ) peptide in senile plaques have been considered key players in the development of Alzheimer's disease (AD). However, the mechanisms underlying the generation and the deposition of Aβ are not clear but emphasis has been given in the role of AβPP protein interactions that regulate its processing and offer a means to manipulate Aβ production. We have previously shown that AβPP interacts with members of the Homer protein family, which leads to inhibition of Aβ generation. Herein, we studied the structural parameters of AβPP/Homer3 interaction by analyzing the sequences and domains that play a role in the formation of the complex. We found that the cytoplasmic tail of AβPP is necessary for the interaction. Regarding Homer3, we report that both the EVH1 protein interacting domain and the polymerization coiled coil domain are essential for the complex assembly. Importantly, phosphorylatio...
Current Alzheimer research, Jan 30, 2013
BRI2, a protein mutated in Familial British and Familial Danish Dementias, interacts with Amyloid... more BRI2, a protein mutated in Familial British and Familial Danish Dementias, interacts with Amyloid Precursor Protein (APP) and reduces the levels of secreted APPβ (sAPPβ), which derives from APP cleavage by β-secretase (BACE1). Exploring the mechanisms of this effect, we obtained data that BRI2 decreases the cellular levels of BACE1 thus reducing the β-cleavage of APP. Deletion of N-terminal cytoplasmic or C-terminal extracellular sequences of BRI2 neither affected its interaction with BACE1 or APP (Fotinopoulou et al., 2005) nor the reduction in the levels of BACE1 and sAPPβ. These results suggest that BRI2 may prevent access of BACE1 to APP and the BRI2/BACE1 interaction may mediate the reduction in BACE1 levels. In support, BRI2 expression induced lysosomal but not proteasomal degradation of BACE1. In parallel, BRI2 expression was also found to reduce BACE1 mRNA levels by 50%. This study adds novel information regarding the mechanism by which BRI2 affects APP processing and BACE1 ...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1997
Appicans are secreted or cell-associated brain chondroitin sulfate proteoglycans produced by glia... more Appicans are secreted or cell-associated brain chondroitin sulfate proteoglycans produced by glia cells and containing Alzheimer amyloid precursor protein (APP) as a core protein. Here, we report that rat C6 glioma cells transfected with appican displayed a dramatic change in their phenotypic appearance compared with untransfected cells or cells transfected with APP. Appican-transfected cells lost the round appearance of the untransfected control C6 cells, acquired a flat morphology, and elaborated more processes than control cells. Untransfected, or APP-transfected C6, cells were completely dissociated from their substrate after 40 min of treatment with cell dissociation solution. Under the same conditions, however, <20% of the appican-transfected C6 cells were dissociated from their substrate, suggesting that the appican-transfected glia cells attach more avidly to their substrate than do untransfected or APP transfected control cells. In contrast, appican-transfected fibroblas...
Molecular psychiatry, 1996
Amyloid beta-peptide (A beta) deposition and loss of cholinergic neurons are characteristics of A... more Amyloid beta-peptide (A beta) deposition and loss of cholinergic neurons are characteristics of Alzheimer's disease. There is evidence that A beta is neurotoxic. The role of signal transduction pathways on A beta-induced toxicity in PC12 cells was investigated. Our results revealed that A beta-induced arachidonic acid was released in a time-dependent manner. Inhibitors of cyclooxygenase (1 microM indomethacin) and lipooxygenase (100 microM nordihydroguairetic acid) protected PC12 cells against A beta-induced toxicity. These data suggest that A beta toxicity is mediated by activation of the arachidonic acid cascade. Furthermore, protein kinase C activators (phorbol ester and 1-oleyl-2-acetyl-glycerol) and tacrine reversed A beta-induced toxicity. These results suggest that A beta toxicity can be modulated by manipulating signal transduction pathways and may provide the basis for novel therapeutic interventions.
Neuroreport, 1998
The majority of early-onset familial Alzheimer's disease (FAD) is associated with mutatio... more The majority of early-onset familial Alzheimer's disease (FAD) is associated with mutations in the presenilin-1 (PS1) gene. We describe a novel Polish PS1 mutation of Pro117Leu, associated with the earliest average age of onset and death so far reported in a PS-linked, FAD kindred. Human kidney 293 and mouse neuroblastoma N2a cells were stably transfected with wild-type and PS1 P117L. There was a significant increase in the amyloid beta42/40 ratio in the N2a P117L PS1 transfected cells compared with N2a transfected with wild-type PS1. What role PS has in the pathogenesis of AD remains to be determined, however, the severity of the clinical picture associated with this PS1 mutation stresses the importance of presenilin.
Journal of Alzheimers Disease, 2013
The amyloid-β protein precursor (AβPP) is a type-1 transmembrane protein involved in Alzheimer&am... more The amyloid-β protein precursor (AβPP) is a type-1 transmembrane protein involved in Alzheimer&amp;#39;s disease (AD). It has become increasingly evident that AβPP, its protein-protein interactions, and its proteolytical fragments may affect calcium homeostasis and vice versa. In addition, there is evidence that calcium dysregulation contributes to AD. To study the role of AβPP in calcium homeostasis, we downregulated its expression in SH-SY5Y cells using shRNA (SH-SY5Y/AβPP-) or increased expression of AβPP695 by transfection (SH-SY5Y/AβPP+). The levels of cytosolic Ca2+ after treatment with thapsigargin, monensin, activation of capacitative calcium entry (CCE), and treatment with SKF, a store operated channel (SOCs) inhibitor, were measured by fura-2AM fluorimetry. SH-SY5Y/AβPP+ cells show reduced response to thapsigargin and reduced CCE, although this reduction is not statistically significant. On the other hand, we found that, relative to SH-SY5Y, SH-SY5Y/AβPP- cells show a significant increase in the response to thapsigargin but not in CCE and their SOCs were more susceptible to SKF inhibition. Additionally, downregulation of AβPP resulted in increased response to monensin that induces calcium release from acidic stores. The increase of calcium release from the endoplasmic reticulum and the acidic stores, when AβPP is downregulated, could be attributed to elevated Ca2+ content or to a dysregulation of Ca2+ transfer through their membranes. These data, along with already existing evidence regarding the role of AβPP in calcium homeostasis and the early occurring structural and functional abnormalities of endosomes, further substantiate the role of AβPP in calcium homeostasis and in AD.
The EMBO Journal, 2002
E-cadherin controls a wide array of cellular behaviors including cell±cell adhesion, differentiat... more E-cadherin controls a wide array of cellular behaviors including cell±cell adhesion, differentiation and tissue development. Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, controls a g-secretase-like cleavage of E-cadherin. This cleavage is stimulated by apoptosis or calcium in¯ux and occurs between human E-cadherin residues Leu731 and Arg732 at the membrane±cytoplasm interface. The PS1/g-secretase system cleaves both the full-length E-cadherin and a transmembrane C-terminal fragment, derived from a metalloproteinase cleavage after the E-cadherin ectodomain residue Pro700. The PS1/ g-secretase cleavage dissociates E-cadherins, b-catenin and a-catenin from the cytoskeleton, thus promoting dis-assembly of the E-cadherin±catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E-cadherin to the cytosol and increases the levels of soluble band a-catenins. Thus, the PS1/g-secretase system stimulates disassembly of the E-cadherin± catenin complex and increases the cytosolic pool of b-catenin, a key regulator of the Wnt signaling pathway.
Proceedings of the National Academy of Sciences, 2001
Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, binds directly t... more 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 p120͞E-cadherin binding, and increasing p120 levels suppresses PS1͞E-cadherin binding. Thus PS1 and p120 bind to and mutually compete for cellular E-cadherin. Furthermore, PS1 stimulates E-cadherin binding to and ␥-catenin, promotes cytoskeletal association of the cad-herin͞catenin complexes, and increases Ca 2؉-dependent cell-cell aggregation. Remarkably, PS1 familial Alzheimer disease mutant ⌬E9 increased neither the levels of cadherin͞catenin 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. P resenilin-1 (PS1) mutations are responsible for most cases of early-onset familial Alzheimer's disease (FAD). PS1 is a transmembrane protein expressed in many tissues, including brain, where it is enriched in neurons (1, 2). PS1 crosses the membrane eight times, with the N terminus, the C terminus, and the large hydrophilic loop all located in the cytoplasm. Most cellular PS1 is cleaved within the large cytoplasmic loop to yield N-terminal fragments (PS1͞NTF) of approximately 30 kDa and C-terminal fragments (PS1͞CTF) of approximately 20 kDa. After cleavage, the PS1 fragments form stable 1:1 heterodimers (3, 4). PS1 facilitates processing of Notch-1 receptor, and amyloid precursor protein (APP) (5, 6) stimulates production of A peptide (6) and may play a role in neuroprotection (7, 8). Mice lacking PS1 (PS1Ϫ͞Ϫ) die shortly after birth with skeletal malformations, impaired neurogenesis, and brain hemorrhage (7). Recently, we reported that PS1 concentrates at synaptic and epithelial cell-cell contact sites, where it forms complexes with the cadherin͞catenin adhesion system (9). Classic cadherins, including E-cadherin and neuronal cadherin (N-cadherin), are a family of type I transmembrane proteins that mediate Ca 2ϩdependent cell-cell adhesion and recognition, and control critical events in neurogenesis, tissue development, and tissue homeostasis (10, 11). These functions are mediated by homophilic interactions of the extracellular region of cadherins. The membrane-distal cytoplasmic sequence of cadherins binds either -catenin or ␥-catenin (plakoglobin), which in turn binds ␣-catenin. The latter protein binds polymerized actin, thus linking the cadherin͞catenin adhesion complex to the cortical cytoskeleton. Cytoskeletal linkage of the cadherin͞catenin complexes is crucial for the full expression of the adhesive functions of surface cadherins (12-14). -Catenin and ␥-catenin, two highly homologous members of the armadillo family of proteins (for a review see ref. 15), bind the same sequence of cytoplasmic cadherin in a mutually exclusive manner (16, 17). The juxtamembrane (membrane-proximal) region of cytoplasmic cadherins binds p120 (also called p120 ctn), a cytosolic protein originally identified as a target for p60 v-src kinase and later shown to regulate cadherin-mediated cell-cell adhesion and tumor metastasis (for a review see ref. 18). Although PS1 binds to the cadherin͞catenin adhesion complex (9), the mechanism and functional consequences of this association remain obscure. Here we show that PS1 binds directly to juxtamembrane cytoplasmic cadherin and inhibits cadherin binding of p120. Furthermore, PS1 stabilizes the cad-herin͞catenin complex, promotes its cytoskeletal association, and stimulates Ca 2ϩ-dependent cell-cell aggregation. In contrast, PS1 FAD mutant ⌬E9 failed to stabilize the cadherin͞ catenin complex and did not stimulate cell-cell aggregation. Materials and Methods Antibodies. Rabbit polyclonal antibody R222 specific for PS1͞ NTF amino acids 2-12 and mouse monoclonal antibody 33B10 specific for PS1͞CTF sequence 331-350 were prepared as described (9). Anti-transferrin receptor antibody was from Zymed, and anti-epithelial cadherin (E-cadherin) antibody H108 (rabbit polyclonal) was from Santa Cruz Biotechnology. Other antibodies against E-cadherin, -catenin, ␥-catenin, ␣-catenin, and p120 were from Transduction Laboratories (Lexington, KY). Unless otherwise stated, ''anti-E-cadherin antibody'' refers to anticytoplasmic E-cadherin antibody. Cell Cultures, Cell Aggregation, Immunoprecipitations (IPs), and Immunoblotting. Unless otherwise stated, cell cultures were grown in DMEM plus 10% FBS, penicillin, and streptomycin in 5% CO 2 at 37°C. Fibroblast cell lines were from wild-type (WT) (PS1 ϩ͞ϩ) and PS1 knockout (PS1Ϫ͞Ϫ) mice (9), and L cells were from the American Type Culture Collection. Stable transfections
Proceedings of the National Academy of Sciences, 1996
The Af3 peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid pr... more The Af3 peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of
Nature Biomedical Engineering, 2018
In the version of this Article originally published, in Fig. 1c-e, on the x axes, the lines label... more In the version of this Article originally published, in Fig. 1c-e, on the x axes, the lines labelled ' Aβ 42 ' and ' Aβ 42 (F19S;L34P)' grouped the data incorrectly; the line labelled Aβ 42 should have grouped the data for Random 1-2 and Clones 1-10, and the line labelled Aβ 42 (F19S;L34P) should have only grouped the data for Random 1-2 on the right end of the plots and blots. These figures have now been corrected in all versions of the Article.
Nature Biomedical Engineering
Protein misfolding and aggregation are common pathological features of several human diseases, in... more Protein misfolding and aggregation are common pathological features of several human diseases, including Alzheimer’s disease and type 2 diabetes. Here, we report an integrated and generalizable bacterial system for the facile discovery of chemical rescuers of disease-associated protein misfolding. In this system, large combinatorial libraries of macrocyclic molecules are biosynthesized in Escherichia coli cells and simultaneously screened for their ability to rescue pathogenic protein misfolding and aggregation using a flow cytometric assay. We demonstrate the effectiveness of this approach by identifying drug-like, head-to-tail cyclic peptides that modulate the aggregation of the Alzheimer’s disease-associated amyloid β peptide. Biochemical, biophysical and biological assays using isolated amyloid β peptide, primary neurons and various established Alzheimer’s disease nematode models showed that the selected macrocycles potently inhibit the formation of neurotoxic amyloid β peptide aggregates. We also applied the system to the identification of misfolding rescuers of mutant Cu/Zn superoxide dismutase—an enzyme linked with inherited forms of amyotrophic lateral sclerosis. Overall, the system enables the identification of molecules with therapeutic potential for rescuing the misfolding of disease-associated polypeptides.A fluorescence-based assay is used to screen cyclic peptides for their activity in preventing protein misfolding, an event that can generate pathogenic aggregates that lead to diseases such as Alzheimer’s disease or amyotrophic lateral sclerosis..
Journal of Alzheimer's Disease
Alterations in tau synaptic distribution are considered to underlie synaptic dysfunction observed... more Alterations in tau synaptic distribution are considered to underlie synaptic dysfunction observed in Alzheimer's disease (AD). In the present study, brain blood hypoperfusion was simulated in mouse brain slices, and tau levels and phosphorylation were investigated in total extracts, as well as in postsynaptic density fractions (PSDs) and non-PSDs obtained through differential extraction and centrifugation. Oxygen deprivation (OD) resulted in tau dephosphorylation at several AD-related residues and activation of GSK3β and phosphatase PP2A. On the contrary, glucose deprivation (GD) did not affect total levels of cellular tau or its phosphorylation despite inactivation of GSK3β. However, tau distribution in PSD and non-PSD fractions and the pattern of tau phosphorylation in these compartments is highly complex. In PSDs, tau was increased under GD conditions and decreased under OD conditions. GD resulted in tau dephosphorylation at Ser199, Ser262, and Ser396 while OD resulted in tau hyperphosphorylation at Ser199 and Ser404. In the non-PSD fraction, GD or OD resulted in lower levels of tau, but the phosphorylation status of tau was differentially affected. In GD conditions, tau was found dephosphorylated at Ser199, Thr205, and Ser404 and hyperphosphorylated at Ser262. However, in OD conditions tau was found hyperphosphorylated at Thr205, SerSer356, Ser396, and Ser404. Combined OD and GD resulted in degradation of cellular tau and dephosphorylation of PSD tau at Ser396 and Ser404. These results indicate that oxygen deprivation causes dephosphorylation of tau, while GD and OD differentially affect distribution of total tau and tau phosphorylation variants in neuronal compartments by activating different mechanisms.
Eur Neuropsychopharmacol, 1996
Neurobiology of Aging, 2000
Mart cases ofearly onset familial Alzheimer's disease (FAD) are cawed by mutations in presendin 1... more Mart cases ofearly onset familial Alzheimer's disease (FAD) are cawed by mutations in presendin 1 gene. We found that in epithelial cells, presenilin I (PSI) protein localizes at cell-cell contact sites and forms complexes with the cadherin-based adhere"\ junctions. The cytoplarmic domain of cell surface cadherin regulates cell-cell adhesion by interacting with soluble protein factors, including p-and y-catenin. We used E-cadherin deletion mutants which lack the p-, and y-catenin binding wquence to show that the PSl/E-cadherin interaction is Independent of the catenin binding. Cross-linking experiments revealed that the cleaved carboxyterminal fragment of PSI binds directly to E-cadherin and an I I amino acid sequence in the cytoplasmic domain of E-cadherin is necessary for this interaction. Furhtermore, absence of PSI destabilizes both the E-cadherin/P-catenin and E-cadherin/ycatenin complexes. Thus. our data shows that PSI binda directly to the cytopla\mic domam of E-cadherin and atabilizs the cadherin/catemn cell-cell adhesmn complex. Adherens junctions regulate cell-cell adhesion/communication end play important roles not only in organogene\is but also in tissue function of adult organisms.
Neurobiology of Aging, 2000
Journal of Alzheimer's disease : JAD, 2015
Amyloid-β protein precursor (AβPP) metabolism and the accumulation of its derivative amyloid-β (A... more Amyloid-β protein precursor (AβPP) metabolism and the accumulation of its derivative amyloid-β (Aβ) peptide in senile plaques have been considered key players in the development of Alzheimer's disease (AD). However, the mechanisms underlying the generation and the deposition of Aβ are not clear but emphasis has been given in the role of AβPP protein interactions that regulate its processing and offer a means to manipulate Aβ production. We have previously shown that AβPP interacts with members of the Homer protein family, which leads to inhibition of Aβ generation. Herein, we studied the structural parameters of AβPP/Homer3 interaction by analyzing the sequences and domains that play a role in the formation of the complex. We found that the cytoplasmic tail of AβPP is necessary for the interaction. Regarding Homer3, we report that both the EVH1 protein interacting domain and the polymerization coiled coil domain are essential for the complex assembly. Importantly, phosphorylatio...
Current Alzheimer research, Jan 30, 2013
BRI2, a protein mutated in Familial British and Familial Danish Dementias, interacts with Amyloid... more BRI2, a protein mutated in Familial British and Familial Danish Dementias, interacts with Amyloid Precursor Protein (APP) and reduces the levels of secreted APPβ (sAPPβ), which derives from APP cleavage by β-secretase (BACE1). Exploring the mechanisms of this effect, we obtained data that BRI2 decreases the cellular levels of BACE1 thus reducing the β-cleavage of APP. Deletion of N-terminal cytoplasmic or C-terminal extracellular sequences of BRI2 neither affected its interaction with BACE1 or APP (Fotinopoulou et al., 2005) nor the reduction in the levels of BACE1 and sAPPβ. These results suggest that BRI2 may prevent access of BACE1 to APP and the BRI2/BACE1 interaction may mediate the reduction in BACE1 levels. In support, BRI2 expression induced lysosomal but not proteasomal degradation of BACE1. In parallel, BRI2 expression was also found to reduce BACE1 mRNA levels by 50%. This study adds novel information regarding the mechanism by which BRI2 affects APP processing and BACE1 ...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1997
Appicans are secreted or cell-associated brain chondroitin sulfate proteoglycans produced by glia... more Appicans are secreted or cell-associated brain chondroitin sulfate proteoglycans produced by glia cells and containing Alzheimer amyloid precursor protein (APP) as a core protein. Here, we report that rat C6 glioma cells transfected with appican displayed a dramatic change in their phenotypic appearance compared with untransfected cells or cells transfected with APP. Appican-transfected cells lost the round appearance of the untransfected control C6 cells, acquired a flat morphology, and elaborated more processes than control cells. Untransfected, or APP-transfected C6, cells were completely dissociated from their substrate after 40 min of treatment with cell dissociation solution. Under the same conditions, however, <20% of the appican-transfected C6 cells were dissociated from their substrate, suggesting that the appican-transfected glia cells attach more avidly to their substrate than do untransfected or APP transfected control cells. In contrast, appican-transfected fibroblas...
Molecular psychiatry, 1996
Amyloid beta-peptide (A beta) deposition and loss of cholinergic neurons are characteristics of A... more Amyloid beta-peptide (A beta) deposition and loss of cholinergic neurons are characteristics of Alzheimer's disease. There is evidence that A beta is neurotoxic. The role of signal transduction pathways on A beta-induced toxicity in PC12 cells was investigated. Our results revealed that A beta-induced arachidonic acid was released in a time-dependent manner. Inhibitors of cyclooxygenase (1 microM indomethacin) and lipooxygenase (100 microM nordihydroguairetic acid) protected PC12 cells against A beta-induced toxicity. These data suggest that A beta toxicity is mediated by activation of the arachidonic acid cascade. Furthermore, protein kinase C activators (phorbol ester and 1-oleyl-2-acetyl-glycerol) and tacrine reversed A beta-induced toxicity. These results suggest that A beta toxicity can be modulated by manipulating signal transduction pathways and may provide the basis for novel therapeutic interventions.
Neuroreport, 1998
The majority of early-onset familial Alzheimer's disease (FAD) is associated with mutatio... more The majority of early-onset familial Alzheimer's disease (FAD) is associated with mutations in the presenilin-1 (PS1) gene. We describe a novel Polish PS1 mutation of Pro117Leu, associated with the earliest average age of onset and death so far reported in a PS-linked, FAD kindred. Human kidney 293 and mouse neuroblastoma N2a cells were stably transfected with wild-type and PS1 P117L. There was a significant increase in the amyloid beta42/40 ratio in the N2a P117L PS1 transfected cells compared with N2a transfected with wild-type PS1. What role PS has in the pathogenesis of AD remains to be determined, however, the severity of the clinical picture associated with this PS1 mutation stresses the importance of presenilin.
Journal of Alzheimers Disease, 2013
The amyloid-β protein precursor (AβPP) is a type-1 transmembrane protein involved in Alzheimer&am... more The amyloid-β protein precursor (AβPP) is a type-1 transmembrane protein involved in Alzheimer&amp;#39;s disease (AD). It has become increasingly evident that AβPP, its protein-protein interactions, and its proteolytical fragments may affect calcium homeostasis and vice versa. In addition, there is evidence that calcium dysregulation contributes to AD. To study the role of AβPP in calcium homeostasis, we downregulated its expression in SH-SY5Y cells using shRNA (SH-SY5Y/AβPP-) or increased expression of AβPP695 by transfection (SH-SY5Y/AβPP+). The levels of cytosolic Ca2+ after treatment with thapsigargin, monensin, activation of capacitative calcium entry (CCE), and treatment with SKF, a store operated channel (SOCs) inhibitor, were measured by fura-2AM fluorimetry. SH-SY5Y/AβPP+ cells show reduced response to thapsigargin and reduced CCE, although this reduction is not statistically significant. On the other hand, we found that, relative to SH-SY5Y, SH-SY5Y/AβPP- cells show a significant increase in the response to thapsigargin but not in CCE and their SOCs were more susceptible to SKF inhibition. Additionally, downregulation of AβPP resulted in increased response to monensin that induces calcium release from acidic stores. The increase of calcium release from the endoplasmic reticulum and the acidic stores, when AβPP is downregulated, could be attributed to elevated Ca2+ content or to a dysregulation of Ca2+ transfer through their membranes. These data, along with already existing evidence regarding the role of AβPP in calcium homeostasis and the early occurring structural and functional abnormalities of endosomes, further substantiate the role of AβPP in calcium homeostasis and in AD.
The EMBO Journal, 2002
E-cadherin controls a wide array of cellular behaviors including cell±cell adhesion, differentiat... more E-cadherin controls a wide array of cellular behaviors including cell±cell adhesion, differentiation and tissue development. Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, controls a g-secretase-like cleavage of E-cadherin. This cleavage is stimulated by apoptosis or calcium in¯ux and occurs between human E-cadherin residues Leu731 and Arg732 at the membrane±cytoplasm interface. The PS1/g-secretase system cleaves both the full-length E-cadherin and a transmembrane C-terminal fragment, derived from a metalloproteinase cleavage after the E-cadherin ectodomain residue Pro700. The PS1/ g-secretase cleavage dissociates E-cadherins, b-catenin and a-catenin from the cytoskeleton, thus promoting dis-assembly of the E-cadherin±catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E-cadherin to the cytosol and increases the levels of soluble band a-catenins. Thus, the PS1/g-secretase system stimulates disassembly of the E-cadherin± catenin complex and increases the cytosolic pool of b-catenin, a key regulator of the Wnt signaling pathway.
Proceedings of the National Academy of Sciences, 2001
Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, binds directly t... more 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 p120͞E-cadherin binding, and increasing p120 levels suppresses PS1͞E-cadherin binding. Thus PS1 and p120 bind to and mutually compete for cellular E-cadherin. Furthermore, PS1 stimulates E-cadherin binding to and ␥-catenin, promotes cytoskeletal association of the cad-herin͞catenin complexes, and increases Ca 2؉-dependent cell-cell aggregation. Remarkably, PS1 familial Alzheimer disease mutant ⌬E9 increased neither the levels of cadherin͞catenin 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. P resenilin-1 (PS1) mutations are responsible for most cases of early-onset familial Alzheimer's disease (FAD). PS1 is a transmembrane protein expressed in many tissues, including brain, where it is enriched in neurons (1, 2). PS1 crosses the membrane eight times, with the N terminus, the C terminus, and the large hydrophilic loop all located in the cytoplasm. Most cellular PS1 is cleaved within the large cytoplasmic loop to yield N-terminal fragments (PS1͞NTF) of approximately 30 kDa and C-terminal fragments (PS1͞CTF) of approximately 20 kDa. After cleavage, the PS1 fragments form stable 1:1 heterodimers (3, 4). PS1 facilitates processing of Notch-1 receptor, and amyloid precursor protein (APP) (5, 6) stimulates production of A peptide (6) and may play a role in neuroprotection (7, 8). Mice lacking PS1 (PS1Ϫ͞Ϫ) die shortly after birth with skeletal malformations, impaired neurogenesis, and brain hemorrhage (7). Recently, we reported that PS1 concentrates at synaptic and epithelial cell-cell contact sites, where it forms complexes with the cadherin͞catenin adhesion system (9). Classic cadherins, including E-cadherin and neuronal cadherin (N-cadherin), are a family of type I transmembrane proteins that mediate Ca 2ϩdependent cell-cell adhesion and recognition, and control critical events in neurogenesis, tissue development, and tissue homeostasis (10, 11). These functions are mediated by homophilic interactions of the extracellular region of cadherins. The membrane-distal cytoplasmic sequence of cadherins binds either -catenin or ␥-catenin (plakoglobin), which in turn binds ␣-catenin. The latter protein binds polymerized actin, thus linking the cadherin͞catenin adhesion complex to the cortical cytoskeleton. Cytoskeletal linkage of the cadherin͞catenin complexes is crucial for the full expression of the adhesive functions of surface cadherins (12-14). -Catenin and ␥-catenin, two highly homologous members of the armadillo family of proteins (for a review see ref. 15), bind the same sequence of cytoplasmic cadherin in a mutually exclusive manner (16, 17). The juxtamembrane (membrane-proximal) region of cytoplasmic cadherins binds p120 (also called p120 ctn), a cytosolic protein originally identified as a target for p60 v-src kinase and later shown to regulate cadherin-mediated cell-cell adhesion and tumor metastasis (for a review see ref. 18). Although PS1 binds to the cadherin͞catenin adhesion complex (9), the mechanism and functional consequences of this association remain obscure. Here we show that PS1 binds directly to juxtamembrane cytoplasmic cadherin and inhibits cadherin binding of p120. Furthermore, PS1 stabilizes the cad-herin͞catenin complex, promotes its cytoskeletal association, and stimulates Ca 2ϩ-dependent cell-cell aggregation. In contrast, PS1 FAD mutant ⌬E9 failed to stabilize the cadherin͞ catenin complex and did not stimulate cell-cell aggregation. Materials and Methods Antibodies. Rabbit polyclonal antibody R222 specific for PS1͞ NTF amino acids 2-12 and mouse monoclonal antibody 33B10 specific for PS1͞CTF sequence 331-350 were prepared as described (9). Anti-transferrin receptor antibody was from Zymed, and anti-epithelial cadherin (E-cadherin) antibody H108 (rabbit polyclonal) was from Santa Cruz Biotechnology. Other antibodies against E-cadherin, -catenin, ␥-catenin, ␣-catenin, and p120 were from Transduction Laboratories (Lexington, KY). Unless otherwise stated, ''anti-E-cadherin antibody'' refers to anticytoplasmic E-cadherin antibody. Cell Cultures, Cell Aggregation, Immunoprecipitations (IPs), and Immunoblotting. Unless otherwise stated, cell cultures were grown in DMEM plus 10% FBS, penicillin, and streptomycin in 5% CO 2 at 37°C. Fibroblast cell lines were from wild-type (WT) (PS1 ϩ͞ϩ) and PS1 knockout (PS1Ϫ͞Ϫ) mice (9), and L cells were from the American Type Culture Collection. Stable transfections
Proceedings of the National Academy of Sciences, 1996
The Af3 peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid pr... more The Af3 peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of