Spiros Efthimiopoulos

A presenilin-1/γ-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions

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 Alzheimers disease, controls a γ‐secretase‐like cleavage of E‐cadherin. This cleavage is stimulated by apoptosis or calcium influx and occurs between human E‐cadherin residues Leu731 and Arg732 at the membrane–cytoplasm interface. The PS1/γ‐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/γ‐secretase cleavage dissociates E‐cadherins, β‐catenin and α‐catenin from the cytoskeleton, thus promoting disassembly of the E‐cadherin–catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E‐cadherin to the cytosol and increases the levels of soluble β‐ and α‐catenins. Thus, the PS1/γ‐secretase system stimulates disassembly of the E‐cadherin– catenin complex and increases the cytosolic pool of β‐catenin, a key regulator of the Wnt signaling pathway.

Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts.

In MDCK cells, presenilin-1 (PS1) accumulates at intercellular contacts where it colocalizes with components of the cadherin-based adherens junctions. PS1 fragments form complexes with E-cadherin, beta-catenin, and alpha-catenin, all components of adherens junctions. In confluent MDCK cells, PS1 forms complexes with cell surface E-cadherin; disruption of Ca(2+)-dependent cell-cell contacts reduces surface PS1 and the levels of PS1-E-cadherin complexes. PS1 overexpression in human kidney cells enhances cell-cell adhesion. Together, these data show that PS1 incorporates into the cadherin/catenin adhesion system and regulates cell-cell adhesion. PS1 concentrates at intercellular contacts in epithelial tissue; in brain, it forms complexes with both E- and N-cadherin and concentrates at synaptic adhesions. That PS1 is a constituent of the cadherin/catenin complex makes that complex a potential target for PS1 FAD mutations.

Presenilin-1 binds cytoplasmic epithelial cadherin, inhibits cadherin/p120 association, and regulates stability and function of the cadherin/catenin adhesion complex

Here we show that presenilin-1 (PS1), a protein involved in Alzheimers 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 Ca2+-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.

A novel Polish presenilin-1 mutation (P117L) is associated with familial Alzheimer's disease and leads to death as early as the age of 28 years.

THE majority of early-onset familial Alzheimers 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 β42/40 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.

Intracellular Cyclic AMP Inhibits Constitutive and Phorbol Ester‐Stimulated Secretory Cleavage of Amyloid Precursor Protein

Abstract: α‐Secretase cleaves the full‐length Alzheimers amyloid precursor protein (APP) within the amyloid β peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP751 to examine whether the α‐secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester‐stimulated secretion of nexin II (NXII), the secreted product of the α‐secretase cleavage of APP751. At 1 µM, forskolin inhibited secretion of NXII by ∼50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9‐dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the α‐secretase cleavage of APP751. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.

Distinct Secretases, a Cysteine Protease and a Serine Protease, Generate the C Termini of Amyloid β-Proteins Aβ1-40 and Aβ1-42, Respectively

Abstract: The carboxy‐terminal ends of the 40‐ and 42‐amino acids amyloid β‐protein (Aβ) may be generated by the action of at least two different proteases termed γ(40)‐ and γ(42)‐secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)‐transfected cell cultures with several dipeptidyl aldehydes including N‐benzyloxycarbonyl‐Leu‐leucinal (Z‐LL‐CHO) and the newly synthesized N‐benzyloxycarbonyl‐Val‐leucinal (Z‐VL‐CHO). All dipeptidyl aldehydes tested inhibited production of both Aβ1‐40 and Aβ1‐42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of γ‐secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two γ‐secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E‐64d. This agent inhibited production of secreted Aβ1‐40, with a concomitant accumulation of its cellular precursor indicating that γ(40)‐secretase is a cysteine protease. In contrast, this treatment increased production of secreted Aβ1‐42. No inhibition of Aβ production was observed with the potent calpain inhibitor I (acetyl‐Leu‐Leu‐norleucinal), suggesting that calpain is not involved. Together, these results indicate that γ(40)‐secretase is a cysteine protease distinct from calpain, whereas γ(42)‐secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the β‐secretase C‐terminal APP fragment and a decrease of the α‐secretase C‐terminal APP fragment, indicating that this mutation shifts APP cleavage from the α‐secretase site to the β‐secretase site.

Enrichment of Presenilin 1 Peptides in Neuronal Large Dense‐Core and Somatodendritic Clathrin‐Coated Vesicles

Abstract: Presenilin 1 is an integral membrane protein specifically cleaved to yield an N‐terminal and a C‐terminal fragment, both membrane‐associated. More than 40 presenilin 1 mutations have been linked to early‐onset familial Alzheimer disease, although the mechanism by which these mutations induce the Alzheimer disease neuropathology is not clear. Presenilin 1 is expressed predominantly in neurons, suggesting that the familial Alzheimer disease mutants may compromise or change the neuronal function(s) of the wild‐type protein. To elucidate the function of this protein, we studied its expression in neuronal vesicular systems using as models the chromaffin granules of the neuroendocrine chromaffin cells and the major categories of brain neuronal vesicles, including the small clear‐core synaptic vesicles, the large dense‐core vesicles, and the somatodendritic and nerve terminal clathrin‐coated vesicles. Both the N‐ and C‐terminal presenilin 1 proteolytic fragments were greatly enriched in chromaffin granule and neuronal large dense‐core vesicle membranes, indicating that these fragments are targeted to these vesicles and may regulate the large dense‐core vesicle‐mediated secretion of neuropeptides and neurotransmitters at synaptic sites. The presenilin 1 fragments were also enriched in the somatodendritic clathrin‐coated vesicle membranes, suggesting that they are targeted to the somatodendritic membrane, where they may regulate constitutive secretion and endocytosis. In contrast, these fragments were not enriched in the small clear‐core synaptic vesicle or in the nerve terminal clathrin‐coated vesicle membranes. Taken together, our data indicate that presenilin 1 proteolytic fragments are targeted to specific populations of neuronal vesicles where they may regulate vesicular function. Although full‐length presenilin 1 was present in crude homogenates, it was not detected in any of the vesicles studied, indicating that, unlike the presenilin fragments, full‐length protein may not have a vesicular function.

Inhibition of neurite outgrowth by familial Alzheimer's disease-linked presenilin-1 mutations

Two (P117L; M146L) familial Alzheimers disease (FAD)-causing presenilin-1 (PS1) mutations have been tested fortheir effect in stably transfected mouse neuroblastoma (N2a) cell lines. The P117L mutation is associated with the earliest onset of AD reported so far (24 years), while the M146L is less pathogenic with the onset at about 43 years. Overexpression of wild-type (wt) PS1 gene was associated with the marked increase in the number and the length of neuritic outgrowths accompanied by accumulation of PS1 immunoreactivity in neurites. The highly pathogenic P117L mutation completely suppressed this effect and the pattern of PS1 immunolabeling resembled a cup structure with all immunoreactivity gathered at one pole of the cell. The effect of less pathogenic M146L mutation was similar, but not as pronounced. These findings suggest that one of the normal functions of PS1 may be the control of neurite outgrowth, and the inhibitory effect of two FAD-linked mutations stresses its importance in the cellular mechanism that leads to the development of Alzheimers disease (AD).

Chondroitin sulfate proteoglycan form of cellular and cell-surface Alzheimer amyloid precursor

The biological function of the amyloid precursor protein (APP) is still not fully understood. Recently, we reported that secreted truncated APP occurs in a chondroitin sulfate proteoglycan form. Here we present evidence that full length APP-chondroitin sulfate proteoglycan is present on the cell surface of C6 glioma cells. In addition, densitometric quantitation of Western blots showed that approximately 50% of the mature cell-associated full length APP is in the proteoglycan form. These findings suggest that the proteoglycan nature of APP may be important for the implementation of its biological function.

THE KUNITZ PROTEASE INHIBITOR FORM OF THE AMYLOID PRECURSOR PROTEIN (KPI/APP) INHIBITS THE PRONEUROPEPTIDE PROCESSING ENZYME PROHORMONE THIOL PROTEASE (PTP): COLOCALIZATION OF KPI/APP AND PTP IN SECRETORY VESICLES

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 K i ,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.