Anastasios Georgakopoulos

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.

Metalloproteinase/Presenilin1 processing of ephrinB regulates EphB-induced Src phosphorylation and signaling.

Bidirectional signaling triggered by interacting ephrinB receptors (EphB) and ephrinB ligands is crucial for development and function of the vascular and nervous systems. A signaling cascade triggered by this interaction involves activation of Src kinase and phosphorylation of ephrinB. The mechanism, however, by which EphB activates Src in the ephrinB‐expressing cells is unknown. Here we show that EphB stimulates a metalloproteinase cleavage of ephrinB2, producing a carboxy‐terminal fragment that is further processed by PS1/γ‐secretase to produce intracellular peptide ephrinB2/CTF2. This peptide binds Src and inhibits its association with inhibitory kinase Csk, allowing autophosphorylation of Src at residue tyr418. EphrinB2/CTF2‐activated Src phosphorylates ephrinB2 and inhibits its processing by γ‐secretase. These data show that the PS1/γ‐secretase system controls Src activation and ephrinB phosphorylation by regulating production of Src activator ephrinB2/CTF2. Accordingly, γ‐secretase inhibitors prevented the EphB‐induced sprouting of endothelial cells and the recruitment of Grb4 to ephrinB. PS1 FAD and γ‐secretase dominant‐negative mutants inhibited the EphB‐induced cleavage of ephrinB2 and Src autophosphorylation, raising the possibility that FAD mutants interfere with the functions of Src and ephrinB2 in the CNS.

A Role for Noncoding Variation in Schizophrenia

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

Ligand binding and calcium influx induce distinct ectodomain/γ-secretase processing pathways of EPHB2 receptor

Binding of EphB receptors to ephrinB ligands on the surface of adjacent cells initiates signaling cascades that regulate angiogenesis, axonal guidance, and neuronal plasticity. These functions require processing of EphB receptors and removal of EphB-ephrinB complexes from the cell surface, but the mechanisms involved are poorly understood. Here we show that the ectodomain of EphB2 receptor is released to extracellular space following cleavage after EphB2 residue 543. The remaining membrane-associated fragment is cleaved by the presenilin-dependent γ-secretase activity after EphB2 residue 569 releasing an intracellular peptide that contains the cytoplasmic domain of EphB2. This cleavage is inhibited by presenilin 1 familial Alzheimer disease mutations. Processing of EphB2 receptor depends on specific treatments: ephrinB ligand-induced processing requires endocytosis, and the ectodomain cleavage is sensitive to peptide inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal but insensitive to metalloproteinase inhibitor GM6001. The ligand-induced processing takes place in endosomes and involves the rapid degradation of the extracellular EphB2. EphrinB ligand stimulates ubiquitination of EphB2 receptor. Calcium influx- and N-methyl-d-aspartic acid-induced processing of EphB2 is inhibited by GM6001 and ADAM10 inhibitors but not by N-benzyloxycarbonyl-Val-Leu-leucinal. This processing requires no endocytosis and promotes rapid shedding of extracellular EphB2, indicating that it takes place at the plasma membrane. Our data identify novel cleavages and modifications of EphB2 receptor and indicate that specific conditions determine the proteolytic systems and subcellular sites involved in the processing of this receptor.

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

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.

FAD mutants unable to increase neurotoxic Aβ 42 suggest that mutation effects on neurodegeneration may be independent of effects on Aβ

Strong support for a primary causative role of the Aβ 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 Aβ 1–42 (Aβ 42). This effect of FAD mutants is also reflected in an increased ratio of peptides Aβ 42 over Aβ 1–40 (Aβ 40). In the present study, we show that several presenilin 1 FAD mutants failed to increase production of Aβ 42 or the Aβ 42/40 ratio. Our data suggest that the mechanism by which FAD mutations promote neurodegeneration and AD may be independent of their effects on Aβ production.

Wild-Type But Not FAD Mutant Presenilin-1 Prevents Neuronal Degeneration by Promoting Phosphatidylinositol 3-Kinase Neuroprotective Signaling

The role of presenilin-1 (PS1) in neuronal phosphatidylinositol 3-kinase (PI3K)/Akt signaling was investigated in primary neuronal cultures from wild-type (WT) and PS1 null (PS1−/−) embryonic mouse brains. Here we show that in PS1−/− cultures, the onset of neuronal maturation coincides with a decrease in the PI3K-dependent phosphorylation-activation of Akt and phosphorylation-inactivation of glycogen synthase kinase-3 (GSK-3). Mature PS1−/− neurons show increased activation of apoptotic caspase-3 and progressive degeneration preceded by dendritic retraction. Expression of exogenous WT PS1 or constitutively active Akt in PS1−/− neurons stimulates PI3K signaling and suppresses both caspase-3 activity and dendrite retraction. The survival effects of PS1 are sensitive to inhibitors of PI3K kinase but insensitive to γ-secretase inhibitors. Familial Alzheimer disease (FAD) mutations suppress the ability of PS1 to promote PI3K/AKT signaling, prevent phosphorylation/inactivation of GSK-3 and promote activation of caspase-3. These mutation effects are reversed upon coexpression of constitutively active Akt. Together, our data indicate that the neuroprotective role of PS1 depends on its ability to activate the PI3K/Akt signaling pathway and that PS1 FAD mutations increase GSK-3 activity and promote neuronal apoptosis by inhibiting the function of PS1 in this pathway. These observations suggest that stimulation of PI3K/Akt signaling may be beneficial to FAD patients.

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

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2 or PIP2] 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 PIP2 and other phosphoinositides. Here, we report a novel requirement for PIP2 in the activation of the epidermal growth factor receptor (EGFR). Down-regulation of PIP2 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 PIP2 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 PIP2 and PIP2-dependent regulation of EGFR activation. A peptide mimicking the EGFR juxtamembrane domain that was assayed by surface plasmon resonance displayed strong binding to PIP2. Neutralization of positively charged amino acids abolished EGFR/PIP2 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 PIP2 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 PIP2 may serve as a general modulator of the activity of many diverse eukaryotic transmembrane proteins through their basic JDs.