Nikolaos K. Robakis

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.

A CBP Binding Transcriptional Repressor Produced by the PS1/ϵ-Cleavage of N-Cadherin Is Inhibited by PS1 FAD Mutations

Presenilin1 (PS1), a protein implicated in Alzheimers 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.

PS1 activates PI3K thus inhibiting GSK‐3 activity and tau overphosphorylation: effects of FAD mutations

Phosphatidylinositol 3‐kinase (PI3K) promotes cell survival and communication by activating its downstream effector Akt kinase. Here we show that PS1, a protein involved in familial Alzheimers disease (FAD), promotes cell survival by activating the PI3K/Akt cell survival signaling. This function of PS1 is unaffected by γ‐secretase inhibitors. Pharmacological and genetic evidence indicates that PS1 acts upstream of Akt, at or before PI3K kinase. PS1 forms complexes with the p85 subunit of PI3K and promotes cadherin/PI3K association. Furthermore, conditions that inhibit this association prevent the PS1‐induced PI3K/Akt activation, indicating that PS1 stimulates PI3K/Akt signaling by promoting cadherin/PI3K association. By activating PI3K/Akt signaling, PS1 promotes phosphorylation/inactivation of glycogen synthase kinase‐3 (GSK‐3), suppresses GSK‐3‐dependent phosphorylation of tau at residues overphosphorylated in AD and prevents apoptosis of confluent cells. PS1 FAD mutations inhibit the PS1‐dependent PI3K/Akt activation, thus promoting GSK‐3 activity and tau overphosphorylation at AD‐related residues. Our data raise the possibility that PS1 may prevent development of AD pathology by activating the PI3K/Akt signaling pathway. In contrast, FAD mutations may promote AD pathology by inhibiting this pathway.

Alzheimer's disease: a re-examination of the amyloid hypothesis

Alzheimers disease (AD) is a neurodegenerative disorder of the brain characterized by the presence of neuritic amyloid plaques and neurofibrillary tangles. Although it most frequently occurs in the elderly, this disorder also afflicts younger patients. The majority of AD cases are late in onset, lack an obvious genetic etiology and are characterized as sporadic, whereas a small percentage of cases are early in onset and segregate strongly within families (FAD), suggesting a genetic etiology. During the past decade it has become evident that the clinical and histopathological phenotypes of this disease are caused by heterogeneous genetic, and probably environmental, factors. Indeed, several genes have been identified that together appear to cause most of the familial forms of the disease, whereas the epsilon4 allele of the apolipoprotein E (apoE) gene has been shown to be a significant risk factor for the late onset forms of AD. Despite this evidence of heterogeneity, it has been suggested that all of these factors work through a common pathway by triggering the deposition of amyloid in the brain, which is ultimately responsible for the neuronal degeneration of AD. This is a controversial theory, however, primarily because there is a poor correlation between the concentrations and distribution of amyloid depositions in the brain and several parameters of AD pathology, including degree of dementia, loss of synapses, loss of neurons and abnormalities of the cytoskeleton.

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.

Amyloid-Independent Mechanisms in Alzheimer's Disease Pathogenesis

Despite the progress of the past two decades, the cause of Alzheimers 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.

The presenilin-1 familial Alzheimer disease mutant P117L impairs neurogenesis in the hippocampus of adult mice

The functions of presenilin 1 (PS1) and how PS1 mutations cause familial Alzheimers disease (FAD) are incompletely understood. PS1 expression is essential for neurogenesis during embryonic development and may also influence neurogenesis in adult brain. We examined how increasing PS1 expression or expressing an FAD mutant would affect neurogenesis in the adult hippocampus. A neuron-specific enolase (NSE) promoter was used to drive neuronal overexpression of either wild-type human PS1 or the FAD mutant P117L in transgenic mice, and the animals were studied under standard-housing conditions or after environmental enrichment. As judged by bromodeoxyuridine (BrdU) labeling, neural progenitor proliferation rate was mostly unaffected by increasing expression of either wild-type or FAD mutant PS1. However, in both housing conditions, the FAD mutant impaired the survival of BrdU-labeled neural progenitor cells leading to fewer new beta-III-tubulin-immunoreactive neurons being generated in FAD mutant animals during the 4-week postlabeling period. The effect was FAD mutant specific in that neural progenitor survival and differentiation in mice overexpressing wild-type human PS1 were similar to nontransgenic controls. Two additional lines of PS1 wild-type and FAD mutant transgenic mice showed similar changes indicating that the effects were not integration site-dependent. These studies demonstrate that a PS1 FAD mutant impairs new neuron production in adult hippocampus by decreasing neural progenitor survival. They also identify a new mechanism whereby PS1 FAD mutants may impair normal neuronal function and may have implications for the physiological functioning of the hippocampus in FAD.

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 comparative study of histological and immunohistochemical methods for neurofibrillary tangles and senile plaques in Alzheimer's disease

SummarySeveral studies have demonstrated that the accurate visualization and quantification of pathological lesions in neurodegenerative disorders depend on the reliability of staining methods. In an attempt to gain a better assessment of the density and distribution of the neuropathological markers of Alzheimers disease, we compared the staining efficiency of a modified thioflavine S protocol for neurofibrillary tangles (NFT) and senile plaques (SP) to different argentic impregnation techniques (Bielchowsky, Gallyas, Globus, Campbell-Switzer-Martin) and to immunohistochemical stainings obtained with two different antibodies against the amyloid β protein A4 and the microtubule-associated tau protein. The modified thioflavine S technique (MTST) detects up to 60% more SP and up to 50% more NFT than the Bielschowsky and Globus methods, respectively. The results obtained with the specific antibodies are comparable to those obtained with the MTST, but these immunotechniques are more expensive and time consuming for routine neuropathological evaluation, and the appropriate antibodies are not always commercially available. Furthermore, the morphological appearance of NFT and SP with MTST is greatly improved when compared to the classical thioflavine S and the increased signal-to-noise ratio between specifically stained structures and background permits an accurate semi-automatic quantification.