The amyloid cascade hypothesis: an updated critical review.

Results from recent clinical trials of antibodies that target amyloid-ß (Aß) for Alzheimer's disease have created excitement and have been heralded as corroboration of the amyloid cascade hypothesis. However, while Aß may contribute to disease, genetic, clinical, imaging and biochemical data suggest a more complex aetiology. 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 Alzheimer's disease, we argue that the data point to Aß playing a minor aetiological role. We also discuss data suggesting that the concerted activity of many pathogenic factors contribute to Alzheimer's disease and propose that evolving multi-factor disease models will better underpin the search for more effective strategies to treat the disease.

PS1 FAD mutants decrease ephrinB2-regulated angiogenic functions, ischemia-induced brain neovascularization and neuronal survival.

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.

What Do Recent Clinical Trials Teach Us About the Etiology of AD.

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/Aß 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 Aß-/γ-secretase-independent functions that can play important roles in AD. Furthermore, non-neuronal brain cells like microglia are implicated in AD pathology.

Open chromatin profiling of human postmortem brain infers functional roles for non-coding schizophrenia loci.

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 from frozen 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 type markers, promoters and enhancers. Relative to those of non-neurons, neuronal OCRs are more 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 postmortem human brain cells and further our understanding of the regulome and the impact of neuropsychiatric disease-associated genetic risk variants.

Presenilin1/γ-secretase protects neurons from glucose deprivation-induced death by regulating miR-212 and PEA15.

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.

The product of the γ-secretase processing of ephrinB2 regulates VE-cadherin complexes and angiogenesis.

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.

Phospholipid dysregulation contributes to ApoE4-associated cognitive deficits in Alzheimer's disease pathogenesis.

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.

Presenilin 1 is necessary for neuronal, but not glial, EGFR expression and neuroprotection via γ-secretase-independent transcriptional mechanisms.

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.

Presenilin-1/γ-secretase controls glutamate release, tyrosine phosphorylation, and surface expression of N-methyl-D-aspartate receptor (NMDAR) subunit GluN2B.

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.

Reduction of synaptojanin 1 accelerates Aß clearance and attenuates cognitive deterioration in an Alzheimer mouse model.

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-ß (Aß). Genetic down-regulation of synj1 reduces both extracellular and intracellular Aß 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 Aß40 and Aß42 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 Aß generation by ß- and γ-secretase cleavage is not affected. Instead, synj1 knockdown increases Aß 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 Aß clearance.

Cell signaling abnormalities may drive neurodegeneration in familial Alzheimer disease.

Presenilins (PSs) are catalytic components of the γ-secretase complex that produces Aß 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 Aß 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 Aß peptides or their products. Treatment of patients with inhibitors of Aß production however, showed no therapeutic benefits while inducing cytotoxicity. Similarly, treatments with anti-Aß 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.

Allelic interference: a mechanism for trans-dominant transmission of loss of function in the neurodegeneration of familial Alzheimer's disease.

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

Revision of Alzheimer's diagnostic criteria or relocation of the Potemkin village.

The recently announced revision of the Alzheimer's disease (AD) diagnostic ATN classification adds to an already existing disregard for clinical assessment the rejection of image-based in vivo assessment of the brain's condition. The revision suggests that the diagnosis of AD should be based solely on the presence of cerebral amyloid-beta and tau, indicated by the "A" and "T". The "N", which stands for neurodegeneration - detected by imaging - should no longer be given importance, except that A+ ± T + = AD with amyloid PET being the main method for demonstrating A+ . We believe this is an artificial and misleading suggestion. It is artificial because it relies on biomarkers whose significance remains obscure and where the detection of "A" is based on a never-validated PET method using a tracer that marks much more than amyloid-beta. It is misleading because many patients without dementia will be falsely classified as having AD, but nonetheless candidates for passive immunotherapy, which may be more harmful than beneficial, and sometimes fatal.

Donanemab, another anti-Alzheimer's drug with risk and uncertain benefit.

Based on "reducing amyloid plaques in the brain", the U.S. Food and Drug Administration has granted accelerated and full approval for two monoclonal anti-Alzheimer's antibodies, aducanumab and lecanemab, respectively. Approval of a third antibody, donanemab, is pending. Moreover, lecanemab and donanemab are claimed to cause delay in the cognitive decline that characterizes the disease. We believe that these findings are subject to misinterpretation and statistical bias. Donanemab is claimed to cause removal of up to 86 % of cerebral amyloid and 36 % delay in cognitive decline compared to placebo. In reality, these are very small changes on an absolute scale and arguably less than what can be achieved with cholinesterase inhibitor/memantine therapy. Moreover, the "removal" of amyloid, based on the reduced accumulation of amyloid-PET tracer, most likely also reflects therapy-related tissue damage. This would also correlate with the minimal clinical effect, the increased frequency of amyloid-related imaging abnormalities, and the accelerated loss of brain volume in treated compared to placebo patients observed with these antibodies. We recommend halting approvals of anti-AD antibodies until these issues are fully understood to ensure that antibody treatment does not cause more harm than benefit to patients.

Passive Alzheimer's immunotherapy: A promising or uncertain option?

The US Food and Drug Administration (FDA)'s recent accelerated approval of two anti-amyloid antibodies for treatment of Alzheimer's disease (AD), aducanumab and lecanemab, has caused substantial debate. To inform this debate, we reviewed the literature on randomized clinical trials conducted with eight such antibodies focusing on clinical efficacy, cerebral amyloid removal, amyloid-related imaging abnormalities (ARIAs) and cerebral volumes to the extent such measurements have been reported. Two antibodies, donanemab and lecanemab, have demonstrated clinical efficacy, but these results remain uncertain. We further argue that the decreased amyloid PET signal in these trials is unlikely to be a one-to-one reflection of amyloid removal, but rather a reflection of increased therapy-related brain damage, as supported by the increased incidence of ARIAs and reported loss of brain volume. Due to these uncertainties of benefit and risk, we recommend that the FDA pauses existing approvals and approval of new antibodies until results of phase 4 studies with these drugs are available to inform on these risk-benefit uncertainties. We recommend that the FDA prioritize FDG PET and detection of ARIAs and accelerated brain volume loss with MRI in all trial patients, and neuropathological examination of all patients who die in these phase 4 trials.

The Anti-Amyloid Monoclonal Antibody Lecanemab: 16 Cautionary Notes.

After the CLARITY-AD clinical trial results of lecanemab were interpreted as positive, and supporting the amyloid hypothesis, the drug received accelerated Food and Drug Administration approval. However, we argue that benefits of lecanemab treatment are uncertain and may yield net harm for some patients, and that the data do not support the amyloid hypothesis. We note potential biases from inclusion, unblinding, dropouts, and other issues. Given substantial adverse effects and subgroup heterogeneity, we conclude that lecanemab's efficacy is not clinically meaningful, consistent with numerous analyses suggesting that amyloid-ß and its derivatives are not the main causative agents of Alzheimer's disease dementia.

Inhibitors of γ-secretase stabilize the complex and differentially affect processing of amyloid precursor protein and other substrates.

γ-Secretase inhibitors (GSIs) are drugs used in research to inhibit production of Aß and in clinical trials to treat Alzheimer's disease (AD). They inhibit proteolytic activities of γ-secretase noncompetitively by unknown mechanisms. Here, we used cortical neuronal cultures expressing endogenous levels of enzymes and substrates to study the effects of GSIs on the structure and function of γ-secretase. We show that GSIs stabilize the interactions between the C-terminal fragment of presenilin (PS-CTF), the central component of the γ-secretase complex, and its partners the APH-1/nicastrin and PS1-NTF/PEN-2 subcomplexes. This stabilization dose-dependently correlates with inhibition of N-cadherin cleavage, a process limited by enzyme availability. In contrast, production of amyloid precursor protein (APP) intracellular domain (AICD) is insensitive to low concentrations of GSIs and is limited by substrate availability. Interestingly, APP is processed by both PS1- and PS2-containing γ-secretase complexes, while N-cadherin and ephrinB1 are processed only by PS1-containing complexes. Paradoxically, low concentrations of GSIs specifically increased the levels of Aß without affecting its catabolism, indicating increased Aß production. Our data reveal a mechanism of γ-secretase inhibition by GSIs and provide evidence that distinct γ-secretase complexes process specific substrates. Furthermore, our observations have implications for GSIs as therapeutics because processing of functionally important substrates may be inhibited at lower concentrations than Aß.

Presenilin1/gamma-secretase promotes the EphB2-induced phosphorylation of ephrinB2 by regulating phosphoprotein associated with glycosphingolipid-enriched microdomains/Csk binding protein.

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.

Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling.

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.