Familial Alzheimer's Disease Presenilin-1 Research Articles

Effects of familial Alzheimer's disease mutations on gamma-secretase activation for cleavage of amyloid precursor protein.

Gamma-secretase, the “proteasome of the membrane”, cleaves within the membrane of 150+ peptide substrates with central roles in biology and medicine, including amyloid precursor protein (APP). Presenilin-1 (PS1) is the catalytic subunit of gamma-secretase and its mutations cause early-onset familial Alzheimer's disease (FAD). However, the exact pathogenic mechanism of FAD remains elusive. Here, we have combined Gaussian accelerated molecular dynamics (GaMD) simulations and biochemical experiments to determine the effects of six representative PS1 FAD mutations on the enzyme-substrate interactions between gamma-secretase and APP. Biochemical experiments showed that all six FAD mutations rendered gamma-secretase less active for the endoproteolytic (ε) cleavage of APP. Distinct low-energy conformational states were identified from the free energy profiles of wildtype and PS1 FAD-mutant gamma-secretase. The PS1 FAD mutants were found to reduce gamma-secretase proteolytic activity by hindering APP residue L49 from proper orientation in the active site and/or disrupting the distance between the catalytic aspartates. Therefore, our complementary experimental and simulation findings provide important mechanistic insights into how PS1 FAD mutations affect structural dynamics and enzyme-substrate interactions of gamma-secretase and APP.

Evidence of beta amyloid independent small vessel disease in familial Alzheimer's disease.

We studied small vessel disease (SVD) pathology in Familial Alzheimer's disease (FAD) subjects carrying the presenilin 1 (PSEN1) p.Glu280Ala mutation in comparison to those with sporadic Alzheimer's disease (SAD) as a positive control for Alzheimer's pathology and Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) bearing different NOTCH3 mutations, as positive controls for SVD pathology. Upon magnetic resonance imaging (MRI) in life, some FAD showed mild white matter hyperintensities and no further radiologic evidence of SVD. In post-mortem studies, total SVD pathology in cortical areas and basal ganglia was similar in PSEN1 FAD and CADASIL subjects, except for the feature of arteriosclerosis which was higher in CADASIL subjects than in PSEN1 FAD subjects. Further only a few SAD subjects showed a similar degree of SVD pathology as observed in CADASIL. Furthermore, we found significantly enlarged perivascular spaces in vessels devoid of cerebral amyloid angiopathy in FAD compared with SAD and CADASIL subjects. As expected, there was greater fibrinogen-positive perivascular reactivity in CADASIL but similar reactivity in PSEN1 FAD and SAD groups. Fibrinogen immunoreactivity correlated with onset age in the PSEN1 FAD cases, suggesting increased vascular permeability may contribute to cognitive decline. Additionally, we found reduced perivascular expression of PDGFRβ AQP4 in microvessels with enlarged PVS in PSEN1 FAD cases. We demonstrate that there is Aβ-independent SVD pathology in PSEN1 FAD, that was marginally lower than that in CADASIL subjects although not evident by MRI. These observations suggest presence of covert SVD even in PSEN1, contributing to disease progression. As is the case in SAD, these consequences may be preventable by early recognition and actively controlling vascular disease risk, even in familial forms of dementia.

PS1 FAD mutant M146V decreases VEGF-induced angiogenesis in brain endothelial cells.

Alzheimer's disease (AD) is a multifactorial and progressive neurodegenerative disease, and the most common form of dementia. Cerebrovascular abnormalities and reduced vascular density are observed in human AD brains. Vascular pathology is an early event that contributes to the progression of AD and it can be caused by impaired angiogenesis. Brain angiogenesis in response to toxic insults is a reparative function regulated by endothelial cells (ECs); impairment in this function would render the brain vulnerable to insults. One of the most potent angiogenic factors, Vascular Endothelial Growth factor (VEGF), promotes angiogenesis by binding VEGFR2 receptor, a type I transmembrane protein (TMP). Most type I TMP are proteolytically processed by γ-secretase, which has presenilin 1 (PS1) as a catalytic subunit. Mutations in PS1 cause Familial AD (FAD) and reduce the activity of γ-secretase. We hypothesize that PS1 FAD mutants decrease the VEGF-induced processing of VEGFR2 and brain angiogenesis. Targeting brain angiogenesis could have therapeutic value in AD. Primary cortical endothelial cells (pCECs) were isolated and cultured from brains of wild type (WT) and PS1 M146V knockin mice, a "humanized" FAD model having the same genotype as in human FAD patients. We performed the in vitro angiogenesis assays sprouting on beads, migration and tube formation using pCEC cultures. Processing of VEGFR2 was studied in VEGFR2-overexpressed HEK293 cells in presence or absence of VEGF-A and γ-secretase inhibitor (RO4929097) with western blotting. We found that VEGFR2 is processed by γ-secretase and that VEGF-induced sprouting, tube formation, migration and angiogenic complexes between VE-cadherin and Rok-α kinase in pCEC are decreased in the presence of γ-secretase inhibitor. We also found that pCEC expressing PS1 FAD mutant M146V do not sprout in response to VEGF as efficiently as WT cells. Our findings suggest that VEGFR2 is processed by γ-secretase and that γ-secretase promotes VEGF-induced angiogenic functions of brain ECs. Our data also show that PS1 FAD mutant decreases angiogenic functions of pCEC. Importantly the effects of PS1 FAD mutants occur in the absence of amyloid plaques and neurofibrillary tangles indicating that PS1 FAD mutants can affect angiogenesis independent of the neuropathological hallmarks of AD.

Decreased Deposition of Beta-Amyloid 1-38 and Increased Deposition of Beta-Amyloid 1-42 in Brain Tissue of Presenilin-1 E280A Familial Alzheimer's Disease Patients.

Familial Alzheimer’s Disease (FAD) caused by Presenilin-1 (PS1) mutations is characterized by early onset, cognitive impairment, and dementia. Impaired gamma secretase function favors production of longer beta-amyloid species in PS1 FAD. The PS1 E280A mutation is the largest FAD kindred under study. Here, we studied beta-amyloid deposits in PS1 E280A FAD brains in comparison to sporadic Alzheimer’s disease (SAD). We analyzed cortices and cerebellum from 10 FAD and 10 SAD brains using immunohistochemistry to determine total beta-amyloid, hyperphosphorylated tau (pTau), and specific beta-amyloid peptides 1-38, 1-40, 1-42, and 1-43. Additionally, we studied beta-amyloid subspecies by ELISA, and vessel pathology was detected with beta-amyloid 1-42 and truncated pyroglutamylated beta-amyloid antibodies. There were no significant differences in total beta-amyloid signal between SAD and FAD. Beta-amyloid 1-38 and 1-43 loads were increased, and 1-42 loads were decreased in frontal cortices of SAD when compared to FAD. Beta-amyloid species assessment by ELISA resembled our findings by immunohistochemical analysis. Differences in beta-amyloid 1-38 and 1-42 levels between SAD and FAD were evidenced by using beta-amyloid length-specific antibodies, reflecting a gamma secretase-dependent shift in beta-amyloid processing in FAD cases. The use of beta-amyloid length-specific antibodies for postmortem assessment of beta-amyloid pathology can differentiate between SAD and PS1 FAD cases and it can be useful for identification of SAD cases potentially affected with gamma secretase dysfunction.

Presenilin1 familial Alzheimer disease mutants inactivate EFNB1- and BDNF-dependent neuroprotection against excitotoxicity by affecting neuroprotective complexes of N-methyl-d-aspartate receptor.

Excitotoxicity is thought to play key roles in brain neurodegeneration and stroke. Here we show that neuroprotection against excitotoxicity by trophic factors EFNB1 and brain-derived neurotrophic factor (called here factors) requires de novo formation of ‘survival complexes’ which are factor-stimulated complexes of N-methyl-d-aspartate receptor with factor receptor and presenilin 1. Absence of presenilin 1 reduces the formation of survival complexes and abolishes neuroprotection. EPH receptor B2- and N-methyl-d-aspartate receptor-derived peptides designed to disrupt formation of survival complexes also decrease the factor-stimulated neuroprotection. Strikingly, factor-dependent neuroprotection and levels of the de novo factor-stimulated survival complexes decrease dramatically in neurons expressing presenilin 1 familial Alzheimer disease mutants. Mouse neurons and brains expressing presenilin 1 familial Alzheimer disease mutants contain increased amounts of constitutive presenilin 1–N-methyl-d-aspartate receptor complexes unresponsive to factors. Interestingly, the stability of the familial Alzheimer disease presenilin 1–N-methyl-d-aspartate receptor complexes differs from that of wild type complexes and neurons of mutant-expressing brains are more vulnerable to cerebral ischaemia than neurons of wild type brains. Furthermore, N-methyl-d-aspartate receptor-mediated excitatory post-synaptic currents at CA1 synapses are altered by presenilin 1 familial Alzheimer disease mutants. Importantly, high levels of presenilin 1–N-methyl-d-aspartate receptor complexes are also found in post-mortem brains of Alzheimer disease patients expressing presenilin 1 familial Alzheimer disease mutants. Together, our data identify a novel presenilin 1-dependent neuroprotective mechanism against excitotoxicity and indicate a pathway by which presenilin 1 familial Alzheimer disease mutants decrease factor-depended neuroprotection against excitotoxicity and ischaemia in the absence of Alzheimer disease neuropathological hallmarks which may form downstream of neuronal damage. These findings have implications for the pathogenic effects of familial Alzheimer disease mutants and therapeutic strategies.

Aβ43-producing PS1 FAD mutants cause altered substrate interactions and respond to γ-secretase modulation.

Abnormal generation of neurotoxic amyloid‐β peptide (Aβ) 42/43 species due to mutations in the catalytic presenilin 1 (PS1) subunit of γ‐secretase is the major cause of familial Alzheimer's disease (FAD). Deeper mechanistic insight on the generation of Aβ43 is still lacking, and it is unclear whether γ‐secretase modulators (GSMs) can reduce the levels of this Aβ species. By comparing several types of Aβ43‐generating FAD mutants, we observe that very high levels of Aβ43 are often produced when presenilin function is severely impaired. Altered interactions of C99, the precursor of Aβ, are found for all mutants and are independent of their particular effect on Aβ production. Furthermore, unlike previously described GSMs, the novel compound RO7019009 can effectively lower Aβ43 production of all mutants. Finally, substrate‐binding competition experiments suggest that RO7019009 acts mechanistically after initial C99 binding. We conclude that altered C99 interactions are a common feature of diverse types of PS1 FAD mutants and that also patients with Aβ43‐generating FAD mutations could in principle be treated by GSMs.

Elevated cellular cholesterol in Familial Alzheimer's presenilin 1 mutation is associated with lipid raft localization of β-amyloid precursor protein.

Familial Alzheimer’s disease (FAD)-associated presenilin 1 (PS1) serves as a catalytic subunit of γ-secretase complex, which mediates the proteolytic liberation of β-amyloid (Aβ) from β-amyloid precursor protein (APP). In addition to its proteolytic role, PS1 is involved in non-proteolytic functions such as protein trafficking and ion channel regulation. Furthermore, postmortem AD brains as well as AD patients showed dysregulation of cholesterol metabolism. Since cholesterol has been implicated in regulating Aβ production, we investigated whether the FAD PS1-associated cholesterol elevation could influence APP processing. We found that in CHO cells stably expressing FAD-associated PS1 ΔE9, total cholesterol levels are elevated compared to cells expressing wild-type PS1. We also found that localization of APP in cholesterol-enriched lipid rafts is substantially increased in the mutant cells. Reducing the cholesterol levels by either methyl-β-cyclodextrin or an inhibitor of CYP51, an enzyme mediating the elevated cholesterol in PS1 ΔE9-expressing cells, significantly reduced lipid raft-associated APP. In contrast, exogenous cholesterol increased lipid raft-associated APP. These data suggest that in the FAD PS1 ΔE9 cells, the elevated cellular cholesterol level contributes to the altered APP processing by increasing APP localized in lipid rafts.

Presenilin 1 promotes trypsin-induced neuroprotection via the PAR2/ERK signaling pathway. Effects of presenilin 1 FAD mutations

Mutants of presenilin 1 (PS1) increase neuronal cell death causing autosomal-dominant familial Alzheimer's disease (FAD). Recent literature shows that treatment of neuronal cultures with low concentrations of trypsin, a member of the serine family of proteases, protects neurons from toxic insults by binding to the proteinase-activated receptor 2 and stimulating survival kinase extracellular signal–regulated kinase (ERK 1/2). Other studies show that PS1 is necessary for the neuroprotective activity of specific neurotrophic factors, such as brain-derived neurotrophic factor, against excitotoxicity and oxidative stress. Here, we show that treatment of mouse cortical neuronal cultures with trypsin activates ERK1/2 and protects neurons against glutamate excitoxicity. The trypsin-dependent ERK activation and neuroprotection requires both alleles of PS1 because neither PS1 knockout nor PS1 hemizygous neuronal cultures can use exogenous trypsin to activate ERK1/2 or increase neuronal survival. The protective effect of PS1 does not depend on its γ-secretase activity because inhibitors of γ-secretase have no effect on trypsin-mediated neuroprotection. Importantly, cortical neuronal cultures either heterozygous or homozygous for PS1 FAD mutants are unable to use trypsin to activate ERK1/2 and rescue neurons from excitotoxicity, indicating that FAD mutants inhibit trypsin-dependent neuroprotection in an autosomal-dominant manner. Furthermore, our data support the theory that PS FAD mutants increase neurodegeneration by inhibiting the ability of neurons to use cellular factors as protective agents against toxic insults.

Attenuated presenilin-1 endoproteolysis enhances store-operated calcium currents in neuronal cells.

Presenilins have been reported to regulate calcium homeostasis in the endoplasmic reticulum, and dysregulation of intracellular calcium has been implicated in the pathogenesis of Alzheimer's disease (AD). Reduced endoproteolysis levels of presenilin-1 (PS1) have been detected in postmortem brains of patients carrying familial Alzheimer's disease PS1 mutations. This study deals with the effect of attenuated endoproteolysis of PS1 on store-operated calcium (SOC) entry in neuronal cells and mouse fibroblasts with double knockouts of PS1 and PS2. Significant enhancement of SOC channel activation has been detected by electrophysiological measurements in cells with reduced PS1 endoproteolysis. The increase in SOC entry was not accompanied by any changes in protein levels of channels subunits or stromal interaction molecule. These data are important for understanding the role of PS1 in AD, apart from its involvement in γ-secretase cleavage of amyloid precursor protein into Aβ. Taking into account that most of familial AD-connected mutations in PS1 are loss-of-function, the observed effects may well be general for familial AD. Reduced endoproteolysis levels of presenilin-1 (PS1) have been detected in postmortem brains of patients carrying familial Alzheimer's disease PS1 mutations. Significant enhancement of SOC channel activation has been detected by electrophysiological measurements in cells with reduced PS1 endoproteolysis. The data obtained shed light on Alzheimer's disease pathogenesis and implicates to the future drugs development.

Possible Role of STIM1 Sensor Signal in Memory Loss Connected with Familial Alzheimer's Disease

Familial Alzheimer's disease (FAD) which leads to memory impairments is caused by mutations in presenilin-1 (PS1) gene in approximately 40% of cases. PS1 is well known as a component of the gamma-secretase enzyme which cleaves APP to A-beta. To become a catalytic part of enzyme PS1 undergoes an endoproteolysis. It was shown that mutations in PS1 gene disrupt endoproteolysis increasing uncleaved protein level in brain tissue of FAD patients. In our study we found effects of FAD PS1 mutants (PS1DE9, PS1 D247A) on activity of store-operated calcium (SOC) channels in mice hippocampal neurons and Neuro2a cell line. Increased uncleaved PS1 levels led to SOC channels hyperactivities detected with direct single-cell electrophysiological measurements and calcium imaging experiments with fura2-AM. The effects were caused by impaired signal transduction from ER to SOC channels in plasmatic membrane. The impaired intracellular signal transduction by STIM1 sensor was revealed in live confocal imaging experiments and proved with STIM1 knock-down. Moreover, a feeding of Drosophila melanogaster transgenes expressing human mutated PS1 in cholinergic nervous system with pharmacological inhibitor of STIM sensor signal transduction 2APB led to rescue of the memory loss detected by courtship based assay with aged animals. Therefore hyperactive STIM1 signal transduction leads to increased SOC channels activity which could be the reason for memory loss in FAD.This work was supported by the program of “Molecular and Cellular Biology” RAS, research grants from the Russian Basic Research Foundation, Russian Scientific Fund and the President of Russia Scholarship.

Characterization and Molecular Profiling of PSEN1 Familial Alzheimer's Disease iPSC-Derived Neural Progenitors

Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes.

Changed membrane integration and catalytic site conformation are two mechanisms behind the increased Aβ42/Aβ40 ratio by presenilin 1 familial Alzheimer-linked mutations

The enzyme complex γ-secretase generates amyloid β-peptide (Aβ), a 37–43-residue peptide associated with Alzheimer disease (AD). Mutations in presenilin 1 (PS1), the catalytical subunit of γ-secretase, result in familial AD (FAD). A unifying theme among FAD mutations is an alteration in the ratio Aβ species produced (the Aβ42/Aβ40 ratio), but the molecular mechanisms responsible remain elusive. In this report we have studied the impact of several different PS1 FAD mutations on the integration of selected PS1 transmembrane domains and on PS1 active site conformation, and whether any effects translate to a particular amyloid precursor protein (APP) processing phenotype. Most mutations studied caused an increase in the Aβ42/Aβ40 ratio, but via different mechanisms. The mutations that caused a particular large increase in the Aβ42/Aβ40 ratio did also display an impaired APP intracellular domain (AICD) formation and a lower total Aβ production. Interestingly, seven mutations close to the catalytic site caused a severely impaired integration of proximal transmembrane/hydrophobic sequences into the membrane. This structural defect did not correlate to a particular APP processing phenotype. Six selected FAD mutations, all of which exhibited different APP processing profiles and impact on PS1 transmembrane domain integration, were found to display an altered active site conformation. Combined, our data suggest that FAD mutations affect the PS1 structure and active site differently, resulting in several complex APP processing phenotypes, where the most aggressive mutations in terms of increased Aβ42/Aβ40 ratio are associated with a decrease in total γ-secretase activity.

Disregulation of Calcium Homeostasis Connected with Familial Alzheimer's Disease

Familial Alzheimer's disease (FAD) caused by mutations in presenilin-1 (PS1) gene in approximately 50% of cases and in Amyloid precursor protein (APP) gene in 13 % cases. It was found that FAD genes mutants affect calcium homeostasis in hippocampal neurons by disrupting Ca2+ storage in the lumen of endoplasmic reticulum (ER). In our study we used electrophysiological recordings with a patch-clamp technique in whole-cell mode and calcium imaging with Fura-2AM to study calcium channels activity, ER calcium load and ER calcium leak. We observed different effects of FAD mutated genes expressions on activity of store-operated and L-type voltage-gated calcium channels in mouse hippocampal neurons, mouse neuroblastoma Neuro2a and human neuroblastoma SK-N-SH cell lines. We found that effects of FAD PS1 mutants on calcium channels were driven by misbalance in activity of ER calcium sensors STIM1 and STIM2. The different effects were connected with calcium sensors STIM1 or STIM2 impaired signal transduction from ER to calcium channels in plasmatic membrane (PM) under control of ER calcium levels. The impaired signal transduction was revealed in live confocal imaging experiments. The enzyme activity of PS1 was involved in regulation of store-operated calcium entry in two ways: the activity of channels was depended on PS1 endoproteolysis level and APP cleavage.This work was supported by the program of “Molecular and Cellular Biology” RAS, research grants from the Russian Basic Research Foundation, grant ERA.Net RUS, research grants from the OPTEC LLC and the President of Russia Scholarship.

The Presenilin-1 ΔE9 Mutation Results in Reduced γ-Secretase Activity, but Not Total Loss of PS1 Function, in Isogenic Human Stem Cells

Presenilin 1 (PS1) is the catalytic core of γ-secretase, which cleaves type 1 transmembrane proteins, including the amyloid precursor protein (APP). PS1 also has γ-secretase-independent functions, and dominant PS1 missense mutations are the most common cause of familial Alzheimer's disease (FAD). Whether PS1 FAD mutations are gain- or loss-of-function remains controversial, primarily because most studies have relied on overexpression in mouse and/or nonneuronal systems. We used isogenic euploid human induced pluripotent stem cell lines to generate and study an allelic series of PS1 mutations, including heterozygous null mutations and homozygous and heterozygous FAD PS1 mutations. Rigorous analysis of this allelic series in differentiated, purified neurons allowed us to resolve this controversy and to conclude that FAD PS1 mutations, expressed at normal levels in the appropriate cell type, impair γ-secretase activity but do not disrupt γ-secretase-independent functions of PS1. Thus, FAD PS1 mutations do not act as simple loss of PS1 function but instead dominantly gain an activity toxic to some, but not all, PS1 functions.

Familial Alzheimer's disease-linked presenilin-1 mutation M146V affects store-operated calcium entry: Does gain look like loss?

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to neuron death and synapse loss in the hippocampus and cortex, with consequent cognitive disability and dementia. Mutations in the presenilin-1 (PS1) gene lead to familial Alzheimer's disease (FAD). Here, we report that the expression of FAD-linked PS1 M146V mutant affects store-operated calcium channel activity (Isoc) in human neuroblastoma SK-N-SH cells. Electrophysiological measurements and calcium imaging experiments have revealed the emergent role of calcium sensor STIM2 in the inhibition of calcium release-activated calcium channel activity (Icrac) and enhancement of intracellular Ca(2+) stores content due to PS1 M146V mutant expression. In general, the results of this study suggest that the pathological inhibition of one type of store-operated calcium channels caused by FAD PS1 mutant expression may be accounted for by preceding gain of spontaneous activity of store-operated calcium channels driven by STIM2.

Presenilin-1 Mutants Connected with Familial Alzheimer's Disease affect Activity of Voltage-Gated Calcium Channels

Familial Alzheimer's disease (FAD) is caused by mutations in presenilin-1 (PS1) gene in approximately 50% of cases. It was found that FAD PS1 mutants disrupt calcium homeostasis in hippocampal neurons disrupting Ca2+ storage in the lumen of endoplasmic reticulum (ER). Recently calcium sensors of ER STIM1 were found to negatively regulate the activity of L-type voltage-gated calcium channels. Therefore it was suggested that FAD PS1 mutants could affect the activity of L-type channels in neurons. To study the activity of voltage-gated calcium channels experiments with a patch-clamp technique in whole-cell mode were performed with human neuroblastoma SK-N-SH cell line transfected with PS1 M146V mutant or PS1 WT. Currents were induced by 10 mV voltage steps per 200 ms from −80 to +40 mV. PS1 M146V mutant expression enchased the amplitude of integral current at positive potentials comparing to cells with expression of PS1 WT and untransfected control cells. Currents were found to be blocked by application 10 uM of nifedipine. Knock-down of STIM1 with shRNA abolished the difference between cells with mutant and PS1 WT expressions but in the same time mock shRNA left the difference unchanged. Knock-down of STIM1 was controlled by western-blot of cell lysates. Expression PS1 M146V enhanced the amplitude of calcium entry in mouse hippocampal neurons induced by depolarization with 140 KCl in calcium imaging experiments with Fura2-AM comparing to PS1 WT expressing cells. It was concluded that PS1 M146V mutant connected with FAD affect activity of L-type calcium channels through the STIM1 calcium sensors.

Presenilins: Role in calcium homeostasis

Mutations in presenilins are responsible for the vast majority of early-onset familial Alzheimer's disease cases. Full-length presenilin structure is composed of nine transmembrane domains which are localized on the endoplasmic reticulum membrane. Upon endoproteolytic cleavage, presenilins assemble into the γ-secretase multiprotein complex and subsequently get transported to the cell surface. There is a wealth of knowledge around the role of presenilins as the catalytic component of γ-secretase, their involvement in amyloid precursor protein processing and generation of neurotoxic β-amyloid species. However recent findings have revealed a wide range of γ-secretase-independent presenilin functions, including involvement in calcium homeostasis. Particularly, familial Alzheimer's disease presenilin mutations have been shown to interfere with the function of several molecular elements involved in endoplasmic reticulum calcium homeostasis. Presenilins modulate the activity of IP3 and Ryanodine receptor channels, regulate SERCA pump function, affect capacitative calcium entry and function per se as endoplasmic reticulum calcium leak conductance pores.

Familial Alzheimer Disease Presenilin-1 Mutations Alter the Active Site Conformation of γ-secretase

Presenilin-1 (PS1) is the catalytic subunit of γ-secretase, and mutations in this protein cause familial Alzheimer Disease (FAD). However, little is known about how these mutations affect the active site of γ-secretase. Here, we show that PS1 mutations alter the S2 subsite within the active site of γ-secretase using a multiple photoaffinity probe approach called "photophore walking." Moreover, we developed a unique in vitro assay with a biotinylated recombinant Notch1 substrate and demonstrated that PS1 FAD mutations directly and significantly reduced γ-secretase activity for Notch1 cleavage. Substitution of the Notch Cys-1752 residue, which interacts with the S2 subsite, with Val, Met, or Ile has little effect on wild-type PS1 but leads to more efficient substrates for mutant PS1s. This study indicates that alteration of this S2 subsite plays an important role in determining the activity and specificity of γ-secretase for APP and Notch1 processing, which provides structural basis and insights on how certain PS1 FAD mutations lead to AD pathogenesis.

Lack of Evidence for Presenilins as Endoplasmic Reticulum Ca2+ Leak Channels

Familial Alzheimer disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutant PS expression has several cellular consequences, including exaggerated intracellular Ca(2+) ([Ca(2+)](i)) signaling due to enhanced agonist sensitivity and increased magnitude of [Ca(2+)](i) signals. The mechanisms underlying these phenomena remain controversial. It has been proposed that PSs are constitutively active, passive endoplasmic reticulum (ER) Ca(2+) leak channels and that FAD PS mutations disrupt this function resulting in ER store overfilling that increases the driving force for release upon ER Ca(2+) release channel opening. To investigate this hypothesis, we employed multiple Ca(2+) imaging protocols and indicators to directly measure ER Ca(2+) dynamics in several cell systems. However, we did not observe consistent evidence that PSs act as ER Ca(2+) leak channels. Nevertheless, we confirmed observations made using indirect measurements employed in previous reports that proposed this hypothesis. Specifically, cells lacking PS or expressing a FAD-linked PS mutation displayed increased area under the ionomycin-induced [Ca(2+)](i) versus time curve (AI) compared with cells expressing WT PS. However, an ER-targeted Ca(2+) indicator revealed that this did not reflect overloaded ER stores. Monensin pretreatment selectively attenuated the AI in cells lacking PS or expressing a FAD PS allele. These findings contradict the hypothesis that PSs form ER Ca(2+) leak channels and highlight the need to use ER-targeted Ca(2+) indicators when studying ER Ca(2+) dynamics.

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.