The Major Risk Factors for Alzheimer's Disease: Age, Sex, and Genes Modulate the Microglia Response to Aß Plaques.

Gene expression profiles of more than 10,000 individual microglial cells isolated from cortex and hippocampus of male and female AppNL-G-F mice over time demonstrate that progressive amyloid-ß accumulation accelerates two main activated microglia states that are also present during normal aging. Activated response microglia (ARMs) are composed of specialized subgroups overexpressing MHC type II and putative tissue repair genes (Dkk2, Gpnmb, and Spp1) and are strongly enriched with Alzheimer's disease (AD) risk genes. Microglia from female mice progress faster in this activation trajectory. Similar activated states are also found in a second AD model and in human brain. Apoe, the major genetic risk factor for AD, regulates the ARMs but not the interferon response microglia (IRMs). Thus, the ARMs response is the converging point for aging, sex, and genetic AD risk factors.

Non-Catalytic Roles of Presenilin Throughout Evolution.

Research into Alzheimer's disease pathology and treatment has often focused on presenilin proteins. These proteins provide the key catalytic activity of the γ-secretase complex in the cleavage of amyloid-ß precursor protein and resultant amyloid tangle deposition. Over the last 25 years, screening novel drugs to control this aberrant proteolytic activity has yet to identify effective treatments for the disease. In the search for other mechanisms of presenilin pathology, several studies have demonstrated that mammalian presenilin proteins also act in a non-proteolytic role as a scaffold to co-localize key signaling proteins. This role is likely to represent an ancestral presenilin function, as it has been described in genetically distant species including non-mammalian animals, plants, and a simple eukaryotic amoeba Dictyostelium that diverged from the human lineage over a billion years ago. Here, we review the non-catalytic scaffold role of presenilin, from mammalian models to other biomedical models, and include recent insights using Dictyostelium, to suggest that this role may provide an early evolutionary function of presenilin proteins.

A γ-Secretase Independent Role for Presenilin in Calcium Homeostasis Impacts Mitochondrial Function and Morphology in Caenorhabditis elegans.

Mutations in the presenilin (PSEN) encoding genes (PSEN1 and PSEN2) occur in most early onset familial Alzheimer's Disease. Despite the identification of the involvement of PSEN in Alzheimer's Disease (AD) ∼20 years ago, the underlying role of PSEN in AD is not fully understood. To gain insight into the biological function of PSEN, we investigated the role of the PSEN homolog SEL-12 in Caenorhabditis elegans. Using genetic, cell biological, and pharmacological approaches, we demonstrate that mutations in sel-12 result in defects in calcium homeostasis, leading to mitochondrial dysfunction. Moreover, consistent with mammalian PSEN, we provide evidence that SEL-12 has a critical role in mediating endoplasmic reticulum (ER) calcium release. Furthermore, we found that in SEL-12-deficient animals, calcium transfer from the ER to the mitochondria leads to fragmentation of the mitochondria and mitochondrial dysfunction. Additionally, we show that the impact that SEL-12 has on mitochondrial function is independent of its role in Notch signaling, γ-secretase proteolytic activity, and amyloid plaques. Our results reveal a critical role for PSEN in mediating mitochondrial function by regulating calcium transfer from the ER to the mitochondria.

Physiological functions of presenilins; beyond γ-secretase.

Presenilin (PS) was identified in screens for mutations causing the early onset forms of familial Alzheimer's disease (FAD) in 1995. As catalytic units of the γ-secretase complex, presenilins participate in the processing of amyloid beta protein (Aß), the main component of deposits in brain of patients with AD. The more than 90 substrates of γ-secretase isolated so far demonstrate its contribution to wide range of cellular processes and signaling events. However, recent findings have revealed numerous γ-secretase-independent presenilin functions, including involvement in calcium homeostasis, endoplasmic reticulum (ER) stress and autophagy. This mini-review attempts to summarize the multiple physiological and pathological functions of presenilin.

Function and dysfunction of presenilin.

The presenilin(PS) genes harbor approximately 90% of the identified mutations linked to familial forms of Alzheimer's disease, and the presenilin (PS) proteins are essential components of the γ-secretase complex involved in the proteolytic cleavage of type I receptors, such as Notch and the amyloid precursor protein. Genetic analysis employing cell type-specific conditional knockout technology highlighted the importance of PS in the adult brain, including learning and memory, synaptic function and age-dependent neuronal survival. In the central synapse, PS regulates neurotransmitter release, short- and long-term synaptic plasticity and calcium homeostasis. However, the molecular mechanisms by which PS maintains these essential functions are less clear. Although many γ-secretase substrates have been identified, their physiological relevance is often unclear. The findings that nicastrin and PS conditional knockout mice exhibit similar deficits in memory and age-dependent neurodegeneration are consistent with the notion that γ-secretase-dependent activities of PS are required for the maintenance of memory and neuronal survival, though the γ-secretase physiological substrates, Notch receptors, are not targets of PS in the adult brain. Thus, despite of the intense interest in PS since its identification in 1995, more work is needed to define the molecular and cellular mechanisms by which PS controls brain functions and the dysfunction conferred by disease-causing mutations.

Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy.

AIMS: Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation-contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. METHODS AND RESULTS: To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. CONCLUSION: These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels.

Hibris, a Drosophila nephrin homolog, is required for presenilin-mediated Notch and APP-like cleavages.

Drosophila Hibris (Hbs), a member of the Nephrin Immunoglobulin Super Family, has been implicated in myogenesis and eye patterning. Here, we uncover a role of Hbs in Notch (N) signaling and γ-secretase processing. Loss of hbs results in classical N-signaling-associated phenotypes in Drosophila, including eye patterning, wing margin, and sensory organ specification defects. In particular, hbs mutant larvae display altered γ-secretase-dependent Notch proteolytic processing. Hbs also interacts molecularly and genetically with Presenilin (Psn) and other components of the γ-secretase complex. This Hbs function appears conserved, as mammalian Nephrin also promotes N signaling in mammalian cells. Our data suggest that Hbs is required for Psn maturation. Consistent with its role in Psn processing, Hbs genetically interacts with the Drosophila ß-amyloid protein precursor-like (Appl) protein, the homolog of mammalian APP, the cleavage of which is associated with Alzheimer's disease. Thus, Hbs/Nephrin appear to share a general requirement in Psn/γ-secretase regulation and associated processes.

Consequences of inhibiting amyloid precursor protein processing enzymes on synaptic function and plasticity.

Alzheimer's disease (AD) is a neurodegenerative disease, one of whose major pathological hallmarks is the accumulation of amyloid plaques comprised of aggregated ß-amyloid (Aß) peptides. It is now recognized that soluble Aß oligomers may lead to synaptic dysfunctions early in AD pathology preceding plaque deposition. Aß is produced by a sequential cleavage of amyloid precursor protein (APP) by the activity of ß- and γ-secretases, which have been identified as major candidate therapeutic targets of AD. This paper focuses on how Aß alters synaptic function and the functional consequences of inhibiting the activity of the two secretases responsible for Aß generation. Abnormalities in synaptic function resulting from the absence or inhibition of the Aß-producing enzymes suggest that Aß itself may have normal physiological functions which are disrupted by abnormal accumulation of Aß during AD pathology. This interpretation suggests that AD therapeutics targeting the ß- and γ-secretases should be developed to restore normal levels of Aß or combined with measures to circumvent the associated synaptic dysfunction(s) in order to have minimal impact on normal synaptic function.

A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2.

Presenilins 1 and 2 (PS1 and PS2) are the catalytic subunits of the γ-secretase complex, and genes encoding mutant PS1 and PS2 variants cause familial forms of Alzheimer's disease. Lee et al. (2010) recently reported that loss of PS1 activity lead to impairments in autophagosomal function as a consequence of lysosomal alkalinization, caused by failed maturation of the proton translocating V0a1 subunit of the vacuolar (H+)-ATPase and targeting to the lysosome. We have reexamined these issues in mammalian cells and in brains of mice lacking PS (PScdko) and have been unable to find evidence that the turnover of autophagic substrates, vesicle pH, V0a1 maturation, or lysosome function is altered compared with wild-type counterparts. Collectively, our studies fail to document a role for presenilins in regulating cellular autophagosomal function. On the other hand, our transcriptome studies of PScdko mouse brains reveal, for the first time, a role for PS in regulating lysosomal biogenesis.

Presenilins and γ-secretase: structure, function, and role in Alzheimer Disease.

Presenilins were first discovered as sites of missense mutations responsible for early-onset Alzheimer disease (AD). The encoded multipass membrane proteins were subsequently found to be the catalytic components of γ-secretases, membrane-embedded aspartyl protease complexes responsible for generating the carboxyl terminus of the amyloid ß-protein (Aß) from the amyloid protein precursor (APP). The protease complex also cleaves a variety of other type I integral membrane proteins, most notably the Notch receptor, signaling from which is involved in many cell differentiation events. Although γ-secretase is a top target for developing disease-modifying AD therapeutics, interference with Notch signaling should be avoided. Compounds that alter Aß production by γ-secretase without affecting Notch proteolysis and signaling have been identified and are currently at various stages in the drug development pipeline.

Presenilin is the molecular target of acidic γ-secretase modulators in living cells.

The intramembrane-cleaving protease γ-secretase catalyzes the last step in the generation of toxic amyloid-ß (Aß) peptides and is a principal therapeutic target in Alzheimer's disease. Both preclinical and clinical studies have demonstrated that inhibition of γ-secretase is associated with prohibitive side effects due to suppression of Notch processing and signaling. Potentially safer are γ-secretase modulators (GSMs), which are small molecules that selectively lower generation of the highly amyloidogenic Aß42 peptides but spare Notch processing. GSMs with nanomolar potency and favorable pharmacological properties have been described, but the molecular mechanism of GSMs remains uncertain and both the substrate amyloid precursor protein (APP) and subunits of the γ-secretase complex have been proposed as the molecular target of GSMs. We have generated a potent photo-probe based on an acidic GSM that lowers Aß42 generation with an IC(50) of 290 nM in cellular assays. By combining in vivo photo-crosslinking with affinity purification, we demonstrated that this probe binds the N-terminal fragment of presenilin (PSEN), the catalytic subunit of the γ-secretase complex, in living cells. Labeling was not observed for APP or any of the other γ-secretase subunits. Binding was readily competed by structurally divergent acidic and non-acidic GSMs suggesting a shared mode of action. These findings indicate that potent acidic GSMs target presenilin to modulate the enzymatic activity of the γ-secretase complex.

Presenilins function in ER calcium leak and Alzheimer's disease pathogenesis.

Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide and is at present, incurable. The accumulation of toxic amyloid-beta (Aß) peptide aggregates in AD brain is thought to trigger the extensive synaptic loss and neurodegeneration linked to cognitive decline, an idea that underlies the 'amyloid hypothesis' of AD etiology in both the familal (FAD) and sporadic forms of the disease. Genetic mutations causing FAD also result in the dysregulation of neuronal calcium (Ca(2+)) handling and may contribute to AD pathogenesis, an idea termed the 'calcium hypothesis' of AD. Mutations in presenilin proteins account for the majority of FAD cases. Presenilins function as catalytic subunits of γ-secretase involved in the generation of Aß peptide. Recently, we discovered that presenilns function as low-conductance, passive ER Ca(2+) leak channels, independent of γ-secretase activity. We further discovered that many FAD mutations in presenilins results in the loss of ER Ca(2+) leak function activity and Ca(2+) overload in the ER. These results provided potential explanation for abnormal Ca(2+) signaling observed in FAD cells with mutations in presenilns. The implications of these findings for understanding AD pathogenesis are discussed in this article.

The many substrates of presenilin/γ-secretase.

The Alzheimer's disease (AD)-associated amyloid-ß protein precursor (AßPP) is cleaved by α-, ß-, and presenilin (PS)/γ-secretases through sequential regulated proteolysis. These proteolytic events control the generation of the pathogenic amyloid-ß (Aß) peptide, which excessively accumulates in the brains of individuals afflicted by AD. A growing number of additional proteins cleaved by PS/γ-secretase continue to be discovered. Similarly to AßPP, most of these proteins are type-I transmembrane proteins involved in vital signaling functions regulating cell fate, adhesion, migration, neurite outgrowth, or synaptogenesis. All the identified proteins share common structural features, which are typical for their proteolysis. The consequences of the PS/γ-secretase-mediated cleavage on the function of many of these proteins are largely unknown. Here, we review the current literature on the proteolytic processing mediated by the versatile PS/γ-secretase complex. We begin by discussing the steps of AßPP processing and PS/γ-secretase complex composition and localization, which give clues to how and where the processing of other PS/γ-secretase substrates may take place. Then we summarize the typical features of PS/γ-secretase-mediated protein processing. Finally, we recapitulate the current knowledge on the possible physiological function of PS/γ-secretase-mediated cleavage of specific substrate proteins.

γ-Secretase component presenilin is important for microglia ß-amyloid clearance.

OBJECTIVE: The cleavage of amyloid precursor protein by γ-secretase is an important aspect of the pathogenesis of Alzheimer's disease. γ-Secretase also cleaves other membrane proteins (eg, Notch), which control cell development and homeostasis. Presenilin 1 and 2 are considered important determinants of the γ-secretase catalytic site. Our aim was to investigate whether γ-secretase can be important for microglial phagocytosis of Alzheimer's disease ß-amyloid. METHODS: We investigated the role of γ-secretase in microglia activity toward ß-amyloid phagocytosis in cell culture using γ-secretase inhibitors and small hairpin RNA and presenilin-deficient mice. RESULTS: We found that γ-secretase inhibitors impair microglial activity as measured in gene expression, protein levels, and migration ability, which resulted in a reduction of soluble ß-amyloid phagocytosis. Moreover, microglia deficient in presenilin 1 and 2 showed impairment in phagocytosis of soluble ß-amyloid. Dysfunction in the γ-secretase catalytic site led to an impairment in clearing insoluble ß-amyloid from brain sections taken from an Alzheimer's disease mouse model when compared to microglia from wild-type mice. INTERPRETATION: We suggest for the first time, a dual role for γ-secretase in Alzheimer's disease. One role is the cleavage of the amyloid precursor protein for pathologic ß-amyloid production and the other is to regulate microglia activity that is important for clearing neurotoxic ß-amyloid deposits. Further studies of γ-secretase-mediated cellular pathways in microglia may provide useful insights into the development of Alzheimer's disease and other neurodegenerative diseases, providing future avenues for therapeutic intervention.

Alzheimer's secretases regulate voltage-gated sodium channels.

BACE1 and presenilin (PS)/γ-secretase are primary proteolytic enzymes responsible for the generation of pathogenic amyloid ß-peptides (Aß) in Alzheimer's disease. We and others have found that ß-subunits of the voltage-gated sodium channel (Na(v)ßs) also undergo sequential proteolytic cleavages mediated by BACE1 and PS/γ-secretase. In a follow-up study, we reported that elevated BACE1 activity regulates total and surface expression of voltage-gated sodium channels (Na(v)1 channels) and thereby modulates sodium currents in neuronal cells and mouse brains. In this review, we focus on the molecular mechanism of how BACE1 and PS/γ-secretase regulate Na(v)1 channels in neuronal cells. We will also discuss potential physiological and pathological roles of BACE1- and PS/γ-secretase-mediated processing of Na(v)ßs in relation to Na(v)1 channel function.

The production ratios of AICDε51 and Aß42 by intramembrane proteolysis of ßAPP do not always change in parallel.

BACKGROUND: During intramembrane proteolysis of ß-amyloid protein precursor (ßAPP) by presenilin (PS)/γ-secretase, ε-cleavages at the membrane-cytoplasmic border precede γ-cleavages at the middle of the transmembrane domain. Generation ratios of Aß42, a critical molecule for Alzheimer's disease (AD) pathogenesis, and the major Aß40 species might be associated with ε48 and ε49 cleavages, respectively. Medicines to downregulate Aß42 production have been investigated by many pharmaceutical companies. Therefore, the ε-cleavages, rather than the γ-cleavage, might be more effective upstream targets for decreasing the relative generation of Aß42. Thus, one might evaluate compounds by analyzing the generation ratio of the ßAPP intracellular domain (AICD) species (ε-cleavage-derived), instead of that of Aß42. METHODS: Cell-free γ-secretase assays were carried out to observe de novo AICD production. Immunoprecipitation/MALDI-TOF MS analysis was carried out to detect the N-termini of AICD species. Aß and AICD species were measured by ELISA and immunoblotting techniques. RESULTS: Effects on the ε-cleavage by AD-associated pathological mutations around the ε-cleavage sites (i.e., ßAPP V642I, L648P and K649N) were analyzed. The V642I and L648P mutations caused an increase in the relative ratio of ε48 cleavage, as expected from previous reports. Cells expressing the K649N mutant, however, underwent a major ε-cleavage at the ε51 site. These results suggest that ε51, as well as ε48 cleavage, is associated with Aß42 production. Only AICDε51, though, and not Aß42 production, dramatically changed with modifications to the cell-free assay conditions. Interestingly, the increase in the relative ratio of the ε51 cleavage by the K649N mutation was not cancelled by these changes. CONCLUSION: Our current data show that the generation ratio of AICDε51 and Aß42 do not always change in parallel. Thus, to identify compounds that decrease the relative ratio of Aß42 generation, measurement of the relative level of Aß42-related AICD species (i.e., AICDε48 and AICDε51) might not be useful. Further studies to reveal how the ε-cleavage precision is decided are necessary before it will be possible to develop drugs targeting ε-cleavage as a means for decreasing Aß42 production.

[GSK-3beta: a central kinase for neurodegenerative diseases?]. / GSK-3bêta :une kinase au coeur des maladies neuro-dégénératives?

Neurodegenerative diseases are more and more prevalent in our aging societies. There is strong evidence that glycogen synthase kinase (GSK)-3b plays a crucial role in Alzheimer's disease (AD). Indeed, it is involved in the regulation of the two major neuropathological hallmarks present in the brains of AD patients. Interestingly, the kinase has been implicated in multiple cellular processes and linked with the pathogenesis and neuronal loss in several neurodegenerative diseases, including Parkinson's and Huntington's diseases, in which abnormally elevated levels of GSK-3b activity have been reported. In this review, we will provide an overview of the current data pointing out the convergent role of GSK-3b in the neuropathological pathways of these diseases. We will also discuss the rationale for the development of specific inhibitors with therapeutic potentials for such devastating human diseases.

Presenilin modulates EGFR signaling and cell transformation by regulating the ubiquitin ligase Fbw7.

The epidermal growth factor receptor (EGFR) and Notch signaling pathways have antagonistic roles during epidermal differentiation and carcinogenesis. The molecular mechanisms regulating the crosstalk between EGFR and Notch during epidermal transformation are largely unknown. We found enhanced EGFR-dependent signaling, proliferation and oncogenic transformation caused by loss of presenilins (PS), the catalytic components of gamma-secretase that generates the Notch1 intracellular domain (NICD). The underlying mechanism for abnormal EGFR signaling in PS-deficient cells involves gamma-secretase-independent transcriptional upregulation of the E3 ubiquitin ligase Fbw7. Fbw7alpha, which targets NICD for degradation, regulates positively EGFR by affecting a proteasome-dependent ubiquitination step essential for constitutive degradation and stability of EGFR. To investigate the pathological relevance of this findings in vivo, we generated a novel epidermal conditional PS-deficient (ePS cDKO) mouse by deleting both PS in keratinocytes of the basal layer of the epidermis. The ePS cDKO mice develop epidermal hyperplasia associated with enhanced expression of both EGFR and Fbw7 and reduced NICD levels in keratinocytes. These findings establish a novel role for PS on epidermal growth and transformation by reciprocally regulating the EGFR and Notch signaling pathways through Fbw7.

Mitochondria, calcium and cell death: a deadly triad in neurodegeneration.

Mitochondrial Ca(2+) accumulation is a tightly controlled process, in turn regulating functions as diverse as aerobic metabolism and induction of cell death. The link between Ca(2+) (dys)regulation, mitochondria and cellular derangement is particularly evident in neurodegenerative disorders, in which genetic models and environmental factors allowed to identify common traits in the pathogenic routes. We will here summarize: i) the current view of mechanisms and functions of mitochondrial Ca(2+) homeostasis, ii) the basic principles of organelle Ca(2+) transport, iii) the role of Ca(2+) in neuronal cell death, and iv) the new information on the pathogenesis of Alzheimer's, Huntington's and Parkinson's diseases, highlighting the role of Ca(2+) and mitochondria.

Presenilin-dependent regulated intramembrane proteolysis and gamma-secretase activity.

Inhibiting the production of amyloid-beta by antagonising gamma-secretase activity is currently being pursued as a therapeutic strategy for Alzheimer's disease (AD). However, early pre-clinical studies have demonstrated that disruption of presenilin-dependent gamma-secretase alters many presenilin-dependent processes, leading to early lethality in several AD model organisms. Subsequently, transgenic animal studies have highlighted several gross developmental side effects arising from presenilin deficiency. Partial knockdown or tissue-specific knockout of presenilins has identified the skin, vascular and immune systems as very sensitive to loss of presenilin functions. A more appreciative understanding of presenilin biology is therefore demanded if gamma-secretase is to be pursued as a therapeutic target. Herein we review the current understanding of gamma-secretase complexes; their regulation, abundance of interacting partners and diversity of substrates. We also discuss regulation of the gamma-secretase complexes, with an emphasis on the functional role of presenilins in cell biology.