Presenilin/γ-Secretase Activity Is Located in Acidic Compartments of Live Neurons

Presenilin (PSEN)/γ-secretase is a protease complex responsible for the proteolytic processing of numerous substrates. These substrates include the amyloid precursor protein (APP), the cleavage of which by γ-secretase results in the production of β-amyloid (Aβ) peptides. However, exactly where within the neuron γ-secretase processes APP C99 to generate Aβ and APP intracellular domain (AICD) is still not fully understood. Here, we employ novel Förster resonance energy transfer (FRET)-based multiplexed imaging assays to directly “visualize” the subcellular compartment(s) in which γ-secretase primarily cleaves C99 in mouse cortex primary neurons (from both male and female embryos). Our results demonstrate that γ-secretase processes C99 mainly in LysoTracker-positive low-pH compartments. Using a new immunostaining protocol which distinguishes Aβ from C99, we also show that intracellular Aβ is significantly accumulated in the same subcellular loci. Furthermore, we found functional correlation between the endo-lysosomal pH and cellular γ-secretase activity. Taken together, our findings are consistent with Aβ being produced from C99 by γ-secretase within acidic compartments such as lysosomes and late endosomes in living neurons.SIGNIFICANCE STATEMENT Alzheimer''s disease (AD) genetics and histopathology highlight the importance of amyloid precursor protein (APP) processing by γ-secretase in pathogenesis. For the first time, this study has enabled us to directly “visualize” that γ-secretase processes C99 mainly in acidic compartments such as late endosomes and lysosomes in live neurons. Furthermore, we uncovered that intracellular β-amyloid (Aβ) is significantly accumulated in the same subcellular loci. Emerging evidence proposes the great importance of the endo-lysosomal pathway in mechanisms of misfolded proteins propagation (e.g., Tau, α-Syn). Therefore, the predominant processing of C99 and enrichment of Aβ in late endosomes and lysosomes may be critical events in the molecular cascade leading to AD.     

Presenilin mutations and their impact on neuronal differentiation in Alzheimer’s disease

The presenilin genes (PSEN1 and PSEN2) are mainly responsible for causing early-onset familial Alzheimer’s disease, harboring ~300 causative mutations, and representing ~90% of all mutations associated with a very aggressive disease form. Presenilin 1 is the catalytic core of the γ-secretase complex that conducts the intramembranous proteolytic excision of multiple transmembrane proteins like the amyloid precursor protein, Notch-1, N- and E-cadherin, LRP, Syndecan, Delta, Jagged, CD44, ErbB4, and Nectin1a. Presenilin 1 plays an essential role in neural progenitor maintenance, neurogenesis, neurite outgrowth, synaptic function, neuronal function, myelination, and plasticity. Therefore, an imbalance caused by mutations in presenilin 1/γ-secretase might cause aberrant signaling, synaptic dysfunction, memory impairment, and increased Aβ₄₂/Aβ₄₀ ratio, contributing to neurodegeneration during the initial stages of Alzheimer’s disease pathogenesis. This review focuses on the neuronal differentiation dysregulation mediated by PSEN1 mutations in Alzheimer’s disease. Furthermore, we emphasize the importance of Alzheimer’s disease-induced pluripotent stem cells models in analyzing PSEN1 mutations implication over the early stages of the Alzheimer’s disease pathogenesis throughout neuronal differentiation impairment.Key Words: familial Alzheimer''s disease, familial Alzheimer''s disease-induced pluripotent stem cells models, induced pluripotent stem cells, neurogenesis, neuronal differentiation, Notch, presenilin 1, PSEN1 mutations, γ-secretase complex     

Retinoic Acid-Elicited RARα/RXRα Signaling Attenuates Aβ Production by Directly Inhibiting γ-Secretase-Mediated Cleavage of Amyloid Precursor Protein

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PEN–2 gene mutation in a familial Alzheimer’s disease case

Genetic evidence indicates a central role of cerebral accumulation of β–amyloid (Aβ) in the pathogenesis of Alzheimer’s disease (AD). Beside presenilin 1 and 2, three other recently discovered proteins (Aph 1, PEN 2 and nicastrin) are associated with γ–secretase activity, the enzymatic complex generating Aβ. Alterations in genes encoding these proteins were candidates for a role in AD. The PEN 2 gene was examined for unknown mutations and polymorphisms in sporadic and familial Alzheimer patients. Samples from age–matched controls (n = 253), sporadic AD (SAD, n = 256) and familial AD (FAD, n = 140) were screened with DHPLC methodology followed by sequencing. Scanning the gene identified for the first time a missense mutation (D90N) in a patient with FAD. Three intronic polymorphisms were also identified, one of which had a higher presence of the mutated allele in AD subjects carrying the allele ε4 of apolipoprotein E than controls. The pathogenic role of the PEN–2 D90N mutation in AD is not clear, but the findings might lead to new studies on its functional and genetic role.*These two authors contributed equally to the paper.     

FAD Mutations in Presenilin-1 or Amyloid Precursor Protein Decrease the Efficacy of a γ-Secretase Inhibitor: Evidence for Direct Involvement of PS1 in the γ-Secretase Cleavage Complex

To investigate the mechanism of regulation of Aß production by familial Alzheimer's disease (FAD)-linked presenilin 1 (PS1), we used a cell-free system that allows de novo Aß generation to examine whether PS1 participates directly in the γ-secretase reaction. Optimal Aß generation in vitro was achieved at mildly acidic pH and could be inhibited by the aspartyl protease inhibitor pepstatin A, consistent with the suggestion that γ-secretase is an aspartyl protease. Dominant negative mutations of the critical transmembrane aspartates in PS1 or full deletion of PS1 did not alter the maturation of APP in the secretory pathway. Instead, PS1 had a direct effect on the inhibition of Aß production by a designed peptidomimetic inhibitor: the inhibition was significantly less effective in cells expressing FAD-causing mutations in either APP or PS1 than in cells expressing the wild-type proteins. Taken together, these findings suggest that PS1 participates physically in a complex with APP during the γ-secretase cleavage event.     

Identification of tetrahydrocarbazoles as novel multifactorial drug candidates for treatment of Alzheimer's disease

Alzheimer''s disease (AD) is a progressive neurodegenerative brain disorder and the most frequent cause of dementia. To date, there are only a few approved drugs for AD, which show little or no effect on disease progression. Impaired intracellular calcium homeostasis is believed to occur early in the cascade of events leading to AD. Here, we examined the possibility of normalizing the disrupted calcium homeostasis in the endoplasmic reticulum (ER) store as an innovative approach for AD drug discovery. High-throughput screening of a small-molecule compound library led to the identification of tetrahydrocarbazoles, a novel multifactorial class of compounds that can normalize the impaired ER calcium homeostasis. We found that the tetrahydrocarbazole lead structure, first, dampens the enhanced calcium release from ER in HEK293 cells expressing familial Alzheimer''s disease (FAD)-linked presenilin 1 mutations. Second, the lead structure also improves mitochondrial function, measured by increased mitochondrial membrane potential. Third, the same lead structure also attenuates the production of amyloid-beta (Aβ) peptides by decreasing the cleavage of amyloid precursor protein (APP) by β-secretase, without notably affecting α- and γ-secretase cleavage activities. Considering the beneficial effects of tetrahydrocarbazoles addressing three key pathological aspects of AD, these compounds hold promise for the development of potentially effective AD drug candidates.     

Alzheimer's disease genetic mutation evokes ultrastructural alterations: Correlation to an intracellular Aβ deposition and the level of GSK-3β-P(Y216) phosphorylated form

Herein we demonstrate that PC12 cells, which overexpress human wild-type amyloid-β precursor protein (AβPPwt) or AβPP bearing double Swedish mutation (AβPPsw), reveal phenotype characteristic for Alzheimer's disease (AD). The examination of cell ultrastructure showed the presence of peptide aggregates within the cells, activation of endosomal–lysosomal system and extensive exocytosis. Furthermore, the autophagy induction was also characteristic hallmark of amyloid-β-induced cytotoxicity. Morphological changes were positively correlated with the extent of phosphorylated glycogen synthase kinase-3β (phospho-Tyr²¹⁶-GSK-3β, GSK-3β-P(Y216)). The activity of GSK-3β is believed to cause tau protein hyper-phosphorylation, increased amyloid-β production and local plaque-associated microglial-mediated inflammatory responses. All of them are symptomatic for AD. In our studies, the highly significant Y216 phosphorylation and over-expression of total GSK-3β were observed in AβPPsw-transfected PC12 cells. In addition, the immuocytochemical analysis showed co-localization of GSK-3β-P(Y216) and amyloid-β deposits. Thus, our data support a functional role of GSK-3β in AβPP processing, further implicating this kinase in the amyloid-β-dependent pathogenesis.     

Mass spectrometric characterization of brain amyloid beta isoform signatures in familial and sporadic Alzheimer’s disease

A proposed key event in the pathogenesis of Alzheimer’s disease (AD) is the formation of neurotoxic amyloid β (Aβ) oligomers and amyloid plaques in specific brain regions that are affected by the disease. The main plaque component is the 42 amino acid isoform of Αβ (Aβ1-42), which is thought to initiate plaque formation and AD pathogenesis. Numerous isoforms of Aβ, e.g., Aβ1-42, Aβ1-40 and the 3-pyroglutamate derivate of Aβ3-42 (pGluAβ3-42), have been detected in the brains of sporadic AD (SAD) and familial AD (FAD) subjects. However, the relative importance of these isoforms in the pathogenesis of AD is not fully understood. Here, we report a detailed study using immunoprecipitation in combination with mass spectrometric analysis to determine the Aβ isoform pattern in the cerebellum, cortex and hippocampus in AD, including subjects with a mutation in the presenilin (M146V) or amyloid precursor protein (KM670/671NL) genes, SAD subjects and non-demented controls. We show that the dominating Aβ isoforms in the three different brain regions analyzed from control, SAD, and FAD are Aβ1-42, pGluAβ3-42, Aβ4-42 and Aβ1-40 of which Aβ1-42 and Aβ4-42 are the dominant isoforms in the hippocampus and the cortex in all groups analyzed, controls included. No prominent differences in Aβ isoform patterns between FAD and SAD patients were seen, underscoring the similarity in the amyloid pathology of these two disease entities.     

Co-expression of β-amyloid precursor protein (βAPP) and apolipoprotein E in cell culture: analysis of βAPP processing

Apolipoprotein E (ApoE) is the major genetic risk factor for Alzheimer's disease (AD). The ApoE4 allele is associated with earlier disease onset and greater cerebral deposition of the amyloid beta peptide (Aβ), the major constituent of senile (amyloid) plaques. The molecular mechanism underlying these effects of ApoE4 remains unclear; ApoE alleles could have different influences on Aβ production, extracellular aggregation, or clearance. Because the missense mutations on chromosomes 14 and 21 that cause familial forms of AD appear to lead to increased secretion of Aβ, it is important to determine whether ApoE4 has a similar effect. Here, we have examined the effects of all three ApoE alleles on the processing of βAPP and the secretion of Aβ in intact cells. We established neural (HS683 human glioma) and non-neural (Chinese hamster ovary) cell culture systems that constitutively secrete both ApoE and Aβ at concentrations like those in human cerebrospinal fluid. βAPP metabolites, generated in the presence of each ApoE allele, were analysed and quantified by two methods: immunoprecipitation and phosphorimaging, and ELISA. We detected no consistent allele-specific effects of ApoE on βAPP processing in either cell type. Our data suggest that the higher amyloid burden found in AD subjects expressing ApoE4 is not due to increased amyloidogenic processing of βAPP, in contrast to findings in AD linked to chromosome 14 or 21. These co-expressing cell lines will be useful in the further search for the effects of ApoE on Aβ aggregation or clearance under physiologically relevant conditions.     

γ-Secretase inhibitors as molecular probes of presenilin function

Mutations in the presenilins cause Alzheimer’s disease (AD) and alter γ-secretase activity to increase the production of the 42-residue amyloid-β peptide (Aβ) found disproportionally in the cerebral plaques that characterize the disease. The serpentine presenilins are required for transmembrane cleavage of both the amyloid-β precursor protein (APP) and the Notch receptor by γ-secretase, and presenilins are biochemically associated with the protease. Inhibitors of γ-secretase have provided critical clues to the function of presenilins. Pharmacological profiling suggested that γ-secretase is an aspartyl protease, leading to the identification of two conserved aspartates important to presenilin’s role in proteolysis. Conversion of transition-state analogue inhibitors of γ-secretase to affinity reagents resulted in specific tagging of the heterodimeric form of presenilins, strongly suggesting that the active site of γ-secretase lies at the interface of the presenilin heterodimer. Heterodimeric presenilin appears to be the catalytic portion of a multi-protein γ-secretase complex.     

Age-related impairment of cerebral blood flow response to KATP channel opener in Alzheimer’s disease mice with presenilin-1 mutation

Local cerebral blood flow (CBF) responses to neuronal activity are essential for cognition and impaired CBF responses occur in Alzheimer’s disease (AD). In this study, regional CBF (rCBF) responses to the K_ATP channel opener diazoxide were investigated in 3xTgAD, WT and mutant Presenilin 1(PS1_M146V) mice from three age groups using Laser-Doppler flowmetry. The rCBF response was reduced early in young 3xTgAD mice and almost absent in old 3xTgAD mice, up to 30%–40% reduction with altered CBF velocity and mean arterial pressure versus WT mice. The impaired rCBF response in 3xTgAD mice was associated with progression of AD pathology, characterized by deposition of intracellular and vascular amyloid-β (Aβ) oligomers, senile plaques and tau pathology. The nitric oxide synthase (NOS) inhibitor N^ω-nitro-L-arginine abolished rCBF response to diazoxide suggesting NO was involved in the mediation of vasorelaxation. Levels of phosphor-eNOS (Ser1177) diminished in 3xTgAD brains with age, while the rCBF response to the NO donor sodium nitroprusside remained. In PS1_M146V mice, the rCBF response to dizoxide reduced and high molecular weight Abeta oligomers were increased indicating PS1_M146V contributed to the dysregulation of rCBF response in AD mice. Our study revealed an Aβ oligomer-associated compromise of cerebrovascular function in rCBF response to diazoxide in AD mice with PS1_M146V mutation.     

The β-secretase, BACE

Evidence suggests that the β-amyloid peptide (Aβ) is central to the pathophysiology of Alzheimer’s disease (AD). Amyloid plaques, primarily composed of Aβ, progressively develop in the brains of AD patients, and mutations in three genes (APP, PS1, and PS2) cause early onset familial AD (FAD) by directly increasing synthesis of the toxic, plaque-promoting Aβ42 peptide. Given the strong association between Aβ and AD, therapeutic strategies to lower the concentration of Aβ in the brain should prove beneficial for the treatment of AD. One such strategy would involve inhibiting the enzymes that generate Aβ. Aβ is a product of catabolism of the large TypeI membrane protein, amyloid precursor protein (APP). Two proteases, called β- and γ-secretase, mediate the endoproteolysis of APP to liberate the Aβ peptide. For over a decade, the molecular identities of these proteases were unknown. Recently, the γ-secretase has been tentatively identified as the presenilin proteins, PS1 and PS2, and the identity of the β-secretase has been shown to be the novel transmembrane aspartic protease, β-site APP cleaving enzyme 1 (BACE1; also called Asp2 and memapsin2). BACE2, a novel protease homologous to BACE1, was also identified, and together the two enzymes define a new family of transmembrane aspartic proteases. BACE1 exhibits all the properties of the β-secretase, and as the key rate-limiting enzyme that initiates the formation of Aβ, BACE1 is an attractive drug target for AD. Here, I review the identification and initial characterization of BACE1 and BACE2, and summarize our current understanding of BACE1 post-translational processing and intracellular trafficking. In addition, I discuss recent studies of BACE1 knockout mice and the BACE1 X-ray structure, and relate implications for BACE1 drug development.     

Familial Alzheimer’s Disease Mutations Differentially Alter Amyloid β-Protein Oligomerization

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Women with the Alzheimer's risk marker ApoE4 lose Aβ-specific CD4+ T cells 10–20 years before men

Adaptive immunity to self-antigens causes autoimmune disorders, such as multiple sclerosis, psoriasis and type 1 diabetes; paradoxically, T- and B-cell responses to amyloid-β (Aβ) reduce Alzheimer''s disease (AD)-associated pathology and cognitive impairment in mouse models of the disease. The manipulation of adaptive immunity has been a promising therapeutic approach for the treatment of AD, although vaccine and anti-Aβ antibody approaches have proven difficult in patients, thus far. CD4⁺ T cells have a central role in regulating adaptive immune responses to antigens, and Aβ-specific CD4⁺ T cells have been shown to reduce AD pathology in mouse models. As these cells may facilitate endogenous mechanisms that counter AD, an evaluation of their abundance before and during AD could provide important insights. Aβ-CD4see is a new assay developed to quantify Aβ-specific CD4⁺ T cells in human blood, using dendritic cells derived from human pluripotent stem cells. In tests of >50 human subjects Aβ-CD4see showed an age-dependent decline of Aβ-specific CD4⁺ T cells, which occurs earlier in women than men. In aggregate, men showed a 50% decline in these cells by the age of 70 years, but women reached the same level before the age of 60 years. Notably, women who carried the AD risk marker apolipoproteinE-ɛ4 (ApoE4) showed the earliest decline, with a precipitous drop between 45 and 52 years, when menopause typically begins. Aβ-CD4see requires a standard blood draw and provides a minimally invasive approach for assessing changes in Aβ biology that may reveal AD-related changes in physiology by a decade. Furthermore, CD4see probes can be modified to target any peptide, providing a powerful new tool to isolate antigen-specific CD4⁺ T cells from human subjects.     

GPR120 Signaling Controls Amyloid-β Degrading Activity of Matrix Metalloproteinases

Alzheimer''s disease (AD) is characterized by the extensive deposition of amyloid-β peptide (Aβ) in the brain. Brain Aβ level is regulated by a balance between Aβ production and clearance. The clearance rate of Aβ is decreased in the brains of sporadic AD patients, indicating that the dysregulation of Aβ clearance mechanisms affects the pathologic process of AD. Astrocytes are among the most abundant cells in the brain and are implicated in the clearance of brain Aβ via their regulation of the blood–brain barrier, glymphatic system, and proteolytic degradation. The cellular morphology and activity of astrocytes are modulated by several molecules, including ω3 polyunsaturated fatty acids, such as docosahexaenoic acid, which is one of the most abundant lipids in the brain, via the G protein-coupled receptor GPR120/FFAR4. In this study, we analyzed the role of GPR120 signaling in the Aβ-degrading activity of astrocytes. Treatment with the selective antagonist upregulated the matrix metalloproteinase (MMP) inhibitor-sensitive Aβ-degrading activity in primary astrocytes. Moreover, the inhibition of GPR120 signaling increased the levels of Mmp2 and Mmp14 mRNAs, and decreased the expression levels of tissue inhibitor of metalloproteinases 3 (Timp3) and Timp4, suggesting that GPR120 negatively regulates the astrocyte-derived MMP network. Finally, the intracerebral injection of GPR120-specific antagonist substantially decreased the levels of TBS-soluble Aβ in male AD model mice, and this effect was canceled by the coinjection of an MMP inhibitor. These data indicate that astrocytic GPR120 signaling negatively regulates the Aβ-degrading activity of MMPs.SIGNIFICANCE STATEMENT The level of amyloid β (Aβ) in the brain is a crucial determinant of the development of Alzheimer''s disease. Here we found that astrocytes, which are the most abundant cell type in the CNS, harbor degrading activity against Aβ, which is regulated by GPR120 signaling. GPR120 is involved in the inflammatory response and obesity in peripheral organs. However, the pathophysiological role of GPR120 in Alzheimer''s disease remains unknown. We found that selective inhibition of GPR120 signaling in astrocytes increased the Aβ-degrading activity of matrix metalloproteases. Our results suggest that GPR120 in astrocytes is a novel therapeutic target for the development of anti-Aβ therapeutics.     

The Conformational Stability of Nonfibrillar Amyloid-β Peptide Oligomers Critically Depends on the C-Terminal Peptide Length

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Mechanisms that lessen benefits of β-secretase reduction in a mouse model of Alzheimer's disease

The β-secretase enzyme BACE1 (β-site amyloid precursor protein-cleaving enzyme 1), which initiates amyloid-β (Aβ) production, is an excellent therapeutic target for Alzheimer''s disease (AD). However, recent evidence raises concern that BACE1-inhibiting approaches may encounter dramatic declines in their abilities to ameliorate AD-like pathology and memory deficits during disease progression. Here, we used BACE1 haploinsufficiency as a therapeutic relevant model to evaluate the efficacy of partial inhibition of this enzyme. Specifically, we crossed BACE1^+/− mice with 5XFAD transgenic mice and investigated the mechanisms by which Aβ accumulation and related memory impairments become less sensitive to rescue by BACE1^+/− reduction. Haploinsufficiency lowered BACE1 expression by ∼50% in 5XFAD mice regardless of age in concordance with reduction in gene copy number. However, profound Aβ plaque pathology and memory deficits concomitant with BACE1 equivalent to wild-type control levels remained in BACE1^+/−·5XFAD mice with advanced age (15–18 months old). Therefore, BACE1 haploinsufficiency is not sufficient to block the elevation of BACE1 expression (approximately twofold), which is also reported to occur during human AD progression, in 5XFAD mice. Our investigation revealed that PERK (PKR-endoplasmic reticulum-related kinase)-dependent activation of eIF2α (eukaryotic translation initiation factor-2α) accounts for the persistent BACE1 upregulation in BACE1^+/−·5XFAD mouse brains at 15–18 months of age. Moreover, BACE1 haploinsufficiency was also no longer able to prevent reduction in the expression of neprilysin, a crucial Aβ-degrading enzyme, in 5XFAD mice with advanced age. These findings demonstrate that partial BACE1 suppression cannot attenuate deleterious BACE1-elevating or neprilysin-reducing mechanisms, limiting its capabilities to reduce cerebral Aβ accumulation and rescue memory defects during the course of AD development.