Eicosapentaenoic acid and docosahexaenoic acid increase the degradation of amyloid-ß by affecting insulin-degrading enzyme.

Omega-3 polyunsaturated fatty acids (PUFAs) have been proposed to be highly beneficial in Alzheimer's disease (AD). AD pathology is closely linked to an overproduction and accumulation of amyloid-ß (Aß) peptides as extracellular senile plaques in the brain. Total Aß levels are not only dependent on its production by proteolytic processing of the amyloid precursor protein (APP), but also on Aß-clearance mechanisms, including Aß-degrading enzymes. Here we show that the omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increase Aß-degradation by affecting insulin-degrading enzyme (IDE), the major Aß-degrading enzyme secreted into the extracellular space of neuronal and microglial cells. The identification of the molecular mechanisms revealed that EPA directly increases IDE enzyme activity and elevates gene expression of IDE. DHA also directly stimulates IDE enzyme activity and affects IDE sorting by increasing exosome release of IDE, resulting in enhanced Aß-degradation in the extracellular milieu. Apart from the known positive effect of DHA in reducing Aß production, EPA and DHA might ameliorate AD pathology by increasing Aß turnover.

Oxidized Docosahexaenoic Acid Species and Lipid Peroxidation Products Increase Amyloidogenic Amyloid Precursor Protein Processing.

One of the main characteristics of Alzheimer's disease (AD) is the ß-amyloid peptide (Aß) generated by ß- and γ-secretase processing of the amyloid precursor protein (APP). Previously it has been demonstrated that polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA), are associated with a reduced risk of AD caused by decreased Aß production. However, in epidemiological studies and nutritional approaches, the outcomes of DHA-dependent treatment were partially controversial. PUFAs are very susceptible to reactive oxygen species and lipid peroxidation, which are increased during disease pathology. In line with published results, lipid peroxidation was elevated in human postmortem AD brains; especially 4-hydroxy-nonenal (HNE) was increased. To investigate whether lipid peroxidation is only a consequence or might also influence the processes leading to AD, we analyzed 7 different oxidized lipid species including 5 oxidized DHA derivatives and the lipid peroxidation products of ω-3 and ω-6 PUFAs, HNE and 4-hydroxy-hexenal, in human neuroblastoma cells and mouse mixed cortical neurons. In the presence of oxidized lipids Aß and soluble ß-secreted APP levels were elevated, whereas soluble α-secreted APP was decreased, suggesting a shift from the nonamyloidogenic to the amyloidogenic pathway of APP processing. Furthermore, ß- and γ-secretase activity was increased by oxidized lipids via increased gene expression and additionally by a direct effect on ß-secretase activity. Importantly, only 1% oxidized DHA was sufficient to revert the protective effect of DHA and to significantly increase Aß production. Therefore, our results emphasize the need to prevent DHA from oxidation in nutritional approaches and might help explain the divergent results of clinical DHA studies.

Tocotrienol Affects Oxidative Stress, Cholesterol Homeostasis and the Amyloidogenic Pathway in Neuroblastoma Cells: Consequences for Alzheimer's Disease.

One of the characteristics of Alzheimer´s disease (AD) is an increased amyloid load and an enhanced level of reactive oxidative species (ROS). Vitamin E has known beneficial neuroprotective effects, and previously, some studies suggested that vitamin E is associated with a reduced risk of AD due to its antioxidative properties. However, epidemiological studies and nutritional approaches of vitamin E treatment are controversial. Here, we investigate the effect of α-tocotrienol, which belongs to the group of vitamin E, on AD-relevant processes in neuronal cell lines. In line with the literature, α-tocotrienol reduced the ROS level in SH-SY5Y cells. In the presence of tocotrienols, cholesterol and cholesterol esters, which have been shown to be risk factors in AD, were decreased. Besides the unambiguous positive effects of tocotrienol, amyloid-ß (Aß) levels were increased accompanied by an increase in the activity of enzymes responsible for Aß production. Proteins and gene expression of the secretases and their components remained unchanged, whereas tocotrienol accelerates enzyme activity in cell-free assays. Besides enhanced Aß production, tocotrienols inhibited Aß degradation in neuro 2a (N2a)-cells. Our results might help to understand the controversial findings of vitamin E studies and demonstrate that besides the known positive neuroprotective properties, tocotrienols also have negative characteristics with respect to AD.

Streptococcus pneumoniae†PBP2x mid-cell localization requires the C-terminal PASTA domains and is essential for cell shape maintenance.

The transpeptidase activity of the essential penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae is believed to be important for murein biosynthesis required for cell division. To study the molecular mechanism driving localization of PBP2x in live cells, we constructed a set of N-terminal GFP-PBP2x fusions under the control of a zinc-inducible promoter. The ectopic fusion protein localized at mid-cell. Cells showed no growth defects even in the absence of the genomic pbp2x, demonstrating that GFP-PBP2x is functional. Depletion of GFP-PBP2x resulted in severe morphological alterations, confirming the essentiality of PBP2x and demonstrating that PBP2x is required for cell division and not for cell elongation. A genetically or antibiotic inactivated GFP-PBP2x still localized at septal sites. Remarkably, the same was true for a GFP-PBP2x derivative containing a deletion of the central transpeptidase domain, although only in the absence of the protease/chaperone HtrA. Thus localization is independent of the catalytic transpeptidase domain but requires the C-terminal PASTA domains, identifying HtrA as targeting GFP-PBP2x derivatives. Finally, PBP2x was positioned at the septum similar to PBP1a and the PASTA domain containing StkP protein, confirming that PBP2x is a key element of the divisome complex.

PS dependent APP cleavage regulates glucosylceramide synthase and is affected in Alzheimer's disease.

BACKGROUND: Gangliosides were found to be associated with Alzheimer's disease (AD). Here we addressed a potential function of γ-secretase (presenilin) dependent cleavage of the amyloid-precursor-protein (APP) in the regulation of ganglioside de novo synthesis. METHODS: To identify a potential role of γ-secretase and APP in ganglioside de novo synthesis we used presenilin (PS) deficient and APP deficient cells and mouse brains, mutated PS as well as transgenic mice and AD post mortem brains. Changes in glucosylceramide synthase (GCS) activity were identified by incorporation of radiolabeled UDP-glucose in glucosylceramide, changes in gene expression via real-time PCR and Western blot analysis. Alterations in ganglioside levels were determined by thin layer chromatography and mass spectrometry. RESULTS: We found that PS and APP deficiency, in vitro and in vivo, resulted in increased GCS gene expression, elevated enzyme activity and thus increased glucosylceramide and total ganglioside level. Using a specific γ-secretase inhibitor revealed that PS proteolytic activity alters ganglioside homeostasis. By the use of mutated PS causing early onset AD in cell culture and transgenic mice we found that GCS is increased in AD, further substantiated by the use of AD post mortem brains, suffering from sporadic AD. CONCLUSION: APP processing regulates ganglioside de novo synthesis and is affected in AD.

Impact of Vitamin D on amyloid precursor protein processing and amyloid-ß peptide degradation in Alzheimer's disease.

Ninety percent of the elderly population has a vitamin D hypovitaminosis, and several lines of evidence suggest that there might be a potential causal link between Alzheimer's disease (AD) and a non-sufficient supply with vitamin D. However, the mechanisms linking AD to vitamin D have not been completely understood. The aim of our study is to elucidate the impact of 25(OH) vitamin D3 on amyloid precursor protein processing in mice and N2A cells utilizing very moderate and physiological vitamin D hypovitaminosis in the range of 20-30% compared to wild-type mice. We found that already under such mild conditions, amyloid-ß peptide (Aß) is significantly increased, which is caused by an increased ß-secretase activity and BACE1 protein level. Additionally, neprilysin (NEP) expression is downregulated resulting in a decreased NEP activity further enhancing the effect of decreased vitamin D on the Aß level. In line with the in vivo findings, corresponding effects were found with N2A cells supplemented with 25(OH) vitamin D3. Our results further strengthen the link between AD and vitamin D3 and suggest that supplementation of vitamin D3 might have a beneficial effect in AD prevention.

A bidirectional link between sulfatide and Alzheimer's disease.

Reduced sulfatide level is found in Alzheimer's disease (AD) patients. Here, we demonstrate that amyloid precursor protein (APP) processing regulates sulfatide synthesis and vice versa. Different cell culture models and transgenic mice models devoid of APP processing or in particular the APP intracellular domain (AICD) reveal that AICD decreases Gal3st1/CST expression and subsequently sulfatide synthesis. In return, sulfatide supplementation decreases Aß generation by reducing ß-secretase (BACE1) and γ-secretase processing of APP. Increased BACE1 lysosomal degradation leads to reduced BACE1 protein level in endosomes. Reduced γ-secretase activity is caused by a direct effect on γ-secretase activity and reduced amounts of γ-secretase components in lipid rafts. Similar changes were observed by analyzing cells and mice brain samples deficient of arylsulfatase A responsible for sulfatide degradation or knocked down in Gal3st1/CST. In line with these findings, addition of sulfatides to brain homogenates of AD patients resulted in reduced γ-secretase activity. Human brain APP level shows a significant negative correlation with GAL3ST1/CST expression underlining the in vivo relevance of sulfatide homeostasis in AD.

The impact of capsaicinoids on APP processing in Alzheimer's disease in SH-SY5Y cells.

The vanilloid capsaicin is a widely consumed spice, known for its burning and "hot" sensation through activation of TRPV1 ion-channels, but also known to decrease oxidative stress, inflammation and influence tau-pathology. Beside these positive effects, little is known about its effects on amyloid-precursor-protein (APP) processing leading to amyloid-ß (Aß), the major component of senile plaques. Treatment of neuroblastoma cells with capsaicinoids (24 hours, 10 µM) resulted in enhanced Aß-production and reduced Aß-degradation, leading to increased Aß-levels. In detailed analysis of the amyloidogenic-pathway, both BACE1 gene-expression as well as protein-levels were found to be elevated, leading to increased ß-secretase-activity. Additionally, γ-secretase gene-expression as well as activity was enhanced, accompanied by a shift of presenilin from non-raft to raft membrane-domains where amyloidogenic processing takes place. Furthermore, impaired Aß-degradation in presence of capsaicinoids is dependent on the insulin-degrading-enzyme, one of the major Aß-degrading-enzymes. Regarding Aß-homeostasis, no differences were found between the major capsaicinoids, capsaicin and dihydrocapsaicin, and a mixture of naturally derived capsaicinoids; effects on Ca2+-homeostasis were ruled out. Our results show that in respect to Alzheimer's disease, besides the known positive effects of capsaicinoids, pro-amyloidogenic properties also exist, enhancing Aß-levels, likely restricting the potential use of capsaicinoids as therapeutic substances in Alzheimer's disease.

Regulatory feedback cycle of the insulin-degrading enzyme and the amyloid precursor protein intracellular domain: Implications for Alzheimer's disease.

One of the major pathological hallmarks of Alzheimer´s disease (AD) is an accumulation of amyloid-ß (Aß) in brain tissue leading to formation of toxic oligomers and senile plaques. Under physiological conditions, a tightly balanced equilibrium between Aß-production and -degradation is necessary to prevent pathological Aß-accumulation. Here, we investigate the molecular mechanism how insulin-degrading enzyme (IDE), one of the major Aß-degrading enzymes, is regulated and how amyloid precursor protein (APP) processing and Aß-degradation is linked in a regulatory cycle to achieve this balance. In absence of Aß-production caused by APP or Presenilin deficiency, IDE-mediated Aß-degradation was decreased, accompanied by a decreased IDE activity, protein level, and expression. Similar results were obtained in cells only expressing a truncated APP, lacking the APP intracellular domain (AICD) suggesting that AICD promotes IDE expression. In return, APP overexpression mediated an increased IDE expression, comparable results were obtained with cells overexpressing C50, a truncated APP representing AICD. Beside these genetic approaches, also AICD peptide incubation and pharmacological inhibition of the γ-secretase preventing AICD production regulated IDE expression and promoter activity. By utilizing CRISPR/Cas9 APP and Presenilin knockout SH-SY5Y cells results were confirmed in a second cell line in addition to mouse embryonic fibroblasts. In vivo, IDE expression was decreased in mouse brains devoid of APP or AICD, which was in line with a significant correlation of APP expression level and IDE expression in human postmortem AD brains. Our results show a tight link between Aß-production and Aß-degradation forming a regulatory cycle in which AICD promotes Aß-degradation via IDE and IDE itself limits its own production by degrading AICD.

Rescue of Hypovitaminosis A Induces Non-Amyloidogenic Amyloid Precursor Protein (APP) Processing.

Retinoic acid, the bioactive metabolite of beta-carotene or vitamin A, plays a pleiotropic, multifunctional role in vertebrate development. Studies in rodents revealed that a diet deficient in vitamin A results in a complex neonatal syndrome (the VAD syndrome), manifested in many organs. In humans, the function of retinoic acid (RA) extends into adulthood, where it has important roles in fertility, vision, and suppression of neoplastic growth. In recent years, it has also been suggested that retinoic acid might potentially act as a therapeutically relevant drug in attenuating or even preventing neurodegenerative diseases such as Alzheimer's disease (AD). Here, we report that VAD leads to an increase in A-beta peptide levels while only minor effects were observed on expression levels of the amyloid precursor protein (APP) processing proteinases in wild type mice. In line with these findings, rescue of hypovitaminosis reduced A-beta amount to baseline and induced sApp-alpha secretion in combination with an increase of alpha-secretase Adam10. By comparing retinoic acid treatment starting from a full nutrition status and a "VAD" situation in human neuroblastoma cells, we show that while intensities of differential gene expression were higher in replenished cells, a large overlap in AD-related, regulated genes was observed. Our data suggest that hypovitaminosis A can contribute to onset or progression of AD by increasing synthesis of A-beta peptides and that several AD-related genes such as ADAM10 or BDNF are regulated by retinoic acid. We suggest that dietary supplementation with retinoic acid derivatives is likely to have a beneficial effect on AD-pathology in individuals with hypovitaminosis and patients with normal vitamin A status.

APP intracellular domain derived from amyloidogenic ß- and γ-secretase cleavage regulates neprilysin expression.

Alzheimer's disease (AD) is characterized by an accumulation of Amyloid-ß (Aß), released by sequential proteolytic processing of the amyloid precursor protein (APP) by ß - and γ-secretase. Aß peptides can aggregate, leading to toxic Aß oligomers and amyloid plaque formation. Aß accumulation is not only dependent on de novo synthesis but also on Aß degradation. Neprilysin (NEP) is one of the major enzymes involved in Aß degradation. Here we investigate the molecular mechanism of NEP regulation, which is up to now controversially discussed to be affected by APP processing itself. We found that NEP expression is highly dependent on the APP intracellular domain (AICD), released by APP processing. Mouse embryonic fibroblasts devoid of APP processing, either by the lack of the catalytically active subunit of the γ-secretase complex [presenilin (PS) 1/2] or by the lack of APP and the APP-like protein 2 (APLP2), showed a decreased NEP expression, activity and protein level. Similar results were obtained by utilizing cells lacking a functional AICD domain (APPΔCT15) or expressing mutations in the genes encoding for PS1. AICD supplementation or retransfection with an AICD encoding plasmid could rescue the down-regulation of NEP further strengthening the link between AICD and transcriptional NEP regulation, in which Fe65 acts as an important adaptor protein. Especially AICD generated by the amyloidogenic pathway seems to be more involved in the regulation of NEP expression. In line, analysis of NEP gene expression in vivo in six transgenic AD mouse models (APP and APLP2 single knock-outs, APP/APLP2 double knock-out, APP-swedish, APP-swedish/PS1Δexon9, and APPΔCT15) confirmed the results obtained in cell culture. In summary, in the present study we clearly demonstrate an AICD-dependent regulation of the Aß-degrading enzyme NEP in vitro and in vivo and elucidate the underlying mechanisms that might be beneficial to develop new therapeutic strategies for the treatment of AD.

Penicillin-binding protein 2x of Streptococcus pneumoniae: the mutation Ala707Asp within the C-terminal PASTA2 domain leads to destabilization.

Streptococcus pneumoniae penicillin-binding protein 2x (PBP2x) is an enzyme involved in the last stages of peptidoglycan assembly and essential for bacterial growth and survival. PBP2x localizes to the division site, a process that depends on its Penicillin-Binding Protein And Serine-Threonine-kinase Associated (PASTA) domains, which was previously demonstrated via GFP-PBP2x in living cells. During this study a mutant strain was isolated in which the GFP-PBP2x fusion protein did not localize at division sites and it contained reduced amounts of the full-length GFP-PBP2x. We now show that this defect is due to a point mutation within the C-terminal PASTA2 domain of PBP2x. The mutant protein was analyzed in detail in terms of beta-lactam binding, functionality, and localization in live cells. We demonstrate that the mutation affects the GFP-tagged PBP2x variant severely and renders it susceptible to the protease/chaperone HtrA.

Upregulation of PGC-1α expression by Alzheimer's disease-associated pathway: presenilin 1/amyloid precursor protein (APP)/intracellular domain of APP.

Cleavage of amyloid precursor protein (APP) by ß- and γ-secretase generates amyloid-ß (Aß) and APP intracellular domain (AICD) peptides. Presenilin (PS) 1 or 2 is the catalytic component of the γ-secretase complex. Mitochondrial dysfunction is an established phenomenon in Alzheimer's disease (AD), but the causes and role of PS1, APP, and APP's cleavage products in this process are largely unknown. We studied the effect of these AD-associated molecules on mitochondrial features. Using cells deficient in PSs expression, expressing human wild-type PS1, or PS1 familial AD (FAD) mutants, we found that PS1 affects mitochondrial energy metabolism (ATP levels and oxygen consumption) and expression of mitochondrial proteins. These effects were associated with enhanced expression of the mitochondrial master transcriptional coactivator PGC-1α and its target genes. Importantly, PS1-FAD mutations decreased PS1's ability to enhance PGC-1α mRNA levels. Analyzing the effect of APP and its γ-secretase-derived cleavage products Aß and AICD on PGC-1α expression showed that APP and AICD increase PGC-1α expression. Accordingly, PGC-1α mRNA levels in cells deficient in APP/APLP2 or expressing APP lacking its last 15 amino acids were lower than in control cells, and treatment with AICD, but not with Aß, enhanced PGC-1α mRNA levels in these and PSs-deficient cells. In addition, knockdown of the AICD-binding partner Fe65 reduced PGC-1α mRNA levels. Importantly, APP/AICD increases PGC-1α expression also in the mice brain. Our results therefore suggest that APP processing regulates mitochondrial function and that impairments in the newly discovered PS1/APP/AICD/PGC-1α pathway may lead to mitochondrial dysfunction and neurodegeneration.

Neprilysin and Aß Clearance: Impact of the APP Intracellular Domain in NEP Regulation and Implications in Alzheimer's Disease.

One of the characteristic hallmarks of Alzheimer's disease (AD) is an accumulation of amyloid ß (Aß) leading to plaque formation and toxic oligomeric Aß complexes. Besides the de novo synthesis of Aß caused by amyloidogenic processing of the amyloid precursor protein (APP), Aß levels are also highly dependent on Aß degradation. Several enzymes are described to cleave Aß. In this review we focus on one of the most prominent Aß degrading enzymes, the zinc-metalloprotease Neprilysin (NEP). In the first part of the review we discuss beside the general role of NEP in Aß degradation the alterations of the enzyme observed during normal aging and the progression of AD. In vivo and cell culture experiments reveal that a decreased NEP level results in an increased Aß level and vice versa. In a pathological situation like AD, it has been reported that NEP levels and activity are decreased and it has been suggested that certain polymorphisms in the NEP gene result in an increased risk for AD. Conversely, increasing NEP activity in AD mouse models revealed an improvement in some behavioral tests. Therefore it has been suggested that increasing NEP might be an interesting potential target to treat or to be protective for AD making it indispensable to understand the regulation of NEP. Interestingly, it is discussed that the APP intracellular domain (AICD), one of the cleavage products of APP processing, which has high similarities to Notch receptor processing, might be involved in the transcriptional regulation of NEP. However, the mechanisms of NEP regulation by AICD, which might be helpful to develop new therapeutic strategies, are up to now controversially discussed and summarized in the second part of this review. In addition, we review the impact of AICD not only in the transcriptional regulation of NEP but also of further genes.