Plant sterols the better cholesterol in Alzheimer's disease? A mechanistical study.

Amyloid-ß (Aß), major constituent of senile plaques in Alzheimer's disease (AD), is generated by proteolytic processing of the amyloid precursor protein (APP) by ß- and γ-secretase. Several lipids, especially cholesterol, are associated with AD. Phytosterols are naturally occurring cholesterol plant equivalents, recently been shown to cross the blood-brain-barrier accumulating in brain. Here, we investigated the effect of the most nutritional prevalent phytosterols and cholesterol on APP processing. In general, phytosterols are less amyloidogenic than cholesterol. However, only one phytosterol, stigmasterol, reduced Aß generation by (1) directly decreasing ß-secretase activity, (2) reducing expression of all γ-secretase components, (3) reducing cholesterol and presenilin distribution in lipid rafts implicated in amyloidogenic APP cleavage, and by (4) decreasing BACE1 internalization to endosomal compartments, involved in APP ß-secretase cleavage. Mice fed with stigmasterol-enriched diets confirmed protective effects in vivo, suggesting that dietary intake of phytosterol blends mainly containing stigmasterol might be beneficial in preventing AD.

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.

Effect of Different Phospholipids on α-Secretase Activity in the Non-Amyloidogenic Pathway of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by extracellular accumulation of amyloid-ß peptide (Aß), generated by proteolytic processing of the amyloid precursor protein (APP) by ß- and γ-secretase. Aß generation is inhibited when the initial ectodomain shedding is caused by α-secretase, cleaving APP within the Aß domain. Therefore, an increase in α-secretase activity is an attractive therapeutic target for AD treatment. APP and the APP-cleaving secretases are all transmembrane proteins, thus local membrane lipid composition is proposed to influence APP processing. Although several studies have focused on γ-secretase, the effect of the membrane lipid microenvironment on α-secretase is poorly understood. In the present study, we systematically investigated the effect of fatty acid (FA) acyl chain length (10:0, 12:0, 14:0, 16:0, 18:0, 20:0, 22:0, 24:0), membrane polar lipid headgroup (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine), saturation grade and the FA double-bond position on α-secretase activity. We found that α-secretase activity is significantly elevated in the presence of FAs with short chain length and in the presence of polyunsaturated FAs, whereas variations in the phospholipid headgroups, as well as the double-bond position, have little or no effect on α-secretase activity. Overall, our study shows that local lipid membrane composition can influence α-secretase activity and might have beneficial effects for AD.

Docosahexaenoic acid reduces amyloid beta production via multiple pleiotropic mechanisms.

Alzheimer disease is characterized by accumulation of the ß-amyloid peptide (Aß) generated by ß- and γ-secretase processing of the amyloid precursor protein (APP). The intake of the polyunsaturated fatty acid docosahexaenoic acid (DHA) has been associated with decreased amyloid deposition and a reduced risk in Alzheimer disease in several epidemiological trials; however, the exact underlying molecular mechanism remains to be elucidated. Here, we systematically investigate the effect of DHA on amyloidogenic and nonamyloidogenic APP processing and the potential cross-links to cholesterol metabolism in vivo and in vitro. DHA reduces amyloidogenic processing by decreasing ß- and γ-secretase activity, whereas the expression and protein levels of BACE1 and presenilin1 remain unchanged. In addition, DHA increases protein stability of α-secretase resulting in increased nonamyloidogenic processing. Besides the known effect of DHA to decrease cholesterol de novo synthesis, we found cholesterol distribution in plasma membrane to be altered. In the presence of DHA, cholesterol shifts from raft to non-raft domains, and this is accompanied by a shift in γ-secretase activity and presenilin1 protein levels. Taken together, DHA directs amyloidogenic processing of APP toward nonamyloidogenic processing, effectively reducing Aß release. DHA has a typical pleiotropic effect; DHA-mediated Aß reduction is not the consequence of a single major mechanism but is the result of combined multiple effects.

Plasmalogens inhibit APP processing by directly affecting γ-secretase activity in Alzheimer's disease.

Lipids play an important role as risk or protective factors in Alzheimer's disease (AD). Previously it has been shown that plasmalogens, the major brain phospholipids, are altered in AD. However, it remained unclear whether plasmalogens themselves are able to modulate amyloid precursor protein (APP) processing or if the reduced plasmalogen level is a consequence of AD. Here we identify the plasmalogens which are altered in human AD postmortem brains and investigate their impact on APP processing resulting in Aß production. All tested plasmalogen species showed a reduction in γ-secretase activity whereas ß- and α-secretase activity mainly remained unchanged. Plasmalogens directly affected γ-secretase activity, protein and RNA level of the secretases were unaffected, pointing towards a direct influence of plasmalogens on γ-secretase activity. Plasmalogens were also able to decrease γ-secretase activity in human postmortem AD brains emphasizing the impact of plasmalogens in AD. In summary our findings show that decreased plasmalogen levels are not only a consequence of AD but that plasmalogens also decrease APP processing by directly affecting γ-secretase activity, resulting in a vicious cycle: Aß reduces plasmalogen levels and reduced plasmalogen levels directly increase γ-secretase activity leading to an even stronger production of Aß peptides.

Plasmalogen synthesis is regulated via alkyl-dihydroxyacetonephosphate-synthase by amyloid precursor protein processing and is affected in Alzheimer's disease.

Lipids play an important role as risk or protective factors in Alzheimer's disease, which is characterized by amyloid plaques composed of aggregated amyloid-beta. Plasmalogens are major brain lipids and controversially discussed to be altered in Alzheimer's disease (AD) and whether changes in plasmalogens are cause or consequence of AD pathology. Here, we reveal a new physiological function of the amyloid precursor protein (APP) in plasmalogen metabolism. The APP intracellular domain was found in vivo and in vitro to increase the expression of the alkyl-dihydroxyacetonephosphate-synthase (AGPS), a rate limiting enzyme in plasmalogen synthesis. Alterations in APP dependent changes of AGPS expression result in reduced protein and plasmalogen levels. Under the pathological situation of AD, increased amyloid-beta level lead to increased reactive oxidative species production, reduced AGPS protein and plasmalogen level. Accordingly, phosphatidylethanol plasmalogen was decreased in the frontal cortex of AD compared to age matched controls. Our findings elucidate that plasmalogens are decreased as a consequence of AD and regulated by APP processing under physiological conditions.

Mechanisms of after-hyperpolarization following activation of fly visual motion-sensitive neurons.

In many neurons, strong excitatory stimulation causes an after-hyperpolarization (AHP) at stimulus offset, which might give rise to activity-dependent adaptation. Graded-potential visual motion-sensitive neurons of the fly Calliphora vicina respond with depolarization and hyperpolarization during motion in their preferred direction and their anti-preferred direction, respectively. A prominent after-response, opposite in sign to the response during motion, is selectively expressed after stimulation with preferred-direction motion. Previous findings suggested that this AHP is generated in the motion-sensitive neurons themselves rather than in presynaptic processing layers. However, it remained unknown whether the AHP is caused by membrane depolarization itself or by another process, e.g. a signaling cascade triggered by activity of excitatory input channels. Here we showed by current injections and voltage clamp that the AHP and a corresponding current are generated directly by depolarization. To test whether the generation of an AHP is linked to depolarization via a Ca(2+)-dependent mechanism, we used photoactivation of a high-affinity Ca(2+) buffer. In accordance with previous findings the AHP was insensitive to manipulation of cytosolic Ca(2+). We propose that membrane depolarization presents a more direction-selective mechanism for the control of AHP than other potential control parameters.

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.

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.

Trans fatty acids enhance amyloidogenic processing of the Alzheimer amyloid precursor protein (APP).

Hydrogenation of oils and diary products of ruminant animals leads to an increasing amount of trans fatty acids in the human diet. Trans fatty acids are incorporated in several lipids and accumulate in the membrane of cells. Here we systematically investigate whether the regulated intramembrane proteolysis of the amyloid precursor protein (APP) is affected by trans fatty acids compared to the cis conformation. Our experiments clearly show that trans fatty acids compared to cis fatty acids increase amyloidogenic and decrease nonamyloidogenic processing of APP, resulting in an increased production of amyloid beta (Aß) peptides, main components of senile plaques, which are a characteristic neuropathological hallmark for Alzheimer's disease (AD). Moreover, our results show that oligomerization and aggregation of Aß are increased by trans fatty acids. The mechanisms identified by this in vitro study suggest that the intake of trans fatty acids potentially increases the AD risk or causes an earlier onset of the disease.

Amyloid precursor protein (APP) mediated regulation of ganglioside homeostasis linking Alzheimer's disease pathology with ganglioside metabolism.

Gangliosides are important players for controlling neuronal function and are directly involved in AD pathology. They are among the most potent stimulators of Aß production, are enriched in amyloid plaques and bind amyloid beta (Aß). However, the molecular mechanisms linking gangliosides with AD are unknown. Here we identified the previously unknown function of the amyloid precursor protein (APP), specifically its cleavage products Aß and the APP intracellular domain (AICD), of regulating GD3-synthase (GD3S). Since GD3S is the key enzyme converting a- to b-series gangliosides, it therefore plays a major role in controlling the levels of major brain gangliosides. This regulation occurs by two separate and additive mechanisms. The first mechanism directly targets the enzymatic activity of GD3S: Upon binding of Aß to the ganglioside GM3, the immediate substrate of the GD3S, enzymatic turnover of GM3 by GD3S was strongly reduced. The second mechanism targets GD3S expression. APP cleavage results, in addition to Aß release, in the release of AICD, a known candidate for gene transcriptional regulation. AICD strongly down regulated GD3S transcription and knock-in of an AICD deletion mutant of APP in vivo, or knock-down of Fe65 in neuroblastoma cells, was sufficient to abrogate normal GD3S functionality. Equally, knock-out of the presenilin genes, presenilin 1 and presenilin 2, essential for Aß and AICD production, or of APP itself, increased GD3S activity and expression and consequently resulted in a major shift of a- to b-series gangliosides. In addition to GD3S regulation by APP processing, gangliosides in turn altered APP cleavage. GM3 decreased, whereas the ganglioside GD3, the GD3S product, increased Aß production, resulting in a regulatory feedback cycle, directly linking ganglioside metabolism with APP processing and Aß generation. A central aspect of this homeostatic control is the reduction of GD3S activity via an Aß-GM3 complex and AICD-mediated repression of GD3S transcription.

Intracellular APP Domain Regulates Serine-Palmitoyl-CoA Transferase Expression and Is Affected in Alzheimer's Disease.

Lipids play an important role as risk or protective factors in Alzheimer's disease (AD), a disease biochemically characterized by the accumulation of amyloid beta peptides (Aß), released by proteolytic processing of the amyloid precursor protein (APP). Changes in sphingolipid metabolism have been associated to the development of AD. The key enzyme in sphingolipid de novo synthesis is serine-palmitoyl-CoA transferase (SPT). In the present study we identified a new physiological function of APP in sphingolipid synthesis. The APP intracellular domain (AICD) was found to decrease the expression of the SPT subunit SPTLC2, the catalytic subunit of the SPT heterodimer, resulting in that decreased SPT activity. AICD function was dependent on Fe65 and SPTLC2 levels are increased in APP knock-in mice missing a functional AICD domain. SPTLC2 levels are also increased in familial and sporadic AD postmortem brains, suggesting that SPT is involved in AD pathology.

The acetylcholine fiber density of the neocortex is altered by isolated rearing and early methamphetamine intoxication in rodents.

Alterations in the cholinergic physiology of the brain were the first to be observed when research on environmental influences on postnatal brain development began 35 years ago. Since then, the effects of isolated rearing (IR) or early pharmacological insults have been shown not only on the physiology, but also the anatomy of a variety of transmitter systems. The cholinergic fiber density, however, still remained to be assessed. We therefore used a histochemical procedure to stain cholinergic fibers in the brains of young adult gerbils reared either in groups in enriched environments or isolated in standard makrolon cages. Half of the animals from each rearing condition had received a single high dose of methamphetamine on postnatal day 14. Fiber densities were measured by computerized image analysis in the medial and orbital prefrontal cortex (PFC), dysgranular and granular insular cortex, sensorimotor cortices, and the entorhinal cortex of both hemispheres. Isolation rearing increased the cholinergic fiber densities in the prefrontal cortices of the left hemisphere and in the entorhinal cortex of the right hemisphere by about 10%, with no effect in the respective contralateral side. The early methamphetamine intoxication showed no influence in prefrontal and entorhinal cortices, but diminished the acetylcholine (ACh) innervation of the forelimb area of cortex in both hemispheres in IR gerbils and of the left hemisphere in ER gerbils, and reduced the acetylcholine innervation in the hindlimb area in both sides in both rearing groups. These results demonstrate that (a) cholinergic fiber density is differentially regulated in different cortical areas and (b) the plasticity of the cholinergic system can only be understood in the interplay with other neuromodulatory innervations.