Guest guest Posted October 10, 2005 Report Share Posted October 10, 2005 I couldnt find the abstract online yet, so in the meantime, heres the PR on it.. http://my.webmd.com/content/article/113/110683?src=RSS_PUBLIC Jeff Quote Link to comment Share on other sites More sharing options...
Guest guest Posted October 11, 2005 Report Share Posted October 11, 2005 --- Jeff Novick <jnovick@...> wrote: Hi All, Being in a rush, the below pdf-available excerpts are the best available version. http://my.webmd.com/content/article/113/110683?src=RSS_PUBLIC Nature Cell Biology, Oct. 9, 2005 advance online edition. Alzheimer's disease: Understanding the lipid connection Letter by Grimm et al. Varying cholesterol levels in mouse models has been shown to affect progress of Alzheimer's disease. Hartmann and colleagues now show that Ab peptide, the amyloid peptide that is produced normally but accumulates markedly during Alzheimer's, controls lipid metabolism by acting on two key enzymes in the pathway. Marcus O. W. Grimm1, Heike S. Grimm1, s J. Pätzold1, Eva G. Zinser1, Riikka Halonen1, Marco Duering1, Jakob-A. Tschäpe1, Bart De Strooper2, Ulrike Müller3, Jie Shen4 & Tobias Hartmann1 Amyloid beta peptide (Aß) has a key role in the pathological process of Alzheimer's disease (AD), but the physiological function of Aß and of the amyloid precursor protein (APP) is unknown1, 2. Recently, it was shown that APP processing is sensitive to cholesterol and other lipids3, 4, 5, 6, 7, 8, 9, 10. Hydroxymethylglutaryl-CoA reductase (HMGR) and sphingomyelinases (SMases) are the main enzymes that regulate cholesterol biosynthesis and sphingomyelin (SM) levels, respectively. We show that control of cholesterol and SM metabolism involves APP processing. A42 directly activates neutral SMase and downregulates SM levels, whereas A40 reduces cholesterol de novo synthesis by inhibition of HMGR activity. This process strictly depends on gamma-secretase activity. In line with altered A40/42 generation, pathological presenilin mutations result in increased cholesterol and decreased SM levels. Our results demonstrate a biological function for APP processing and also a functional basis for the link that has been observed between lipids and Alzheimer's disease (AD). One of the main characteristics of Alzheimer's disease (AD) is the accumulation of amyloid beta peptide (A), which is associated with neurodegeneration1, 2. A production is initiated by the gamma-secretase cleavage of amyloid precursor protein (APP), which results in production of the APP C-terminal fragment C99. This fragment is further cleaved by gamma-secretase to generate A40 and A42. The active centre of the gamma-secretase complex is formed by presenilin-1 or -2 (PS1 or PS2)11. As the gamma-secretase cleavage site is centred within the transmembrane domain, membrane composition may considerably influence Aß generation12. Such proteolytic events of regulated intra-membrane proteolysis (RIP) are involved in numerous cellular regulatory events, including lipid homeostasis13. So far, the physiological role of APP and its proteolytic fragments, including A, have mainly been conceived as being brain specific. However, the ubiquitous production of APP, Aß and PS indicates that their functions are also ubiquitous. Recently, strong evidence has accumulated that links AD and Aß generation with lipid homeostasis5, and it has been suggested that cellular lipid metabolism controls APP processing5. Studies have shown that Aß production is sensitive to cholesterol levels and lipid trafficking. In vitro enhanced cholesterol levels lead to increased Aß production14. Furthermore, cholesterol depletion reduces gamma-secretase activity and results in reduced Aß generation3, 4, 6, 7, 8, 9. Controversially, one report found a correlation between mildly decreased cholesterol levels and increased Aß production in Chinese hamster ovary cells15. In vivo, Aß production and deposition are tightly associated with changes in cholesterol levels6, 10. Lipid levels vary in response to changes in diet, and physical or neuronal activity. Similarly, Aß levels change in response to several conditions, including cholesterol and other factors16, 17. Brain cholesterol levels increase during the progression of AD18 and hypercholesterolaemia is an early risk factor in the development of AD19. Consequently, cholesterol-lowering drugs are being considered as a potential treatment for AD16, 20, 21. Nevertheless, the cellular mechanisms that link lipids, Aß generation and AD are poorly understood. To investigate the role of PS in lipid regulation, we used mouse embryonic fibroblast cells that were devoid of PS1 and PS2 (MEF PS1/2-/-)11 and expression-level-matched, A-producing PS re-transfected MEF PS1/2-/- control cells (MEF PS1r and MEF PS2r, respectively) (see Supplementary Information, Fig. S1). The absence of PS resulted in increased cellular levels of cholesterol and sphingomyelin (SM), but not phosphatidylcholine (PC) or total lipid-associated phosphate (Fig. 1a). The catabolic sphingomyelinase (SMase) activities were downregulated, anabolic SM-synthase activity was upregulated and the SM-to-ceramide ratio was increased in PS-deficient cells in accordance with the altered neutral SMase (nSMase) and SM-synthase activities (Fig. 1a, see Supplementary Information, Fig. S2a). Absence of PS1 is lethal and, as PS2 is able to replace PS1 (for example, in nSMase activity, see Fig. 1c), brains from conditional PS1 knockout mice, crossed to PS2 knockout mice, were used to evaluate PS function in lipid homeostasis in vivo. In these viable mice, PS1 remains expressed in some neurons and other cells22 and brain Aß levels in these mice are reduced to 20 & #8722;50%, depending on age and Aß species23. As before, absence of PS in neurons resulted in increased cholesterol and SM levels, decreased SMase activity, but unchanged PC levels (Fig. 1b). Dependence on gamma-secretase proteolytic activity was investigated using the gamma-secretase activity inhibitor L-685,458. This treatment reduced nSMase activity in MEF PS1r and PS2r cells to similar levels as those of PS1/2-/- cells (Fig. 1c). By contrast, this inhibitor treatment had no effect on MEF cells that were devoid of PS (Fig. 1c). Treatment of wild-type mouse neurons decreased nSMase activity and increased SM levels (Fig. 1c). Accordingly, in inhibitor-treated PS-expressing cells, SM levels were increased. Cholesterol levels increased in MEF PS1/2-/- cells (Fig. 1a), whereas no significant difference was observed between inhibitor-treated MEF PS1r and MEF PS1/2-/- cells (see Supplementary Information, Fig. S2b). It was established previously that cholesterol influences Aß production. To investigate Aß putative influence of SM on Aß production, we inhibited nSMase activity in neurons. This resulted in increased SM levels and decreased intracellular and secreted Aß (Fig. 2a). This was confirmed in COS7 cells using additional inhibitors and direct SM exposure (see Supplementary Information, Fig. S3a, 3b). Reduced gamma-secretase cleavage causes C99 accumulation, which was observed with increased SM levels (see Supplementary Information, Fig. S3c). Our results implicate the involvement of a gamma-secretase substrate and that its cleavage is essential in PS-mediated lipid regulation. MEF APP/APLP2-/- and MEF wild-type cells were analysed to evaluate whether APP or APLP2 might be involved in this regulatory cascade. SM, nSMase activity and cholesterol levels responded to the absence of APP/APLP2 in the same way as they did to the absence of PS/gamma-secretase activity (Fig. 2b). The same was observed in APP-/- mouse brain (Fig. 2b), suggesting that an in vivo function of APP exists in lipid homeostasis. Unlike in wild-type cells, inhibition of gamma-secretase activity did not alter cholesterol in MEF APP/APLP2-/- cells (Fig. 2c). This seems to rule out the existence of other gamma-secretase substrates that are able to replace the APP/APLP2 function in cholesterol regulation in these cells. The combined necessity of APP and gamma-secretase activity in this process indicates that a proteolytic APP fragment is involved in lipid homeostasis (Fig. 2b, c). The most prominent peptide that is derived from APP gamma-secretase cleavage is A. To determine a possible function of Aß in cholesterol and SM regulation, we analysed key enzymes and metabolites in the respective pathways. Analysing SM metabolism in MEF APP/APLP2-/- cells showed a decrease in nSMase activity compared with MEF wild-type cells (Fig. 2b). When exposed to A-containing conditioned media from human SH-SY5Y cells, the nSMase enzymatic activity was partially restored in MEF APP/APLP2-/- cells (Fig. 2d, left). To investigate whether this might be caused by A, cell homogenates of MEF APP/APLP2-/- cells were exposed to synthetic A1 & #8722;40/A1 & #8722;42 at a physiological ratio24, resulting in enhanced nSMase activity (Fig. 2d, right). To determine whether SMase is a molecular target for regulation by Aß and to evaluate whether this interaction might be direct, purified human placenta nSMase was exposed to synthetic Aß in a biochemical assay that was free of other cellular components. Covering the physiological concentration range of A24, the effect reached a maximum at peptide levels that are typically found in cerebrospinal fluid. The effect decreased thereafter, probably due to augmented peptide aggregation (Fig. 2e). An effect of Aß on the other assay components was excluded (see Supplementary Information, Fig. S4a); A42 & #8722;1 (inverse A) was inactive and aggregated Aß had only residual stimulatory activity, presumably due to remnant non-aggregated peptide. An excess of A40 reduced nSMase activation by A42, indicating A42 specificity (see Supplementary Information, Fig. S4b). To evaluate the involvement of oxidative stress in nSMase activation by Aß peptides, the production of reactive oxygen species (ROS) was investigated. No significant differences in ROS were observed when wild-type and rescue cells were compared with the respective knockout cells (Fig. 2f). Incubation of MEF PS1/2-/- with Aß in the concentration range used above did not result in a concentration-dependent increase of ROS (Fig. 2e). To investigate the regulation of cholesterol metabolism, MEF APP/APLP2-/- cells were treated with A, which reversed the effects that had been induced by the absence of APP/APLP2 (see Supplementary Information, Fig. S4c). To ascertain the mechanism of A in cholesterol homeostasis, MEF APP/APLP2-/- cells were treated with the two Aß species separately and de novo synthesized cholesterol was analysed. In contrast to nSMase activity with A40, which showed only a minor effect, de novo cholesterol synthesis responded already to low levels of A40 and partially restored wild-type levels (Fig. 2g, left). To identify the enzyme in the cholesterol biosynthetic pathway targeted by A, MEF APP/APLP2-/- cells were incubated with the hydroxymethylglutaryl-CoA reductase (HMGR) substrate HMG-CoA, or with mevalonate, the HMGR product. Increased de novo cholesterol synthesis was observed upon incubation with HMG-CoA, but not with mevalonate (Fig. 2g, right). Genotype-specific alterations in cellular cholesterol release could be excluded (Fig. 2h). This identifies HMGR as a target in the regulation of cholesterol de novo synthesis by APP/A. The different effects of A40 (HMGR inhibition) and A42 (SMase activation) allow for a specific prediction of their activities in a cell-biological context. A change in A40/42 ratio should lead to altered lipid levels. Familial PS mutations (PS-FAD) strongly increase A42 and mildly decrease A40 levels, without a relevant impact on the total A levels. Expression of PS-FAD mutations resulted in increased cholesterol and decreased SM levels, which was further confirmed by increased nSMase activity (Fig. 3). As predicted by the previous experiments, changes in the A40/42 ratio alter lipid homeostasis. In summary, our in vivo and in vitro data are consistent with a model (Fig. 4) in which the cascade of gamma-secretase, APP processing and A peptides downregulate cholesterol and sphingolipid levels, which, in turn, regulate A generation. The molecular targets for A in the cholesterol and SM metabolic pathways are HMGR and nSMase, respectively. The role of A in lipid homeostasis implies that its precursor APP has a function in lipid homeostasis, which is supported by altered lipid homeostasis in APP-/- mice. We assume that additional gamma-secretase substrates and cleavage products contribute to lipid homeostasis. This is supported by partial reversal of the lipid phenotype in cells following addition of A (Fig. 2g). Multiple gamma-secretase products exist. Our data identify the involvement of A, but do not rule out the involvement of other fragments. In the MEF APP/APLP2-/- cells, lipid homeostasis could not be altered by inhibition of gamma-secretase proteolytic activity. This identifies that APP/APLP2 in the MEF model are essential substrates. In addition, it rules out that, in the absence of APP/APLP2, other putative gamma-secretase substrates have a detectable effect on SM and cholesterol homeostasis. The gamma-secretase-dependent regulation of lipid homeostasis is likely to be influenced by additional factors, as indicated by the other proteases that are involved in APP RIP and gamma-secretase-independent regulatory cycles13. In agreement with the ubiquitous presence of APP, A and PS25, 26, the mechanism described here was found in cells of different origin. Stimulation of SMase by addition of synthetic A peptides in the absence of any other modulating factors in a cell-free assay indicates that A42 directly interacts with nSMase. This is consistent with the presence of an nSMase luminal/extracellular domain and the subcellular localizations of nSMases and A. Physiological A42 concentrations were not previously investigated in this context, but high concentrations of A42 or fibrillary A42 were reported to cause oxidative stress, to affect lipid levels, but not cellular cholesterol content, and to stimulate nSMase in vitro by oxidative stress27, 28. This may provide an alternative explanation for the direct interaction of A with SMase. ROS effects were previously observed to start occurring at A concentrations, coinciding with the highest A42 levels used here. However, at this peptide concentration, nSMase activity already declined in our system. We observed, at the comparatively low A concentrations used here, only marginal differences in ROS levels. This indicates that either low A levels produce ROS levels below our detection limit or that, in agreement with previous work28, low A concentrations do not trigger sufficient ROS production to explain the pronounced increase in SMase activity observed here. Reduced nSMase activity in the presence of additional A40 (see Supplementary Information, Fig. S4b) and in the absence of a dose-dependent response of ROS to A indicates that a ROS-independent interaction is more likely. This may represent a remarkable difference between the physiological and pathological function of A at different peptide concentrations. Properties of synthetic peptides may not reflect the in vivo situation. The cellular experiments with PS-FAD mutations were carried out on an endogenous APP background, without synthetic peptides or protein overexpression. As these experiments confirmed the conclusions drawn from the use of synthetic peptides, we have to assume that the experiments with synthetic peptides gave sufficiently valid mechanistic information. Cholesterol, gamma-secretase and A represent factors in a regulatory cycle: Cholesterol enhances gamma-secretase activity, thereby yielding more A. A40 suppresses HMGR activity, leading to decreased cholesterol levels. The absence of APP/APLP2 influences cholesterol de novo synthesis at the level of HMGR but not downstream of it, clearly identifying HMGR as the target. In principle, the structure of HMGR would allow a direct interaction with A, but it is currently unknown whether or not this actually occurs. After the discovery of the lipid-controlled RIP mechanism of the sterol regulatory element binding protein (SREBP) with site-1 protease and site-2 protease (S2P), our findings characterize a second proteolytic system in lipid homeostasis. Although there are no sequence homologies, the functional analogies to the system presented here are striking13. Both systems act via HMGR, but an important difference is that APP processing reduces, whereas SREBP processing upregulates, cholesterol de novo synthesis. In a further analogy, the example of S2P suggests that not all gamma-secretase substrates are processed lipid-dependently29. The effect of PS-FAD mutations in SM homeostasis is inversed compared with cholesterol homeostasis, validating the stronger activation of nSMase by A42 and the downregulation of de novo cholesterol synthesis by A40. Notably, this differential regulation rules out SM homeostasis as a causative mediator of PS-dependent cholesterol levels. Active involvement of APP cleavage, A, gamma-secretase activity and the effect of pathological PS-FAD mutations in lipid homeostasis provides a functional context for APP processing that has direct relevance for AD and may provide a rational basis for therapy. Methods ... Al Pater, PhD; email: old542000@... __________________________________ Start your day with - Make it your home page! http://www./r/hs Quote Link to comment Share on other sites More sharing options...
Guest guest Posted October 11, 2005 Report Share Posted October 11, 2005 --- Jeff Novick <jnovick@...> wrote: Hi All, Being in a rush, the below pdf-available excerpts are the best available version. http://my.webmd.com/content/article/113/110683?src=RSS_PUBLIC Nature Cell Biology, Oct. 9, 2005 advance online edition. Alzheimer's disease: Understanding the lipid connection Letter by Grimm et al. Varying cholesterol levels in mouse models has been shown to affect progress of Alzheimer's disease. Hartmann and colleagues now show that Ab peptide, the amyloid peptide that is produced normally but accumulates markedly during Alzheimer's, controls lipid metabolism by acting on two key enzymes in the pathway. Marcus O. W. Grimm1, Heike S. Grimm1, s J. Pätzold1, Eva G. Zinser1, Riikka Halonen1, Marco Duering1, Jakob-A. Tschäpe1, Bart De Strooper2, Ulrike Müller3, Jie Shen4 & Tobias Hartmann1 Amyloid beta peptide (Aß) has a key role in the pathological process of Alzheimer's disease (AD), but the physiological function of Aß and of the amyloid precursor protein (APP) is unknown1, 2. Recently, it was shown that APP processing is sensitive to cholesterol and other lipids3, 4, 5, 6, 7, 8, 9, 10. Hydroxymethylglutaryl-CoA reductase (HMGR) and sphingomyelinases (SMases) are the main enzymes that regulate cholesterol biosynthesis and sphingomyelin (SM) levels, respectively. We show that control of cholesterol and SM metabolism involves APP processing. A42 directly activates neutral SMase and downregulates SM levels, whereas A40 reduces cholesterol de novo synthesis by inhibition of HMGR activity. This process strictly depends on gamma-secretase activity. In line with altered A40/42 generation, pathological presenilin mutations result in increased cholesterol and decreased SM levels. Our results demonstrate a biological function for APP processing and also a functional basis for the link that has been observed between lipids and Alzheimer's disease (AD). One of the main characteristics of Alzheimer's disease (AD) is the accumulation of amyloid beta peptide (A), which is associated with neurodegeneration1, 2. A production is initiated by the gamma-secretase cleavage of amyloid precursor protein (APP), which results in production of the APP C-terminal fragment C99. This fragment is further cleaved by gamma-secretase to generate A40 and A42. The active centre of the gamma-secretase complex is formed by presenilin-1 or -2 (PS1 or PS2)11. As the gamma-secretase cleavage site is centred within the transmembrane domain, membrane composition may considerably influence Aß generation12. Such proteolytic events of regulated intra-membrane proteolysis (RIP) are involved in numerous cellular regulatory events, including lipid homeostasis13. So far, the physiological role of APP and its proteolytic fragments, including A, have mainly been conceived as being brain specific. However, the ubiquitous production of APP, Aß and PS indicates that their functions are also ubiquitous. Recently, strong evidence has accumulated that links AD and Aß generation with lipid homeostasis5, and it has been suggested that cellular lipid metabolism controls APP processing5. Studies have shown that Aß production is sensitive to cholesterol levels and lipid trafficking. In vitro enhanced cholesterol levels lead to increased Aß production14. Furthermore, cholesterol depletion reduces gamma-secretase activity and results in reduced Aß generation3, 4, 6, 7, 8, 9. Controversially, one report found a correlation between mildly decreased cholesterol levels and increased Aß production in Chinese hamster ovary cells15. In vivo, Aß production and deposition are tightly associated with changes in cholesterol levels6, 10. Lipid levels vary in response to changes in diet, and physical or neuronal activity. Similarly, Aß levels change in response to several conditions, including cholesterol and other factors16, 17. Brain cholesterol levels increase during the progression of AD18 and hypercholesterolaemia is an early risk factor in the development of AD19. Consequently, cholesterol-lowering drugs are being considered as a potential treatment for AD16, 20, 21. Nevertheless, the cellular mechanisms that link lipids, Aß generation and AD are poorly understood. To investigate the role of PS in lipid regulation, we used mouse embryonic fibroblast cells that were devoid of PS1 and PS2 (MEF PS1/2-/-)11 and expression-level-matched, A-producing PS re-transfected MEF PS1/2-/- control cells (MEF PS1r and MEF PS2r, respectively) (see Supplementary Information, Fig. S1). The absence of PS resulted in increased cellular levels of cholesterol and sphingomyelin (SM), but not phosphatidylcholine (PC) or total lipid-associated phosphate (Fig. 1a). The catabolic sphingomyelinase (SMase) activities were downregulated, anabolic SM-synthase activity was upregulated and the SM-to-ceramide ratio was increased in PS-deficient cells in accordance with the altered neutral SMase (nSMase) and SM-synthase activities (Fig. 1a, see Supplementary Information, Fig. S2a). Absence of PS1 is lethal and, as PS2 is able to replace PS1 (for example, in nSMase activity, see Fig. 1c), brains from conditional PS1 knockout mice, crossed to PS2 knockout mice, were used to evaluate PS function in lipid homeostasis in vivo. In these viable mice, PS1 remains expressed in some neurons and other cells22 and brain Aß levels in these mice are reduced to 20 & #8722;50%, depending on age and Aß species23. As before, absence of PS in neurons resulted in increased cholesterol and SM levels, decreased SMase activity, but unchanged PC levels (Fig. 1b). Dependence on gamma-secretase proteolytic activity was investigated using the gamma-secretase activity inhibitor L-685,458. This treatment reduced nSMase activity in MEF PS1r and PS2r cells to similar levels as those of PS1/2-/- cells (Fig. 1c). By contrast, this inhibitor treatment had no effect on MEF cells that were devoid of PS (Fig. 1c). Treatment of wild-type mouse neurons decreased nSMase activity and increased SM levels (Fig. 1c). Accordingly, in inhibitor-treated PS-expressing cells, SM levels were increased. Cholesterol levels increased in MEF PS1/2-/- cells (Fig. 1a), whereas no significant difference was observed between inhibitor-treated MEF PS1r and MEF PS1/2-/- cells (see Supplementary Information, Fig. S2b). It was established previously that cholesterol influences Aß production. To investigate Aß putative influence of SM on Aß production, we inhibited nSMase activity in neurons. This resulted in increased SM levels and decreased intracellular and secreted Aß (Fig. 2a). This was confirmed in COS7 cells using additional inhibitors and direct SM exposure (see Supplementary Information, Fig. S3a, 3b). Reduced gamma-secretase cleavage causes C99 accumulation, which was observed with increased SM levels (see Supplementary Information, Fig. S3c). Our results implicate the involvement of a gamma-secretase substrate and that its cleavage is essential in PS-mediated lipid regulation. MEF APP/APLP2-/- and MEF wild-type cells were analysed to evaluate whether APP or APLP2 might be involved in this regulatory cascade. SM, nSMase activity and cholesterol levels responded to the absence of APP/APLP2 in the same way as they did to the absence of PS/gamma-secretase activity (Fig. 2b). The same was observed in APP-/- mouse brain (Fig. 2b), suggesting that an in vivo function of APP exists in lipid homeostasis. Unlike in wild-type cells, inhibition of gamma-secretase activity did not alter cholesterol in MEF APP/APLP2-/- cells (Fig. 2c). This seems to rule out the existence of other gamma-secretase substrates that are able to replace the APP/APLP2 function in cholesterol regulation in these cells. The combined necessity of APP and gamma-secretase activity in this process indicates that a proteolytic APP fragment is involved in lipid homeostasis (Fig. 2b, c). The most prominent peptide that is derived from APP gamma-secretase cleavage is A. To determine a possible function of Aß in cholesterol and SM regulation, we analysed key enzymes and metabolites in the respective pathways. Analysing SM metabolism in MEF APP/APLP2-/- cells showed a decrease in nSMase activity compared with MEF wild-type cells (Fig. 2b). When exposed to A-containing conditioned media from human SH-SY5Y cells, the nSMase enzymatic activity was partially restored in MEF APP/APLP2-/- cells (Fig. 2d, left). To investigate whether this might be caused by A, cell homogenates of MEF APP/APLP2-/- cells were exposed to synthetic A1 & #8722;40/A1 & #8722;42 at a physiological ratio24, resulting in enhanced nSMase activity (Fig. 2d, right). To determine whether SMase is a molecular target for regulation by Aß and to evaluate whether this interaction might be direct, purified human placenta nSMase was exposed to synthetic Aß in a biochemical assay that was free of other cellular components. Covering the physiological concentration range of A24, the effect reached a maximum at peptide levels that are typically found in cerebrospinal fluid. The effect decreased thereafter, probably due to augmented peptide aggregation (Fig. 2e). An effect of Aß on the other assay components was excluded (see Supplementary Information, Fig. S4a); A42 & #8722;1 (inverse A) was inactive and aggregated Aß had only residual stimulatory activity, presumably due to remnant non-aggregated peptide. An excess of A40 reduced nSMase activation by A42, indicating A42 specificity (see Supplementary Information, Fig. S4b). To evaluate the involvement of oxidative stress in nSMase activation by Aß peptides, the production of reactive oxygen species (ROS) was investigated. No significant differences in ROS were observed when wild-type and rescue cells were compared with the respective knockout cells (Fig. 2f). Incubation of MEF PS1/2-/- with Aß in the concentration range used above did not result in a concentration-dependent increase of ROS (Fig. 2e). To investigate the regulation of cholesterol metabolism, MEF APP/APLP2-/- cells were treated with A, which reversed the effects that had been induced by the absence of APP/APLP2 (see Supplementary Information, Fig. S4c). To ascertain the mechanism of A in cholesterol homeostasis, MEF APP/APLP2-/- cells were treated with the two Aß species separately and de novo synthesized cholesterol was analysed. In contrast to nSMase activity with A40, which showed only a minor effect, de novo cholesterol synthesis responded already to low levels of A40 and partially restored wild-type levels (Fig. 2g, left). To identify the enzyme in the cholesterol biosynthetic pathway targeted by A, MEF APP/APLP2-/- cells were incubated with the hydroxymethylglutaryl-CoA reductase (HMGR) substrate HMG-CoA, or with mevalonate, the HMGR product. Increased de novo cholesterol synthesis was observed upon incubation with HMG-CoA, but not with mevalonate (Fig. 2g, right). Genotype-specific alterations in cellular cholesterol release could be excluded (Fig. 2h). This identifies HMGR as a target in the regulation of cholesterol de novo synthesis by APP/A. The different effects of A40 (HMGR inhibition) and A42 (SMase activation) allow for a specific prediction of their activities in a cell-biological context. A change in A40/42 ratio should lead to altered lipid levels. Familial PS mutations (PS-FAD) strongly increase A42 and mildly decrease A40 levels, without a relevant impact on the total A levels. Expression of PS-FAD mutations resulted in increased cholesterol and decreased SM levels, which was further confirmed by increased nSMase activity (Fig. 3). As predicted by the previous experiments, changes in the A40/42 ratio alter lipid homeostasis. In summary, our in vivo and in vitro data are consistent with a model (Fig. 4) in which the cascade of gamma-secretase, APP processing and A peptides downregulate cholesterol and sphingolipid levels, which, in turn, regulate A generation. The molecular targets for A in the cholesterol and SM metabolic pathways are HMGR and nSMase, respectively. The role of A in lipid homeostasis implies that its precursor APP has a function in lipid homeostasis, which is supported by altered lipid homeostasis in APP-/- mice. We assume that additional gamma-secretase substrates and cleavage products contribute to lipid homeostasis. This is supported by partial reversal of the lipid phenotype in cells following addition of A (Fig. 2g). Multiple gamma-secretase products exist. Our data identify the involvement of A, but do not rule out the involvement of other fragments. In the MEF APP/APLP2-/- cells, lipid homeostasis could not be altered by inhibition of gamma-secretase proteolytic activity. This identifies that APP/APLP2 in the MEF model are essential substrates. In addition, it rules out that, in the absence of APP/APLP2, other putative gamma-secretase substrates have a detectable effect on SM and cholesterol homeostasis. The gamma-secretase-dependent regulation of lipid homeostasis is likely to be influenced by additional factors, as indicated by the other proteases that are involved in APP RIP and gamma-secretase-independent regulatory cycles13. In agreement with the ubiquitous presence of APP, A and PS25, 26, the mechanism described here was found in cells of different origin. Stimulation of SMase by addition of synthetic A peptides in the absence of any other modulating factors in a cell-free assay indicates that A42 directly interacts with nSMase. This is consistent with the presence of an nSMase luminal/extracellular domain and the subcellular localizations of nSMases and A. Physiological A42 concentrations were not previously investigated in this context, but high concentrations of A42 or fibrillary A42 were reported to cause oxidative stress, to affect lipid levels, but not cellular cholesterol content, and to stimulate nSMase in vitro by oxidative stress27, 28. This may provide an alternative explanation for the direct interaction of A with SMase. ROS effects were previously observed to start occurring at A concentrations, coinciding with the highest A42 levels used here. However, at this peptide concentration, nSMase activity already declined in our system. We observed, at the comparatively low A concentrations used here, only marginal differences in ROS levels. This indicates that either low A levels produce ROS levels below our detection limit or that, in agreement with previous work28, low A concentrations do not trigger sufficient ROS production to explain the pronounced increase in SMase activity observed here. Reduced nSMase activity in the presence of additional A40 (see Supplementary Information, Fig. S4b) and in the absence of a dose-dependent response of ROS to A indicates that a ROS-independent interaction is more likely. This may represent a remarkable difference between the physiological and pathological function of A at different peptide concentrations. Properties of synthetic peptides may not reflect the in vivo situation. The cellular experiments with PS-FAD mutations were carried out on an endogenous APP background, without synthetic peptides or protein overexpression. As these experiments confirmed the conclusions drawn from the use of synthetic peptides, we have to assume that the experiments with synthetic peptides gave sufficiently valid mechanistic information. Cholesterol, gamma-secretase and A represent factors in a regulatory cycle: Cholesterol enhances gamma-secretase activity, thereby yielding more A. A40 suppresses HMGR activity, leading to decreased cholesterol levels. The absence of APP/APLP2 influences cholesterol de novo synthesis at the level of HMGR but not downstream of it, clearly identifying HMGR as the target. In principle, the structure of HMGR would allow a direct interaction with A, but it is currently unknown whether or not this actually occurs. After the discovery of the lipid-controlled RIP mechanism of the sterol regulatory element binding protein (SREBP) with site-1 protease and site-2 protease (S2P), our findings characterize a second proteolytic system in lipid homeostasis. Although there are no sequence homologies, the functional analogies to the system presented here are striking13. Both systems act via HMGR, but an important difference is that APP processing reduces, whereas SREBP processing upregulates, cholesterol de novo synthesis. In a further analogy, the example of S2P suggests that not all gamma-secretase substrates are processed lipid-dependently29. The effect of PS-FAD mutations in SM homeostasis is inversed compared with cholesterol homeostasis, validating the stronger activation of nSMase by A42 and the downregulation of de novo cholesterol synthesis by A40. Notably, this differential regulation rules out SM homeostasis as a causative mediator of PS-dependent cholesterol levels. Active involvement of APP cleavage, A, gamma-secretase activity and the effect of pathological PS-FAD mutations in lipid homeostasis provides a functional context for APP processing that has direct relevance for AD and may provide a rational basis for therapy. Methods ... Al Pater, PhD; email: old542000@... __________________________________ Start your day with - Make it your home page! http://www./r/hs Quote Link to comment Share on other sites More sharing options...
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