Statins Boost α-Secretase, but Not Through Cholesterol
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In efforts to interfere with Aβ peptide production, most of the attention has been lavished on the β- and γ-secretase enzymes. Could, however, their neglected cousin α-secretase prove to be the better target? In an article published January 10, 2005, in the journal Public Library of Science Medicine, Sam Gandy’s group of Thomas Jefferson University, Philadelphia, with colleagues elsewhere, report evidence suggesting that statin drugs can boost α-secretase cleavage of AβPP via the Rho/ROCK1 protein phosphorylation pathway. If confirmed, this data could offer new insight into how one might tip the scales away from β cleavage of AβPP. It could also help explain the mechanism underlying the apparent beneficial effect of statin drugs on Alzheimer disease.
Recent evidence has shown that upregulating α-secretase reduces brain levels of Aβ in APP transgenic mice (see ARF related news story). Moreover, "shedding" of the α-secretase-cleaved APP ectodomain appears to be stimulated by statin drugs (see Parvathy et al., 2004). In the current paper, first author Steve Pedrini and colleagues take aim at one of the candidate pathways for mediating this effect (for review of pathways to boosting α-secretase cleavage, see Allinson et al., 2003).
Statins reduce cholesterol levels, at least in part, by inhibiting the ability of the enzyme hydroxymethylglutaryl-coenzyme A (HMGCoA) reductase to promote the conversion of HMGCoA into the cholesterol-synthesis intermediate mevalonic acid. But in doing this, statins also reduce levels of other intermediates on the way to cholesterol, including isoprenoids such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate. Pedrini and colleagues point out that these lipids play other regulatory roles. For example, they are critical in activating the Rho family of GTPases, which in turn activate Rho-associated coiled-coil containing kinases (ROCKs). By inhibiting HMGCoA, then, statins might ultimately reduce ROCK-mediated protein phosphorylation. One member of this family, ROCK1, has been implicated in the regulation of γ-secretase cleavage (Zhou et al., 2003; but see also γ-secretase news story from the 2004 Society for Neuroscience meeting). Pedrini and colleagues suggest that the isoprenoid-Rho-ROCK1 pathway might also be involved in the statin-induced shedding of soluble α-secretase-cleaved AβPP (sAβPPα).
Working in mouse neuroblastoma cells transfected with the gene for APP carrying the Swedish AD-causing mutation, the researchers find evidence consistent with this hypothesis. A dominant-negative form of ROCK1 increased sAβPPα, as did an inhibitor of the enzyme required for isoprenylation of Rho. Conversely, a constitutively active ROCK1 was able to abolish statin-induced sAβPPα shedding. Further implicating the isoprenoid portion of this pathway in statins' actions on α cleavage, the researchers abolished statin-induced shedding of sAβPPα by adding mevalonic acid, effectively bypassing the statin inhibition of HMGCoA reductase. On the other hand, inhibiting the isoprenylation of Rho mimicked the effects of statins on sAβPPα.
"Taken together, these results suggest the existence of a reciprocal relationship between isoprenoid-mediated Rho/ROCK signaling and sAPPa shedding, i.e., activation of ROCK1 blocks basal and stimulated shedding while ROCK1 inhibition apparently relieves a tonic negative influence exerted on shedding by ROCK1 activity," write the authors. Whether statins will therefore block ROCK1 activity in neurons is something the authors are now investigating.—Hakon Heimer.
See also:
Synopsis: How Statins May Protect Against Alzheimer Disease PLoS Med 2(1):e22.
Q&A with author Sam Gandy
Q: There has been mention of the Rho-ROCK pathway possibly boosting Aβ42 production by effects on γ-secretase. How does this relate to your results?
A:We haven't yet addressed it directly.
Many papers (e.g., Zhou et al., 2003) used Y27632 (nominally a ROCK inhibitor) to imply ROCK actions. Surprisingly, we found opposing results on statin-activated α-secretion when we used dominant-negative ROCK vs. Y27632. The predicted result was that they would show identical effects. Since our conditionally active ROCK had effects that were the opposite of those of dominant-negative ROCK, we chose to pursue the results from those molecules that were in "agreement" and, for now, set aside the results with the drug (which we discuss in the paper).
The Rho/ROCK pathway is also very state-dependent. In fact, there are examples of ROCK activators and ROCK inhibitors doing the exact same thing even in the same system, rather than having opposing effects. Presumably there is some moment-to-moment balance of which pathways prevail. Obviously, there is a lot we don't know about ROCK regulation.
There may be real conflicts in predicted results because different cells or different cellular states were not controlled for. There may also be conflicting data depending on whether ROCK's role is implicated by drug or by molecular biology.
So, the short answer is that we must look directly at Aβ, and we must look at neurons. We are doing this now. If it seems like a mess, it is. The results are too unpredictable to guess. We just have to do the experiments.
Q: What about the effects of isoprenoid pathway (pathways?) on γ- vs. α-secretase? Could it be affecting both? Different isoprenoids? Is all that still to be investigated?
A:Again, the short answer is yes, multiple pathways could be differentially regulated, and we just have to do the experiments.
α- vs. β-secretase "competition" controls levels of Aβ only, while γ-secretase can control either levels or 40/42 ratio. It's really probably just the absolute level of Aβ42 that is the most important. Standardizing to Aβ40 was devised in the early 1990s by Steve Younkin and Dennis Selkoe as a convenient way of comparing one dish to another. Aβ42 is so vanishingly low in amount, especially before highly sensitive ELISAs were developed, that the standardization was crucial for getting interpretable data. The Aβ42 signal and variations could easily get lost in the background noise were it not for this innovative standardization technique. Even so, Aβ42 is the real culprit (this is also the title of a highly cited review by Steve Younkin in Annals of Neurology).
Theoretically, if you hyperactivate α-secretase enough, you might get so little Aβ made that the ratio wouldn't matter. Hyperactivating α-secretase would be tantamount to BACE inhibition (which, using the small-molecule BACE1 approach, has been very difficult so far due to the large catalytic pocket in BACE). The trouble in developing BACE inhibitors has provided some impetus for revisiting the strategy of indirect BACE inhibition by hyperactivating α-secretase.
There is some disagreement over what ROCK does to γ-secretase: Some say it inhibits generation of 42 specifically (Zhou et al., 2003). But at least one group at the Alzheimer Congress in Philadelphia and/or at the SFN Meeting in San Diego reported that ROCK controlled both Aβ40 and Aβ42. I haven't heard an update on how that has sorted out.
Among known isoprenylated substrates, farnesylation is most common, but it may be that we just don't know all the geranylgeranyl substrates yet. Certainly different Rhos can differentially regulate the same reaction: I can't immediately think of an example where farnesylation causes "reaction A" to proceed in one direction and geranylgeranylation causes the reverse. These pathways are receiving a lot of attention in oncology research, where a farnesyl transferase inhibitor is in human clinical trials.
Q: What are the principle dietary sources of isoprenoids?
A: Squalene is the most readily identifiable dietary isoprenoid (on MedLine), and olive oil is a rich source. Phytosteroids and oxidized sterols seem to be included in this class, as well. Dietary isoprenoids exert complex actions on cholesterol metabolism at the level of both HMGCoA reductase and LDL, and have been proposed as adjuncts to statins in ASCVD and as anti-neoplastics, but their anti-neoplastic properties still fall in the realm of alternative medicine.
Given the vicissitudes of this pathway, I would not hazard a guess as to which way they would modulate α- and γ-secretases. If I really wanted to know, I would do the experiment that Steve Paul suggests: feed some olive oil to a plaque-forming mouse, or a triple transgenic with both plaque and tangle etiology, and see what happens to pathology and behavior. Maybe the olive oil industry would support such a study? (Only half kidding!)
References
News Citations
Paper Citations
- Parvathy S, Ehrlich M, Pedrini S, Diaz N, Refolo L, Buxbaum JD, Bogush A, Petanceska S, Gandy S. Atorvastatin-induced activation of Alzheimer's alpha secretase is resistant to standard inhibitors of protein phosphorylation-regulated ectodomain shedding. J Neurochem. 2004 Aug;90(4):1005-10. PubMed.
- Allinson TM, Parkin ET, Turner AJ, Hooper NM. ADAMs family members as amyloid precursor protein alpha-secretases. J Neurosci Res. 2003 Nov 1;74(3):342-52. PubMed.
- Zhou Y, Su Y, Li B, Liu F, Ryder JW, Wu X, Gonzalez-DeWhitt PA, Gelfanova V, Hale JE, May PC, Paul SM, Ni B. Nonsteroidal anti-inflammatory drugs can lower amyloidogenic Abeta42 by inhibiting Rho. Science. 2003 Nov 14;302(5648):1215-7. PubMed.
External Citations
Further Reading
No Available Further Reading
Primary Papers
- Pedrini S, Carter TL, Prendergast G, Petanceska S, Ehrlich ME, Gandy S. Modulation of statin-activated shedding of Alzheimer APP ectodomain by ROCK. PLoS Med. 2005 Jan;2(1):e18. PubMed.
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Comments
Boston University School of Medicine
Statins are known to increase secretion of APP, but the mechanism by which this occurs is poorly understood [1]. The current manuscript by Pedrini et al. focuses on the effect of statins on Rho and Rho-associated coiled-coil containing kinase 1 (ROCK). The group observes that a constitutively active ROCK prevented the actions of statins on APPsα. This suggests that inhibition of ROCK plays an important role in the mechanism of action of statins. They also performed the converse experiment, and examined how dominant-negative ROCK affects secretion of APPaα. Unfortunately, this is a point where the group's story strays. The dominant-negative ROCK increases APPsα secretion on cells not exposed to statins, but does not increase the actions of statins; thus, the effects of dominant-negative ROCK are not strictly opposite to those of the constitutively active ROCK. These data suggest that ROCK can modulate the effects of statins, but do not explicitly prove that statins act on APPsα through ROCK. Nonetheless, this is a very interesting story which nicely integrates Rho signaling into the mechanism of action of statins.
References:
Kojro E, Gimpl G, Lammich S, Marz W, Fahrenholz F. Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the alpha -secretase ADAM 10. Proc Natl Acad Sci U S A. 2001 May 8;98(10):5815-20. PubMed.
University of Wisconsin-Madison
Since the appearance of the first epidemiological and animal studies claiming a connection between cholesterol and Alzheimer disease, at least four different aspects of cholesterol metabolism have been directly linked to AD neuropathology:
(i) clustering of APP and BACE1 into lipid rafts, which facilitates β cleavage of APP (1);
(ii) intracellular cholesterol distribution, which is able to activate the amyloidogenic processing of APP (2);
(iii) ozonolysis of cholesterol, which generates peroxi-derivatives of cholesterol that accelerate the aggregation of Aβ monomers (3), and
(iv) Aβ-mediated oxidation of membrane cholesterol, which liberates H2O2 and aggravates oxidative stress (4).
Therefore, strategies aimed at the modulation of cholesterol metabolism/distribution in the brain have received wide attention for the prevention of AD. Among those, statins seem to be especially welcome, mostly because they are already available, have been widely studied for their role in the prevention of atherosclerosis, and are overall very safe. Statins were introduced as pharmacological inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase, the rate-limiting enzyme in the biosynthesis of cholesterol, but they were soon shown to do more than just that, including stimulation of bone osteogenesis and inhibition of growth/invasion of certain types of cancers. Some of these effects could be related to their cholesterol-lowering activity, some cannot (at least for now).
This story seems to hold even when we switch to the “molecular” effects of statins: They do more than just inhibit HMGCoA reductase. In this new study by Pedrini et al., a post-translational modification involving isoprenoids—and not cholesterol itself—is shown to affect α cleavage of APP. As the paper points out, isoprenoid (farnesyl and geranylgeranyl) moieties, too, originate from the cholesterol biosynthetic pathway, just a few steps downstream from HMGCoA reductase and a few steps upstream of cholesterol (for review, see 5). This paper is a continuation of previous work from the senior author, Sam Gandy, who has been investigating the mechanisms that regulate α cleavage of APP for a long time. Here, the authors show that inhibition of cholesterol biosynthesis increases α cleavage of APP through a mechanism that is in part independent of cholesterol itself. They used elegant biochemical approaches, including HMGCoA reductase and farnesyl-transferase inhibition in the presence or absence of mevalonate. Since mevalonate is able to bypass HMG-CoA reductase but not farnesyl-transferase inhibition, they managed to identify a novel form of regulation of APP processing that requires isoprenoids. The authors went on to show that such an event seems to involve post-translational modulation of the Rho family of GTPases and Rho-associated coiled-coil containing kinases (ROCKs).
The effect produced by ROCK is completely abolished after deletion of both the pleckstrin homology and the Rho-binding domains, and after inhibition of the kinase activity. The specific roles of the different domains of ROCK or the possible interaction between Rho GTPases and ROCK itself are not explored in detail. However, since a conformational change of ROCK is required for the functional activation of the kinase activity of the protein, it is likely that the Rho-binding domain is necessary for the Rho-mediated activation of ROCK. Therefore, statin-mediated inhibition of the cholesterol biosynthetic pathway may also lead to decreased transfer of isoprenoid moieties to Rho proteins, thereby decreasing their functional activity.
Unfortunately, the paper did not describe what happens to β cleavage of APP or to the production of Aβ peptides. It would be interesting to see whether or not Rho/ROCK proteins can also influence β cleavage, either by diverting APP from the β to the α pathway, or by directly affecting β cleavage of APP. In this regard, it is very tempting to try to find a possible connection with the loe phenotype observed in D. melanogaster (6). Flies are not able to generate cholesterol; the HMG-CoA reductase-dependent pathway stops immediately after the generation of isoprenoids. This pathway is under the inhibitory control of AMP-activated kinase (AMPK), which blocks the biosynthesis of both fatty acids and isoprenoids, and the hydrolysis of diet-derived cholesterol esters. Disruption of AMPK (loe phenotype) in D. melanogaster leads to a marked decrease in the shedding of APPL, the fly homolog of human APP. This event is in part due to the increased levels of isoprenoids, because statin-mediated inhibition of isoprenoid biosynthesis was able to partially recover APPL processing. It seems that both Pedrini et al. and Tschape et al. have found a connection between isoprenoids and APP processing, a connection that has been conserved throughout evolution but that can differ in some aspects, probably because of different molecules situated between isoprenoids and APP. The identification of those molecules will be the next stop…and Sam Gandy and Suzana Petanceska will certainly satisfy our curiosity.
See also:
Jackson, S. M. et al. (1997). Signaling molecules derived from the cholesterol biosynthetic pathway. In: Subcellular Biochemistry. Cholesterol: its functions and metabolism in biology and medicine. Bittman, R, ed. (Plenum Press New York, NY), pp. 1-21.
References:
Ehehalt R, Keller P, Haass C, Thiele C, Simons K. Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts. J Cell Biol. 2003 Jan 6;160(1):113-23. PubMed.
Puglielli L, Konopka G, Pack-Chung E, Ingano LA, Berezovska O, Hyman BT, Chang TY, Tanzi RE, Kovacs DM. Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid beta-peptide. Nat Cell Biol. 2001 Oct;3(10):905-12. PubMed.
Zhang Q, Powers ET, Nieva J, Huff ME, Dendle MA, Bieschke J, Glabe CG, Eschenmoser A, Wentworth P, Lerner RA, Kelly JW. Metabolite-initiated protein misfolding may trigger Alzheimer's disease. Proc Natl Acad Sci U S A. 2004 Apr 6;101(14):4752-7. PubMed.
Opazo C, Huang X, Cherny RA, Moir RD, Roher AE, White AR, Cappai R, Masters CL, Tanzi RE, Inestrosa NC, Bush AI. Metalloenzyme-like activity of Alzheimer's disease beta-amyloid. Cu-dependent catalytic conversion of dopamine, cholesterol, and biological reducing agents to neurotoxic H(2)O(2). J Biol Chem. 2002 Oct 25;277(43):40302-8. PubMed.
Tschäpe JA, Hammerschmied C, Mühlig-Versen M, Athenstaedt K, Daum G, Kretzschmar D. The neurodegeneration mutant löchrig interferes with cholesterol homeostasis and Appl processing. EMBO J. 2002 Dec 2;21(23):6367-76. PubMed.
National Institute on Aging
Gary Landreth's paper in the current issue of The Journal of Neuroscience on statins reducing Aβ-induced microglial inflammatory responses is very elegant work (Cordle and Landreth, 2005). This study shows that statin treatment of microglia and monocytes leads to robust reduction of Aβ-induced Il1β and inducible nitric oxide synthase expression, as well as reduction of nitric oxide production. As isoprenoids and the Rac and Rho-GTPases are implicated as mediators of these effects, this study complements the findings by Pedrini et al.
Furthermore, in 2002, Barbara Cordell's group provided evidence that ApoE secretion from glia requires a prenylated protein entity, and that the reduction of ApoE secretion by statins is due to inhibition of the synthesis of isoprenoids (Naidu et al., 2002).
In 2003, we discussed possible mechanisms by which statins can reduce brain amyloidosis (Petanceska et al., 2003). We hypothesized that the pleiotropic, lipid-independent effects of statins (specifically their antiinflammatory, antioxidant, and vascular effects), which are a result of inhibition of isoprenoid synthesis, can contribute to their in-vivo ability to attenuate brain Aβ deposition.
Together with the findings of the Cordell group, the new data provided by Pedrini et al. suggest that even the effects of statins on ApoE secretion and APP processing, which were believed to be solely mediated by the lipid-lowering activity of statins, are at least in part lipid-independent and a result of inhibition of isoprenoid synthesis.
References:
Cordle A, Landreth G. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors attenuate beta-amyloid-induced microglial inflammatory responses. J Neurosci. 2005 Jan 12;25(2):299-307. PubMed.
Naidu A, Xu Q, Catalano R, Cordell B. Secretion of apolipoprotein E by brain glia requires protein prenylation and is suppressed by statins. Brain Res. 2002 Dec 20;958(1):100-11. PubMed.
Petanceska S, Pappolla M, Refolo LM. Modulation of Alzheimer's amyloidosis by statins: Mechanisms of action. Curr Med Chem Immun Endoc & Metab Agents. 2003 Aug;3(1):1-7.
Solo practitioner and independent researcher; Founder, National Institute of Good Health
As Sam Gandy says regarding his research on statin effects in Alzheimer disease: "If it seems like a mess, it is." Hippocrates said, "Every disease has a nature of its own, and each arises from its own natural cause." Why, 2,000 years later, is modern science unable to find a simple "natural cause" for AD?
Are we asking the right questions? Is this a modern disease, with a modern cause? How common are AD lesions in preserved brains from the 19th century? Should we examine the Yerkes and Corsellis collections?
The cholesterol-AD story has confused beginnings, and a messy ending. What government would consider mass-medicating its ageing population with statins to prevent AD, knowing that its best and most dedicated scientists had failed to find a preventable cause of the disease?
Those who prefer intervention over prevention will protest that the environmental origins are so murky and multifactorial that treatment and prevention must perforce be piecemeal. It would come as a great shock to such thinking if a simple, preventable cause of the disease were found, which at a stroke would wipe out drug development programs and all further research on the disease.
By piecing together the available facts on this disease, it is possible to reach an inductive conclusion, that the simple common cause is Wesson steam-deodorization of polyunsaturated vegetable oils, an industrial process that, since 1900, has been removing some 30 percent of the neuroprotective vitamin E from common frying and salad oils. Reduced antioxidant protection of dietary omega-6 essential fatty acids (linoleic acid in oils) exposes the long-chain EFA of the brain and retina to lipid peroxidation. A major product of arachidonic acid breakdown in neuronal synapses is 4-hydroxynonenal (4-HNE), which is known to inactivate ion-motive ATPases, and glucose and glutamate transporters.
In addition, there is an intriguing possibility that 4-HNE may inactivate α-secretase, by forming adducts with vulnerable amino acids at the catalytic site. Such inactivation would be a key mechanism in a refined oil hypothesis, since it would account for slow β amyloid accumulation.
I propose that my suggested mechanism of HNE-induced inactivation of α-secretase be tested in some laboratory somewhere, by some scientist who retains a native sense of curiosity about causes of disease, unspoilt by commercial temptations.
If the prediction is proved correct, government would welcome the breakthrough, which would finally pin down the most critical mechanism in the refined oil hypothesis, paving the way for legislation requiring food oil processors to increase the vitamin E content of refined oils to natural levels (at least 0.6 mg per gm of EFA).
Mayo Clinic Jacksonville
This manuscript confirms and extends a previous study showing that statin treatment can increase the release of sAPPα [1]. The biochemical mechanism by which HMG-CoA reductase inhibition leads to this increase isn’t fully understood. The authors present intriguing data that suggests the small GTPase pathway may be involved. First, a farnesyltransferase inhibitor was shown to increase statin-induced sAPP shedding, implying a farnesylated GTPase may be involved. They then looked at dominant-negative (DN) and constitutively active (CA) forms of ROCK, which is an effector protein kinase of the small GTPase Rho. CA ROCK decreases sAPP release while the DN form increases sAPP release. These results suggest that statin-mediated sAPP shedding could be mediated by isoprenoids, which can regulate the amount of membrane-associated Rho and thus the extent of ROCK activation.
As the authors acknowledge in the discussion, there are a couple of inconsistencies in the data that are confusing. Their data suggests that the effects of statins are mediated at the plasma membrane. They also conclude that this effect may be mediated through a farnesylated form of Rho via ROCK. There are three isoforms of Rho (A,B,C) [2]. RhoA and C are only geranylgeranylated and located mainly at the plasma membrane. RhoB can be farnesylated or geranylgeranylated and is found primarily in the endosomes, suggesting a spatial disconnect. One critical issue is the specificity of the farnesyl transferase inhibitor that was used. If this effect is specific, treatment with FPP and not GGPP should block the increase in sAPP.
Since the small GTPase pathway is so complex, DN and CA forms of these proteins can often have unexpected effects. It would be informative to look directly at the isoforms of Rho, as well as other GTPases that are theoretically not involved in sAPP processing.
Finally, the ROCK inhibitor Y-27632 had no effect on sAPP release. This unexpected result could be a result of multiple activities since it is known that this compound can affect multiple kinases [3]. A variety of more specific and potent ROCK inhibitors have now been developed that can be screened to more thoroughly probe this effect [4].
Despite these issues, this manuscript provides an intriguing association between the alpha secretase processing of APP and the isoprenoid pathway, which has also been recently implicated in γ-secretase processing. A paper by Zhou et al. suggests that NSAIDs mediate their Aβ42 lowering effect through inhibition Rho [5]. We presented data at the 2004 Society for Neuroscience meeting suggesting that NSAIDs do not act through Rho. Instead, our data suggests that NSAIDs, as well as isoprenoids, directly target the γ-secretase complex to modulate Aβ production.
How these effects and the isoprenoid pathway interact with all APP processing pathways remains to be determined. Clearly, the isoprenoid pathway and the numerous GTPases that are influenced by these metabolites are complex and incompletely understood. Moreover, almost nothing is known about isoprenoid metabolism in the brain (besides the fact that the enzymes that regulate it are abundant). Further research into the role of isoprenoids and small GTPase in APP metabolism and Alzheimer’s disease is required and may provide important insight into disease mechanism and novel therapeutic strategies.
References:
Kojro E, Gimpl G, Lammich S, Marz W, Fahrenholz F. Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the alpha -secretase ADAM 10. Proc Natl Acad Sci U S A. 2001 May 8;98(10):5815-20. PubMed.
Wheeler AP, Ridley AJ. Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility. Exp Cell Res. 2004 Nov 15;301(1):43-9. PubMed.
Breitenlechner C, Gassel M, Hidaka H, Kinzel V, Huber R, Engh RA, Bossemeyer D. Protein kinase A in complex with Rho-kinase inhibitors Y-27632, Fasudil, and H-1152P: structural basis of selectivity. Structure. 2003 Dec;11(12):1595-607. PubMed.
Sasaki Y, Suzuki M, Hidaka H. The novel and specific Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probing molecule for Rho-kinase-involved pathway. Pharmacol Ther. 2002 Feb-Mar;93(2-3):225-32. PubMed.
Zhou Y, Su Y, Li B, Liu F, Ryder JW, Wu X, Gonzalez-DeWhitt PA, Gelfanova V, Hale JE, May PC, Paul SM, Ni B. Nonsteroidal anti-inflammatory drugs can lower amyloidogenic Abeta42 by inhibiting Rho. Science. 2003 Nov 14;302(5648):1215-7. PubMed.
Massachusetts General Hospital / Harvard Medical School
Pedrini et al. identified two connected pathways with ROCK1 as the central player. Their findings indicate that ROCK1 inhibits α-secretase activity; two different statins inhibit ROCK1 via reducing isoprenylation of the Rho GTPases. Thus, statins could activate α-secretase, at least in part, via inhibition of ROCK1.
Regulation of α-secretase and γ-secretase (Zhou et al. 2003) activities by the Rho/ROCK1 phosphorylation pathway may provide interesting clues to the neuronal function of the secretases. The role of the Rho GTPases in cell motility and axon guidance is well established. In neuronal cell lines, RhoA/ROCK are activated in response to repulsive cues and lead to growth cone collapse. In contrast, attractive cues activate Cdc42 and Rac GTPases, which, in turn, promote extension of axons to appropriate targets. The growth cone integrates multiple signals to produce coordinated changes in cytoskeletal dynamics. These changes are mediated by signaling via the C-terminal tails of axon guidance molecules, such as DCC, N-cadherin, NCAM, LAR, ephrinA/B, by activating either the Rho/ROCK (repulsion) or the Cdc42 and Rac (attraction) pathways. Interestingly, many of the signaling proteins are substrates for α-secretase-like and γ-secretase cleavages. The studies by Pedrini and Zhou suggest that the RhoA/ROCK pathway may regulate α- and γ-secretase activities to produce specific coordinated changes in growth cone collapse.
The work of Pedrini et al. adds to our understanding of the mechanisms by which intracellular lipid metabolism regulates secretase activities. Isoprenoids line up with membrane cholesterol, cholesteryl-esters, phospholipids, and ceramide in regulating APP processing. The identification of the downstream effector, ROCK1, for isoprenoid-mediated regulation of α-secretase sets this pathway apart from the others. This pathway is likely to account, at least in part, for the Aβ-lowering effects of statins by activating α-secretase. Cholesterol-lowering effects of statins have recently come under scrutiny by Abad-Rodriguez et al., (J. Cell Biol, 2004). This paper shows that slightly reduced membrane cholesterol leads to elevated Aβ production, instead of a decrease. More than a 35 percent reduction in membrane cholesterol is required to achieve inhibition of Aβ generation. These findings already suggest the existence of at least two different pathways by which statins may regulate APP processing. Meanwhile, reduction of cholesteryl-esters is accompanied by an increase in membrane cholesterol, and yet Aβ generation is decreased (Puglielli et al, 2001; Hutter-Paier et al., 2004). Clearly, APP processing is not simply modulated by levels of membrane cholesterol, but is influenced by the complex interplay of a number of lipid and protein components of the cell. How exactly isoprenoids fit into this interplay will likely be the subject of further studies from the laboratory of Sam Gandy and of others investigating the role of lipids in regulating Aβ production.
Indiana University School of Medicine
Clincial evidence suggests that long- term use of statins is associated with a decreased risk of Alzheimer disease (AD). As these drugs block the synthesis of cholesterol, much research has been focused on the importance of cholesterol metabolism in the pathogenesis of AD. Recently, it has been appreciated that statins can also exert biological effects independently of cholesterol. HMGCoA inhibition also blocks the production of isoprenyl precursors, and these isoprenyl groups are required for the proper function of Rho family GTPases. For example, it has been shown that inhibition of Rho contributes to the in vitro antiinflammatory effects of statins (Cordle et al., 2005).
In their recent paper, Pedrini et al. address an important issue by looking at cholesterol-independent effects of statins on APP metabolism. This group has previously shown that, in vitro, treatment of neuroblastoma cells with statins leads to an increase in shedding of sAPPα (Parvathy et al., 2004). In the present work, they expand on this theme by showing that the effects of statins on APP metabolism are independent of cholesterol, and by identifying Rho-associated coiled-coil containing kinase (ROCK) as a possible downstream signaling target that may be disrupted by statin treatment.
The authors show that statins increase levels of holo-APP about twofold, yet increase sAPPα shedding three- to fourfold. These data suggest that inhibition of Rho family proteins preferentially drives the α-secretase pathway, though the mechanism remains undetermined. The most interesting data in the paper suggest that ROCK could be the key regulator of APP metabolism in this paradigm. ROCK is a kinase that is activated upon Rho activation. Thus, inhibition of Rho by statins could block ROCK activation and thus relieve a constitutive inhibitory influence exerted by this pathway. By using dominant-negative (DN) and dominant-active (DA) ROCK constructs, Pedrini et al. show that a DN ROCK increases shedding of sAPPα and that DA ROCK decreases sAPPα shedding. While not conclusive, these data suggest that ROCK regulates APP metabolism, and that statins may increase sAPPα shedding via inhibition of ROCK activity. These findings are consistent with our finding that broadly acting inhibitors of Rho proteins, such as Toxin A of C. difficile and isoprenyltransferase inhibitors (unpublished data), elevate sAPPα levels. Paradoxically, Pedrini et al. found that the well-documented ROCK inhibitor Y27632 blocked statin-induced sAPP generation, a finding which remains unexplained.
At face value, it seems as though the increased shedding of sAPPα upon statin treatment would ameliorate the disease process, as an increase in non-amyloidogenic APP processing is usually associated with a decrease in amyloidogenic processing. However, Pedrini et al. demonstrated that treatment with statins results in a twofold increase in holo-APP. We and Bob Vassar’s lab have shown that this results in a corresponding increase in Aβ peptide levels. Pedrini et al. do not show the effect of statins on Aβ levels. Thus, the statin-mediated elevation of cellular APP levels results in an increase in steady-state holo-APP levels, with a commensurate increase in both sAPPα and Aβ production. The data by Pedrini et al. suggest that sAPPα may be preferentially increased, but it is unclear if this phenomenon is separable from increased Aβ production. Thus, it is unclear whether the Rho-ROCK pathway will become an appropriate therapeutic target.
References:
Cordle A, Landreth G. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors attenuate beta-amyloid-induced microglial inflammatory responses. J Neurosci. 2005 Jan 12;25(2):299-307. PubMed.
Parvathy S, Ehrlich M, Pedrini S, Diaz N, Refolo L, Buxbaum JD, Bogush A, Petanceska S, Gandy S. Atorvastatin-induced activation of Alzheimer's alpha secretase is resistant to standard inhibitors of protein phosphorylation-regulated ectodomain shedding. J Neurochem. 2004 Aug;90(4):1005-10. PubMed.
Sam Gandy’s group’s study underscores an emerging role for isoprenoid-mediated regulation of APP processing and its possible relationship to Alzheimer disease pathogenesis. Over a year ago, we reported that GGPP, one of the isoprenoids synthesized in the mevalonate biosynthetic pathway, preferentially increases the generation of the more amyloidogenic Aβ species, Aβ42 (Zhou et al., 2003). Based on our experiments using dominant-negative and constitutively active Rho, as well as the ROCK inhibitor Y27632, we concluded that GGPP mediates an increase of Aβ42 through activation of the Rho/ROCK pathway, possibly by modulating γ-secretase.
In our opinion, the most important finding reported in our paper is the one showing that physiological lipids, such as GGPP, can regulate the generation of the amyloidogenic species Aβ42. Interestingly, isoprenoids are generated not only endogenously but also can be taken up through the diet. Thus, dietary isoprenoids could also regulate APP processing and Aβ synthesis and contribute to AD pathogenesis.
At last year’s International Conference on Alzheimer’s and Related Diseases in Philadelphia, Todd Golde’s group reported that they had confirmed the effect of GGPP on Aβ generation. However, based on their finding that the generation of Aβ can also be increased by GGPP in the isolated lipid rafts, they suggested that isoprenoids may act directly on the γ-secretase complex instead of through a Rho/ROCK signaling pathway.
In the present paper, Steve Pedrini and colleagues performed a series of elegant experiments demonstrating that isoprenoids regulate APPα shedding through modulating ROCK activity. However, the consequence of modulating APPα shedding by ROCK on Aβ generation is still under investigation by this group. As Dr. Gandy said in the Q&A, the effect of small G-proteins and their effectors on certain cellular functions, such as APP processing, is complicated because of “some moment-to-moment balance of which pathways prevail.” Add to that interwoven and feedback signal transduction pathways controlled by these small G-proteins, and the studies are truly complex to both perform and interpret.
Regardless of the exact mechanism, the fundamental question of whether long-term, high-dose consumption of dietary isoprenoids could impact central APP processing, Aβ synthesis, and AD neuropathology should be addressed. Experiments designed to feed APP transgenic or wild-type mice isoprenoid-supplemented food daily for many months, and then looking for effects on APP processing/brain neuropathology should prove informative. If dietary isoprenoids indeed aggravate the progress of brain amyloid deposition in APP transgenic mice, one might reasonably speculate on their possible role in contributing to the pathogenesis of AD.
References:
Zhou Y, Su Y, Li B, Liu F, Ryder JW, Wu X, Gonzalez-DeWhitt PA, Gelfanova V, Hale JE, May PC, Paul SM, Ni B. Nonsteroidal anti-inflammatory drugs can lower amyloidogenic Abeta42 by inhibiting Rho. Science. 2003 Nov 14;302(5648):1215-7. PubMed.
Solo practitioner and independent researcher; Founder, National Institute of Good Health
I sincerely thank Alzforum for publishing my provocative comment on AD and cholesterol, albeit somewhat sanitized of its original pungency! If my theory about refined oils causing sporadic AD is correct, then "stripped" oil (containing little or no vitamin E, after prolonged heating) would be a good means of inducing neuronal lipid peroxidation in culture, which should generate both measurable 4-hydroxynonenal and reduced formation of secreted APP (sAPP), along with a mysterious rise in Aβ. My best wishes go to anybody who may care to do this experiment! Let us fortify ourselves with three observations that should encourage us:
1. Safflower oil, given as 20 percent of the diet, caused learning impairment in weaned rat pups (Harman et al., 1976). When the experiment was repeated with vitamin E supplementation, no harmful effects were seen on learning. Harman's safflower oil may have been typical steam-refined oil, which has about 0.45 mg of vitamin E per gm of essential fatty acids, compared with 0.65 mg in cottonseed oil, 0.36 mg in corn oil, and a miserly 0.28 mg in soya oil (Herting and Drury, 1963). Lipid peroxidation is seen in animal experiments when the level drops below 0.6 mg. Even if Harman had used cold pressed safflower oil, an initially adequate vitamin E level would have been reduced by the deep-freeze cold-storage he mentions in his paper. More recently, Greg Cole at UCLA has found that safflower oil (source unstated) aggravates transgenically induced AD pathology in mice.
2. M K Horwitt, in the only human vitamin E deprivation trial ever done (at the Elgin mental hospital in Illinois, during the 1960s) observed increased H2O2-induced red blood cell haemolysis after giving stripped corn oil, in one phase of the trial. Such haemolysis is considered to reflect a membrane weakened by lipid peroxidation, so this test might be a good clinical test for current brain peroxidation, due to early Alzheimer's, or to current refined oil consumption at any age. Other markers of brain peroxidation include F2 isoprostanes in blood and urine, and expired air pentane or ethane, as seen in children with attention deficit hyperactivity disorder (Nutritional Neuroscience, Sept 2003)—another refined oil syndrome, arising in pregnancy and aggravated postnatally by refined oils in the child's diet.
3. In hundreds of my patients exposed to refined frying and salad oils, or oily cakes and dips, I have observed and described a typical "refined oil syndrome," consisting of short-term memory impairment, night blindness, and characteristic glare sensitivity (easily provoked with a clinical pen-torch). Vitamin E rapidly corrects the memory deficit, but fish oil is required to improve the visual symptoms.
I did a small pilot study in 1993, finding that 12 patients diagnosed with AD had all used refined oils for decades, compared with 20 controls with excellent memories, none of whom had any regular exposure to refined oils (Peers, 1993). It is time we found out what these oils can do in the laboratory!
References:
HARMAN D, Hendricks S, Eddy DE, Seibold J. Free radical theory of aging: effect of dietary fat on central nervous system function. J Am Geriatr Soc. 1976 Jul;24(7):301-7. PubMed.
HERTING DC, DRURY EJ. VITAMIN E CONTENT OF VEGETABLE OILS AND FATS. J Nutr. 1963 Dec;81:335-42. PubMed.
Peers R. Alzheimer's disease and vegetable oils. N Z Med J. 1993 Nov 10;106(967):481. PubMed.
Please see our commentary on this important study at PLoS Medicine eLetters page
BRG1 and BRM are subunits of the SWI/SNF chromatin remodeling complex which have been implicated in the regulation of gene expression, cell cycle control, and oncogenesis.
The Liu group [1] reports that the BAF (BRG1 associated factor) complex results in promoter activation of CSF-1 and promotes Z-DNA formation. A conformational change from B-DNA to Z-DNA in the hippocampus in AD is reported by Suram et al. [2], as is increased serum CSF-1 [3]. This might lead us to expect increased BRG1 in AD, and consequently increased ROCK1.
The Emerson group [4] reports that BRG1 binds to zinc finger proteins through a unique N-terminal domain that is not present in BRM. BRM interacts with two ankyrin repeat proteins that are critical components of Notch signal transduction. SWI/SNF BRG1 complexes, but not BRM, bind to the CREB transcription factor only when CREB is phosphorylated. DYRK1A, a gene in the Down syndrome critical region, has been found to phosphorylate CREB.
The findings by the Emerson lab would seem to provide a targeted therapy in AD as well as DS. They state that they can screen for molecules that block the association between chromatin remodeling complexes and the specific transcription factors with which they interact. Might this be more beneficial than statins alone, which have inhibited ROCK1?
It's interesting that amyloid-β precursor protein forms a transcriptionally active complex with the chromatin remodeling enzyme,Tip60.
References:
Liu R, Liu H, Chen X, Kirby M, Brown PO, Zhao K. Regulation of CSF1 promoter by the SWI/SNF-like BAF complex. Cell. 2001 Aug 10;106(3):309-18. PubMed.
Suram A, Rao KS, Latha KS, Viswamitra MA. First evidence to show the topological change of DNA from B-dNA to Z-DNA conformation in the hippocampus of Alzheimer's brain. Neuromolecular Med. 2002;2(3):289-97. PubMed.
Kong QL, Zhang JM, Zhang ZX, Ge PJ, Xu YJ, Mi RS, Zhao YH, Sui YP, He W. [Serum levels of macrophage colony stimulating factor in the patients with Alzheimer's disease]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2002 Jun;24(3):298-301. PubMed.
University of Massachusetts Medical School
The role of statins in modifying both cholesterol- and isoprenoid-related Abeta production is of consierable interest, as reported here. Alternatively, however, the effects of statins on endothelial integrity and function (via increase of eNOS and decrease of Endothelin-1, e.g.) may be especially important in sporadic Alzheimer's disease. There is extensive evidence for the key role of vascular risk factors in sporadic AD; and endothelial-secreted cytokines have been shown (for example) to be important for development and division of neural stem cells. The pleiotropic effects of statins raise many possibilities regarding which of their effects on cholesterol, Abeta, or other signalling pathways may account for their effectiveness in vascular disorders, and their potential efficacy in AD may well involve more than Abeta.
References:
Breteler MM. Vascular risk factors for Alzheimer's disease: an epidemiologic perspective. Neurobiol Aging. 2000 Mar-Apr;21(2):153-60. PubMed.
Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D'Agostino RB, Wilson PW, Wolf PA. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N Engl J Med. 2002 Feb 14;346(7):476-83. PubMed.
Shen Q, Goderie SK, Jin L, Karanth N, Sun Y, Abramova N, Vincent P, Pumiglia K, Temple S. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells. Science. 2004 May 28;304(5675):1338-40. Epub 2004 Apr 1 PubMed.
Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem. 1998 Sep 11;273(37):24266-71. PubMed.
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