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...  Read more

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:
1. 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. Epub 2001 Apr 17. Abstract

View all comments by Benjamin Wolozin

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...  Read more

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.

References:
1. 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. Epub 2003 Jan 06. Abstract

2. 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. Abstract

3. Zhang Q, Powers ET, Nieva J, Huff ME, Dendle MA, Bieschke J, Glabe CG, Eschenmoser A, Wentworth P Jr, 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. Epub 2004 Mar 19. Abstract

4. 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. Epub 2002 Aug 20. Abstract

5. 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.

6. Tschape JA, Hammerschmied C, Muhlig-Versen M, Athenstaedt K, Daum G, Kretzschmar D. The neurodegeneration mutant lochrig interferes with cholesterol homeostasis and Appl processing. EMBO J. 2002 Dec 2;21(23):6367-76. Abstract

View all comments by Luigi Puglielli

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...  Read more

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.

View all comments by Suzana Petanceska

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...  Read more

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).

View all comments by Robert Peers

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...  Read more

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:
1. 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, 98:5815-20. Abstract

2. Wheeler AP, Ridley AJ. Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility. Exp Cell Res 2004, 301:43-9. Abstract

3. 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 (Camb) 2003, 11:1595-607. Abstract

4. 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, 93:225-32. Abstract

5. 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, 302:1215-7. Abstract

View all comments by Thomas Kukar

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...  Read more

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.

View all comments by Dora M. Kovacs

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...  Read more

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. Abstract

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. Abstract

View all comments by Gary Landreth

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., Science 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...  Read more

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., Science 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.

View all comments by Steven Paul
View all comments by Yan Zhou

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,...  Read more

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!

View all comments by Robert Peers

Please see our commentary on this important study at PLoS Medicine eLetters page

View all comments by Alexei R. Koudinov

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...  Read more

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:
1. 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. Abstract

2. 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. Abstract

3. 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. Chinese. Abstract

4. http://www.salk.edu/otm/alltech.html

View all comments by Mary Reid

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, M. Vascular risk factors for Alzheimer's disease: an epidemiologic perspective. Neurobiol Aging. 2000, 21:153-60. Seshadri, S. et al. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. New Engl. J Med,2000; 346:476-483 Shen, Q et al. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells. Science, 2004; 304:1338-1340 Laufs, U and Liao, JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem, 1998; 273: 24266-71

View all comments by David Drachman

I enjoyed reading your news article on "Aging, Acetate, and Aβ: Sirtuins Regulate Metabolism and More." I would like to point your attention to our article, "Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2" (published online in PNAS on June 20, 2006), which describes the connection among mitochondria, sirtuins, and acetyl-CoA synthetase 2.

View all comments by Bjoern Schwer

Calorie restriction (CR) or dietary restriction (about 60 percent of ad libitum or normal calorie consumption) has been known to possess numerous useful benefits for aging (Cohen et al., 2004; Wood et al., 2004) and age-related disorders such as Alzheimer disease (Mattson et al., 2003; Patel et al., 2005). The recent paper by Qin et al. is a valuable addition to the growing literature on the beneficial effects of CR on AD mechanisms. Qin et al. explains how CR relates to the activation of the mammalian sirtuin protein SIRT1 and, in turn, how this activation promotes a non-amyloidogenic, α-secretase pathway for amyloid precursor protein (APP) processing and reduces amyloid-β production in Tg2576 mice. The authors also elegantly utilized viral transfection systems to show that SIRT1 expression in Tg2576 neurons and CHO-APPswe cells significantly attenuates the production of amyloid-β peptides. Most interestingly, they demonstrated that increased SIRT1 expression following a CR regimen reduces expression levels of the Rho kinase ROCK1, and that reduced ROCK1 levels...  Read more

Calorie restriction (CR) or dietary restriction (about 60 percent of ad libitum or normal calorie consumption) has been known to possess numerous useful benefits for aging (Cohen et al., 2004; Wood et al., 2004) and age-related disorders such as Alzheimer disease (Mattson et al., 2003; Patel et al., 2005). The recent paper by Qin et al. is a valuable addition to the growing literature on the beneficial effects of CR on AD mechanisms. Qin et al. explains how CR relates to the activation of the mammalian sirtuin protein SIRT1 and, in turn, how this activation promotes a non-amyloidogenic, α-secretase pathway for amyloid precursor protein (APP) processing and reduces amyloid-β production in Tg2576 mice. The authors also elegantly utilized viral transfection systems to show that SIRT1 expression in Tg2576 neurons and CHO-APPswe cells significantly attenuates the production of amyloid-β peptides. Most interestingly, they demonstrated that increased SIRT1 expression following a CR regimen reduces expression levels of the Rho kinase ROCK1, and that reduced ROCK1 levels somehow activate the non-amyloidogenic processing of APP (Qin et al., 2006). Perhaps a subsequent challenge in CR-related research is to demonstrate a clear link between a decrease in the expression of ROCK1 and the increase in the activity of α-secretase.

Can CR serve as a reliable treatment for AD?
The answer to this question is not simple. On a positive note, CR-associated mechanisms are the most known and reliable pathways that promote anti-aging effects in diverse groups of organisms ranging from yeasts to mammals. These effects seem to be consistently associated with an increased expression of SIRT1 (Bordone and Guarente, 2005).

Although “eat less, age well, and remember well” appears to be the new mantra in cutting-edge research on human aging, there are some unfavorable aspects to using CR as a therapy to treat AD patients (Anekonda and Reddy, 2006; Anekonda, 2006). First, eating less is not a popular treatment, as it involves giving up favorite tastes. Second, at least for now, there are not many research articles showing the long-term benefits of eating less in humans (Dirks and Leeuwenburg, 2006). Third, inappropriate CR may have severe adverse effects in humans (reviewed in Dirks and Leeuwenburg, 2006).

What is needed is advice on the amount of calorie restriction that individuals need, as determined by scientific studies.

Can CR mimetics serve as a reliable treatment for AD?
People do not need to give up their favorite tastes in order to gain the healthful benefits from CR. Trans-resveratrol (simply resveratrol) found in the skin of purple grapes and in 70 or so other plant species, when ingested in a predetermined regimen, mimics the effects of CR on a diverse group of organisms (Howitz et al., 2003; Laming et al., 2004; Wood et al., 2004). Resveratrol operates by triggering an increased expression of SIRT1. Resveratrol not only possesses numerous therapeutic benefits in both animal models and humans (reviewed in Baur and Sinclair, 2006), but also interferes favorably in multiple pathways associated with AD pathology (reviewed in Anekonda, 2006).

Can resveratrol or any other herbal equivalents be used as reliable therapeutics for healthy aging or age-related disorders? Herbal compounds may have some side effects that need to be clarified before they are used as therapies. A given herb can possess dozens of pharmacologically useful compounds, but the effects of these compounds need to be substantiated through scientific testing. The composition of active compounds in plants varies, depending on the growth environment, resulting in inconsistent pharmacological performance. It is tedious, time-consuming work, defining the bioavailability of each phytochemical useful in treating AD (reviewed in Anekonda and Reddy, 2005). In addition, the ability of the herbal compounds to cross the blood-brain barrier, any toxic side effects, or any useful synergistic effects must be carefully defined before they are used in treatment of AD.

For now, it appears that both CR and CR-mimetics require long-term testing on humans to define their safety. Even before considering CR therapies, it is perhaps essential to understand the critical mechanisms associated with CR in AD. To this end, the Qin et al. paper is a step forward.

References:
Cohen HY, Miller C, Bitterman KJ, Wall NR, Hekking B, Kessler B, Howitz KT, Gorospe M, de Cabo R, Sinclair DA. Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science. 2004 Jul 16;305(5682):390-2. Epub 2004 Jun 17. Abstract

Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D. Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature. 2004 Aug 5;430(7000):686-9. Epub 2004 Jul 14. Erratum in: Nature. 2004 Sep 2;431(7004):107. Abstract

Mattson MP, Duan W, Guo Z. Meal size and frequency affect neuronal plasticity and vulnerability to disease: cellular and molecular mechanisms. J Neurochem. 2003 Feb;84(3):417-31. Review. Abstract

Patel NV, Gordon MN, Connor KE, Good RA, Engelman RW, Mason J, Morgan DG, Morgan TE, Finch CE. Caloric restriction attenuates Abeta-deposition in Alzheimer transgenic models. Neurobiol Aging. 2005 Jul;26(7):995-1000. Epub 2004 Nov 25. Abstract

Qin W, Yang T, Ho L, Zhao Z, Wang J, Chen L, Thiyagarajan M, Macgrogan D, Rodgers JT, Puigserver P, Sadoshima J, Deng H H, Pedrini S, Gandy S, Sauve A, Pasinetti GM. Neuronal SIRT1 activation as a novel mechanism underlying the prevention of Alzheimer's disease amyloid neuropathology by calorie restriction. J Biol Chem. 2006 Jun 2; [Epub ahead of print] Abstract

Bordone L, Guarente L. Calorie restriction, SIRT1 and metabolism: understanding longevity. Nat Rev Mol Cell Biol. 2005 Apr;6(4):298-305. Review. Abstract

Anekonda TS, Reddy PH. Neuronal protection by sirtuins in Alzheimer's disease. J Neurochem. 2006 Jan;96(2):305-13. Epub 2005 Oct 7. Review. Abstract

Anekonda TS. Resveratrol-A boon for treating Alzheimer's disease? Brain Res Brain Res Rev. 2006 Jun 9; [Epub ahead of print] Abstract

Dirks AJ, Leeuwenburgh C. Caloric restriction in humans: potential pitfalls and health concerns. Mech Ageing Dev. 2006 Jan;127(1):1-7. Epub 2005 Oct 13. Review. Abstract

Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, Zipkin RE, Chung P, Kisielewski A, Zhang LL, Scherer B, Sinclair DA. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature. 2003 Sep 11;425(6954):191-6. Epub 2003 Aug 24. Abstract

Lamming DW, Wood JG, Sinclair DA. Small molecules that regulate lifespan: evidence for xenohormesis. Mol Microbiol. 2004 Aug;53(4):1003-9. Review. Abstract

Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev Drug Discov. 2006 Jun;5(6):493-506. Epub 2006 May 26. Review. Abstract

Anekonda TS, Reddy PH. Can Herbs Provide a New Generation of Drugs for Treating Alzheimer’s Disease? Brain Res Brain Res Rev. 2005 Dec 15;50(2):361-76. Epub 2005 Nov 2. Abstract

View all comments by Thimmappa Anekonda

Methylation and Tau
The wealth of reports in the last decade confirming an association between homocysteine and Alzheimer disease hint that disturbed methylation might somehow relate to AD pathology (Smith, 2008; McCaddon and Hudson, 2007). A link between impaired methylation and neurofibrillary tangle formation was first proposed by Scott and Vafai in 2002 (Vafai and Stock, 2002). In support of this elegant hypothesis Obeid et al. found an association between phospho-tau and the ratio of the methyl donor S-adenosylmethionine (SAM) and its demethylated product S-adenosylhomocysteine (SAH) in the CSF of 182 patients with various neurological disorders (Obeid et al., 2007).

SAH is a potent inhibitor of methyltransferase reactions, and last year Sontag et al. found that exposing neuroblastoma cells to SAH led to reduced methylation of PP2A (Sontag et al., 2007). Sontag’s group now show that folate deprivation downregulates PP2A carboxymethyltransferase expression in these cells, ultimately resulting in cell death. Protection is afforded by overexpressing either the...  Read more

Methylation and Tau
The wealth of reports in the last decade confirming an association between homocysteine and Alzheimer disease hint that disturbed methylation might somehow relate to AD pathology (Smith, 2008; McCaddon and Hudson, 2007). A link between impaired methylation and neurofibrillary tangle formation was first proposed by Scott and Vafai in 2002 (Vafai and Stock, 2002). In support of this elegant hypothesis Obeid et al. found an association between phospho-tau and the ratio of the methyl donor S-adenosylmethionine (SAM) and its demethylated product S-adenosylhomocysteine (SAH) in the CSF of 182 patients with various neurological disorders (Obeid et al., 2007).

SAH is a potent inhibitor of methyltransferase reactions, and last year Sontag et al. found that exposing neuroblastoma cells to SAH led to reduced methylation of PP2A (Sontag et al., 2007). Sontag’s group now show that folate deprivation downregulates PP2A carboxymethyltransferase expression in these cells, ultimately resulting in cell death. Protection is afforded by overexpressing either the methyltransferase or the Balpha regulatory subunit of PP2A, whereas knockdown of either protein accelerates the toxicity of folate deprivation. Reduced SAM and elevated SAH concentrations in folate deficient mice are also associated with enhanced tau phosphorylation in susceptible brain regions.

Importantly, this work suggests an association between methylation status and one of the key pathological features of Alzheimer disease. Impaired methylation is likely to have other widespread effects in the nervous system. For example, alterations in the SAM/SAH ratio are also associated with PS1 and BACE upregulation and β amyloid deposition (Fuso et al., 2008). ”Tau-ists” and ”Bap-tists”’ should both be aware that the ”Meth-odists” may well be on to something!

References:
Fuso A, Nicolia V, Cavallaro RA, Ricceri L, D'Anselmi F, Coluccia P, Calamandrei G, Scarpa S. B-vitamin deprivation induces hyperhomocysteinemia and brain S-adenosylhomocysteine, depletes brain S-adenosylmethionine, and enhances PS1 and BACE expression and amyloid-beta deposition in mice. Mol Cell Neurosci. 2008 Apr;37(4):731-46. Abstract

McCaddon, A. and Hudson, P. 2007. Alzheimer's disease, oxidative stress and B-vitamin depletion. Future Neurology. 2.537-47.

Obeid R, Kasoha M, Knapp JP, Kostopoulos P, Becker G, Fassbender K, Herrmann W. Folate and methylation status in relation to phosphorylated tau protein(181P) and beta-amyloid(1-42) in cerebrospinal fluid. Clin Chem. 2007 Jun;53(6):1129-36. Abstract

Smith AD. The worldwide challenge of the dementias: a role for B vitamins and homocysteine? Food Nutr Bull. 2008 Jun;29(2 Suppl):S143-72. Abstract

Sontag E, Nunbhakdi-Craig V, Sontag JM, Diaz-Arrastia R, Ogris E, Dayal S, Lentz SR, Arning E, Bottiglieri T. Protein phosphatase 2A methyltransferase links homocysteine metabolism with tau and amyloid precursor protein regulation. J Neurosci. 2007 Mar 14;27(11):2751-9. Abstract

Sontag JM, Nunbhakdi-Craig V, Montgomery L, Arning E, Bottiglieri T, Sontag E. Folate deficiency induces in vitro and mouse brain region-specific downregulation of leucine carboxyl methyltransferase-1 and protein phosphatase 2A B(alpha) subunit expression that correlate with enhanced tau phosphorylation. J Neurosci. 2008 Nov 5;28(45):11477-87. Abstract

Vafai SB, Stock JB. Protein phosphatase 2A methylation: a link between elevated plasma homocysteine and Alzheimer's Disease. FEBS Lett. 2002 May 8;518(1-3):1-4. Abstract

View all comments by Andrew McCaddon

One must be careful when calling nicotinamide an "inhibitor" in this experiment. While it is true that our lab showed that nicotinamide is a direct inhibitor of SIRT1 enzyme, it is also a precursor of NAD+, and NAD+ is a co-substrate (i.e., activator) of SIRT1.

In vivo, there is an abundant enzyme called Nampt in cells and serum that initiates the conversion of nicotinamide to NAD+. Therefore we should entertain the possibility that nicotinamide is activating SIRT1 in vivo, not inhibiting it. This would fit with other papers showing that SIRT1 is neuroprotective.

View all comments by David Sinclair

The experimental dose used in the study was 200 mg/kg/day. This would translate to a daily dose of nearly 14,000 mg for a 70 kg (154 lb.) person. Yet in the proposed clinical trial the experimental group will be receiving a daily dose of 3,000 mg. How does one explain the lower dose being used in the clinical trial?

View all comments by William Polsky

I am responding to William Polsky's comment on computation of the human dose of nicotinamide.

Following the publication of a study on the use of resveratrol in mice to improve their health and maximum lifespan, the press reported that a human would have to consume an enormous amount of wine or supplements to gain similar benefits. This statement shows a lack of understanding of the appropriate criteria for dosage translations between species.

There are a number of acceptable ways to compute the human equivalent dose from animal studies. The key is to consider energy-expenditure differences between species. Energy expenditure is a measure of metabolic rate. The method favored by the FDA (see www.fda.gov/cber/gdlns/dose.htm) uses the body surface area (BSA) normalization method. Basal metabolic rate is directly related to surface area. As the FDA notes, the BSA method correlates well across several mammalian species with several parameters of biology, including oxygen utilization, caloric expenditure, basal...  Read more

I am responding to William Polsky's comment on computation of the human dose of nicotinamide.

Following the publication of a study on the use of resveratrol in mice to improve their health and maximum lifespan, the press reported that a human would have to consume an enormous amount of wine or supplements to gain similar benefits. This statement shows a lack of understanding of the appropriate criteria for dosage translations between species.

There are a number of acceptable ways to compute the human equivalent dose from animal studies. The key is to consider energy-expenditure differences between species. Energy expenditure is a measure of metabolic rate. The method favored by the FDA (see www.fda.gov/cber/gdlns/dose.htm) uses the body surface area (BSA) normalization method. Basal metabolic rate is directly related to surface area. As the FDA notes, the BSA method correlates well across several mammalian species with several parameters of biology, including oxygen utilization, caloric expenditure, basal metabolism, blood volume, circulating plasma proteins, and renal function. However, there are important differences, such as different sensitivities, that make the BSA method a guide rather than a rule.

A recent article in the FASEB Journal criticized the media for its misunderstanding (or ignorance of) what a human equivalent dose would be for the amount of resveratrol used in the Sinclair mouse study to which the comment refers (1). Immediately after that paper was published, the popular press—along with a contingent of the scientific community—voiced concerns regarding the relevance to humans of the resveratrol dose used by the researchers. Almost without exception, the press scaled the amount of resveratrol given to the mice—22.4 mg per kg of body weight—to humans on a straight weight basis. According to their reports, a person weighing 175 lb. (about 80 kg) would have to ingest 22.4 x 80 = 1,792 mg/day. Furthermore, the media typically wrote that to get that much resveratrol from red wine (using an estimate of 2 mg of resveratrol per bottle), a person would have to drink 896 bottles per day.

Pharmacology 101 teaches us, however, that ratios involving body weight, energy expenditure, and body surface area are far more realistic than weight ratios alone in scaling dosages from one species to another. This has been known for over a century, and the relevant scaling factors are familiar to most scientists. The media concluded that the human equivalent dose of the Sinclair study was ridiculously large and impractical.

This does an injustice to the researchers, not to mention impede implementation. It's frustrating considering that resveratrol has been found to be safe in extremely large doses.

Returning to the article in the FASEB Journal, the authors assert that the mouse dose in the Sinclair study should be multiplied by the appropriate mouse/human scaling factor of 3/37, which gives a value of 1.82 mg per kg per day. Using the 80-kg person as an example again, the human dosage would therefore be 1.82 x 80 = 146 mg/day, an amount easily achieved with supplements, but not so easily with wine (73 bottles!). But the mice were not fed wine.

We do not know for certain if resveratrol can do for humans what it does for mice and other creatures, but the upside potential is great, and there does not appear to be a downside as yet.

Applying this line of reasoning to nicotinamide yields about 1,298 mg/day for a 80-kg person (200 * 0.081 * 80).

References:
1. Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J 2007 Oct 17. [Epub ahead of print] Abstract

View all comments by Will Block

Tauopathies are a group of diseases characterized by accumulation of tau protein. Tau protein has a novel physiological function in the brain—stabilizing the neurons. Alterations in the amount or the structure of tau protein might destabilize the microtubules, thus causing changes in subcellular structures like the lysosomes (1) or the mitochondria (2). Tau can be structurally modified by phosphorylation, glycosylation, oxidation, and crosslinking. These pathological forms of tau tend to form self-aggregates and thus forming the neurofibrilary tangles (NFTs). NFTs are typical findings in all tauopathies containing paired PHF comprising hyperphosphorylated tau (3).

Alzheimer disease (AD) is the best known tauopathy that is characterized by accumulation of NFTs in the brain. In an animal model of neurodegenerative diseases, mice developed progressive accumulation of NFTs, neuronal loss, and memory decline (4). Suppressing the transgenic tau caused improvement in memory function, and neuron numbers stabilized. Unexpectedly, NFTs continued to accumulate. The authors...  Read more

Tauopathies are a group of diseases characterized by accumulation of tau protein. Tau protein has a novel physiological function in the brain—stabilizing the neurons. Alterations in the amount or the structure of tau protein might destabilize the microtubules, thus causing changes in subcellular structures like the lysosomes (1) or the mitochondria (2). Tau can be structurally modified by phosphorylation, glycosylation, oxidation, and crosslinking. These pathological forms of tau tend to form self-aggregates and thus forming the neurofibrilary tangles (NFTs). NFTs are typical findings in all tauopathies containing paired PHF comprising hyperphosphorylated tau (3).

Alzheimer disease (AD) is the best known tauopathy that is characterized by accumulation of NFTs in the brain. In an animal model of neurodegenerative diseases, mice developed progressive accumulation of NFTs, neuronal loss, and memory decline (4). Suppressing the transgenic tau caused improvement in memory function, and neuron numbers stabilized. Unexpectedly, NFTs continued to accumulate. The authors concluded that tau accumulation rather than NFTs can cause cognitive decline or neuronal death in this model of tauopathy (4). Although, these results can not be extrapolated to humans without further research, they might suggest that lowering tau protein can improve cognitive function in humans as well (4).

A very tempting hypothesis that would have implications for prevention and treatment of tauopathies is that lowering the modified forms of tau protein might protect the brain. The degree of tau phosphorylation and the phosphorylated residues regulate binding of tau to microtubules, thus affecting its function. There is a state of balance between kinase-mediated phosphorylation and phosphatase-mediated dephosphorylation. Tau can be phosphorylated by several kinases (proline and non-proline directed; glycogen synthase kinase 3, Cdk5, MAP kinase, JNK, PKC, calmodulin kinase II). Protein phosphatase 2A (PP2A) is the most important phosphatase in the brain acting on most phosphorylated sites of tau. PP2A is composed of three subunits: A, B, and C. The subunit Bα is involved in substrate recognition and is considered the regulatory subunit. The binary enzyme AC must be methylated on Leu-309 to be able to join the B subunit and form the functional enzyme. The methylation is mediated by leucine carboxy methyltransferase-1 (LCMT-1), an S-adenosyl methionine (SAM) dependent enzyme.

In the recently published study by Sontag et al., the authors conducted several elegant experiments aiming at testing the effect of folate deprivation on the expression of hyperphosphorylated tau protein, PP2A enzyme and the methyltransferase required for its activation (5). The authors found that folate deficiency caused disturbed methylation potential in brain tissues. Furthermore, enhanced tau phosphorylation and cell death were related to downregulation of LCMT-1 and subsequent loss of ABαC complex. Folate deficiency caused an enhanced expression of the non-methylated form of PP2A enzyme. This condition did not change the existing PP2A, suggesting that binding of the ABαC complex is stable against demethylation by PME-1. In a previous study by Sontag et al., the authors found that mice fed a high methionine/low folate diet had higher brain S-adenosylhomocysteine (SAH) and lower expression of LCMT-1 (also called PP2A methyl transferase, or PPMT), thus leading to severe decrease of the steady state of PP2A (6). Another recent study found that SH-SY5Y neuroblastoma cells grown in a folate-deficient medium showed a decrease in the phosphatase activity, and this effect was reversible by adding SAM to the folate-deficient medium (7). Another interesting mechanism explaining the effect of folate deficiency on phosphorylated tau protein is related to enhancing activity of one or more of the kinases responsible for tau phosphorylation in the brain (7). This effect on the kinases is thought to be related to activating NMDA channels and thereby Ca++-dependent kinase pathways by homocysteine (Hcy). Therefore, folate deficiency is causally related to accumulation of tau protein; this effect is at least partly mediated by disturbed methylation status.

The study by Sontag et al. opens new perspectives for future research dealing with the pathophysiology of dementia or studies aiming at developing protective or therapeutic measures (5). Methyl group metabolism is regulated by micronutrients such as folate, vitamin B12, and vitamin B6. Several methyl donors in human diet have been identified, such as methionine and choline. The role of folate ingested with the diet is to convert Hcy into methionine in the presence of methyl cobalamin. Methionine is activated in the presence of ATP and further converted into SAM. SAM is a methyl donor that participates in numerous biological reactions including DNA methylation, and metabolism of neurotransmitters and phospholipids. Betaine, a product of the nutrient choline, is an alternative methyl donor in the conversion of Hcy into methionine via homocysteine betaine methyltransferase. However, this pathway has probably no or a limited role in Hcy remethylation in the brain. Furthermore, all factors that cause accumulation of Hcy (renal insufficiency, vitamin B deficiency, and alcoholism and liver disorders) might cause disturbed methylation potential. This metabolic condition implies increased S-adenosyl homocysteine (SAH) that acts as a potent inhibitor of methyltransferases by preventing SAM binding.

Deficiency of micronutrients is common in elderly people. Among other environmental factors, both hyperhomocysteinemia and B-vitamin deficiencies have been linked to increased risk for dementia and other age-related neurodegenerative diseases. Therefore, enhancing B vitamin status has a great potential to prevent metabolic conditions, associated memory disorders, and dementia. Future studies should test in-vivo the role of micronutrients in preventing or reversing phospho-tau accumulation in the brain. It is equally important in such studies to control for all sources of methyl donors in the diet.

References:
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