A Case of “Heads Win, Tails Don’t Lose”
The progress in uncovering the function of amyloid precursor protein (APP) has remained frustratingly slow due to the presence of APP-related proteins APLP1 and APLP2 and also because APP is continuously processed into three major fragments (paradoxically, intense focus on pathology of Aβ may also have contributed to this situation). However, the tide now seems to be shifting as more and more in vivo studies are shedding light on APP function in animal models ranging from C. elegans to mice (1). Employing an elegant approach, the Muller group used reverse genetics to express APP fragments in mice in which endogenous APP had been deleted (APP-KO). The “knock-in” strategy they used is superior to regular transgene-expression techniques since the endogenous regulatory elements still remain in place. The authors knocked-in either APPsα or APP lacking the last 15 residues of the cytoplasmic domain and subjected the resultant transgenic animals to a battery of behavioral tests. The authors report (2) that expression...  Read more

A Case of “Heads Win, Tails Don’t Lose”
The progress in uncovering the function of amyloid precursor protein (APP) has remained frustratingly slow due to the presence of APP-related proteins APLP1 and APLP2 and also because APP is continuously processed into three major fragments (paradoxically, intense focus on pathology of Aβ may also have contributed to this situation). However, the tide now seems to be shifting as more and more in vivo studies are shedding light on APP function in animal models ranging from C. elegans to mice (1). Employing an elegant approach, the Muller group used reverse genetics to express APP fragments in mice in which endogenous APP had been deleted (APP-KO). The “knock-in” strategy they used is superior to regular transgene-expression techniques since the endogenous regulatory elements still remain in place. The authors knocked-in either APPsα or APP lacking the last 15 residues of the cytoplasmic domain and subjected the resultant transgenic animals to a battery of behavioral tests. The authors report (2) that expression of APPsα, which lacks the transmembrane and the cytoplasmic domain of APP “…grossly attenuated or completely rescued the prominent deficits of APP-KO mice.” Although, the single APP-KO mice show only modest phenotypic changes (compared to the triple KO animals lacking all three proteins) and the behavioral testing can be affected by mixed genetic background of KI mice (ES cells from 129/Ola were injected in C57BL/6 mice), this paper clearly shows that expression of APP ectoplasmic domain rescues the deficits examined here. Does it mean that the APP cytoplasmic domain is dispensable and not relevant to APP function?

The answer to this question is a “no”! The cytoplasmic domain of APP represents the most conserved region of the protein through the evolution and shares a very high degree of homology with that of APLP1 and APLP2. The “GYENPTY” motif in APP cytoplasmic domain which allows binding of Fe65 (and several other adapter proteins) to APP is completely conserved in all APP family members. Ring, Muller, and colleagues expressed APP fragments in single APP-KO mice which expressed endogenous APLP1 and APLP2. The high degree of homology in the cytoplasmic region and conserved protein-protein interactions make it almost certain that the function of the “missing” APP cytoplasmic domain was replaced by the counterparts of APLP1 and APLP2. Indeed, APLP2 alone is capable of replacing APP, and APLP1 functions if postnatal lethality, observed in triple KO animals, is used as a functional readout. A crucial experiment to be performed, as the authors concede, will be to test whether APPsα rescues the deficits observed when all three APP family members are deleted. These experiments, although not trivial, must be on the drawing board.

While we await results from such experiments, it should be noted that the present results do make two important contributions to our knowledge base. It has long been believed (3) that APP ectodomain has neuroprotective and growth-promoting properties, and this notion has been supported by numerous in vitro tissue culture studies. The present studies provide an in vivo proof of concept by showing that APPsα was able to restore normal brain weight. Secondly, the results of Ring et al. argue strongly against a physiological role for Aβ as has been previously suggested (4). There is no Aβ made in APPSα-KI mice, and there is no likelihood that an “Aβ”-like fragment could mask the deficiency of Aβ function; the Aβ region, unlike the cytoplasmic domain, is completely dissimilar in APP and APLP1/APLP2. So, while the field may continue to debate the role of Aβ in pathology, the question of a “normal” function of Aβ may be put to rest.

References:
1. Zheng H, Koo EH. The amyloid precursor protein: beyond amyloid. Mol Neurodegener. 2006 Jul 3;1:5. Abstract

2. Ring S, Weyer SW, Kilian SB, Waldron E, Pietrzik CU, Filippov MA, Herms J, Buchholz C, Eckman CB, Korte M, Wolfer DP, Muller UC. The secreted beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice. J Neurosci. 2007 Jul 18;27(29):7817-26. Abstract

3. Saitoh T, Sundsmo M, Roch JM, Kimura N, Cole G, Schubert D, Oltersdorf T, Schenk DB. Secreted form of amyloid beta protein precursor is involved in the growth regulation of fibroblasts. Cell. 1989 Aug 25;58(4):615-22. Abstract

4. Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R. APP processing and synaptic function. Neuron. 2003 Mar 27;37(6):925-37. Abstract

View all comments by Sanjay W. Pimplikar

This is a carefully crafted and cautiously interpreted study showing that the α-secretase derived extracellular domain of APP (APPsα) is sufficient for rescue of the APP knockout (KO) mouse phenotypes that have been described so far. The beauty of this work is that a knock-in strategy was used, thus Ring et al. ensured that their conclusions would not be confounded by ectopic and/or excessive expression of APPsα and all isoforms of APP can be generated. Importantly, APPsα can rescue learning deficits of aged mice in the Morris water maze test and impaired long-term potentiation (LTP) in the CA3/CA1 pathway of hippocampal slices obtained from aged APP knockout mice to the same extent as an APP allele lacking the C-terminal 15 amino acid residues. Furthermore, the absence of the APP C-terminus did not affect behavior in the probe test, suggesting that retention of the learned behavior does not require the APP C-terminus.

Although these data do not preclude a role for the intracellular domain of APP and its related family members, APLP1 and APLP2, in normal...  Read more

This is a carefully crafted and cautiously interpreted study showing that the α-secretase derived extracellular domain of APP (APPsα) is sufficient for rescue of the APP knockout (KO) mouse phenotypes that have been described so far. The beauty of this work is that a knock-in strategy was used, thus Ring et al. ensured that their conclusions would not be confounded by ectopic and/or excessive expression of APPsα and all isoforms of APP can be generated. Importantly, APPsα can rescue learning deficits of aged mice in the Morris water maze test and impaired long-term potentiation (LTP) in the CA3/CA1 pathway of hippocampal slices obtained from aged APP knockout mice to the same extent as an APP allele lacking the C-terminal 15 amino acid residues. Furthermore, the absence of the APP C-terminus did not affect behavior in the probe test, suggesting that retention of the learned behavior does not require the APP C-terminus.

Although these data do not preclude a role for the intracellular domain of APP and its related family members, APLP1 and APLP2, in normal brain development, they do show that the APP C-terminus is not essential for APP-dependent learning and memory in the adult mouse brain. However, recently Ma et al. (PNAS 2007) reported that the γ-secretase derived intracellular domain of APP (AICD) was the only APP fragment that correlated with enhanced memory observed in the Morris water maze test for mice that overexpress WT APP. We can conclude from these two studies that the APP ectodomain in the form of APPsα is essential for learning, that the APP C-terminus is not required for retention of the learned behavior, and that AICD may play a role in enhancing memory of the learned behavior.

It has also previously been hypothesized that Aβ, on the basis of its ability to depress excitatory synaptic transmission and the ability of neuronal activation to increase secreted Aβ, may be part of a negative feedback loop for neuronal activity (Kamenetz et al., 2003). Since APPsα can completely rescue the learning deficits of the APP KO mice, the authors conclude that Aβ deficiency does not contribute to this phenotype. Although the Ring et al. study does not negate the proposed role for Aβ in regulation of neuronal activity and it remains possible that future studies will reveal a phenotypic manifestation for Aβ deficiency that is related to the proposed effects of Aβ on neuronal activity, there currently are no obvious consequences of Aβ deficiency for spatial learning and memory in mice.

View all comments by Suzanne Guenette

I stand in awe of this painstaking, comprehensive study from Ulrike Müller and coworkers. This is probably as close as we can get to the function of APP and the amyloid peptides in vivo—and to the problem of their physiological function and pathological role. The latter is evident and indisputable from what we have learned over the years since Glenner and Wong identified the amyloid peptides in 1984, but the normal function of APP and Aβ remains controversial. The data collected and documented in our own mouse models corroborated those of others (e.g., Kamenetz et al, 2003), and it led us to compare the amyloid peptides to totally unrelated but equally real and mysterious objects with unknown functions, i.e., the pentagonal dodecaeder from Gallo-Roman times (see comment by Dewachter and Van Leuven, 2005).

I fully agree that this study leaves little to the imagination about the amyloid peptides exerting major...  Read more

I stand in awe of this painstaking, comprehensive study from Ulrike Müller and coworkers. This is probably as close as we can get to the function of APP and the amyloid peptides in vivo—and to the problem of their physiological function and pathological role. The latter is evident and indisputable from what we have learned over the years since Glenner and Wong identified the amyloid peptides in 1984, but the normal function of APP and Aβ remains controversial. The data collected and documented in our own mouse models corroborated those of others (e.g., Kamenetz et al, 2003), and it led us to compare the amyloid peptides to totally unrelated but equally real and mysterious objects with unknown functions, i.e., the pentagonal dodecaeder from Gallo-Roman times (see comment by Dewachter and Van Leuven, 2005).

I fully agree that this study leaves little to the imagination about the amyloid peptides exerting major physiological actions in brain. Perhaps one could or should examine platelets and their related (patho)physiology in these novel mice, but I suspect that few researchers will wage money on that horse!

The AICD, on the other hand, remains a subject of discussion, although this study strengthens the case against it being essential for neuronal well-being. To untangle that knot, the possible experimental approaches in vivo are rather limited. Further, as they must involve APLP1 and APLP2, they are fraught with technical obstacles and “project-breaking” pitfalls. We hope that the group of Ulrike Müller can continue along the path they are on, since they appear alone to have the needed tools—and courage!

View all comments by Fred Van Leuven

Excellent review with an extensive coverage of APP, APLP1 and APLP2 knockout mice and the putative roles of APP under physiological conditions.

View all comments by Jurgen Gotz

Is anyone thinking of doing studies with this drug on PSP patients who only have tau tangles and do not have amyloid plaques at all?

View all comments by joanna connolly

Methylene blue most likely decreases the hyperphosphorylation of tau proteins by inhibiting the formation of peroxynitrites (peroxynitrites form through the combination of superoxides and inducible nitric oxides). Methylene blue accepts electrons from various oxidases, thus limiting the formation of superoxides (and thus peroxynitrites).

Peroxynitrites play a critical role in the progression of Alzheimer disease. Peroxynitrites result in high GSK3 activity, which in turn causes the hyperphosphorylation of tau proteins. By largely inactivating protein kinase B (AKT) through tyrosine nitration and largely inactivating most forms of protein kinase C through cysteine oxidation of G proteins, peroxynitrites inhibit the two pathways by which GSK3 is inactivated. Peroxynitrites also decrease the protein kinase C mediated uptake of choline through muscarinic receptors and choline acetyltransferase activity. Thus, peroxynitrites cause large deficits in the memory storing compound acetylcholine.

Researchers should study the efficacy of other peroxynitrite inhibitors in combination...  Read more

Methylene blue most likely decreases the hyperphosphorylation of tau proteins by inhibiting the formation of peroxynitrites (peroxynitrites form through the combination of superoxides and inducible nitric oxides). Methylene blue accepts electrons from various oxidases, thus limiting the formation of superoxides (and thus peroxynitrites).

Peroxynitrites play a critical role in the progression of Alzheimer disease. Peroxynitrites result in high GSK3 activity, which in turn causes the hyperphosphorylation of tau proteins. By largely inactivating protein kinase B (AKT) through tyrosine nitration and largely inactivating most forms of protein kinase C through cysteine oxidation of G proteins, peroxynitrites inhibit the two pathways by which GSK3 is inactivated. Peroxynitrites also decrease the protein kinase C mediated uptake of choline through muscarinic receptors and choline acetyltransferase activity. Thus, peroxynitrites cause large deficits in the memory storing compound acetylcholine.

Researchers should study the efficacy of other peroxynitrite inhibitors in combination with or separate from methylene blue. Rosemary holds high promise in this regard. Rosemary can be inhaled directly into the brain, it decreases homocysteine levels (high homocysteine levels contribute to the formation of peroxynitrites), it directly scavenges peroxynitrites, and it limits tyrosine nitration. If you stop the formation of peroxynitrites, you stop the progression of Alzheimer disease.

References:
Alkam T, Nitta A, Mizoguchi H, Itoh A, Nabeshima T. A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory induced by Abeta(25-35). Behav Brain Res. 2007 Jun 18;180(2):139-45. Abstract

Guermonprez L, Ducrocq C, Gaudry-Talarmain YM. Inhibition of acetylcholine synthesis and tyrosine nitration induced by peroxynitrite are differentially prevented by antioxidants. Mol Pharmacol. 2001 Oct;60(4):838-46. Abstract

Heydrick SJ, Reed KL, Cohen PA, Aarons CB, Gower AC, Becker JM, Stucchi AF. Intraperitoneal administration of methylene blue attenuates oxidative stress, increases peritoneal fibrinolysis, and inhibits intraabdominal adhesion formation. J Surg Res. 2007 Dec;143(2):311-9. Abstract

Zhang YJ, Xu YF, Liu YH, Yin J, Li HL, Wang Q, Wang JZ. Peroxynitrite induces Alzheimer-like tau modifications and accumulation in rat brain and its underlying mechanisms. FASEB J. 2006 Jul;20(9):1431-42. Abstract

View all comments by Lane Simonian

I’d like to make two major points about this paper from Hickman and colleagues.

First, I don’t view changing gene expression in microglia as a dysfunction. Microglia change their protein synthesis all the time, including surface receptors and proinflammatory cytokines, especially when activated under conditions of acute injury. The changes reported here for PS1-APP mice seem to be a result of the genetic manipulations in these mice since they do not occur in wild-type cells.

Second, the role of microglia in amyloid clearance remains controversial. Certainly, the histopathology of human brain does not support a role for microglia in amyloid clearance, especially when it comes to early diffuse amyloid, which does not seem to elicit any kind of response from microglia, i.e., the cells remain in their resting state.

That said, I do feel that understanding the role of microglia in AD is key to devising new treatments. I also think that microglial dysfunction plays a role, but in the sense that microglia themselves are subject to degeneration, resulting in a dysfunction...  Read more

I’d like to make two major points about this paper from Hickman and colleagues.

First, I don’t view changing gene expression in microglia as a dysfunction. Microglia change their protein synthesis all the time, including surface receptors and proinflammatory cytokines, especially when activated under conditions of acute injury. The changes reported here for PS1-APP mice seem to be a result of the genetic manipulations in these mice since they do not occur in wild-type cells.

Second, the role of microglia in amyloid clearance remains controversial. Certainly, the histopathology of human brain does not support a role for microglia in amyloid clearance, especially when it comes to early diffuse amyloid, which does not seem to elicit any kind of response from microglia, i.e., the cells remain in their resting state.

That said, I do feel that understanding the role of microglia in AD is key to devising new treatments. I also think that microglial dysfunction plays a role, but in the sense that microglia themselves are subject to degeneration, resulting in a dysfunction that manifests itself in a loss of neuroprotection. In other words, I think that neurodegeneration in AD results from neglect (due to a loss of microglia) rather than from aggression (due to neuroinflammation).

View all comments by Wolfgang Streit

There is just a small handful of information about methylene blue and Alzheimer's (see Atamna et al., 2008; Necula et al., 2007; Taniguchi et al., 2005; Wischik et al., 1996).

As an interesting and somewhat related concept, the use of phenothiazines for prion diseases has been investigated at UC San Francisco. Apparently phenothiazines were derived from methylene blue—not everyone knew that, perhaps.

A press release from UCSF said:

"In [Korth's] current study, he set out by identifying classes of drugs that were known to cross the blood-brain barrier to the brain, and then tested their ability to inhibit prion formation in the cultured mouse neuroblastoma cells.

"He identified only one class that met both criteria: phenothiazines, a group of tricyclic drugs used to treat psychosis. He then determined that a phenothiazine containing a particular side chain structure was the most effective. This was chlorpromazine.

"When he discovered that phenothiazines were derived from methylene blue, a dye used in England in the 1850s, he examined other derivatives...  Read more

There is just a small handful of information about methylene blue and Alzheimer's (see Atamna et al., 2008; Necula et al., 2007; Taniguchi et al., 2005; Wischik et al., 1996).

As an interesting and somewhat related concept, the use of phenothiazines for prion diseases has been investigated at UC San Francisco. Apparently phenothiazines were derived from methylene blue—not everyone knew that, perhaps.

A press release from UCSF said:

"In [Korth's] current study, he set out by identifying classes of drugs that were known to cross the blood-brain barrier to the brain, and then tested their ability to inhibit prion formation in the cultured mouse neuroblastoma cells.

"He identified only one class that met both criteria: phenothiazines, a group of tricyclic drugs used to treat psychosis. He then determined that a phenothiazine containing a particular side chain structure was the most effective. This was chlorpromazine.

"When he discovered that phenothiazines were derived from methylene blue, a dye used in England in the 1850s, he examined other derivatives of the dye and determined that one, quinacrine, had a similar tricyclic scaffold and the same side chain structure as chlorpromazine."

References:
Press Release

Korth C, May BC, Cohen FE, Prusiner SB. Acridine and phenothiazine derivatives as pharmacotherapeutics for prion disease. Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9836-41. Abstract

Atamna H, Nguyen A, Schultz C, Boyle K, Newberry J, Kato H, Ames BN. Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB J. 2008 Mar;22(3):703-12. Epub 2007 Oct 10. Abstract

Necula M, Breydo L, Milton S, Kayed R, van der Veer WE, Tone P, Glabe CG. Methylene blue inhibits amyloid Abeta oligomerization by promoting fibrillization. Biochemistry. 2007 Jul 31;46(30):8850-60. Epub 2007 Jun 27. Abstract

Taniguchi S, Suzuki N, Masuda M, Hisanaga S, Iwatsubo T, Goedert M, Hasegawa M. Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins. J Biol Chem. 2005 Mar 4;280(9):7614-23. Epub 2004 Dec 17. Abstract

Wischik CM, Edwards PC, Lai RY, Roth M, Harrington CR. Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):11213-8. Abstract

View all comments by P.F. Jennings

How Does Rember^TM Work?
How exactly does Rember work? We have been puzzling over this in recent days, and are finding it difficult to believe that a drug so remarkably successful (yes, we know the caveats) could act on only one of the many problems in AD brain.

Rember is methylene blue, we are told. Methylene blue is a redox dye, which means it transports electrons. This is what mitochondria do. Methylene blue has been found to restore cognition to animals with dysfunctional cytochrome oxidase (Callaway et al., 2002), which is of great interest because cytochrome oxidase transports electrons in mitochondria and is low in AD brain (Mutisya et al., 1994).

Haem synthesis is another potential target of methylene blue. Very recently Atamna et al. (2008) found that methylene blue delays cellular senescence and improves haem synthesis. Haem is made in mitochondria and involves reduction of iron (III) to iron (II) by the electron transport chain, and specifically by cytochrome oxidase (Williams et al., 1976). In fact, cytochrome oxidase is itself a haem...  Read more

How Does Rember^TM Work?
How exactly does Rember work? We have been puzzling over this in recent days, and are finding it difficult to believe that a drug so remarkably successful (yes, we know the caveats) could act on only one of the many problems in AD brain.

Rember is methylene blue, we are told. Methylene blue is a redox dye, which means it transports electrons. This is what mitochondria do. Methylene blue has been found to restore cognition to animals with dysfunctional cytochrome oxidase (Callaway et al., 2002), which is of great interest because cytochrome oxidase transports electrons in mitochondria and is low in AD brain (Mutisya et al., 1994).

Haem synthesis is another potential target of methylene blue. Very recently Atamna et al. (2008) found that methylene blue delays cellular senescence and improves haem synthesis. Haem is made in mitochondria and involves reduction of iron (III) to iron (II) by the electron transport chain, and specifically by cytochrome oxidase (Williams et al., 1976). In fact, cytochrome oxidase is itself a haem enzyme, which means a defect in haem synthesis could feed back on itself in a vicious circle. Quite possibly the tangles that Rember is targeting would not develop in the first place if mitochondria were working properly to make resources available for breaking down faulty proteins before they become a problem. Rember dissolves tangles in vitro, like some other redox dyes (Wischik et al., 1996). Tau-tau interaction is thought to be the target, but it might not be the only one. According to Yamamoto et al. (2002), tangles isolated from AD brain can be dissolved by reduction of iron (III) to iron (II), which mirrors what methylene blue might be doing in haem synthesis (see above), and in methaemoglobinaemia, where it does indeed reduce iron (III) to iron (II) (Bradberry, 2003). Iron (III) can aggregate hyperphosphorylated tau via the phosphate groups, say Yamamoto et al., but iron (II) cannot. Iron is a problem in AD brain (Smith et al., 1997), and perhaps its ability to aggregate tau is just as important as its promotion of oxidative stress.

The success of Rember might have even wider significance. Very recently Leslie Klevay published a paper in Medical Hypotheses entitled “Alzheimer's disease as copper deficiency” (Klevay, 2008). Klevay is best known for the copper deficiency theory of heart disease (Klevay, 2000). Heart disease shares important characteristics with AD, not least high serum homocysteine (Whincup et al., 1999) and low cytochrome oxidase activity (Burke and Poyton, 1998).

Cytochrome oxidase is a copper enzyme as well as a haem enzyme. Copper is required for other aspects of iron metabolism besides haem synthesis, including iron efflux from the brain (Xu et al., 2004). Homocysteine metabolism, too, is intimately associated with that of copper (Bethin et al., 1995a and 1995b). Methylation reactions are inhibited by S-adenosylhomocysteine (SAH), which is broken down by SAH hydrolase, a copper protein. Another key enzyme in the pathway, methionine synthase, may require copper in addition to vitamin B12 (Tamura et al., 1999). Most significantly, copper and protein methylation are involved in NGF-dependent neurite outgrowth, and so is SAH hydrolase (Birkaya and Aletta, 2005).

High homocysteine means problems with methylation reactions. And here is the link with tangles in AD brain: methylation is needed for assembly of the phosphatase primarily responsible for dephosphorylating P-tau, PP2A (Vafai and Stock, 2002). Obeid et al. (2007) found correlations in neurological patients between CSF P-tau and homocysteine, SAH and the SAM/SAH ratio, and they suggest the link is through PP2A.

Copper is low in the modern diet, being largely removed during refining of grains, and Table 1 of the 2006 paper by Morris et al. shows an astonishing correlation between copper intake and cognitive function. It was recently found, most intriguingly, that Aβ peptides 1-40 and 1-42 are members of the Ecto-nox family of copper-dependent redox oscillators (Markert et al., 2004), which suggests they are not just toxic cellular junk.

Methylene blue is a kind of redox oscillator, too. All kinds of biological processes involve redox oscillations, almost certainly including neurite extension and axonal transport. Tangles are produced when these processes malfunction. Even if Rember doesn't turn out to work quite as well as it appears, it will still have made a major contribution to AD research.

References:
Atamna H, Nguyen A, Schultz C, Boyle K, Newberry J, Kato H, Ames BN. Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB J. 2008 Mar;22(3):703-12. Abstract

Bethin KE, Petrovic N, Ettinger MJ. Identification of a major hepatic copper binding protein as S-adenosylhomocysteine hydrolase. J Biol Chem. 1995a Sep 1;270(35):20698-702. Abstract

Bethin KE, Cimato TR, Ettinger MJ. Copper binding to mouse liver S-adenosylhomocysteine hydrolase and the effects of copper on its levels. J Biol Chem. 1995b Sep 1;270(35):20703-11. Abstract

Birkaya B, Aletta JM. NGF promotes copper accumulation required for optimum neurite outgrowth and protein methylation. J Neurobiol. 2005 Apr;63(1):49-61. Abstract

Bradberry SM. Occupational methaemoglobinaemia. Mechanisms of production, features, diagnosis and management including the use of methylene blue. Toxicol Rev. 2003;22(1):13-27. Abstract

Burke PV, Poyton RO. Structure/function of oxygen-regulated isoforms in cytochrome c oxidase. J Exp Biol. 1998 Apr;201(Pt 8):1163-75. Abstract

Callaway NL, Riha PD, Wrubel KM, McCollum D, Gonzalez-Lima F. Methylene blue restores spatial memory retention impaired by an inhibitor of cytochrome oxidase in rats. Neurosci Lett. 2002 Oct 31;332(2):83-6. Abstract

Klevay LM. Dietary copper and risk of coronary heart disease. Am J Clin Nutr. 2000 May;71(5):1213-4. Abstract

Klevay LM. Alzheimer's disease as copper deficiency. Med Hypotheses. 2008;70(4):802-7. Abstract

Markert C, Morré DM, Morré DJ. Human amyloid peptides Abeta1-40 and Abeta1-42 exhibit NADH oxidase activity with copper-induced oscillations and a period length of 24 min. Biofactors. 2004;20(4):207-21. Abstract

Morris MC, Evans DA, Tangney CC, Bienias JL, Schneider JA, Wilson RS, Scherr PA. Dietary copper and high saturated and trans fat intakes associated with cognitive decline. Arch Neurol. 2006 Aug;63(8):1085-8. (Note that the conclusions of this paper appear to be at odds with the data in its Table 1.) Abstract

Mutisya EM, Bowling AC, Beal MF. Cortical cytochrome oxidase activity is reduced in Alzheimer's disease. J Neurochem. 1994 Dec;63(6):2179-84. Abstract

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 MA, Harris PL, Sayre LM, Perry G. Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci U S A. 1997 Sep 2;94(18):9866-8. Abstract

Tamura T, Hong KH, Mizuno Y, Johnston KE, Keen CL. Folate and homocysteine metabolism in copper-deficient rats. Biochim Biophys Acta. 1999 May 24;1427(3):351-6. 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

Williams DM, Loukopoulos D, Lee GR, Cartwright GE. Role of copper in mitochondrial iron metabolism. Blood. 1976 Jul;48(1):77-85. Abstract

Wischik CM, Edwards PC, Lai RY, Roth M, Harrington CR. Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):11213-8. Abstract

Whincup PH, Refsum H, Perry IJ, Morris R, Walker M, Lennon L, Thomson A, Ueland PM, Ebrahim SB. Serum total homocysteine and coronary heart disease: prospective study in middle aged men. Heart. 1999 Oct;82(4):448-54. Abstract

Xu X, Pin S, Gathinji M, Fuchs R, Harris ZL. Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis. Ann N Y Acad Sci. 2004 Mar;1012:299-305. Abstract

View all comments by Jane Karlsson

PubMed lists six peer-reviewed publications showing preclinical research in which methylene blue facilitates memory and one in which it prevents neurodegeneration by its combined action as a brain metabolic enhancer and antioxidant. Below is a list of these publications:

Wrubel KM, Riha PD, Maldonado MA, McCollum D, Gonzalez-Lima F. The brain metabolic enhancer methylene blue improves discrimination learning in rats. Pharmacol Biochem Behav. 2007 Apr;86(4):712-7. Epub 2007 Mar 6. Abstract

Wrubel KM, Barrett D, Shumake J, Johnson SE, Gonzalez-Lima F. Methylene blue facilitates the extinction of fear in an animal model of susceptibility to learned helplessness. Neurobiol Learn Mem. 2007 Feb;87(2):209-17. Epub 2006 Oct 2. Abstract

Zhang X, Rojas JC, Gonzalez-Lima F. Methylene blue prevents neurodegeneration caused by rotenone in the retina. Neurotox Res. 2006 Jan;9(1):47-57. Abstract

Riha PD, Bruchey AK, Echevarria DJ,...  Read more

PubMed lists six peer-reviewed publications showing preclinical research in which methylene blue facilitates memory and one in which it prevents neurodegeneration by its combined action as a brain metabolic enhancer and antioxidant. Below is a list of these publications:

Wrubel KM, Riha PD, Maldonado MA, McCollum D, Gonzalez-Lima F. The brain metabolic enhancer methylene blue improves discrimination learning in rats. Pharmacol Biochem Behav. 2007 Apr;86(4):712-7. Epub 2007 Mar 6. Abstract

Wrubel KM, Barrett D, Shumake J, Johnson SE, Gonzalez-Lima F. Methylene blue facilitates the extinction of fear in an animal model of susceptibility to learned helplessness. Neurobiol Learn Mem. 2007 Feb;87(2):209-17. Epub 2006 Oct 2. Abstract

Zhang X, Rojas JC, Gonzalez-Lima F. Methylene blue prevents neurodegeneration caused by rotenone in the retina. Neurotox Res. 2006 Jan;9(1):47-57. Abstract

Riha PD, Bruchey AK, Echevarria DJ, Gonzalez-Lima F. Memory facilitation by methylene blue: dose-dependent effect on behavior and brain oxygen consumption. Eur J Pharmacol. 2005 Mar 28;511(2-3):151-8. Abstract

Gonzalez-Lima F, Bruchey AK. Extinction memory improvement by the metabolic enhancer methylene blue. Learn Mem. 2004 Sep-Oct;11(5):633-40. Abstract

Callaway NL, Riha PD, Bruchey AK, Munshi Z, Gonzalez-Lima F. Methylene blue improves brain oxidative metabolism and memory retention in rats. Pharmacol Biochem Behav. 2004 Jan;77(1):175-81. Abstract

Callaway NL, Riha PD, Wrubel KM, McCollum D, Gonzalez-Lima F. Methylene blue restores spatial memory retention impaired by an inhibitor of cytochrome oxidase in rats. Neurosci Lett. 2002 Oct 31;332(2):83-6. Abstract

View all comments by Francisco Gonzalez-Lima

The timely report by Hickman, Allison, and El Khoury presents an interesting interpretation of the interplay between microglia and cerebral amyloidosis. It has long been established that Tg2576 mice manifest microglial activation concomitant with Abeta deposition, and that before plaques develop these animals have very little microgliosis (see for example Benzing et al., 1999). These authors have performed a related study in the APPPS1 mice developed by Joanna Jankowsky and David Borchelt (Jankowsky et al., 2001) and find a similar phenomenon.

They open their abstract by stating that “Early microglial accumulation in Alzheimer’s disease (AD) delays disease progression by promoting clearance of beta-amyloid (Abeta) before formation of senile plaques”. However, I'd like to note that this is a controversial statement, for which the authors do not present experimental evidence. Early ultrastructural studies from Henryk Wisniewski and Jerzy Wegiel actually suggested the opposite, that early microglial activation is a key factor in promoting progression of cerebral...  Read more

The timely report by Hickman, Allison, and El Khoury presents an interesting interpretation of the interplay between microglia and cerebral amyloidosis. It has long been established that Tg2576 mice manifest microglial activation concomitant with Abeta deposition, and that before plaques develop these animals have very little microgliosis (see for example Benzing et al., 1999). These authors have performed a related study in the APPPS1 mice developed by Joanna Jankowsky and David Borchelt (Jankowsky et al., 2001) and find a similar phenomenon.

They open their abstract by stating that “Early microglial accumulation in Alzheimer’s disease (AD) delays disease progression by promoting clearance of beta-amyloid (Abeta) before formation of senile plaques”. However, I'd like to note that this is a controversial statement, for which the authors do not present experimental evidence. Early ultrastructural studies from Henryk Wisniewski and Jerzy Wegiel actually suggested the opposite, that early microglial activation is a key factor in promoting progression of cerebral amyloidosis (Wisniewski and Wegiel, 1994). Further, observations in Tg2576 mice do not support an association between reactive microglia and diffuse beta-amyloid deposits (only between activated microglia and mature Abeta deposits, Benzing et al., 1999). This seems partially at odds with the authors’ contention.

Certainly, there are at least two interpretations for the observation of microglial activation occurring in tight temporal and spatial association with more mature beta-amyloid plaques. 1) That, as the authors contend, reactive microglia begin to clear non-deposited (i.e., soluble oligomeric) forms of Abeta and then become easily overwhelmed, or 2) that the microglia become activated in response to “seeds” (e.g., protofibrils or fibrils) of beta-amyloid plaques and then, via chronic, low-level production of pro-inflammatory cytokines and acute-phase reactants, contribute to plaque maturation. Probably the most direct support for the latter interpretation comes from the NSAID epidemiologic literature, where use of NSAIDs is associated with reduced microglial activation in humans (Mackenzie and Munoz, 1998) and as much as 50 percent reduced risk for AD (in t’Veld et al., 2001; Szekely et al., 2004). More recently at ICAD 2008, John Breitner showed impressively that naproxen given during the randomized controlled ADAPT trial for approximately 2 years duration and followed-up for another ~2 years results in reduced incidence of AD.

But the real “meat” of the work by Hickman et al. comes from their FACS approach as applied to single brain cell suspensions from APPPS1 mice of different ages. Using this approach, the authors show that CD11b+ (presumed microglial) cells from younger APPPS1 animals (from 1.5 to 3 months old, before obvious manifestation of beta-amyloid plaques) appear remarkably similar to age-matched wild-type controls when measuring mRNA for the microglial Abeta uptake receptors SRA, CD36, and RAGE. Similar results where observed on the Abeta-degrading enzymes Insulysin, Neprilysin, and MMP9.

However, a different pattern of results emerged when considering older (from 8 to 14 months) animals; in this case, APPPS1 mice had significant reductions in both the Abeta phagocytosis receptors and the Abeta degrading enzymes. Interestingly, these same older APPPS1 mice demonstrated up-regulation of mRNA for the pro-inflammatory cytokines IL-1beta and TNF-alpha, suggesting that these microglia are undergoing a phenotype “shift” from Abeta phagocytic, non-inflammatory to Abeta anti-phagocytic, pro-inflammatory.

We have suggested something similar when we defined microglial activation as a continuum of responses ranging from productive (pro-phagocytic and anti-inflammatory) to deleterious (anti-phagocytic and pro-inflammatory) (Town et al., 2005). We agree with the authors that, to ensure productive clearance of Abeta by phagocytes such as microglia, therapies should promote an anti-inflammatory, pro-phagocytic phenotype. As in-vitro proof-of-concept, the authors show that treatment of N9 microglia with TNF-alpha reduces expression of SRA and CD36 and opposes Abeta uptake by these cells. We have observed something very similar when blocking the pro-inflammatory CD40-CD40L interaction on microglia – we then see increased Abeta uptake and clearance by microglia and reduced pro-inflammatory antigen presenting cell function (Tan, Town et al., 1999; Townsend, Town et al., 2005).

One further issue that deserves mentioning is the origin of the CD11b+ cells that the authors have nicely characterized. It has now been clearly demonstrated that peripheral macrophages do enter brains of AD mice (Stalder et al., 2005; Simard et al., 2006; El Khoury et al., 2007), and most recently we have shown that boosting their brain entry by blocking TGF-betaRII signaling on these cells reduces AD-like pathology (Town et al., 2008). One wonders what percentage of the CD11b+ cells described by the authors are from the periphery. Nancy Ruddle has routinely used CD45int (brain-resident microglia) versus CD45hi (blood-borne macrophages) FACS staining to discriminate between the two populations (Juedes and Ruddle, 2001), and we have recently employed her protocol for this purpose (Town et al., 2008). The key question remains of whether these blood-borne macrophages are more efficient Abeta phagocytes than their long-term CNS-resident microglial cousins.

References:
Benzing WC, Wujek JR, Ward EK, Shaffer D, Ashe KH, Younkin SG, Brunden KR. Evidence for glial-mediated inflammation in aged APP(SW) transgenic mice. Neurobiol Aging. 1999 Nov-Dec;20(6):581-9. Abstract

El Khoury J, Toft M, Hickman SE, Means TK, Terada K, Geula C, Luster AD. Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease. Nat Med. 2007 Apr;13(4):432-8. Epub 2007 Mar 11. Abstract

in t' Veld BA, Ruitenberg A, Hofman A, Launer LJ, van Duijn CM, Stijnen T, Breteler MM, Stricker BH. Nonsteroidal antiinflammatory drugs and the risk of Alzheimer's disease. N Engl J Med. 2001 Nov 22;345(21):1515-21. Abstract

Jankowsky JL, Slunt HH, Ratovitski T, Jenkins NA, Copeland NG, Borchelt DR. Co-expression of multiple transgenes in mouse CNS: a comparison of strategies. Biomol Eng. 2001 Jun;17(6):157-65. Abstract

Juedes AE, Ruddle NH. Resident and infiltrating central nervous system APCs regulate the emergence and resolution of experimental autoimmune encephalomyelitis. J Immunol. 2001 Apr 15;166(8):5168-75. Abstract

Simard AR, Soulet D, Gowing G, Julien JP, Rivest S. Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron. 2006 Feb 16;49(4):489-502. Abstract

Stalder AK, Ermini F, Bondolfi L, Krenger W, Burbach GJ, Deller T, Coomaraswamy J, Staufenbiel M, Landmann R, Jucker M. Invasion of hematopoietic cells into the brain of amyloid precursor protein transgenic mice. J Neurosci. 2005 Nov 30;25(48):11125-32. Abstract

Szekely CA, Thorne JE, Zandi PP, Ek M, Messias E, Breitner JC, Goodman SN. Nonsteroidal anti-inflammatory drugs for the prevention of Alzheimer's disease: a systematic review. Neuroepidemiology. 2004 Jul-Aug;23(4):159-69. Abstract

Tan J, Town T, Paris D, Mori T, Suo Z, Crawford F, Mattson MP, Flavell RA, Mullan M. Microglial activation resulting from CD40-CD40L interaction after beta-amyloid stimulation. Science. 1999 Dec 17;286(5448):2352-5. Abstract

Town T, Nikolic V, Tan J. The microglial "activation" continuum: from innate to adaptive responses. J Neuroinflammation. 2005 Oct 31;2:24. Abstract

Town T, Laouar Y, Pittenger C, Mori T, Szekely CA, Tan J, Duman RS, Flavell RA. Blocking TGF-beta-Smad2/3 innate immune signaling mitigates Alzheimer-like pathology. Nat Med. 2008 Jun;14(6):681-7. Abstract

Townsend KP, Town T, Mori T, Lue LF, Shytle D, Sanberg PR, Morgan D, Fernandez F, Flavell RA, Tan J. CD40 signaling regulates innate and adaptive activation of microglia in response to amyloid beta-peptide. Eur J Immunol. 2005 Mar;35(3):901-10. Abstract

Wisniewski HM, Wegiel J. The role of microglia in amyloid fibril formation. Neuropathol Appl Neurobiol. 1994 Apr;20(2):192-4. Abstract

View all comments by Terrence Town

Is methylene blue, a rather old drug, finally on the way to becoming a cure? Speculation and criticism come by the dozen.

The blue urine may enhance placebo effects. Therefore it would be worthwhile to investigate human brain penetration before we start to speculate, and well before we inject or swallow it in larger numbers. Iodine-labeled methylene blue did not reach the brain within 14h, but the additional iodine may have interfered with brain penetration (Link et al.,1996). Therefore an 11C-labeled methylene blue would be far more appropriate. Strange enough: 11C-labeled methylene blue has been available at the University of Aberdeen since 2003 (Schweiger et al, 2003)!

So where are the data? Was the brain penetration of methylene blue disclosed at the ICAD?

References:
Link EM, Costa DC, Lui D, Ell PJ, Blower PJ, Spittle MF. Targeting disseminated melanoma with radiolabelled methylene blue: Comparative bio-distribution studies in man and animals. Acta Oncol. 1996;35(3):331-41. Abstract

Schweiger L, Craib S, Welch A, Sharp P. Radiosynthesis of [N-methyl-11C]methylene blue. Journal of Labelled Compounds and Radiopharmaceuticals, 2003 Nov;46,(13):1221-1228. Abstract

View all comments by Boris Schmidt

The demographic study of Krister Håkansson of Växjö University, Växjö, and Karolinska Institutet, Stockholm, Sweden, is very interesting. The effects of pair bonding and attachment have substantial effect on hormonal induction and neuroprotection.

Oxytocin is a hormone that has been studied to a great extent in the recent years, from both a psychological stance—learning about attachment—and the steroidal effects oxytocin has on the brain (Carter et al., 2005, and Carter, 2007).

Calza et al. (1997) showed that to a great extent corticotrophin-releasing hormone neurons of the paraventricular nucleus remain high in numbers in the elderly. However, the number of oxytocin neurons and vasopressin neurons decrease in the elderly. Thus, the induction of the hormone oxytocin with a rekindling of bonding and attachment is evident in the increase and maintenance of the number of oxytocin and vasopressin neurons, i.e., affecting both vascular circulation and neuroprotection from corticotrophin stress effects, which are excitotoxicity and cell death.

The involvement...  Read more

The demographic study of Krister Håkansson of Växjö University, Växjö, and Karolinska Institutet, Stockholm, Sweden, is very interesting. The effects of pair bonding and attachment have substantial effect on hormonal induction and neuroprotection.

Oxytocin is a hormone that has been studied to a great extent in the recent years, from both a psychological stance—learning about attachment—and the steroidal effects oxytocin has on the brain (Carter et al., 2005, and Carter, 2007).

Calza et al. (1997) showed that to a great extent corticotrophin-releasing hormone neurons of the paraventricular nucleus remain high in numbers in the elderly. However, the number of oxytocin neurons and vasopressin neurons decrease in the elderly. Thus, the induction of the hormone oxytocin with a rekindling of bonding and attachment is evident in the increase and maintenance of the number of oxytocin and vasopressin neurons, i.e., affecting both vascular circulation and neuroprotection from corticotrophin stress effects, which are excitotoxicity and cell death.

The involvement of the hypothalamic-pituitary-adrenal axis in stress is greater when there is no marriage support in the widowed or bachelors. Love and attachment increase oxytocin levels and thus reduce neurodegeneration (Czlonkowska et al., 2003) given a cascade of neurohumoral protection.

In an interdisciplinary conference, Carter (2007) delivered a talk about the effects of oxytocin, one of which was the "rewiring of the brain" after childbirth. Mother-child bonding and love affected the brain significantly. She also indicated that, generally, oxytocin reduces muscle mass. That seems to confound the mortality rate data; muscle mass is indicated in longevity and that was the finding with the Belgian Blue cow.

There seems to be a paradox in mortality rate that is related to the effects of oxytocin's neuroprotection (increased brain mass) and that which affects muscles (reduced mass) in Alzheimer's patients or the elderly in general. These are important for demographical research.

References:
Calzà L, Pozza M, Coraddu F, Farci G, Giardino L. Hormonal influences on brain ageing quality: focus on corticotropin releasing hormone-, vasopressin- and oxytocin-immunoreactive neurones in the human brain. J Neural Transm. 1997;104(10):1095-100. Abstract

Carter, C.S., Ahnert, L. Groosmann, K. E., Hardy, S. B., Lamb, M. E., Porges, S. W., et al. (Eds.).(2005). Attachment and bonding: A new synthesis. Cambridge, MA: MIT Press.

Carter CS. Sex differences in oxytocin and vasopressin: implications for autism spectrum disorders? Behav Brain Res. 2007 Jan 10;176(1):170-86. Abstract

Carter, C.A. (2007, March 30 - April 1). A Mother's Love. Presented at "Seven Dimensions of Emotion: Integrating Biological, Clinical, and Cultural Perspectives on Fear, Disgust, Empathy, Grief, Anger, Love and Hope." FPR-UCLA 3rd Interdisciplinary Conference. Korn Convovation Hall, UCLA.

Czlonkowska A, Ciesielska A, Joniec I. Influence of estrogens on neurodegenerative processes. Med Sci Monit. 2003 Oct;9(10):RA247-56. Abstract

View all comments by Kiumars Lalezarzadeh

This treatment should be combined with the cognishunt apparatus to clear the spinal fluid of the released plaque.

View all comments by john doe

This report states that we had pooled randomization arms post-hoc in our efficacy analyses, which was not true. All of our analyses respected the original randomization, and the study remained double blind through to the end, i.e., two years. The primary analysis was conducted as pre-specified, and achieved statistical significance at the 24-week and 50-week time points. The effect was about an 84 percent reduction in the observed rate of progression over one year, regardless of how the analysis was conducted and which of several imputation methods was used in the ITT analysis.

View all comments by Claude Wischik