The epidemiologic link between elevated blood homocysteine (HC) and Alzheimer disease (see ARF related news story) has spurred the search for the mechanisms by which this ubiquitous amino acid might damage neurons. HC has been directly linked to AD by experiments showing it sensitizes neurons to the toxic effects of soluble amyloidβ (Aβ) peptides (see, e.g., Ho et al., 2001; Kruman et al., 2002; and Alzforum live discussion). Now, to add insult to injury, new results suggest that a metabolite of HC, homocysteic acid (HA), can act via γ-secretase to raise levels of intracellular Aβ peptides.

In a paper published May 16 in the Journal of Neuroscience Research online, Tohru Hasegawa in Saga, Japan, and collaborators show that high concentrations of HA increase intracellular Aβ1-42, but not Aβ1-40, in cultured cortical neurons and in APP-expressing CHO cells. The researchers also report that a γ-secretase inhibitor can protect neurons from the toxic effects of HA, suggesting a role for Aβ production in the neurotoxicity of HA. The physiologic significance of the finding, however, remains to be seen, as the increases in Aβ are elicited at concentrations that exceed the levels of HA the researchers could measure in plasma or CSF.

For the study, Hasegawa treated cultured cortical neurons with homocysteine and homocysteic acid, and measured Aβ1-42 and A β1-40 in the culture medium or in cell lysates. In cells treated with 1 microM or more HA, they detected an increase in Aβ42, but not Aβ40, associated with cells. In CHO cells expressing human APP (Swedish mutation), intracellular Aβ1-42 was elevated after exposure to 10 microM HA. Cortical neuron cultures treated with 1 microM HA lost about half the neurons within 48 hours, but the cells could be completely protected by pretreatment with the γ-secretase inhibitor LY-411,575, suggesting a role for Aβ production in neuronal toxicity of HA.

When the researchers measured the concentration of HA in normal individuals and AD patients, they found both groups had CSF levels in the 100 nM range. Plasma levels were undetectable. They did see the expected difference in both plasma and CSF levels of HC, where AD patients had CSF levels of HC around 700 nM, nearly twice as high as unaffected individuals.

Despite the widely different concentrations of HA present in vivo and in vitro, the results are intriguing. HA is produced from HC in the brain, and can promote calcium influx via NMDA receptors, a signaling pathway recently shown to increase Aβ production (Pierrot, 2004). Increased oxidative stress could also increase Aβ production. Compared to the cortical cultures studied here, HA has been shown to be more potently toxic for hippocampal neurons (Lockhart, 2000). If the results hold up, they may suggest a “potential therapeutic benefit of agents that modify the production and neurotoxic actions of HA and homocysteine,” the authors conclude.—Pat McCaffrey.

Reference:
Hasegawa T, Ukai W, Jo DG, Xu X, Mattson MP, Nakagawa M, Araki W, Saito T, Yamada T. Homocysteic acid induces intraneuronal accumulation of neurotoxic Aβ42: Implications for the pathogenesis of Alzheimer's disease. J Neurosci Res. 2005 May 16;80(6):869-876 [Epub ahead of print] Abstract

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  1. Homocysteine and AD: More Than Meets the Eye
    Hyoung-gon Lee, Mark A. Smith, Barney Dwyer, Aki Nunomura, George Perry, Xiongwei Zhu Increased levels of plasma homocysteine (HC), a key metabolic intermediate in sulfur amino acid metabolism, have been associated with several disorders including Alzheimer disease (AD). While HC is toxic in cell culture models including primary cortical neurons, the mechanism of HC toxicity and the role of HC in disease pathogenesis remain unclear. Hasegawa and colleagues hypothesized that homocysteic acid (HA), an oxidant product of HC, might play an important role in the pathogenesis of AD by regulating amyloid-β (Aβ) production. They demonstrate that HA dramatically decreases the extracellular level of Aβ42 but increases the intracellular level of Aβ42 in primary cortical neurons and APP-overexpressing CHO cells, and they suggest that this is associated with HA toxicity. This finding led them to show that a γ-secretase inhibitor prevents HA toxicity. While the level of HC is increased both in plasma and CSF in AD, there is no change in HA levels. However, interestingly, increasing HC can effectively enhance HA toxicity manyfold, presumably also via the regulation of Aβ.

    These interesting findings shed some new light on how increased levels of HC may contribute to the pathogenesis of AD—increased HC enhances HA toxicity, which results in intraneuronal accumulation of Aβ42 and subsequent neuronal death. However, such a conclusion warrants tempering. Indeed, it is ambiguous in this study whether intracellular Aβ42 actually leads to neuronal death, since there was a threefold increase of intracellular Aβ42 in ten μM HA-treated cells, yet no apparent neuronal death at this concentration. Nevertheless, it raises the question of how HA regulates Aβ42. As suggested by the authors, Aβ upregulation might involve oxidative stress, and this is consistent with in vivo and in vitro studies showing that oxidative stress precedes Aβ accumulation (Nunomura et al., 2001; Lee et al., 2004). Thus, it will be of interest to directly determine whether HA affects Aβ accumulation by oxidative stress and also the sequence of events regarding the elevation of HC and Aβ accumulation in AD patients. Recently we proposed that elevated HC may participate in a vicious cycle involving iron dysregulation, resulting in oxidative stress and other early events seen in AD (Dwyer et al., 2004). The increased vulnerability against HA by the elevated level of HC may also contribute to such a vicious cycle and enhance oxidative damage. This article provides a solid foundation for such studies.

    References:

    . Homocysteine and Alzheimer's disease: a modifiable risk?. Free Radic Biol Med. 2004 Jun 1;36(11):1471-5. PubMed.

    . Challenging the amyloid cascade hypothesis: senile plaques and amyloid-beta as protective adaptations to Alzheimer disease. Ann N Y Acad Sci. 2004 Jun;1019:1-4. PubMed.

    . Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol. 2001 Aug;60(8):759-67. PubMed.

  2. Homocysteine, oxidative stress, and AD: An even more vicious cycle!
    Commenting on Hasegawa et al., Dwyer and collaborators note that elevated homocysteine may participate in a vicious cycle involving iron dysregulation, resulting in oxidative stress seen in AD (Dwyer et al., 2004). Their proposed mechanism suggests that localized heme deficiency in AD brain could result in loss of cystathionine β-synthase redox responsiveness and incur increased homocysteine during periods of oxidative stress.

    It is also important to note that the other major route of homocysteine metabolism, the methionine synthase reaction, is also exquisitely sensitive to oxidative stress. We have proposed a complementary mechanism whereby such stress impairs methionine synthase activity (McCaddon et al 2002; McCaddon and Kelly, 1992, and see Alzheimer Research Forum "A cobalaminergic hypothesis.")

    Taken together, these two mechanisms suggest that it might be important to address oxidative stress as well as B vitamin deficiency in cognitively impaired patients presenting with hyperhomocysteinaemia. We have observed promising clinical effects using such an approach in several recent cases [McCaddon A, Davies G. Int.J.Ger.Psych. 2005 (in press)]. A randomized controlled clinical trial is now underway to formally evaluate this novel synergistic approach in these patients.

    References:

    . Homocysteine and Alzheimer's disease: a modifiable risk?. Free Radic Biol Med. 2004 Jun 1;36(11):1471-5. PubMed.

    . Functional vitamin B(12) deficiency and Alzheimer disease. Neurology. 2002 May 14;58(9):1395-9. PubMed.

    . Alzheimer's disease: a 'cobalaminergic' hypothesis. Med Hypotheses. 1992 Mar;37(3):161-5. PubMed.

    . Co-administration of N-acetylcysteine, vitamin B12 and folate in cognitively impaired hyperhomocysteinaemic patients. Int J Geriatr Psychiatry. 2005 Oct;20(10):998-1000. PubMed.

  3. I look at the results reported bearing in mind that homocysteine is one of the products of S-adenosylmethionine metabolism. It has been recently reported by my group (Scarpa et al., 2003 and Fuso et al., 2005) that both PS1 and BACE are regulated by DNA methylation and that accumulation of homocysteine, obtained by starvation of B12 and folate in the culture medium, increased amyloid production. As far as amyloid release (Fig. 1) and the ratio between intracellular and extracellular concentrations of the two Aβ species, my comment is that HA administration, by changing the methylation status of membrane lipids, among several other events, could decrease the fluidity of the membranes and therefore the secretion. Consequently, amyloid accumulates inside the cells (Fig. 3A and 4).

    I think it is important to look carefully at the main metabolism in which homocysteine is involved. The main product in the pathway is S-adenosylmethionine, the donor of all the methylation reactions. The accumulation of homocysteine, either pathologic or administered (please note that Frauscher, in the paper cited by the authors, used the thiolactone that produces both HC and HA), reverses the reaction controlled by S-adenosylhomocysteine-hydrolase, blocking the methylation reaction cycle and therefore inducing a generalized hypomethylation.

    References:

    . Presenilin 1 gene silencing by S-adenosylmethionine: a treatment for Alzheimer disease?. FEBS Lett. 2003 Apr 24;541(1-3):145-8. PubMed.

    . S-adenosylmethionine/homocysteine cycle alterations modify DNA methylation status with consequent deregulation of PS1 and BACE and beta-amyloid production. Mol Cell Neurosci. 2005 Jan;28(1):195-204. PubMed.

  4. Zou et al. (1) report that γ-secretase is involved in the processing of megalin. In view of the fact that megalin binds cubilin, the receptor for B12-intrinsic factor complex, and mediates uptake of the vitamin B12-transcobalamin complex (2), what are the implications for AD?

    References:

    . Linking receptor-mediated endocytosis and cell signaling: evidence for regulated intramembrane proteolysis of megalin in proximal tubule. J Biol Chem. 2004 Aug 13;279(33):34302-10. PubMed.

    . Receptors of the low density lipoprotein (LDL) receptor family in man. Multiple functions of the large family members via interaction with complex ligands. Biol Chem. 1998 Aug-Sep;379(8-9):951-64. PubMed.

  5. Does homocysteic acid also induce expression of HERP ( Homocysteine- and endoplasmic reticulum stress-inducible protein, ubiquitin-like domain-containing, 1)? Sai et al. (1) report that HERP increases the generation of amyloid beta-protein (Abeta) and that Herp interacts with presenilin (PS).

    Herp is an endoplasmic reticulum (ER)-stress-inducible membrane protein, which has a ubiquitin-like domain (ULD). However, its biological function is as yet unknown. Previously, we reported that a high expression level of Herp in cells increases the generation of amyloid beta-protein (Abeta) and that Herp interacts with presenilin (PS). Here, we addressed the role of the ULD of Herp in Abeta generation and intracellular Herp stability. We found that the ULD is not essential for the enhancement of Abeta generation by Herp expression and the interaction of Herp with PS, but is involved in the rapid degradation of Herp, most likely via the ubiquitin/proteasome pathway. Thus, the ULD of Herp most likely plays a role in the regulation of the intracellular level of Herp under ER stress. 

    References:

    . The ubiquitin-like domain of Herp is involved in Herp degradation, but not necessary for its enhancement of amyloid beta-protein generation. FEBS Lett. 2003 Oct 9;553(1-2):151-6. PubMed.

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Paper Citations

  1. . Homocysteine potentiates beta-amyloid neurotoxicity: role of oxidative stress. J Neurochem. 2001 Jul;78(2):249-53. PubMed.
  2. . Folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize them to amyloid toxicity in experimental models of Alzheimer's disease. J Neurosci. 2002 Mar 1;22(5):1752-62. PubMed.
  3. . Intraneuronal amyloid-beta1-42 production triggered by sustained increase of cytosolic calcium concentration induces neuronal death. J Neurochem. 2004 Mar;88(5):1140-50. PubMed.
  4. . Inhibition of L-homocysteic acid and buthionine sulphoximine-mediated neurotoxicity in rat embryonic neuronal cultures with alpha-lipoic acid enantiomers. Brain Res. 2000 Feb 14;855(2):292-7. PubMed.
  5. . Homocysteic acid induces intraneuronal accumulation of neurotoxic Abeta42: implications for the pathogenesis of Alzheimer's disease. J Neurosci Res. 2005 Jun 15;80(6):869-76. PubMed.

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  1. . Homocysteic acid induces intraneuronal accumulation of neurotoxic Abeta42: implications for the pathogenesis of Alzheimer's disease. J Neurosci Res. 2005 Jun 15;80(6):869-76. PubMed.

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  1. . Homocysteic acid induces intraneuronal accumulation of neurotoxic Abeta42: implications for the pathogenesis of Alzheimer's disease. J Neurosci Res. 2005 Jun 15;80(6):869-76. PubMed.