The interesting paper by Bart De Strooper and colleagues (1) points to nitrosative modification of the γ-secretase complex in aged neuronal cultures with consequent elevated Aβ(1-42) formation. The authors opine that aging, the major risk factor for Alzheimer's disease (AD), with consequent mitochondrial dysfunction (especially that of manganese superoxide dismutase), couples oxidative and nitrosative stress and AD via this nitrosative modification of the γ-secretase complex. The research is well done and the conclusions certainly seem to be supported by the data.

However, a few comments appear to be in order regarding this interesting paper.

1. Protein nitration is a formal oxidation, consistent with the well-known protein oxidation in brains of subjects with AD and amnestic mild cognitive impairment (MCI) (2,3).

2. This work involves neuronal cultures. In AD brain, of course, many other cell types are involved and may significantly contribute to the nitrosative modification of γ-secretase in vivo. For example, microglia, when activated, secrete inducible nitric...  Read more

The interesting paper by Bart De Strooper and colleagues (1) points to nitrosative modification of the γ-secretase complex in aged neuronal cultures with consequent elevated Aβ(1-42) formation. The authors opine that aging, the major risk factor for Alzheimer's disease (AD), with consequent mitochondrial dysfunction (especially that of manganese superoxide dismutase), couples oxidative and nitrosative stress and AD via this nitrosative modification of the γ-secretase complex. The research is well done and the conclusions certainly seem to be supported by the data.

However, a few comments appear to be in order regarding this interesting paper.

1. Protein nitration is a formal oxidation, consistent with the well-known protein oxidation in brains of subjects with AD and amnestic mild cognitive impairment (MCI) (2,3).

2. This work involves neuronal cultures. In AD brain, of course, many other cell types are involved and may significantly contribute to the nitrosative modification of γ-secretase in vivo. For example, microglia, when activated, secrete inducible nitric oxide synthase, which catalyzes formation of nitric oxide (NO), and, in the presence of superoxide radical (much of which emanates from mitochondria), peroxynitrite is formed by radical-radical recombination. In the presence of carbon dioxide, peroxynitrite undergoes a series of reactions that result in nitration of protein-resident tyrosine residues, especially in an oxidative environment such as the AD brain (2,3). Tyrosine nitration is highly detrimental to neurons, for example, blocking receptor tyrosine kinase phosphorylation and subsequent cellular signaling events, due to the steric hindrance associated with the NO2 functionality in the 3-position of tyrosine.

3. Our laboratory showed that, in the brains of subjects with both AD and amnestic mild cognitive impairment (obtained with very short postmortem intervals), 3-nitrotyrosine was elevated (3,4), and we were the first to use redox proteomics to identify excessively nitrated brain proteins in both disorders (5-7). Accordingly, the interesting results shown in the De Strooper paper are a nice follow-up of already well-established findings in both disorders. Interestingly, that amnestic MCI brain demonstrates elevated 3-nitrotyrosine is consistent with the notion that this oxidative modification occurs early in the progression of AD (2-7).

4. Mitochondria are known to be altered in AD and MCI (8), and MnSOD is an oxidatively modified protein in this dementing disorder (9) and nitrated in the APP/PS-1 human double mutant knock-in mouse model of AD (10).

In summary, De Strooper and colleagues make a nice contribution to the literature, enhancing already well-established concepts.

References:
1. F.X. Guix, T. Wahle, K. Vennekens, S.A.L. Chavez-Gutierrez, G.Ill-Raga, E. Ramos, C. Guardia-Laguarta, A. Lleó, M. Arimon, O. Berezovska, F.J. Muñoz, C.G. Dotti, and B.De Strooper (2012). Modification of -Secretase by Nitrosative Stress Links Neuronal Aging to Sporadic Alzheimer’s Disease. EMBO Mol Med. Abstract

2. D.A. Butterfield, J. Drake, C. Pocernich and A. Castegna (2001) Evidence of Oxidative Damage in Alzheimer's Disease Brain: Central Role of Amyloid β-Peptide. Trends Molec Med 7, 548-554. Abstract

3. D.A. Butterfield, T. Reed, S. F. Newman and R. Sultana (2007). Roles of Amyloid β-Peptide-Associated Oxidative Stress and Brain Protein Modifications in the Pathogenesis of Alzheimer’s Disease and Mild Cognitive Impairment. Free Radic Biol Med 43, 658-677. Abstract

4. D.A. Butterfield, T. Reed, M. Perluigi, Carlo De Marco, Raffaella Coccia, J.N. Keller, W.R. Markesbery and R. Sultana (2007) Elevated Levels of 3-Nitrotyrosine in Brain from Subjects with Amnestic Mild Cognitive Impairment: Implications for the Role of Nitration in the Progression of Alzheimer’s Disease. Brain Res 1148, 243-248. Abstract

5. A. Castegna, V. Thongboonkerd, J.B. Klein, B. Lynn, W.R. Markesbery and D.A. Butterfield (2003) Proteomic Identification of Nitrated Proteins in Alzheimer's Disease Brain. J Neurochem 85, 1394-1401. Abstract

6. R. Sultana, D. Boyd-Kimball, H.F. Poon, J. Cai, W.M. Pierce, J.B. Klein, W.R. Markesbery and D.A. Butterfield (2006) Identification of Nitrated Proteins in Alzheimer's Disease Brain Using a Redox Proteomics Approach. Neurobiol Dis 22, 76-87. Abstract

7. R. Sultana, T. Reed, M. Perluigi, R. Coccia, W.M. Pierce and D.A. Butterfield (2007) Proteomic Identification of Nitrated Brain Proteins in Amnestic Mild Cognitive Impairment: A Regional Study. J Cell Molec Med 11, 839-851. Abstract

8. R. Sultana and D.A. Butterfield (2009) Oxidatively Modified, Mitochondria-Relevant Brain Proteins in Subjects with Alzheimer Disease and Mild Cognitive Impairment. J. Bioenerget Biomembr 41, 441-446. Abstract

9. D.A. Butterfield, M. Perluigi, T. Reed, T. Muharib, C. Hughes, R. Robinson and R. Sultana (2012) Redox Proteomics in Selected Neurodegenerative Disorders. From its Infancy to Future Applications. Antioxid Redox Signal. 2012 Jan 18. Abstract

10. M. Anantharaman, J. Tangpong, J.N. Keller, M.P. Murphy, W.R. Markesbery, K.K. Kiningham, and D.K. St Clair (2006) Beta-Amyloid Mediated Nitration of Manganese Superoxide Dismutase: Implications for Oxidative Stress in a APPNHL/NHL X PS-1P264L/P264L Double Knock-In Mouse Model of Alzheimer’s Disease. Am J Pathol 168, 1608-1618. Abstract

View all comments by Allan Butterfield

There appear to be several links of nitrosative stress (related to nitric oxide) and AD, and one exciting pathway is described here in this new and elegant work from Bart De Strooper's laboratory concerning nitration of γ-secretase. In this case, peroxynitrite (formed by reaction of nitric oxide [NO] and superoxide anion) nitrates a tyrosine residue on PS1.

Other types of nitrosative stress in AD lead to S-nitrosylation (reaction of NO with a critical cysteine thiol on a protein), as our group reported for Drp1 in Science in 2009 (see Cho et al., 2009). Additional such reactions are emerging in AD and related diseases, and will be published soon.

Please note that the chemistry of the events occurring here is important, but often confused in the literature: The new findings of De Strooper and colleagues report nitration of a tyrosine residue on PS1. In contrast, S-nitrosation (or what Jonathan Stamler and I have termed S-nitrosylation because of its regulation of protein function being akin to phosphorylation) involves reaction...  Read more

There appear to be several links of nitrosative stress (related to nitric oxide) and AD, and one exciting pathway is described here in this new and elegant work from Bart De Strooper's laboratory concerning nitration of γ-secretase. In this case, peroxynitrite (formed by reaction of nitric oxide [NO] and superoxide anion) nitrates a tyrosine residue on PS1.

Other types of nitrosative stress in AD lead to S-nitrosylation (reaction of NO with a critical cysteine thiol on a protein), as our group reported for Drp1 in Science in 2009 (see Cho et al., 2009). Additional such reactions are emerging in AD and related diseases, and will be published soon.

Please note that the chemistry of the events occurring here is important, but often confused in the literature: The new findings of De Strooper and colleagues report nitration of a tyrosine residue on PS1. In contrast, S-nitrosation (or what Jonathan Stamler and I have termed S-nitrosylation because of its regulation of protein function being akin to phosphorylation) involves reaction of NO with a cysteine residue (probably involving the thiolate anion of the cysteine residue).

View all comments by Stuart A. Lipton

This is a very interesting paper, carefully studying levels of secreted Aβ peptides with time in culture in wild-type neurons and noting a marked elevation of Aβ42 secretion between 21 and 28 days in vitro (DIV). The authors go on to define a mechanistic reason for such an increase in Aβ secretion and elevated 42/40 ratio by showing higher levels of the γ-secretase complex and nitrosylation of presenilin fragments concomitant with elevated nitrosative stress between 21 and 28 DIV. They demonstrate experimentally that PS1 nitrotyrosination elevates the 42/40 ratio, and show that levels of nitrosylated PS1-N-terminal fragments are elevated in AD brain. Here, it would have been interesting to also compare young versus old healthy control brains. Remarkably, using a reporter of PS1 conformation (GFP-PS1-RFP construct), they show that nitrosylated PS1 attains a conformation that resembles the conformational change seen with PS1 FAD mutations. They also link falling superoxide dismutase 2 (SOD2) levels to the increases in nitrosylation in aging cultured neurons and provide additional...  Read more

This is a very interesting paper, carefully studying levels of secreted Aβ peptides with time in culture in wild-type neurons and noting a marked elevation of Aβ42 secretion between 21 and 28 days in vitro (DIV). The authors go on to define a mechanistic reason for such an increase in Aβ secretion and elevated 42/40 ratio by showing higher levels of the γ-secretase complex and nitrosylation of presenilin fragments concomitant with elevated nitrosative stress between 21 and 28 DIV. They demonstrate experimentally that PS1 nitrotyrosination elevates the 42/40 ratio, and show that levels of nitrosylated PS1-N-terminal fragments are elevated in AD brain. Here, it would have been interesting to also compare young versus old healthy control brains. Remarkably, using a reporter of PS1 conformation (GFP-PS1-RFP construct), they show that nitrosylated PS1 attains a conformation that resembles the conformational change seen with PS1 FAD mutations. They also link falling superoxide dismutase 2 (SOD2) levels to the increases in nitrosylation in aging cultured neurons and provide additional supportive data using SOD2 knockout neurons and SOD2+/- mice.

This excellent and comprehensive paper brings together leading experts in AD and γ-secretase with colleagues that have pioneered cultured neurons as a model for aging. How aging acts as the most important risk factor for the development of AD remains a critical, unanswered question. Studies by numerous groups have supported the role of oxidative stress (OS) in the age-related risk of AD, and a few studies, including one of ours, have shown that OS modulates Aβ. Cell culture models have major advantages when trying to elucidate underlying biological mechanisms, and are much needed as complementary models to work on living animals and humans. When we began using neurons with time in culture as a model for AD (Takahashi et al., 2004) we were worried about using embryonic neurons to model an age-related disease. Over the years, we have been amazed by how much selective AD-like synapse and β amyloid alterations are reflected in "aging" AD transgenic neurons in culture. One can keep in mind that AD transgenic mice can develop behavioral dysfunction and Aβ pathology in a matter of months, which is also a very different time frame from the decades for the human disease.

In summary, elucidating how aging can induce AD is of major importance, and that oxidative stress and aging can directly impact γ-secretase and the 42/40 ratio is therefore highly interesting. Not atypical for the field, intraneuronal Aβ receives no mention. The question is not whether, but when, this will change. The relationship between the extra- and intracellular pools of Aβ is turning out to be quite complex. I hope the authors consider comparing their results in aging wild-type neurons to FAD mutant neurons. Surprisingly, aging AD-prone neurons eventually cannot cope with or fail to secrete their elevated Aβ (Tampellini et al., 2011).

View all comments by Gunnar K. Gouras