NO Laughing Matter—Nitrosylation of Isomerase Spells Trouble for Neurons

If the number of diseases associated with protein aggregates is anything to go by, keeping proteins in their native state is not a simple task. One of the many molecules that have evolved to deal with the problem is an enzyme called protein disulphide isomerase (PDI). Ensconced in the endoplasmic reticulum (ER), PDI ensures that disulphide bonds in secretory proteins are properly cross-linked. Because disulphide bonds provide the only opportunity for protein side chains to covalently interact with each other, PDI performs a uniquely important task that raises an equally important question: Is PDI activity ever compromised, and what happens if it is?

In last Thursday’s Nature, Stuart Lipton at the Burnham Institute for Medical Research in La Jolla, California; Eliezer Masliah at the University of California, San Diego; Yasuyuki Nomura at Hokkaido University, Sapporo, Japan; and their colleagues report that inactive forms of PDI are present in brain samples taken from Alzheimer and Parkinson disease patients. More specifically, first author Takashi Uehara and coworkers have found that thiol groups at the active site of PDI are chemically modified by addition of a nitric oxide (NO) group. Because the authors also found that this modification both inactivates PDI and exacerbates accumulation of ubiquitinated proteins in cells, the findings hint that inactivation of the isomerase may compound the pathology of neurodegenerative diseases.

Using in vitro reactions, Uehara and colleagues first found that PDI could be inactivated by NO donors such as S-nitrosocysteine (SNOC). The isomerase becomes S-nitrosylated at any of four cysteine thiols present in two active site domains that lie at the N- and C-terminals of the protein. To find out if this modification has any physiological significance, the authors looked for S-nitrosylated PDI (SNO-P) in dopaminergic SH-SY5Y cells that had been treated with rotenone—this mitochondrial inhibitor, which induces a Parkinson disease-like pathology in animals, also leads to increases in NO. Finding that SNO-P does indeed form in the rotenone-treated cells, the authors then tested the human brain samples.

Of course, rotenone is not likely to be the cause of SNO-P in the human brain, but there are plenty of other factors that might increase NO production, one of them being excessive stimulation of N-methyl-D-aspartate (NMDA) receptors. When the researchers exposed primary cortical neurons to NMDA, they detected SNO-P, polyubiquitinated proteins, and signs of an activated unfolded protein response (UPR)—up-regulation of UPR proteins CHOP and XBP-1 (see ARF related news story). These events could all be prevented by overexpressing active PDI or treating the cells with NO blockers. The authors also found that wild-type, but not an isomerase-negative PDI, could attenuate Lewy body-like inclusions when synphilin was overexpressed in SH-SY5Y cells. And they found that this protection from synphilin aggregation was abolished by NO or SNOC.

All told, these findings suggest that PDI may be more than a mere housekeeping protein. It may also help protect cells from various forms of stress. In fact, to see just how versatile PDI might be, Uehara and colleagues overexpressed the isomerase in SH-SY5Y cells that were treated with either the ER toxin thapsigargin or the proteasome inhibitor MG132. In both cases the isomerase reduced the number of cells that underwent apoptosis by about half.

“Our data demonstrate a previously unrecognized relationship between NO and protein misfolding in degenerative disorders, showing that PDI can be a target of NO after mitochondrial insult in cellular models of PD and in human neurodegenerative diseases,” write the authors. The data also suggest yet another way that NMDA receptors, which have been linked to calcium toxicity, can contribute to cell death.—Tom Fagan

Comments

  1. Amyloid-β peptides induce DNA fragmentation: an alternative pathway yet to be understood in Alzheimer disease pathology
    The paper (Prestwich et al., 2005) has fascinated us and made us believe that we are in the right direction toward exploring an alternative pathway for the pathogenesis of the Alzheimer disease. Our earlier finding of amyloid-β peptides binding to and inducing conformational change in DNA (Hegde et al., 2004) led us to study the effect of amyloid-β peptides on DNA integrity. That other amyloidogenic peptides, such as α-synuclein and prion, also were found to bind to DNA (Veer Bala Gupta et al., 2006), made us argue that there is a common mechanism of action of these peptides at work in neurodegeneration. In this perspective, we highlighted an interesting mechanism of different molecular forms (monomer-oligomer aggregates) of amyloid-β and α-synuclein binding to DNA and inducing DNA damage (Hegde et al., 2004, Abstract). It also gives us insight into understanding the different events taking place at different stages of the disease in view of the changing conformation of these peptides in brain.

    References:

    Prestwich EG, Roy MD, Rego J, Kelley SO. Oxidative DNA strand scission induced by peptides. Chem Biol. 2005 Jun;12(6):695-701. PubMed.

    Hegde ML, Anitha S, Latha KS, Mustak MS, Stein R, Ravid R, Rao KS. First evidence for helical transitions in supercoiled DNA by amyloid Beta Peptide (1-42) and aluminum: a new insight in understanding Alzheimer's disease. J Mol Neurosci. 2004;22(1-2):19-31. PubMed.

    Gupta VB, Hegde ML, Rao KS. Role of protein conformational dynamics and DNA integrity in relevance to neuronal cell death in neurodegeneration. Curr Alzheimer Res. 2006 Sep;3(4):297-309. PubMed.

    Hegde ML, Anitha S, Jagannatha KS. Are Monomer-Oligomer Aggregates of Amyloidogenic Peptides Toxic Species in Neurodegeneration. Neurobiol Aging 2004; 25(Suppl 2):S170.

    View all comments by K.S. Jagannatha Rao

  2. I partially agree with the suggestion that villagers in India who consume mustard oil didn't have AD in old age, also because it was found in current research that turmeric and mustard oil users are less susceptible to AD, but I disagree with his query that we can't relate AD with age as we have certain data for its proof. Age is the most important known risk factor for AD. The number of people with the disease doubles every 5 years beyond age 65. Middle-aged women are at greater risk than men (AD). A recent study shows that high blood pressure dramatically increases this risk, foretelling a potential epidemic of dementia as baby boomers enter their later years. Research at Boston University School of Medicine tracked 4,883 people under evaluation for the Framingham Heart Study. Forty years' worth of data revealed that one in four suffers from AD. Men of the same age are slightly less susceptible, having a one in six chance of AD. Combined, these risk factors threaten one out of every two older women and one in three of their male peers.

    I agree with Mr. Ranganath Rao that trace metals increase risk of AD, as we have with us certain data for lead-related risk of AD. Exposure to lead increases the risk for developing AD, according to a study done at Case Western University Medical School's department of neurology. Individuals in work environments with high levels of lead are three to four times as likely to have Alzheimer's as their unexposed peers later in life. The study offers the first conclusive evidence of a link between on-the-job hazards and AD. Scientists at Case Western examined the work histories of Alzheimer patients and compared them to the histories of healthy elderly people. Dr. Elisabeth Koss and her colleagues discovered that individuals with the highest levels of lead exposure were up to four times as likely to have Alzheimer's as those who had minimal work-related exposure. The research team reported their findings at the 52nd Annual Meeting of the American Academy of Neurology. Even after taking into account a number of other factors that can influence the development of the disease, results showed that 14 percent of Alzheimer patients had experienced lead exposure at work, as compared to just 6 percent of individuals without the disorder. However, no association was made between exposure to other common workplace toxins—such as aluminum, copper, iron, zinc, and solvents—and an increased risk for developing the disease. Typically, lead exposure occurs either by inhaling lead dust (the most toxic mode of transmission) or by absorbing lead through the skin. Jobs that involve smelting and casting lead, working with lead-based paints or inks, making stained glass, or manufacturing products including batteries, lead-glazed pottery, ammunition, lead pipes, and electronics parts place workers at serious risk for high lead exposure. At-home risks also exist where lead is contained in drinking water and soil, or in older homes that have peeling lead-based paint.

    See also:

    D. F. Swabb and E. Fliers, Brain Res. 1985;140:566.

    References:

    Ito T, Yamadera H, Ito R, Endo S. [Effects of bright light on cognitive disturbances in Alzheimer-type dementia]. Nihon Ika Daigaku Zasshi. 1999 Aug;66(4):229-38. PubMed.

    Brusco LI, Márquez M, Cardinali DP. Monozygotic twins with Alzheimer's disease treated with melatonin: Case report. J Pineal Res. 1998 Dec;25(4):260-3. PubMed.

    Hughes CP, Berg L, Danziger WL, Coben LA, Martin RL. A new clinical scale for the staging of dementia. Br J Psychiatry. 1982 Jun;140:566-72. PubMed.

    Rosen WG, Mohs RC, Davis KL. A new rating scale for Alzheimer's disease. Am J Psychiatry. 1984 Nov;141(11):1356-64. PubMed.

    Volicer L, Harper DG, Manning BC, Goldstein R, Satlin A. Sundowning and circadian rhythms in Alzheimer's disease. Am J Psychiatry. 2001 May;158(5):704-11. PubMed.

    Bliwise DL, Hughes M, McMahon PM, Kutner N. Observed sleep/wakefulness and severity of dementia in an Alzheimer's disease special care unit. J Gerontol A Biol Sci Med Sci. 1995 Nov;50(6):M303-6. PubMed.

    View all comments by Sumit Gupta

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References

News Citations

  1. World Alzheimer Conference 2000: Presenilin Roundup

Further Reading

Primary Papers

  1. Uehara T, Nakamura T, Yao D, Shi ZQ, Gu Z, Ma Y, Masliah E, Nomura Y, Lipton SA. S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration. Nature. 2006 May 25;441(7092):513-7. PubMed.

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