A new mouse model has achieved a long-sought pathological prize—the production of hyperphosphorylated and aggregated tau proteins in a background of elevated Aβ. The trick to linking Aβ to tau’s tangles, it turns out, is to get rid of nitric oxide (NO). In a paper published August 14 in PNAS online, Hana Dawson and colleagues from Duke University, Durham, North Carolina, report that knocking out the gene for inducible nitric oxide synthase (NOS2) in an APP transgenic mouse leads to the hyperphosphorylation and aggregation of endogenous mouse tau protein and neuronal death.

The results, showing that the decrease in NOS exacerbates Aβ production and generates pathological tau species, run contrary to previous work finding NOS knockouts are protected against amyloid pathology (Nathan et al., 2005). In general, NO has been considered harmful to neurons (see ARF related news story and ARF news story), but the new results suggest that under some circumstances, NO may actually protect them.

The new mouse, generated by first author Carol Colton and coworkers, resulted from crossing the APPsw transgenic line Tg2576 with a NOS2 knockout. The progeny lacked the iNOS protein and displayed total NOS activity of about one-third that of APPsw mice. Hyperphosphorylated tau appeared in the soma and dendrites of cortical and hippocampal neurons in the APPsw/NOS-/- animals—phospho-tau was not seen in the APPsw or NOS-/- mice. Aggregated tau was detected by immunostaining or electron microscopy after filtration of brain lysates, and in intact tissue using thioflavin F staining.

Knockout of NOS2 also enhanced amyloid pathology in the mice. Total brain Aβ levels were five to six times higher in APPsw/NOS2-/- compared to APPsw littermates. The increase was mostly due to insoluble Aβ peptides. These results suggest that NO might influence Aβ generation or clearance by an unknown mechanism.

Finally, the mice showed neuron loss, which is not normally seen in APPsw mice. In three of four APPsw/NOS2-/- animals, cortical neurodegeneration was apparent after staining with Fluoro-Jade C, an anionic fluorescein derivative that specifically stains degenerating neurons (Schmued et al., 2005). Some neurons appeared to be undergoing apoptosis, as activated caspase-3 was detected in hippocampal neurons. Tau cleavage was also elevated in the same neurons compared to those in the APPsw mice.

The mechanism by which loss of NO promotes the accumulation of phospho-tau remains a mystery, but the authors propose two possibilities. Nitrosylation of tau inhibits tangle formation, so lack of NO might contribute to tau aggregation. Also, NO activates the Akt kinase, which inhibits tau phosphorylation via GSK3, and loss of NOS might release this inhibition.

In contrast to these results, NOS2 knockout in a double transgenic APPsw and mutant human presenilin background was previously reported to result in fewer plaques, longer lifespan, and less microglial activation. The authors of the current report speculate that the presenilin mutation, which has been implicated in NO-mediated toxicity (Hashimoto et al., 2004), could account for the different results.

As NO is generally accepted as a danger to neurons (see ARF related news story), one contribution of this work is to shade the view a bit to consider that the levels and timing of NO production may determine its impact. In light of the results that lowering NO over the life of a mouse causes the appearance of tau pathology, it could be that the induction of iNOS documented in Alzheimer disease (see Meda et al., 1995) might even represent a protective response, the authors speculate.—Pat McCaffrey

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  1. Nitric oxide signaling via the second messenger molecule cGMP is essential
    for normal physiological brain function. NO itself is produced by the NO
    synthase (NOS) family of enzymes, and NO bioactivity is also exerted by
    metabolites of NO and by cGMP-independent pathways. In many brain regions,
    activation of NOS and NO/cGMP signaling is a consequence of activation of
    glutamatergic excitatory amino acid receptors and cholinergic muscarinic
    receptor subtypes. The NO/sGC/cGMP signal transduction system is
    considered to be important for modulating synaptic transmission and
    plasticity in brain regions such as the hippocampus and cerebral cortex,
    which are critical for learning and memory. We have long argued that NO
    plays a decisive role in signal transduction cascades that are compromised
    in AD, and therefore that drugs delivering NO bioactivity represent targets for
    AD therapy. Acceptance of this argument has been impeded by a popular view
    that NO is neurotoxic and causative in diseases such as AD.

    NO, before realization of its essential role in human physiology, was best
    known as a toxic atmospheric pollutant, and it is easy to demonstrate NO
    toxicity toward brain cells in vitro. Excitotoxic neurodegeneration as
    occurs in ischemic stroke has been linked with increased NO levels. It
    has been proposed that disease states where chronic inflammation is a
    causative factor would benefit from the use of inhibitors of iNOS, which is
    induced under such conditions and can produce high levels of cellular NO.
    Some researchers have espoused a simplistic paradigm of “bad” iNOS and “good” nNOS and eNOS. NO has been accepted as a causative factor
    in brain diseases despite many reports demonstrating
    anti-neurodegenerative properties and even neuroprotection in response to
    insults such as amyloid-β (Aβ) neurotoxicity. There is also 130 years of
    drug therapy with nitrates, drug sources of NO bioactivity, to support the
    safety of NO and NO-based medications.

    Recently, 1) nitrates have been reported to reverse cognition deficits
    induced by cholinergic neurodegeneration and reduce amyloid load in
    transgenic AD mouse models, and 2) direct links have been shown from
    amyloid protein to neuronal dysfunction mediated by damage to NO signaling
     networks.

    The present work of Colton and coworkers serves to emphasize the
    fallacy in the presumption that NO and/or iNOS causes
    neurodegeneration. The work draws further links between loss of brain NO
    bioactivity and both buildup of amyloid and hyperphosphorylated tau
    deposits, the key pathological brain markers of AD. The Colton transgenic
    mouse model superimposes an iNOS knockout on a standard AD transgenic
    model leading to a pathophysiology that mimics aspects of human AD:

    • formation of hyperphosphorylated tau in hippocampal neurons
    • intracellular tangle-like aggregates
    • increased amyloid load and plaques
    • widespread cortical neuronal damage

    Interpretation of data from NOS knockout transgenics is especially difficult because knockout of one isozyme leads to compensatory changes in the remaining two isoforms. In this transgenic,
    the researchers took care to note that eNOS protein levels increased and
    nNOS levels fell, which may have also contributed to the observed
    pathology. The observation of apoptotic cell death and raised caspase
    activity may provide a pathway for formation of neurofibrillary tangles of
    hyperphosphorylated tau initiated by loss of NO bioactivity. There remain
    many questions to answer, in particular the mechanism of loss of iNOS
    activity in the early stages of AD, but the work provides another strong
    supporting plank for the argument that drugs that reinforce NO bioactivity
    represent a valid and urgent approach to AD.

  2. Antioxidant Defenses—Nitric Oxide, Amyloid-β and Tau Phosphorylation: A Zero Sum Game
    This paper is exceptionally well executed and may lead to a paradigm shift. Dogma indicates that nitric oxide, amyloid-β, and tau phosphorylation are all bad. By deleting nitric oxide synthase 2 (NOS2) in APP transgenic lines, the authors find increased amyloid-β and tau phosphorylation. In our opinion, one can reconcile these data only by viewing nitric oxide, amyloid-β, and tau phosphorylation as protective antioxidants (Smith et al., 2002; Lee et al., 2005; Castellani et al., 2006; Lee et al., 2006). Mutations in APP cause oxidative stress in vitro (Marques et al., 2003), in animal models (Pappolla et al., 1998; Smith et al., 1998), and in humans (Nunomura et al., 2004). Such oxidative stress leads to cellular adaptations, including increases in amyloid-β, tau phosphorylation, and nitric oxide. In the Colton study, by deleting NOS2, remaining cellular adaptations (amyloid-β and tau phosphorylation) are increased, though in this case not sufficiently to prevent neurodegeneration.

    References:

    . Antioxidant protection and neurodegenerative disease: the role of amyloid-beta and tau. Am J Alzheimers Dis Other Demen. 2006 Mar-Apr;21(2):126-30. PubMed.

    . Tau phosphorylation in Alzheimer's disease: pathogen or protector?. Trends Mol Med. 2005 Apr;11(4):164-9. PubMed.

    . Amyloid beta: the alternate hypothesis. Curr Alzheimer Res. 2006 Feb;3(1):75-80. PubMed.

    . Neurotoxic mechanisms caused by the Alzheimer's disease-linked Swedish amyloid precursor protein mutation: oxidative stress, caspases, and the JNK pathway. J Biol Chem. 2003 Jul 25;278(30):28294-302. PubMed.

    . Neuronal RNA oxidation is a prominent feature of familial Alzheimer's disease. Neurobiol Dis. 2004 Oct;17(1):108-13. PubMed.

    . Evidence of oxidative stress and in vivo neurotoxicity of beta-amyloid in a transgenic mouse model of Alzheimer's disease: a chronic oxidative paradigm for testing antioxidant therapies in vivo. Am J Pathol. 1998 Apr;152(4):871-7. PubMed.

    . Amyloid-beta and tau serve antioxidant functions in the aging and Alzheimer brain. Free Radic Biol Med. 2002 Nov 1;33(9):1194-9. PubMed.

    . Amyloid-beta deposition in Alzheimer transgenic mice is associated with oxidative stress. J Neurochem. 1998 May;70(5):2212-5. PubMed.

    View all comments by Xiongwei Zhu
  3. Colton and colleagues reported that crossing the APPsw transgenic line with the NOS2-/- mouse led to the novel Tg2576/NOS2-/- bigenic mouse which recapitulates the key pathological features of Alzheimer disease (AD), that is, β amyloid deposition, accumulation of hyperphosphorylated tau, and neuronal loss.

    The significance of these results is twofold: they indicate that nitric oxide (NO) plays a role in the development of AD pathological hallmarks and they highlight the neuroprotective properties of NO. This view is supported by other findings obtained using NO-releasing derivatives of anti-inflammatory and antioxidant compounds (reviewed in Gasparini et al., 2004; 2005). In particular, HCT 1026 and NCX 2216, two NO-releasing derivatives of the nonsteroidal anti-inflammatory drug flurbiprofen, have been investigated in neuroinflammation and AD transgenic models. Besides showing improved anti-inflammatory activity (Prosperi et al., 2001; 2004), these compounds have additional properties of potential benefit for AD. For example, it has been shown that chronic administration of both HCT 1026 and NCX 2216 reduce β amyloid load in APPsw/presenilin 1 mutant double transgenic mice (Jantzen 2002; Van Groen and Kadish, 2005). Besides this, they also have peculiar activities that are mediated by NO. Specifically, these compounds act as PPARγ agonists on cultured rat microglia (Bernardo et al., 2005; 2006), inhibit Nf-kB activation (Fratelli et al., 2003), attenuate the loss of cholinergic cells following LPS-induced neuroinflammation, and reduce LPS-induced caspase-3, -8 and -9 activity in rat brain (Wenk et al., 2000).

    It is worth noting that the kinetics of NO release and its concentration at the tissue level are critical to achieve such effects. The above-mentioned compounds release small amounts of NO with slow kinetics. Fast NO donors do not share the same effects (Gasparini, unpublished observations), indicating that the amount of NO and the timing are important to determine the neuroprotective effects of NO.

    Altogether, these results show that approaches focusing on NO bioavailability and biogenesis could represent a valuable therapeutic strategy for AD treatment.

    References:

    . Nuclear receptor peroxisome proliferator-activated receptor-gamma is activated in rat microglial cells by the anti-inflammatory drug HCT1026, a derivative of flurbiprofen. J Neurochem. 2005 Feb;92(4):895-903. PubMed.

    . Dynamic regulation of microglial functions by the non-steroidal anti-inflammatory drug NCX 2216: implications for chronic treatments of neurodegenerative diseases. Neurobiol Dis. 2006 Apr;22(1):25-32. PubMed.

    . Inhibition of nuclear factor-kappaB by a nitro-derivative of flurbiprofen: a possible mechanism for antiinflammatory and antiproliferative effect. Antioxid Redox Signal. 2003 Apr;5(2):229-35. PubMed.

    . Non-steroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease: old and new mechanisms of action. J Neurochem. 2004 Nov;91(3):521-36. PubMed.

    . Activity of flurbiprofen and chemically related anti-inflammatory drugs in models of Alzheimer's disease. Brain Res Brain Res Rev. 2005 Apr;48(2):400-8. PubMed.

    . Microglial activation and beta -amyloid deposit reduction caused by a nitric oxide-releasing nonsteroidal anti-inflammatory drug in amyloid precursor protein plus presenilin-1 transgenic mice. J Neurosci. 2002 Mar 15;22(6):2246-54. PubMed.

    . Comparison between flurbiprofen and its nitric oxide-releasing derivatives HCT-1026 and NCX-2216 on Abeta(1-42)-induced brain inflammation and neuronal damage in the rat. Int J Immunopathol Pharmacol. 2004 Sep-Dec;17(3):317-30. PubMed.

    . NO-flurbiprofen attenuates excitotoxin-induced brain inflammation, and releases nitric oxide in the brain. Jpn J Pharmacol. 2001 Jun;86(2):230-5. PubMed.

    . Transgenic AD model mice, effects of potential anti-AD treatments on inflammation and pathology. Brain Res Brain Res Rev. 2005 Apr;48(2):370-8. PubMed.

    . Mechanisms to prevent the toxicity of chronic neuroinflammation on forebrain cholinergic neurons. Eur J Pharmacol. 2000 Aug 18;402(1-2):77-85. PubMed.

  4. Reply to Lee, Perry, Smith, and Zhu
    My colleagues and I are delighted to see the enthusiastic interest in our mouse model for Alzheimer disease. The APPsw NOS2-/- mouse represents a novel approach to understanding the relationship between amyloid and tau pathology. The concept that NO serves as an antioxidant and promotes cell survival has been well developed in a number of physiological systems, such as the cardiovascular system. Thus, it is timely and appropriate that the role of NO as a neuroprotective agent, rather than as an “unrelenting killer” be more thoroughly explored in chronic neurodegenerative diseases. In many ways, the physiological adaptations to a long-lasting disease state are central to this study. As stated by Lee, Perry, Smith, and Zhu, multiple mechanisms including the formation of Aβ may serve to maintain a normal brain redox balance during chronic disease. Through its ability to bind reactive copper, Aβ peptide can serve as a Fenton-type oxidant in the presence of ascorbate (Dikalov et al., 2004) or as an “antioxidant” through modification of its histidine groups (Schoneich, 2004). Thus, what has appeared to be a destructive redox molecule may in fact reduce toxic levels of reactive copper under certain conditions. In our mouse, the ability of nitric oxide to counteract H2O2 and lipid peroxidation is lost, and thus the redox balance is likely to be tipped in favor of oxidation over time, regardless of the source of oxidation. NO’s ability to regulate cGMP, caspases, and kinases, however, greatly extends the level at which NO can alter cell function.

    When and why NO falls and/or NOS fails is of great interest. Both Blaylock and Jansson suggest different, but potentially interesting mechanisms for NOS destruction in the brain. We favor the view that NO is siphoned away initially by reactive scavengers, such as the iron associated with heme oxygenase 1 activity or other reactive NO scavengers. The essential question to neurodegenerative disease, however, is “can NO be successfully replaced and tissue destruction consequently slowed?” The use of NO NSAIDs as described by Gasparini is one such category of agents that have been used. However, in the case of flurbiprofen it may be difficult to separate NO’s contributions from the ferulic acid group. Other therapeutic candidates are clearly being developed by Thatcher and his group. Although we would caution that the ubiquitous nature of NO makes targeting to select tissue locations a critical issue, there is the possibility that useful new therapeutics can be developed from this concept. It would be interesting to know if the incidence and severity of AD are altered in patient populations who have used long-term nitroglycerin as a therapy for cardiovascular disease.

    References:

    . Cupric-amyloid beta peptide complex stimulates oxidation of ascorbate and generation of hydroxyl radical. Free Radic Biol Med. 2004 Feb 1;36(3):340-7. PubMed.

    . Selective Cu2+/ascorbate-dependent oxidation of alzheimer's disease beta-amyloid peptides. Ann N Y Acad Sci. 2004 Mar;1012:164-70. PubMed.

    View all comments by Carol Colton
  5. A practical problem with transgenic animals is that most focus on single aspects of the biology of AD such as amyloid. This paper broadens the scope of inquiry to a second variable, in this case, nitric oxide. It is of interest that aluminum, which epidemiology identifies as a risk factor in AD, has significant effects on NOS levels. Bondy et al. (1998) found that aluminum treatment induces NOS in the rat brain over 3 days and 3 weeks. Rodella et al. (2006) reported that aluminum exposure impaired the glutamate-nitric oxide-cGNP pathway and reduced numbers of nitroxidergic neurons in the rat somatosensory cortex with the largest effects seen after 3 months. Aluminum appeared to downregulate NOS in the 1-3-month period, and then decreased the NPY system at 6 and 12 months that is colocalized with NOS in cortical neurons. Kim (2003) found a decrease in nNOS immunoreactive neurons in rat pups after perinatal exposure.

    References:

    . Aluminum treatment induces nitric oxide synthase in the rat brain. Neurochem Int. 1998 Jul;33(1):51-4. PubMed.

    . Exposure to aluminium changes the NADPH-diaphorase/NPY pattern in the rat cerebral cortex. Arch Histol Cytol. 2006 Mar;69(1):13-21. PubMed.

    . Perinatal exposure to aluminum alters neuronal nitric oxide synthase expression in the frontal cortex of rat offspring. Brain Res Bull. 2003 Aug 30;61(4):437-41. PubMed.

  6. I found this study to be most interesting. Of particular interest from other studies is the suggestion that ionic mercury may play a role in Alzheimer's disease. Recent studies have shown that mercury in submicromolar concentrations inhibits glutamate transporters, triggering excitotoxicity.

    The finding that NO protects neurons from tau pathology is of interest. While NO is produced during excitotoxicity, the mechanism of toxiciity is assumed to be the production of peroxynitrite in the face of elevated superoxide production. By suppressing mitochondrial energy production, excitotoxicity is greatly increased. This study provides more evidence for mercury's involvement in this process, since it has been shown that mercury reduces NOS activity. This would block NO's protective effects, without blocking excitotoxicity.

  7. This paper is highly interesting in demonstrating a role for NO in the pathogenesis of Alzheimer-like neuropathology in transgenic mice. However, the seemingly new aspect of NO's protective role in neuropathology is not so new. Already in 1999, Willenborg and coworkers reviewed the controversial role of NO in the pathogenesis of a rodent model of multiple sclerosis—experimental autoimmune encephalomyelitis (EAE). We and others have shown that NO may ameliorate the EAE disease course by exerting immunomodulatory functions (Kahl et al., 2003 and 2004). However, the effects were very dependent on the type of EAE model, with NO mediating CNS damage in other models (reviewed by Willenborg et al., 1999).

    Another aspect is that findings from rodents regarding NOS type 2 cannot easily be transferred to the human situation, as—like Colton and coworkers also mention in their discussion—the expression of NOS-2 appears to be under much tighter control than in rodents.

    Finally, one must not forget that NO has a large range of physiological functions, such as vasodilation and neurotransmission. Thus, modulating the NO system at any point may lead to compensatory processes or side effects.

    Taken together, the role of NO in the pathogenesis of amyloid plaque and tau aggregate formation warrants further studies, especially in vitro investigations in human cell systems and comparative analyses in different rodent models of Alzheimer neuropathology.

    References:

    . Experimental autoimmune encephalomyelitis in mice with a targeted deletion of the inducible nitric oxide synthase gene: increased T-helper 1 response. Neurosci Lett. 2004 Mar 18;358(1):58-62. PubMed.

    . Protective role of the cytokine-inducible isoform of nitric oxide synthase induction and nitrosative stress in experimental autoimmune encephalomyelitis of the DA rat. J Neurosci Res. 2003 Jul 15;73(2):198-205. PubMed.

    . Our shifting understanding of the role of nitric oxide in autoimmune encephalomyelitis: a review. J Neuroimmunol. 1999 Dec;100(1-2):21-35. PubMed.

References

News Citations

  1. NO Parkin—A Simple Modification Arrests Ligase
  2. New Role for NO in Neuronal Apoptosis
  3. NO Laughing Matter—Nitrosylation of Isomerase Spells Trouble for Neurons

Paper Citations

  1. . Protection from Alzheimer's-like disease in the mouse by genetic ablation of inducible nitric oxide synthase. J Exp Med. 2005 Nov 7;202(9):1163-9. PubMed.
  2. . Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons. Brain Res. 2005 Feb 21;1035(1):24-31. PubMed.
  3. . Amino- and carboxyl-terminal mutants of presenilin 1 cause neuronal cell death through distinct toxic mechanisms: Study of 27 different presenilin 1 mutants. J Neurosci Res. 2004 Feb 1;75(3):417-28. PubMed.
  4. . Activation of microglial cells by beta-amyloid protein and interferon-gamma. Nature. 1995 Apr 13;374(6523):647-50. PubMed.

Further Reading

No Available Further Reading

Primary Papers

  1. . NO synthase 2 (NOS2) deletion promotes multiple pathologies in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2006 Aug 22;103(34):12867-72. PubMed.