Individually, both nitric oxide (NO) and zinc (Zn+) have been implicated in triggering neurodegeneration (see related news for NO) and Zn2+). But who would have thought that the two small molecules act in concert to activate apoptosis? That's the conclusion Stuart Lipton and colleagues propose in a report in the February 5 Neuron.

Previous work had shown that Zn2+ may spill from intracellular stores in response to increases in NO (see, for example, Cuajungco and Lees, 1998). To test if this trigger works in neurons, first author Ella Bossy-Wetzel and colleagues at the Burnham Institute and the University of California, both in La Jolla, exposed cultures of cerebrocortical neurons to S-nitrosocysteine (SNOC), a commonly used NO generator. Bossy-Wetzel found that SNOC caused more than a 10-fold increase in the amount of free zinc in the neuron, as judged by the fluorescence of the Zn2+ indicator Newport Green. Adding the extracellular metal chelator EDTA had no effect on this spike, indicating that the metal must be coming from intracellular stores, possibly metalloproteinases (see ARF related news story). Then, the scientists used a slightly more sophisticated fluorescent probe, Rhod-zin3, which has little affinity for calcium and superior kinetics (those interested in its properties could check out Sensi et al., 2003). This experiment indicated that the metal was mobilized within 10 minutes of adding SNOC, and that it was initially localized to the mitochondria. This hinted at apoptotic pathways, some of which require mitochondrial proteins.

But is any of this in-vitro work physiologically relevant? Quite possibly, argue the authors. That’s because NMDA (N-methyl-D-aspartate), an agonist of the glutamatergic receptor of the same name, mimics the effect of SNOC, and it is known that intense stimulation of the NMDA receptor activates neuronal nitric oxide synthase (nNOS), which in turn generates increased NO.

Next, Bossy-Wetzel and colleagues used a cell-free system to investigate how the zinc spike may affect the mitochondria. They found that adding even small amounts (0.1 micromolar) of the metal blocked respiration by almost 20 percent, while one micromolar zinc blocked it by 70 percent. Zinc also made the organelles swell up and release cytochrome c, a known trigger of the apoptotic pathway.

That's not all. The authors also suggest that zinc may be a prime player in NO-induced activation of p38 MAPK, a cytoplasmic kinase known as a powerful neurodegenerative trigger (see, most recently, Xie et al., 2004). When the scientists added NO to neurons in the presence of the zinc chelator TPEN, p38 remained inactive.

Overall, the results suggest that NO and zinc cooperate to deliver a double whammy to the cell. Zinc released by the gas causes mitochondrial damage, such as respiration block and cytochrome c leakage, and it activates the p38 MAPK. How the latter contributes to neurodegeneration is debated, but the authors postulated that an expulsion of monovalent ions might drive rapid shrinking of the cell. To test this hypothesis, the authors measured intracellular potassium concentrations before and after inducing cell death with NO. They found that potassium was first lost in distal neurites, then in proximal neurites and cell bodies. The process took only about 20 minutes, and blocking potassium efflux with tetraethylammonium prevented it, confirming the authors’ suspicions. When Bossy-Wetzel carried out the same experiment in neurons expressing a dominant negative form of p38, the potassium channel was unaffected and the cells maintained their volume. Thus, NO may cause cell death by a combination of respiratory stress, loss of osmotic pressure, and activation of apoptotic pathways, all mediated by the little metal ion, zinc.

Where do mitochondria intersect with these pathways? The answer may lie in the organelle’s prime function, respiration. When the respiratory chain is interrupted (as with the insecticide rotenone, a toxin used to mimic neuronal loss in Parkinson’s disease—see ARF related news story), reducing equivalents build up and respiratory chain components begin passing electrons to whatever receptor is most accessible, usually oxygen. Highly reactive oxygen species (ROS, such as superoxide and hydroxyl radicals) are thus formed. Besides inactivating essential macromolecules, ROS also convert NO to peroxynitrite, which is even more reactive than the gas itself. A theoretical chain of events, perhaps, but in support the authors found that superoxide scavengers, such as superoxide dismutase, prevent the deleterious effects of NO on neurons, including p38 and potassium channel activation. Being a respiratory chain inhibitor, zinc fits this picture. It is ironic, if not surprising, that the respiratory chain, so heavily reliant on heavy metals to shunt electrons, should be scuppered by zinc.—Tom Fagan


  1. This is a very interesting finding in line with our publicaion. We have published already our finding on trace metal increase in moderately affected AD brain compared to control. In particular, we found that Zn was higher than other elements Si, Cu, Mg, Ca, Fe, Al , Fe, etc. However Fe, & Al was extremely elevated only in severe AD. Publiction is available In Alzeimr's Reports Vol 2, No. 4, 1999, pp 241-246.
    With regards,
    R.V.Rao & K.S.J.Rao

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News Citations

  1. New Role for NO in Neuronal Apoptosis
  2. Synaptic Zinc Fingered As Critical In Plaque Formation
  3. Novel Cell-Based Parkinson's Model Shows Promise

Paper Citations

  1. . Nitric oxide generators produce accumulation of chelatable zinc in hippocampal neuronal perikarya. Brain Res. 1998 Jul 13;799(1):118-29. PubMed.
  2. . A new mitochondrial fluorescent zinc sensor. Cell Calcium. 2003 Sep;34(3):281-4. PubMed.
  3. . Activated glia induce neuron death via MAP kinase signaling pathways involving JNK and p38. Glia. 2004 Jan 15;45(2):170-9. PubMed.

Further Reading

No Available Further Reading

Primary Papers

  1. . Crosstalk between nitric oxide and zinc pathways to neuronal cell death involving mitochondrial dysfunction and p38-activated K+ channels. Neuron. 2004 Feb 5;41(3):351-65. PubMed.