Think Zinc—Mice Missing Key Ion Transporter Develop AD-like Problems

Might amyloid plaques suck the life out of neurons by trapping metal ions required for synaptic transmission? A study in this week’s Journal of Neuroscience lends support for this intriguing idea. Led by Ashley Bush of the University of Melbourne, Victoria, Australia, researchers analyzed mice lacking a transporter that controls synaptic zinc levels, and found that with age they develop neurophysiological and cognitive deficits resembling Alzheimer disease. “They are a phenocopy of AD transgenics, really,” Bush told ARF.

The mice have a 20 percent reduction of total zinc and virtually none in synapses due to their genetic deficiency in zinc transporter-3 (ZnT3), a protein that loads the metal ion into synaptic vesicles (Cole et al., 1999). Interestingly, learning and memory had seemed fine in the ZnT3 knockout mice when initially characterized about a decade ago (Cole et al., 2001). “We doubted that that could possibly be the case,” Bush said, given that extracellular zinc interacts with a host of synaptic proteins and thus was believed to be important for learning and memory. First author Paul Adlard and colleagues suspected the ZnT3 knockout mice had been tested too young (i.e., at six to 10 weeks of age), and that cognitive problems might crop up as the mice got older.

The current study confirmed their hunch. In the Morris water maze, which gauges spatial learning and memory by how quickly animals remember the location of a submerged platform, three-month-old ZnT3 knockouts did just as well as their wild-type counterparts. However, by the time they were six months old, the knockouts were taking more than twice as long as age-matched wild-type mice to find the escape platform. The researchers did additional tests to show that these differences could not be attributed to visual or motor shortcomings in the ZnT3-deficient animals.

In addition to the cognitive impairment, ZnT3 knockout mice had age-associated reductions in a slew of synaptic and plasticity-related proteins, as revealed by Western blot of brain tissue. “It’s quite an impressive list of things that go wrong,” Bush said. “By six months of age, all of the favorite substrates for memory are changed, most conspicuously the NR2B subunit of the NMDA receptor (NMDAR2b).” ZnT3 knockout mice also had reduced levels of NMDAR2a, presynaptic (SNAP25) and post-synaptic (PSD95) markers, AMPA receptor subunits, and several trophic factors. Some of these deficits were detectable at three months but became more dramatic by six months of age.

At any age, the ZnT3 knockouts had roughly a third less hippocampal zinc than their wild-type counterparts. However, even the wild-type mice had a 23 percent drop in brain zinc levels between three and six months, indicating that some zinc loss occurs with age. As it turns out, aging brains gradually lose ZnT3, too. The researchers found that this occurs in both mice (1.9 and 8.7 months of age) and people (48-91 years old). This was a surprise, perhaps even a tad counterintuitive, given that ZnT3 regulates synaptic zinc levels and hence might be expected to rise to counteract age-associated cognitive decline, Bush said. Analysis of AD brain tissue showed even sharper decreases in brain ZnT3 levels, relative to age-matched healthy people.

Though puzzling, these findings did suggest that the losses of zinc and ZnT3 are important in aging and disease pathophysiology. In their paper, the researchers propose a model for what they think is happening. “Normally, ZnT3 loads zinc into glutamatergic vesicles, and that zinc is released during neurotransmission and hits post-synaptic targets such as the NMDA receptor,” Bush said. “We believe that zinc plays an important role in conditioning post-synaptic targets in a manner analogous to that recently reported for magnesium.” That study, published in the 28 January issue of Neuron, showed that raising brain magnesium improves cognition in rats, and that these benefits may arise from magnesium-mediated NMDA receptor changes that boost LTP (see ARF related news story).

“The mechanism emerging from both papers is that the NMDAR likes to see a sustained level of magnesium or zinc in order to upregulate concentrations of the NR2B subunit,” Bush said. “If you take away the magnesium or zinc as a consequence of aging, the NR2B levels drop, and you lose LTP. And in AD, what happens is that in addition to the drop in ZnT3, you've got amyloid soaking up the zinc. As a result, the NR2B levels fall even further.” Bush noted a recent paper suggesting that soluble Aβ oligomers may interfere with synaptic transmission by sequestering available zinc away from nearby NMDA receptors (Deshpande et al., 2009). He and colleagues have preliminary data consistent with this model. In studies with hippocampal slices, they show that synaptic zinc uptake is slow in aging rodents. “The zinc pools outside the cell, and the intracellular compartment is deficient,” Bush said. “And the amyloid is this great big zinc fly trap.”

This mechanism could relate to the action of the zinc/copper ligand PBT2, Bush told ARF. This compound boosts cognition in AD transgenic mice (Adlard et al., 2008 and ARF related news story) and has shown promise in the clinic, reducing cerebrospinal fluid Aβ42 levels and improving certain cognitive measures in a Phase 2a trial of mild AD patients (see ARF related news story; Lannfelt et al., 2008). “We think our drug takes the zinc out of the amyloid and puts it back into the cell,” said Bush, who co-founded the Australian biotech company Prana Biotechnology Ltd. that is developing PBT2. “We are hoping to proceed with the next stage of clinical trials in the coming year.”—Esther Landhuis

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References

News Citations

  1. Mind Your Magnesium—Oral Compound Boosts Cognition in Rats
  2. Improving Cognition in Mice: Copper Ionophore Shows Some Mettle
  3. Anti-Amyloid Drug Clears Phase 2a Hurdle

Paper Citations

  1. Cole TB, Wenzel HJ, Kafer KE, Schwartzkroin PA, Palmiter RD. Elimination of zinc from synaptic vesicles in the intact mouse brain by disruption of the ZnT3 gene. Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1716-21. PubMed.
  2. Cole TB, Martyanova A, Palmiter RD. Removing zinc from synaptic vesicles does not impair spatial learning, memory, or sensorimotor functions in the mouse. Brain Res. 2001 Feb 9;891(1-2):253-65. PubMed.
  3. Deshpande A, Kawai H, Metherate R, Glabe CG, Busciglio J. A role for synaptic zinc in activity-dependent Abeta oligomer formation and accumulation at excitatory synapses. J Neurosci. 2009 Apr 1;29(13):4004-15. PubMed.
  4. Adlard PA, Cherny RA, Finkelstein DI, Gautier E, Robb E, Cortes M, Volitakis I, Liu X, Smith JP, Perez K, Laughton K, Li QX, Charman SA, Nicolazzo JA, Wilkins S, Deleva K, Lynch T, Kok G, Ritchie CW, Tanzi RE, Cappai R, Masters CL, Barnham KJ, Bush AI. Rapid restoration of cognition in Alzheimer's transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Abeta. Neuron. 2008 Jul 10;59(1):43-55. PubMed.
  5. Lannfelt L, Blennow K, Zetterberg H, Batsman S, Ames D, Harrison J, Masters CL, Targum S, Bush AI, Murdoch R, Wilson J, Ritchie CW, . Safety, efficacy, and biomarker findings of PBT2 in targeting Abeta as a modifying therapy for Alzheimer's disease: a phase IIa, double-blind, randomised, placebo-controlled trial. Lancet Neurol. 2008 Sep;7(9):779-86. PubMed.

Further Reading

Papers

  1. Lee JY, Cole TB, Palmiter RD, Suh SW, Koh JY. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice. Proc Natl Acad Sci U S A. 2002 May 28;99(11):7705-10. PubMed.
  2. Stoltenberg M, Bush AI, Bach G, Smidt K, Larsen A, Rungby J, Lund S, Doering P, Danscher G. Amyloid plaques arise from zinc-enriched cortical layers in APP/PS1 transgenic mice and are paradoxically enlarged with dietary zinc deficiency. Neuroscience. 2007 Dec 5;150(2):357-69. PubMed.
  3. Adlard PA, Cherny RA, Finkelstein DI, Gautier E, Robb E, Cortes M, Volitakis I, Liu X, Smith JP, Perez K, Laughton K, Li QX, Charman SA, Nicolazzo JA, Wilkins S, Deleva K, Lynch T, Kok G, Ritchie CW, Tanzi RE, Cappai R, Masters CL, Barnham KJ, Bush AI. Rapid restoration of cognition in Alzheimer's transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Abeta. Neuron. 2008 Jul 10;59(1):43-55. PubMed.
  4. Lannfelt L, Blennow K, Zetterberg H, Batsman S, Ames D, Harrison J, Masters CL, Targum S, Bush AI, Murdoch R, Wilson J, Ritchie CW, . Safety, efficacy, and biomarker findings of PBT2 in targeting Abeta as a modifying therapy for Alzheimer's disease: a phase IIa, double-blind, randomised, placebo-controlled trial. Lancet Neurol. 2008 Sep;7(9):779-86. PubMed.
  5. Deshpande A, Kawai H, Metherate R, Glabe CG, Busciglio J. A role for synaptic zinc in activity-dependent Abeta oligomer formation and accumulation at excitatory synapses. J Neurosci. 2009 Apr 1;29(13):4004-15. PubMed.

Primary Papers

  1. Adlard PA, Parncutt JM, Finkelstein DI, Bush AI. Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic and memory deficits of Alzheimer's disease?. J Neurosci. 2010 Feb 3;30(5):1631-6. PubMed.

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