Christopher J. Frederickson

Histochemically-reactive zinc in amyloid plaques, angiopathy, and degenerating neurons of Alzheimer's diseased brains

Excess brain zinc has been implicated in Alzheimers neuropathology. Here we evaluated that hypothesis by searching the brains of Alzheimers patients for abnormal zinc deposits. Using histochemical methods, we found vivid Zn2+ staining in the amyloid deposits of dense-core (senile) plaques, in the amyloid angiopathy surrounding diseased blood vessels, and in the somata and dendrites of neurons showing the characteristic neurofibrillary tangles (NFT) of Alzheimers. In contrast, brains from age-matched, non-demented subjects showed only occasional staining for Zn2+ in scattered neurons and possible plaques. A role of abnormal zinc metabolism in Alzheimers neuropathology is suggested.

Induction of Mossy Fiber→CA3 Long-Term Potentiation Requires Translocation of Synaptically Released Zn2+

The mammalian CNS contains an abundance of chelatable Zn2+ sequestered in the vesicles of glutamatergic terminals. These vesicles are particularly numerous in hippocampal mossy fiber synapses of the hilar and CA3 regions. Our recent observation of frequency-dependent Zn2+ release from mossy fiber synaptic terminals and subsequent entry into postsynaptic neurons has prompted us to investigate the role of synaptically released Zn2+ in the induction of long-term potentiation (LTP) in field CA3 of the hippocampus. The rapid removal of synaptically released Zn2+ with the membrane-impermeable Zn2+ chelator CaEDTA (10 mm) blocked induction of NMDA receptor-independent mossy fiber LTP by high-frequency electrical stimulation (HFS) in rat hippocampal slices. Mimicking Zn2+ release by bath application of Zn2+ (50–100 μm) without HFS induced a long-lasting potentiation of synaptic transmission that lasted more than 3 hr. Moreover, our experiments indicate the effects of Zn2+ were not attributable to its interaction with extracellular membrane proteins but required its entry into presynaptic or postsynaptic neurons. Co-released glutamate is also essential for induction of LTP under physiological conditions, in part because it allows Zn2+ entry into postsynaptic neurons. These results indicate that synaptically released Zn2+, acting as a second messenger, is necessary for the induction of LTP at mossy fiber→CA3 synapses of hippocampus.

Evidence that synaptically-released zinc contributes to neuronal injury after traumatic brain injury

Prior evidence indicates that synaptically-released zinc enters postsynaptic neurons in toxic excess during ischemia and seizures. In addition, prevention of this zinc translocation has been shown to be neuroprotective in both ischemia and seizures. Here we show evidence that the same translocation of zinc from presynaptic boutons into postsynaptic neurons occurs after mechanical injury to the brain. Specifically, using a rat model of traumatic brain injury, we show that trauma is associated with (i) loss of zinc from presynaptic boutons (ii) appearance of zinc in injured neurons, and (iii) neuroprotection by intraventricular administration of a zinc chelator just prior to brain impact. The possible use of zinc chelators for neuroprotection after head trauma is considered.

Synaptic release of zinc from brain slices : Factors governing release, imaging, and accurate calculation of concentration

Cerebrocortical neurons that store and release zinc synaptically are widely recognized as critical in maintenance of cortical excitability and in certain forms of brain injury and disease. Through the last 20 years, this synaptic release has been observed directly or indirectly and reported in more than a score of publications from over a dozen laboratories in eight countries. However, the concentration of zinc released synaptically has not been established with final certainty. In the present work we have considered six aspects of the methods for studying release that can affect the magnitude of zinc release, the imaging of the release, and the calculated concentration of released zinc. We present original data on four of the issues and review published data on two others. We show that common errors can cause up to a 3000-fold underestimation of the concentration of released zinc. The results should help bring consistency to the study of synaptic release of zinc.

Fluorescence microscopy of stimulated Zn(II) release from organotypic cultures of mammalian hippocampus using a carbonic anhydrase-based biosensor system.

We demonstrate here that electrical stimulation of organotypic cultures of rat hippocampus results in the prompt release of significant amounts of Zn(II) by a fluorescence microscopic method. The fluorescence imaging of free Zn(II) is achieved using a highly selective biosensing indicator system consisting of human apo-carbonic anhydrase II (apoCAII) and a fluorescent aryl sulfonamide inhibitor of the enzyme, ABD-N. The apoenzyme and ABD-N in the absence of Zn(II) exhibit weak, reddish fluorescence typical of the ABD-N alone; when Zn(II) is added it binds to the apoenzyme (K(D) = 4 pM), which strongly promotes binding of ABD-N to the holoenzyme (K(D) = 0.9 microM). Binding of ABD-N to the holoenzyme results in a 9-fold increase in apparent quantum yield, significant blue shifts in excitation and emission, an increase in average fluorescence lifetime, a 4-fold increase in the ratio of intensities at 560 and 680 nm, and a large increase in anisotropy. Prior to stimulation, cultures immersed in phosphate-buffered saline with glucose and apoCAII with ABD-N emitted negligible fluorescence, but within 20 s after electrical stimulation a diffuse cloud of greenish fluorescence emerged and subsequently covered most of the culture, indicating release of zinc into the extracellular medium.

Amyloid-β Metal Interaction and Metal Chelation

Alzheimer’s disease (AD) is associated with the abnormal aggregation of amyloid-beta (Aβ) protein. Aβ and its precursor protein (APP) interact with metal ions such as zinc, copper and iron. Evidence shows that these metals play a role in the precipitation and cytotoxicity of Aβ. Despite recent advances in AD research, there is a lack of therapeutic agents to hinder the apparent aggregation and toxicity of Aβ. Recent studies show that drugs with metal chelating properties could produce a significant reversal of amyloid-β plaque deposition in vitro and in vivo. Here we discuss the interaction of Aβ with metals, metal dyshomeostasis in the CNS of patients with AD, and the potential therapeutic effects of metal chelators.

Clioquinol effects on tissue chelatable zinc in mice.

Recent evidence for the involvement of zinc in the formation of β-amyloid plaques in the brain in Alzheimers disease has led to the establishment of new therapeutic strategies for the degenerative disorder based on metal chelation. The present experiment was conducted on a membrane-permeable zinc chelator, clioquinol (CQ), that has shown potential in initial studies on a mouse model of Alzheimers disease [1]. The degree of chelatable zinc in mice treated with CQ, delivered by two different routes, was measured using complementary protocols for identifying chelatable zinc: 6-methoxy-8-quinolyl-p-toluenesulfonamide (TSQ) histofluorescence, and selenite autometalography. Mice injected intraperitoneally with CQ showed a dramatic reduction in chelatable zinc in brain, testis, and pancreas. In contrast, mice given CQ orally showed no significant change in levels of chelatable zinc in these tissues. This suggests that CQ administered orally to patients with Alzheimers disease should not significantly perturb chelatable zinc levels in key organs and may be used over long periods without adverse endocrinological and reproductive effects related to zinc deficiency. In contrast, CQ injected intraperitoneally may be used not only as a tool for investigating chelatable zinc pools but also in a clinical context. For example, injected CQ could be employed in situations requiring the rapid buffering of excessive chelatable zinc following ischemic episodes or brain trauma. Thus, our findings indicate that CQ has considerable potential as a versatile scientific and clinical tool used for selective modulation of zinc pools.

Imaging Zinc: Old and New Tools

As recently as 20 years ago, all zinc in biological systems was believed to be tightly bound to proteins, and the idea of imaging zinc was considered heretical. Beginning with Maskes research with dithizonate staining of the hippocampus in the 1950s, however, zinc-sensitive dyes have indicated that, in mammalian cells, free zinc can exist in at least three separate pools. These pools include vesicular zinc sequestered in presynaptic vesicles and secretory granules, zinc released from these vesicles into the extracellular space after physiological stimulation, and transient increases in zinc in cells in the regions where extracellular release of zinc has occurred. This Perspective covers the zinc-imaging tools, from dithizonate to the newest FRET-based sensors, that have galvanized biomedical science.

Loss of vesicular zinc and appearance of perikaryal zinc after seizures induced by pilocarpine.

The condition of status epilepticus induced by systemic administration of kainic acid (KA) causes an apparent translocation of vesicular zinc from presynaptic boutons into postsynaptic neurons. The accumulation of zinc in the somata has been identified as a contributing cause of neuronal injury. We show here that another form of status epilepticus, induced by administration of the muscarinic agonist pilocarpine, produces changes in zinc that are essentially the same as those produced by the kainic acid-induced seizures. Moreover, neurons that develop zinc staining after pilocarpine seizures are the same that shown degenerative changes. This result suggests that the loss of zinc from presynaptic boutons and the appearance of zinc in postsynaptic somata may both occur in seizures per se, regardless of etiology.

Protective effects of zinc chelation in traumatic brain injury correlate with upregulation of neuroprotective genes in rat brain

Chelation of excessive neuronal zinc ameliorates zinc neurotoxicity and reduces subsequent neuronal injury. To clarify the molecular mechanisms of this neuroprotective effect, we used a focused cDNA array of stress-response genes with zinc chelation (calcium EDTA) in our rat model of fluid percussion brain injury at 2 h, 24 h, and 7 days after injury. In parallel experiments, we compared neuronal cell death in TUNEL-stained brain sections in traumatized rats with and without calcium EDTA treatment. Zinc chelation induced the expression of several neuroprotective genes; neuroprotective gene expression correlated with substantially decreased numbers of TUNEL-positive cells.