Nitric oxide causes apparent release of zinc from presynaptic boutons.

One of us showed previously [Cuajungco and Lees (1998) Brain Res. 799, 188-129] that nitric oxide injected into the cerebrum in vivo causes zinc staining to appear in the somata of neurons and suggested that this staining of somata might be accompanied by a depletion (release) of zinc from axon terminals. In the present study, we confirm earlier results and report that there is a dramatic loss (apparent release) of histologically reactive zinc from the boutons of zinc-containing axons induced by infusion of nitric oxide into the brain in vivo. Rats were anesthetized with halothane and a cannula was inserted into the hippocampus. Either nitric oxide donor (spermineNONOate, 100 mM/2 microl) or control (spermine, 100 mM/2 l) was infused into the hippocampus or the cerebellar cortex. Two hours after infusion, N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) staining for zinc in the brains revealed that sperminenitric oxide, but not control (spermine only) produced up to 95% depletion of zinc staining from the zinc-containing boutons. TSQ-positive neurons were also conspicuous throughout injection sites, in both the cerebral cortex and in the cerebellar cortex, where the Purkinje neurons were especially vivid, despite the scarcity of zinc-containing axonal boutons. It is suggested that the TSQ-stainable zinc in somata might represent intracellular stores mobilized from within or permeating extracellular stores.

Cloning, characterization, and genomic structure of the mouse Ikbkap gene.

Our laboratory recently reported that mutations in the human I-kappaB kinase-associated protein (IKBKAP) gene are responsible for familial dysautonomia (FD). Interestingly, amino acid substitutions in the IKAP correlate with increased risk for childhood bronchial asthma. Here, we report the cloning and genomic characterization of the mouse Ikbkap gene, the homolog of human IKBKAP. Like its human counterpart, Ikbkap encodes a protein of 1332 amino acids with a molecular weight of approximately 150 kDa. The Ikbkap gene product, Ikap, contains 37 exons that span approximately 51 kb. The protein shows 80% amino acid identity with human IKAP. It shows very high conservation across species and is homologous to the yeast Elp1/Iki3p protein, which is a member of the Elongator complex. The Ikbkap gene maps to chromosome 4 in a region that is syntenic to human chromosome 9q31.3. Because no animal model of FD currently exists, cloning of the mouse Ikbkap gene is an important first step toward creating a mouse model for FD. In addition, cloning of Ikbkap is crucial to the characterization of the putative mammalian Elongator complex.

Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia.

Familial dysautonomia (FD; also known as "Riley-Day syndrome"), an Ashkenazi Jewish disorder, is the best known and most frequent of a group of congenital sensory neuropathies and is characterized by widespread sensory and variable autonomic dysfunction. Previously, we had mapped the FD gene, DYS, to a 0.5-cM region on chromosome 9q31 and had shown that the ethnic bias is due to a founder effect, with >99.5% of disease alleles sharing a common ancestral haplotype. To investigate the molecular basis of FD, we sequenced the minimal candidate region and cloned and characterized its five genes. One of these, IKBKAP, harbors two mutations that can cause FD. The major haplotype mutation is located in the donor splice site of intron 20. This mutation can result in skipping of exon 20 in the mRNA of patients with FD, although they continue to express varying levels of wild-type message in a tissue-specific manner. RNA isolated from lymphoblasts of patients is primarily wild-type, whereas only the deleted message is seen in RNA isolated from brain. The mutation associated with the minor haplotype in four patients is a missense (R696P) mutation in exon 19, which is predicted to disrupt a potential phosphorylation site. Our findings indicate that almost all cases of FD are caused by an unusual splice defect that displays tissue-specific expression; and they also provide the basis for rapid carrier screening in the Ashkenazi Jewish population.

Evidence that the beta-amyloid plaques of Alzheimer's disease represent the redox-silencing and entombment of abeta by zinc.

Abeta binds Zn(2+), Cu(2+), and Fe(3+) in vitro, and these metals are markedly elevated in the neocortex and especially enriched in amyloid plaque deposits of individuals with Alzheimer's disease (AD). Zn(2+) precipitates Abeta in vitro, and Cu(2+) interaction with Abeta promotes its neurotoxicity, correlating with metal reduction and the cell-free generation of H(2)O(2) (Abeta1-42 > Abeta1-40 > ratAbeta1-40). Because Zn(2+) is redox-inert, we studied the possibility that it may play an inhibitory role in H(2)O(2)-mediated Abeta toxicity. In competition to the cytotoxic potentiation caused by coincubation with Cu(2+), Zn(2+) rescued primary cortical and human embryonic kidney 293 cells that were exposed to Abeta1-42, correlating with the effect of Zn(2+) in suppressing Cu(2+)-dependent H(2)O(2) formation from Abeta1-42. Since plaques contain exceptionally high concentrations of Zn(2+), we examined the relationship between oxidation (8-OH guanosine) levels in AD-affected tissue and histological amyloid burden and found a significant negative correlation. These data suggest a protective role for Zn(2+) in AD, where plaques form as the result of a more robust Zn(2+) antioxidant response to the underlying oxidative attack.

Alzheimer's disease, beta-amyloid protein and zinc.

Alzheimer's disease (AD) is characterized by amyloid deposits within the neocortical parenchyma and the cerebrovasculature. The main component of these predominantly extracellular collections, Abeta, which is normally a soluble component of all biological fluids, is cleaved out of a ubiquitously expressed parent protein, the amyloid protein precursor (APP), one of the type 1 integral membrane glycoproteins. Considerable evidence has indicated that there is zinc dyshomeostasis and abnormal cellular zinc mobilization in AD. We have characterized both APP and Abeta as copper/zinc metalloproteins. Zinc, copper and iron have recently been reported to be concentrated to 0.5 to 1 mmol/L in amyloid plaque. In vitro, rapid Abeta aggregation is mediated by Zn(II), promoted by the alpha-helical structure of Abeta, and is reversible with chelation. In addition, Abeta produces hydrogen peroxide in a Cu(II)/Fe(III)-dependent manner, and the hydrogen peroxide formation is quenched by Zn(II). Moreover, zinc preserves the nontoxic properties of Abeta. Although the zinc-binding proteins apolipoprotein E epsilon4 allele and alpha(2)-macroglobulin have been characterized as two genetic risk factors for AD, zinc exposure as a risk factor for AD has not been rigorously studied. Based on our findings, we envisage that zinc may serve twin roles by both initiating amyloid deposition and then being involved in mechanisms attempting to quench oxidative stress and neurotoxicity derived from the amyloid mass. Hence, it remains debatable whether zinc supplementation is beneficial or deleterious for AD until additional studies clarify the issue.

Metal chelation as a potential therapy for Alzheimer's disease.

Alzheimer's disease is a rapidly worsening public health problem. The current lack of effective treatments for Alzheimer's disease makes it imperative to find new pharmacotherapies. At present, the treatment of symptoms includes use of acetylcholinesterase inhibitors, which enhance acetylcholine levels and improve cognitive functioning. Current reports provide evidence that the pathogenesis of Alzheimer's disease is linked to the characteristic neocortical amyloid-beta deposition, which may be mediated by abnormal metal interaction with A beta as well as metal-mediated oxidative stress. In light of these observations, we have considered the development of drugs that target abnormal metal accumulation and its adverse consequences, as well as prevention or reversal of amyloid-beta plaque formation. This paper reviews recent observations on the possible etiologic role of A beta deposition, its redox activity, and its interaction with transition metals that are enriched in the neocortex. We discuss the effects of metal chelators on these processes, list existing drugs with chelating properties, and explore the promise of this approach as a basis for medicinal chemistry in the development of novel Alzheimer's disease therapeutics.

Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction.

Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Abeta amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H(2)O(2) generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Abeta in cell culture. The potentiation of toxicity is greatest for Abeta1-42 > Abeta1-40 >> mouse/rat Abeta1-40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H(2)O(2) in cell-free assays and to exhibit amyloid pathology. The copper complex of Abeta1-42 has a highly positive formal reduction potential ( approximately +500-550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Abeta-mediated oxidative damage in Alzheimer's disease.

The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction.

Oxidative stress markers characterize the neuropathology both of Alzheimer's disease and of amyloid-bearing transgenic mice. The neurotoxicity of amyloid A beta peptides has been linked to peroxide generation in cell cultures by an unknown mechanism. We now show that human A beta directly produces hydrogen peroxide (H2O2) by a mechanism that involves the reduction of metal ions, Fe(III) or Cu(II), setting up conditions for Fenton-type chemistry. Spectrophotometric experiments establish that the A beta peptide reduces Fe(III) and Cu(II) to Fe(II) and Cu(I), respectively. Spectrochemical techniques are used to show that molecular oxygen is then trapped by A beta and reduced to H2O2 in a reaction that is driven by substoichiometric amounts of Fe(II) or Cu(I). In the presence of Cu(II) or Fe(III), A beta produces a positive thiobarbituric-reactive substance (TBARS) assay, compatible with the generation of the hydroxyl radical (OH.). The amounts of both reduced metal and TBARS reactivity are greatest when generated by A beta 1-42 >> A beta 1-40 > rat A beta 1-40, a chemical relationship that correlates with the participation of the native peptides in amyloid pathology. These findings indicate that the accumulation of A beta could be a direct source of oxidative stress in Alzheimer's disease.

Zinc and Alzheimer's Disease: An Update.

Zinc is an essential metal in human nutrition. A growing number of observations have implicated zinc in the pathophysiology of Alzheimer's disease (AD). Zinc binds and interacts with both the amyloid-ß peptide (the major component of senile plaques) and its larger parent protein known as amyloid precursor protein. Nevertheless, the pathophysiological position of zinc in AD remains unclear. Recently, much research has been published that supports the involvement of zinc in AD pathophysiology. Consistent reports show cerebral zinc to be elevated in selected regions of AD brain, and is highly concentrated within senile plaques. Quantitative assessment of zinc in plaques estimates the zinc concentration to be enriched to the near millimolar concentration range. Furthermore, zinc is also detected in neurons that are positively identified to contain neurofibrillary tangles. This paper reviews current available data that indicate a role for zinc in AD pathology.

Diverse effects of metal chelating agents on the neuronal cytotoxicity of zinc in the hippocampus.

Abnormal metabolism of metal ions such as zinc may contribute to neuropathology. Complexing zinc could reduce this pathology. Thus, to examine the effectiveness of metal chelating agents in vivo, a model system was used. This involved determining the ability of chelating agents to prevent neuronal death caused by zinc chloride injected into the rat hippocampus. Significant protection against zinc toxicity was obtained with pyrithione, inositol hexakisphosphate, ethylenediamine tetraacetate (EDTA) and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). The affinity of these agents for zinc varied between 106 M-1 and 1018 M-1. Thus, the affinity for zinc within this range does not appear to be a major factor affecting the ability of chelators to provide neuroprotection. While almost complete protection was found with EDTA and TPEN given simultaneously with zinc chloride, poor protection was obtained if TPEN was given before or after zinc chloride. Other agents either did not protect against zinc-induced neuronal death (zincon), or exacerbated zinc toxicity (BTC-5N and about 40% of rats injected with a combination of zinc chloride and diethylenetriamine pentaacetate [DTPA]). Rats showing increased damage after zinc plus BTC-5N or DTPA suffered wet dog-like shakes (WDS), suggesting that these zinc chelate complexes can induce seizures resulting in seizure-related damage. In contrast, in the 60% of rats treated with zinc chloride and DTPA that had no WDS, there was about an 80% reduction in the size of the zinc-induced lesion. The ability of chelators to cross cell membranes was examined by determining whether Timm's staining for vesicular zinc was reduced following the injection of a chelator into the hippocampus. TPEN and pyrithione reduced Timm's staining for zinc. However, cell permeability was not necessary for a chelator to protect against zinc toxicity.

Effect of metal chelating agents on the direct and seizure-related neuronal death induced by zinc and kainic acid.

The ability of metal chelating agents to affect seizure-induced neuronal death caused by intra-amygdaloid injections of kainic acid was investigated. N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), diethyldithiocarbamate (DEDTC) and diphenylthiocarbazone (dithizone), administered simultaneously or within 30 min of a kainate injection, all failed to affect the amount of neuronal loss in the ipsilateral hippocampus. This failure was not due to an inability to complex endogenous zinc as all these chelating agents quenched staining for endogenous zinc by the Timm method. However, the period for which this quenching occurred was short for DEDTC and dithizone (a maximum of 1.5 h) although it lasted for 8 h with TPEN. TPEN, but not DEDTC or dithizone prevented the neuronal loss caused by intra-hippocampal injections of zinc chloride. In the presence of diazepam to prevent seizures, co-injection of TPEN and kainate into the hippocampus also failed to prevent the direct cytotoxicity of kainate. Endogenous zinc, released from mossy fibres in the hippocampus by seizure activity, does not appear to modify seizure activity sufficiently to alter the extent of the resulting neuronal death.

Nitric oxide generators produce accumulation of chelatable zinc in hippocampal neuronal perikarya.

While zinc is essential for health, it has also been implicated in the neuropathology of several disease states such as Alzheimer's disease, epilepsy and cerebral ischemia. Recent studies have shown that oxidative and nitrosylative stresses can liberate zinc from metalloproteins in vitro. Thus, nitric oxide (NO.), a radical molecule which serves as a retrograde messenger, was studied for its effects on the in vivo accumulation of zinc in neurons. Three NO. -donors, sodium nitroprusside (SNP; >/=5 nmol), spermine-nitric oxide complex (SPER-NO;

Zinc and Alzheimer's disease: is there a direct link?

Zinc is an essential trace element in human biology, but is neurotoxic at high concentrations. Several studies show that zinc promotes aggregations of beta-amyloid protein, the main component of the senile plaques typically found in Alzheimer's disease brains. In other neurological disorders where neurons appear to be dying by apoptosis (gene-directed cell death), chelatable zinc accumulates in the perikarya of neurons before, or during degeneration. As there is evidence for apoptotic death of neurons in Alzheimer's disease, an involvement of zinc in this process needs to be investigated. Zinc interacts with enzymes and proteins, including transcription factors, which are critical for cell survival and could be linked to apoptotic processes. While controversial, some studies indicate that total tissue zinc is markedly reduced in several brain regions of Alzheimer's patients. At face value, it seems that a paradox exists between reports of a decrease in zinc in the Alzheimer's brain and the putative link to aberrant high zinc levels promoting plaque formation. An hypothesis to explain this inconsistency is presented. Neuropathological changes mediated by endogenous or exogenous stressors may be relevant factors affecting abnormal zinc metabolism. This paper reviews current investigations that suggest a role of zinc in the etiology of Alzheimer's disease.

Zinc metabolism in the brain: relevance to human neurodegenerative disorders.

Zinc is an important trace element in biology. An important pool of zinc in the brain is the one present in synaptic vesicles in a subgroup of glutamatergic neurons. In this form it can be released by electrical stimulation and may serve to modulate responses at receptors for a number of different neurotransmitters. These include both excitatory and inhibitory receptors, particularly the NMDA and GABA(A) receptors. This pool of zinc is the only form of zinc readily stained histochemically (the chelatable zinc pool), but constitutes only about 8% of the total zinc content in the brain. The remainder of the zinc is more or less tightly bound to proteins where it acts either as a component of the catalytic site of enzymes or in a structural capacity. The metabolism of zinc in the brain is regulated by a number of transport proteins, some of which have been recently characterized by gene cloning techniques. The intracellular concentration may be mediated both by efflux from the cell by the zinc transporter ZrT1 and by complexing with apothionein to form metallothlonein. Metallothionein may serve as the source of zinc for incorporation into proteins, including a number of DNA transcription factors. However, zinc is readily released from metallothionein by disulfides, increasing concentrations of which are formed under oxidative stress. Metallothionein is a very good scavenger of free radicals, and zinc itself can also reduce oxidative stress by binding to thiol groups, decreasing their oxidation. Zinc is also a very potent inhibitor of nitric oxide synthase. Increased levels of chelatable zinc have been shown to be present in cell cultures of immune cells undergoing apoptosis. This is very reminiscent of the zinc staining of neuronal perikarya dying after an episode of ischemia or seizure activity. Thus a possible role of zinc in causing neuronal death in the brain needs to be fully investigated. intraventricular injections of calcium EDTA have already been shown to reduce neuronal death after a period of ischemia. Pharmacological doses of zinc cause neuronal death, and some estimates indicate that extracellular concentrations of zinc could reach neurotoxic levels under pathological conditions. Zinc is released in high concentrations from the hippocampus during seizures. Unfortunately, there are contrasting observations as to whether this zinc serves to potentiate or decrease seizure activity. Zinc may have an additional role in causing death in at least some neurons damaged by seizure activity and be involved in the sprouting phenomenon which may give rise to recurrent seizure propagation in the hippocampus. In Alzheimer's disease, zinc has been shown to aggregate beta-amyloid, a form which is potentially neurotoxic. The zinc-dependent transcription factors NF-kappa B and Sp1 bind to the promoter region of the amyloid precursor protein (APP) gene. Zinc also inhibits enzymes which degrade APP to nonamyloidogenic peptides and which degrade the soluble form of beta-amyloid. The changes in zinc metabolism which occur during oxidative stress may be important in neurological diseases where oxidative stress is implicated, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). Zinc is a structural component of superoxide dismutase 1, mutations in which give rise to one form of familiar ALS. After HIV infection, zinc deficiency is found which may be secondary to immune-induced cytokine synthesis. Zinc is involved in the replication of the HIV virus at a number of sites. These observations should stimulate further research into the role of zinc in neuropathology.

Prevention of zinc neurotoxicity in vivo by N,N,N',N'-tetrakis (2-pyridylmethyl) ethylene-diamine (TPEN).

Intrahippocampal injections of zinc chloride (10 nmol) produce lesions in the rat hippocampus. A heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylene-diamine (TPEN), co-injected with zinc chloride in vivo, totally prevented zinc-induced neuronal loss. Furthermore, intrahippocampal injections of TPEN (10 nmol) were non-toxic when compared with controls. TPEN injections in vivo quenched the in vitro staining of brain sections for zinc (Timm's stain), particularly the staining of the hippocampal mossy fibres. This demonstrates that TPEN is taken up by zinc-containing neurones in vivo. The use of TPEN to block the actions of zinc could help define the possible role of zinc in the pathology of various neurological disorders.