Chelatable Zn Research Articles

Brain zinc chelation by diethyldithiocarbamate increased the behavioral and mitochondrial damages in zebrafish subjected to hypoxia.

The increase in brain levels of chelatable zinc (Zn) in dysfunctions involving oxygen deprivation has stimulated the treatment with Zn chelators, such as diethyldithiocarbamate (DEDTC). However, DEDTC is a redox-active compound and it should be better evaluated during hypoxia. We use the hypoxia model in zebrafish to evaluate DEDTC effects. The exploratory behavior, chelatable Zn content, activities of mitochondrial dehydrogenases, reactive species levels (nitric oxide, superoxide anion, hydroxyl radical scavenger capacity) and cellular antioxidants (sulfhydryl, superoxide dismutase) of zebrafish brain were assessed after recovery, with or without 0.2 mM DEDTC. The increased brain levels of chelatable Zn induced by hypoxia were mitigated by DEDTC. However, the novel tank task indicated that DEDTC did further enhance the exploratory deficit caused by hypoxia. Furthermore, these behavioral impairments caused by DEDTC were more associated with a negative action on mitochondrial activity and brain oxidative balance. Thus, due to apparent pro-oxidant action of DEDTC, our data do not support its use for neuroprotection in neuropathologies involving oxygen deprivation.

How does zebrafish support new strategies for neuroprotection and neuroregeneration in hypoxia-related diseases?

Hypoxia is a condition found commonly in several disorders, such as ischemia, asthma, anemia and neonatal hypoxia. Individuals subjected suddenly to high altitude or extreme exercise are also challenged to low oxygen (O2) levels. Since the brain presents elevated basal O2 consumption, this organ is readily affected by hypoxia. For this reason, cerebral hypoxia has been investigated in different species in order to elucidate the involved molecular mechanisms. Based on these models, it is now understood that many of the neural ischemic events occur in cerebral hypoxia, such as excessive release of chelatable Zn from neurons (Udayabanu et al., 2012), glial activation (Li et al., 2010), depletion in mitochondrial activity, increase in reactive oxygen species and cell necrosis (see review in Lukyanova and Kirova, 2015). Moreover, it has been recently reported that chelatable Zn contribute also to the development of the pathophysiology of hypoxia, triggering neuroinflammatory and oxidative stress responses observed in hypoxia (Malairaman et al., 2014). Thus, potential compounds have been tested on hypoxia models to evaluate new neuroregenerative and neuroprotective therapies able to replace the loss of neural cells and decrease the progression of secondary injuries caused by hypoxia, respectively. To advance the research in hypoxia, we have supported the development of a new model in zebrafish. These animals have been used in large scale screening of neuroprotective drugs, because they present features, such as easy maintenance, low operating cost and functional conservation of mammalian brain areas. Moreover, the sharing of many human genes, including those encoding to hypoxia-inducible factors (HIFs), further supports the adoption of zebrafish to evaluate the effects of hypoxia.

Modulation of the chelatable Zn pool in the brain by diethyldithiocarbamate is associated with behavioral impairment in adult zebrafish

DEDTC leads to a buildup of DEDTC in the brain with consequent chelation of reactive Zn and behavioral impairment of zebrafish.

Development of Zn<sup>2+</sup> Selective Fluorescent Probes for Biological Applications

Zn(2+) is an essential element for life and is known to play important roles in biological processes including gene expression, apoptosis, enzyme regulation, immune system and neurotransmission. To investigate physiological roles of free or chelatable Zn(2+) in living cells, Zn(2+)-selective fluorescent probes are valuable tools. A variety of fluorescent probes based on quinoline, BF2 chelated dipyrromethene, fluorescein, etc. has been developed recently. In principle, such tools can provide useful information about zinc biology. However, most of the fluorescent probes presented so far possess a fluorescent core and a separate part for binding to Zn(2+) within the molecule, so that the molecular weight is usually large and the molecules are hydrophobic. As a result, the applications of such molecules in biological systems often face difficulties. Therefore, we need to develop a new class of fluorescent probes for Zn(2+) with improved molecular characteristics. If the initial core structure is small enough, the fluorescent probes may still be molecular weight below 500 with desirable physico-chemical properties, even after the modifications. In this review, we described novel low-molecular-weight fluorescent probes for Zn(2+) based on pyridine-pyridone. Small modification of pyridine-pyridone core structure brought about a marked improvement such as aqueous solubility, affinity toward Zn(2+), and fluorescence ON/OFF switching. Fluorescence images of Zn(2+) in cells showed that the pyridine-pyridone probe can be used in biological applications.

Zn7Metallothionein-3 and the Synaptic Vesicle Cycle: Interaction of Metallothionein-3 with the Small GTPase Rab3A

In the central nervous system, a large amount of chelatable Zn(2+) is sequestered in presynaptic vesicles of certain glutamatergic nerve terminals. The exo-endocytotic cycle of synaptic vesicles is strictly linked to the small GTPase Rab3A. Metallothionein-3 (Zn(7)MT-3) has been proposed to be involved in the intracellular trafficking of Zn(2+) in zinc-containing neurons, but its role in this process is not understood. By using affinity precipitation and surface plasmon resonance analysis, we show that Zn(7)MT-3 binds reversibly to Rab3A.GDP (K(D) = 2.6 microM), but not to Rab3A.GTP. The binding of Zn(7)MT-3 to Rab3A.GDP is specific as no binding was observed with the metal-free form of MT-3. Mutational studies of Rab3A mapped the interaction site to the effector binding site of the protein. This location is further supported by the kinetics of GDP exchange, which was found to be unaffected by binding of Zn(7)MT-3 to Rab3A.GDP. The interaction of Zn(7)MT-3 with Rab3A indicates that Zn(7)MT-3 is not merely a cellular Zn(2+) buffer, but actively participates in synaptic vesicle trafficking upstream of vesicle fusion.

Development of a zinc ion-selective luminescent lanthanide chemosensor for biological applications.

Detection of chelatable zinc (Zn(2+)) in biological studies has attracted much attention recently, because chelatable Zn(2+) plays important roles in many biological systems. Lanthanide complexes (Eu(3+), Tb(3+), etc.) have excellent spectroscopic properties for biological applications, such as long luminescence lifetimes of the order of milliseconds, a large Stoke's shift of >200 nm, and high water solubility. Herein, we present the design and synthesis of a novel lanthanide sensor molecule, [Eu-7], for detecting Zn(2+). This europium (Eu(3+)) complex employs a quinolyl ligand as both a chromophore and an acceptor for Zn(2+). Upon addition of Zn(2+) to a solution of [Eu-7], the luminescence of Eu(3+) is strongly enhanced, with high selectivity for Zn(2+) over other biologically relevant metal cations. One of the important advantages of [Eu-7] is that this complex can be excited with longer excitation wavelengths (around 340 nm) as compared with previously reported Zn(2+)-sensitive luminescent lamthanide sensors, whose excitation wavelength is at too high an energy level for biological applications. The usefulness of [Eu-7] for monitoring Zn(2+) changes in living HeLa cells was confirmed. This novel Zn(2+)-selective luminescent lanthanide chemosensor [Eu-7]should be an excellent lead compound for the development of a range of novel luminescent lanthanide chemosensors for biological applications.

A novel, cell-permeable, fluorescent probe for ratiometric imaging of zinc ion.

Zn(2+) plays important roles in various biological systems; as a result, the development of tools that can visualize chelatable Zn(2+) has attracted much attention recently. We report here newly synthesized fluorescent sensors for Zn(2+), ZnAF-Rs, whose excitation maximum is shifted by Zn(2+) under physiological conditions. Thus, these sensors enable ratiometric imaging, which is a technique to reduce artifacts by minimizing the influence of extraneous factors on the fluorescence of a probe. Ratiometric measurement can provide precise data, and some probes allow quantitative detection. ZnAF-Rs are the first ratiometric fluorescent sensors for Zn(2+) that enable quantitative analysis under physiological conditions. ZnAF-Rs also possess suitable K(d) for applications, and high selectivity against other biologically relevant cations, especially Ca(2+). Using these probes, changes of intracellular Zn(2+) concentration in cultured cells were monitored successfully. We believe that these probes will be extremely useful in studies on the biological functions of Zn(2+).

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

The mammalian CNS contains an abundance of chelatable Zn(2+) 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 Zn(2+) release from mossy fiber synaptic terminals and subsequent entry into postsynaptic neurons has prompted us to investigate the role of synaptically released Zn(2+) in the induction of long-term potentiation (LTP) in field CA3 of the hippocampus. The rapid removal of synaptically released Zn(2+) with the membrane-impermeable Zn(2+) chelator CaEDTA (10 mm) blocked induction of NMDA receptor-independent mossy fiber LTP by high-frequency electrical stimulation (HFS) in rat hippocampal slices. Mimicking Zn(2+) release by bath application of Zn(2+) (50-100 microm) without HFS induced a long-lasting potentiation of synaptic transmission that lasted more than 3 hr. Moreover, our experiments indicate the effects of Zn(2+) 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 Zn(2+) entry into postsynaptic neurons. These results indicate that synaptically released Zn(2+), acting as a second messenger, is necessary for the induction of LTP at mossy fiber-->CA3 synapses of hippocampus.