Latha M. Malaiyandi

Direct visualization of mitochondrial zinc accumulation reveals uniporter-dependent and -independent transport mechanisms

Current evidence suggests that zinc kills neurons by disrupting energy production, specifically by inhibiting mitochondrial function. However it is unclear if the inhibitory effect requires zinc accumulation, and if so, precisely how zinc enters mitochondria. Here, using fluorescence microscopy to visualize individual rat brain mitochondria, we detected matrix zinc uptake using the fluorophore FluoZin‐3. Fluorescence increased rapidly in mitochondria treated with micromolar free zinc, and was quickly returned to baseline by membrane permeant chelation. Zinc uptake occurred through the calcium uniporter, because depolarization or uniporter blockade reduced fluorescence changes. However, increased fluorescence under these conditions suggests that zinc can enter through a uniporter‐independent pathway. Fluorescence steadily declined over time and was unaffected by acidification or phosphate depletion, suggesting that zinc precipitation is not a mechanism for reducing matrix zinc. Uniporter blockade with ruthenium red also did not change the rate of zinc loss. Instead, zinc appears to exit the matrix through a novel efflux pathway not yet identified. Interestingly, dye‐loaded mitochondria showed no fluorescence increase after treatment with strong oxidants, arguing against oxidant‐labile intra‐mitochondrial zinc pools. This study is the first to directly demonstrate zinc accumulation in individual mitochondria and provides insight about mechanisms mediating mitochondrial zinc uptake and efflux.

Zn2+ Inhibits Mitochondrial Movement in Neurons by Phosphatidylinositol 3-Kinase Activation

Mitochondria have been identified as targets of the neurotoxic actions of zinc, possibly through decreased mitochondrial energy production and increased reactive oxygen species accumulation. It has been hypothesized that impairment of mitochondrial trafficking may be a mechanism of neuronal injury. Here, we report that elevated intraneuronal zinc impairs mitochondrial trafficking. At concentrations just sufficient to cause injury, zinc rapidly inhibited mitochondrial movement without altering morphology. Zinc chelation initially restored movement, but the actions of zinc became insensitive to chelator in <10 min. A search for downstream signaling events revealed that inhibitors of phosphatidylinositol (PI) 3-kinase prevented this zinc effect on movement. Moreover, transient inhibition of PI 3-kinase afforded neuroprotection against zinc-mediated toxicity. These data illustrate a novel mechanism that regulates mitochondrial trafficking in neurons and also suggest that mitochondrial trafficking may be closely coupled to neuronal viability.

Divergent consequences arise from metallothionein overexpression in astrocytes: Zinc buffering and oxidant-induced zinc release

Excessive accumulation of the heavy metal zinc is cytotoxic. As a consequence, cellular vulnerability to zinc‐induced injury may be regulated by the abundance of proteins that maintain intracellular free zinc concentrations ([Zn2+]i). In this study, we overexpressed the zinc‐binding protein metallothionein‐II (MT) in astrocytes to assess its impact as (1) an acute zinc buffering mechanism, and (2) an oxidant‐releasable zinc pool. Overexpression of MT in primary astrocyte cultures was accomplished using an adenoviral vector. Using the zinc‐sensitive fluorescent indicator mag‐fura‐2, we monitored [Zn2+]i after stimulating zinc influx or oxidant treatment. With MT overexpression, we observed an acute buffering effect manifested as a dampening of stimulus‐induced increases in [Zn2+]i. In contrast, we also saw enhanced zinc release with application of the sulfhydryl oxidizing agent 2,2′‐dithiodipyridine. These results indicate that overexpression of a zinc‐binding protein can quickly diminish [Zn2+]i following zinc influx, but elevate [Zn2+]i under conditions of oxidative stress, providing protective yet potentially endangering effects.

NXY-059 maintains Akt activation and inhibits release of cytochrome C after focal cerebral ischemia

Stroke is the third leading cause of death in the US, with a prevalence of 750,000 patients per year, and a social cost estimated at

Drugs to Treat Head and Neck Cancers: Mechanisms of Action

50 billion. Current therapeutics are targeted at restoring blood flow rather than on preventing the actual mechanisms associated with neuronal cell death. Here, we show that, following transient (2 h) middle cerebral artery occlusion (tMCAO) in male, Wistar rats, neuronal damage determined using MAP-2 staining increased progressively after the tMCAO. Notably, such neuronal degeneration was first associated with a decrease in p-Akt in both the focus and penumbra of the infarct region and, later with an increase in cytosolic cytochrome C levels in cortical neurons in the infarct area. These findings implicate that Akt alterations and consequent release of cytochrome C are involved in neuronal death. To further address this issue, NXY-059 (disodium 4-[(tert.-butylimino)methyl]benzene-1,3-disulfonate N-oxide) administered i.v. (30 mg/kg bolus, followed by 30 mg/kg/h infusion for up to 24 h), commencing 1 h after reperfusion, not only prevented the increase in infarct area but also attenuated the postreperfusion increase in neuronal cytosolic cytochrome C and the postperfusion decrease in neuronal p-Akt. Thus, NXY-059, by preventing mitochondrial cytochrome C release by maintaining activation of the Akt pathway, appears to protect neurons from damage after ischemia.

M3-subtype muscarinic receptor activation stimulates intracellular calcium oscillations and aldosterone production in human adrenocortical HAC15 cells

This chapter presents the anticancer agents used for the treatment of head and neck squamous cell carcinomas (HNSCC), emphasizing the mechanisms of action of the various drug classes. Current therapies for HNSCC can be broadly divided into four categories: (1) DNA damaging agents, (2) Antimetabolites that interfere with DNA synthesis, (3) Antimitotic agents that interfere with cell division, (4) Agents that target pathways whose dysregulation are critical for tumorigenesis, including apoptosis and angiogenesis. Agents from the first three groups interfere with cell division and are therefore fundamentally non-selective. Most of their significant adverse effects result from the damage they inflict on normal cells that divide or remodel rapidly. Targeted therapies in contrast have greater potential to selectively inhibit transformed cells while sparing normal tissues. All HNSCC therapies are affected by resistance mechanisms that decrease drug efficacy. Typical mechanisms of tumor resistance include reduced drug uptake, increased drug efflux, rapid metabolism, and overexpression/mutation of target enzymes and receptors. Resistance can be pre-empted using combination chemotherapy regimens in which several anticancer agents are given simultaneously. These agents are also used in multimodal therapies, i.e. as a complement to surgery and/or radiation. Indeed, most HNSCC is treated with multimodal therapy and combination chemotherapy. Intravenous injection is the typical route of administration, however a few can be given orally. We also discuss several compounds in various stages of investigation.

Detecting zinc and cadmium with fura ratiometric probes

A previous body of work in bovine and rodent models shows that cholinergic agonists modulate the secretion of steroid hormones from the adrenal cortex. In this study we used live-cell Ca2+ imaging to investigate cholinergic activity in the HAC15 human adrenocortical carcinoma cell line. The cholinergic agonists carbachol and acetylcholine triggered heterogeneous Ca2+ oscillations that were strongly inhibited by antagonists with high affinity for the M3 muscarinic receptor subtype, while preferential block of M1 or M2 receptors was less effective. Acute exposure to carbachol and acetylcholine modestly elevated aldosterone secretion in HAC15 cells, and this effect was also diminished by M3 inhibition. HAC15 cells expressed relatively high levels of mRNA for M3 and M2 receptors, while M1 and M5 mRNA were much lower. In conclusion, our data extend previous findings in non-human systems to implicate the M3 receptor as the dominant muscarinic receptor in the human adrenal cortex.

A reevaluation of neuronal zinc measurements: artifacts associated with high intracellular dye concentration.

Abstract. Fluorescent probes such as fura-2 and its relatives mag-fura-2 and fura-2FF are used to detect changes in intracellular ions such as Ca2+ and Mg2+. However, they are sufficiently non-selective to record changes in other species such as Zn2+, Cd2+, Fe2+ and other transition metals. In the present study we have characterized the responses of these three dyes to Zn2+ and Cd2+, in a buffered salt solution using spectrofluorophotometry. Fura-2, mag-fura-2 and fura-2FF generally reacted to increasing concentrations of Zn2+ or Cd2+ with a shift in the excitation spectra. We found that for a given probe the Zn2+ and Cd2+ excitation spectra were broadly similar, but isosbestic and peak wavelengths were useful in distinguishing between the two metals.