Manganese neurotoxicity and the role of reactive oxygen species.

Manganese (Mn) is an essential dietary nutrient, but an excess or accumulation can be toxic. Disease states, such as manganism, are associated with overexposure or accumulation of Mn and are due to the production of reactive oxygen species, free radicals, and toxic metabolites; alteration of mitochondrial function and ATP production; and depletion of cellular antioxidant defense mechanisms. This review focuses on all of the preceding mechanisms and the scientific studies that support them as well as providing an overview of the absorption, distribution, and excretion of Mn and the stability and transport of Mn compounds in the body.

Manganese transport via the transferrin mechanism.

Excessive manganese (Mn) uptake by brain cells, particularly in regions like the basal ganglia, can lead to toxicity. Mn(2+) is transported into cells via a number of mechanisms, while Mn(3+) is believed to be transported similarly to iron (Fe) via the transferrin (Tf) mechanism. Cellular Mn uptake is therefore determined by the activity of the mechanisms transporting Mn into each type of cell and by the amounts of Mn(2+), Mn(3+) and their complexes to which these cells are exposed; this complicates understanding the contributions of each transporter to Mn toxicity. While uptake of Fe(3+) via the Tf mechanism is well understood, uptake of Mn(3+) via this mechanism has not been systematically studied. The stability of the Mn(3+)Tf complex allowed us to form and purify this complex and label it with a fluorescent (Alexa green) tag. Using purified and labeled Mn(3+)Tf and biophysical tools, we have developed a novel approach to study Mn(3+)Tf transport independently of other Mn transport mechanisms. This approach was used to compare the uptake of Mn(3+)Tf into neuronal cell lines with published descriptions of Fe(3+) uptake via the Tf mechanism, and to obtain quantitative information on Mn uptake via the Tf mechanism. Results confirm that in these cell lines significant Mn(3+) is transported by the Tf mechanism similarly to Fe(3+)Tf transport; although Mn(3+)Tf transport is markedly slower than other Mn transport mechanisms. This novel approach may prove useful for studying Mn toxicity in other systems and cell types.

An analysis of the effects of Mn2+ on oxidative phosphorylation in liver, brain, and heart mitochondria using state 3 oxidation rate assays.

Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays--a measure of ATP production--under rapid phosphorylation conditions to explore sites of Mn(2+) inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn(2+) inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn(2+) inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F1F0 ATP synthase. In mitochondria fueled by either succinate or glutamate+malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn(2+) inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.

Cyclophilin D interacts with Bcl2 and exerts an anti-apoptotic effect.

Cyclophilin D (CypD) is a mitochondrial immunophilin and a key positive regulator of the mitochondrial permeability transition (MPT). Several reports have shown that CypD is overexpressed in various tumors, where it has an anti-apoptotic effect. Because the MPT is a cell death-inducing phenomenon, we hypothesized that the anti-apoptotic effect of CypD is independent of the MPT but is due to its interaction with some key apoptosis regulator, such as Bcl2. Our data indicate that CypD indeed interacts with Bcl2 as confirmed with co-immunoprecipitation, pulldown, and mammalian two-hybrid assays. A cyclophilin D inhibitor, cyclosporine A, disrupts the CypD-Bcl2 interaction. CypD enhances the limiting effect of Bcl2 on the tBid-induced release of cytochrome c from mitochondria, which is not mediated via the MPT. Gain- and loss-of-function experiments confirm that CypD has a limiting effect on cytochrome c release from mitochondria and that such an effect of CypD is cyclosporine A- and Bcl2-dependent. On a cellular level, overexpression or knockdown of CypD respectively decreases or increases cytochrome c release from mitochondria and overall cell sensitivity to apoptosis progressing via the "intrinsic" pathway. Therefore, we here describe a novel function of CypD as a Bcl2 collaborator and an inhibitor of cytochrome c release from mitochondria independent of the MPT. This function of CypD may explain the anti-apoptotic effect of this protein observed in various cancer cells. The fact that some tumors overexpress CypD suggests that this may be an additional mechanism of suppression of apoptosis in cancer.

Characteristics and possible functions of mitochondrial Ca(2+) transport mechanisms.

Mitochondria produce around 92% of the ATP used in the typical animal cell by oxidative phosphorylation using energy from their electrochemical proton gradient. Intramitochondrial free Ca(2+) concentration ([Ca(2+)](m)) has been found to be an important component of control of the rate of this ATP production. In addition, [Ca(2+)](m) also controls the opening of a large pore in the inner mitochondrial membrane, the permeability transition pore (PTP), which plays a role in mitochondrial control of programmed cell death or apoptosis. Therefore, [Ca(2+)](m) can control whether the cell has sufficient ATP to fulfill its functions and survive or is condemned to death. Ca(2+) is also one of the most important second messengers within the cytosol, signaling changes in cellular response through Ca(2+) pulses or transients. Mitochondria can also sequester Ca(2+) from these transients so as to modify the shape of Ca(2+) signaling transients or control their location within the cell. All of this is controlled by the action of four or five mitochondrial Ca(2+) transport mechanisms and the PTP. The characteristics of these mechanisms of Ca(2+) transport and a discussion of how they might function are described in this paper.

Nanoparticle (NP) uptake by type I alveolar epithelial cells and their oxidant stress response.

Mammalian cells take up nanoparticles (NPs) and some NPs increase ROS. We use imaging and measure ROS in parallel to evaluate NP-cell interactions with type I-like alveolar epithelial cells exposed to NPs at 1.2 µg/cm(2) . Titanium dioxide (Ti0(2)), gold (Au), silver (Ag), and manganese (Mn) were internalized by R3-1 cells; copper (Cu) NPs were observed at the cell surface only. TiO(2) and Au did not increase cell death but Mn and Cu did, with surviving cells recovering after initial Cu exposure. Ag NPs caused 80% of R3-1 cells to lift off the slides within one hour. Amplex Red was used to report H(2)O(2) production after exposure to 0.4 µg/cm(2) TiO(2), Au, Cu, Mn and Ag. TiO(2), Au, and Ag caused no significant increase in H(2)O(2) while Cu and Mn increased H(2)O(2). NPs that give up electrons, increase ROS production and cause cell death in R3-1 cells.

Role of cyclophilin D in the resistance of brain mitochondria to the permeability transition.

The mitochondrial permeability transition (MPT) is involved in both necrosis and apoptosis. Cyclophilin D (CypD) is an important component of the MPT. Brain mitochondria are more resistant to the MPT when compared to heart or liver mitochondria. We found that this increased resistance correlates with low expression of CypD in brain when compared to heart or liver. In newborn rats, sensitivity of brain mitochondria to the MPT and CypD expression are significantly higher than in mature animals. In an in vitro model of neuronal development, mitochondria in differentiated neuronal-like cells exert a higher calcium threshold toward MPT induction and express significantly less CypD when compared to undifferentiated precursor cells. Gain and loss of function experiments confirm the role of CypD in sensitivity to the MPT. Together our data indicate that the increased calcium threshold of brain mitochondria to the MPT correlates with low expression of CypD in brain; and that neuronal cells lose CypD during differentiation and become less sensitive to the MPT induction. This may be a protection mechanism that raises the threshold of brain tissue against injuries.

Speciation of manganese in cells and mitochondria: a search for the proximal cause of manganese neurotoxicity.

Recent studies of speciation of manganese (Mn) in brain mitochondria, neuron-like cells, and astrocytes are reviewed. No evidence is found for oxidation of Mn(2+) complexes to a Mn(3+) complex. The only evidence for any Mn(3+) complex is found in a spectrum essentially identical to that of mitochondrial manganese superoxide dismutase (MnSOD). While this does not prove that no Mn(3+) is produced in these tissues by oxidation of Mn(2+), it does suggest that formation of an active Mn(3+) complex by oxidation of Mn(2+) probably does not play as important a role in Mn toxicity as has been suggested earlier. Since these results suggest that we should look elsewhere for the proximal causes of Mn neurotoxicity, we consider the possibilities that Mn(3+) may be transported into the cell via transferrin and that Mn(2+) may inhibit Ca(2+)-activation and control of the rate of ATP production by oxidative phosphorylation.

Determining the oxidation states of manganese in NT2 cells and cultured astrocytes.

Excessive brain manganese (Mn) can produce a syndrome called "manganism", which correlates with loss of striatal dopamine and cell death in the striatum and globus pallidus. The prevalent hypothesis for the cause of this syndrome has been oxidation of cell components by the strong oxidizing agent, Mn(3+), either formed by oxidation of intracellular Mn(2+) or transported into the cell as Mn(3+). We have recently used X-ray absorption near edge structure spectroscopy (XANES) to determine the oxidation states of manganese complexes in brain and liver mitochondria and in nerve growth factor (NGF)-induced and non-induced PC12 cells. No evidence was found for stabilization or accumulation of Mn(3+) complexes because of oxidation of Mn(2+) by reactive oxygen species in these tissues. Here we extend these studies of manganese oxidation state to cells of brain origin, human neuroteratocarcinoma (NT2) cells and primary cultures of rat astrocytes. Again we find no evidence for stabilization or accumulation of any Mn(3+) complex derived from oxidation of Mn(2+) under a range of conditions.

Determining the oxidation states of manganese in PC12 and nerve growth factor-induced PC12 cells.

Excessive brain Mn can produce toxicity with symptoms resembling parkinsonism. This syndrome, called "manganism," correlates with loss of dopamine in the striatum and cell death in the striatum and globus pallidus. A common hypothesis is that cell damage in Mn toxicity is caused by oxidation of important cell components by Mn3+. Determination of the amount of Mn3+ present, under a range of conditions, in neuronal cells and brain mitochondria represents an important step in evaluating the "damage through oxidation by Mn3+ hypothesis." In an earlier paper we used X-ray absorption near-edge structure (XANES) spectroscopy to determine the amount of Mn2+ and Mn3+ in brain mitochondria under a range of conditions. Here we extend the study to investigate the evidence for formation of Mn3+ through oxidation of Mn2+ by ROS in PC12 cells and in PC12 cells induced with nerve growth factor (NGF) to display a phenotype more like that of neurons. Although the results suggest that very small amounts of Mn3+ might be present at low Mn levels, probably in Mn superoxide dismutase, Mn3+ is not stabilized by complex formation in these cells and therefore does not accumulate to detectable amounts.

Diazoxide-mediated preconditioning against apoptosis involves activation of cAMP-response element-binding protein (CREB) and NFkappaB.

Treatment of various types of cells with the mitochondrial ATP-sensitive K+ channel opener, diazoxide, preconditions cells to subsequent injuries and inhibits apoptosis. The mechanism of such preconditioning is not well understood. We have studied the effect of diazoxide pretreatment on mitochondrial morphology and function in HL60 cells and on susceptibility of these cells to apoptosis. We have found that diazoxide pretreatment inhibited etoposide-induced apoptosis and mitochondrial dysfunction. Diazoxide induced moderate mitochondrial swelling and increase in the cytosolic fraction of mitochondrial intermembrane proteins including cytochrome c without any significant effect on the oxidative phosphorylation function or membrane potential. Possibly as an adaptive response, total protein and mRNA levels of cytochrome c and of the anti-apoptotic Bcl-2 family member, Bcl-xl, increased. These effects coincided with activation of the transcription factors cAMP-response element-binding protein (CREB) and NFkappaB. The gene encoding cytochrome c carries the cAMP-response element (CRE), and the gene encoding Bcl-xl carries both the CRE and NFkappaB response elements. The inability of etoposide to trigger apoptosis in preconditioned cells was most likely because of prosurvival signaling by CREB and NFkappaB, which included up-regulation of cytochrome c and Bcl-xl. All described effects were reversed by a specific mitochondrial ATP-sensitive K+ channel inhibitor, 5-hydroxydecanoate, proving the specificity of the action of diazoxide. Preconditioning was also reversed by a specific NFkappaB inhibitor, SN50, proving the importance of this transcription factor for the phenomenon of preconditioning. CREB and NFkappaB were activated most likely in response to an observed elevation in cytosolic calcium following diazoxide treatment. We, therefore, conclude that diazoxide-mediated preconditioning against apoptosis involves activation of the pro-survival transcription factors CREB and NFkappaB.

Calcium and mitochondria.

The literature suggests that the physiological functions for which mitochondria sequester Ca(2+) are (1). to stimulate and control the rate of oxidative phosphorylation, (2). to induce the mitochondrial permeability transition (MPT) and perhaps apoptotic cell death, and (3). to modify the shape of cytosolic Ca(2+) pulses or transients. There is strong evidence that intramitochondrial Ca(2+) controls both the rate of ATP production by oxidative phosphorylation and induction of the MPT. Since the results of these processes are so divergent, the signals inducing them must not be ambiguous. Furthermore, as pointed out by Balaban [J. Mol. Cell. Cardiol. 34 (2002 ) 11259-11271], for any repetitive physiological process dependent on intramitochondrial free Ca(2+) concentration ([Ca(2+)](m)), a kind of intramitochondrial homeostasis must exist so that Ca(2+) influx during the pulse is matched by Ca(2+) efflux during the period between pulses to avoid either Ca(2+) buildup or depletion. In addition, mitochondrial Ca(2+) transport modifies both spatial and temporal aspects of cytosolic Ca(2+) signaling. Here, we look at the amounts of Ca(2+) necessary to mediate the functions of mitochondrial Ca(2+) transport and at the mechanisms of transport themselves in order to set up a hypothesis about how the mechanisms carry out their roles. The emphasis here is on isolated mitochondria and on general mitochondrial properties in order to focus on how mitochondria alone may function to fulfill their physiological roles even though the interactions of mitochondria with other organelles, particularly with endoplasmic and sarcoplasmic reticulum [Sci. STKE re1 (2004) 1-9], may also influence this story.

Determination of the oxidation states of manganese in brain, liver, and heart mitochondria.

Excess brain manganese can produce toxicity with symptoms that resemble those of Parkinsonism and causes that remain elusive. Manganese accumulates in mitochondria, a major source of superoxide, which can oxidize Mn2+ to the powerful oxidizing agent Mn3+. Oxidation of important cell components by Mn3+ has been suggested as a cause of the toxic effects of manganese. Determining the oxidation states of intramitochondrial manganese could help to identify the dominant mechanism of manganese toxicity. Using X-ray absorbance near edge structure (XANES) spectroscopy, we have characterized the oxidation state of manganese in mitochondria isolated from brain, liver, and heart over concentrations ranging from physiological to pathological. Results showed that (i) spectra from different model manganese complexes of the same oxidation state were similar to each other and different from those of other oxidation states and that the position of the absorption edge increases with oxidation state; (ii) spectra from intramitochondrial manganese in isolated brain, heart and liver mitochondria were virtually identical; and (iii) under these conditions intramitochondrial manganese exists primarily as a combination of Mn2+ complexes. No evidence for Mn3+ was detected in samples containing more than endogenous manganese levels, even after incubation under conditions promoting reactive oxygen species (ROS) production. While the presence of Mn3+ complexes cannot be proven in the spectrum of endogenous mitochondrial manganese, the shape of this spectrum could suggest the presence of Mn3+ near the limit of detection, probably as MnSOD.

Bcl-2 prevents abnormal mitochondrial proliferation during etoposide-induced apoptosis.

At the late stage of etoposide-induced apoptosis in HL-60 cells, marked by condensation of chromatin, mitochondria increase in numbers. There is also a drastic increase in mitochondrial DNA content. This increase in mitochondrial numbers and DNA content is an indicator of mitochondrial proliferation during apoptosis. These proliferating mitochondria exhibit abnormal morphology and are impaired, which is demonstrated by decrease in mitochondrial membrane potential and ATP content. The described apoptosis-induced abnormal mitochondrial proliferation was inhibited by overexpression of Bcl-2 protein, which also diminishes mitochondrial impairment. The increase in mitochondrial DNA levels correlated with elevated expression of one of the regulators of mitochondrial DNA replication, mtSSB. Our data suggest that proliferation of mitochondria may be an integral part of a cascade of apoptotic events.

Bcl-2 and tBid proteins counter-regulate mitochondrial potassium transport.

The mechanism of cytochrome c release from mitochondria in apoptosis remains obscure, although it is known to be regulated by bcl-2 family proteins. Here we describe a set of novel apoptotic phenomena--stimulation of the mitochondrial potassium uptake preceding cytochrome c release and regulation of such potassium uptake by bcl-2 family proteins. As a result of increased potassium uptake, mitochondria undergo moderate swelling sufficient to release cytochrome c. Overexpression of bcl-2 protein prevented the mitochondrial potassium uptake as well as cytochrome c release in apoptosis. Bcl-2 was found to upregulate the mitochondrial potassium efflux mechanism--the K/H exchanger. Specific activation of the mitochondrial K-uniporter led to cytochrome c release, which was inhibited by bcl-2. tBid had an opposite effect-it stimulated mitochondrial potassium uptake resulting in cytochrome c release. The described counter-regulation of mitochondrial potassium transport by bcl-2 and Bid suggests a novel view of a mechanism of cytochrome c release from mitochondria in apoptosis.

Parathyroid hormone-related peptide regulation of chick tibial growth plate chondrocyte maturation requires protein kinase A.

Regulation of phenotype in chick tibial growth plate chondrocytes (GPCs) by parathyroid hormone-related peptide (PTHrP) is facilitated via signaling through three pathways: protein kinase A (PKA), protein kinase C (PKC) and inositol-1,4,5-trisphosphate-induced Ca2+ transients. To establish the underlying signaling specificity for PTHrP-regulation of chondrocyte maturation, we examined the separate involvement of each of these three pathways in the PTHrP regulation of key hallmarks of GPC phenotype: stimulation of proliferation and proteoglycan synthesis and reduction of alkaline phosphatase activity and type X collagen expression. Mimicking the PTHrP stimulation either of PKC with 1-oleoyl 2-acetyl glycerol or of a Ca2+ pulse with 65 mM KCl did not lead to PTHrP-like effects on any of the four markers examined. Also, inhibition of PKC with myr-psiPKC or blockade of Ca2+ signals with an intracellular chelator did not inhibit PTHrP action. However, PKA activation with dibutyryl cAMP mimicked PTHrP and blockade of PTHrP stimulation of PKA with H-89 inhibited the regulatory action of the factor. These data demonstrate that although activation of PKC or Ca2+ signals is not required, the cylic AMP-dependent A kinase is required for PTHrP to regulate key hallmarks of GPC phenotype.

Growth plate chondrocyte maturation is regulated by basal intracellular calcium.

Among the cellular events that are associated with the process of endochondral ossification is an incremental increase in chondrocyte basal intracellular free Ca(2+) concentration ([Ca(2+)](i)) from 50 to 100 nM. To determine if this rise in [Ca(2+)](i) functionally participates in the maturational process of growth plate chondrocytes (GPCs), we examined its effect on several markers of hypertrophy, including annexin V, bone morphogenetic protein-6, type X collagen, and indian hedgehog. Expression of these genes was determined under conditions either where the Ca(2+) chelator EGTA was used to deplete extracellular Ca(2+) and lower [Ca(2+)](i) to < 50 nM or where the extracellular addition of 5 mM CaCl(2) was used to elevate [Ca(2+)](i) to > 100 nM. Although no effect on the expression of these genes was observed following treatment with 5 mM CaCl(2), 4 mM EGTA significantly inhibited their expression. This effect was recapitulated in sternal chondrocytes and was reversed following withdrawal of EGTA. Based on these findings, we hypothesized that the EGTA-induced suppression of these genes was mediated by a factor whose expression is responsive to changes in basal [Ca(2+)](i). Since EGTA mimicked the effect of parathyroid hormone-related peptide (PTHrP) on GPC maturation, we examined the effect of low [Ca(2+)](i) on PTHrP expression. Suggesting that low [Ca(2+)](i) suppression of hypertrophy was PTHrP-dependent in GPCs, (a) treatment with 4 mM EGTA increased PTHrP expression, (b) the EGTA effect was rescued by blocking PTHrP binding to its receptor with the competitive antagonist TIP(7-39), and (c) EGTA could mimic the PTHrP stimulation of AP-1 binding to DNA. Additionally, PTHrP promoter analysis identified a domain (-1498 to -862, relative to the start codon) involved with conferring Ca(2+) sensitivity to the PTHrP gene. These findings underscore the importance of cellular Ca(2+) in GPC function and suggest that PTHrP action in the growth plate is at least partially regulated by changes in basal [Ca(2+)](i).

Bcl-2 sensitive mitochondrial potassium accumulation and swelling in apoptosis.

During etoposide-induced apoptosis in HL-60 cells, cytochrome c release was associated with mitochondrial swelling caused by increased mitochondrial potassium uptake. The mitochondrial permeability transition was also observed; however, it was not the primary cause of mitochondrial swelling. Potassium uptake and swelling of mitochondria were blocked by bcl-2 overexpression. As a result, cytochrome c release was reduced, and apoptosis delayed. Residual cytochrome c release in the absence of swelling in bcl-2 expressing cells could be due to observed Bax translocation into mitochondria. This study suggests several novel aspects of apoptotic signaling: (1) potassium related swelling of mitochondria; (2) inhibition of mitochondrial potassium uptake by bcl-2; (3) co-existence within one system of multiple mechanisms of cytochrome c release: mitochondrial swelling and swelling-independent permeabilization of the outer mitochondrial membrane.