Mitochondrial inhibition and oxidative stress: reciprocating players in neurodegeneration.

Although the etiology for many neurodegenerative diseases is unknown, the common findings of mitochondrial defects and oxidative damage posit these events as contributing factors. The temporal conundrum of whether mitochondrial defects lead to enhanced reactive oxygen species generation, or conversely, if oxidative stress is the underlying cause of the mitochondrial defects remains enigmatic. This review focuses on evidence to show that either event can lead to the evolution of the other with subsequent neuronal cell loss. Glutathione is a major antioxidant system used by cells and mitochondria for protection and is altered in a number of neurodegenerative and neuropathological conditions. This review also addresses the multiple roles for glutathione during mitochondrial inhibition or oxidative stress. Protein aggregation and inclusions are hallmarks of a number of neurodegenerative diseases. Recent evidence that links protein aggregation to oxidative stress and mitochondrial dysfunction will also be examined. Lastly, current therapies that target mitochondrial dysfunction or oxidative stress are discussed.

Oxidative stress during energy impairment in mesencephalic cultures is not a downstream consequence of a secondary excitotoxicity.

Past studies have shown that inhibiting energy metabolism with malonate in mesencephalic cultures damages neurons by mechanisms involving N-methyl-D-aspartate receptors and free radicals. Overstimulation of N-methyl-D-aspartate receptors is known to produce free radicals. This study was, therefore, carried out to determine if N-methyl-D-aspartate receptor activation triggered by energy impairment was a significant contributor to the oxidative stress generated during energy inhibition. Exposure of mesencephalic cultures to malonate for the minimal time required to produce toxicity, i.e. 6h, resulted in an increase in the efflux of both oxidized and reduced glutathione, and a decrease in tissue levels of reduced glutathione. In contrast, exposure to 1mM glutamate for 1h caused an increased efflux of reduced glutathione, but no changes in intra- or extracellular oxidized glutathione or intracellular reduced glutathione. Blocking N-methyl-D-aspartate receptors with MK-801 (0.5 microM) during malonate exposure did not modify malonate-induced alterations in glutathione status or free radical generation as monitored by dihydrochlorofluorescein diacetate and dihydrorhodamine 123 fluorescence. In contrast, the increase in dihydrorhodamine fluorescence caused by glutamate was completely blocked by MK-801. Reduction of tissue glutathione with a 24h pretreatment with 10 microM buthionine sulfoxamine, as shown previously, greatly potentiated malonate-induced toxicity to dopamine and GABA neurons, but had no potentiating effect on toxicity due to glutamate. The findings indicate that although oxidative stress mediates damage due either to energy deprivation or excitotoxicity, N-methyl-D-aspartate receptor over-stimulation does not contribute significantly to the oxidative stress that is incurred during malonate exposure.

Action of the anti-ischemic agent ifenprodil on N-methyl-D-aspartate and kainate-mediated excitotoxicity.

The efficacy of ifenprodil to antagonize N-methyl-D-aspartate (NMDA) and kainate (KA)-induced acute excitotoxicity was evaluated in embryonic day 13 chick retina. Incubation with either 50 microM NMDA or KA produced a characteristic histological lesion and release of endogenous gamma-aminobutyric acid (GABA). Ifenprodil potently attenuated NMDA-induced GABA efflux by 80% (IC50, 1.26 microM). Histology showed protection of all but a subpopulation of amacrine neurons and processes even at 500 microM ifenprodil. MK-801 and CGS 19755, uncompetitive and competitive NMDA antagonists, respectively, protected all NMDA-sensitive amacrines, including the ifenprodil-resistant population, whilst CNQX, a non-NMDA glutamate receptor antagonist, was ineffective. Ifenprodil was less effective versus KA, requiring 10-100-fold higher concentrations to significantly attenuate GABA release. The potent antagonism of NMDA-mediated acute excitotoxicity by ifenprodil may explain its efficacy as an anti-ischemic agent. Ifenprodil does, however, leave unprotected a subpopulation of NMDA-susceptible neurons suggesting a heterogeneity in the NMDA receptor population.

Transferrin in chick retina: distribution and location during development.

Chick retinas from embryonic day 6 (E6) to 3 weeks post-hatching were examined for the presence and location of endogenous transferrin. Immunocytochemistry revealed that transferrin was differentially distributed in retinal layers. Furthermore, the pattern of transferrin distribution changed with developmental age. At day E6, transferrin was found in 2 distinct bands which were located in the area of the Müller cell end-feet. By day E9, additional regions of transferrin immunoreactivity could be found in the inner and outer plexiform layers (IPL, OPL) and the nerve fiber layer (NFL). These latter 3 bands (IPL, OPL and NFL) became more prominent from E9 until E17 as the synaptic layers and nerve fiber layer increased in density and maturation. Perikarya in the nuclear layers size, density and maturation. Perikarya in the nuclear layers were negative. At day E17 and later, the newly forming outer segments of photoreceptor cells were strongly reactive for transferrin while the somas of the photoreceptor cells, in the ONL, were negative. Retinas from chicks 1 day to 3 weeks post-hatching retained strong immunoreactivity for transferrin in the photoreceptor cell outer segments and OPL, lessened immunoreactivity in the IPL and loss of immunoreactivity in the NFL. Iron distribution in the retina for all ages examined showed only 2 bands that locally corresponded to the Müller cell end-feet. Iron stores were not found in the synaptic layers or photoreceptor cell outer segments. These studies suggest an iron storage function for retinal glia and a role for transferrin in neuronal development and differentiation.

Damage to dopaminergic nerve terminals in mice by combined treatment of intrastriatal malonate with systemic methamphetamine or MPTP.

The mechanisms involved in methamphetamine (METH)-induced damage to nigrostriatal dopaminergic neurons in experimental animals are unknown. We have examined the possibility that perturbations in energy metabolism contribute to METH-induced toxicity by investigating the effects of systemic METH treatment in mice which received a unilateral intrastriatal infusion of malonate, a metabolic inhibitor which decreases ATP levels. Malonate (1-4 mumol) produced a dose-dependent decrease in striatal dopamine (DA). The combined treatment of intrastriatal malonate with systemic METH resulted in greater damage to dopaminergic neurons than by METH or malonate treatment alone. In parallel studies, MPTP was administered to mice which received intrastriatal infusions of saline or malonate. Similar to results obtained with METH, decreases in striatal DA content and tyrosine hydroxylase (TH) activity were greatest in MPTP-treated mice infused with malonate. The present results lend credence to the hypothesis that METH-induced increases in energy utilization create a state of metabolic stress for DA neurons which may ultimately contribute to the neurodegenerative effects of METH. Moreover, the finding that combined malonate and MPTP treatment produced greater damage than either substance alone is consistent with the hypothesis that perturbations in energy metabolism contribute to the neuronal death produced by MPP+.

Treatment of mice with methamphetamine produces cell loss in the substantia nigra.

Studies were conducted to determine if treatment of mice with methamphetamine (METH) would produce a loss of dopaminergic cells in the substantia nigra. The number of TH+/Nissl-stained was significantly decreased in both Swiss-Webster (S-W) and C57bl mice (approx. cell loss of 40% and 45%, respectively) 5-8 days after treatment with METH. In these same mice there was a corresponding decrease in neostriatal dopamine (DA) content (90% and 92%, respectively). In parallel studies, treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produced similar neuropathological effects. The finding that nigral cell loss occurs after METH treatment indicates that the METH-treated mouse may be a very relevant model of Parkinson's disease (PD).

Transferrin can alter physiological properties of retinal neurons.

The role of transferrin as a possible neurotransmitter was examined in cultured chick retinal cells. Brief exposure to transferrin caused a dramatic and transient increase in intracellular calcium levels in approximately 20% of the total population of cultured retinal neurons. The increase in intracellular calcium was observed in cell bodies and neuronal processes. Electrophysiological analysis of a subset of the population, bipolar-like neurons, demonstrated that more than half of these cells responded to the application of transferrin with a transient membrane depolarization. Under voltage clamp conditions, the currents evoked by transferrin were similar to glutamate in that they both displayed non-linear voltage dependence. Furthermore, acute transferrin exposure resulted in a 200% increase in the amount of Na+ independent [3H]glutamate binding observed in these cultures. These results suggest that transferrin may function as a neurotransmitter or neuromodulator in the developing vertebrate nervous system.

Inhibition of striatal energy metabolism produces cell loss in the ipsilateral substantia nigra.

This study examined whether damage to dopamine (DA) nerve terminals via inhibition of energy metabolism in the striatum would result in the retrograde loss of cell bodies in the substantia nigra. Infusion of 2 micromol malonate into the left striatum of rats resulted in a 67% loss of striatal DA and a 40% loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. No change in the number of Nissl-positive-TH-negative neurons was observed. These findings demonstrate the retrograde destruction of DA cell bodies in the substantia nigra resulting from energy impairment at their terminal projection site.

Ultrastructural cytochemistry of monoamines in cultured human fetal sympathetic neurons.

The presence and storage of adrenergic neurotransmitter (monoamines) in cultured human fetal sympathetic neurons was investigated by chromate-dichromate cytochemistry, formaldehyde-induced fluorescence and potassium permanganate fixation. Monoamines were specifically identified in the neurons by the presence of an electron dense precipitate following cytochemical treatment. Reaction product was found in cell somas and processes in all chromate-dichromate treated cultures. The size range and morphology of the precipitate indicated a vesicular storage site within large dense core vesicles. Neurons fluoresced after treatment with formaldehyde vapors, further confirming the presence of monoamines. When potassium permanganate was employed as the fixative, occasional positive dense core vesicles were found but their frequency was greatly reduced from that seen in the chromate-dichromate treated cultures. These findings show that cultured human fetal sympathetic neurons retain an adrenergic phenotype during long-term serum-free culture. In addition, the storage site for the adrenergic neurotransmitter in the developing neuron is within large dense core vesicles. The lack of dense core vesicles in potassium permanganate fixed material is believed to be due to the depletion of monoamines during fixation.

In vivo vulnerability of dopamine neurons to inhibition of energy metabolism.

In vitro studies indicate that mesencephalic dopamine neurons are more vulnerable than other neurons to impairment of energy metabolism. Such findings may have bearing on the loss of dopamine neurons in Parkinson's disease, in which mitochondrial deficiencies have been identified, but would only be relevant if the selective vulnerability were maintained in vivo. To examine this, rats were stereotaxically administered various concentrations of the succinate dehydrogenase inhibitor, malonate (0.25-4 mumol), either into the left substantia nigra or striatum. One week following injection, dopamine and gamma-aminobutyric acid (GABA) levels in the mesencephalon and striatum were measured. Intranigral injection of malonate caused nigral dopamine and GABA to be comparably reduced at all doses tested. The 50% dose level for malonate vs. dopamine and GABA loss was 0.39 and 0.42 mumol, respectively. Tyrosine hydroxylase immunocytochemistry of the midbrains of rats which received an intranigral injection of malonate showed normal staining with 0.25 mumol malonate, but almost complete loss of tyrosine hydroxylase positive nigral pars compacta cells with 1 mumol malonate. Intrastriatal injection of malonate produced a loss of both tyrosine hydroxylase activity and dopamine. In contrast to what was seen in substantia nigra, there was a greater loss of dopamine than GABA in striatal regions nearest the injection site. In striatal regions most distal to the injection site, and which received the lowest concentration of malonate due to diffusion, dopamine levels were significantly reduced with all doses of malonate (0.5-4 mumol), whereas GABA levels were unaffected. Intrastriatal coinfusion of succinate along with malonate completely prevented the loss of dopamine and GABA indicating that succinate dehydrogenase inhibition was the cause of toxicity. These findings indicate that dopamine terminals in the striatum of adult rats are selectively more vulnerable than are the GABA neurons to a mild energy impairment.

Developmental differences in antagonism of NMDA toxicity by the polyamine site antagonist ifenprodil.

Antagonists of 4 distinct regulatory sites on the N-methyl-D-aspartate (NMDA) receptor were tested for their ability to attenuate NMDA-mediated acute excitotoxicity in isolated chick retina of various embryonic ages between days 11 and 19 in ovo. Acute excitotoxicity was monitored by histology and by release of endogenous gamma-aminobutyric acid (GABA) into the medium during 30 min of incubation with 50 microM NMDA. The uncompetitive PCP channel site antagonist, MK-801, the competitive antagonist, CGS 19755, and the strychnine-insensitive glycine site antagonist, 7-chlorokynurenate, completely blocked NMDA-induced cell swelling and increased GABA release at all ages tested. Potencies versus NMDA were MK-801 greater than CGS 19755 greater than 7-chlorokynurenate with IC50S of 0.02, 0.62, and 15 microM, respectively. NMDA antagonism by the polyamine site antagonist, ifenprodil, differed from other classes of antagonists in several respects. At the earlier embryonic ages tested (E12-13) ifenprodil provided differential protection; completely blocking somal and neuritic swelling in most but not all inner nuclear layer neurons and inner plexiform processes. In dose-response studies, ifenprodil attenuated the NMDA-induced increase in medium GABA at all ages tested with an Imax of 10 microM. Ifenprodil, however, showed a decreased ability to completely protect some NMDA-sensitive neurons. This was reflected both histologically and by GABA release. Maximal attenuation of NMDA evoked GABA release was 83, 80, 62 and 50% at days E12, 13, 15 and 19, respectively. Histologically, differential protection was seen at E12 and 13, in limited areas at E15, and was no longer present at E19.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamate and kainate are not directly toxic to developing amacrine cells: analysis in a Lucifer Yellow-labeled population.

Lucifer Yellow (LY)-labeled retinal amacrine cells were examined for toxin sensitivity to glutamate (Glu) or kainate (KA) and for high-affinity uptake (HAU) of Glu. In vitro, a 24-h exposure to either toxin caused a 50% loss of neurons with no loss of LY neurons. A 1-h exposure of intact retina to the toxins resulted in a two-fold increase in non-viable cells with no change in LY neurons. While histological damage within the retina was seen following toxin exposure, the LY population appeared unaffected. HAU in vitro was found in 5% of neurons but never colocalized with LY neurons. These studies show that Glu and KA do not directly affect the LY-labeled amacrine cells and support the hypothesis that their eventual loss may be an indirect consequence of toxin exposure.

Lucifer Yellow labeling of embryonic chick retinal amacrine cells in vitro.

Lucifer Yellow (LY) selectively labels embryonic chick amacrine cells from days 11 until 14 in vivo. Its usefulness as an in vitro marker was investigated. In vivo labeling and subsequent culturing was not possible due to dye leakage. Neurons, however, could be labeled at various times in vitro. The number of neurons labeled with LY in vitro was consistent with the number of neurons expected to be labeled and increased when selected areas of the retina known to be rich in LY-labeled neurons were used in culturing. Neurons plated at times when labeling was not found in vivo (Embryonic day 8, E8) began to label only at times that were equivalent to times when labeling was found in vivo (E10-E11). This suggests that the selectivity of labeling is preserved in vitro and that LY can be used as an in vitro marker for retinal amacrine cells.

Acute excitotoxicity in chick retina caused by the unusual amino acids BOAA and BMAA: effects of MK-801 and kynurenate.

beta-N-Oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) were tested for their ability to produce acute excitotoxicity in in embryonic chick retina. gamma-Aminobutyric acid (GABA) release and histology were monitored in retina treated with various concentrations of BOAA, BMAA, kainate (KA), N-methyl-D-aspartate (NMDA), or glutamate. BOAA and BMAA caused retinal lesions similar to those produced by the excitatory amino acids. BOAA was slightly less potent than KA, whereas BMAA had a potency similar to glutamate. BOAA, like KA and NMDA, caused a dose-dependent increase in GABA release. Addition of the NMDA antagonist (+)-MK-801, completely blocked acute excitotoxicity caused by NMDA or BMAA but was ineffective against KA or BOAA. Kynurenate, a nonspecific glutamate receptor antagonist, and DIDS, a Cl- channel blocker, were effective in blocking all agonist-induced toxicity. It is concluded that BOAA and BMAA cause excitotoxic damage in retina; BOAA induces toxicity through a non-NMDA type glutamate receptor and BMAA through the NMDA receptor.

Transferrin concentration and location during formation of chick retina: developmental correlates.

The amount of transferrin in chick retina was measured during development and compared to transferrin location seen immunocytochemically. Between embryonic day 6 (E6), and 5 days post hatching, two periods occur in which transferrin concentrations rise sharply and decline. During the first, transferrin concentration rises 5-fold between E6 and 10, then rapidly declines by E14. A second increase begins on E17 and peaks by E19-20. Immunocytochemical findings demonstrate that during the first rise in concentration, transferrin is located primarily in neuritic layers. Later in development, when levels again increase, newly forming photoreceptor outer segments are strongly transferrin positive. These findings are discussed in light of developmental events occurring during retinal maturation.

Activity at the GABA transporter contributes to acute cellular swelling produced by metabolic impairment in retina.

The role of the GABA transporter in acute toxicity in chick retina due to metabolic inhibition was investigated by the use of several substrate (nipecotic acid, THPO) and nonsubstrate (SKF 89976A, NO711) GABA transport inhibitors. Metabolic stress-induced acute toxicity in the retina is characterized by swelling of distinct populations of retinal neurons and selective release of GABA into the medium. Inhibitor concentrations were based on that needed to attenuate 14C-GABA uptake at its approximate KM concentration by > or = 70%. Under basal conditions, substrate, but not nonsubstrate, inhibitors increased extracellular GABA, but did not cause histological swelling per se. Under conditions of glycolytic inhibition, nonsubstrate, but not substrate, inhibitors significantly attenuated acute toxicity. Metabolic stress-induced acute toxicity was not altered by the GABA agonist muscimol, nor did muscimol reverse the protective effects of nonsubstrate transport inhibitors, suggesting that an increase in extracellular GABA during metabolic stress was not a component of the acute phase of toxicity. The results indicate that during metabolic inhibition, activity at the GABA transporter contributes to acute cellular swelling.

Characterization of intracellular elevation of glutathione (GSH) with glutathione monoethyl ester and GSH in brain and neuronal cultures: relevance to Parkinson's disease.

Parkinson's disease (PD) is associated with loss of total glutathione (GSH) which may contribute to progressive cell death. Peripheral GSH administration has been used clinically with reported benefits. Despite this, there is little specific information to characterize its cellular uptake or clearance, brain elevation with peripheral delivery or neuroprotective efficacy in PD models. The current study was carried out to provide this information using in vitro and in vivo approaches. In rat mesencephalic culture, the monoethyl ester of GSH (GEE), but not GSH (1-10 mM, 24 h) produced a dose-dependent elevation in GSH. The half-life for clearance was 10.14 h and was not different in cells depleted of GSH prior to loading. Elevation of GSH with GEE protected neurons from oxidative stress with H2O2 or metabolic stress with the complex I and II inhibitors MPP+ and malonate, respectively. To determine if peripheral administration of GEE could elevate brain GSH levels, rats were administered 0.1-50 mg/kg/day GEE via osmotic minipump either subcutaneously (sc) or via a cannula placed into the left cerebral ventricle (icv) for 28 days. Only central delivery of GEE resulted in significant elevations of brain GSH. Elevation of brain GSH by icv infusion of GEE was examined for its neuroprotective effects against chronic central delivery of MPP+. Infusion of 0.142 mg/kg/day MPP+ for 28 days caused a selective ipsilateral loss of striatal dopamine. Co-infusion of MPP+ with 10 mg/kg/day GEE significantly protected against striatal dopamine loss. These findings show that the ethyl ester of GSH but not GSH per se can elevate intracellular GSH, that brain elevation of GSH requires central delivery of the ethyl ester and that this elevation provides neuroprotection against oxidative stress or chronic mitochondrial impairment.