Margaret T. T. Wong-Riley

Cytochrome oxidase: an endogenous metabolic marker for neuronal activity

The brain is composed of a heterogeneous population of neurons whose physiological characteristics often elude morphological identification. The tight coupling between neuronal activity and oxidative energy metabolism forms the basis for the use of cytochrome oxidase as an endogenous metabolic marker for neurons. In the past decade, cytochrome oxidase histo- and cytochemistry have provided a window to view the regional, cellular and subcellular functional diversity among neurons. These methods have shown that the entire neuron is often not metabolically homogeneous; most of the oxidative activity is usually found in dendrites. They have also revealed the dynamic metabolic responses of developing and mature neurons to altered functional demands.

Therapeutic photobiomodulation for methanol-induced retinal toxicity

Methanol intoxication produces toxic injury to the retina and optic nerve, resulting in blindness. The toxic metabolite in methanol intoxication is formic acid, a mitochondrial toxin known to inhibit the essential mitochondrial enzyme, cytochrome oxidase. Photobiomodulation by red to near-IR radiation has been demonstrated to enhance mitochondrial activity and promote cell survival in vitro by stimulation of cytochrome oxidase activity. The present studies were undertaken to test the hypothesis that exposure to monochromatic red radiation from light-emitting diode (LED) arrays would protect the retina against the toxic actions of methanol-derived formic acid in a rodent model of methanol toxicity. Using the electroretinogram as a sensitive indicator of retinal function, we demonstrated that three brief (2 min, 24 s) 670-nm LED treatments (4 J/cm2), delivered at 5, 25, and 50 h of methanol intoxication, attenuated the retinotoxic effects of methanol-derived formate. Our studies document a significant recovery of rod- and cone-mediated function in LED-treated, methanol-intoxicated rats. We further show that LED treatment protected the retina from the histopathologic changes induced by methanol-derived formate. These findings provide a link between the actions of monochromatic red to near-IR light on mitochondrial oxidative metabolism in vitro and retinoprotection in vivo. They also suggest that photobiomodulation may enhance recovery from retinal injury and other ocular diseases in which mitochondrial dysfunction is postulated to play a role.

Histochemical localization of cytochrome oxidase in the hippocampus: Correlation with specific neuronal types and afferent pathways

Cytochrome oxidase was histochemically localized in the hippocampus and dentate gyrus of various species of mammals. The most intense staining was observed within stratum moleculare of areas CA1-3 and the outer molecular layer of the dentate gyrus, as well as the somatic and basal dendritic layers of CA3. These regions correspond to the synaptic terminal fields of major excitatory afferent pathways to the hippocampus. The somata of CA3 pyramidal cells and various interneurons were more intensely stained than CA1 pyramidal cells and dentate granule cells, and these levels appeared to correlate positively with their reported rates of spontaneous firing. At the electron-microscopic level, the highest concentrations of densely reactive mitochondria were localized within the distal apical dendritic profiles of principal cells (granule and pyramidal) and certain interneurons (pyramidal basket and stratum pyramidale interneurons). The specific layers in which these structures were found are known to receive intense excitatory input from the perforant pathway. High concentrations of reactive mitochondria were also observed within the somata and proximal dendrites of CA3 pyramidal cells and various interneurons, confirming our light-microscopic observations. These results demonstrated that not only can soma and dendrites of the same cell have disparate but distinct levels of cytochrome oxidase activity, but the pattern of reactivity within a neurons apical and basal dendrites, or even within specific dendritic segments of the same dendrite can be quite different. While the levels of somatic reactivity correlate with reported levels of spontaneous and/or synaptic activity, the degree of dendritic and somatic staining appeared to be more closely related to the intensity of convergent and/or pathway-specific excitatory synaptic input.

Effect of NASA Light-Emitting Diode Irradiation on Molecular Changes for Wound Healing in Diabetic Mice

OBJECTIVE The purpose of this study was to assess the changes in gene expression of near-infrared light therapy in a model of impaired wound healing. BACKGROUND DATA Light-Emitting Diodes (LED), originally developed for NASA plant growth experiments in space, show promise for delivering light deep into tissues of the body to promote wound healing and human tissue growth. In this paper we present the effects of LED treatment on wounds in a genetically diabetic mouse model. MATERIALS AND METHODS Polyvinyl acetal (PVA) sponges were subcutaneously implanted in the dorsum of BKS.Cg-m +/+ Lepr(db) mice. LED treatments were given once daily, and at the sacrifice day, the sponges, incision line and skin over the sponges were harvested and used for RNA extraction. The RNA was subsequently analyzed by cDNA array. RESULTS Our studies have revealed certain tissue regenerating genes that were significantly upregulated upon LED treatment when compared to the untreated sample. Integrins, laminin, gap junction proteins, and kinesin superfamily motor proteins are some of the genes involved during regeneration process. These are some of the genes that were identified upon gene array experiments with RNA isolated from sponges from the wound site in mouse with LED treatment. CONCLUSION We believe that the use of NASA light-emitting diodes (LED) for light therapy will greatly enhance the natural wound healing process, and more quickly return the patient to a preinjury/illness level of activity. This work is supported and managed through the Defense Advanced Research Projects Agency (DARPA) and NASA Marshall Space Flight Center-SBIR Program.

Light-emitting diode treatment reverses the effect of TTX on cytochrome oxidase in neurons.

Light close to and in the near-infrared range has documented benefits for promoting wound healing in human and animals. However, mechanisms of its action on cells are poorly understood. We hypothesized that light treatment with a light-emitting diode array at 670 nm (LED) is therapeutic in stimulating cellular events involving increases in cytochrome oxidase activity. LED was administered to cultured primary neurons whose voltage-dependent sodium channels were blocked by tetrodotoxin. The down-regulation of cytochrome oxidase activity by TTX was reverted to control levels by LED. LED alone also up-regulated enzyme activity. Thus, the results are consistent with our hypothesis that LED has a stimulating effect on cytochrome oxidase in neurons, even when they have been functionally silenced by TTX.

The effect of impulse blockage on cytochrome oxidase activity in the cat visual system.

Our previous studies have indicated that sensory deprivation in the developing and mature nervous system could bring about an adjustment in oxidative metabolism of neurons demonstrable by cytochrome oxidase (C.O.) histochemistry45,46,49. Since sensory deprivation potentially involves changes in several parameters, such as natural stimuli, action potentials and axoplasmic transport, we wished to know if the cessation of primary afferent impulses could produce enzymatic adjustment in the postsynaptic neurons. Tetrodotoxin (TTX), in a dosage determined by Stryker (personal communication) to block action potentials without blocking axoplasmic transport, was injected intravitreally into one eye of 10 adult cats every 3 days for total periods of 1, 2, 4, 5.5 or 6 weeks respectively. After perfusion, the brains were processed for C.O. histochemistry. The results indicated that: (1) a decreased level of C.O. activity was observed in the lateral geniculate (LGN) laminae innervated by the injected eye as well as in area 17 of all animals; (2) changes were discernible after 1 week, which was the earliest time examined, but became progressively more prominent up to 6 weeks; (3) within the LGN, the ipsilateral lamina A1 consistently appeared more severely affected than the contralateral lamina A; (4) the monocular segment of the affected lamina A exhibited a high level of enzyme activity, indicating that its oxidative metabolism might be sustained by other factor(s) or synaptic activity; (5) within the binocular representation of the striate cortex, a banding pattern of high and low C.O. activity in lamina IV became progressively more prominent with longer survivals. In cats subjected to 4 weeks of TTX treatment and 6 weeks of recovery, the enzymatic levels in the affected LGN and cortical laminae were comparable to that of the normal. This indicated that the dosage used apparently did not damage the neural pathways. Thus, impulse blockage can cause a decrease in the level of cytochrome oxidase activity in the affected postsynaptic neurons, and the maintenance of functional and enzymatic integrity of postsynaptic neurons in the adult is dependent upon viable presynaptic impulse conduction.

Nuclear Respiratory Factor 1 Regulates All Ten Nuclear-encoded Subunits of Cytochrome c Oxidase in Neurons *

Cytochrome c oxidase (COX) is one of only four bigenomic proteins in mammalian cells, having ten subunits encoded in the nuclear genome and three in the mitochondrial DNA. The mechanism of its bigenomic control is not well understood. The ten nuclear subunits are on different chromosomes, and the possibility of their coordinate regulation by the same transcription factor(s) deserves serious consideration. The present study tested our hypothesis that nuclear respiratory factor 1 (NRF-1) serves such a role in subunit coordination. Following in silico analysis of murine nuclear-encoded COX subunit promoters, electrophoretic mobility shift and supershift assays indicated NRF-1 binding to all ten promoters. In vivo chromatin immunoprecipitation assays also showed NRF-1 binding to all ten promoters in murine neuroblastoma cells. Site-directed mutagenesis of putative NRF-1 binding sites confirmed the functionality of NRF-1 binding on all ten COX promoters. These sites are highly conserved among mice, rats, and humans. Silencing of NRF-1 with RNA interference reduced all ten COX subunit mRNAs and mRNAs of other genes involved in mitochondrial biogenesis. We conclude that NRF-1 plays a significant role in coordinating the transcriptional regulation of all ten nuclear-encoded COX subunits in neurons. Moreover, NRF-1 is known to activate mitochondrial transcription factors A and B, thereby indirectly regulating the expressions of the three mitochondrial-encoded COX subunits. Thus, NRF-1 and our previously described NRF-2 prove to be the two key bigenomic coordinators for transcriptional regulation of all cytochrome c oxidase subunits in neurons. Possible interactions between the NRFs will be investigated in the future.

Near-infrared light via light-emitting diode treatment is therapeutic against rotenone- and 1-methyl-4-phenylpyridinium ion-induced neurotoxicity.

Parkinsons disease is a common progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Mitochondrial dysfunction has been strongly implicated in the pathogenesis of Parkinsons disease. Thus, therapeutic approaches that improve mitochondrial function may prove to be beneficial. Previously, we have documented that near-infrared light via light-emitting diode (LED) treatment was therapeutic to neurons functionally inactivated by tetrodotoxin, potassium cyanide (KCN), or methanol intoxication, and LED pretreatment rescued neurons from KCN-induced apoptotic cell death. The current study tested our hypothesis that LED treatment can protect neurons from both rotenone- and MPP(+)-induced neurotoxicity. Primary cultures of postnatal rat striatal and cortical neurons served as models, and the optimal frequency of LED treatment per day was also determined. Results indicated that LED treatments twice a day significantly increased cellular adenosine triphosphate content, decreased the number of neurons undergoing cell death, and significantly reduced the expressions of reactive oxygen species and reactive nitrogen species in rotenone- or MPP(+)-exposed neurons as compared with untreated ones. These results strongly suggest that LED treatment may be therapeutic to neurons damaged by neurotoxins linked to Parkinsons disease by energizing the cells and increasing their viability.

Neuronal expression of nuclear and mitochondrial genes for cytochrome oxidase (CO) subunits analyzed by in situ hybridization: comparison with CO activity and protein

Cytochrome oxidase (CO) is a mitochondrial energy-generating enzyme of the oxidative phosphorylation pathway. In neurons, CO activity varies among different cells and compartments (perikarya, dendrites, axons, and terminals) according to their physiological activity and metabolic requirements. Regulation of enzyme protein levels, rather than enzyme turnover number, largely accounts for local variations in CO activity (Hevner and Wong-Riley, 1989, 1990). In the present study, we examined how CO activity and protein levels are related to mitochondrial DNA (mtDNA) and CO subunit mRNA levels in neurons and neuronal compartments. Mammalian CO comprises 13 subunits (Kadenbach et al., 1983), of which three are encoded in mtDNA and 10 in nuclear genes. We studied one mitochondrial-encoded mRNA [subunit I (COI)], two nuclear- encoded mRNAs (COIV, COVIII), and mtDNA, using in situ hybridization to determine their distributions in monkey hippocampus, cerebellum, and primary visual cortex. We compared their distributions with those of CO activity and protein, determined by histochemistry and immunohistochemistry, respectively. In all regions, the local content of mtDNA was similar, but not identical, to the activity and amount of CO. Expression of COI mRNA was not proportional to mtDNA abundance or CO activity and protein, but instead was highest in cell bodies, lower in dendrites, and undetectable in axon terminals. COIV and COVIII mRNAs were detected exclusively in perikarya and proximal dendrites. Thus, the nuclear-encoded subunits of CO are probably translated mainly in neuronal cell bodies and allocated to other compartments posttranslationally. Regulation of CO was studied in two monkeys treated by monocular tetrodotoxin (TTX) injection, a procedure that blocks impulses from one eye. In those animals, cortical changes in CO activity were correlated with changes in mtDNA and in COI, COIV, and COVIII mRNA. Our results suggest that neuronal CO is synthesized and assembled mainly in cell bodies and indicate that both nuclear and mitochondrial CO subunit genes are regulated by neuronal activity.

Cytochrome oxidase in Alzheimer's disease: Biochemical, histochemical, and immunohistochemical analyses of the visual and other systems

Defects in oxidative metabolism have been implicated in Alzheimers disease (AD). The present study evaluated the level of cytochrome oxidase (C.O.), an indicator of neuronal oxidative capacity, in various brain regions of post-mortem AD and control patients. We found a statistically significant reduction in C.O. levels in all cortical areas examined, including the primary and secondary visual cortices. In addition, all layers of the dorsal lateral geniculate nucleus and sublaminae of the primary visual cortex in AD cases examined suffered a reduction in their relative C.O. activity and protein amount. Our results suggest a generalized suppression of oxidative metabolism throughout the cortex, as well as in a major subcortical visual center in AD. Such hypometabolism may form the basis for not only deficits in higher cortical functions, but also a variety of visual dysfunctions known to occur in AD.