Huan Ling Liang

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.

PRETREATMENT WITH NEAR-INFRARED LIGHT VIA LIGHT-EMITTING DIODE PROVIDES ADDED BENEFIT AGAINST ROTENONE- AND MPP+- INDUCED NEUROTOXICITY

Parkinsons disease (PD) is a movement disorder caused by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to nigrostriatal degeneration. The inhibition of mitochondrial respiratory chain complex I and oxidative stress-induced damage have been implicated in the pathogenesis of PD. The present study used these specific mitochondrial complex I inhibitors (rotenone and 1-methyl-4-phenylpyridinium or MPP(+)) on striatal and cortical neurons in culture. The goal was to test our hypothesis that pretreatment with near-infrared light (NIR) via light-emitting diode (LED) had a greater beneficial effect on primary neurons grown in media with rotenone or MPP(+) than those with or without LED treatment during exposure to poisons. Striatal and visual cortical neurons from newborn rats were cultured in a media with or without 200 nM of rotenone or 250 microM of MPP(+) for 48 h. They were treated with NIR-LED twice a day before, during, and both before and during the exposure to the poison. Results indicate that pretreatment with NIR-LED significantly suppressed rotenone- or MPP(+)-induced apoptosis in both striatal and cortical neurons (P<0.001), and that pretreatment plus LED treatment during neurotoxin exposure was significantly better than LED treatment alone during exposure to neurotoxins. In addition, MPP(+) induced a decrease in neuronal ATP levels (to 48% of control level) that was reversed significantly to 70% of control by NIR-LED pretreatment. These data suggest that LED pretreatment is an effective adjunct preventative therapy in rescuing neurons from neurotoxins linked to PD.

Bigenomic functional regulation of all 13 cytochrome c oxidase subunit transcripts in rat neurons in vitro and in vivo

Cytochrome c oxidase is a multisubunit, bigenomically encoded inner mitochondrial membrane protein. Its enzymatic activity and amount in the brain vary with metabolic demands, but the precise regulation of all 13 subunits to form a functional holoenzyme in a 1:1 stoichiometry is not well understood. To determine if all 13 subunit transcripts were coordinately regulated by functional alteration in neurons, cultured primary neurons were depolarized by potassium chloride for 5-24 h, or tetrodotoxin inactivated for 2-6 days. In vivo studies were done on rats monocularly enucleated for 4 days to 2 weeks. Expressions of cytochrome c oxidase subunit mRNAs were measured by real-time quantitative polymerase chain reaction. Results showed that in vitro, all 13 transcripts were significantly up-regulated after 5 h of depolarizing stimulation. With tetrodotoxin blockade, however, the three mitochondrial-encoded transcripts were down-regulated earlier than the 10 nuclear ones (2 days versus 4 days). In vivo, all three mitochondrial-encoded subunit mRNAs were also down-regulated earlier than the nuclear ones in deprived visual cortex (4 days versus 1 week after monocular enucleation). Cytochrome c oxidase activity and protein levels were significantly decreased in parallel after 4 days of deprivation in vitro and 1 week in vivo. Our results are consistent with a coordinated mechanism of up-regulation of all 13 transcripts in response to functional stimulation, but an earlier and more severe down-regulation of the mitochondrial transcripts than the nuclear ones in response to functional deprivation. Thus, the mitochondrial subunits may play a more important role in regulating cytochrome c oxidase protein amount and activity in neurons. Our results also point to the need of all 13 subunits to form a functional holoenzyme.

Ultrastructural study of depolarization‐induced translocation of NRF‐2 transcription factor in cultured rat visual cortical neurons

Nuclear respiratory factor (NRF)‐2 or GA‐binding protein is a potential transcriptional, bigenomic coordinator of mitochondrial and nuclear‐encoded subunits of cytochrome oxidase genes. It is composed of an α subunit that binds DNA and a β subunit that has the transactivating domain. Previously, we found that the level of NRF‐2 paralleled that of cytochrome oxidase under normal and functionally altered states. The goal of our present study was to increase the resolution to the ultrastructural level and to quantify changes before and after depolarizing stimulation. We used a pre‐embedding immunogold–silver method for the two subunits of NRF‐2 in cultured rat visual cortical neurons. NRF‐2α and β were normally located in both the nucleus and the cytoplasm. In the nucleus, both subunits were associated primarily with euchromatin rather than heterochromatin, consistent with active involvement in transcription. In the cytoplasm, they were associated mainly with free ribosomes and occasionally with the Golgi apparatus and the outer membrane of the nuclear envelope. Labelling was not found in the mitochondria, confirming the specificity of the antibodies. Neuronal depolarization by KCl for 5 h induced a six‐ to seven‐fold increase in the nuclear‐to‐cytoplasmic ratio of both subunits (P < 0.001) without increases in total labelling densities. These results strongly indicate that both NRF‐2α and NRF‐2β respond to increased neuronal activity by translocating from the cytoplasm to the nucleus, where they engage in transcriptional activation of target genes. Our results also indicate that the cytoplasmic to nuclear movement of transcription factors is a dynamic process induced by neuronal activity.

Activity-dependent transcriptional regulation of nuclear respiratory factor-1 in cultured rat visual cortical neurons.

Nuclear respiratory factor 1 is a transcription factor involved in the regulation of mitochondrial biogenesis by activating the transcription of subunit genes of cytochrome oxidase and other respiratory enzymes. Very little is known of its role in neurons. To determine if neuronal activity regulates nuclear respiratory factor 1 expression, cultured primary neurons from postnatal rat visual cortex were subjected to 20 mM KCl depolarizing treatment for 1, 3, 5, and 7 h, or exposed to 7 h of KCl followed by withdrawal for 1, 3, 5, and 7 h. Nuclear respiratory factor 1 expression was analyzed by immunoblots, immunocytochemistry, quantitative electron microscopy, real-time quantitative PCR, and in situ hybridization. Nuclear respiratory factor 1 protein was expressed at relatively low basal levels in both the nucleus, where it was associated primarily with euchromatin, and in the cytoplasm, where it was localized to free ribosomes and occasionally to the Golgi apparatus and the outer nuclear membrane. Depolarizing treatment progressively up-regulated both nuclear respiratory factor 1 protein and mRNA in a time-dependent manner, increasing above controls after 1 h and remaining high at 3, 5, and 7 h. Both nuclear and cytoplasmic mRNA levels increased with stimulation, and there was an apparent cytoplasmic-to-nuclear translocation of protein. Following the withdrawal of KCl, both nuclear respiratory factor 1 message and protein were significantly reduced after 1 h. The message returned to basal levels by 5 h and the protein by 7 h. These results strongly indicate that the expression and compartmental redistribution of nuclear respiratory factor 1 protein and mRNA in visual cortical neurons are dynamic processes tightly controlled by neuronal activity.

Nuclear respiratory factor 1 co‐regulates AMPA glutamate receptor subunit 2 and cytochrome c oxidase: tight coupling of glutamatergic transmission and energy metabolism in neurons

Neuronal activity, especially of the excitatory glutamatergic type, is highly dependent on energy from the oxidative pathway. We hypothesized that the coupling existed at the transcriptional level by having the same transcription factor to regulate a marker of energy metabolism, cytochrome c oxidase (COX) and an important subunit of alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid glutamate receptors, GluR2 (Gria2). Nuclear respiratory factor 1 (NRF‐1) was a viable candidate because it regulates all COX subunits and potentially activates Gria2. By means of in silico analysis, electrophoretic mobility shift and supershift, chromatin immunoprecipitation, and promoter mutational assays, we found that NRF‐1 functionally bound to Gria2 promoter. Silencing of NRF‐1 with small interference RNA prevented the depolarization‐stimulated up‐regulation of Gria2 and COX, and over‐expression of NRF‐1 rescued neurons from tetrodotoxin‐induced down‐regulation of Gria2 and COX transcripts. Thus, neuronal activity and energy metabolism are tightly coupled at the molecular level, and NRF‐1 is a critical agent in this process.

p38 mitogen‐activated protein kinase and calcium channels mediate signaling in depolarization‐induced activation of peroxisome proliferator‐activated receptor gamma coactivator‐1α in neurons

Peroxisome proliferator‐activated receptor gamma coactivator 1α (PGC‐1α) coactivates a number of transcription factors critical for mitochondrial biogenesis. Previously, we found that the expression of PGC‐1α is governed by neuronal activity, but the signaling mechanism is poorly understood. The present study aimed at testing our hypothesis that depolarizing activation of PGC‐1α in neurons is mediated by p38 mitogen‐activated protein kinase (MAPK) and calcium channels. Cultured primary neurons and N2a cells were depolarized with 20 mM KCl for varying times, and increases in PGC‐1α mRNA and protein levels were found after 0.5 and 1 hr of stimulation, respectively. These levels returned to those of controls after the withdrawal of KCl. Significantly, 15 min of KCl stimulation induced an up‐regulation of both p38 MAPK and phosphorylated p38 MAPK that were suppressed by 30 min of pretreatment with SB203580, a blocker of p38 MAPK that also blocked the up‐regulation of PGC‐1α by KCl. Likewise, 30 min of pretreatment with nifedipine, a calcium channel blocker, also prevented the up‐regulation of PGC‐1α mRNA and proteins by KCl. Furthermore, a knockdown of p38 MAPK with small interference hairpin RNA significantly suppressed PGC‐1α mRNA and protein levels. Our results indicate that both p38 MAPK and calcium play important roles in mediating signaling in depolarization‐induced activation of PGC‐1α at the protein and message levels in neurons.

Transcriptional coupling of synaptic transmission and energy metabolism: role of nuclear respiratory factor 1 in co-regulating neuronal nitric oxide synthase and cytochrome c oxidase genes in neurons.

Neuronal activity is highly dependent on energy metabolism; yet, the two processes have traditionally been regarded as independently regulated at the transcriptional level. Recently, we found that the same transcription factor, nuclear respiratory factor 1 (NRF-1) co-regulates an important energy-generating enzyme, cytochrome c oxidase, as well as critical subunits of glutamatergic receptors. The present study tests our hypothesis that the co-regulation extends to the next level of glutamatergic synapses, namely, neuronal nitric oxide synthase, which generates nitric oxide as a downstream signaling molecule. Using in silico analysis, electrophoretic mobility shift assay, chromatin immunoprecipitation, promoter mutations, and NRF-1 silencing, we documented that NRF-1 functionally bound to Nos1, but not Nos2 (inducible) and Nos3 (endothelial) gene promoters. Both COX and Nos1 transcripts were up-regulated by depolarizing KCl treatment and down-regulated by TTX-mediated impulse blockade in neurons. However, NRF-1 silencing blocked the up-regulation of both Nos1 and COX induced by KCl depolarization, and over-expression of NRF-1 rescued both Nos1 and COX transcripts down-regulated by TTX. These findings are consistent with our hypothesis that synaptic neuronal transmission and energy metabolism are tightly coupled at the molecular level.

Quantitative immuno-electron microscopic analysis of depolarization-induced expression of PGC-1α in cultured rat visual cortical neurons

Peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC- 1alpha) is a coactivator of nuclear receptors and other transcription factors that regulate several metabolic processes, including mitochondrial biogenesis, energy homeostasis, respiration, and gluconeogenesis. PGC-1alpha plays a vital role in stimulating genes that are important to oxidative metabolism and other mitochondrial functions in brown adipose tissue and skeleton muscles, but the significance of PGC-1alpha in the brain remains elusive. The goal of our present study was to determine by means of quantitative immuno-electron microscopy the expression of PGC-1alpha in cultured rat visual cortical neurons under normal conditions as well as after depolarizing stimulation for varying periods of time. Our results showed that: (a) PGC-1alpha was normally located in both the nucleus and the cytoplasm. In the nucleus, PGC-1alpha was associated mainly with euchromatin rather than heterochromatin, consistent with active involvement in transcription. In the cytoplasm, it was associated mainly with free ribosomes. (b) Neuronal depolarization by KCl for 0.5 h induced a significant increase in PGC-1alpha labeling density in both the nucleus and the cytoplasm (P<0.01). The heightened expression continued after 1 and 3 h of depolarizing treatment (P<0.01), but decreased from 5 h onward and returned to baseline level by 10 h. These results indicate that PGC-1alpha responds very early to increased neuronal activity by synthesizing more proteins in the cytoplasm and translocating them to the nucleus for gene activation. PGC-1alpha level in neurons is, therefore, tightly regulated by neuronal activity.

Quantitative immuno-electron microscopic analysis of nuclear respiratory factor 2 alpha and beta subunits: Normal distribution and activity-dependent regulation in mammalian visual cortex

The macaque visual cortex is exquisitely organized into columns, modules, and streams, much of which can be correlated with its metabolic organization revealed by cytochrome oxidase (CO). Plasticity in the adult primate visual system has also been documented by changes in CO activity. Yet, the molecular mechanism of regulating this enzyme remains not well understood. Being one of only four bigenomic enzymes in mammalian cells, the transcriptional regulation of this enzyme necessitates a potential bigenomic coordinator. Nuclear respiratory factor 2 (NRF-2) or GA-binding protein is a transcription factor that may serve such a critical role. The goal of the present study was to determine if the two major subunits of NRF-2, 2alpha and 2beta, had distinct subcellular distribution in neurons of the rat and monkey visual cortex, if major metabolic neuronal types in the macaque exhibited different levels of the two subunits, and if they would respond differently to monocular impulse blockade. Quantitative immuno-electron microscopy was used. In both rats and monkeys, nuclear labeling of alpha and beta subunits was mainly over euchromatin rather than heterochromatin, consistent with their active participation in transcriptional activity. Cytoplasmic labeling was over free ribosomes, the Golgi apparatus, and occasionally the nuclear envelope, signifying sites of synthesis and possible posttranslational modifications. The density of both subunits was much higher in the nucleus than in the cytoplasm for all neurons examined, again indicating that their major sites of cellular action is in the nucleus.