Neuroprotection and Neuroregeneration Strategies Using the rNAION Model: Theory, Histology, Problems, Results and Analytical Approaches.

Nonarteritic anterior ischemic optic neuropathy (NAION) is the most common cause of sudden optic nerve (ON)-related vision loss in humans. Study of this disease has been limited by the lack of available tissue and difficulties in evaluating both treatments and the window of effectiveness after symptom onset. The rodent nonarteritic anterior ischemic optic neuropathy model (rNAION) closely resembles clinical NAION in its pathophysiological changes and physiological responses. The rNAION model enables analysis of the specific responses to sudden ischemic axonopathy and effectiveness of potential treatments. However, there are anatomic and genetic differences between human and rodent ON, and the inducing factors for the disease and the model are different. These variables can result in marked differences in lesion development between the two species, as well as in the possible responses to various treatments. These caveats are discussed in the current article, as well as some of the species-associated differences that may be related to ischemic lesion severity and responses.

Oligodendrocyte death, neuroinflammation, and the effects of minocycline in a rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION).

Purpose: Optic nerve (ON) damage following nonarteritic anterior ischemic optic neuropathy (NAION) and its models is associated with neurodegenerative inflammation. Minocycline is a tetracycline derivative antibiotic believed to exert a neuroprotective effect by selective alteration and activation of the neuroinflammatory response. We evaluated minocycline's post-induction ability to modify early and late post-ischemic inflammatory responses and its retinal ganglion cell (RGC)-neuroprotective ability. Methods: We used the rodent NAION (rNAION) model in male Sprague-Dawley rats. Animals received either vehicle or minocycline (33 mg/kg) daily intraperitoneally for 28 days. Early (3 days) ON-cytokine responses were evaluated, and oligodendrocyte death was temporally evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. Cellular inflammation was evaluated with immunohistochemistry, and RGC preservation was compared with stereology of Brn3a-positive cells in flat mounted retinas. Results: Post-rNAION, oligodendrocytes exhibit a delayed pattern of apoptosis extending over a month, with extrinsic monocyte infiltration occurring only in the primary rNAION lesion and progressive distal microglial activation. Post-induction minocycline failed to improve retinal ganglion cell survival compared with the vehicle treated (893.14 vs. 920.72; p>0.9). Cytokine analysis of the rNAION lesion 3 days post-induction revealed that minocycline exert general inflammatory suppression without selective upregulation of cytokines associated with the proposed alternative or neuroprotective M2 inflammatory pathway. Conclusions: The pattern of cytokine release, extended temporal window of oligodendrocyte death, and progressive microglial activation suggests that selective neuroimmunomodulation, rather than general inflammatory suppression, may be required for effective repair strategies in ischemic optic neuropathies.

Synergistic Protection of Retinal Ganglion Cells (RGCs) by SARM1 Inactivation with CNTF in a Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy.

We evaluated whether inhibiting sterile alpha and (Toll/interleukin receptor (TIR)) motif-containing 1 (SARM1) activity protects retinal ganglion cells (RGCs) following ischemic axonopathy (rodent nonarteritic anterior ischemic optic neuropathy: rNAION) by itself and combined with ciliary neurotrophic factor (CNTF). Genetically modified SARM1(-) rats were rNAION-induced in one eye and compared against equivalently induced wild-type animals of the same background. Optic nerve (ON) diameters were quantified using optical coherence tomography (SD-OCT). RGCs were quantified 30 d post-induction using retinal stereology for Brn3a(+) nuclei. ON sections were analyzed by TEM and immunohistochemistry. SARM1(-)(-) and WT animals were then bilaterally sequentially rNAION-induced. One eye received intravitreal vehicle injection following induction; the contralateral side received CNTF and was analyzed 30 d post-induction. Inhibiting SARM1 activity suppressed axonal collapse following ischemic axonopathy. SARM1(-) animals significantly reduced RGC loss, compared with WT animals (49.4 ± 6.8% RGC loss in SARM1(-) vs. 63.6 ± 3.2% sem RGC loss in WT; Mann-Whitney one-tailed U-test, (p = 0.049)). IVT-CNTF treatment vs. IVT-vehicle in SARM1(-) animals further reduced RGC loss by 24% at 30 d post-induction, but CNTF did not, by itself, improve long-term RGC survival in WT animals compared with vehicle (Mann-Whitney one-tailed t-test; p = 0.033). While inhibiting SARM1 activity is itself neuroprotective, combining SARM1 inhibition and CNTF treatment generated a long-term, synergistic neuroprotective effect in ischemic neuropathy. Combinatorial treatments for NAION utilizing independent neuroprotective mechanisms may thus provide a greater effect than individual treatment modalities.

Adaptation of microplate-based respirometry for hippocampal slices and analysis of respiratory capacity.

Multiple neurodegenerative disorders are associated with altered mitochondrial bioenergetics. Although mitochondrial O(2) consumption is frequently measured in isolated mitochondria, isolated synaptic nerve terminals (synaptosomes), or cultured cells, the absence of mature brain circuitry is a remaining limitation. Here we describe the development of a method that adapts the Seahorse Extracellular Flux Analyzer (XF24) for the microplate-based measurement of hippocampal slice O(2) consumption. As a first evaluation of the technique, we compared whole-slice bioenergetics with previous measurements made with synaptosomes or cultured neurons. We found that mitochondrial respiratory capacity and O(2) consumption coupled to ATP synthesis could be estimated in cultured or acute hippocampal slices with preserved neural architecture. Mouse organotypic hippocampal slices oxidizing glucose displayed mitochondrial O(2) consumption that was well coupled, as determined by the sensitivity to the ATP synthase inhibitor oligomycin. However, stimulation of respiration by uncoupler was modest (<120% of basal respiration) compared with previous measurements in cells or synaptosomes, though enhanced slightly (to ∼150% of basal respiration) by acute addition of the mitochondrial complex I-linked substrate pyruvate. These findings suggest a high basal utilization of respiratory capacity in slices and a limitation of glucose-derived substrate for maximal respiration. The improved throughput of microplate-based hippocampal respirometry over traditional O(2) electrode-based methods is conducive to neuroprotective drug screening. When coupled with cell type-specific pharmacology or genetic manipulations, the ability to measure O(2) consumption efficiently from whole slices should advance our understanding of mitochondrial roles in physiology and neuropathology.

Approaches to Potentiated Neuroprotective Treatment in the Rodent Model of Ischemic Optic Neuropathy.

Nonarteritic anterior ischemic optic neuropathy (NAION) commonly causes sudden optic nerve (ON)-related vision loss. The rodent NAION model (rAION) closely resembles NAION in presentation and physiological responses. We identified early rAION-associated optic nerve head (ONH) inflammatory gene expression responses and the anti-inflammatory prostaglandin PGJ2's effects on those responses. We hypothesized that blocking pro-inflammatory prostaglandin (PGE2) production by inhibiting monoacylglycerol lipase or cyclooxygenase activity and co-administering PGJ2 would potentiate RGC survival following ischemic neuropathy. Deep sequencing was performed on vehicle- and PGJ2-treated ONHs 3d post-rAION induction. Results were compared against responses from a retinal ischemia model. Animals were treated with PGJ2 and MAGL inhibitor KML29, or PGJ2 + COX inhibitor meloxicam. RGC survival was quantified by stereology. Tissue PG levels were quantified by ELISA. Gene expression was confirmed by qPCR. PGJ2 treatment nonselectively reduced inflammatory gene expression post-rAION. KML29 did not reduce PGE2 1d post-induction and KML29 alone increased RGC loss after rAION. Combined treatments did not improve ONH edema and RGC survival better than reported with PGJ2 alone. KML29's failure to suppress PGE2 ocular synthesis, despite its purported effects in other CNS tissues may result from alternative PG synthesis pathways. Neither KML29 nor meloxicam treatment significantly improved RGC survival compared with vehicle. While exogenous PGJ2 has been shown to be neuroprotective, treatments combining PGJ2 with these PG synthesis inhibitors do not enhance PGJ2's neuroprotection.

Anti-NOGO Antibody Neuroprotection in a Rat Model of NAION.

Purpose: To evaluate the efficacy and mechanisms of anti-NOGO receptor monoclonal antibody 11C7mAb in a rat model of nonarteritic anterior ischemic optic neuropathy (rNAION). Methods: The rNAION was induced in one eye of 20 Long-Evans rats, which were studied in 10 groups of two rats, each group containing a sham rat receiving intravitreal injections of vehicle and a treatment rat receiving intravitreal injections of 11C7mAb. Fellow eyes served as naïve controls. The rats were tested using flash electroretinograms (ERGs), flash visual evoked potentials (VEPs), and optical coherence tomography (OCT). Thirty days after induction, they were euthanized, and the eyes were prepared for immunohistochemistry (two groups), hematoxylin and eosin staining (two groups) or transmission electron microscopy (TEM; six groups). Results: Ninety-five percent of the VEP amplitude was preserved in eyes treated with 11C7mAb, versus 69% in the control eyes. Immunohistochemistry revealed a large reduction in microglia and extrinsic macrophages with axon sparing. In addition to axon preservation, TEM also showed reduced extracellular debris and only slight myelin damage compared with the vehicle-treated animals. There were no significant differences in retinal ganglion cell counts, nor was there a difference in optic nerve swelling as measured by OCT. ERGs were used to exclude eyes with retinal vascular occlusions, an occasional complication of the induction technique. Conclusions: The 11C7mAb preserves optic nerve integrity and reduces inflammation in rNAION. Translational Relevance: This study evaluates the efficacy of an anti-NOGO receptor antibody in a rat model of NAION, a disorder that currently has no universally-acknowledged treatment.

Biomarkers of lesion severity in a rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION).

The rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION) is similar in many of its pathophysiological responses to clinical NAION. Like human NAION, there is significant variability in the severity of the lesion produced, and little is known of the parameters associated with rNAION induction severity or if pre- or early post-induction biomarkers can be identified that enable prediction of lesion severity and ultimate loss of retinal ganglion cells (RGCs). Adult male Sprague-Dawley outbred rats were evaluated for various parameters including physiological characteristics (heart rate, respiratory rate, temperature, hematocrit [Hct]), optic nerve head (ONH) appearance, pre- and post-induction mean diameter, and intravenous fluorescein and indocyanine green angiographic patterns of vascular leakage at 5 hours post-induction, performed using a spectral domain-optical coherence tomography (SD-OCT) instrument. Early changes were correlated with ultimate RGC loss by Brn3a (+) immunohistology. RGC loss also was correlated with the relative level of laser exposure. The severity of ONH edema 2d, but not 5hr, post induction was most closely associated with the degree of RGC loss, revealing a threshold effect, and consistent with a compartment syndrome where a minimum level of capillary compression within a tight space is responsible for damage. RGC loss increased dramatically as the degree of laser exposure increased. Neither physiological parameters nor the degree of capillary leakage 5hr post induction were informative as to the ultimate degree of RGC loss. Similar to human NAION, the rNAION model exhibits marked variability in lesion severity. Unlike clinical NAION, pre-induction ONH diameter likely does not contribute to ultimate lesion severity; however, cross-sectional ONH edema can be used as a biomarker 2d post-induction to determine randomization of subjects prior to inclusion in specific neuroprotection or neuroregeneration studies.

Real-time visualization of cytoplasmic calpain activation and calcium deregulation in acute glutamate excitotoxicity.

Although calpain (EC 3.4.22) protease activation was suggested to contribute to excitotoxic delayed calcium deregulation (DCD) via proteolysis of Na+/Ca2+ exchanger 3 (NCX3), cytoplasmic calpain activation in relation to DCD has never been visualized in real-time. We employed a calpain fluorescence resonance energy transfer substrate to simultaneously image calpain activation and calcium deregulation in live cortical neurons. A calpain inhibitor-sensitive decline in fluorescence resonance energy transfer was observed at 39 +/- 5 min after the occurrence of DCD in neurons exposed to continuous glutamate (100 microM). Inhibition of calpain by calpeptin did not delay the onset of DCD, recovery from DCD-like reversible calcium elevations, or cell death despite inhibiting alpha-spectrin processing by > 90%. NCXs reversed during glutamate exposure, the NCX antagonist KB-R7943 prolonged the time to DCD, and significant NCX3 cleavage following 90 min of glutamate exposure was not observed. Our findings suggest that robust calpain activation associated with acute glutamate toxicity occurs only after a sustained loss in calcium homeostasis. Processing of NCX3 or other calpain substrates is unlikely to be the primary cause of acute excitotoxicity in cortical neurons. However, a role for calpain as a contributing factor or in response to milder glutamate insults is not excluded.

Topical Trabodenoson Is Neuroprotective in a Rodent Model of Anterior Ischemic Optic Neuropathy (rNAION).

PURPOSE: Nonarteritic anterior ischemic optic neuropathy (NAION) is the leading cause of sudden optic nerve-related vision loss currently without effective treatment. We evaluated the neuroprotective potential of ocular (topical) delivery of trabodenoson, a selective A1 receptor mimetic, in a rodent model of NAION (rNAION). METHODS: Daily topical delivery of 3% trabodenoson or vehicle administered in both eyes 3 days prior to rNAION induction and for 21 days post induction. Retinal appearance and optic nerve head (ONH) edema was evaluated using spectral-domain optical coherence tomography (SD-OCT). Retinal function was evaluated before and after induction by ganzfeld electroretinography (ERG). Brn3a(+) retinal ganglion cells (RGCs) were quantified by stereology. Axonal ultrastructure was evaluated by electron microscopy. RESULTS: Trabodenoson-treated eyes had significantly reduced optic nerve (ON) edema compared with vehicle-treated eyes (ANOVA, P < 0.05). Electrophysiologically, there was a nonsignificant trend toward b-wave and oscillatory potential (OP) preservation in the trabodenoson-treated eyes. RGC counts were higher in trabodenoson-treated eyes compared to vehicle (74% versus 47% of the contralateral eye; two-tailed t-test; P = 0.01), as were ON axons. No overt morphologic differences in cell inflammation were observed between vehicle- and trabodenoson-treated ONHs, but trabodenoson-treated ONHs revealed increased expression of astrocyte-related neuroprotective responses. CONCLUSIONS: Trabodenoson preserves RGCs in the rodent NAION model. While previous clinical trials focused on trabodenoson's ocular antihypertensive effect, our data suggest trabodenoson's primary target may be both the retina and ONH. Selective adenosine A1 agonists may prove an appropriate neuroprotective adjunctive for ischemia-related ON diseases such as NAION and glaucoma. TRANSLATIONAL RELEVANCE: RGC and ON neuroprotection in ischemic neuropathies may be achievable by topical administration of A1 adenosine agonists rather than by simply relying on intraocular pressure reduction.

The IFN-beta and retinoic acid-induced cell death regulator GRIM-19 is upregulated during focal cerebral ischemia.

The induction of GRIM-19 has been shown to be essential for interferon-beta (IFN-beta)-induced and retinoic acid (RA)-induced tumor cell death. We have studied the localization and levels of GRIM-19 in IFN/RA-induced cell death in neural cells and in focal cerebral ischemia. Exposure to IFN/RA caused a approximately 15-fold increase in GRIM-19 protein levels and induced >50% cell death in human neuroblastoma SH-SY5Y cells. In rats subjected to permanent focal cerebral ischemia, increased oxidative stress, as well as increased GRIM mRNA levels (32-fold) and increased GRIM-19 (>50%) protein levels were noted in the ipsilateral (affected) hemisphere compared with the contralateral (unaffected) hemisphere. These results suggest that GRIM-19 may play a role in ischemia-induced neuronal cell death.

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION).

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

Dendrimers Target the Ischemic Lesion in Rodent and Primate Models of Nonarteritic Anterior Ischemic Optic Neuropathy.

INTRODUCTION: Polyamidoamine dendrimer nanoparticles (~ 4 nanometers) are inert polymers that can be linked to biologically active compounds. These dendrimers selectively target and accumulate in inflammatory cells upon systemic administration. Dendrimer-linked compounds enable sustained release of therapeutic compounds directly at the site of damage. The purpose of this study was to determine if dendrimers can be used to target the optic nerve (ON) ischemic lesion in our rodent and nonhuman primate models of nonarteritic anterior ischemic optic neuropathy (NAION), a disease affecting >10,000 individuals in the US annually, and for which there currently is no effective treatment. METHODS: NAION was induced in male Long-Evans rats (rNAION) and in one adult male rhesus monkey (pNAION) using previously described procedures. Dendrimers were covalently linked to near-infrared cyanine-5 fluorescent dye (D-Cy5) and injected both intravitreally and systemically (in the rats) or just systemically (in the monkey) to evaluate D-Cy5 tissue accumulation in the eye and optic nerve following induction of NAION. RESULTS: Following NAION induction, Cy-5 dendrimers selectively accumulated in astrocytes and circulating macrophages. Systemic dendrimer administration provided the best penetration of the ON lesion site when injected shortly after induction. Systemic administration 1 day post-induction in the pNAION model gave localization similar to that seen in the rats. CONCLUSIONS: Dendrimers selectively target the ischemic ON lesion after induction of both rNAION and pNAION. Systemic nanoparticle-linked therapeutics thus may provide a powerful, targeted and safe approach to NAION treatment by providing sustained and focused treatment of the cells directly affected by ischemia.

Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry.

Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by "bioenergetics-based profiling" may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.

Rodent anterior ischemic optic neuropathy (rAION) induces regional retinal ganglion cell apoptosis with a unique temporal pattern.

PURPOSE: Nonarteritic anterior ischemic optic neuropathy (NAION) results in optic nerve damage with retinal ganglion cell (RGC) loss. An NAION model, rodent anterior ischemic optic neuropathy (rAION), was used to determine AION-associated mechanisms of RGC death and associated regional retinal changes. METHODS: rAION was induced in male Wistar rats, and the retinas analyzed at various times after induction. RGCs were positively identified by both retrograde fluorogold labeling and brain-expressed X-linked protein-1/2 (Bex1/2) immunoreactivity. RGC death was analyzed by fluorescein-tagged annexin-V labeling (FITC-annexin-V), as well as by terminal nucleotide nick-end labeling (TUNEL). Retinal flatmount preparations enabled regional retinal analysis of labeled dying cells. Apoptosis pathway activation was confirmed by Western analysis, with an antibody that recognizes cleaved caspase-3. RESULTS: Post-rAION, RGCs die by apoptosis over a longer period than previously recognized. Cleaved caspase-3 immunoreactivity was greatest between 11 and 15 days. rAION-induced RGC death occurs regionally, with sparing of large contiguous regions of RGCs. CONCLUSIONS: rAION results in later RGC death than in traumatic optic nerve damage models. Apoptosis, measured by FITC-annexin, occurs maximally in the second to third week after infarct. Cleaved caspase-3 activation confirms that after rAION, RGCs undergo apoptosis by the caspase activation pathway. The regional pattern in dying RGCs after rAION implies that a measure of retinotopic organization occurs in the rodent optic nerve. The prolonged period from insult to death suggests that the window for successful treatment after ON infarct may be longer than previously recognized.

Postischemic oxidative stress promotes mitochondrial metabolic failure in neurons and astrocytes.

Oxidative stress and mitochondrial dysfunction have been closely associated in many subcellular, cellular, animal, and human studies of both acute brain injury and neurodegenerative diseases. Our animal models of brain injury caused by cardiac arrest illustrate this relationship and demonstrate that both oxidative molecular modifications and mitochondrial metabolic impairment are exacerbated by reoxygenation of the brain using 100% ventilatory O(2) compared to lower levels that maintain normoxemia. Numerous molecular mechanisms may be responsible for mitochondrial dysfunction caused by oxidative stress, including oxidation and inactivation of mitochondrial proteins, promotion of the mitochondrial membrane permeability transition, and consumption of metabolic cofactors and intermediates, for example, NAD(H). Moreover, the relative contribution of these mechanisms to cell injury and death is likely different among different types of brain cells, for example, neurons and astrocytes. In order to better understand these oxidative stress mechanisms and their relevance to neurologic disorders, we have undertaken studies with primary cultures of astrocytes and neurons exposed to O(2) and glucose deprivation and reoxygenation and compared the results of these studies to those using a rat model of neonatal asphyxic brain injury. These results support the hypothesis that release and or consumption of mitochondrial NAD(H) is at least partially responsible for respiratory inhibition, particularly in neurons.

Cyclosporin A increases mitochondrial calcium uptake capacity in cortical astrocytes but not cerebellar granule neurons.

Isolated brain mitochondria are a heterogeneous mixture from different cell types and these subsets may have differing sensitivities to Ca2+-induced membrane permeability transition (MPT) and to inhibition of the MPT by cyclosporin A (CsA). This study tested the hypothesis that mitochondria within primary cultures of astrocytes and neurons exhibit different energy-dependent Ca2+ uptake capacities and different degrees to which CsA increases their uptake capacity. Astrocytes and neurons were suspended in a cytosol-like medium containing respiratory substrates, ATP, and Mg2+ in the presence of digitonin to selectively permeabilize the plasma membrane. Uptake of added Ca2+ by mitochondria within the cells was measured by Calcium Green 5N fluorescent monitoring of the medium [Ca2+]. Permeabilized astrocytes had a fourfold higher Ca2+ uptake capacity, relative to neurons and a twofold higher content based on relative contents of mitochondria assessed by measurements of mitochondrial DNA and cytochrome oxidase subunit 1 protein. In astrocytes the Ca2+ uptake capacity was increased twofold by preincubation with 2-5 microM CsA, while in neurons CsA had no effect. Similar results were obtained using measurements of the effects of added Ca2+ on mitochondrial membrane potential. FK506, a drug similar to CsA but without MPT inhibitory activity, had no effect on either cell type. These results are consistent with the presence of a calcium-induced MPT in astrocytes, even in the presence of ATP, and indicate that the MPT in cerebellar granule neurons is resistant to CsA inhibition. Some of the protective effects of CsA in vivo may therefore be mediated by preservation of mitochondrial functional integrity within astrocytes.

Regulation of mitochondrial gene expression by energy demand in neural cells.

Mitochondrial DNA (mtDNA) encodes critical subunit proteins of the oxidative phosphorylation (OXPHOS) complex that generates ATP. This study tested the hypothesis that mitochondrial gene expression in neural cells is regulated by energy demand, as modified via stimulation of cellular sodium transport. Exposure of PC12S cells to the sodium ionophore monensin (250 nm) for 1-6 h caused a 13-60% decrease in cellular ATP (from 15 to 5 nmol per mg protein at 6 h). Levels of mitochondrial DNA-encoded mRNAs (mt-mRNAs) increased significantly (150%) within the first hour of exposure to monensin, and then decreased significantly (50%) at 3-4 h. Levels of mtDNA-encoded 12S rRNA and nuclear DNA-encoded OXPHOS subunit mRNAs were not significantly affected. Exposure of primary cerebellar neuronal cultures to the excitatory amino acid glutamate caused a similar rapid and significant increase followed by a significant decrease in cell mt-mRNA levels. The monensin-induced initial increase in mt-mRNA levels was abolished by pretreatment with actinomycin D or by reducing extracellular sodium ion concentration. The monensin-induced delayed reduction in mt-mRNA levels was accelerated in the presence of actinomycin D, and was accompanied by a 67% reduction in the half-life (from 3.6 to 1.2 h). Exposure of PC12S cells to 2-deoxy-d-glucose significantly decreased cellular ATP levels (from 14.2 to 7.1 nmol per mg protein at 8 h), and increased mt-mRNA levels. These results suggest a physiological transcriptional mechanism of regulation of mitochondrial gene expression by energy demand and a post-transcriptional regulation that is independent of energy status of the cell.

RNase-L regulates the stability of mitochondrial DNA-encoded mRNAs in mouse embryo fibroblasts.

Accelerated decrease in the levels of mitochondrial DNA-encoded mRNA (mt-mRNA) occurs in neuronal cells exposed either to the excitatory amino acid, glutamate or to the sodium ionophore, monensin, suggesting a role of mitochondrial RNase(s) on the stability of mt-mRNAs. Here we report that in mouse embryo fibroblasts that are devoid of the interferon-regulated RNase, RNase-L, the monensin-induced decrease in the half-life of mt-mRNA was reduced. In monensin (250 nM)-treated RNase-L(+/+) cells the average half-life of mt-mRNA, determined after termination of transcription with actinomycin D, was found to be 3h, whereas in monensin-treated RNase-L(-/-) cells the half-life of mt-mRNA was >6h. In contrast, the stability of nuclear DNA-encoded beta-actin mRNA was unaffected. Induction of RNase-L expression in mouse 3T3 fibroblasts further decreased the monensin-induced reduction in mt-mRNA half-life to 1.5h. The results indicate that the RNase-L-dependent decrease in mtDNA-encoded mRNA transcript levels occurs through a decrease in the half-life of mt-mRNA, and that RNase-L may play a role in the stability of mt-mRNA.

Bilobalide, a component of the Ginkgo biloba extract (EGb 761), protects against neuronal death in global brain ischemia and in glutamate-induced excitotoxicity.

In this study, the effect of bilobalide, a purified terpene lactone component of the Ginkgo biloba extract (EGb 761), and EGb 761 against ischemic injury and against glutamate-induced excitotoxic neuronal death was compared. In the case of ischemic injury, neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in the hippocampal regions of gerbils was measured. A significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons at 7-days of reperfusion after 5 min of transient global forebrain ischemia. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected hippocampal CA1 neurons against ischemia-induced neuronal death and reductions in COX III mRNA. In rat cerebellar neuronal cultures, addition of bilobalide or EGb 761 protected in a dose-dependent manner against glutamate-induced excitotoxic neuronal death [effective concentration (EC50) = 5 microg/ml (12 microM) forbilobalide and 100 microg/ml for EGb 761]. These results suggest thatboth EGb 761 and bilobalide protect against ischemia-induced neuronal death in vivo and glutamate-induced neuronal death in vitro by synergistic mechanisms involving anti-excitotoxicity, inhibition of free radical generation, scavenging of reactive oxygen species, and regulation of mitochondrial gene expression.