Hsiang-Shu Yin

The mechanisms of neuronal death produced by mitochondrial toxin 3-nitropropionic acid: the roles of N-methyl-D-aspartate glutamate receptors and mitochondrial calcium overload

Previous studies showed that 3-nitropropionic acid, an irreversible inhibitor of succinate dehydrogenase, produced neuronal death secondary to perturbed intracellular calcium homeostasis. However, the response of intramitochondrial calcium ([Ca(2+)](m)) to 3-nitropropionic acid remains unknown. In this study, we investigated the roles of and relationships among [Ca(2+)](m) overload, mitochondrial reactive oxygen species, and mitochondrial membrane depolarization in 3-nitropropionic acid-induced neuronal death. Following 1 mM 3-nitropropionic acid treatment on primary rat neuronal cultures, there was a gradual increase of [Ca(2+)](m) beginning at 2-4 h post 3-nitropropionic acid application, and a twofold increase of mitochondrial reactive oxygen species at 4 h. These were followed by mitochondrial membrane depolarization at 6-8 h post-treatment. By inhibiting [Ca(2+)](m) uptake, Ruthenium Red attenuated the production of reactive oxygen species, and prevented the 3-nitropropionic acid-induced mitochondrial membrane depolarization and 70% of apoptotic neuronal death (P<0.001). Inhibition of caspase activation attenuated the elevation of [Ca(2+)](m) (P<0.001), indicating that caspase activation plays a role in the elevation of [Ca(2+)](m). MK-801, an antagonist of N-methyl-D-aspartate (NMDA) glutamate receptors, prevented 3-nitropropionic acid-induced [Ca(2+)](m) elevation, caspase-3 activation, mitochondrial depolarization, and neuronal death. We conclude that the activation of NMDA glutamate receptor contributes to mitochondrial alterations induced by 3-nitropropionic acid. Inhibition of its activation and [Ca(2+)](m) overload with subsequent mitochondrial membrane depolarization can therefore attenuate the neuronal death induced by 3-nitropropionic acid.

In-Vitro Effects of Dexamethasone on Cellular Proliferation, Apoptosis, and Na+-K+-ATPase Activity of Bovine Corneal Endothelial Cells

Purpose: To assess the in-vitro effects of dexamethasone (DEX) on the proliferation, apoptosis, and Na+-K+-ATPase activity of bovine corneal endothelial cells. Methods: Bovine corneal endothelial cells were cultured with DEX ranging from 10−10 to 10−3 M. The effect of DEX on the proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay. Apoptosis and necrosis were detected by staining with fluorescein-conjugated annexin V and propidium iodide, followed by flow cytometry. The effect of DEX on Na+-K+-ATPase activity was evaluated using non-isotopic methods. Results: DEX did not affect cellular proliferation or induce apoptosis/necrosis from 10−10 to 10−5 M. At 10−4 and 10−3 M, DEX significantly decreased proliferation and increased apoptosis and/or necrosis. DEX significantly increased the Na+-K+-ATPase activity from 10−8 to 10−6 M, with the maximal effect at 10−6 M (p < 0.01); this effect was inhibited by RU38486, an antiglucocorticoid molecule. Conclusions: Bovine corneal endothelial cells express glucocorticoid receptor (GR) mRNA and protein. DEX decreases cell proliferation and induces cellular apoptosis and/or necrosis at high concentrations. DEX also increases the Na+-K+-ATPase activity at certain concentrations.

Toxicity of tunicamycin to cultured brain neurons: Ultrastructure of the degenerating neurons

Our previous study has shown that tunicamycin irreversibly downregulates the expression of GABAAR and causes cell death in cultured brain neurons by biochemical and light microscopic methods. In this study, we examined mechanisms underlying the degeneration of the neurons mainly employing electron microscopic analysis. Cultured neurons derived from embryonic chicken brains were incubated with 5 μg/ml of tunicamycin (TM) for 24 h, followed by continual incubation or removal of TM for additional 3 h or 24 h. Neurons treated with TM for 24 h showed dilated rough endoplasmic reticulum (rER), nuclear envelope and components of Golgi apparatus, in addition to the degranulation of rER and disaggregation of ribosomal rosettes. In neurons subjected to the prolonged incubation, some ribosomes reattached to the membranes of rER; the polyribosomes reappeared, and the swelling of Golgi apparatus subsided. However, the distention of rER persisted, and an uncommon spindle‐like structure appeared in the perikarya. This structure is implicated to involve the neuronal degeneration. Moreover, extracellular cell debris was increased with time of incubation. The ratio of the light neurons, defined as containing lower cytoplasmic matrix density than the untreated control, decreased from 28% at 3 h to 3% at 24 h after the removal of TM, and 45% at further 3 h to 6% at further 24 h incubation of TM, whereas dense neurons only appeared in the two 24 h groups, as 44% and 34%. The light neurons resemble necrotic cells, but the dense neurons exhibit distinct morphological features from necrosis and apoptosis. The gel electrophoresis assay revealed the absence of DNA fragmentation in all cultures. In addition, whole cell recordings exhibited a 40% decrease of the GABA‐elicited current in the neurons exposed to TM for 24 h. The results indicate irreversible toxicity of chronic TM treatment to the neurons and suggest differential mechanisms for the neuronal death among various populations of cells. It is evident that the N‐glycosylation plays a critical role for neuronal survival. J. Cell. Biochem. 74:638–647, 1999.

Changes in the Gene Expression of GABAA Receptor α1 and α2 Subunits and Metabotropic Glutamate Receptor 5 in the Basal Ganglia of the Rats with Unilateral 6-Hydroxydopamine Lesion and Embryonic Mesencephalic Grafts

By using an animal model of parkinsonism, we examined the expression of GABA(A) receptor (R) and metabotropic glutamate receptor (mGluR) 5 in the basal ganglia after transplantation with dopamine-rich tissue. The adult rats were unilaterally lesioned by the injection of 6-hydroxydopamine to their left medial forebrain bundles. At 5-10 weeks following the dopaminergic denervation, the levels of GABA(A)R in the left caudate-putamen and globus pallidus were about 20 and 16% lower than that of the right intact (control) sides, as shown by [3H]flunitrazepam binding autoradiography on the brain sections. However, the receptor density increased to around 132 and 130% of control levels in the entopeduncular nucleus and substantia nigra pars reticulata of the lesioned sides. Furthermore, in situ hybridization analysis exhibited parallel trends of changes in the levels of the GABA(A)R alpha1 and alpha2 subunit and mGluR5 mRNAs in the neurons of the brain regions with that of the proteins detected by the binding assay. A number of the rats 5 weeks postlesion were transplanted with the ventral mesencephalon of the embryonic rat into their left striata. Five weeks later, the changes in the [3H]flunitrazepam binding seemed to be recovered by approximately 50-63% on the grafted sides of the areas. Moreover, the transplantation appeared to produce a nearly complete reversal of the lesion-induced alterations in the levels of the mRNAs. Thus, the data indicate the mechanism of gene regulation for the modified expression of the receptors and could implicate the participation of the receptors in the pathogenesis of Parkinsons disease.

Extrinsic Inhibitory Innervation to Rubral Neurons in Rat Brain-Stem Slices

Synaptic connections between the neurons in the red nucleus (RN) and its extrinsic neurons were studied using rat brain-stem slices. Intracellular records were obtained from the RN neurons. Ipsilateral stimuli to areas in the dorsolateral mesencephalic reticular formation (DLMRF) or substantia nigra (SN) elicited monosynaptic hyperpolarizing postsynaptic potentials (PSPs) in about 95% of RN neurons recorded. The hyperpolarizing PSPs could be reversibly blocked by bicuculline, indicating that they were GABAA receptor-mediated-Cl(-)-inhibitory PSPs. The sites of most inhibitory synapses arising from DLMRF and SN are possibly located on the proximal half of the soma-dendritic membrane of RN neurons, according to the analysis of the IPSPs with Ralls model. In addition, tracing dyes were employed to examine the morphological pathways. After rhodamine B, a retrograde tracer, was applied to the RN in brain slices, the cell bodies of a number of neurons in DLMRF and SN were labeled. These labeled neurons were also immunopositive for glutamic acid decarboxylase (GAD) as revealed from double labeling with an anti-GAD antiserum. The anterograde tracer, tetramethylrhodamine dextran, was applied to the DLMRF or SN and taken up by many neurons in the areas. A portion of these cells extended their processes toward and terminated within the RN. Moreover, electron microscopic examination confirmed that the tetramethylrhodamine dextran-decorated synaptic terminals were present in the RN. The results indicate that the rubral neurons receive direct GABAA receptor-mediated inhibitory inputs from neurons in the DLMRF and SN, which may participate in modulation of rubral outputs.

Effects of axotomy on lamprey spinal neurons.

Abstract The central axonal processes of dorsal cells of larval sea lampreys were cut by a high spinal transection. Subsequent histologic changes included a loss of cytoplasmic basophilia, nuclear eccentricity, the development of a perinuclear basophilic shell, and a slight decrease in cell diameter and increase in nuclear diameter resulting in a 26% increase in the ratio of nucleus to cell diameter. These changes were maximal at 3 weeks and were related to the proximity of the cell to the transection site. Electrophysiologic changes included increased input resistance, increased voltage and current thresholds for spike initiation, decreased maximal rate of rise of action potentials, increased spike width, and increased spike overshoot. There was also an increase in axonal conduction velocity and a reduction in resting membrane potential. The electrophysiologic changes tended to lag behind the histologic changes by 1 to 2 weeks and were not clearly related to the proximity of the cell to the transection site. The cellular electrophysiologic changes could result from a decrease in the densities of sodium and potassium channels of the membrane, but there is no evidence yet to support this or any other specific hypothesis.

Electrophysiologic evidence of regeneration of lamprey spinal neurons.

Morphologic evidence has shown that the anteriorly projecting axons of giant interneurons (GIs) can regenerate after spinal transection in larval sea lampreys (19). In the present study, we showed that the regenerating neurites of GIs were electrically excitable. We also showed evidence for regeneration of descending afferent connections to GIs. Spinal cords were transected at the level of the cloaca. After at least 70 days recovery, GIs located 1.5 to 17.0 mm below the scar were impaled with microelectrodes. Stimulating electrodes were placed at various distances above the scar. Six of 13 GIs located 4 to 17 mm below the scar could be activated antidromically. For 1 GI, the rostralmost point of stimulation which elicited these responses was 13.5 mm above the scar. For the others, the range was 0.5 to 4.5 mm. Estimated average conduction velocity in regenerated neurites was 0.50 m/s compared with 1.94 m/s for the parent axon. Twelve GIs could be orthodromically activated by fixed-latency EPSPs. The most rostral point of stimulation that could elicit such responses was 0.5 to 8.5 mm above the scar. There was an inverse relationship between the farthest distance of stimulation and the distance of the GI from the scar. These findings are consistent with the hypothesis that regeneration of axons across a spinal transection is limited to neurons whose cell bodies are situated within 1 to 2 cm from the transection, and that regenerating neurites grow only a few millimeters beyond the scar.

Acrylamide disturbs the subcellular distribution of GABAA receptor in brain neurons

Mechanisms underlying the action of acrylamide on neurons were studied by monitoring the expression of GABAA receptor (R) in cultured brain neurons derived from chicken embryos. In situ trypsinization of the neurons and 3H‐flunitrazepam binding assay were employed to examine the subcellular distribution of GABAAR. A 3‐h exposure of the cultured neurons to 10 mM of acrylamide raised reversibly the proportion of intracellular (trypsin‐resistant) 3H‐flunitrazepam binding sites by about 48% and decreased cell surface binding 24% from respective control values, without altering total cellular binding and the affinity of the ligand. Moreover, the acrylamide treatment induced more intense perikaryal immunostaining of GABAAR α subunit proteins than that in control neurons but did not change the total level of cellular α immunostain, in accordance with the binding data. In the cell bodies of acrylamide‐treated neurons, the level of neurofilament‐200 kDa proteins was similar to control, whereas the tubulin protein content was significantly lowered approximately 51% from control, as revealed by quantifying the immunostained cytoskeletal elements. In addition, electron microscopic observations found reductions in the numbers of microtubules and neurofilaments in the perikarya of acrylamide‐treated neurons. As exhibited by the 3H‐leucine and 3H‐monosaccharide incorporation experiments, the exposure to acrylamide inhibited the rate of general protein synthesis in the culture by 21%, while the rate of glycosylation remained unaltered. Furthermore, in situ hybridization analysis showed that acrylamide did not modify the expression of GABAAR α subunit mRNAs. Taken together, these data suggest that acrylamide may downregulate the microtubular system and disintegrate neurofilaments, and thereby block the intracellular transport of GABAAR, resulting in the accumulation of intracellular receptors. J. Cell. Biochem. 85: 561–571, 2002.

Regulation of the subcellular distribution and gene expression of GABAA receptor by microtubules and microfilaments in cultured brain neurons

Mechanisms underlying the intracellular transport of γ‐aminobutyric acidA receptor (GABAAR) were examined in the cultured neurons derived from chicken embryo brains. In situ trypsinization of the cultures and 3H‐flunitrazepam (FNZ) binding assay were employed to determine the cell surface and intracellular distribution of the receptor. A 3‐h treatment of the cells with 1 μM of colchicine, a microtubule depolymerizer, reversibly raised the proportion of intracellular GABAAR density by about 36% and decreased that of the cell surface receptors by 18% from respective control values, whereas the 3‐h incubation with 2 μM of cytochalasin D, a microfilament disrupter, did not cause significant changes. These treatments failed to alter the total number of the 3H‐FNZ binding sites of the neurons and the affinity of the ligand. Moreover, the exposure to colchicine seemed to produce a stronger cytoplasmic immunostaining of the GABAAR α subunits in many neurons without affecting the total cellular level of the proteins, in accordance with the increased fraction of intracellular 3H‐FNZ binding. However, in the neurons exposed to cytochalasin D, there was an increase of around 28% in the total content of α1+51kDa proteins. In addition, the colchicine or cytochalasin D treatment inhibited approximately 21 or 18% of the rate of general protein synthesis in the culture. Notably, in situ hybridization assay showed that the GABAAR α1 or α2 mRNA was present in 92 ± 2% or 94 ± 2% of the cytochalasin D‐treated neurons, both of which were higher than 71 ± 2–74 ± 3% of the control and colchicine‐treated cells. The data suggest that by regulating the intracellular transport, the microtubular system participates in the maintenance of normal subcellular distribution of GABAAR in the neurons. By contrast, the organization of microfilaments may play a role in modulating the gene expression of GABAAR subunits. J. Cell. Biochem. 83: 291–303, 2001.

Changes in the microtubule proteins in the developing and transected spinal cords of the bullforg tadpole: Induction of microtubule-associated protein 2c and enhanced levels of Tau and tubulin in regenerating central axons

The distribution of tubulin, microtubule-associated protein 2 and Tau in the spinal cords of bullfrog tadpoles during development and after transection was studied. alpha-Tubulin or beta-tubulin immunoreactivity was present in the axons, neuronal perikarya and dendrites, as revealed by immunocytochemistry. The axonal staining intensity of the tubulins in the tadpoles was significantly stronger than that in the adult bullfrog. Microtubule-associated protein 2 immunoreactivity was localized largely to dendrites and expanded from distal to proximal dendrites with time; a high-molecular-weight microtubule-associated protein 2 was seen on the immunoblots of cord homogenates throughout development Tau1 stained mainly the axons. Two-dimensional gel immunoblotting disclosed that the tadpole contained a greater number of isoforms of Tau than the frog. Complete transection of the spinal cords of stage IV tadpoles was followed by regeneration of the damaged cord region. The levels of tubulin and Tau immunoreactivity in the regenerating axons of the ventral fasciculi were generally increased. Strikingly, microtubule-associated protein 2 immunoreactivity appeared in the regenerating axons and the chromatolytic cell bodies of axotomized motor neurons, paralleling the induction of microtubule-associated protein 2c in the regenerating cord segment shown by immunoblotting. The chromatolytic cell bodies were also markedly labeled by Tau1, whereas the high-molecular-weight microtubule-associated protein 2 diminished on the immunoblots, in accordance with the reduced level of staining for the dendrites. It is apparent that the changes in the cytoskeletal proteins in the regenerating axons mostly recapitulated their developmental patterns. Moreover, the data indicate a close relationship between tubulin and microtubule-associated proteins in axonal growth as well as providing evidence for similar molecular mechanisms underlying successful regeneration for central and peripheral axons.