Reaction of small heat-shock proteins to different kinds of cellular stress in cultured rat hippocampal neurons

Upregulation of small heat-shock proteins (sHsps) in response to cellular stress is one mechanism to increase cell viability. We previously described that cultured rat hippocampal neurons express five of the 11 family members but only upregulate two of them (HspB1 and HspB5) at the protein level after heat stress. Since neurons have to cope with many other pathological conditions, we investigated in this study the expression of all five expressed sHsps on mRNA and protein level after sublethal sodium arsenite and oxidative and hyperosmotic stress. Under all three conditions, HspB1, HspB5, HspB6, and HspB8 but not HspB11 were consistently upregulated but showed differences in the time course of upregulation. The increase of sHsps always occurred earlier on mRNA level compared with protein levels. We conclude from our data that these four upregulated sHsps (HspB1, HspB5, HspB6, HspB8) act together in different proportions in the protection of neurons from various stress conditions.     

Transneuronal degeneration in the Rolando substance of the primate spinal cord evoked by axotomy-induced transganglionic degenerative atrophy of central primary sensory terminals

Summary Transection of the sciatic nerve in Rhesus monkeys and the consequent transganglionic degenerative atrophy (TDA) of central terminals of primary afferents result in transneuronal degeneration of substantia gelatinosa (SG) cells. Severe degeneration is characterized by an increased electron density of the nucleus and by conspicuous shrinkage of the cytoplasm, mitochondrial swelling, dilation of cisterns of the rough-surfaced endoplasmic reticulum, accumulation of free ribosomes and an electron-dense material in the cytoplasm. In the mild form, dilation of cisternal elements of the endoplasmic reticulum, swollen mitochondria and accumulation of free ribosomes takes place. About 10% of SG cells in segment L₅ undergo the severe form whereas the rest shows signs of the mild form. Cytoplasmic alterations that occur during transneuronal degeneration seem to start at the level of subsurface cisterns. Dendrites and axons of transneuronally degenerating SG cells also show a conspicuous electron density. By analyzing the synaptic relationships of such darkened dendrites, connections in the upper dorsal horn can be deciphered. Modular units of the primary nociceptive analyzer that evaluate noxious and innocuous inputs on the basis of thin versus thick (AC/A) afferent activity and subjecting them to descending control appear to be recruited from structurally dispersed elements of synaptic glomeruli. These are arranged alongside dendritic processes of large antenna cells which relay impulses to projection cells of the spinothalamic tract.     

A review of the in vitro and in vivo neurochemical characterization of the NMDA/PCP/Glycine/Ion channel receptor macrocomplex

Conclusions Current neurochemical studies of the NMDA receptor macromolecular complex are yielding new insights into the interactions of the subunits of this complex and the associated potential clinical benefits of selective modulation of these subnits. Such studies offer the great potential for a new generation of pharmacotherapies for a wide range of CNS disorders, including stroke, a condition for which there is currently no effective pharmacological treatment. However, it is essential to understand that the first generation products in this area may not be optimal pharmacotherapies, such that haracterization of possible receptor subtypes and understanding the molecular biology of the component proteins of the receptor complex will be crucial in the design of the optimal pharmacological modulators of the NMDA receptor complex.Special issue dedicated to Dr. Erminio Costa     

Acetazolamide inhibits the recovery from triethyl tin intoxication: Putative role of carbonic anhydrase in dehydration of central myelin

The vacuolar degeneration of central myelin was produced in Sprague-Dawley rats by oral administration of triethyl tin. The wet weight of brain stems which seems to reflect the degree of accumulation of water increased during the administration of the toxin, whereas the activity of 2, 3-cyclic nucleotide 3-phosphodiesterase altered less remarkably. When TET was withdrawn from the drinking water, the rats showed a dramatic clinical improvement along with reduction in wet weight of brain stems. Treatment with acetazolamide following TET inhibited the clinical improvement and reduction in wet weight of brain stems. The present results indicates that central myelin has plasticity in recovering from the vacuolar degeneration by removing the accumulated fluid and carbonic anhydrase is possibly involved in the dehydration, of myelin in such a recovery phase.     

Structure of retinular cells in a Drosophila melanogaster visual mutant,rdgA, at early stages of degeneration

Summary A Drosophila visual mutant rdgA has photoreceptive cells which degenerate gradually after eclosion. Fine structure of the retinular cells of rdgA ^KS60 and rdgA ^K014 was studied during early stages of degeneration to determine the initial morphological defects. The retinular cells of these two alleles showed the following structural abnormality within 1 day after eclosion: (1) rhabdomeres were small and irregular in shape; (2) cisternae of the rough endoplasmic reticulum were more numerous than those in normal retinular cells; (3) submicrovillar cisternae were absent; and (4) lysosomes were fewer than normal. Three-dimensional reconstruction of serial sections of the ommatidia showed that the degeneration of mutant rhabdomeres proceeds more rapidly in regions remote from the nuclei. These results suggest that the process of turnover of rhabdomeric microvilli is abnormal in rdgA. We also confirmed an increase of lysosomes and destruction of cellular organelles, as reported by previous investigators at more advanced stages of degeneration.     

Energy metabolism and selective neuronal vulnerability following global cerebral ischemia

A short period of global ischemia results in the death of selected subpopulations of neurons. Some advances have been made in understanding events which might contribute to the selectivity of this damage but the cellular changes which culminate in neuronal death remain poorly defined. This overview examines the metabolic state of tissue in the post-ischemic period and the relationship of changes to the development of damage in areas containing ischemia-susceptible neurons. During early recirculation there is substantial recovery of ATP, phosphocreatine and related metabolites in all brain regions. However, this recovery does not signal restitution of normal energy metabolism as reductions of the oxidative metabolism of glucose are seen in many areas and may persist for several days. Furthermore, decreases in pyruvate-supported respiration develop in mitochondria from at least one ischemia-susceptible region at times coincident with the earliest histological evidence of ischemia-induced degeneration. These mitochondrial changes could simply be an early marker of irreversible damage but the available evidence is equally consistent with these contributing to the degenerative process and offering a potential site for therapeutic intervention.Submitted as an Overview article for the volume of Neurochemical Research in honor of Alan N. Davison.     

The involvement of ethanol in the free radical reaction of 6-hydroxydopamine

ESR spin trapping technique was used to detect and analyze free radical formation. When 6-hydroxydomine (6-OHDA) was incubated alone or in the presence of a free radical generating system (H₂O₂ and FeSO₄), hydroxyl free radicals were observed in a concentration-dependent manner. Glutathione was found to be the most effective scavenger of the ESR signal when compared with vitamin E or Mannitol. The addition of ethanol resulted in the formation of the pure hydroxyethyl free radicals. The amount of hydroxyethyl free radicals in the system was dependent upon the concentration of ethanol and the formation of hydroxyethyl free radicals correlated well with the extent of lipid peroxidation and the loss of enzymic activity of the membrane-bound (Na⁺, K⁺)-ATPase. We suggest that in the biological system ethanol may potentiate the neurotoxicity of 6-OHDA with the formation of hydroxyethyl free radicals, which are longer-lived and far more damaging to membranes that the hydroxyl radicals. These data lead us to further hypothesize that the neuronal degeneration caused by 6-OHDA and other compounds that generate free radicals could be potentiated in the presence of ethanol.     

Effect of Unilateral Decortication on Choline Acetyltransferase Activity in the Nucleus Basalis and Other Areas of the Rat Brain

Acetyl-coenzyme A: choline O-acetyltransferase (EC 2.3.1.6) (ChAT) enzyme activity was measured in the nucleus basalis and other microscopically identified brain areas at various times after unilateral cortical lesions were made in the rat. Initially, a significant decrease in ChAT activity was detected in the nucleus basalis ipsilateral to the lesion. However, after 120 days ChAT activity had apparently recovered, as levels of the enzyme at that time were not significantly different from control values. No changes in ChAT activity could be detected in any of the other brain areas similarly studied. The significance of these findings and their relationship to the morphological changes seen in neurones of the nucleus basalis after cortical lesions are discussed.     

Changes in Protein Content of Goldfish Optic Nerve During Degeneration and Regeneration Following Nerve Crush

Abstract: After the goldfish optic nerve was crushed, the total amount of protein in the nerve decreased by about 45% within 1 week as the axons degenerated, began to recover between 2 and 5 weeks as axonal regeneration occurred, and had returned to nearly normal by 12 weeks. Corresponding changes in the relative amounts of some individual proteins were investigated by separating the proteins by two-dimensional gel electrophoresis and performing a quantitative analysis of the Coomassie Brilliant Blue staining patterns of the gels. In addition, labelling patterns showing incorporation of [³H]proline into individual proteins were examined to differentiate between locally synthesized proteins (presumably produced mainly by the glial cells) and axonal proteins carried by fast or slow axonal transport. Some prominent nerve proteins, ON1 and ON2 (50–55 kD, pI ～6), decreased to almost undetectable levels and then reappeared with a time course corresponding to the changes in total protein content of the nerve. Similar changes were seen in a protein we have designated NF (～130 kD, pI ～5.2). These three proteins, which were labelled in association with slow axonal transport, may be neurofilament constituents. Large decreases following optic nerve crush were also seen in the relative amounts of α- and β-tubulin, which suggests that they are localized mainly in the optic axons rather than the glial cells. Another group of proteins, W2, W3, and W4 (35–45 kD, pI 6.5–7.0), which showed a somewhat slower time course of disappearance and were intensely labelled in the local synthesis pattern, may be associated with myelin. A small number of proteins increased in relative amount following nerve crush. These included some, P1 and P2 (35–40 kD, pIs 6.1–6.2) and NT (～50 kD, pI ～5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (～45 kD, pI ～4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (～60 and 65 kD respectively, pIs 4.5–5.0), that are carried mainly by slow axonal transport. Other proteins, including actin, that showed no net changes in relative amount (but presumably changed in absolute amount in direct proportion to the changes in total protein content of the nerve), are apparently distributed in both the neuronal and nonneuronal compartments of the nerve.     

Activation of Cl− Currents in Cultured Rat Retinal Pigment Epithelial Cells by Intracellular Applications of Inositol-1,4,5-triphosphate: Differences Between Rats with Retinal Dystrophy (RCS) and Normal Rats

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl⁻-currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 μm inositol-1,4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl⁻ currents, blockable by 1 mm 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). IP3 activated Cl⁻ currents in the presence of a high concentration of the calcium chelator BAPTA (10 mm) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10⁻⁵ m Ca²⁺ via the patch pipette also led to a transient activation of Cl⁻ currents. When the cells were preincubated in a bath solution containing thapsigargin (1 μm) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca²⁺ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl⁻-current activation induced by IP3 which could be reduced by nifedipine (1 μm). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3. Received: 17 July 1995/Revised: 31 January 1996