Macrolide antibiotics protect neurons in culture against the N-methyl-D-aspartate (NMDA) receptor-mediated toxicity of glutamate.

The immunosuppressive macrolide FK-506 has been shown to protect neurons in culture against glutamate excitotoxicity. This effect was attributed to the binding of immunosuppressants to calcineurin-inhibiting immunophilins. We now report that also the non-immunosuppressive macrolide antibiotics protect neurons in culture against NMDA- but not kainate-mediated excitotoxicity. The effect was structure-dependent: larger macrolide rings were more active. Macrolides did not affect the 3-(2-carboxypiperazin-4yl)-propyl-1-phosphonic acid (CPP) binding or the NMDA-mediated calcium influx.

NMDA-stimulated expression of BDNF mRNA in cultured cerebellar granule neurones.

The brain-derived neurotrophic factor (BDNF) affects the developing cerebellar granule cells. Exposure of 9-11-day-old primary cultures of rat cerebellar granule neurones for 3 h to a more depolarizing medium (additional 15-30 mM KCl) stimulated the release of glutamate and increased the BDNF mRNAs levels. This BDNF and mRNA upregulation was inhibited by dizocilpine (MK-801), the noncompetitive blocker of N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors, and mimicked by NMDA. Continuous (up to 5 h) culture exposure to non-toxic NMDA concentration resulted in a prolonged increase in BDNF mRNA expression and enhanced neuronal resistance to glutamate toxicity. The latter effect of NMDA was attenuated by cycloheximide, a protein synthesis inhibitor. The mechanisms responsible for NMDA-triggered BDNF upregulation and neuroprotection might be important in the compensatory response of brain to excitotoxicity.

Trans-azetidine-2,4-dicarboxylic acid activates neuronal metabotropic receptors.

The expression of metabotropic glutamate receptors (mGluRs) in primary cultures of cerebellar granule neurones can be: (i) modulated by the degree of depolarization during the culture period, rendering neurones differently sensitive to agonist-stimulated inositol phosphate (IP) hydrolysis; (ii) down-regulated by specific mGluR agonists. In this culture the new rigid glutamate analogue, (+/-)-trans-azetidine-2,4-dicarboxylic acid (t-ADA) and the known mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) stimulated IP formation in line with the depolarization-modified expression of mGluR1. However, the two compounds caused different patterns of mGluR down-regulation. The effects of t-ADA and 1S,3R-ACPD were also tested on transformed human embryonic kidney 293 cells transfected with mGluR1. Only 1S,3R-ACPD, but not t-ADA, stimulated IP hydrolysis, suggesting that t-ADA acts on a subtype of metabotropic receptors different from mGluR1. Hence, t-ADA might prove useful in differentiating the function of various mGluR subtypes.

Effects of nerve growth factor on neuronal plasticity of the kitten visual cortex.

1. The effect of intraventricular administration of nerve growth factor (NGF) by means of a cannula-minipump system was studied in kittens monocularly deprived during the critical period. The ocular dominance of area 17 neurones of NGF-treated and control kittens was determined by conventional extracellular recordings. The soma size of cells in A and A1 laminae of the lateral geniculate nucleus (LGN) was also evaluated in Cresyl Violet preparations. 2. Binocularly responsive neurons were found to be significantly more numerous in NGF-treated than in control kittens. The shrinkage of cells from the deprived LGN laminae normally observed in control kittens was prevented by NGF administration. 3. Following an initial period of monocular deprivation (MD) kittens subsequently treated with NGF showed a substantial recovery of functional binocular connections. 4. These findings indicate that the administration of NGF during the period of deprivation reduces the amblyopic effects of MD, while its administration to kittens with both eyes open following the initial deprivation promotes recovery of the deprived eye. 5. Neurotrophic factors may contribute to the regulation of experience-dependent modifications of synaptic connectivity in the visual cortex.

Functional evidence for a L-AP3-sensitive metabotropic receptor different from glutamate metabotropic receptor mGluR1.

The efficacy of mGluR agonists quisqualate and 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) in stimulating the inositol phosphate (IP) formation in primary cultures of cerebellar granule neurons correlated with mGluR1 mRNA expression and was affected by the medium KCl content. L-2-Amino-3-phosphonopropionic acid (L-AP3) mimicked the stimulatory action of mGluR agonists. Maximal stimulatory doses of mGluR agonist 1S,3R-ACPD and L-AP3 were additive, suggesting the action of L-AP3 on a receptor different from mGluR1. Indeed, in embryonic kidney 293 cells transfected with mGluR1 cDNA quisqualate and 1S,3R-ACPD but not L-AP3 stimulated the IP formation.

Carboxyl domain of glutamate receptor directs its coupling to metabolic pathways.

Of the six metabotropic glutamate receptors (mGluRs) only mGluR1 and mGluR5, which possess a large carboxyl terminal domain, are positively linked to phosphoinositide (PI) hydrolysis. We expressed a 3' deletion of mGluR1 alpha (mGluR1T) lacking the terminal 290 codons and the full length mGluR1 alpha cDNAs in human embryonic kidney 293 cells. Agonist stimulation of both mGluR1 alpha and mGluR1T stimulated PI hydrolysis. Glutamate activation of PI hydrolysis was reduced by pertussis toxin when mediated via mGluR1 alpha, while mGluR1T required the presence of extracellular Ca2+. Glutamate-mediated reduction of adenylyl cyclase stimulation by forskolin occurred only in mGluR1T-expressing cells. The results suggest that the carboxyl terminal extension directs the coupling of mGluR1 with different signal transduction pathways.

A subpopulation of cerebellar granule neurons in culture expresses a functional mGluR1 metabotropic glutamate receptor: effect of depolarizing growing conditions.

Homogeneous neuronal cultures of cerebellar granule neurons express different levels of metabotropic glutamate receptor mGluR1 mRNA depending on the concentration of KCl present during the culture period. We have studied the effect of KCl on mGluR1 expression at the single neuron level by measuring: i) the effect of mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid, tACPD, on intracellular free calcium concentration, [Ca2+]i; ii) the immunocytochemical quantitation of mGluR1 alpha protein. tACPD-induced increases in cytoplasmic free Ca2+ were pertussis toxin-sensitive. The number of tACPD-responding and mGluR1 alpha-positive neurons was higher in cultures grown in the presence of 15 mM than 25 mM KCl, but it never exceeded 50% of the cells. The measurement of basal intracellular free Ca2+ under the respective growing condition revealed a bigger subpopulation of cells with high basal Ca2+ concentration in neurons maintained in 25 mM KCl respect to 15 mM KCl. Hence, in primary cultures of cerebellar granule neurons, depolarization (possibly via Ca(2+)-regulated mechanisms) modulates the expression of a functional mGluR1 only in a subpopulation of cells and thus may account for a functional heterogeneity of morphologically similar cells.

Pathological phosphorylation causes neuronal death: effect of okadaic acid in primary culture of cerebellar granule cells.

We have investigated the role of protracted phosphatase inhibition and the consecutive protracted protein phosphorylation on neuronal viability. We found that in primary cultures of cerebellar granule neurons, the protracted (24-h) inhibition of the serine/threonine protein phosphatases 1 and 2A (EC 3.1.3.16) by treatment of the cultures with okadaic acid (OKA; 5-20 nM) caused neurotoxicity that could be inhibited by the protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) or by the previous down-regulation of the neuronal protein kinase C (PKC; ATP:protein phosphotransferase; EC 2.7.1.37). PKC was down-regulated by exposure of the cultures for 24 h to 100 nM phorbol 12-myristate 13-acetate (TPA). The effect of the drugs used in the viability studies on the pattern of protein phosphorylation was measured by quantitative autoradiography. In particular, the 50- and 80-kDa protein bands showed dramatic changes in the degree of phosphorylation: increase by OKA and brief TPA treatment; decrease by H7 or 24 h of TPA treatment; and inhibition of the OKA-induced increase by H7 or 24 h of TPA treatment. The results suggest that the protracted phosphorylation, in particular that mediated by PKC, may lead to neuronal death and are in line with our previous suggestion that prolonged PKC translocation is operative in glutamate neurotoxicity.

Distribution of protein gene product 9.5 (PGP 9.5) in the vertebrate retina: evidence that immunoreactivity is restricted to mammalian horizontal and ganglion cells.

Using light microscopic immunocytochemistry, we have studied the distribution of protein gene product 9.5 (PGP 9.5), a neuron-specific protein first extracted from human brain (Doran et al., '83:J. Neurochem. 40:1542-1547), in the vertebrate retina. Retinas were obtained from frog, chicken, rat, rabbit, cow, cat, dog, and human. No immunoreactivity was observed in frog and only a faint staining was present in chicken. In mammalian retinas, a strong positive reaction was restricted to horizontal and ganglion cells, with minor interspecies variations. Immunostaining was present throughout the cell body and the dendritic tree in horizontal cells. At the level of retinal ganglion cells, immunolabel was particularly abundant in cell bodies and axons forming the optic nerve. Only the main dendrites were stained, the remainder of the dendritic tree giving rise to a diffuse punctate reaction in the inner plexiform layer. In rats, displaced amacrine cells, which are known to contribute largely (40-50%) to the total neuronal population within the ganglion cell layer (Perry, '81: Neuroscience 6:931-944) were not immunoreactive, as demonstrated from (i) analysis of the morphology, cell size and cell density of immunoreactive neurons in wholemounts; (ii) colocalization of retrograde label and PGP 9.5 immunoreactivity in about 80% of ganglion cells after injection of peroxidase into the optic nerve; and (iii) reduction of immunoreactivity in the inner plexiform and ganglion cell layers following optic nerve transection. Western blot analysis of extracts from rabbit retinas indicated that the immunoreactive species is PGP 9.5 or a closely related molecule. Recent studies have demonstrated that PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase (Wilkinson et al., '89:Science 246:670-673). The present results, therefore, suggest that differences in the ubiquitination process exist between retinal neurons.

Expression of NGF receptor and NGF receptor mRNA in the developing and adult rat retina.

Nerve growth factor (NGF) has been recently found to rescue axotomized retinal ganglion cells (RGCs) of the adult rat from degeneration. Because the trophic effect of NGF involves a receptor-coupling event, the characterization and cellular localization of the NGF receptor (NGFR) in the retina are essential to understanding the possible specific action of NGF in this district of the central nervous system. We report here that the NGFR mRNA is expressed in fetal, neonatal, and adult rat retina. Using monoclonal antibody 192-IgG to immunoprecipitate and immunohistochemically identify NGFR, we also found that the NGFR from the retina has a molecular weight identical to that of the NGFR from PC12 cells. The NGFR is localized on RGCs and Müller cells. Finally, following ligation of the optic nerve, NGFR-immunopositive material was found to accumulate both distal and proximal to the site of ligation, suggesting that RGC axons anterogradely and retrogradely transport the NGFR. These data raise the possibility that NGF may play a specific role in rat RGCs.

Schwann cells promote the survival of rat retinal ganglion cells after optic nerve section.

Schwann cells (SCs) are known to play an important role for the regeneration of mammalian peripheral nerves. Their effect is likely due to the production of neuronotrophic and/or supportive factors. Here we study the effect of intraocular transplant of SCs on the survival of rat retinal ganglion cells (RGCs) after the intracranial section of the optic nerve. SCs were injected intraocularly in adult hooded rats. Surviving RGCs were retrogradely labeled with horseradish peroxidase applied to the proximal stump of the optic nerve. Results show that intraocular transplants of SCs promote the survival of a large number of RGCs for periods as long as 9 and 14 weeks after optic nerve section. In experimental retinae, surviving RGCs were 2- to 8-fold more numerous than in controls. This finding suggests that SCs are the source of factors that promote the survival of RGCs. Nerve growth factor is produced by SCs, and the intraocular injection of nerve growth factor has been previously shown to promote RGC survival. The rescuing effect of SCs on RGCs is greater than that obtained by intraocular injection of nerve growth factor. This greater effect may be due to the action of other neurotrophic factors produced by SCs or by transplanted SCs producing NGF in a sustained fashion.

Effect of NGF on the survival of rat retinal ganglion cells following optic nerve section.

The ability of NGF (2.5S subunit) to support the survival of adult rat retinal ganglion cells (RGCs) and optic nerve fibers after intracranial section of the optic nerve was investigated. NGF was injected intraocularly at a dose of 3 micrograms/injection every 2.3 d from the day of axotomy to analysis. Control animals received cytochrome c injections. The survival of RGCs was analyzed in whole-mounted retinas after either cresyl violet staining or labeling with HRP applied to the proximal stump of the optic nerve. Survival times were 5 and 7 weeks. Diameter distribution and number of myelinated optic nerve fibers were assessed in ultrathin cross sections of the optic nerve. We found that RGCs surviving axotomy were much more numerous following NGF treatment compared with controls. Large-size cells were, in particular, preserved by NGF treatment. The quantitative ultrastructural studies indicated that the number of myelinated optic nerve fibers at 5 and 7 weeks postaxotomy was significantly greater in the NGF group with respect to the cytochrome c group. In agreement with the results obtained at the level of the RGCs, large-diameter axons were, in particular, preserved. We conclude that NGF injected intraocularly is effective in promoting the survival of RGCs and optic nerve fibers at least for a period as long as 7 weeks after intracranial section of the optic nerve.