A role for gangliosides in astroglial cell differentiation in vitro.

Rat cerebral astroglial cells in culture display specific morphological and biochemical behaviors in response to exogenously added gangliosides. To examine a potential function for endogenous gangliosides in the processes of astroglial cell differentiation, we have used the B subunit of cholera toxin as a ganglioside-specific probe. The B subunit, which is multivalent and binds specifically to GM1 ganglioside on the cell surface, induced a classical star-shaped (stellate) morphology in the astroglial cells and inhibited DNA synthesis in a dose-dependent manner. The morphological response was massive and complete within 2 h, with an ED50 of 0.8 nM, and appeared to depend on the direct interaction of the B subunit with GM1 on the cell surface. A B subunit-evoked inhibition of DNA synthesis and cell division (ED50 = 0.2 nM) was observed when the cells were stimulated with defined mitogens, such as epidermal growth factor and basic fibroblast growth factor. Maximal inhibition approached 80% within 24 h. The effects of the B subunit were unrelated to increases in cAMP. These observations, taken together with previous studies, demonstrate that both endogenously occurring plasma membrane gangliosides and exogenously supplied gangliosides can influence the differentiative state (as judged by morphological and growth behaviors) of astroglial cells in vitro.

Nigericin-induced transient changes in rat-liver mitochondria.

Addition of nigericin to mitochondria oxidizing succinate in a choline- and Tris-supplemented, low-KCl medium leads to a transient matrix acidification, followed by a return of pHin to values very close to pHout. The initial inhibition of stimulated respiration is gradually relieved as pHin returns to higher values. Matrix realkalinization depends on the operation of the H+ pumps and on the electrogenic influx of cations and efflux of anions. The process leads to replacement of much of the matrix K+ by other cations. Throughout the acidification/realkalinization cycle delta mu H variations, if any, are small, even though there are profound changes in the relative contributions of its two components, delta psi and delta pH.

Tissue-specific modulation of the mitochondrial calcium uniporter by magnesium ions.

This paper analyzes the kinetics of the Ca2+ uniporter of mitochondria from rat heart, kidney and liver operating in a range of Ca2+ concentrations near the steady-state value (1-4 microM). Heart mitochondria exhibit the lowest activity, and physiological Mg2+ concentrations inhibit the mitochondrial Ca2+ uniporter by approx. 50% in heart and kidney, and by 20% in liver. At physiological Ca2+ and Mg2+ concentrations the external free Ca2+ maintained by respiring mitochondria in vitro is higher in heart and kidney with respect to liver mitochondria. This behaviour could represent an adaptation of different mitochondria to their specific intracellular environment.

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.

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.

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.

Photochemical stroke and brain-derived neurotrophic factor (BDNF) mRNA expression.

In situ hybridization and Northern blotting were used to study the expression of brain-derived neurotrophic factor (BDNF) mRNA in the rat brain following photochemical stroke. A focal thrombotic lesion of the sensorimotor cortex was produced by intravenously injecting the light-sensitive dye rose bengal and exposing the skull to a controlled beam of light. Four hours after the light exposure the level of BDNF mRNA was increased in the hippocampus and cortex ipsilateral and perifocal to the lesion. The stroke-induced BDNF mRNA increase was prevented by the non-competitive glutamate receptor blocker dizocilpine (MK-801). The results indicate that the activation of N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors is involved in the stroke-triggered stimulation of BDNF mRNA increase.

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.

Phospholipids can influence the interconversion of flat epithelial-like and stellate process-bearing astroglial cells in culture: relationships between molecular structure and biological activity.

Secondary cultures of neonatal rat astroglial cells, maintained in a serum-free, chemically defined medium were treated with several agents known to elevate intracellular cyclic AMP levels in these cells. Earlier studies had shown such drugs to induce a process-bearing (stellate) morphology in the astroglial cells, a response that is antagonized or reversed by the presence of exogenously added gangliosides. As a next step in understanding the basis for such an influence on cell morphologics, we have examined in more detail the molecular specificity of this response. In particular, a variety of phospholipids have been used in substitution of GM1 ganglioside. Natural phosphatidic acid (PA), which physicochemically displays lipophilic and hydrophilic bipolarity as does GM1, was fully active in mimicking the effects of GM1. The ED50 for the morphologic effect of PA was 10 microM, similar to that of GM1. Synthetic PAs (oleic, stearic, palmitic, myristic) had no effect up to 50 microM. Relatively long fatty acid chains were thus required for a PA effect. Other phospholipids including phosphatidylserine could not replace PA. Exposure of the cells to phospholipase D to generate endogenous PA from other phospholipids elicited the morphological response as well. These results indicate that the ability of exogenously supplied lipid molecules to modulate astroglial cell behaviors can be assigned, in functional terms, to a class of molecules having the appropriate balance (which includes PA and GM1) between their hydrophobic and hydrophilic domains.

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.

Depolarization- and agonist-regulated expression of neuronal metabotropic glutamate receptor 1 (mGluR1).

In established 8-12-day-old primary cultures of differentiated rat cerebellar granule neurons the level of metabotropic glutamate receptor 1 (mGluR1) mRNA and the sensitivity of cultures to the agonist-stimulated inositol phosphate (IP) formation was reversibly modified by changing the depolarizing properties of the medium, i.e. the medium KCl concentration. The mGluR1 mRNA content was suppressed by increasing the medium KCl content and elevated by decreasing it. The mGluR agonist quisqualate inhibited the mGluR1 expression. This is the first direct demonstration of a differential expression of neuronal mGluR1 in an established neuronal culture. The model can be used to study the molecular mechanism of neuronal plasticity.

Ganglioside-mediated protection from glutamate-induced neuronal death.

Glutamate, an excitatory amino acid (EAA), plays an important role in neuron to neuron signaling by binding to specific receptors. When, during neuronal firing, quanta of glutamate are released from the nerve terminal, they interact with the receptors for a few milliseconds and, thereafter, glutamate is promptly cleared by appropriate mechanisms. The neurotoxic action of glutamate arises from its capacity to trigger a pathophysiological chain of events when it acts continuously and abusively on its receptors (e.g., during cerebral edema associated with trauma, ischemia, stroke). In primary cultures of cerabellar granule neurons the abusive stimulation of EAA receptors by glutamate amplifies pathologicaly two early intracellular signals: free cytosolic Ca++ and the translocation of protein kinase C (PKC) from cytosol to neuronal membrane. Both of these signals persist unabated even after removal of glutamate from the incubation medium. Natural gangliosides and their semisynthetic derivatives protect neurons from glutamate toxicity by blocking the consequences of receptor abuse but they leave physiological responses to glutamate unaffected; hence they represent a prototype of a "receptor abuse dependent antagonist" (RADA).

Pathways for Ca2+ efflux in heart and liver mitochondria.

1. Two processes of Ruthenium Red-insensitive Ca2+ efflux exist in liver and in heart mitochondria: one Na+-independent, and another Na+-dependent. The processes attain maximal rates of 1.4 and 3.0 nmol of Ca2+.min-1.mg-1 for the Na+-dependent and 1.2 and 2.0 nmol of Ca2+.min-1.mg-1 for the Na+-independent, in liver and heart mitochondria, respectively. 2. The Na+-dependent pathway is inhibited, both in heart and in liver mitochondria, by the Ca2+ antagonist diltiazem with a Ki of 4 microM. The Na+-independent pathway is inhibited by diltiazem with a Ki of 250 microM in liver mitochondria, while it behaves as almost insensitive to diltiazem in heart mitochondria. 3. Stretching of the mitochondrial inner membrane in hypo-osmotic media results in activation of the Na+-independent pathway both in liver and in heart mitochondria. 4. Both in heart and liver mitochondria the Na+-independent pathway is insensitive to variations of medium pH around physiological values, while the Na+-dependent pathway is markedly stimulated parallel with acidification of the medium. The pH-activated, Na+-dependent pathway maintains the diltiazem sensitivity. 5. In heart mitochondria, the Na+-dependent pathway is non-competitively inhibited by Mg2+ with a Ki of 0.27 mM, while the Na+-independent pathway is less affected; similarly, in liver mitochondria Mg2+ inhibits the Na+-dependent pathway more than it does the Na+-independent pathway. In the presence of physiological concentrations of Na+, Ca2+ and Mg2+, the Na+-independent and the Na+-dependent pathways operate at rates, respectively, of 0.5 and 1.0 nmol of Ca2+.min-1.mg-1 in heart mitochondria and 0.9 and 0.2 nmol of Ca2+.min-1.mg-1 in liver mitochondria. It is concluded that both heart and liver mitochondria possess two independent pathways for Ca2+ efflux operating at comparable rates.

Intrinsic uncoupling of mitochondrial proton pumps. 1. Non-ohmic conductance cannot account for the nonlinear dependence of static head respiration on delta microH.

The passive membrane conductance LH1 of rat liver mitochondria has been measured and compared with the quantity nJesh/delta microHsh (n = H+/e stoichiometry; Jesh = rate of electron transfer in static head) over a delta microH range. The two curves approach each other only in the lower part of the range, while they sharply diverge at large values of delta microH. Thus nJesh/delta microHsh cannot be considered to be a measure of LH1 in the upper delta microH region. Only a fraction of the static head electron flow is accounted for by futile proton cycling via leaks. Contaminating open membrane fragments or completely leaky mitochondria can be responsible for only a small part of the residual rate of oxygen consumption. We conclude that a large part of static head respiration must have yet another cause and propose it to be intrinsic uncoupling of the respiratory chain enzymes.

Inhibition of DNA synthesis in C6 glioma cells following cellular incorporation of GM1 ganglioside and choleragenoid exposure.

The B subunit of cholera toxin, which is multivalent and binds specifically to GM1 ganglioside on the cell surface, has previously been used as a ganglioside-specific probe to regulate DNA synthesis in thymocytes and fibroblasts. To explore in more detail this growth-regulatory action of gangliosides, C6 glioma cells (which are GM1 ganglioside deficient) were used as a model system. When cultures of C6 cells were first treated with GM1, followed by exposure to the B subunit, proliferation was inhibited, as measured by 3H-labeled thymidine incorporation into DNA. Pretreatment of the cells with 50 microM GM1 for 15 min (followed by washing with fetal calf serum) and incubation with 1 microgram/ml of B subunit for 21 h was sufficient to reduce DNA synthesis to 15% of control values (and confirmed by autoradiographic analysis), although maximal inhibition could be achieved with as little as 30 min exposure to B, followed by washing. Furthermore, the B subunit inhibited the response of the C6 cells to basic fibroblast growth factor only following GM1 pretreatment. The B subunit-induced inhibition of DNA synthesis was specific for the ganglioside GM1, and was unrelated to increases of cyclic AMP. These results demonstrate that cell-incorporated GM1 ganglioside may act as a receptor capable of undergoing a specific ligand interaction, subsequently affecting molecular processes at the nuclear level.

Delayed increase of Ca2+ influx elicited by glutamate: role in neuronal death.

The mechanism of delayed neurotoxicity, triggered by glutamate, was studied in 7-8-day-old primary cultures of rat cerebellar granule cells. Treatment of cultures for 15 min with 50 microM glutamate in Mg2+ -free medium, followed by removal of the excitoxin, resulted in neuronal death, which started to appear 2-3 hr after the termination of glutamate treatment. The number of dead neurons increased gradually in the next few hours and 80-85% of neurons were found dead 24 hr later. Antagonists of N-methyl-D-aspartate-sensitive glutamate receptors (phencyclidine) or 1.2 mM MgCl2, but not the antagonist of N-methyl-D-asparatate-insensitive glutamate receptors (6-cyano-7-nitroquinoxaline-2,3-dione), abolished the neurotoxic effect of kainate. Development of glutamate-induced neuronal death depends strongly on Ca2+. Removal of extracellular Ca2+ (with 1mM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) immediately after the termination of glutamate exposure and before the appearance of the early signs of neuronal death (post-glutamate period) dramatically reduced neuronal degeneration. Neurotoxic concentrations of glutamate induced sustained increase of 45Ca2+ uptake in the post-glutamate period. The delayed increase of 45Ca2+ uptake, as well as the delayed neurotoxicity, were not affected by post-glutamate treatment with phencyclidine, dibenzocyclohepteneimine; DL-2-amino-5-phosphonovalerate, or MgCl2 or with voltage-dependent Ca2+ channel blockers (nitrendipine, verapamil, diltiazem). Neurotoxic concentrations of glutamate also induced a delayed sustained increase of [3H]phorbol-12,13-dibutyrate binding, reflecting an increased translocation of protein kinase C (PKC) from cytosol to the cell membrane during the post-glutamate period. Pretreatment of neurons with the ganglioside GT1b (trisialosylgangliotetraglycosylceramide), followed by removal of free GT1b from the incubation medium, prevented PKC translocation, the sustained increase of 45Ca2+ uptake in the post-glutamate period, and the delayed neuronal death. We suggest that the sustained activation and translocation of PKC primed by glutamate receptor stimulation may be the triggering event causing the protracted increase of neuronal Ca2+ influx. This influx is insensitive to voltage-dependent Ca2+ channel blockers and glutamate receptor antagonists. It appears that this delayed increase of Ca2+ influx may be important in causing neuronal death.

Gangliosides prevent glutamate and kainate neurotoxicity in primary neuronal cultures of neonatal rat cerebellum and cortex.

Using a sensitive histofluorescence staining method that allows for a quantitation of neuronal death, we compared the protective effects of gangliosides (a group of naturally occurring glycosphingolipids), phencyclidine (PCP), and MK-801 (dibenzocyclohepteneimine) on glutamate- and kainate-induced neuronal death in primary cultures of cortical and cerebellar neurons prepared from neonatal rats. PCP and MK-801 block neurotoxicity induced by glutamate doses 50 times higher than the LD50 (LD50 in Mg2+-free medium, 10 microM) but only partially block the kainate neurotoxicity (LD50 in presence of Mg2+, 100 microM). In contrast, pretreatment with gangliosides (GT1b greater than GD1b greater than GM1) results in complete and insurmountable protection against the neurotoxicity elicited by glutamate or kainate. In primary cultures of cerebellar granule cells gangliosides, unlike PCP and MK-801, fail to block glutamate-gated cationic currents and the glutamate-evoked increase of (i) inositol phospholipid hydrolysis (ii) c-fos mRNA content, and (iii) nuclear accumulation of c-fos protein. Protection of glutamate neurotoxicity by gangliosides does not require their presence in the incubation medium; however, it is proportional to the amount of glycosphingolipid accumulated in the neuronal membranes. The ganglioside concentration (30-60 microM) that blocks glutamate-elicited neuronal death also prevents glutamate- and kainate-induced protein kinase C translocation from cytosol to neuronal membranes.

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.

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.