Marco Favaron

Glutamate Neurotoxicity Is Independent of Calpain I Inhibition in Primary Cultures of Cerebellar Granule Cells

Glutamate‐induced neurotoxicity and calpain activity were studied in primary cultures of rat cerebellar granule neurons and glial cells. Calpain activation, as monitored by quantitative immunoblotting of spectrin, required micromolar concentrations of Ca2+ in neuronal homogenates (calpain I) and millimolar Ca2+ concentrations in glial homogenates (calpain II). Glutamate‐induced toxicity and calpain activation were observed in neuronal, but not in glial, cultures. In neurons, calpain I activation by glutamate was dose‐dependent and persisted after withdrawal of neurotoxic doses of glutamate. Natural (GM1) and semisynthetic (LIGA4) gangliosides or the glutamate receptor blocker MK‐801 prevented calpain I activation and delayed neuronal death elicited by glutamate. GM1 and LIGA4 had no effect on calpain I activity in neuronal homogenates, however. Furthermore, two calpain I inhibitors (leupeptin and N‐acetyl‐Leu‐Leu‐ norleucinal) prevented glutamate‐induced spectrin degradation, but failed to affect glutamate neurotoxicity. These results thus suggest that glutamate‐induced neurotoxicity is independent of calpain I activation.

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.

Pathological Phosphorylation Causes Neuronal Death: Effect of Okadaic Acid in Primary Culture of Cerebellar Granule Cells

Abstract: 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 l‐(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.

Macrolide antibiotics protect neurons in culture against theN-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.

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.

Inhibition of DNA Synthesis in C6 Glioma Cells Following Cellular Incorporation of GM1 Ganglioside and Choleragenoid Exposure

Abstract: The B subunit of cholera toxin, which is multiva‐lent 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 fibro‐blasts. 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 μM GM1 for 15 min (followed by washing with fetal calf serum) and incubation with 1 μ/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 GM 1, 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.

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.

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.

Protection by Natural and Semisynthetic Gangliosides from Ca2+-Dependent Neurotoxicity Caused by Excitatory Amino Acid (EAA) Neurotransmitters

Neuronal death is a frequent occurrence during nervous system ontogenesis and continues, though at a slower rate, throughout life. EAA neurotransmitters, when added (in appropriate concentrations) to primary neuronal cultures, are the only neurotransmitters known to cause neuronal death by destabilizing homeostasis of free Ca2+ (Olney, 1969; Coyle, 1987; Vaccarino et al., 1987; Favaron et al., 1988; Connor et al., 1988). In neurons, EAA increase Ca2+ influx through specific cationic channel activation, thereby stimulating Ca2+-dependent enzymes, including protein kinase C (PKC) (Wroblewski and Danysz, 1989). Presumably, these actions of EAAs can be mediated at NMDA-, kainate- or quisqualate-sensitive synaptic receptors (Wroblewski and Danysz, 1989). Normally, intraneuronal Ca2+ homeostasis is maintained by the equilibration of free Ca2+, with Ca2+ compartmentalized in endoplasmic reticulum and mitochondria and the operation of channels and pumps allowing Ca2+ influx (cationic channels) and efflux (Nat-Ca2+ exchanger and ATPase-operated Ca2+ pumps) (Carafoli, 1987). It is, therefore, not surprising that many have attempted to evaluate whether EAA receptors are also operative in physiologically programmed neuronal death. In central nervous system, an alteration of EAA transmission may occur in a variety of acute (stroke, ischemia, and CNS traumas) or chronic (autoimmune responses, Huntington’s chorea, amyotrophic lateral sclerosis (ALS), olivopontocerebellar atrophy, lathyrism, and the Guamanian complex) neuropathological processes (Rothman, 1984; Kostic et al., 1989; Spencer et al., 1987; Plaitakis et al., 1988).

Phospholipids can influence the interconversion of flat epitheliallike 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.