Arylsulfonamides as a new class of cannabinoid CB1 receptor ligands: identification of a lead and initial SAR studies.

High-throughput screening of the UCB sample collection identified the piperidinyl-sulfonyl benzoic ester 1 as a novel agonist for CB(1) receptor with nanomolar affinity. We report here the pharmacological profile of compound 1 as well as preliminary biological activities in pain model. Diverse close analogs of 1 were purchased and the structure-affinity relationships among this novel class are discussed.

Solubilization and immunopurification of rat brain synaptic vesicle protein 2A with maintained binding properties.

This study reports the solubilization of the rat synaptic vesicle protein SV2A, the brain binding site for the antiepileptic drug levetiracetam (LEV), and its characterization. N-dodecyl-beta-D-maltoside (DDM) was the best detergent at achieving a high percentage of SV2A solubilization and at maintaining the binding characteristics of a tritiated form of a more potent analogue of LEV, [3H]ucb 30889 ((2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide). Scatchard analysis revealed that approximately 25% of SV2A proteins from brain membranes are solubilized by DDM under optimal conditions. Competition binding experiments with a variety of LEV analogues indicated that [3H]ucb 30889 labels the same binding site in both crude homogenates and soluble extracts, with still high stereoselectivity. After immunoprecipitation of SV2A from solubilized rat brain membranes, binding properties of [3H]ucb 30889 to SV2A and association with synaptotagmin I were maintained. The two other isoforms SV2B and SV2C were found to be co-immunoprecipitated with SV2A. The solubilization and immunopurification of SV2A with unmodified ligand affinities and synaptotagmin I interaction provides the starting point for future protein-protein interactions and structural studies.

Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a preferential loss of the dopaminergic neurons of the substantia nigra pars compacta. Although the etiology of PD is unknown, major biochemical processes such as oxidative stress and mitochondrial inhibition are largely described. However, despite these findings, the actual therapeutics are essentially symptomatical and are not able to block the degenerative process. Recent histological studies performed on brains from PD patients suggest that nigral cell death could be apoptotic. However, since post-mortem studies do not allow precise determination of the sequence of events leading to this apoptotic cell death, the molecular pathways involved in this process have been essentially studied on experimental models reproducing the human disease. These latter are created by using neurotoxic compounds such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or dopamine (DA). Extensive study of these models have shown that they mimick, in vitro and in vivo, the histological and/or the biochemical characteristics of PD and thus help to define important cellular actors of cell death presumably critical for the nigral degeneration. This review reports recent data concerning the biochemical and molecular apoptotic mechanisms underlying the experimental models of PD and correlates them to the phenomena occurring in human disease.

The relationship between differentiation and survival in PC12 cells treated with cyclic adenosine monophosphate in the presence of epidermal growth factor or nerve growth factor.

We have asked whether treatment of PC12 cells with cyclic adenosine monophosphate (cAMP) and epidermal growth factor (EGF) results, like treatment with cAMP and nerve growth factor (NGF), in irreversible neuronal differentiation characterized by irreversible neurite extension, loss of serum-dependence, and death by apoptosis after trophic factor withdrawal. Although EGF alone, unlike NGF, did not cause morphological differentiation or prevent cell death, synergy between a cAMP-mediated signal transduction pathway and a pathway activated by the EGF receptor tyrosine kinase resulted in the same irreversible differentiation. EGF/cAMP-differentiated cells required cAMP to survive, but NGF, through a TrkA-dependent mechanism, could substitute for cAMP. The cyclin-dependent kinase inhibitors olomoucine and roscovitine also promoted survival of the irreversibly differentiated cells, by a mechanism that must be determined, since cell death was not associated with nuclear (3)H-thymidine accumulation, an index of mitotic activity.

Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death.

Mitochondrial free calcium levels measured by Rhod-2 fluorescence and ultrastructure were examined during cell death in nerve growth factor (NGF)-differentiated PC12 cells that were 1) exposed to C2-ceramide, 2) deprived of serum to induce endogenous ceramide production, or 3) treated with calcium ionophore A23187. Rhod-2 fluorescence in mitochondria and also in the nucleolus increased to a maximum within 3 hours after C2-ceramide treatment or serum withdrawal. In A23187-treated cells, Rhod-2 fluorescence remained at baseline levels. In all three models, enlargement of the endoplasmic reticulum was the first ultrastructural alteration, followed by mitochondrial shrinkage in ionophore-treated cells, but by mitochondrial swelling in the ceramide-dependent models, in which rupture of the outer mitochondrial membrane and unfolding of the inner membrane were frequently seen. Dihydro-C2-ceramide, which did not cause cell death, had no effect on cellular ultrastructure. NGF, which inhibits ceramide-dependent cell death, prevented the effects of serum deprivation on mitochondrial ultrastructure but not on endoplasmic reticulum morphology or Rhod-2 fluorescence. Nuclear shrinkage with loss of nuclear membrane integrity, characterized by nuclear pores, free or surrounded by electron-dense filaments, was a late event in ceramide-dependent cell death. Chromatin condensation and other morphological features associated with apoptosis were seen in only a few atypical cells. Ceramide-mediated cell death, therefore, did not involve classical apoptosis but was mediated by a reproducible series of events beginning in the endoplasmic reticulum, followed by the mitochondria, and then the nucleus. NGF-dependent cell death inhibition intervenes at the mitochondrial level, not by blocking the increase in Rhod-2 fluorescence but by preventing the ultrastructural changes that follow.

Neuropharmacologic aspects of apoptosis: significance for neurodegenerative diseases.

The cause of neuronal death in Parkinson's, Alzheimer's, and other neurodegenerative diseases is not known, except in some hereditary forms of these disorders in which a mutated gene has been identified. Even in these cases, the molecular mechanisms that underlie the loss of specific populations of neurons have not been determined, although it is highly probable that apoptosis is involved. Some of the biochemical events that occur during apoptosis have been elucidated. We focus in this review on the role played by the proapoptotic caspases, the antiapoptotic proteins of the Bcl-2 family, and the apoptosis associated signal transducers such as ceramide, calcium, and reactive nitrogen or oxygen species. The role of the mitochondria and the possible implication of cell cycle regulators will also be addressed. Of particular interest are the endogenous inhibitory mechanisms and the pharmacologic agents that can be used to block apoptosis signaling cascades, because they offer models for the development of therapeutic strategies designed to prevent the evolution of pathologic neurodegeneration.

Mechanisms of apoptosis in PC12 cells irreversibly differentiated with nerve growth factor and cyclic AMP.

PC12 cells treated with cAMP become irreversibly differentiated and die by apoptosis when deprived of trophic support, instead of dedifferentiating and reentering the cell cycle. To approach the molecular mechanism underlying the cAMP-induced switch from differentiation/proliferation to apoptosis, we compared three sequential markers of a candidate apoptogenic signal transduction pathway (ceramide, free radicals and NF-kappaB), after trophic factor withdrawal in PC12 cells before and after irreversible differentiation. Serum withdrawal increased ceramide and free radical production regardless of the state of differentiation of the cells. It was followed by cell death, however, only in the absence of NGF and/or cAMP, and was no longer required for apoptosis in NGF/cAMP-differentiated cells. NGF and cAMP withdrawal sufficed. NF-kappaB was activated by NGF withdrawal in reversibly differentiated PC12 cells during dedifferentiation and reentry into the cell cycle, whereas in NGF/cAMP-differentiated cells, it was activated, at a late stage of the apoptotic process, concomitantly with cell death. These results show that a serum factor inhibits ceramide-dependent apoptosis upstream of ceramide and free radical production, whereas NGF- and cAMP-dependent mechanisms inhibit apoptosis either downstream or parallel to these events. After terminal differentiation by cAMP, apoptosis appears to be initiated from the second site, consistent with the serum independence of these cells and the absence of ceramide and free radical production when the NGF/cAMP-dependent inhibitions are released. The differential regulation of NF-kappaB appears to be an important step in the switch from mitosis to apoptosis that occurs during irreversible differentiation of PC12 cells by cAMP.

[Neuronal death caused by apoptosis in Parkinson disease]. / La mort neuronale par apoptose dans la maladie de Parkinson.

The identity of the neuronal populations (dopaminergic, noradrenergic, serotoninergic, cholinergic) that die in Parkinson's disease is well established. The cause of this degeneration, and the mechanism by which it takes place is still unknown, although there is data, at least for the dopaminergic neurons, suggesting that oxidative stress might play a role. In addition, recent ultrastructural studies of dopaminergic neurons in patients with Parkinson's disease have shown that these neurons die by apoptosis, and immunocytochemical studies have shown that the cytokine TNF-alpha, observed in microglial cells in the substantia nigra of patients post-mortem, might play a role, as might the transcription factor NF-kappa B, which is translocated into the nucleus of dopaminergic neurons in patients, a sign of its activation. We have developed an in vitro model of dopaminergic cell death that accounts for these observations. In both differentiated PC12 cells and primary cultures of mesencephalic neurons, we have shown that when the sphingomyelin-dependent signaling pathway is activated, these cells die by apoptosis, preceded by the production of superoxide radicals in the mitochondria and the nuclear translocation of NF-kappa B. TNF-alpha is known to induce all three such events: apoptosis, activation of the sphingomyelin pathway, free radical production. Our results suggest that the superoxide radicals are used as signalling molecules within the sphingomyelin pathway. These observations may help to explain the origin of the evidence, in postmortem brain from parkinsonian patients, for oxidative stress, hypothesized to be an etiological factor in this disease.