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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., (Copyright 1999 Elsevier Science B.V.)

When rabbits are injected with tissue homogenates of white matter from bovine corpus callosum, an antiserum is produced which reacts with the surface membrane of 20-30% of all cells obtained by trypsin-dissociation of cerebellum from 10-day-old mice. The antigen or set of antigens recognized by this antiserum is detectable on embryonic, early postnatal, and adult mouse brain, but not on liver, spleen, kidney, thymus and sperm. The antigen is expressed in different regions of the brain and also, in decreased amounts, on retina. In histological sections of cerebellum from 21-day-old mice the antigen is predominantly localized in white matter tracts. Whereas nervous tissue from chicken and rabbit does not carry detectable levels of the antigen, rat, bovine and human brains are antigen-positive.

The molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were studied in frontal cortex (grey and white matter), postmortem, and in cerebrospinal fluid (CSF) of demented patients with Parkinson's disease compared to controls and non-demented parkinsonians. In the frontal cortex, AChE activity decreased significantly in both demented and non-demented parkinsonian subjects compared to controls; the 10S form of the enzyme was significantly lower in demented parkinsonians than in the non-demented subjects. The decrease in AChE activity was correlated with a decrease in choline acetyltransferase activity thought to reflect lesion of cholinergic neurones in the substantia innominata which innervate the cerebral cortex. BChE activity decreased only in the non-demented parkinsonians; in the demented subjects, BChE activity was at control levels. Similar results were obtained with grey and white matter, although absolute levels of the two enzymes were different in the two types of tissue, suggesting that the enzymes were affected in the cholinergic neurones before transport to cortical nerve terminals. No decreases in AChE or BChE activity were observed in the CSF of the patients studied. On the contrary, AChE and BChE levels were significantly higher in demented parkinsonian patients compared to the non-demented subjects.

Alpha 1, alpha 2, beta 1 and beta 2 adrenergic receptors were measured in the pre-frontal cortex of parkinsonian patients post-mortem. The number of beta 2 receptors was the same in control and parkinsonian subjects. alpha 1 and beta 1 receptors increased in number, particularly in demented parkinsonian patients, while alpha 2 receptors decreased. The affinity constants were unchanged. The modifications seem to be related to lesion of the noradrenergic pathway from the locus coeruleus to the cortex. The relationship between this lesion and the symptoms of dementia and depression in parkinsonian patients is discussed.

CAT activity was decreased in the frontal cortex and the substantia innominata of parkinsonian subjects, post-mortem. The decrease was greater in the frontal cortex of parkinsonians with dementia. The density of muscarinic cholinergic receptors increased in the cortex. This increase was inversely correlated with tremor. The effects on these parameters of both neuronal degeneration and anticholinergic therapy are discussed.

Monoclonal antibodies which immunocytochemically label Lewy bodies on sections of substantia nigra from subjects with Parkinson's disease were produced by immunization of mice with substantia nigra and locus coeruleus containing Lewy bodies from parkinsonian subjects post-mortem. Tests of specificity indicate that the antibodies do not recognize the same antigen. One of the antibodies (G7) immunocytochemically labels only Lewy bodies, the other (G9) also faintly labels the cell bodies of nigral dopaminergic neurons and cerebellar Purkinje cells in both normal and parkinsonian brains. Absorption experiments show, however, that the G7 antigen is present in normal substantia nigra and the G9 antigen in normal substantia nigra and Purkinje cells. Neither of the antibodies seems to be directed against neurofilament protein. Immunoblots after two-directional electrophoresis indicate that antibody G7 labels a protein with an iso-electric point around 5.6 and a mol. wt. of approximately 40 kdalton, whereas the protein labeled by antibody G9 has an iso-electric point of near 8 and a mol. wt. above 70 kdalton.

Two antigens, G7 and G9, which are labelled by monoclonal antibodies in Lewy bodies in brains from patients with Parkinson's disease, were characterized by two-dimensional electrophoresis, followed by electroblotting, in order to explore their possible relationship with neuronal degeneration in this disease. The G7 antigen was found in the substantia nigra of subjects with Parkinson's disease and progressive supranuclear palsy, as well as in normal subjects. It was also found in the dopaminergic nucleus paranigralis and the locus coeruleus. The G9 antigen was found in the substantia nigra and locus coeruleus, but also in the caudate nucleus, terminal region of the nigrostriatal dopaminergic neurons, and in the cortex and cerebellum, terminal regions of the noradrenergic neurons in the locus coeruleus. The identity of the antigens remains unknown. They do not correspond to tyrosine hydroxylase or neurofilaments previously detected in Lewy bodies, or to other cytoskeletal proteins. Nor are they related to the presence of neuromelanin in the cells that degenerate in Parkinson's disease. The proteins, or at least the epitopes labelled by the antibodies, are found in normal brain, suggesting that these proteins do not play a causal role in the formation of the Lewy bodies in degenerating neurons in Parkinson's disease. The G7 antigen is absent from the cholinergic substantia innominata, where Lewy bodies are also found, indicating that these antigens are not essential for the formation of the corpuscle.

The concentrations of somatostatin in the cortex, hippocampus and caudate nucleus of subjects with Parkinson's disease were determined by radioimmunoassay. Somatostatin levels in the frontal cortex were significantly reduced in Parkinsonian subjects who were slightly or severely demented compared to controls and to non-demented Parkinsonians. Significant reductions were also observed in the hippocampus and entorhinal cortex of severely demented subjects.

[3H]Quinuclidinylbenzilate ([3H]QNB) binding and choline acetyltransferase (CAT) activity were studied in post-mortem brains from control and Parkinsonian subjects. CAT levels were reduced in the cortex and hippocampus of Parkinsonians. The apparent affinity of [3H]QNB for the muscarinic receptor was higher in both the caudate nucleus and the frontal cortex. Receptor density increased only in the frontal cortex. These changes are discussed in relation to dementia and mental disturbances following anticholinergic treatment frequently observed in Parkinson's disease.