Expression of Bcl-2 in adult human brain regions with special reference to neurodegenerative disorders.

The expression of the protooncogene bcl-2, an inhibitor of apoptosis in various cells, was examined in the adult human brain. Several experimental criteria were used to verify its presence; mRNA was analyzed by northern blot with parallel experiments in mouse tissues, by RNase protection, and by in situ hybridization histochemistry. Bcl-2 protein was detected by western blot analysis and immunohistochemistry. Two bcl-2 mRNA species were identified in the human brain. The pattern of distribution of bcl-2 mRNA at the cellular level showed labeling in neurons but not glia. The in situ hybridization signal was stronger in the pyramidal neurons of the cerebral cortex and in the cholinergic neurons of the nucleus basalis of Meynert than in the Purkinje neurons of the cerebellum. Both melanized and nonmelanized neurons were labeled in the substantia nigra. In the striatum, bcl-2 mRNA was detected in some but not all neurons. In the regions examined for Bcl-2 protein, the expression pattern correlated with the mRNA results. In patients with Alzheimer's and Parkinson's diseases, quantification of bcl-2 mRNA in the nucleus basalis of Meynert and substantia nigra, respectively, showed that the expression was unaltered compared with controls, raising the possibility that the expression of other components of apoptosis is modulated.

Altered expression of amyloid protein precursor mRNA in the rat hippocampus following trimethyltin intoxication: an in situ hybridization study.

Previous studies have reported increased levels of amyloid protein precursor (APP) and APP mRNA in the hippocampus and basal forebrain of patients with Alzheimer's disease. Similar changes have been found in the brains of aged rodents and transgenic mice. It now appears that alterations in the expression of individual isoforms of APP mRNA may have a role to play in amyloid-pathogenesis. Here we examined the effect of acute administration of the limbic system neurotoxin trimethyltin (TMT) (8 mg/kg i.p.) on APP-751 and APP-695 mRNA expression in the rat hippocampus (CA1, CA2, CA3 and CA4) using in situ hybridization techniques. We found that following TMT treatment the expression of APP-751 mRNA was increased in CA1 pyramidal cells while that of APP-695 mRNA remained unchanged. TMT also increased the numbers of APP-751 and APP-695 mRNA positively hybridized cells in the CA1 pyramidal layer. These findings suggest that an alteration in APP mRNA expression is involved in the response of the rodent brain to TMT intoxication.

Clinical and pathological features in hydrocarbon-induced parkinsonism.

A neuropathological examination was performed on a patient with parkinsonism induced by prolonged exposure to a mixture of aliphatic hydrocarbons, mainly n-hexane and halogenated compounds. The patient developed a rapid-course disease that progressed even after withdrawal from the toxic exposure. Pathological examination and immunohistochemical analysis of the brain revealed severe and widespread dopaminergic neuronal loss, associated with severe gliosis, in the substantia nigra, and almost complete loss of tyrosine hydroxylase immunostaining in the striatum. No Lewy bodies were detected. Neuronal loss was also observed in the periaqueductal gray matter, locus ceruleus, and pedunculopontine nucleus. These changes, combined with the moderate anemia due to marrow suppression, and the mild axonal neuropathy observed in vivo, are suggestive of a hydrocarbon toxic insult.

Neurotrophin receptors and selective loss of cholinergic neurons in Alzheimer disease.

The most consistent neuropathological finding in Alzheimer disease (AD) is the loss of cholinergic neurons of the nucleus basalis of Meynert (NbM). Using immunohistochemistry, we have previously shown that cholinergic neurons located in the ventral striatum were affected, whereas those of the caudate nucleus, putamen, and mesencephalon were spared. Since cholinergic neurons that degenerate in AD are sensitive to NGF and those that are spared are not, it has been hypothesized that the loss of neurotrophins receptors may play a role in the death of cholinergic neurons in AD. Using immunohistochemistry, we have detected the presence of TrkA on most cholinergic neurons from the NbM, on some from those of the striatum, but not on those of the mesencephalon in the human brain. In AD patients, the number of neurons that expressed TrkA was markedly decreased in the NbM very likely as a consequence of cholinergic neuronal loss. In the striatum, despite the loss of high-affinity NGF binding previously reported, no loss of TrkA was observed. Taken together, these results suggest a decreased expression of NGF receptors on the striatal cholinergic neurons in AD. This loss may contribute, when it reaches a crucial threshold, to the death of cholinergic neurons occurring in AD.

Loss of striatal high affinity NGF binding sites in progressive supranuclear palsy but not in Parkinson's disease.

125I-Nerve growth factor (NGF) binding sites were analyzed by autoradiography in the striatum of 3 control subjects, 3 patients with Parkinson's disease and 3 patients with progressive supranuclear palsy. A high level of 125I-NGF binding was observed (0.3-0.4 fmol/mg of tissue equivalent) in the striatum and the nucleus basalis of Meynert of control patients. Pockets of lower 125I-NGF binding corresponding to acetylcholinesterase-poor striosomes were detected in the striatum of control subjects and patients with Parkinson's disease or progressive supranuclear palsy. When compared to controls, the density of 125I-NGF binding sites was reduced by 30% in the striatum of patients with progressive supranuclear palsy but not reduced in that of patients with Parkinson's disease. 125I-NGF binding was not significantly decreased in the nucleus basalis of Meynert in either diseases. Since NGF receptors are thought to be localized on cholinergic neurons in the striatum, the decrease in NGF binding is compatible with the loss of cholinergic neurons reported in the striatum from PSP patients.

Low affinity nerve growth factor receptor, adrenal transplant and Parkinson's disease.

Immunohistochemistry of low-affinity nerve growth factor receptor (LNGFR) was performed postmortem in the striatum and the adrenal gland of a parkinsonian patient with an adrenal to brain transplantation. Few LNGFR-positive fibers were observed in the necrotic graft. By contrast numerous immunostained fibers were detected in a restricted zone of the host striatum adjacent to the graft, corresponding to a selective zone of sprouting of tyrosine hydroxylase-positive fibers. This suggests that nerve growth factor or related growth factors may promote sprouting of catecholaminergic fibers in the host striatum.

Autoradiographic study of [125I]epidermal growth factor-binding sites in the mesencephalon of control and parkinsonian brains post-mortem.

Epidermal growth factor (EGF) is assumed to act as a neurotrophic factor on dopaminergic nigrostriatal neurons in cell cultures and animal brain. This led us to consider its possible role in the pathophysiology of Parkinson's disease. An autoradiographic study of the distribution of EGF-binding sites was performed in the mesencephalon of controls and patients with Parkinson's disease, a neurodegenerative disease associated with dramatic damage to the mesostriatal dopaminergic neurons. Scatchard analysis revealed a single type of binding sites with a high affinity constant, in the various mesencephalic dopaminergic areas examined. The characteristics and density of [125I]EGF-binding sites were similar in controls and parkinsonian patients. This suggests that EGF receptors in the mesencephalon are unaffected in Parkinson's disease and may therefore contribute to the increased activity and survival of the remaining dopaminergic neurons.

Does loss of nerve growth factor receptors precede loss of cholinergic neurons in Alzheimer's disease? An autoradiographic study in the human striatum and basal forebrain.

The mechanism by which cholinergic neurons degenerate in Alzheimer's disease is not known. Some of these neurons depend, however, on trophic support from NGF via a membrane receptor. We have analyzed the state of these receptors by autoradiography, with 125I-NGF as the ligand, in the caudate nucleus, putamen, ventral striatum, nucleus basalis of Meynert, and nucleus tegmenti pedunculopontinus of six patients with Alzheimer's disease and five controls, matched for age and postmortem delay. The binding characteristics were similar in the striatum (including caudate nucleus, putamen, and ventral striatum) and basal forebrain of control subjects and patients with Alzheimer's disease (Kd = 2.5-4 x 10(-11) M). In control brains, high levels of 125I-NGF binding were observed in the basal forebrain and striatum (0.32-0.49 fmol/mg tissue equivalent), but no specific binding was detected in the nucleus tegmenti pedunculopontinus. NGF binding sites were distributed heterogeneously in the striatum with patches of low density, corresponding to AChE-poor striosomes, surrounded by a matrix in which receptor density was significantly greater. In Alzheimer's disease, the density of NGF receptors was markedly decreased in the caudate nucleus, putamen, ventral striatum, and nucleus basalis of Meynert. In contrast, AChE staining decreased less in the nucleus basalis of Meynert in all Alzheimer's disease patients, and in the ventral striatum of those most severely affected. These results indicate that if NGF receptors are located on cholinergic neurons, receptor loss and the consequent decrease in trophic support may precede cell degeneration in Alzheimer's disease. The relationship between the loss of these receptors and the pathogenesis of the disease remains to be determined.

Decreased choline acetyltransferase mRNA expression in the nucleus basalis of Meynert in Alzheimer disease: an in situ hybridization study.

The subnormal choline acetyltransferase (ChoAcTase) activity in the cerebral cortex of patients with Alzheimer disease (AD) is thought to originate from the loss of cholinergic neurons in the nucleus basalis of Meynert (nbM). To examine possible changes in the functional activity of the remaining cholinergic neurons in the nbM of patients with AD, the level of expression of ChoAcTase mRNA was evaluated. A procedure for double-labeling cholinergic neurons to detect ChoAcTase mRNA and the corresponding protein in the same cell was developed, taking advantage of an anti-ChoAcTase antibody and the recently isolated cDNA complementary to a sequence of the human ChoAcTase mRNA. In the study of three controls and four patients with AD, the presence of both ChoAcTase mRNA and protein was observed in the same large neurons in both nbM and putamen. Specificity of in situ hybridization was further supported by the absence of neuronal staining with a sense probe. In AD patients a subnormal level of expression of ChoAcTase mRNA per cholinergic cell was detected in the nbM but not in the putamen. Our data support the hypothesis that expression of ChoAcTase mRNA might be down-regulated in the surviving cholinergic neurons in the nbM of patients with AD, raising the possibility of functional restoration by stimulating ChoAcTase synthesis.

Human neuronal cells in culture: from concepts to basic methodology.

The paper reviews some conceptual and methodological aspects of the tissue culture models which, during the past three decades, demonstrated a remarkable mimicry of many important structures and functions of the mammalian Central Nervous System (CNS) and related peripheral sensory and motor elements. Emphasis is placed on an original human neuronal tissue culture model obtained from selective CNS areas. The different cell types were identified and the neurotrophic interactions preliminary characterized. Neuropathological findings suggest hypothesis that can be fully tested using in vitro human models of affected cerebral specific areas.

Human neuronal cell viability demonstrated in culture after cryopreservation.

A human neuronal cell freezing technique has been developed. The results indicate that human fetal neuronal cells can be frozen with 7%-10% dimethyl sulfoxide (DMSO) as cryoprotectant. The survival of isolated thawed cells has been evaluated in culture. Cells have been frozen at -196 degrees C for 370 days without loss of viability. Average recovery rate of frozen cells was 62% of the recovery rate of cultured unfrozen controls. Thawed cells show neurite outgrowth and maintain both cellular markers such as neuron specific enolase (NSE) and neurochemical characteristics (GABA synthesis). Morphological integrity of cryopreserved neurons has been confirmed at ultrastructural level.

Cryopreservation of human fetal adrenal medullary cells.

Cryopreservation of human fetal adrenomedullary cells was performed, after dissociation of the gland and purification of chromaffin aggregates, using 10% dymethyl sulfoxide as cryoprotectant. More than 90% of thawed cultured clusters appear morphologically intact, showing extensive neurite outgrowth and absence of appreciable cellular loss. Immunoreactivity for tyrosine hydroxylase and response to nerve growth factor were demonstrated. These findings indicate that human medullary cells maintain their distinctive functional characteristics after cryopreservation.

Clinical and neurophysiologic features in paraneoplastic polyneuropathy.

Three of 8,954 in- patients have been selected as affected by paraneoplastic polyneuropathy. In all of them the polyneuropathy had a steadily progressive course, with symptoms beginning in the lower limbs and spreading to the upper limbs in a few months. An increase in protein content of the cerebrospinal fluid was evident in each case. No other possible causes of polyneuropathy were found, and the association with malignancy was histologically proved in all 3 cases. A bronchogenic ("oat cell") carcinoma was present in the first patient, who had an almost exclusively motor neuropathy. An osteosarcoma was diagnosed in the second case, and its association with a polyneuropathy seems to be exceptional. A sigmoid adenocarcinoma was discovered in the third patient. Neurophysiologic investigations were indicative of a polyneuropathy with predominant axonic involvement in all 3 cases.