Parvalbumin-containing interneurons of the human cerebral cortex express nicotinic acetylcholine receptor proteins.

Cholinergic fibers from the basal forebrain are known to contact cholinoceptive cortical pyramidal neurons. Recent electrophysiological studies have revealed that nicotinic acetylcholine receptors are also present in human cerebrocortical interneurons. A direct visualization of nicotinic receptor subunits in cortical interneurons has, however, not yet been performed. We have applied double-immunofluorescence using antibodies against parvalbumin --a marker for the Chandelier and basket cell subpopulation of interneurons--and to the alpha4 and alpha7 subunit proteins of the nicotinic acetylcholine receptor. The vast majority of the parvalbuminergic interneurons was immunoreactive for the alpha4 and the alpha7 nicotinic acetylcholine receptor. Provided these receptors would be functional--as suggested by recent electrophysiological findings--the connectivity pattern of cholinergic afferents appears much more complex than thought before. Not only direct cholinergic impact on cortical projection neurons but also the indirect modulation of these by cholinergic corticopetal fibers contacting intrinsic cortical cells would be possible.

Expression of nicotinic acetylcholine receptors in Alzheimer's disease: postmortem investigations and experimental approaches.

Nicotinic ligand binding studies have shown rather early that the cholinoceptive system is affected in Alzheimer's disease (AD). Today, molecular histochemistry enables one to study the nicotinic acetylcholine receptor (nAChR) subunit expression on the cellular level in human autopsy brains, in animal models and in in vitro approaches, thus deciphering the distribution of nAChRs and their role as potential therapeutic targets. The studies on the nAChR expression in the frontal and temporal cortex of AD patients and age-matched controls could demonstrate that both, the numbers of alpha4- and alpha7-immunoreactive neurons and the quantitative amount, in particular of the alpha4 protein, were markedly decreased in AD. Because the number of the corresponding mRNA expressing neurons was unchanged these findings point to a translational/posttranslational rather than a transcriptional event as an underlying cause. This assumption is supported by direct mutation screening of the CHRNA4 gene which showed no functionally important mutations. To get more insight into the underlying mechanisms, two model systems organotypic culture and primary hippocampal culture - have been established, both allowing to mimic nAChR expression in vitro. In ongoing studies the possible impact of beta-amyloid (Abeta) on nAChR expression is tested. Preliminary results obtained from primary cultures point to an impaired nAChR expression following Abeta exposure.

Expression of the alpha4 isoform of the nicotinic acetylcholine receptor in the fetal human cerebral cortex.

Nicotinic acetylcholine receptors are likely to play an important role in neuronal migration during development. Furthermore, the alpha4 receptor subunit gene is related to a hereditary juvenile form of epilepsy. Only little information is available, however, on the expression of cerebrocortical nicotinic acetylcholine receptors during human fetal development. Using non-isotopic in situ hybridization and immunohistochemistry, we have studied the distribution of the alpha4 subunit of the nicotinic acetylcholine receptor mRNA and protein in the human frontal cortex at middle (17-24 weeks of gestation) and late (34-42 weeks of gestation) fetal stages. Both, alpha4 receptor mRNA and alpha4 receptor protein were observed beginning during week 17-18 of gestation. At this time of development, a few weakly labeled mRNA-containing cells were present mainly in the ventricular zone, the subplate and the cortical plate. A similar distribution pattern was found for the receptor protein. Around week 38 of gestation, the distribution in the cerebral cortex of alpha4 subunit-containing cells was similar to that of adult human cortices with the highest densities of labeled neurons found in layers II/III, followed by layers V and VI. Nicotinic acetylcholine receptor-containing neurons appear rather early in human fetal development. Given functional maturity, they may interact during cortical development with acetylcholine released from corticopetal fibers or other yet unknown sources subserving the process of neuronal migration and pathfinding.

Gene expression of the nicotinic acetylcholine receptor alpha 4 subunit in the frontal cortex in Parkinson's disease patients.

Cognitive impairment in Parkinson's disease is accompanied by a marked decrease of cerebrocortical nicotinic receptors. To study the putative site of impaired receptor synthesis, frontal cortices of Parkinson patients with cognitive dysfunction have been screened for the expression of the nicotinic receptor alpha 4 subunit gene. Quantitative assessment of alpha 4 mRNA-expressing neurons did not show significant differences between patients and controls. Therefore, decreased nicotinic receptor sites cannot be attributed to alterations at the transcriptional level of the alpha 4 gene. Alternative causes have to be searched for at the translational and/or postranslational level.

Expression of the tumour suppressor gene CADM1 is associated with favourable outcome and inhibits cell survival in neuroblastoma.

Cell adhesion molecule 1 (CADM1) is a putative tumour suppressor gene, which is downregulated in many solid tumours. In neuroblastoma, loss of CADM1 expression has recently been found in disseminated tumours with adverse outcome, prompting us to investigate its role in neuroblastoma tumour progression. Oligonucleotide-microarray analysis of 251 neuroblastoma specimens demonstrated that CADM1 downregulation is associated with unfavourable prognostic markers like disseminated stage 4, age >18 months, MYCN amplification and chromosome 11q alterations (P<0.001 each). Furthermore, low CADM1 expression was significantly correlated with unfavourable gene expression-based classification (P<0.001) and adverse patient outcome (P<0.001). Bisulphite sequencing and genetic analysis of 18 primary neuroblastomas suggested that neither haploinsufficiency nor hypermethylation is regularly involved in CADM1 gene silencing in neuroblastoma, which is in contrast to results obtained in other malignancies. In addition, no mutations disrupting the CADM1 reading frame were found in 25 primary neuroblastomas. Over-expression of CADM1 in neuroblastoma cells resulted in significant reduction of proliferation, viability and colony formation in soft agar. Collectively, our results suggest that downregulation of CADM1 tumour suppressor gene expression is a critical event in neuroblastoma pathogenesis resulting in tumour progression and unfavourable patient outcome.

Cellular expression of alpha7 nicotinic acetylcholine receptor protein in the temporal cortex in Alzheimer's and Parkinson's disease--a stereological approach.

Cognitive deficits in Alzheimer's and Parkinson's disease are closely related to disturbed cholinergic transmission. The decrease of nicotinic acetylcholine receptor protein has been assessed by Western blotting and immunohistochemistry. Stereology, however, has not been used to assess numbers of receptor-expressing human cerebrocortical neurons. Our approach applies a combination of alpha7 subunit-immunohistochemistry with a stereological technique using defined stretches of pial surface as reference standard. The number of alpha7 subunit protein-expressing neurons in the Alzheimer temporal cortices amounted to approximately half of that of controls while numbers in Parkinson patients lay in between. No differences in the total number of neurons were seen. These results corroborate nonstereological studies on Alzheimer cortices and for the first time show a similar decrease in receptor expression in Parkinson's disease. They provide evidence that not only Alzheimer dementia but also cognitive deficits in Parkinson's disease may be related to decreased nicotinic receptor expression.

Expression of nicotinic acetylcholine receptor subunits in the cerebral cortex in Alzheimer's disease: histotopographical correlation with amyloid plaques and hyperphosphorylated-tau protein.

Impairment of cholinergic transmission and decreased numbers of nicotinic binding sites are well-known features accompanying the cognitive dysfunction seen in Alzheimer's disease (AD). In order to elucidate the underlying cause of this cholinoceptive dysfunction, the expression of two pharmacologically different nicotinic acetylcholine receptor (nAChR) subunits (alpha4, alpha7) was studied in the cerebral cortex of Alzheimer patients as compared to controls. Patch-clamp recordings of 14 dissociated neurons of control cortices showed responses suggesting the existence of alpha4- and alpha7-containing functional nAChRs in the human cortex. In cortices of Alzheimer patients and controls, the pattern of distribution and the number of alpha4 and alpha7 mRNA-expressing neurons were similar, whereas at the protein level a decrease in the density of alpha4- and alpha7-expressing neurons of approximately 30% was observed in Alzheimer patients. The histotopographical correlation of nAChR expression with accompanying pathological changes, e.g. accumulation of hyperphosphorylated-tau (HP-tau) protein and beta-amyloid showed that neurons in the vicinity of beta-amyloid plaques bore both nAChR transcripts. Neurons heavily labelled for HP-tau, however, expressed little or no alpha4 and alpha7 mRNA. These results point to an impaired synthesis of nAChRs on the protein level as a possible cause of the cholinoceptive deficit in AD. Further investigations need to elucidate whether interactions of HP-tau with nAChR mRNA, or alterations in the quality of alpha4 and alpha7 transcripts give rise to decreased protein expression at the level of individual neurons.