Ulrich Schütz

Quantitative assessment of nicotinic acetylcholine receptor proteins in the cerebral cortex of Alzheimer patients

Cholinergic transmission has for long been known to be one of the most severely affected systems in Alzheimers disease (AD), resulting clinically in massive cognitive deficits. The molecular basis of this dysfunction--on both the pre- and the postsynaptic sites--is still a matter of ongoing investigations. Here, we report on the quantitative assessment of nicotinic acetylcholine receptor isoform expression in AD vs. control cortices. For both subunit proteins assessed, the alpha4 and the alpha7 isoform, highly significant decreases in diseased vs. normal cortices were observed. Both alpha4 and alpha7 subunits are known to be important constituents in hetero- (alpha4beta2) and homooligomeric (alpha7) receptor subtypes. Their decreased expression may contribute to the decreased nicotinic binding known to be accompanied by AD and severe cognitive deficits. The quantitative assessment of nicotinic acetylcholine receptor expression will help to determine those subunits suited as targets for pharmacological stimulation.

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 (α4, α7) 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 α4‐ and α7‐containing functional nAChRs in the human cortex. In cortices of Alzheimer patients and controls, the pattern of distribution and the number of α4 and α7 mRNA‐expressing neurons were similar, whereas at the protein level a decrease in the density of α4‐ and α7‐expressing neurons of ≈ 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 β‐amyloid showed that neurons in the vicinity of β‐amyloid plaques bore both nAChR transcripts. Neurons heavily labelled for HP‐tau, however, expressed little or no α4 and α7 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 α4 and α7 transcripts give rise to decreased protein expression at the level of individual neurons.

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 Alzheimers 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.

Tau-Mediated Nuclear Depletion and Cytoplasmic Accumulation of SFPQ in Alzheimer's and Pick's Disease

Tau dysfunction characterizes neurodegenerative diseases such as Alzheimers disease (AD) and frontotemporal lobar degeneration (FTLD). Here, we performed an unbiased SAGE (serial analysis of gene expression) of differentially expressed mRNAs in the amygdala of transgenic pR5 mice that express human tau carrying the P301L mutation previously identified in familial cases of FTLD. SAGE identified 29 deregulated transcripts including Sfpq that encodes a nuclear factor implicated in the splicing and regulation of gene expression. To assess the relevance for human disease we analyzed brains from AD, Picks disease (PiD, a form of FTLD), and control cases. Strikingly, in AD and PiD, both dementias with a tau pathology, affected brain areas showed a virtually complete nuclear depletion of SFPQ in both neurons and astrocytes, along with cytoplasmic accumulation. Accordingly, neurons harboring either AD tangles or Pick bodies were also depleted of SFPQ. Immunoblot analysis of human entorhinal cortex samples revealed reduced SFPQ levels with advanced Braak stages suggesting that the SFPQ pathology may progress together with the tau pathology in AD. To determine a causal role for tau, we stably expressed both wild-type and P301L human tau in human SH-SY5Y neuroblastoma cells, an established cell culture model of tau pathology. The cells were differentiated by two independent methods, mitomycin C-mediated cell cycle arrest or neuronal differentiation with retinoic acid. Confocal microscopy revealed that SFPQ was confined to nuclei in non-transfected wild-type cells, whereas in wild-type and P301L tau over-expressing cells, irrespective of the differentiation method, it formed aggregates in the cytoplasm, suggesting that pathogenic tau drives SFPQ pathology in post-mitotic cells. Our findings add SFPQ to a growing list of transcription factors with an altered nucleo-cytoplasmic distribution under neurodegenerative conditions.

Loss of nicotinic acetylcholine receptor subunits α4 and α7 in the cerebral cortex of Parkinson patients

Cerebral cortical cholinergic deficits, represented by a decrease in choline acetyltransferase activity, severe losses of nicotinic binding sites as well as cell degeneration in the basal forebrain can be observed in neurodegenerative diseases such as Parkinsons disease and Alzheimers disease. The potential role of nicotinic acetylcholine receptor subunits as pharmacological targets for the treatment of cognitive deficits raises the question as to what extent these subunits are affected in neurodegenerative diseases. We here report on a significant decrease of the alpha4 and the alpha7 nicotinic acetylcholine receptor subunit in cortices of Parkinson patients which turns out to be similar to recent findings in Alzheimer patients.

Improved outcome of facial nerve repair in rats is associated with enhanced regenerative response of motoneurons and augmented neocortical plasticity

Within a recent study on the vibrissae motor performance after facial nerve repair in strains of blind (SD/RCS) and sighted (SD) rats we found that, despite persisting myotopic disorganization in the facial nucleus, the blind animals fully restored vibrissal whisking. Here we searched for morphological substrates of better recovery in the regenerating motoneurons and in the cerebral motor cortex. Expression analyses of the neurite growth‐related proteins f‐actin, neuronal class III β‐tubulin and plasticity‐related gene‐1, and stereological estimates of growth cone densities revealed a more vigorous regenerative response in the proximal nerve stump of blind SD/RCS rats compared with SD animals at 5–7 days after buccal nerve transection. Using c‐Fos immunoreactivity as a marker for neuronal activation, we found that the volume of the cortex acutely responding to nerve transection (facial muscles reactive volume, FMRV) in both hemispheres of intact sighted rats was twofold smaller than that measured in blind animals. One month after transection and suture of the right facial nerve (FFA) we found a twofold increase in the FMRV in both rat strains compared with intact animals. The FMRV in SD/RCS animals, but not in SD rats, returned to the values in intact rats 2 months after FFA. Our findings suggest that enhanced plasticity in the CNS and an augmented regenerative response of the injured motoneurons contribute to better functional recovery in blind rats.

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.

Classical Alzheimer features and cholinergic dysfunction : towards a unifying hypothesis?

Objective – Our autopsy studies show possible links between classical Alzheimer pathology and decreased expression of nicotinic acetylcholine receptors. For further elucidation we are now using in vitro models. We report preliminary evidence for the impact of β‐amyloid on nicotinic receptor expression in hippocampal dissociation culture. Methods – Cultures (E18 rats) were grown in a serum‐free medium and incubated at 8 days in vitro for 3 days with 1 µM Aβ1–42. Expression of α4, α7, and β2 nicotinic receptor subunit protein was assessed immunohistochemically and rated semiquantitatively. Results – Aβ1–42 incubation resulted in a massive reduction of α4 protein‐expressing neurons, this effect was less pronounced for the α7 and β2 subunit protein. Conclusion – These findings provide first evidence for a direct impact of classical Alzheimer pathology features on nicotinic receptor expression in vitro. Our model will be useful for testing the potential of drugs to stop or reverse these effects.

Expression of the α4 isoform of the nicotinic acetylcholine receptor in the fetal human cerebral cortex

Abstract Nicotinic acetylcholine receptors are likely to play an important role in neuronal migration during development. Furthermore, the α4 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 α4 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, α4 receptor mRNA and α4 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 α4 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.

Mutation screening of the Chrna4 and Chrnb2 nicotinic cholinergic receptor genes in Alzheimer's disease

Potential genomic changes leading to decreased nicotine binding, crucial for cognitive dysfunction in Alzheimers disease (AD), have not yet been studied. A search for mutations of the genes coding for the most widely distributed nicotinic receptor subtype alpha4beta2 (CHRNA4/CHRNB2) has been performed in AD patients by screening the coding regions of both genes by single strand conformation analysis and heteroduplex analysis of fibroblast-derived genomic DNA. Polymorphisms in CHRNA4, none of which led to amino acid changes in the predicted sequence, were found in three patients. Although the other receptor subunits have yet to be screened, it appears likely that the reduction of nicotine binding sites in AD is not due to genomic changes.