Hannsjörg Schröder

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures.

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti‐receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit‐deficient mice should be ideal tools for testing the specificity of subunit‐directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the α3‐, α4‐, α7‐, β2‐, and β4‐nicotinic acetylcholine receptor subunits on brain tissues of the respective knock‐out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild‐type and knock‐out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.

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.

Neuronal IGF-1 resistance reduces Aβ accumulation and protects against premature death in a model of Alzheimer’s disease

Alzheimers disease (AD) is characterized by progressive neurodegeneration leading to loss of cognitive abilities and ultimately to death. Postmortem investigations revealed decreased expression of cerebral insulin‐like growth factor (IGF)‐1 receptor (IGF‐1R) and insulin receptor substrate (IRS) proteins in patients with AD. To elucidate the role of insulin/IGF‐1 signaling in AD, we crossed mice expressing the Swedish mutation of amyloid precursor protein (APPSW, Tg2576 mice) as a model for AD with mice deficient for either IRS‐2, neuronal IGF‐1R (nIGF‐1R−/−), or neuronal insulin receptor (nIR−/−), and analyzed survival, glucose, and APP metabolism. In the present study, we show that IRS‐2 deficiency in Tg2576 mice completely reverses premature mortality in Tg2576 females and delays β‐amyloid (Aβ) accumulation. Analysis of APP metabolism suggested that delayed Aβ accumulation resulted from decreased APP processing. To delineate the upstream signal responsible for IRS‐2‐mediated disease protection, we analyzed mice with nIGF‐1R or nIR deficiency predominantly in the hippocampus. Interestingly, both male and female nIGF‐1R−/−Tg2576 mice were protected from premature death in the presence of decreased Aβ accumulation specifically in the hippocampus formation. However, neuronal IR deletion had no influence on lethality of Tg2576 mice. Thus, impaired IGF‐1/IRS‐2 signaling prevents premature death and delays amyloid accumulation in a model of AD.—Freude, S., Hettich, M. M., Schumann, C., Stohr, O., Koch, L., Kohler, C., Udelhoven, M., Leeser, U., Müller, M., Kubota, N., Kadowaki, T., Krone, W., Schroder, H., Bruning, J. C., Schubert, M. Neuronal IGF‐1 resistance reduces Aβ accumulation and protects against premature death in a model of Alzheimers disease. FASEB J. 23, 3315–3324 (2009). www.fasebj.org

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.

Nicotinic Receptor Function in the Mammalian Central Nervous Systema

The diversity of neuronal nicotinic receptors (nAChRs) in addition to their possible involvement in such pathological conditions as Alzheimers disease have directed our research towards the characterization of these receptors in various mammalian brain areas. Our studies have relied on electrophysiological, biochemical, and immunofluorescent techniques applied to cultured and acutely dissociated hippocampal neurons, and have been aimed at identifying the various subtypes of nAChRs expressed in the mammalian central nervous system (CNS), at defining the mechanisms by which CNS nAChR activity is modulated, and at determining the ion permeability of CNS nAChR channels. Our findings can be summarized as follows: (1) hippocampal neurons express at least three subtypes of CNS nAChRs--an alpha 7-subunit-bearing nAChR that subserves fast-inactivating, alpha-BGT-sensitive currents, which are referred to as type IA, and alpha 4 beta 2 nAChR that subserves slowly inactivating, dihydro-beta-erythroidine-sensitive currents, which are referred to as type II, and an alpha 3 beta 4 nAChR that subserves slowly inactivating, mecamylamine-sensitive currents, which are referred to as type III; (2) nicotinic agonists can activate a single type of nicotinic current in olfactory bulb neurons, that is, type IA currents; (3) alpha 7-subunit-bearing nAChR channels in the hippocampus have a brief lifetime, a high conductance, and a high Ca2+ permeability; (4) the peak amplitude of type IA currents tends to rundown with time, and this rundown can be prevented by the presence of ATP-regenerating compounds (particularly phosphocreatine) in the internal solution; (5) rectification of type IA currents is dependent on the presence of Mg2+ in the internal solution; and (6) there is an ACh-insensitive site on neuronal and nonneuronal nAChRs through which the receptor channel can be activated. These findings lay the groundwork for a better understanding of the physiological role of these receptors in synaptic transmission in the CNS.

In vivo study of acetylcholine esterase in basal forebrain, amygdala, and cortex in mild to moderate Alzheimer disease

It is currently unclear whether impairment of the cholinergic system is present in Alzheimer disease (AD) already at an early stage and to what extent it depends on degeneration of the nucleus basalis of Meynert (nbM). We examined acetylcholine esterase activity in vivo in the nbM, the amygdala, and cerebral neocortex. Measurements were performed in normal controls and in patients with mild to moderate AD with positron emission tomography (PET) and C-11-labeled N-methyl-4-piperidyl-acetate (MP4A) which is a specific substrate of AChE. AChE activity was reduced significantly in amygdala and cerebral cortex. In contrast, AChE activity and glucose metabolism appeared preserved or even increased in the nbM. The results support the concept that neocortical and amygdaloid functional changes of the cholinergic system are an early and leading event in AD, rather than the consequence of neurodegeneration of basal nuclei.

Cellular and subcellular localization of the 2B-subunit of the NMDA receptor in the adult rat telencephalon

NMDA receptors (NR) are encoded by a family of genes including those of the NR1 and NR2A-D subunits. In situ hybridization has revealed that NR1, comprising eight splice variants, is ubiquitously expressed in the central nervous system (CNS) while the expression of NR2 isoforms is restricted to particular CNS regions. We report on the cellular and ultrastructural distribution of the NR2B polypeptide in rat telencephalon. In the telencephalon, the hippocampus represented the most intensively immunolabeled region. Here, predominantly the CA pyramidal neurons were heavily stained. Intense immunoreactivity (IR) was also detected in cortical neurons, in particular in pyramidal-like ones of layers II/III and V. On the ultrastructural level, the NR2B subunit was present not only in synaptic complexes where it usually was present in postsynaptic sites but in addition could be located at extrasynaptic sites. Furthermore, preliminary evidence indicates a presynaptic location of NR2B in some rare cases. NR2B antigen distribution is consistent with that of corresponding transcripts. Indeed, NR2B immunoreactivity coincides largely with that for NR1, indicating that both subunits are coexpressed in numerous cortical and hippocampal neurons.

Immunohisto- and cytochemical localization of cortical nicotinic cholinoceptors in rat and man.

A monoclonal antibody (WF 6) raised against purified Torpedo nicotinic acetylcholine receptor was applied to study the cellular and subcellular receptor distribution in human and rat neocortex. In both species, immunostaining was most prominent in perikarya and dendrites of the projection neurons in layers III and V. In layer VI fusiform cells displayed immunoreactivity while in layers I, II and IV some round-shaped cells were immunostained. Subcellularly, immunoprecipitate was found in neuronal perikarya, dendrites and in the postsynaptic thickenings, indicating intracellular sites of synthesis, transport and membrane incorporation of receptor protein. The results suggest that WF 6-immunocytochemistry is a useful tool to label nicotinic cholinergic receptors rendering new information about the specific cell-type and subcellular receptor distribution hardly obtainable by using conventional receptor autoradiography.

Nicotinic acetylcholine receptors in Alzheimer's disease.

Nicotinic cholinoceptive dysfunction associated with cognitive impairment is a leading neurochemical feature of Alzheimers disease. There-fore, nicotinic acetylcholine receptors have attracted considerable interest as potential therapeutic targets. The deficit of nicotine binding sites in Alzheimers disease may be related to alterations of nicotinic receptor synthesis on the levels of (i) transcription, (ii) translation and post-translational modifications, (iii) receptor transport and turnover, including membrane insertion. Current approaches aim at the elucidation of molecular changes at all three levels. Although a comprehensive picture has not yet been achieved, currently available data can be summarized as follows: (i) there are no changes at the level of transcription of subunit mRNAs studied so far, (ii) evidence is accumulating for a distinct decrease on the protein level in the expression especially of the alpha 4-subunit, and (iii) preliminary findings point to a possible correlation of cytoskeletal changes (hyperphosphorylation of tau-protein) with decreased nicotinic acetylcholine receptor expression.

Cellular distribution of nicotinic acetylcholine receptor subunit mRNAs in the human cerebral cortex as revealed by non-isotopic in situ hybridization

The pharmacology of telencephalic nicotinic acetylcholine receptors (nAChRs) has become an important issue in recent years. While in the human brain a direct pharmacological assessment is difficult to achieve the visualization of nAChRs has been enabled by histochemical techniques providing an ever increasing and improving resolution. Receptor autoradiography was used to visualize binding sites on the level of cortical layers whereas immunohistochemistry has allowed for the cell type-specific and ultrastructural localization of receptor protein. Further investigations have to elucidate the cellular sites of NAChR biosynthesis by visualizing subunit-specific transcripts. Using autopsy samples of the human precentral cortex (Area 4) as a paradigm we have applied digoxigenin-labeled cRNA probes to localize transcripts for the alpha 3- and alpha 4-1-subunits of the nAChR. In accordance with findings in the monkey cortex, the alpha 3-subunit seems to be expressed mainly in pyramidal neurons of layers III-VI of the human cerebral cortex. Transcripts for the alpha 4-1-subunit, by contrast, appear to be present in a large number of neurons throughout all layers of the cerebral cortex, consonant with its ubiquitous distribution in the rodent brain. The present findings show that also in human autopsy brains the cell type-specific detection of nAChR transcripts is possible. For the future, this technique will enable to investigate the expression of receptor transcripts in diseased human brains as compared to controls.