Neuronal Ca2+ sensor VILIP-1 leads to the upregulation of functional alpha4beta2 nicotinic acetylcholine receptors in hippocampal neurons.

The neuronal Ca2+-sensor protein VILIP-1, known to affect clathrin-dependent receptor trafficking, has been shown to interact with the cytoplasmic loop of the alpha4-subunit of the alpha4beta2 nicotinic acetylcholine receptor (nAChR), which is the most abundant nAChR subtype with high-affinity for nicotine in the brain. The alpha4beta2 nAChR is crucial for nicotine addiction and the beneficial effects of nicotine on cognition. Its dysfunction has been implicated in frontal lobe epilepsy, Alzheimer's disease and schizophrenia. Here we report that overexpression of VILIP-1 enhances ACh responsiveness, whereas siRNA against VILIP-1 reduces alpha4beta2 nAChR currents of hippocampal neurons. The underlying molecular mechanism likely involves enhanced constitutive exocytosis of alpha4beta2 nAChRs mediated by VILIP-1. The two interaction partners co-localize in a Ca2+-dependent manner with syntaxin-6, a Golgi-SNARE protein involved in trans-Golgi membrane trafficking. Thus, we speculate that regulation of VILIP-1-expression might modulate surface expression of ligand-gated ion channels, such as the alpha4beta2 nAChRs, possibly comprising a novel form of physiological up-regulation of ligand-gated ion channels.

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 alpha3-, alpha4-, alpha7-, beta2-, and beta4-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.

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.

Modulation of nicotinic receptor activity in the central nervous system: a novel approach to the treatment of Alzheimer disease.

Impaired cholinergic function in the central nervous system is an early feature of Alzheimer disease (AD). Currently, cholinergic deficit is usually corrected by increasing the amount of acetylcholine in the synapse by inhibiting acetylcholinesterase (AChE). One of the most consistent cholinergic deficits in AD is the reduced expression of nicotinic acetylcholine receptors (nAChR) in the brain. Since these receptors are essential for learning and memory, restoring nicotinic cholinergic function is a promising approach to treating AD. Allosteric modulation of nAChR is a novel approach, which circumvents development of tolerance through long-term use of conventional nicotinic agonists. Allosteric modulators interact with receptor-binding sites distinct from those capable of recognizing the natural agonist. Positive allosteric modulation of nAChR activity has no effect on conductance of single channels; instead, by facilitating channel opening, it potentiates responses evoked by the interaction of the natural agonist with presynaptic and postsynaptic nAChR. Allosteric modulation of nAChR activity could therefore potentially produce a significant benefit in AD. One such allosteric modulator is galantamine. In addition to increasing nAChR activity, galantamine also inhibits AChE. This novel, dual mechanism of action distinguishes galantamine from many other AChE inhibitors. Galantamine has been shown to improve cognitive and daily function for at least 6 months in placebo-controlled trials, and to maintain these functions at baseline levels for at least 12 months in a 6-month open-label extension study. Galantamine has positive effects on nAChR expression, which are likely to contribute to its sustained efficacy in the treatment of AD patients.

Differentiation-associated apoptosis of neural stem cells is effected by Bcl-2 overexpression: impact on cell lineage determination.

Apoptosis is an integral part of neural development. To elucidate the importance of programmed cell death on cell lineage determination we utilized murine PCC7-Mzl cells, a model system for neural differentiation. Treatment of pluripotent PCC7-Mzl stem cells with 0.1 microM all-trans retinoic acid (RA) causes a cease of proliferation and an initiation of differentiation into neurons, glial cells and fibroblasts. Simultaneously, a fraction of the cell culture (ca. 25%) dies within 24 h by apoptosis. We transfected PCC7-Mzl cells with the human bcl-2 cDNA and generated PCC7-Mz-Bcl-2 cell lines expressing two- to tenfold higher levels of Bcl-2 than parental cells. Overexpression of Bcl-2 resulted in hypophosphorylation of the retinoblastoma (Rb) protein and consequently prolonged the doubling time of the culture from 18 h to 23 h. RA-induced apoptosis was drastically reduced to 3 to 15% depending on the level of Bcl-2 expression. RA-induced caspase activation, cytochrome c release from the mitochondria to the cytosol and DNA fragmentation was completely blocked. Furthermore, treating Bcl-2 cultures with ceramide (10 microM), a second messenger mediating the RA-initiated death signal in parental cells, no longer caused DNA laddering. Bcl-2 overexpression did not interfere with the potential of PCC7-Mz cells to develop into neurons, glial cells and fibroblasts. However, the relative distribution of cell types in the culture was shifted such that the fraction of neurons was reduced to half (from 60 to 30%) with a concomitant increase in the number of glial and fibroblastoid cells. Furthermore, Bcl-2-overexpressing neurons, but not neurons of parental or mock-transfected PCC7-Mzl cultures, were able to grow as single cells.

Pharmacokinetic rationale for switching from donepezil to galantamine.

Galantamine, the most recently approved acetylcholinesterase inhibitor (AChEI) for use in the United States, has allosteric modulating activity at nicotinic receptors and inhibits acetylcholinesterase. This dual mechanism of action may make galantamine an attractive option for patients with Alzheimer's disease who have not benefited from their current therapy; thus, methods for switching patients from donepezil or rivastigmine to galantamine are needed. Protocols for switching patients from one AChEI to another must consider both the time required for washout of the first drug and the rate of dose escalation of the second drug. Both issues depend on the pharmacodynamics, pharmacokinetics, and pharmacology of the drugs under consideration. Because the common property of the drugs considered here is their acetylcholinesterase inhibitory activity, it seems reasonable to keep this activity at or below the activity achieved by the first drug at all times. In addition, the patient's condition should be monitored to avoid deterioration resulting from subtherapeutic drug concentrations during the switch. The switching protocol proposed here has been based on an analysis of mean plasma concentrations of donepezil following administration of a single dose and on the established pharmacokinetics of galantamine.

The pharmacological rationale for treating vascular dementia with galantamine (Reminyl).

There is considerable evidence indicating that, as in Alzheimer's disease, the central cholinergic system is impaired in vascular dementia (VaD). Using lessons learned from Alzheimer's disease research, it has been proposed that enhancement of the cholinergic system is a rational approach to treating the symptoms of VaD. Galantamine's dual mode of action may provide a greater chance of success in treating patients with Alzheimer's disease through enhanced efficacy on the cognitive, functional and behavioural aspects of dementia. Trials are currently underway to see if this broad spectrum of efficacy extends to patients with Alzheimer's disease with cerebrovascular disease ('mixed' dementia) or VaD, as well as other conditions, and the results are eagerly awaited.

Allosteric sensitization of nicotinic receptors by galantamine, a new treatment strategy for Alzheimer's disease.

Cholinesterase inhibitors are the only approved drug treatment for patients with mild to moderately severe Alzheimer's disease. Interestingly, the clinical potency of these drugs does not correlate well with their activity as cholinesterase inhibitors, nor is their action as short lived as would be expected from purely symptomatic treatment. A few cholinesterase inhibitors, including galantamine, produce beneficial effects even after drug treatment has been terminated. These effects assume modes of action other than mere esterase inhibition and are capable of inducing systemic changes. We have recently discovered a mechanism that could account, at least in part, for the above-mentioned unexpected properties of some cholinesterase inhibitors. We have found that a subgroup of cholinesterase inhibitors, including galantamine but excluding tacrine, directly interacts with nicotinic acetylcholine receptors. These compounds, named allosterically potentiating ligands, sensitize nicotinic receptors by increasing the probability of channel opening induced by acetylcholine and nicotinic agonists and by slowing down receptor desensitization. The allosterically potentiating ligand action, which is not necessarily associated with cholinesterase inhibition, has been demonstrated by whole-cell patch-clamp recordings to occur in natural murine and human neurons and in murine and human cell lines expressing various subtypes of neuronal nicotinic acetylcholine receptors.

Allosteric modulation of nicotinic receptors as a treatment strategy for Alzheimer's disease.

Impairment of the central cholinergic system has a pivotal role in the cognitive decline observed in patients with Alzheimer's disease (AD). One of the most prominent cholinergic deficits is the reduced number of nicotinic acetylcholine receptors (nAChR) in the brain. Since these receptors are important for memory and learning, enhancing nicotinic neurotransmission is a promising treatment strategy for AD. The two most common approaches to correcting these cholinergic deficits are to increase the synaptic availability of acetylcholine (ACh) by inhibiting acetylcholinesterase (AChE), or to mimic the effects of ACh (nicotinic agonists) by acting directly on nicotinic receptors. Clinical studies suggest that AChE inhibitors produce only short-term symptomatic improvement. Similarly, long-term use of nicotinic agonists may induce desensitization of nicotinic receptors, leading to tolerance and therefore limiting the duration of efficacy. Allosteric modulation of nAChR is a novel approach, which circumvents the development of tolerance. Allosteric modulators bind to a site on nAChR that is different to the binding site of the natural agonist, ACh. This allosteric interaction amplifies the actions of ACh at post- and presynaptic nAChR. In particular, presynaptic nAChR are capable of modulating the release of ACh and other neurotransmitters, such as glutamate, serotonin and GABA, which may contribute to symptoms of the illness. Allosteric modulation of nAChR could therefore produce significant therapeutic benefit in AD. One of the most potent of these allosteric modulators is galantamine. As well as modulating nAChR, galantamine inhib- its AChE. The extent to which the clinical benefits of galantamine are attributable specifically to its nicotinic effects is uncertain and requires further investigation. However, galantamine maintains patients' level of cognitive and daily function for at least 1 year, which has not been reported for other AChE inhibitors. Galantamine's modulatory effects on nAChR may influence transcriptional regulation, resulting in an increased synthesis of nAChR. This may account for galantamine's sustained efficacy.

Neuronal nicotinic receptors in synaptic functions in humans and rats: physiological and clinical relevance.

The present report describes the participation of nicotinic receptors (nAChRs) in controlling the excitability of local neuronal circuitries in the rat hippocampus and in the human cerebral cortex. The patch-clamp technique was used to record responses triggered by the non-selective agonist ACh and the alpha7-nAChR-selective agonist choline in interneurons of human cerebral cortical and rat hippocampal slices. Evidence is provided that functional alpha7- and alpha4beta2-like nAChRs are present on somatodendritic and/or preterminal/terminal regions of interneurons in the CA1 field of the rat hippocampus and in the human cerebral cortex and that activation of the different nAChR subtypes present in the preterminal/terminal areas of the interneurons triggers the tetrodotoxin-sensitive release of GABA. Modulation by nAChRs of GABAergic transmission, which can result either in inhibition or disinhibition of pyramidal neurons, depends both on the receptor subtype present in the interneurons and on the agonist acting upon these receptors. Not only do alpha7 nAChRs desensitize faster than alpha4beta2 nAChRs, but also alpha7 nAChR desensitization induced by ACh lasts longer than that induced by choline. These mechanisms, which appear to be retained across species, might explain the involvement of nAChRs in cognitive functions and in such neurological disorders as Alzheimer's disease and schizophrenia.

Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer's disease.

One of the most prominent cholinergic deficit in Alzheimer's disease (AD) is the reduced number of nicotinic acetylcholine receptors (nAChR) in the hippocampus and cortex of AD patients, as compared to age-matched controls. This deficit results in reduced nicotinic cholinergic excitation which may not only impair postsynaptic depolarization but also presynaptic neurotransmitter release and Ca2+-dependent intracellular signaling, including transcriptional activity. Presently, the most common approach to correct the nicotinic cholinergic deficit in AD is the application of cholinesterase inhibitors. Due to the resulting increase in synaptic acetylcholine levels, both in concentration and time, additional nAChR molecules, e.g. those more distant from the ACh release sites, could be activated. As an obvious disadvantage, this approach affects cholinergic neurotransmission as a whole, including muscarinic neurotransmission. As a novel and alternative approach, a treatment strategy which exclusively targets nicotinic receptors is suggested. The strategy is based on a group of modulating ligands of nicotinic receptors, named allosterically potentiating ligands (APL), which increase the probability of channel opening induced by ACh and nicotinic agonists, and in addition decrease receptor desensitization. The action of APL on nicotinic receptors is reminiscent of that of benzodiazepines on GABA(A) receptors and of that of glycine on the NMDA-subtype of glutamate receptor. Representative nicotinic APL are the plant alkaloids physostigmine, galanthamine and codeine, and the neurotransmitter serotonin (5HT). The potentiating effect of APL on nicotinic neurotransmission has been shown by whole-cell patch-clamp studies in natural murine and human neurons, and in murine and human cell lines expressing various subtypes of neuronal nAChR.

Production of ceramides causes apoptosis during early neural differentiation in vitro.

To investigate signal transduction pathways leading to apoptosis during the early phase of neurogenesis, we employed PCC7-Mz1 cells, which cease to proliferate and begin to differentiate into a stable pattern of neurons, astroglial cells, and fibroblasts upon incubation with retinoic acid (RA). As part of lineage determination, a sizable fraction of RA-treated cultures die by apoptosis. Applying natural long-chain C(16)-ceramides as well as membrane-permeable C(2)/C(6)-ceramide analogs caused apoptosis, whereas the biologically nonactive C(2)-dihydroceramide did not. Treating PCC7-Mz1 stem cells with a neutral sphingomyelinase or with the ceramidase inhibitor N-oleoylethanolamine elevated the endogenous ceramide levels and concomitantly induced apoptosis. Addition of RA caused an increase in ceramide levels within 3-5 h, which reached a maximum (up to 3.5-fold of control) between days 1 and 3 of differentiation. Differentiated PCC7-Mz1 cells did not respond with ceramide formation and apoptosis to RA treatment. The acidic sphingomyelinase contributed only weakly and the neutral Mg(2+)-dependent and Mg(2+)-independent sphingomyelinases not at all to the RA-mediated production of ceramides. However, ceramide increase was sensitive to the ceramide synthase inhibitor fumonisin B(1), suggesting a crucial role for the de novo synthesis pathway. Enzymatic assays revealed that ceramide synthase activity remained unaltered, whereas serine palmitoyltransferase (SPT), a key enzyme in ceramide synthesis, was activated approximately 2.5-fold by RA treatment. Activation of SPT seemed to be mediated via a post-translational mechanism because levels of the mRNAs coding for the two SPT subunits were unaffected. Expression of marker proteins shows that ceramide regulates apoptosis, rather than differentiation, during early neural differentiation.

alpha7 nicotinic acetylcholine receptors and modulation of gabaergic synaptic transmission in the hippocampus.

The present report provides new findings regarding modulation of gamma-aminobutyric acid (GABA) transmission by alpha7 nicotinic receptor activity in CA1 interneurons of rat hippocampal slices. Recordings were obtained from tight-seal cell-attached patches of the CA1 interneurons, and agonists were delivered to the neurons via a modified U-tube. Application for 6 s of the alpha7 nicotinic receptor-selective agonist choline (> or =1 mM) to all CA1 interneurons tested triggered action potentials that were detected as fast current transients. The activity triggered by choline terminated well before the end of the agonist pulse, was blocked by the alpha7 nicotinic receptor antagonist methyllycaconitine (50 nM) and was concentration dependent; the higher the concentration of choline the higher the frequency of events and the shorter the delay for detection of the first event. In 40% of the neurons tested, choline-triggered action potentials decreased in amplitude progressively until no more events could be detected despite the presence of the agonist. Primarily, this finding could be explained by Na(+)-channel inactivation associated with membrane depolarization induced by alpha7 nicotinic receptor activation. In 60% of the neurons, the amplitude of choline-induced action potentials was sustained at the intial level, but again the activity did not last as long as the agonist pulse, in this case apparently because of agonist-induced receptor desensitization. These results altogether demonstrate that agonists interacting with alpha7 nicotinic receptors, including the natural transmitter acetylcholine and its metabolite choline, influence GABAergic transmission, not only by activating these receptors, but also by controlling the rate of Na(+)-channel inactivation and/or by inducing receptor desensitization.

Allosteric modulation of nicotinic acetylcholine receptors as a treatment strategy for Alzheimer's disease.

The basic symptoms of Alzheimer's dementia, i.e., a loss in cognitive function, are due to impaired nicotinic cholinergic neurotransmission. To compensate for this impairment by drug treatment, blockers of the acetylcholine-degrading enzyme acetylcholinesterase are applied, even though this approach obviously is prone to many side-effects, including those of muscarinic nature. We have recently described a novel class of nicotinic acetylcholine receptor ligands which, similar to the action of benzodiazepines on GABA(A) receptors, allosterically potentiate submaximal nicotinic responses. The sensitizing effect is a consequence of facilitated channel opening in the presence of allosterically potentiating ligand (APL). Representative members of this class of ligands are the plant alkaloids physostigmine, galanthamine, and codeine. Because APLs could enhance nicotinic neurotransmission under conditions of reduced secretion and/or increased degradation of acetylcholine or reduced acetylcholine-sensitivity of nicotinic acetylcholine receptors, they could have a preventive and corrective action on impaired but still functioning nicotinic neurotransmission.

Ordered networks of rat hippocampal neurons attached to silicon oxide surfaces.

The control of neuronal cell position and outgrowth is of fundamental interest in the development of applications ranging from cellular biosensors to tissue engineering. We have produced rectangular networks of functional rat hippocampal neurons on silicon oxide surfaces. Attachment and network formation of neurons was guided by a geometrical grid pattern of the adhesion peptide PA22-2 which matches in sequence a part of the A-chain of laminin. PA22-2 was applied by contact printing onto the functionalised silicon oxide surface and was immobilised by hetero-bifunctional cross-linking with sulfo-GMBS. Geometric pattern matching was achieved by microcontact printing using a polydimethylsiloxane (PDMS) stamp. In this way the produced grid pattern ranged from 3 to 20 microm in line width and from 50 to 100 microm in line distances. As shown by atomic force microscopy (AFM), line widths and line distances of the peptide pattern differ less than 0.5 microm from the used PDMS stamp. The height of the layer of immobilised PA22-2 was approximately 3.5 nm implying the layer to be monomolecular. Immobilised PA22-2 was capable of binding anti-PA22-2 antibodies indicating that the function of the peptide was not compromised by immobilisation. Rat hippocampal neurons, cultured at low density in serum-free medium, were applied to the growth matrix of PA22-2-coated substrates and, within 1-3 h of culture, formed a network-like pattern that more or less matched the printed grid. Reliability and reproducibility of neuronal network formation depended on the geometry, line width and node diameter of the grid pattern. The immobilised neurons showed resting membrane potentials comparable with controls and, already after 1 day of culture, were capable of eliciting action potentials. The suitability of the immobilised neurons for the study of man-made neural networks and for multi-site recordings from a functional neuronal network is discussed.

Galantamine is an allosterically potentiating ligand of the human alpha4/beta2 nAChR.

Galantamine (Reminyl) is a novel drug treatment for mild to moderate Alzheimer's disease (AD). Originally established as a reversible inhibitor of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), galantamine also acts as an allosterically potentiating ligand (APL) on nicotinic acetylcholine receptors (nAChR). Having previously established this second mode of action on nAChRs from murine brain, we demonstrate here the same action of galantamine on the most abundant nAChR in the human brain, the alpha4/beta2 subtype. This nAChR-sensitizing action is not a common property of all, or most, AChE inhibitors, as is shown by the absence of this effect for other therapeutically applied AChE inhibitors including tacrine, metrifonate, rivastigmine and donepezil. The possible benefits for therapy of AD of an APL action on nicotinic receptors is discussed.

Expression of functional alpha7 nicotinic acetylcholine receptor during mammalian muscle development and denervation.

We have studied, on the transcriptional, protein and functional level, the expression of alpha7 nicotinic acetylcholine receptors (nAChR) in the course of rat muscle development, denervation and renervation. At foetal day 13, alpha7 nAChR expression was observed in somites and developing muscles of the back, but not yet in migrating myoblasts. Two days later, concomitant with myoblast aggregation, the alpha7 isoform began to be expressed in isolated myoblasts, with the highest level of expression in the frontal zone of the migrating wave. On foetal day 18, a time when the myoblasts in the upper hindleg have fused, alpha7 nAChR expression was most prominent in the outer layer of muscle tissue. The highest level of expression was observed in the first postnatal week, when practically all muscle cells stained positively for alpha7 protein. During the following weeks, alpha7 nAChR expression slowly decreased and practically disappeared in adult hindleg muscle. Following chronic denervation of adult Soleus muscle fibres, expression of alpha7 nAChR returned within 2-4 weeks. Electrophysiological measurements showed that the alpha7 nAChR of chronically denervated soleus muscle fibres were functional and, in particular, that they could be activated by choline. The presence of the alpha7 nAChR in developing and denervated muscle suggests a role for this nicotinic receptor in neuronal pathfinding and/or endplate stabilization.

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.

Analysis of NO synthase expression in neuronal, astroglial and fibroblast-like derivatives differentiating from PCC7-Mz1 embryonic carcinoma cells.

We studied the expression of the NO synthase isoforms in an in vitro model of neural development using RT-PCR, Western blot and immunohistochemistry. Murine PCC7-Mz1 cells (Jostock et al., Eur. J. Cell Biol. 76, 63-76, 1998) differentiate in the presence of all-trans retinoic acid and dibutyryl cAMP along the neural pathway into neuron-like, fibroblast-like and astroglia-like cells. Undifferentiated cells showed immunofluorescent staining for neuronal-type NOS I and endothelial-type NOS III. This expression pattern was retained in those cells differentiating into neurofilament- and tau protein-positive neuronal cells. Thymocyte alloantigen (Thy1.2/CD 90.2)-positive fibroblasts, appearing around day 3, and glial fibrillary acidic protein (GFAP)-positive astroglial cells, appearing after day 6 of differentiation, stained negative for any NOS isoform. Starting at day 6 of differentiation, expression of inducible-type NOS II could be stimulated with cytokines in a subset of cells, which may represent activated astrocytes. NOS II was always undetectable in non-induced cultures. These data indicate that the ability of stem cells to express NOS I and NOS III is only retained when the cells differentiate along the neuronal lineage, while a small subpopulation of cells acquires the ability to express NOS II in response to cytokines.