Isolation and characterization of neurotoxic astrocytes derived from adult triple transgenic Alzheimer's disease mice.

Alzheimer's disease has been considered mostly as a neuronal pathology, although increasing evidence suggests that glial cells might play a key role in the disease onset and progression. In this sense, astrocytes, with their central role in neuronal metabolism and function, are of great interest for increasing our understanding of the disease. Thus, exploring the morphological and functional changes suffered by astrocytes along the course of this disorder has great therapeutic and diagnostic potential. In this work we isolated and cultivated astrocytes from symptomatic 9-10-months-old adult 3xTg-AD mice, with the aim of characterizing their phenotype and exploring their pathogenic potential. These "old" astrocytes occurring in the 3xTg-AD mouse model of Alzheimer's Disease presented high proliferation rate and differential expression of astrocytic markers compared with controls. They were neurotoxic to primary neuronal cultures both, in neuronal-astrocyte co-cultures and when their conditioned media (ACM) was added into neuronal cultures. ACM caused neuronal GSK3ß activation, changes in cytochrome c pattern, and increased caspase 3 activity, suggesting intrinsic apoptotic pathway activation. Exposure of neurons to ACM caused different subcellular responses. ACM application to the somato-dendritic domain in compartmentalised microfluidic chambers caused degeneration both locally in soma/dendrites and distally in axons. However, exposure of axons to ACM did not affect somato-dendritic nor axonal integrity. We propose that this newly described old 3xTg-AD neurotoxic astrocytic population can contribute towards the mechanistic understanding of the disease and shed light on new therapeutical opportunities.

Distinct small non-coding RNA landscape in the axons and released extracellular vesicles of developing primary cortical neurons and the axoplasm of adult nerves.

Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored.We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons invivo. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5'-tRHs compose the great majority of tRNA-derived fragments observed invitro, a shift to 3'-tRNAs is observed in mature axons invivo.The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication.

Vinculin is required for neuronal mechanosensing but not for axon outgrowth.

Integrin receptors are transmembrane proteins that bind to the extracellular matrix (ECM). In most animal cell types integrins cluster together with adaptor proteins at focal adhesions that sense and respond to external mechanical signals. In the central nervous system (CNS), ECM proteins are sparsely distributed, the tissue is comparatively soft and neurons do not form focal adhesions. Thus, how neurons sense tissue stiffness is currently poorly understood. Here, we found that integrins and the integrin-associated proteins talin and focal adhesion kinase (FAK) are required for the outgrowth of neuronal processes. Vinculin, however, whilst not required for neurite outgrowth was a key regulator of integrin-mediated mechanosensing of neurons. During growth, growth cones of axons of CNS derived cells exerted dynamic stresses of around 10-12 Pa on their environment, and axons grew significantly longer on soft (0.4 kPa) compared to stiff (8 kPa) substrates. Depletion of vinculin blocked this ability of growth cones to distinguish between soft and stiff substrates. These data suggest that vinculin in neurons acts as a key mechanosensor, involved in the regulation of growth cone motility.

Functional Genomics of Axons and Synapses to Understand Neurodegenerative Diseases.

Functional genomics studies through transcriptomics, translatomics and proteomics have become increasingly important tools to understand the molecular basis of biological systems in the last decade. In most cases, when these approaches are applied to the nervous system, they are centered in cell bodies or somatodendritic compartments, as these are easier to isolate and, at least in vitro, contain most of the mRNA and proteins present in all neuronal compartments. However, key functional processes and many neuronal disorders are initiated by changes occurring far away from cell bodies, particularly in axons (axopathologies) and synapses (synaptopathies). Both neuronal compartments contain specific RNAs and proteins, which are known to vary depending on their anatomical distribution, developmental stage and function, and thus form the complex network of molecular pathways required for neuron connectivity. Modifications in these components due to metabolic, environmental, and/or genetic issues could trigger or exacerbate a neuronal disease. For this reason, detailed profiling and functional understanding of the precise changes in these compartments may thus yield new insights into the still intractable molecular basis of most neuronal disorders. In the case of synaptic dysfunctions or synaptopathies, they contribute to dozens of diseases in the human brain including neurodevelopmental (i.e., autism, Down syndrome, and epilepsy) as well as neurodegenerative disorders (i.e., Alzheimer's and Parkinson's diseases). Histological, biochemical, cellular, and general molecular biology techniques have been key in understanding these pathologies. Now, the growing number of omics approaches can add significant extra information at a high and wide resolution level and, used effectively, can lead to novel and insightful interpretations of the biological processes at play. This review describes current approaches that use transcriptomics, translatomics and proteomic related methods to analyze the axon and presynaptic elements, focusing on the relationship that axon and synapses have with neurodegenerative diseases.

PDCD4 regulates axonal growth by translational repression of neurite growth-related genes and is modulated during nerve injury responses.

Programmed cell death 4 (PDCD4) protein is a tumor suppressor that inhibits translation through the mTOR-dependent initiation factor EIF4A, but its functional role and mRNA targets in neurons remain largely unknown. Our work identified that PDCD4 is highly expressed in axons and dendrites of CNS and PNS neurons. Using loss- and gain-of-function experiments in cortical and dorsal root ganglia primary neurons, we demonstrated the capacity of PDCD4 to negatively control axonal growth. To explore PDCD4 transcriptome and translatome targets, we used Ribo-seq and uncovered a list of potential targets with known functions as axon/neurite outgrowth regulators. In addition, we observed that PDCD4 can be locally synthesized in adult axons in vivo, and its levels decrease at the site of peripheral nerve injury and before nerve regeneration. Overall, our findings demonstrate that PDCD4 can act as a new regulator of axonal growth via the selective control of translation, providing a target mechanism for axon regeneration and neuronal plasticity processes in neurons.

Spatiotemporal regulation of GSK3ß levels by miRNA-26a controls axon development in cortical neurons.

Both the establishment of neuronal polarity and axonal growth are crucial steps in the development of the nervous system. The local translation of mRNAs in the axon provides precise regulation of protein expression, and is now known to participate in axon development, pathfinding and synaptic formation and function. We have investigated the role of miR-26a in early stage mouse primary cortical neuron development. We show that micro-RNA-26a-5p (miR-26a) is highly expressed in neuronal cultures, and regulates both neuronal polarity and axon growth. Using compartmentalised microfluidic neuronal cultures, we identified a local role for miR-26a in the axon, where the repression of local synthesis of GSK3ß controls axon development and growth. Removal of this repression in the axon triggers local translation of GSK3ß protein and subsequent transport to the soma, where it can impact axonal growth. These results demonstrate how the axonal miR-26a can regulate local protein translation in the axon to facilitate retrograde communication to the soma and amplify neuronal responses, in a mechanism that influences axon development.

Mitochondrial impairment activates the Wallerian pathway through depletion of NMNAT2 leading to SARM1-dependent axon degeneration.

Wallerian degeneration of physically injured axons involves a well-defined molecular pathway linking loss of axonal survival factor NMNAT2 to activation of pro-degenerative protein SARM1. Manipulating the pathway through these proteins led to the identification of non-axotomy insults causing axon degeneration by a Wallerian-like mechanism, including several involving mitochondrial impairment. Mitochondrial dysfunction is heavily implicated in Parkinson's disease, Charcot-Marie-Tooth disease, hereditary spastic paraplegia and other axonal disorders. However, whether and how mitochondrial impairment activates Wallerian degeneration has remained unclear. Here, we show that disruption of mitochondrial membrane potential leads to axonal NMNAT2 depletion in mouse sympathetic neurons, increasing the substrate-to-product ratio (NMN/NAD) of this NAD-synthesising enzyme, a metabolic fingerprint of Wallerian degeneration. The mechanism appears to involve both impaired NMNAT2 synthesis and reduced axonal transport. Expression of WLDS and Sarm1 deletion both protect axons after mitochondrial uncoupling. Blocking the pathway also confers neuroprotection and increases the lifespan of flies with Pink1 loss-of-function mutation, which causes severe mitochondrial defects. These data indicate that mitochondrial impairment replicates all the major steps of Wallerian degeneration, placing it upstream of NMNAT2 loss, with the potential to contribute to axon pathology in mitochondrial disorders.

Biological Significance of microRNA Biomarkers in ALS-Innocent Bystanders or Disease Culprits?

MicroRNAs (miRNAs) represent potential biomarkers for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). However, whether expression changes of individual miRNAs are simply an indication of cellular dysfunction and degeneration, or actually promote functional changes in target gene expression relevant to disease pathogenesis, is unclear. Here we used bioinformatics to test the hypothesis that ALS-associated miRNAs exert their effects through targeting genes implicated in disease etiology. We documented deregulated miRNAs identified in studies of ALS patients, noting variations in participants, tissue samples, miRNA detection or quantification methods used, and functional or bioinformatic assessments (if performed). Despite lack of experimental standardization, overlap of many deregulated miRNAs between studies was noted; however, direction of reported expression changes did not always concur. The use of in silico predictions of target genes for the most commonly deregulated miRNAs, cross-referenced to a selection of previously identified ALS genes, did not support our hypothesis. Specifically, although deregulated miRNAs were predicted to commonly target ALS genes, random miRNAs gave similar predictions. To further investigate biological patterns in the deregulated miRNAs, we grouped them by tissue source in which they were identified, indicating that for a core of frequently detected miRNAs, blood/plasma/serum may be useful for future profiling experiments. We conclude that in silico predictions of gene targets of deregulated ALS miRNAs, at least using currently available algorithms, are unlikely to be sufficient in informing disease pathomechanisms. We advocate experimental functional testing of candidate miRNAs and their predicted targets, propose miRNAs to prioritise, and suggest a concerted move towards protocol standardization for biomarker identification.

Impact of voluntary exercise and housing conditions on hippocampal glucocorticoid receptor, miR-124 and anxiety.

BACKGROUND: Lack of physical activity and increased levels of stress contribute to the development of multiple physical and mental disorders. An increasing number of studies relate voluntary exercise with greater resilience to psychological stress, a process that is highly regulated by the hypothalamic-pituitary-adrenal (HPA) axis. However, the molecular mechanisms underlying the beneficial effects of exercise on stress resilience are still poorly understood. Here we have studied the impact of long term exercise and housing conditions on: a) hippocampal expression of glucocorticoid receptor (Nr3c1), b) epigenetic regulation of Nr3c1 (DNA methylation at the Nr3c1-1F promoter and miR-124 expression), c) anxiety (elevated plus maze, EPM), and d) adrenal gland weight and adrenocorticotropic hormone receptor (Mc2r) expression. RESULTS: Exercise increased Nr3c1 and Nr3c1-1F expression and decreased miR-124 levels in the hippocampus in single-housed mice, suggesting enhanced resilience to stress. The opposite was found for pair-housed animals. Bisulfite sequencing showed virtually no DNA methylation in the Nr3c1-1F promoter region. Single-housing increased the time spent on stretch attend postures. Exercise decreased the time spent at the open arms of the EPM, however, the mobility of the exercise groups was significantly lower. Exercise had opposite effects on the adrenal gland weight of single and pair-housed mice, while it had no effect on adrenal Mc2r expression. CONCLUSIONS: These results suggest that exercise exerts a positive impact on stress resilience in single-housed mice that could be mediated by decreasing miR-124 and increasing Nr3c1 expression in the hippocampus. However, pair-housing reverses these effects possibly due to stress from dominance disputes between pairs.

Drosophila CLIP-190 and mammalian CLIP-170 display reduced microtubule plus end association in the nervous system.

Axons act like cables, electrically wiring the nervous system. Polar bundles of microtubules (MTs) form their backbones and drive their growth. Plus end-tracking proteins (+TIPs) regulate MT growth dynamics and directionality at their plus ends. However, current knowledge about +TIP functions, mostly derived from work in vitro and in nonneuronal cells, may not necessarily apply to the very different context of axonal MTs. For example, the CLIP family of +TIPs are known MT polymerization promoters in nonneuronal cells. However, we show here that neither Drosophila CLIP-190 nor mammalian CLIP-170 is a prominent MT plus end tracker in neurons, which we propose is due to low plus end affinity of the CAP-Gly domain-containing N-terminus and intramolecular inhibition through the C-terminus. Instead, both CLIP-190 and CLIP-170 form F-actin-dependent patches in growth cones, mediated by binding of the coiled-coil domain to myosin-VI. Because our loss-of-function analyses in vivo and in culture failed to reveal axonal roles for CLIP-190, even in double-mutant combinations with four other +TIPs, we propose that CLIP-190 and -170 are not essential axon extension regulators. Our findings demonstrate that +TIP functions known from nonneuronal cells do not necessarily apply to the regulation of the very distinct MT networks in axons.

Caspase-8-mediated PAR-4 cleavage is required for TNFα-induced apoptosis.

The tumor suppressor protein prostate apoptosis response-4 (PAR-4) is silenced in a subset of human cancers and its down-regulation serves as a mechanism for cancer cell survival following chemotherapy. PAR-4 re-expression selectively causes apoptosis in cancer cells but how its pro-apoptotic functions are controlled and executed precisely is currently unknown. We demonstrate here that UV-induced apoptosis results in a rapid caspase-dependent PAR-4 cleavage at EEPD131G, a sequence that was preferentially recognized by caspase-8. To investigate the effect on cell growth for this cleavage event we established stable cell lines that express wild-type-PAR-4 or the caspase cleavage resistant mutant PAR-4 D131G under the control of a doxycycline-inducible promoter. Induction of the wild-type protein but not the mutant interfered with cell proliferation, predominantly through induction of apoptosis. We further demonstrate that TNFα-induced apoptosis leads to caspase-8-dependent PAR-4-cleavage followed by nuclear accumulation of the C-terminal PAR-4 (132-340) fragment, which then induces apoptosis. Taken together, our results indicate that the mechanism by which PAR-4 orchestrates the apoptotic process requires cleavage by caspase-8.

Regulation of axon growth by the JIP1-AKT axis.

The polarisation of developing neurons to form axons and dendrites is required for the establishment of neuronal connections leading to proper brain function. The protein kinase AKT and the MAP kinase scaffold protein JNK-interacting protein-1 (JIP1) are important regulators of axon formation. Here we report that JIP1 and AKT colocalise in axonal growth cones of cortical neurons and collaborate to promote axon growth. The loss of AKT protein from the growth cone results in the degradation of JIP1 by the proteasome, and the loss of JIP1 promotes a similar fate for AKT. Reduced protein levels of both JIP1 and AKT in the growth cone can be induced by glutamate and this coincides with reduced axon growth, which can be rescued by a stabilized mutant of JIP1 that rescues AKT protein levels. Taken together, our data reveal a collaborative relationship between JIP1 and AKT that is required for axon growth and can be regulated by changes in neuronal activity.

microRNA-9 regulates axon extension and branching by targeting Map1b in mouse cortical neurons.

The capacity of neurons to develop a long axon and multiple dendrites defines neuron connectivity in the CNS. The highly conserved microRNA-9 (miR-9) is expressed in both neuronal precursors and some post-mitotic neurons, and we detected miR-9 expression in the axons of primary cortical neurons. We found that miR-9 controlled axonal extension and branching by regulating the levels of Map1b, an important protein for microtubule stability. Following microfluidic separation of the axon and the soma, we found that miR-9 repressed Map1b translation and was a functional target for the BDNF-dependent control of axon extension and branching. We propose that miR-9 links regulatory signaling processes with dynamic translation mechanisms, controlling Map1b protein levels and axon development.

Characterisation of a new regulator of BDNF signalling, Sprouty3, involved in axonal morphogenesis in vivo.

During development, many organs, including the kidney, lung and mammary gland, need to branch in a regulated manner to be functional. Multicellular branching involves changes in cell shape, proliferation and migration. Axonal branching, however, is a unicellular process that is mediated by changes in cell shape alone and as such appears very different to multicellular branching. Sprouty (Spry) family members are well-characterised negative regulators of Receptor tyrosine kinase (RTK) signalling. Knockout of Spry1, 2 and 4 in mouse result in branching defects in different organs, indicating an important role of RTK signalling in controlling branching pattern. We report here that Spry3, a previously uncharacterised member of the Spry family plays a role in axonal branching. We found that spry3 is expressed specifically in the trigeminal nerve and in spinal motor and sensory neurons in a Brain-derived neurotrophin factor (BDNF)-dependent manner. Knockdown of Spry3 expression causes an excess of axonal branching in spinal cord motoneurons in vivo. Furthermore, Spry3 inhibits the ability of BDNF to induce filopodia in Xenopus spinal cord neurons. Biochemically, we show that Spry3 represses calcium release downstream of BDNF signalling. Altogether, we have found that Spry3 plays an important role in the regulation of axonal branching of motoneurons in vivo, raising the possibility of unexpected conservation in the involvement of intracellular regulators of RTK signalling in multicellular and unicellular branching.

Mouse ACF7 and drosophila short stop modulate filopodia formation and microtubule organisation during neuronal growth.

Spectraplakins are large actin-microtubule linker molecules implicated in various processes, including gastrulation, wound healing, skin blistering and neuronal degeneration. Expression data for the mammalian spectraplakin ACF7 and genetic analyses of the Drosophila spectraplakin Short stop (Shot) suggest an important role during neurogenesis. Using three parallel neuronal culture systems we demonstrate that, like Shot, ACF7 is essential for axon extension and describe, for the first time, their subcellular functions during axonal growth. Firstly, both ACF7 and Shot regulate the organisation of neuronal microtubules, a role dependent on both the F-actin- and microtubule-binding domains. This role in microtubule organisation is probably the key mechanism underlying the roles of Shot and ACF7 in growth cone advance. Secondly, we found a novel role for ACF7 and Shot in regulating the actin cytoskeleton through their ability to control the formation of filopodia. This function in F-actin regulation requires EF-hand motifs and interaction with the translational regulator Krasavietz/eIF5C, indicating that the underlying mechanisms are completely different from those used to control microtubules. Our data provide the basis for the first mechanistic explanation for the role of Shot and ACF7 in the developing nervous system and demonstrate their ability to coordinate the organisation of both actin and microtubule networks during axonal growth.

The JIP1 scaffold protein regulates axonal development in cortical neurons.

The development of neuronal polarity is essential for the determination of neuron connectivity and for correct brain function. The c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1) is highly expressed in neurons and has previously been characterized as a regulator of JNK signaling.JIP1 has been shown to localize to neurites in various neuronal models, but the functional significance of this localization is not fully understood [1-4]. JIP1 is also a cargo of the motor protein kinesin-1, which is important for axonal transport [2, 4]. Here we demonstrate that before primary cortical neurons become polarized, JIP1 specifically localizes to a single neurite and that after axonal specification,it accumulates in the emerging axon. JIP1 is necessary for normal axonal development and promotes axonal growth dependent upon its binding to kinesin-1 and via a newly described interaction with the c-Abl tyrosine kinase. JIP1associates with and is phosphorylated by c-Abl, and the mutation of the c-Abl phosphorylation site on JIP1 abrogates its ability to promote axonal growth. JIP1 is therefore an important regulator of axonal development and is a key target of c-Abl-dependent pathways that control axonal growth.

Targeted deletion of the mitogen-activated protein kinase kinase 4 gene in the nervous system causes severe brain developmental defects and premature death.

The c-Jun NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase (MAPK) involved in the regulation of various physiological processes. Its activity is increased upon phosphorylation by the MAPK kinases MKK4 and MKK7. The early embryonic death of mice lacking an mkk4 or mkk7 gene has provided genetic evidence that MKK4 and MKK7 have nonredundant functions in vivo. To elucidate the physiological role of MKK4, we generated a novel mouse model in which the mkk4 gene could be specifically deleted in the brain. At birth, the mutant mice were indistinguishable from their control littermates, but they stopped growing a few days later and died prematurely, displaying severe neurological defects. Decreased JNK activity in the absence of MKK4 correlated with impaired phosphorylation of a subset of physiologically relevant JNK substrates and with altered gene expression. These defects resulted in the misalignment of the Purkinje cells in the cerebellum and delayed radial migration in the cerebral cortex. Together, our data demonstrate for the first time that MKK4 is an essential activator of JNK required for the normal development of the brain.

Cellular responses to nicotinic receptor activation are decreased after prolonged exposure to galantamine in human neuroblastoma cells.

In this study, we have examined cellular responses of neuroblastoma SH-SY5Y cells after chronic treatment with galantamine, a drug used to treat Alzheimer's disease that has a dual mechanism of action: inhibition of acetylcholinesterase and allosteric potentiation of nicotinic acetylcholine receptors (nAChR). Acute experiments confirmed that maximum potentiation of nicotinic responses occurs at 1 microM galantamine; hence this concentration was chosen for chronic treatment. Exposure to 1 microM galantamine for 4 days decreased Ca(2+) responses (by 19.8+/-3.6%) or [(3)H]noradrenaline ([(3)H]NA) release (by 23.9+/-3.3%) elicited by acute application of nicotine. KCl-evoked increases in intracellular Ca(2+) were also inhibited by 10.0+/-1.9% after 4 days' treatment with galantamine. These diminished responses are consistent with the downregulation of downstream cellular processes. Ca(2+) responses evoked by activation of muscarinic acetylcholine receptors were unaffected by chronic galantamine treatment. Exposure to the more potent acetylcholinesterase inhibitor rivastigmine (1 microM) for 4 days failed to alter nicotine-, KCl-, or muscarinic receptor-evoked increases in intracellular Ca(2+). These observations support the hypothesis that chronic galantamine exerts its effects through interaction with nAChR in this cell line. Exposure to 10 microM nicotine for 4 days produced decreases in acute nicotine- (18.0+/-3.5%) and KCl-evoked Ca(2+) responses (10.6+/-2.5%) and nicotine-evoked [(3)H]NA release (26.0+/-3.3%) that are comparable to the effects of a corresponding exposure to galantamine. Treatment with 1 microM galantamine did not alter numbers of [(3)H]epibatidine-binding sites in SH-SY5Y cells, in contrast to 62% upregulation of these sites in response to 10 microM nicotine. Thus, chronic galantamine acts at nAChR to decrease subsequent functional responses to acute stimulation with nicotine or KCl. This effect appears to be independent of the upregulation of nAChR-binding sites.

Nicotinic acetylcholine receptors and the regulation of neuronal signalling.

Neuronal nicotinic acetylcholine (nACh) receptors in the brain are more commonly associated with modulatory events than mediation of synaptic transmission. nACh receptors have a high permeability for Ca(2+), and Ca(2+) signals are pivotal in shaping nACh receptor-mediated neuromodulatory effects. In this review, we consider the mechanisms through which nACh receptors convert rapid ionic signals into sustained, wide-ranging phenomena. The complex Ca(2+) responses that are generated after activation of nACh receptors can transmit information beyond the initial domain and facilitate the interface with many intracellular processes. These mechanisms underlie the diverse repertoire of neuronal activities of nicotine in the brain, from the enhancement of learning and memory, to addiction and neuroprotection.

The allosteric potentiation of nicotinic acetylcholine receptors by galantamine is transduced into cellular responses in neurons: Ca2+ signals and neurotransmitter release.

Neuronal nicotinic acetylcholine receptors (nAChR) modulate a variety of cellular responses, including Ca2+ signals and neurotransmitter release, which can influence neuronal processes such as synaptic efficacy and neuroprotection. In addition to receptor activation through the agonist binding site, an allosteric modulation of nAChR has also been described for a novel class of allosteric ligands. Of these, the acetylcholinesterase inhibitor and Alzheimer drug galantamine represents the prototypical allosteric ligand, based on its potentiation of nAChR-evoked single-channel and whole-cell currents. The aim of this study was to establish whether the allosteric potentiation of nAChR currents is transduced in downstream cellular responses to nAChR activation, namely increases in intracellular Ca2+ and [3H]noradrenaline release. In SH-SY5Y cells, galantamine potentiated nicotine-evoked increases in intracellular Ca2+ and [3H]noradrenaline release with a bell-shaped concentration-response profile; maximum enhancement of nicotine-evoked responses occurred at 1 muM galantamine. This potentiation was blocked by mecamylamine, whereas galantamine had no effect on these measures in the absence of nicotine. Galantamine did not compete for radioligand binding to the agonist binding sites of several nAChR subtypes, consistent with an allosteric mode of action. Unlike galantamine, the acetylcholinesterase inhibitors rivastigmine and donepezil did not potentiate nAChR-mediated responses, whereas donepezil was a reasonably potent inhibitor of nicotine- and KCl-evoked increases in Ca2+. nAChR-mediated [3H]noradrenaline release from hippocampal slices was also potentiated by galantamine, with an additional component attributable to acetylcholinesterase inhibition and subsequent increase in acetylcholine. These results indicate that the allosteric regulation of nAChR results in the potentiation of receptor-dependent cellular processes relevant to many of the physiological consequences of nAChR activation.