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Objective: Synovitis is a widely accepted sign of osteoarthritis (OA), characterised by tissue hyperplasia, where increased infiltration of immune cells and proliferation of resident fibroblasts adopt a pro-inflammatory phenotype, and increased the production of pro-inflammatory mediators that are capable of sensitising and activating sensory nociceptors, which innervate the joint tissues. As such, it is important to understand the cellular composition of synovium and their involvement in pain sensitisation to better inform the development of effective analgesics., Methods: Studies investigating pain sensitisation in OA with a focus on immune cells and fibroblasts were identified using PubMed, Web of Science and SCOPUS., Results: In this review, we comprehensively assess the evidence that cellular crosstalk between resident immune cells or synovial fibroblasts with joint nociceptors in inflamed OA synovium contributes to peripheral pain sensitisation. Moreover, we explore whether the elucidation of common mechanisms identified in similar joint conditions may inform the development of more effective analgesics specifically targeting OA joint pain., Conclusion: The concept of local environment and cellular crosstalk within the inflammatory synovium as a driver of nociceptive joint pain presents a compelling opportunity for future research and therapeutic advancements., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

We report the first total syntheses of simonsol F (3), simonsinol (5), fargenin (4), and macranthol (6) in addition to syntheses of simonsol C (2), simonsol G (1), and honokiol (14). The syntheses are based upon a phosphonium ylide-mediated cascade reaction and upon natural product isomerization reactions which proceed through Cope rearrangements of putative biosynthetic dienone intermediates. As a corollary of the natural product isomerization reactions, we propose an alternative biosynthesis of honokiol (14), simonsinol (5), and macranthol (6) which unites the natural products in this family under a single common precursor, chavicol (7). Finally, we demonstrate that simonsol C (2) and simonsol F (3) promote axonal growth in primary mouse cortical neurons., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

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 GSK3β 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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 in vivo . 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 in vitro , a shift to 3'-tRNAs is observed in mature axons in vivo .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.

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., (Copyright © 2021. Published by Elsevier Inc.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Di Paolo, Garat, Eastman, Farias, Dajas-Bailador, Smircich and Sotelo-Silveira.)

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., (© 2020 Di Paolo et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

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 GSK3β controls axon development and growth. Removal of this repression in the axon triggers local translation of GSK3β 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., Competing Interests: Competing interestsThe authors declare they have no competing interests or other interests that might be perceived to influence the results and discussion reported in this paper., (© 2020. Published by The Company of Biologists Ltd.)

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 WLD S 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., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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.

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., (© 2015 Beaven et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

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.

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.

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.

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.

Notch1 signaling has a critical function in maintaining a balance among cell proliferation, differentiation, and apoptosis. Our earlier work showed that the Notch1 intracellular domain interferes with the scaffolding function of c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1), yet the effect of JIP1 for Notch1-recombining binding protein suppressor of hairless (RBP-Jk) signaling remains unknown. Here, we show that JIP1 suppresses Notch1 activity. JIP1 was found to physically associate with either intracellular domain of Notch1 or RBP-Jk and interfere with the interaction between them. Furthermore, we ascertained that JIP1 caused the cytoplasmic retention of RBP-Jk through an interaction between the C-terminal region of JIP1 including Src homology 3 domain and the proline-rich domain of RBP-Jk. We also found that RBP-Jk inhibits JIP1-mediated activation of the JNK1 signaling cascade and cell death. Our results suggest that direct protein-protein interactions coordinate cross-talk between the Notch1-RBP-Jk and JIP1-JNK pathways.

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 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.

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.

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.

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.

Activation of neuronal nicotinic acetylcholine receptors (nAChR) by nicotine has been suggested to protect neurons against a hypoxic insult. The objective of this study was to examine the nature of cell death induced by acute hypoxia in rat primary cortical cultures and the neuroprotective potential of nicotine in ameliorating these processes. Neuronal cell death induced by a 4-h exposure to hypoxia (0.1% O(2)) was apoptotic, as shown by TUNEL staining and assays monitoring DNA strand breaks and caspase-3/7 activity. The presence of nicotine (10 microM) during the hypoxic insult protected a subpopulation of susceptible neurones against DNA damage and apoptosis induced by oxygen deprivation. This protective effect of nicotine was prevented by a 30-min pre-incubation with either 100 nM alpha-bungarotoxin or 1 microM dihydro-beta-erythroidine, but not 1 microM atropine, suggesting that activation of at least two subtypes of nAChR, alpha7 and beta2* nAChR, is involved in mediating nicotine neuroprotection.

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate many cellular mechanisms, such as cell survival and memory processing, which are also influenced by the serine/threonine protein kinases ERK1/2. In SH-SY5Y cells and hippocampal neurones, nicotine (100 microM) increased the activity of ERK1/2. This effect was Ca2+ dependent, and prevented by the alpha7 nAChR antagonist alpha-bungarotoxin (alpha-Bgt) and an inhibitor (PD98059) of the upstream kinase MEK. To determine the intervening steps linking Ca2+ entry to MEK-ERK1/2 activation, inhibitors of Ca2+-dependent kinases were deployed. In SH-SY5Y cells, selective blockers for PKC (Ro 31-8220), CaM kinase II (KN-62) or PI3 kinase (LY 294002) failed to inhibit the nicotine-evoked increase in ERK1/2 activity. In contrast, two structurally different inhibitors of PKA (KT 5720 and H-89) completely prevented the nicotine-dependent increase in ERK1/2 activity. Inhibition of the nicotine-evoked increase in ERK1/2 activity by H-89 was also observed in hippocampal cultures. Down stream of PKA, the activity of B-Raf was significantly decreased by nicotine in SH-SY5Y cells, as determined by direct measurement of MEK1 phosphorylation or in vitro kinase assays, whereas the modulation of MEK1 phosphorylation by Raf-1 tended to increase. Thus, this study provides evidence for a novel signalling route coupling the stimulation of alpha7 nAChR to the activation of ERK1/2, in a Ca2+ and PKA dependent manner.

Neuronal nicotinic acetylcholine receptors (nAChR) have been suggested to play a role in a variety of modulatory and regulatory processes, including neuroprotection. Here we have characterized the neuroprotective effects of nicotine against an excitotoxic insult in primary hippocampal cultures. Exposure of hippocampal neurons to 200 microM NMDA for 1 h decreased cell viability by 25+/-5%, an effect blocked by NMDA receptor antagonists. Nicotine (10 microM) counteracted the NMDA-induced cell death when co-incubated with NMDA or when present subsequent to the NMDA treatment. Nicotine protection was prevented by 1 microM MLA, confirming that it was mediated by nAChR, and by 1 microM alpha-bungarotoxin, demonstrating that the alpha7 nAChR subtype was responsible. Both the NMDA evoked neurotoxicity and nicotine neuroprotection were Ca(2+)-dependent. In Fura-2-loaded hippocampal neurons, nicotine (10 microM) and NMDA (200 microM) acutely increased intracellular resting Ca(2+) from 70 nM to 200 and 500 nM, respectively. Responses to NMDA were unaffected by the presence of nicotine. (45)Ca(2+) uptake after a 1 h exposure to nicotine or NMDA also demonstrated quantitative differences between the two drugs. This study demonstrates that the alpha7 subtype of nAChR can support neuronal survival after an excitotoxic stimulus, through a Ca(2+) dependent mechanism that operates downstream of NMDA receptor activation.

The dopaminergic and antioxidant properties of pukateine [(R)-11-hydroxy-1,2-methylenedioxyaporphine, PUK], a natural aporphine derivative, were analyzed in the rat central nervous system. At dopamine (DA) D1 ([3H]-SCH 23390) and D2 ([3H]-raclopride) binding sites, PUK showed IC50 values in the submicromolar range (0.4 and 0.6 microM, respectively). When the uptake of tritiated dopamine was assayed by using a synaptosomal preparation, PUK showed an IC50 = 46 microM. In 6-hydroxydopamine unilaterally denervated rats, PUK (8 mg/kg but not 4 mg/kg) elicited a significant contralateral circling, a behavior classically associated with a dopaminergic agonist action. When perfused through a microdialysis probe inserted into the striatum, PUK (340 microM) induced a significant increase in dopamine levels. In vitro experiments with a crude rat brain mitochondrial suspension showed that PUK did not affect monoamine oxidase activities, at concentrations as high as 100 microM. PUK potently (IC50 = 15 microM) and dose-dependently inhibited the basal lipid peroxidation of a rat brain membrane preparation. As a whole, PUK showed a unique profile of action, comprising an increase in extracellular DA, an agonist-like interaction with DA receptors, and antioxidant activity. Thus, PUK may be taken as a lead compound for the development of novel therapeutic strategies for Parkinson disease.

To study the involvement of oxidative stress in 6-OHDA neurotoxicity, we investigated the production of the hydroxyl free radical (OH.) in the substantia nigra (SN) and the striatum (CS) several moments after intranigral injection of the neurotoxin, with or without an added episode of hypoxia (30 min, 95% N2, 5% O2). We utilized the hydroxylation of salicylate to 2,3 dihydroxybenzoic acid (2,3 DHBA) as indication of OH. production. When 2.3 DHBA levels were not modified, the levels of 2,5 DHBA were taken as an indication of cytochrome P450 (CYP 450) metabolism. 6-OHDA alone did not increase the production of 2,3 DHBA in the SN. 2,5 DHBA increased significantly after 120 min and was high up to 24 h. An episode of hypoxia (60 min after 6-OHDA injection) significantly worsened the decrease of dopamine (DA) in the striatum assessed 8 days after injection of 6-OHDA in the SN. Hypoxia performed 60 min and 24 h before or 24 h after 6-OHDA did not show any additional effect on striatal DA levels. Contrary to results obtained after 6-OHDA alone, 2,3 DHBA increased significantly 120 min after the injection, when the hypoxia-reoxygenation was added to the 6-OHDA treatment. Our data are showing a relationship between the increase in OH. production and a concomitant worsening of neuronal degeneration. As a whole, the results support the idea that neurons undergoing 6-OHDA neurotoxicity have their antioxidant defences affected and that oxidative stress is actually an important eliciting factor in 6-OHDA dependant neurodegeneration. However, OH. may not be the main radical species involved in this process. Additionally, 6-OHDA also appeared to provoke a long-term increase in CYP 450 activity., (Copyright 1998 Elsevier Science B.V.)

Snake neurotoxins (NTX) have proven to be valuable tools for the characterisation of muscular nicotinic acetylcholine receptor structure and function. It is very likely that they could also be utilised to identify subtypes of neuronal nicotinic receptors controlling specific functions within the central nervous system. In this study we examined the effects of long alpha NTX (alpha-bungarotoxin, alpha-Bgt, and alpha-cobratoxin, alpha-Cbt) and short alpha NTX (alpha-erabutoxin a, alpha-Ebt) as well as the anticholinesterase toxin fasciculin-2 (FAS), on the nicotine-evoked release of dopamine (DA) in the striatum, using the in vivo push-pull technique. The short toxins alpha-Ebt and FAS blocked the extracellular increase of DA evoked by nicotine at 4.2 microM concentrations and alpha-Ebt was more potent, as reflected by the blockade at the lower dose of 0.42 microM. In contrast, the long toxins showed a different profile of action. Alpha-Cbt did not show any blockade of the nicotine-evoked release of DA at the doses studied while alpha-Bgt did block it only at the higher dose (4.2 microM) These results indicate that short neurotoxins show a stronger interaction with striatal nicotinic receptors subtypes controlling DA release when compared to the long ones. This interaction of short neurotoxin polypeptides and presynaptic receptors may permit the further elucidation of the particular nicotinic receptor populations responsible for the modulation of striatal DA release.

In the rat striatum, acetylcholine (ACh) increases dopamine (DA) release. The role of increased cholinergic activity provoked by acetylcholinesterase inhibitors (AChEi) on DA release is currently under revision after recent papers have shown a blockade of nicotinic transmission by AChEi in vitro. To study the effects of AChEi in vivo, Fasciculin2 (FAS), a peptidergic AChEi, and physostigmine (PHY), a classical carbamate AChEi, were applied through push-pull or microdialysis cannulae respectively, to the striatum of rats, alone or with ACh. Extracellular concentrations of DA were assessed by HPLC with electrochemical detection. Alone, the AChEi studied did not provoke changes in basal extracellular levels of DA, in the different doses studied. ACh (100 microM, 1 and 5 mM) applied through the push-pull cannulae in basal conditions provoked a dose-dependent increase of extracellular DA. This effect was not observed with ACh in concentrations of 100 microM and 1 mM if FAS (0.4 and 4.2 microM) was applied first. Higher concentrations of ACh (5 mM) evoked a partial response after FAS 0.42 microM, an effect still blocked by FAS at 4.2 microM. PHY 50 microM applied through microdialysis completely blocked the increase in DA release provoked by ACh 10, 20 mM, while at ACh 30 mM, PHY 50 microM only partially blocked the evoked increase. A partial blockade was also observed with PHY 20 microM, on the three different concentrations of ACh. On the other hand PHY 10 microM did not block any of the ACh doses perfused. These results showed that AChEi like FAS and PHY interfere with the ACh-evoked DA release in the striatum.