Ubiquitination regulates the degradation, membrane trafficking, and transcription of proteins. At mammalian synapses, the ubiquitinproteasome system (UPS) influences synaptic transmission and plasticity. Nicotine also has the ability to affect synaptic function via mechanisms that remain partially unknown. We found that nicotine, at concentrations achieved by smokers, reduced proteasomal activity, produced accumulation of ubiquitinated synaptic proteins, and increased total protein levels. In particular, a 24 h exposure to nicotine decreased proteasome-dependent degradation of the ␣7 nicotinic acetylcholine receptor (nAChR) subunit, as indicated by the accumulation of ubiquitinated ␣7. The same nicotine treatment increased the levels of the AMPA glutamate receptor subunit GluR1, the NMDA receptor subunit NR2A, the metabotropic receptor mGluR1␣, the plasticity factor Homer-1A, and the scaffolding postsynaptic density protein PSD-95, whereas the levels of another scaffolding protein, Shank, were reduced. These changes were associated with an inhibition of proteasomal chymotrypsin-like activity by nicotine. The nAChR antagonist mecamylamine was only partially able to block the effects of nicotine on the UPS, indicating that nAChR activation does not completely explain nicotine-induced inhibition of proteasomal catalytic activity. A competition binding assay suggested a direct interaction between nicotine and the 20S proteasome. These results suggest that the UPS might participate in nicotine-dependent synaptic plasticity.
Smokers often report an anxiolytic effect of cigarettes. In addition, stress-related disorders such as anxiety, posttraumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the a5 nicotinic acetylcholine receptor subunit in anxiety-related responses, control and a5 subunit null mice (a5 2/2 ) were subjected to the open field activity (OFA), light-dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, a5 2/2 behaved like wild-type controls. In the EPM, female a5 2/2 mice displayed an anxiolytic-like phenotype, while male a5 2/2 mice were undistinguishable from littermate controls. We studied the hypothalamus-pituitary-adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin-releasing factor. Consistent with an anxiolyticlike phenotype, female a5 2/2 mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of a5, we treated cultured NTera 2 cells with progesterone and found that a5 protein levels were upregulated. In addition, brain levels of a5 mRNA increased upon progesterone injection into ovariectomized wild-type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolyticlike in wild-type mice, but no cycle-dependent fluctuations in anxiety levels were found in a5 2/2 females. Thus, a5-containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone-dependent modulation of a5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.
Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the ␣3 and ␣4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with ␣3-containing nAChRs (␣3* nAChRs) expressed in HEK293 cells, PC12 cells, and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the ␣32 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining, and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of ␣3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the ␣3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of ␣3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the ␣3 subunit and, consequently, the number of receptors at the cell surface.
The medial habenula (MHb) densely expresses nicotinic acetylcholine receptors (nAChRs) and participates in nicotine-related behaviors such as nicotine withdrawal and regulating nicotine intake. Although specific nAChR subunits are identified as being involved in withdrawal behavior, the cellular mechanisms through which nicotine acts to cause this aversive experience is unclear. Here, we demonstrate an interaction between the nicotinic and neurokinin signaling systems that may form the basis for some symptoms experienced during nicotine withdrawal. Using patch-clamp electrophysiology in mouse brain slices, we show that nicotine (1 M) increases intrinsic excitability in MHb neurons. This nicotine-induced phenomenon requires ␣5-containing nAChRs and depends on intact neurokinin signaling. The effect is blocked by preincubation with neurokinin 1 (NK1; L-732138, 10 M) and NK3 (SB222200, 2 M) antagonists and mimicked by NK1 (substance P, 100 nM) and NK3 (neurokinin B [NKB], 100 nM) agonists. Microinjections (1 l) of L-732138 (50 nM) and SB222200 (100 nM) into the MHb induces withdrawal behavior in chronic nicotine-treated (8.4 mg/kg/d, 2 weeks) mice. Conversely, withdrawal behavior is absent with analogous microinjections into the lateral habenula of nicotine-treated mice or in mice chronically treated with a vehicle solution. Further, chronic nicotine reduces nicotine's acute modulation of intrinsic excitability while sparing modulation by NKB. Our work elucidates the interplay between two neuromodulatory signaling systems in the brain through which nicotine acts to influence intrinsic excitability. More importantly, we document a neuroadaptation of this mechanism to chronic nicotine exposure and implicate these mechanisms collectively in the emergence of nicotine withdrawal behavior.
Ubiquitination is a key event for protein degradation by the proteasome system, membrane protein internalization, and protein trafficking among cellular compartments. Few data are available on the role of the ubiquitin-proteasome system (UPS) in the trafficking of neuronal nicotinic acetylcholine receptors (nAChRs). Experiments conducted in neuron-like differentiated rat pheochromocytoma cells (PC12 cells) show that the α3, β2, and β4 nAChR subunits are ubiquitinated and that their ubiquitination is necessary for degradation. A 24-h treatment with the proteasome inhibitor PS-341 increased the total levels of α3 and the two β subunits in both whole cell lysates and fractions enriched for the ER/Golgi compartment. nAChR subunit upregulation was also detected in plasma membrane-enriched fractions. Inhibition of the lysosomal degradation machinery by E-64 had a significantly smaller effect on nAChR turnover. The present data, together with previous results showing that the α7 nAChR subunit is a target of the UPS, point to a prominent role of the proteasome in nAChR trafficking.
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