α6* nicotinic acetylcholine receptor expression and function in a visual salience circuit.

Nicotinic acetylcholine receptors (nAChRs) containing α6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus ceruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of α6-containing nAChRs, we created and studied transgenic mice expressing a variant α6 subunit with green fluorescent protein (GFP) fused in-frame in the M3-M4 intracellular loop. In α6-GFP transgenic mice, α6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong α6* nAChR expression in glutamatergic retinal axons, which terminate in superficial superior colliculus (sSC), we also found α6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch-clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive α6* nAChRs, we confirmed functional, postsynaptic α6* nAChR expression. Further, sSC GABAergic neurons expressing α6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitive α6* nAChRs by ACh. α6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of α6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that α6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that α6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway.

Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.

We employed a pH-sensitive GFP analog, superecliptic phluorin, to observe aspects of nicotinic acetylcholine receptor (nAChR) trafficking to the plasma membrane (PM) in cultured mouse cortical neurons. The experiments exploit differences in the pH among endoplasmic reticulum (ER), trafficking vesicles, and the extracellular solution. The data confirm that few α4ß4 nAChRs, but many α4ß2 nAChRs, remain in neutral intracellular compartments, mostly the ER. We observed fusion events between nAChR-containing vesicles and PM; these could be quantified in the dendritic processes. We also studied the ß4R348C polymorphism, linked to amyotrophic lateral sclerosis (ALS). This mutation depressed fusion rates of α4ß4 receptor-containing vesicles with the PM by ∼2-fold, with only a small decrease in the number of nAChRs per vesicle. The mutation also decreased the number of ER exit sites, showing that the reduced receptor insertion results from a change at an early stage in trafficking. We confirm the previous report that the mutation leads to reduced agonist-induced currents; in the cortical neurons studied, the reduction amounts to 2-3-fold. Therefore, the reduced agonist-induced currents are caused by the reduced number of α4ß4-containing vesicles reaching the membrane. Chronic nicotine exposure (0.2 µM) did not alter the PM insertion frequency or trafficking behavior of α4ß4-laden vesicles. In contrast, chronic nicotine substantially increased the number of α4ß2-containing vesicle fusions at the PM; this stage in α4ß2 nAChR up-regulation is presumably downstream from increased ER exit. Superecliptic phluorin provides a tool to monitor trafficking dynamics of nAChRs in disease and addiction.

Nicotine exploits a COPI-mediated process for chaperone-mediated up-regulation of its receptors.

Chronic exposure to nicotine up-regulates high sensitivity nicotinic acetylcholine receptors (nAChRs) in the brain. This up-regulation partially underlies addiction and may also contribute to protection against Parkinson's disease. nAChRs containing the α6 subunit (α6* nAChRs) are expressed in neurons in several brain regions, but comparatively little is known about the effect of chronic nicotine on these nAChRs. We report here that nicotine up-regulates α6* nAChRs in several mouse brain regions (substantia nigra pars compacta, ventral tegmental area, medial habenula, and superior colliculus) and in neuroblastoma 2a cells. We present evidence that a coat protein complex I (COPI)-mediated process mediates this up-regulation of α6* or α4* nAChRs but does not participate in basal trafficking. We show that α6ß2ß3 nAChR up-regulation is prevented by mutating a putative COPI-binding motif in the ß3 subunit or by inhibiting COPI. Similarly, a COPI-dependent process is required for up-regulation of α4ß2 nAChRs by chronic nicotine but not for basal trafficking. Mutation of the putative COPI-binding motif or inhibition of COPI also results in reduced normalized Förster resonance energy transfer between α6ß2ß3 nAChRs and εCOP subunits. The discovery that nicotine exploits a COPI-dependent process to chaperone high sensitivity nAChRs is novel and suggests that this may be a common mechanism in the up-regulation of nAChRs in response to chronic nicotine.

Transcriptional regulation by nicotine in dopaminergic neurons.

Dopaminergic neurons in the substantia nigra pars compacta (SNc) degenerate in Parkinson's disease. These neurons robustly express several nicotinic acetylcholine receptor (nAChR) subtypes. Smoking appears to be neuroprotective for Parkinson's disease but the mechanism is unknown. To determine whether chronic nicotine-induced changes in gene expression contribute to the neuroprotective effects of smoking, we develop methods to measure the effect of prolonged nicotine exposure on the SNc neuronal transcriptome in an unbiased manner. Twenty neurons were collected using laser-capture microscopy and transcriptional changes were assessed using RNA deep sequencing. These results are the first whole-transcriptome analyses of chronic nicotine treatment in SNc neurons. Overall, 129 genes were significantly regulated: 67 upregulated, 62 downregulated. Nicotine-induced relief of endoplasmic reticulum (ER) stress has been postulated as a potential mechanism for the neuroprotective effects of smoking. Chronic nicotine did not significantly affect the expression of ER stress-related genes, nor of dopamine-related or nAChR genes, but it did modulate expression of 129 genes that could be relevant to the neuroprotective effects of smoking, including genes involved in (1) the ubiquitin-proteasome pathway, (2) cell cycle regulation, (3) chromatin modification, and (4) DNA binding and RNA regulation. We also report preliminary transcriptome data for single-cell dopaminergic and GABAergic neurons isolated from midbrain cultures. These novel techniques will facilitate advances in understanding the mechanisms taking place at the cellular level and may have applications elsewhere in the fields of neuroscience and molecular biology. The results give an emerging picture of the role of nicotine on the SNc and on dopaminergic neurons.

Characterizing functional α6ß2 nicotinic acetylcholine receptors in vitro: mutant ß2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells.

α6* nicotinic acetylcholine receptors (nAChRs) are highly expressed in mesostriatal and nigrostriatal dopaminergic systems, and participate in motor control, reward, and learning and memory. In vitro functional expression of α6* nAChRs is essential for full pharmacological characterization of these receptors and for drug screening, but has been challenging. We expressed eGFP-tagged-α6 and ß2 nAChR subunits in Neuro-2a cells, leading to functional channels. Inward currents were elicited with 300 µM ACh in 26% (5/19) of cells with evenly expressed α6-eGFP in cytoplasm and periphery. We dramatically increased chances of detecting functional α6-eGFPß2 nAChRs by (i) introducing two endoplasmic reticulum (ER) export-enhancing mutations into ß2 subunits, and (ii) choosing cells with abundant Sec24D-mCherry-labeled ER exit sites. Both manipulations also modestly increased α6-eGFPß2 nAChR current amplitude. α6-eGFPß2 nAChRs were also activated by nicotine and by TC-2403. The α6-eGFPß2 currents were desensitized by 1µM nicotine, blocked by α-conotoxin MII, partially inhibited by dihydro-ß-erythroidine, and potentiated by extracellular Ca(2+). Single-channel recordings showed that α6-eGFPß2 nAChRs had similar single-channel conductance to, but longer open time than, α4-eGFPß2 nAChRs. These methods provide avenues for developing cell lines expressing subtypes of α6* nAChRs for both pharmacological study and drug screening.

Nicotine up-regulates alpha4beta2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning.

The up-regulation of α4ß2* nicotinic acetylcholine receptors (nAChRs) by chronic nicotine is a cell-delimited process and may be necessary and sufficient for the initial events of nicotine dependence. Clinical literature documents an inverse relationship between a person's history of tobacco use and his or her susceptibility to Parkinson's disease; this may also result from up-regulation. This study visualizes and quantifies the subcellular mechanisms involved in nicotine-induced nAChR up-regulation by using transfected fluorescent protein (FP)-tagged α4 nAChR subunits and an FP-tagged Sec24D endoplasmic reticulum (ER) exit site marker. Total internal reflection fluorescence microscopy shows that nicotine (0.1 µM for 48 h) up-regulates α4ß2 nAChRs at the plasma membrane (PM), despite increasing the fraction of α4ß2 nAChRs that remain in near-PM ER. Pixel-resolved normalized Förster resonance energy transfer microscopy between α4-FP subunits shows that nicotine stabilizes the (α4)(2)(ß2)(3) stoichiometry before the nAChRs reach the trans-Golgi apparatus. Nicotine also induces the formation of additional ER exit sites (ERES). To aid in the mechanistic analysis of these phenomena, we generated a ß2(enhanced-ER-export) mutant subunit that mimics two regions of the ß4 subunit sequence: the presence of an ER export motif and the absence of an ER retention/retrieval motif. The α4ß2(enhanced-ER-export) nAChR resembles nicotine-exposed nAChRs with regard to stoichiometry, intracellular mobility, ERES enhancement, and PM localization. Nicotine produces only small additional PM up-regulation of α4ß2(enhanced-ER-export) receptors. The experimental data are simulated with a model incorporating two mechanisms: (1) nicotine acts as a stabilizing pharmacological chaperone for nascent α4ß2 nAChRs in the ER, eventually increasing PM receptors despite a bottleneck(s) in ER export; and (2) removal of the bottleneck (e.g., by expression of the ß2(enhanced-ER-export) subunit) is sufficient to increase PM nAChR numbers, even without nicotine. The data also suggest that pharmacological chaperoning of nAChRs by nicotine can alter the physiology of ER processes.