Nicotine is a potent inhibitor of the immune response and is protective against experimental autoimmune encephalomyelitis (EAE). Initial studies suggested that the cholinergic system modulates inflammation via the α7‐nicotinic acetylcholine receptor (nAChR) subtype. We recently have shown that effector T cells and myeloid cells constitutively express mRNAs encoding nAChR α9 and β2 subunits and found evidence for immune system roles for non‐α7‐nAChRs. In the present study, we assessed the effects of nAChR α9 or β2 subunit gene deletion on EAE onset and severity, with or without nicotine treatment. We report again that disease onset is delayed and severity is attenuated in nicotine‐treated, wild‐type mice, an effect that also is observed in α9 subunit knock‐out (KO) mice irrespective of nicotine treatment. On the other hand, β2 KO mice fail to recover from peak measures of disease severity regardless of nicotine treatment, despite retaining sensitivity to nicotine's attenuation of disease severity. Prior to disease onset, we found significantly less reactive oxygen species production in the central nervous system (CNS) of β2 KO mice, elevated proportions of CNS myeloid cells but decreased ratios of CNS macrophages/microglia in α9 or β2 KO mice, and some changes in iNOS, TNF‐α and IL‐1β mRNA levels in α9 KO and/or β2 KO mice. Our data thus suggest that β2*‐ and α9*‐nAChRs, in addition to α7‐nAChRs, have different roles in endogenous and nicotine‐dependent modulation of immune functions and could be exploited as therapeutic targets to modulate inflammation and autoimmunity.
The plant alkaloid methyllycaconitine (MLA) is considered to be a selective antagonist of the alpha7 subtype of neuronal nicotinic acetylcholine receptor (nAChR). However, 50 nM MLA partially inhibited (by 16%) [(3)H]dopamine release from rat striatal synaptosomes stimulated with 10 microM nicotine. Other alpha7-selective antagonists had no effect. Similarly, MLA (50 nM) inhibited [(3)H]dopamine release evoked by the partial agonist (2-chloro-5-pyridyl)-9-azabicyclo[4.2.1]non-2-ene (UB-165) (0.2 microM) by 37%. In both cases, inhibition by MLA was surmountable with higher agonist concentrations, indicative of a competitive interaction. At least two subtypes of presynaptic nAChR can modulate dopamine release in the striatum, and these nAChR are distinguished by their differential sensitivity to alpha-conotoxin-MII (alpha-CTx-MII). MLA was not additive with a maximally effective concentration of alpha-CTx-MII (100 nM) in inhibiting [(3)H]dopamine release elicited by 10 microM nicotine or 0.2 microM UB-165, suggesting that both toxins act at the same site. This was confirmed in quantitative binding assays with (125)I-alpha-CTx-MII, which displayed saturable specific binding to rat striatum and nucleus accumbens with B(max) values of 9.8 and 16.5 fmol/mg of protein, and K(d) values of 0.63 and 0.83 nM, respectively. MLA fully inhibited (125)I-alpha-CTx-MII binding to striatum and nucleus accumbens with a K(i) value of 33 nM, consistent with the potency observed in the functional assays. We speculate that MLA and alpha-CTx-MII interact with a presynaptic nAChR of subunit composition alpha3/alpha6beta2beta3* on dopamine neurons. The use of MLA as an alpha7-selective antagonist should be exercised with caution, especially in studies of nAChR in basal ganglia.
The activation of neuronal nicotinic acetylcholine receptors (nAChRs) has been implicated in the activity-dependent development and plasticity of retina and the refinement of retinal projections. Pharmacological and functional studies have also indicated that different presynaptic nAChRs can have a modulatory function in retinotectal synapses. We biochemically and pharmacologically identified the multiple nAChR subtypes expressed on retinal afferents of the superior colliculus (SC) and lateral geniculate nucleus (LGN). We found that the α6β2* and α4(nonα6)β2* nAChRs are the major receptor populations expressed in both SC and LGN. In addition, the LGN contains two minor populations of α2α6β2* and α3β2* subtypes, whereas the SC contains a relatively large population of a new native subtype, the α3β2(α5/β3) nAChR. This subtype binds the α-conotoxin MII with an affinity 50 times lower than that of the native α6β2* subtype. Studies of tissues obtained from eye-enucleated animals allowed the identification of nAChRs expressed by retinal afferents: in SC α6β2*, α4α6β2*, and α3β2* (approximately 45, 35, and 20%, respectively), in LGN, α4α6β2*, α6β2*, α4β2*, α2α6β2*, and α3β2* (approximately 40, 30, 20, 5, and 5%, respectively). In both regions, more than 50% of nAChRs were not expressed by retinal afferents and belonged to the α4β2* (90%) or α4α5β2* (10%) subtypes. Moreover, studies of the SC tissues obtained from wild-type and α4, α6, and β3 knockout mice confirmed and extended the data obtained in rat tissue and allowed a comprehensive dissection of the composition of nAChR subtypes present in this retinorecipient area.
mRNAs for the neuronal nicotinic acetylcholine receptor (nAChR) α6 and β3 subunits are abundantly expressed and colocalized in dopaminergic cells of the substantia nigra and ventral tegmental area. Studies using subunit-null mutant mice have shown that α6- or β3-dependent nAChRs bind α-conotoxin MII (α-CtxMII) with high affinity and modulate striatal dopamine release. This study explores the effects of β3 subunit-null mutation on striatal and midbrain nAChR expression, composition, and pharmacology. Ligand binding and immunoprecipitation experiments using subunit-specific antibodies indicated that β3-null mutation selectively reduced striatal α6* nAChR expression by 76% versus β3+/+ control. Parallel experiments showed a smaller reduction in both midbrain α3* and α6* nAChRs (34 and 42% versus β3+/+ control, respectively). Sedimentation coefficient determinations indicated that residual α6* nAChRs in β3–/– striatum were pentameric, like their wild-type counterparts. Immunoprecipitation experiments on immunopurified β3* nAChRs demonstrated that almost all wild-type striatal β3* nAChRs also contain α4, α6, and β2 subunits, although a small population of non-β3 α6* nAChRs is also expressed. β3 subunit incorporation seemed to increase α4 participation in α6β2* complexes. 125I-Epibatidine competition binding studies showed that the α-CtxMII affinity of α6* nAChRs from the striata of β3–/– mice was similar to those isolated from β3+/+ animals. Together, the results of these experiments show that the β3 subunit is important for the correct assembly, stability and/or transport of α6* nAChRs in dopaminergic neurons and influences their subunit composition. However, β3 subunit expression is not essential for the expression of α6*, high-affinity α-CtxMII binding nAChRs.
Initiated by findings that Alzheimer's disease is associated with a profound loss of cholinergic markers in human brain, decades of studies have examined the interactions between specific subtypes of nicotinic acetylcholine receptors and amyloid-β [derived from the amyloid precursor protein (APP), which is cleaved to yield variable isoforms of amyloid-β]. We review the evolving understanding of amyloid-β's roles in Alzheimer's disease and pioneering studies that highlighted a role of nicotinic acetylcholine receptors in mediating important aspects of amyloid-β's effects. This review also surveys the current state of research into amyloid-β / nicotinic acetylcholine receptor interactions. The field has reached an exciting point in which common themes are emerging from the wide range of prior research and a range of accessible, relevant model systems are available to drive further progress. We highlight exciting new areas of inquiry and persistent challenges that need to be considered while conducting this research. Studies of amyloid-β and the nicotinic acetylcholine receptor populations that it interacts with provide opportunities for innovative basic and translational scientific breakthroughs related to nicotinic receptor biology, Alzheimer's disease, and cholinergic contributions to cognition more broadly.
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