Lynx1 and Aβ1–42 bind competitively to multiple nicotinic acetylcholine receptor subtypes

AbstractLynx1 regulates synaptic plasticity in the brain by regulating nicotinic acetylcholine receptors (nAChRs). It is not known to which extent Lynx1 can bind to endogenous nAChR subunits in the brain or how this interaction is affected by Alzheimer's disease pathology. We apply affinity purification to demonstrate that a water-soluble variant of human Lynx1 (Ws-Lynx1) isolates α3, α4, α5, α6, α7, β2, and β4 nAChR subunits from human and rat cortical extracts, and rat midbrain and olfactory bulb extracts, suggesting that Lynx1 forms complexes with multiple nAChR subtypes in the human and rodent brain. Incubation with Ws-Lynx1 decreases nicotine-mediated extracellular signal–regulated kinase phosphorylation in PC12 cells and striatal neurons, indicating that binding of Ws-Lynx1 is sufficient to inhibit signaling downstream of nAChRs. The effect of nicotine in PC12 cells is independent of α7 or α4β2 nAChRs, suggesting that Lynx1 can affect the function of native non-α7, non-α4β2 nAChR subtypes. We further show that Lynx1 and oligomeric β-amyloid1–42 compete for binding to several nAChR subunits, that Ws-Lynx1 prevents β-amyloid1–42–induced cytotoxicity in cortical neurons, and that cortical Lynx1 levels are decreased in a transgenic mouse model with concomitant β-amyloid and tau pathology. Our data suggest that Lynx1 binds to multiple nAChR subtypes in the brain and that this interaction might have functional and pathophysiological implications in relation to Alzheimer's disease

Functional interaction between Lypd6 and nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain‐containing 6 (Lypd6) with nAChRs in human brain extracts, identifying Lypd6 as a novel regulator of nAChR function. Using protein cross‐linking and affinity purification from human temporal cortical extracts, we demonstrate that Lypd6 is a synaptically enriched membrane‐bound protein that binds to multiple nAChR subtypes in the human brain. Additionally, soluble recombinant Lypd6 protein attenuates nicotine‐induced hippocampal inward currents in rat brain slices and decreases nicotine‐induced extracellular signal‐regulated kinase phosphorylation in PC12 cells, suggesting that binding of Lypd6 is sufficient to inhibit nAChR‐mediated intracellular signaling. We further show that perinatal nicotine exposure in rats (4 mg/kg/day through minipumps to dams from embryonic day 7 to post‐natal day 21) significantly increases Lypd6 protein levels in the hippocampus in adulthood, which did not occur after exposure to nicotine in adulthood only. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain, and that Lypd6 is dysregulated by nicotine exposure during early development. [Image: see text] Regulatory proteins of the Lynx family modulate the function of nicotinic receptors (nAChRs). We report for the first time that the Lynx protein Lypd6 binds to nAChRs in human brain extracts, and that recombinant Lypd6 decreases nicotine‐induced ERK phosphorylation and attenuates nicotine‐induced hippocampal inward currents. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain

Perinatal nicotine treatment induces transient increases in NACHO protein levels in the rat frontal cortex

The nicotinic acetylcholine receptor (nAChR) regulator chaperone (NACHO) was recently identified as an important regulator of nAChR maturation and surface expression. Here we show that NACHO levels decrease during early postnatal development in rats. This decrease occurs earlier and to a greater degree in the frontal cortex compared with the hippocampus. We further show that rats exposed to nicotine during pre- and postnatal development exhibit significantly higher NACHO levels in the frontal cortex at postnatal day (PND) 21, but not at PND60. Repeated exposure to nicotine selectively during early (PND8-14) or late (PND54-60) postnatal stages did not affect NACHO protein levels in the frontal cortex or hippocampus. Neither did exposure to high doses of the selective α7 nAChR agonists SSR180711, A-582941, or PNU-282987. However, we found significantly increased NACHO protein levels in the frontal cortex of PND36 rats after a single exposure to a combination of nicotine and the type II α7 nAChR positive allosteric modulator (PAM) PNU-120596, but not the type I PAM AVL-3288. These findings suggest that exposure to nAChR agonism affects NACHO protein levels, and that this effect is more pronounced during pre- or early postnatal development. The effect of PNU-120596 further suggests that the increase in NACHO expression is caused by activation rather than desensitization of nAChRs