Fig 2: Lypd6 binds multiple nAChR subunits in the human brain. (a) Cortical homogenates from human temporal cortex (input) were affinity purified using magnetic beads covalently coupled with recombinant human Lypd6 (Lypd6) or non-coupled beads (ctrl). Also shown is the remaining homogenate after affinity purification (output). Samples were submitted to gel electrophoresis and western blotting followed by detection of nAChR subunits and the GluR2 AMPA receptor subunit. (b) Representative images of western blots of Lypd6 affinity purification of human temporal cortex homogenates pre-incubated with 100 nM a-BTX or vehicle [phosphate-buffered saline (PBS)] for 30 min. (c) Quantification of data represented in (b), showing that a-BTX interferes with binding of the a7 nAChR subunit to Lypd6-coupled beads (n = 5). Values are normalized to their individual vehicle and represented as mean ± SEM. **p < 0.01 indicates statistical difference from affinity purification in the absence of a-BTX in a ratio paired t-test.

Fig 3: Lypd6 reversibly attenuates nAChR-induced membrane responses in CA1 hippocampal interneurons. (a) Bright field image of the stratum radiatum of the CA1 hippocampal region where recordings were conducted (white arrow indicates recorded cell). Lypd6 (6 µM) was applied via a 2nd patch pipette (black arrow) close to the recorded cell and nicotine was applied via a 3rd pipette (gray arrow). Higher magnification image of a CA1 interneuron recorded for this study (white arrow). The electrode delivering nicotine is also visible (gray arrow). (b) Whole-cell voltage clamp recordings from a CA1 interneuron showing no effect on induction of inward current by application of Lypd6. (c) An example of whole-cell voltage clamp recordings from a CA1 interneuron showing induction of inward current by application of nicotine (arrow) and attenuation of this current when applied in the presence of Lypd6 (Nic + Lypd6). Nicotine responses were not attenuated with a 3-min interval between applications, indicating that reductions induced in the presence of Lypd6 were not because of cell run down, but were specific to the presence of the protein. (d) An example of whole-cell voltage clamp recordings from a CA1 interneuron, pre-incubated in Lypd6 for 5 min before first application of nicotine, showing that the effect of Lypd6 is lost upon wash-out. The recording examples shown in (c) and (d) are not representing the mean values, but were rather chosen as high-amplitude samples to clearly demonstrate the inhibitory effect of Lypd6. (e) Graphs of mean responses to nicotine (ncells = 5) and to the non-subtype-specific nAChR agonist DMPP (ncells = 3) from the population of cells recorded, indicating that when Lypd6 is present, the amplitude of inward currents induced by activation of nAChRs is significantly reduced. Data are displayed as each cell's response to the agonist in the presence of Lypd6 normalized to the same cell's response before the addition of Lypd6. Values are represented as mean ± SEM. *p < 0.05, **p < 0.01, indicates statistical difference between groups in a paired t-test.

Fig 4: Perinatal nicotine exposure increases hippocampal Lypd6 levels. (a) Quantification of Lypd6, Lynx1, and Ly6H protein levels as well as the ß2 nAChR subunit from frontal cortex (FC) and hippocampus (HIP) of male rats exposed to nicotine from embryonic day 7 to post-natal day (PND) 21 through osmotic minipumps implanted into pregnant dams. Animals were killed at PND7 (n = 5), 21 (n = 10), or 60 (n = 13). Main effect of nicotine treatment in a two-way anova with age and treatment as the fixed factors is indicated in the graphs. *p < 0.05 indicates statistical difference in a Sidak-corrected multiple comparisons test. (b) Representative western blot images of protein levels from FC and HIP at the different ages. (c) Quantification of Lypd6 and GluR2 protein levels in hippocampal tissue from the P60 groups described (a), showing that perinatal nicotine exposure increases hippocampal Lypd6 levels in nuclear/endosomal fractions. Tissue was fractionated using differential centrifugation into a pellet 1 (P1, nuclear) and pellet 2 (P2, crude synaptosome) fraction or cross-linked with bis(sulfosuccinimidyl) suberate (BS 3). Data are normalized to actin levels and the level of the untreated, non-fractionated homogenate was set to 1 (dashed line). *p < 0.05, **p < 0.01, ***p < 0.001 indicates statistical difference from homogenate in a ratio paired t-test, # p < 0.05 indicates statistical difference between vehicle- and nicotine-treated groups in an unpaired t-test. (d) Representative western blot images of protein levels shown in (c).

Fig 5: Distribution and developmental expression of Lypd6. (a–e) Representative images and analysis of western blots showing Lypd6 protein levels in: (a) different organs of adult male rats (n = 2) in relation to actin, (b) cortical tissue from adult male rats, rat cortical primary neurons cultured for 7 or 14 days in vitro (neuron DIV7 and 14, respectively), rat primary microglia cultured for 15 days in vitro, and rat primary astroglia cultured for 18 days in vitro (n = 3) in relation to actin, (c) cerebrospinal fluid (CSF), and soluble and membrane fractions of cortical tissue from adult male rats. The membrane-bound proteins, PSD-95 and GluR2, and the soluble protein extracellular signal-regulated kinase 1 (ERK) are used as controls for effective fractionation (n = 4), (d) FC and HIP tissue fractionated using differential centrifugation into a pellet 1 (P1, nuclear) and pellet 2 (P2, crude synaptosome) fraction. Data are normalized to actin levels and the level of the non-fractionated homogenate (Hom) was set to 1 (dashed line). *p < 0.05, **p < 0.01 indicates statistical difference between non-fractionated homogenate and the P2 fraction, # p < 0.05 indicates statistical difference between the P1 and P2 fractions in a Bonferroni-corrected ratio paired t-test (n = 5), (e) minced tissue extracts from the FC or HIP of adult male rats cross-linked with bis(sulfosuccinimidyl) suberate (BS 3) or vehicle (Veh). The staining for GluR2 shows that cross-linking with BS 3 reduces the intensity of the GluR2 band at 95 kDa, and produces a smear of high-molecular weight cross-linked proteins. Data are normalized to actin levels and the vehicle group was set to 1. **p < 0.01, ***p < 0.001 indicates statistical difference between vehicle- and BS 3-treated tissue in a Bonferroni-corrected ratio paired t-test (n = 9–11). (f) Lypd6 mRNA levels in frontal cortex (FC) and hippocampus (HIP) from male rats killed at post-natal day 1, 7, 14, 26, 42, or 60 (n = 5–6). The values were normalized to GAPDH levels.