Addiction associated N40D mu-opioid receptor variant modulates synaptic function in human neurons.

MOR N40D expressing inhibitory human neurons exhibit more robust suppression of inhibitory synaptic transmission by DAMGO.(A) Oct4 (green), Tra 1-60 (red), and DAPI (blue) ICC for N40 and D40 subject iPS cells depicting pluripotency (B) Sequencing confirming homozygous A118 or G118 genotype of human iPS cell lines (C) MAP2 (green) and Synapsin (red) ICC of iN cells generated from N40 and D40 subject iPS cells (D) MAP2 (green) and VGAT (red) ICC of induced inhibitory neuronal (iN) cells generated from N40 and D40 subject iPS cells (E-G) Both N40 and D40 iN cells exhibit PTX sensitive spontaneous IPSCs whose frequency (N40 vs D40: N.S.) and amplitude (N40 vs D40: N.S.) are unaffected by MOR N40D substitution (H) Representative traces of action potentials induced by step current injections (from −20 to +75 pA, 5 pA increments) during current clamp recordings from one N40 and D40 cell line (I) Quantification of induced action potentials in inhibitory iNs cells illustrating that neuronal excitability is unchanged as a consequence of MOR N40D (N40 vs D40: N.S. at all current injections) (J) Representative traces of sIPSCs recorded to increasing concentrations of DAMGO in N40 and D40 iN cells (K) Quantification of inhibition of sIPSC frequency in individual subject derived N40 and D40 iN cells (L) Merged data of the four N40 and three D40 subject lines illustrates that D40 iN cells exhibit stronger suppression of IPSC frequency compared to N40 iN cells (M-N) sIPSC frequency response to a single concentration of 10 ¼M DAMGO; data is normalized to control (N40 vs control: p<0.001, D40 vs control: p<0.001). Summary graphs are shown as individual cell lines or merged data of either four N40 patients (red bars) and three D40 patients (blue bars) (N40 vs D40: p <0.01). Data are depicted as means ± SEM. Numbers of cells/Number of independently generated cultures analyzed are depicted in bars. Paired t-test was used to evaluate within genotype statistical differences and one-way ANOVA was used to evaluate between genotype statistical differences (*p <0.05, **p <0.01, ***p <0.001).

Human neurons from two sets of independently targeted isogenic human stem cell lines for OPRM1 A118G validate differential DAMGO response observed in patient cell lines.(A) OPRM1 Targeting Strategy 1: Structure of OPRM1 gene on chromosome 6 and schematic overview of CRISPR/Cas9 gene targeting strategy to knock-in homozygous G118 alleles into human H1 embryonic stem cell (H1ES) in which sgRNA targets donor strand. In the 140bp ssODN, we inserted a T to C mutation to incorporate OPRM1 GG118, synonymous G to A for PAM mutation, and synonymous G to C and G to A mutations to create a BamHI restriction enzyme site. (B) Sequencing of original H1ES control cell line carrying homozygous A118 (N40) alleles, and two isolated clones carrying homozygous OPRM1 G118 (D40) alleles (Clone 9-2-17, Clone 9-2-18). (C) OPRM1 Targeting Strategy 2: Structure of OPRM1 gene on chromosome 6 and an independent CRISPR/Cas9 gene targeting strategy to correct 03SF patient line (originally homozygous G118 expressing MOR D40) to homozygous A118 (N40). We designed a 200 nt template strand to knock-in homozygous A118 alleles, containing mutations to generate a HpaI restriction enzyme site for screening (D) Sequencing of passage-matched, uncorrected 03SF patient cell line carrying homozygous D40 alleles (C12) and two gene-corrected clones (Clone A10, D11) carrying homozygous OPRM1 A118 (N40) alleles after subcloning. (E) ICC of MAP2 (green) and Synapsin (red) of iN cells produced from gene-targeted ES cells and iPS cells. (F) Immunofluorescence of MAP2 (green) and VGAT (red) of iN cells produced from gene-targeted ES cells and iPS cells. (G-I) Relative mRNA levels of OPRM1 as well as markers for inhibitory subtype specificity (GAD1, VGAT) measured by quantitative RT-PCR; mRNA levels are normalized to Synapsin I. Data are represented as means of three independently differentiated batches of iNs from each patient iPS cell line. (J) Representative traces of sIPSCs recorded to increasing concentrations of DAMGO in N40 and D40 iN isogenic iN cells derived from ES cells. (K-L) Quantification of sIPSC frequency (H1 vs control: p <0.05, Clone 17 vs control: p <0.001, Clone 18 vs control: p <0.001, N40 vs control: p <0.05, D40 vs control: p <0.001, N40 vs D40: p <0.05) and amplitude in response to 6 ¼M DAMGO (H1 vs control: N.S., Clone 17 vs control: N.S., Clone 18 vs control: N.S., N40 vs control: N.S., D40 vs. control: N.S., N40 vs D40: N.S.) (M) Representative traces of sIPSCs recorded to increasing concentrations of DAMGO in N40 and D40 iN isogenic iN cells derived from iPS cells. (N-O) Quantification of sIPSC frequency (A10: DAMGO vs control: p <0.01, D11: DAMGO vs control: N.S., C12: DAMGO vs control: p <0.001, N40: DAMGO vs control: p <0.01, D40: DAMGO vs control: p <0.001, N40 vs D40: p <0.05) and amplitude in response to 6 ¼M DAMGO (A10: DAMGO vs control: N.S., D11: DAMGO vs control: N.S., C12: DAMGO vs control: N.S., N40: DAMGO vs control: N.S., D40: DAMGO vs. control: N.S., N40 vs D40: N.S.). Data are depicted as means ± SEM. Numbers of cells/Number of independently generated cultures analyzed are depicted in bars. Paired t-test was used to evaluate within genotype statistical differences and one-way ANOVA was used to evaluate between genotype statistical differences (*p <0.05, **p <0.01, ***p <0.001).

D40 iN cells exhibit greater inhibition of synaptic release and intrinsic excitability.(A) Representative traces of mIPSCs in one N40 (A10) and one D40 (C12) cell line derived iN cells recorded at a 0 mV holding potential and their response to DAMGO. (B-C) Quantification of mIPSC frequency (A10: DAMGO vs control p <0.01, C12: DAMGO vs control p <0.001) and amplitude (A10 and C12: DAMGO vs control p >0.05) in A10 and C12 iN cells normalized to before DAMGO application. (DAMGO effect on frequency: A10 vs C12: p < 0.05, DAMGO effect on amplitude: A10 vs C12: N.S.) (D) Representative traces of evoked IPSCs from one N40 (A10) and one D40 (C12) cell line derived iN cells (E) Quantification of evoked IPSC amplitude in A10 and C12 iN cells normalized to before DAMGO application (A10: DAMGO vs control p <0.05, C12: DAMGO vs control p <0.001, A10 vs C12: p <0.001). Data are depicted as means ± SEM. Numbers of cells/Number of independently generated cultures analyzed are depicted in bars. Paired t-test was used to evaluate within genotype statistical differences and one-way ANOVA was used to evaluate between genotype statistical differences (*p <0.05, **p <0.01, ***p <0.001).

D40 iN cells exhibit a sustained decrease in intrinsic excitability.(A) Representative traces of repetitive action potentials generated from depolarizing current injections in one N40 (A10) cell line derived iN and one D40 (C12) cell line derived iN, and their response to DAMGO (B-D) Summary graphs of DAMGO effect on AP Number (A10: DAMGO vs control: N.S., C12: DAMGO vs control p <0.001), amplitude (A10: DAMGO vs control p <0.01, C12: DAMGO vs control p <0.001) and firing threshold (A10: DAMGO vs control: N.S., C12: DAMGO vs control: N.S.). Data normalized to before DAMGO application reveals DAMGO preferentially decreases intrinsic excitability of D40 iNs but not N40 iNs (DAMGO effect on frequency: A10 vs C12 p <0.01) with no effect on amplitude (A10 vs C12: N.S.) or firing threshold (A10 vs C12: N.S.) (E) Representative traces depicting the effect of DAMGO on spontaneous action potential firing in one D40 (C12) and one N40 (A10) cell line derived iN (F) Quantification of number of spontaneous action potentials fired before and after DAMGO application in N40 and D40 iNs represented as a time course (G) Quantification of resting membrane potential before and after DAMGO application in N40 and D40 iNs represented as a time course (H) Representative traces of individual Action Potentials before and after DAMGO in N40 and D40 iN cells (J-K) Summary graphs depicting that DAMGO causes a trending increase in the after hyperpolarization potential (AHP) amplitude in D40 iN cells compared to N40 iN cells (A10: DAMGO vs control: N.S., C12: DAMGO vs control: p <0.05, A10 vs C12: N.S.) with no effect on firing threshold (A10: DAMGO vs control: N.S., C12: DAMGO vs control: N.S., A10 vs C12: N.S.) or half width (A10: DAMGO vs control: N.S., C12: DAMGO vs control: N.S., A10 vs C12: N.S.). Data are depicted as means ± SEM. Numbers of cells/Number of independently generated cultures analyzed are depicted in bars. Paired t-test was used to evaluate within genotype statistical differences and one-way ANOVA was used to evaluate between genotype statistical differences (*p <0.05, **p <0.01, ***p <0.001).

Differential N-glycosylation of human MOR carrying N40D variants.(A) Immunoblot (IB) of recombinant human FLAG-MOR N-terminal region-human Fc (amino acids 1-67) expressed as a soluble entity in HEK 293 cells. Both peptides (N40 and D40) were separated by size on an SDS-PAGE gel and detection was accomplished using an anti-FLAG antibody. Note that the untreated D40 sample (lane 2) is already of lower molecular weight compared to the N40 (lane 1) and after PNGase F digestion (lanes 3-6) both samples show an identical migration pattern, indicating complete removal of the N-linked glycans. PNGase reactions were carried out in two ways. Specifically, either the peptide was denatured prior to enzyme application (lane 3 and 4) or the native peptide was treated with PNGase F (lanes 5 and 6). Glycan removal was equally effective both ways tested. (B) Recombinant human FLAG-MOR N-terminal region-human Fc peptide was purified from the culture media of HEK293S (wild-type) (lanes 1-4) or HEK293S GnT1- (lanes 5-8). SDS-PAGE followed by Coomassie staining revealed that under conditions where glycosylation is defective or abolished N40 and D40 MOR peptide migrate at the same molecular weight (lanes 5 and 6) regardless of PNGase F treatment.