The schizophrenia risk gene product miR-137 alters presynaptic plasticity

Abstract

Noncoding variants in the human MIR137 gene locus increase schizophrenia risk with genome-wide significance. However, the functional consequence of these risk alleles is unknown. Here we examined induced human neurons harboring the minor alleles of four disease-associated single nucleotide polymorphisms in MIR137. We observed increased MIR137 levels compared to those in major allele-carrying cells. microRNA-137 gain of function caused downregulation of the presynaptic target genes complexin-1 (Cplx1), Nsf and synaptotagmin-1 (Syt1), leading to impaired vesicle release. In vivo, miR-137 gain of function resulted in changes in synaptic vesicle pool distribution, impaired induction of mossy fiber long-term potentiation and deficits in hippocampus-dependent learning and memory. By sequestering endogenous miR-137, we were able to ameliorate the synaptic phenotypes. Moreover, reinstatement of Syt1 expression partially restored synaptic plasticity, demonstrating the importance of Syt1 as a miR-137 target. Our data provide new insight into the mechanism by which miR-137 dysregulation can impair synaptic plasticity in the hippocampus.

Figure 1. MIR137 gain of function affects presynaptic targets

(a) Location of the four disease-associated SNPs (red) in the human genome. Black bar: MIR137 coding region (Chr1:98,511,626–727, UCSC genome browser, GRCh37/hg19). (b) Schematic overview for direct induction of human fibroblasts to neurons. Fluorescence-activated cell sorting (FACS). (c) Fold-change differences of MIR137 determined by qRT-PCR. W_Fibroblasts = 19, ^nsP = 0.1802, r = −0.33, W_{InducedNeurons} = 65, *P = 0.0157, r = −0.40; box plot shows, in ascending order, the lowest maximum value, the first quartile, median, third quartile and the highest maximum value, with dots representing outliers; n, analyzed samples obtained from three reprogramming from each fibroblast line. (d–g) Confirmation of in silico predicted miR-137 presynaptic target genes. (d) Schematic overview of the luciferase assay. CDS, coding DNA sequence. (e) Luciferase activity normalized to ΔmiR-137_OE. Light gray and dark orange, deleted miR-137-binding site. Syn3 has two predicted miR-137-binding sites, s1 and s2. t_psiCheck2(53.51) = 0.40, ^nsP = 0.6932, r = 0.06; t_Ezh2(40.7) = −8.94, ***P < 0.001, r = 0.81; F_Cplx1(3,74) = 6.73, ***P < 0.001; F_Nsf(3,54) = 97.32, ***P < 0.001; F_Syn3-s1(3,75) = 16.67, ***P < 0.001; F_Syn3-s2(3,108) = 8.47, ***P < 0.001; F_Syt1(3,181) = 67.12, ***P < 0.001, F_Hcrt(3,85) = 14.12, ***P < 0.001, F_Syt7(3,74) = 37.38, ***P < 0.001, F_Stx17(3,80) = 3.44, *P = 0.0207, post hoc pairwise comparison of ΔmiR-137_OE-miR-137_OE after Benjamini and Hochberg-adusted P value for multiple comparisons: Cplx1 (P = 0.0006), Nsf (P < 0.001), Syn3-s1 (P < 0.001), Syn3-s2 (P = 0.00016), Syt1 (P < 0.001), Hcrt (P = 0.0029), Syt7 (P < 0.001), Stx17 (P = 0.043); n, number of experiments, each in triplicate. (f,g) mRNA levels of miR-137 target genes in HT22 (f) and N2a (g) cells transfected with a commercially available miR-137 mimic normalized to a mimic-control. HT22: t_Cplx1(6.98) = −3.58, **P = 0.0090, r = 0.81; W_Nsf = 0, **P = 0.0022, r = −0.89; t_Syn3(9.71) = −3.40, **P = 0.007, r = 0.74; t_Syt1(3.21) = −2.99, ^nsP = 0.0535, r = 0.86; W_Hcrt = 15, ^nsP = 0.4452, r = −0.21; t_Syt7(6.06) = −1.02, ^nsP = 0.3481, r = 0.38; t_Stx17(4.31) = 0.51, ^nsP = 0.6337, r = 0.24; N2a: t_Cplx1(9.10) = −6.18, ***P < 0.001, r = 0.90; t_Nsf(4.43) = −3.01, *P = 0.3453, r = 0.82; t_Syn3(8.99) = −6.23, ***P < 0.001, r = 0.90; W_Syt1 = 0, *P = 0.0286, r = −0.77, t_Hcrt(10) = −3.15, *P = 0.0103, r = −0.71; t_Syt7(6.27) = 0.91, ^nsP = 0.3962, r = 0.34 t_Stx17(9.96) = −2.47, *P = 0.033, r = 0.62; n, number of experiments. (h) mRNA levels in induced neurons by qRT-PCR. t_CPLX1 (5.97) = 2.56, *P = 0.043, r = 0.72; t_NSF (17.89) = 2.85, *P = 0.0107, r = 0.56; t_SYN3 (11.29) = 3.00, *P = 0.0118, r = 0.67; t_SYT1 (13.40) = 3.45, **P = 0.004, r = 0.69; t_PCLO (22.95) = 1.05, ^nsP = 0.3034, r = 0.22, t_STX8 (20.36) = −0.49, ^nsP = 0.7013, r = 0.22, t_SYNJ1 (18.94) = −1.21, ^nsP = 0.2398, r = 0.27; n, number of samples from at least three reprogrammings of each fibroblast line. (i) FM4-64 imaging analysis, H(1) = 38.15, ***P < 0.001; n, number of analyzed cells, usually one cell per coverslip from two reprogramming from each fibroblast line. ^ns, not significant. Error bars, s.e.m, W, Wilcoxon-rank sum and signed rank tests, r, effect size, H, Kruskal-Wallis rank sum test.

Figure 2. Recapitulation of miR-137 gain of function by overexpressing miR-137 in the mouse dentate gyrus

(a) Schematic overview of the lentivirus delivery constructs, expressing the miR-137 precursor under the RPPH1 promoter (miR-137_OE). ΔmiR-137_OE lacks mature miR-137 region. LTR, long terminal repeat. (b) Schematic overview of stereotactic virus injection into the murine dorsal dentate gyrus. (c) Tile-scan of hippocampi injected with either miR-137_OE (left) or ΔmiR-137_OE (right), labeled against mCherry (magenta), the mossy fiber marker zinc transporter 3 (ZnT3, green) and the nuclear dye DAPI (blue). DG, dentate gyrus. Representative images of at least three animals. Scale bars, 100 μm. (d) miR-137 expression in ΔmiR-137_OE-, miR-137_OE-injected, and naive animals, H(2) = 7.68, *P = 0.0215, post hoc pairwise comparison after Tukey (t_{ΔmiR-137OE-miR-137OE} = 2.53, *P = 0.0471, t_{miR-137OE-naive} = −2.72, *P = 0.0312, t_{ΔmiR-137OE-naive} = 0.261, ^nsP = 0.796), box plot shows, in ascending order, the lowest maximum value, the first quartile, median, third quartile and the highest maximum value, with dots representing outliers; n, number of dissected dorsal DG regions from at least three animals. (e) Quantified protein levels of miR-137 target genes in mossy fiber-CA3 pathway for ΔmiR-137_OE and miR-137_OE, t_Cplx1(5.43) = −6.46, ***P < 0.01, r = 0.94; t_Nsf(6.47) = −3.43, *P = 0.0123, r = 0.80; t_Syn3(9.89) = −3.00, *P = 0.0135, r = 0.69; t_Syt1(7.21) = −2.47, *P = 0.0418, r = 0.68; n, dorsal DG-CA3 region of at least three animals. Right, cropped western blot images. Full-length blots are presented in Supplementary Figure 9. ns, not significant. Error bars, s.e.m.

Figure 3. Morphological and functional alterations are evident at the mossy fiber synapse of miR-137_OE

(a,b) Ultrastructural analysis of the mossy fiber presynaptic terminal. (a) Representative images of virus-transduced synapse. Black arrowhead, gold-particles labeling mCherry; orange arrowhead, gap in the vesicle pool in miR-137_OE synapse; *, active zone; scale bar, 100 nm. (b) Vesicle distribution for ΔmiR-137_OE (black, n = 84) and miR-137_OE (orange, n = 112), V = 0.26, F(11,184) = 5.81, ***P < 0.001; with post hoc analysis shown: F_Docked(1,194) = 2.91, ^nsP = 0.0892; F_50nm(1,194) = 6.83, *P = 0.0097; F_100nm(1,194) = 37.7, ***P < 0.001; F_150nm(1,194) = 32.5, ***P < 0.001; F_200nm(1,194) = 12.4, ***P < 0.001; F_250nm(1,194) = 0.87, ^nsP = 0.3517; F_300nm(1,194) = 1.57, ^nsP 0.211; F_350nm(1,194) = 4.00, *P = 0.0471; F_400nm(1,194) = 27.3, ***P < 0.001; F_450nm(1,194) = 7.46, **P = 0.0069; n, number of analyzed synapses of at least three animals. (c) Schematic overview of the electrophysiological paradigm for acute hippocampal slice recording; magenta, presumptive mCherry virus expression. (d) fEPSP amplitude for 1-Hz sustained stimulation, H(1) = 67.34, ***P < 0.001. Representative traces embedded; gray traces, response to first stimulation; n, number of analyzed hippocampal slice preparation from at least three animals. ns, not significant. Error bars, s.e.m, V, multivariate analysis of variance.

Figure 4. miR-137 overexpression in the dentate gyrus causes impairment in hippocampus-dependent learning

(a) LTP recording for miR-137_OE and control ΔmiR-137_OE, F(1,1060) = 144.6, ***P < 0.001. Arrows indicate application of either high frequency stimulation or DCG-IV. Representative fEPSP traces inset; gray traces, baseline levels before stimulation. Right bar chart, LTP magnitude calculated by averaging fEPSP amplitude of the last 10 min (50–60 min) recording; W = 7283, ***P < 0.001, r = −0.42. (b) Input-output curve from ΔmiR-137_OE (black, n = 12) and miR-137_OE (orange, n = 14), H(1) = 0.02, ^nsP = 0.8774. Shaded area, 95% confidence interval; n, number of analyzed hippocampal slice preparation from at least three animals. (c,d) Fear conditioning. Percentage freezing for contextual (c; t(17.76) = 3.87, **P = 0.0011, r = 0.68) and cue response (d; t(22) = 1.90, ^nsP = 0.0709, r = 0.38). (e–g) Morris water maze. (e) Escape latency, F(1,117) = 14.32, ***P < 0.001; post hoc analysis shown: W_Day1 = 30, ^nsP = 0.5535, r = −0.14; W_Day2 = 28.5, ^nsP = 0.495, r = −0.16; W_Day3 = 29, ^nsP = 0.5309, r = −0.15; W_Day4 = 17.5, ^nsP = 0.0831, r = −0.41; W_Day5 = 14, *P = 0.0380, r = −0.50; W_Day6 = 16.5, ^nsP = 0.0673, r = −0.43; W_Day7 = 7, **P = 0.061, r = −0.65. (f) No difference in swim velocity, t(13.51) = −0.66, ^nsP = 0.5224, r = 0.18. (g) Probe trial, ΔmiR-137_OE mice prefer target quadrant, F(3,28) = 10.16, ***P < 0.001. This effect was not observed for miR-137_OE. A significant main effect exists for the target quadrant between ΔmiR-137_OE and miR-137_OE, F(7, 60) = 7.24, *P = 0.0438; n, number of animals. ns, not significant. Error bars, s.e.m.

Figure 5. Sequestration of endogenous miR-137 leads to phenotype reversal. Sponge_Control (black), Sponge_miR-137 (blue)

(a) Relative mRNA levels of miR-137 target genes in transfected N2a cells, t_Cplx1(8.80) = 3.02, *P = 0.0149, r = 0.71; t_Nsf(2.46) = 9.00, *P = 0.036, r = 0.64; t_Syn3(7.70) = −3.24, *P = 0.0125, r = 0.76; t_Syt1(7.96) = 3.00 *P = 0.0173, r = 0.73; n, number of experiments. (b) Relative mRNA levels in transduced induced minor allele SNP neurons, W_CPLX1 = 27, **P = 0.0064, r = −0.53; W_NSF = 13, ***P < 0.001, r = −0.70; W_SYN3 = 117, ***P < 0.001, r = −0.71; W_SYT1 = 66.5 *P = 0.0209, r = −0.39; t_PCLO(4.82) = 0.52 ^nsP = 0.6282, r = 0.23; t_STX8(5.63) = 1.54 ^nsP = 0.1782, r = 0.54; t_SYNJ1(5.56) = 1.06 ^nsP = 0.3338, r = 0.41; Sponge_Control, n = 11; Sponge_miR-137, n = 18; number of transduced minor allele SNP fibroblast lines with either construct from three reprogramming. (c) FM_4-64 imaging analysis, H(1) = 152.06, ***P < 0.001; n, number of analyzed transduced induced neurons of the two minor allele fibroblast lines from three reprogramming. (d) miR-137 target genes protein level in mossy fiber-CA3 pathway, t_Cplx1(21.97) = −2.69, *P = 0.0140, r = 0.5; t _Nsf(29.0) = −2.36, *P = 0.0251, r = 0.40; t_Syn3(16.11) = 3.43, **P = 0.0034, r = 0.65; t_Syt1(21.41) = −2.17, *P = 0.0415, r = 0.42; n, number of dissected dorsal DG-CA3 regions from at least three animals. Right, cropped western blot image. Full-length blots are presented in Supplementary Figure 9. (e,f) Ultrastructural analysis of mossy fiber presynaptic terminal. (e) Representative images of virus-transduced synapse labeled with gold particles (arrowhead); *, active zone; scale bars, 100 nm. (f) Vesicle distribution from the synaptic release site for Sponge_Control (n = 50) and Sponge_miR-137 (n = 43), V = 0.25, F(11,91) = 2.40, *P = 0.0125, post hoc analysis: F_Docked(1,91) = 4.01, *P = 0.0483; F_50nm(1,91) = 4.13, *P = 0.045; F_100nm(1,91) = 1.68, ^nsP = 0.1977; F_150nm(1,91) = 3.04, ^nsP = 0.0845; F_200nm(1,91) = 2.18, ^nsP = 0.1429; F_250nm(1,91) = 0.09, ^nsP = 0.7687; F_300nm(1,91) = 0.29, ^nsP = 0.5886; F_350nm(1,91) = 3.22, ^nsP = 0.0759; F_400nm(1,91) = 0.55, ^nsP = 0.4619; F_450nm(1,91) = 1.53, ^nsP = 0.2188; n, number of analyzed synapses from at least three animals. (g) fEPSP amplitude for 1 Hz sustained stimulation, F(1,305) = 77.73, ***P < 0.001. Representative traces embedded; gray trace, response to first stimulation; n, number of analyzed hippocampal slices from at least three animals. (h) LTP recording in DG-CA3 mossy fiber pathway, H(1) = 125.92, ***P < 0.001. Arrows, application of either high frequency stimulation or DCG-IV. Inset, representative fEPSP traces; gray traces, baseline before stimulation. Right bar chart, LTP magnitude for the last 10 min (50–60 min) recording, W = 341, ***P < 0.001, r = −0.67; n, number of analyzed hippocampal slices from at least three animals. (i,j) Fear conditioning. Freezing percentage for context (i; t(17.0) = −1.43, ^nsP = 0.1707, r = 0.33) and cue (j; t(16.21) = 0.16, ^nsP = 0.873, r = 0.04). n, number of animals. ^ns, not significant. Error bars, s.e.m.

Figure 6. Impact of alteration of synaptotagmin-1 (Syt1) expression under miR-137 gain of function

ΔmiR-137_OE-Venus (gray), miR-137_OE-Venus (red), miR-137-Syt1-Venus (green) and ΔmiR-137_OE-Syt1-Venus (purple), (a) Schematic overview of bicistronic expression vectors. LTR, long terminal repeat. (b,c) Ultrastructural analysis of the mossy fiber terminal. (b) Representative image of Venus-immunopositive synapse (black arrowhead). Red arrowheads. gap in the vesicle pool in miR-137_OE-Venus synapse; *, active zone; scale bar, 100 nm. (c) Vesicle distribution from the synaptic release site for ΔmiR-137_OE-Venus (n = 46), miR-137_OE-Venus (n = 47), miR-137-Syt1-Venus (n = 70) and ΔmiR-137_OE-Syt1-Venus (n = 58); n, number of analyzed synapses of at least three animals, V_gray-red = 0.72, F(11,97) = 22.9, ***P < 0.001, V_gray-green = 0.66, F(11,100) = 17.7, ***P < 0.001, V_gray-purple = 0.57, F(11,102) = 12.3, ***P < 0.001. (d) fEPSP amplitude during 1 Hz sustained stimulation, H(3) = 217.81 ***P < 0.001. Multiple comparison test after Kruskal-Wallis showed *P < 0.05 for all combinations. Inset, representative traces; gray traces, baseline before stimulation; n_gray = 5, n_red = 7, n_green = 4, n_purple = 4; number of hippocampal slices from at least three animals. (e) Right, mossy fiber LTP recording; arrows, application of high frequency stimulation or DCG-IV. Insets, representative traces; gray, baseline before stimulation. Left, magnitude of potentiation during the last 10 min (50–60 min) recording, H(3) = 106.41, ***P < 0.001, multiple comparison two-tailed test after Kruskal-Wallis, *P < 0.05; n, number of analyzed hippocampal slices from at least 3 animals. (f) Input-output curve of fEPSP against fiber volley amplitude at the mossy fiber-CA3 synapse, H(3) = 3.85, ^nsP = 0.2776. Shaded area, 95% confidence interval; n_gray = 15, n_red = 15, n_purple = 15, n_green = 15); n, number of analyzed hippocampal slices from at least three animals. (g) Freezing level % for contextual fear conditioning, F(3,34) = 4.53, **P = 0.0089; post hoc Tukey analysis: tmiR-137-miR137-Syt1 = 3.57, **P = 0.0060; tΔmiR-137-miR137 = −2.28, ^nsP = 0.1236, tΔmiR-137-miR137-_Syt1 = 1.03, ^nsP = 0.7317, tΔmiR-137-ΔmiR137-Syt1 = −0.93, ^nsP = 0.789, t_{miR-137-ΔmiR137-Syt1} = 1.37, ^nsP = 0.5278, t_{miR-137-Syt1-ΔmiR137-Syt1} = −2.07, ^nsP = 0.1825; n, number of animals. ns, not significant. Error bars, s.e.m.