Title: mir-1202: A Primate Specific and Brain Enriched mirna Involved in Major Depression and Antidepressant Treatment. Authors: Juan Pablo Lopez 1, Raymond Lim 3, Cristiana Cruceanu 1, Liam Crapper 1, Caroline Fasano 2, Benoit Labonte 1, Gilles Maussion 1, Jennie P Yang 1, Volodymyr Yerko 1, Erika Vigneault 2, Salah El Mestikawy 2, Naguib Mechawar 1, Paul Pavlidis 3, Gustavo Turecki 1. Affiliations: 1 McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada. 2 Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada. 3 Department of Psychiatry, University of British Columbia, Vancouver, B.C, Canada. Supplementary Information
Supplementary Tables and Figures Supplementary Table 1. Postmortem Brains. Prefrontal cortex (BA44) brain tissue was obtained in collaboration with the Quebec Coroner s Office from the Quebec Suicide Brain Bank (QSBB). All individuals were male and groups were matched for age, ph and refrigeration delay. Psychological autopsies were performed post-mortem on both cases (n=14) and controls (n=11) based on DSM-IV criteria. The control group had no history of suicidal behavior or major mood or psychotic disorders. We found no significant differences between any of these variables in the experimental groups. Normality was assessed by Shapiro-Wilk normality tests, and statistical differences between groups were analyzed using Student s t-test (two-sided). Controls MDD Statistical Test (P value) Sample 11 14 N/A Sex Male Male N/A Age 37±4.7 37±3.5 0.99 RD 27.7±4.1 32.8±4.1 0.39 Brain ph 6.6±0.1 6.6±0.1 0.72 RIN 6.2±0.2 6.5±0.2 0.28 Page 2 of 17
Supplementary Table 2. mir-1202 Target Prediction. Gene targets of mir-1202 were predicted using five mirna target prediction databases: mirwalk, microrna.org, RNA22, RNA Hybrid and TargetScan. Only genes predicted by all 5 databases were chosen. We then cross-referenced these genes with our existing microarray expression libraries and only genes that were expressed in human brain and up-regulated in PFC of depressed subjects were selected for further validation. mirna Gene Databases FC Regulation hsa-mir-1202 RRN3 5 1,1 UP hsa-mir-1202 TOX 5 1,1 UP hsa-mir-1202 ZFHX4 5 1,1 UP hsa-mir-1202 EIF2S1 5 1,5 UP hsa-mir-1202 MRPS35 5 1,1 UP hsa-mir-1202 SELT 5 1,2 UP hsa-mir-1202 GRM4 5 1,1 UP hsa-mir-1202 HTR2A 5 1,2 UP hsa-mir-1202 NUMBL 5 1,3 UP hsa-mir-1202 NTNG1 5 1,1 UP hsa-mir-1202 CHSY3 5 1,1 UP hsa-mir-1202 PFTK1 5 1,1 UP hsa-mir-1202 TMEM66 5 1,1 UP hsa-mir-1202 RALBP1 5 1,1 UP hsa-mir-1202 FOXJ2 5 1,2 UP hsa-mir-1202 GAS8 5 N/A NO INFO hsa-mir-1202 HLA-DRA 5 N/A NO INFO hsa-mir-1202 ARG1 5 N/A NO INFO hsa-mir-1202 MS4A12 5 N/A NO INFO hsa-mir-1202 EML4 5 N/A NO INFO hsa-mir-1202 CAND1 5 N/A NO INFO hsa-mir-1202 KRTAP17-1 5 N/A NO INFO hsa-mir-1202 ZDHHC21 5-1,2 DOWN hsa-mir-1202 PUM2 5-1,1 DOWN hsa-mir-1202 FTSJ1 5-1,1 DOWN hsa-mir-1202 OSBPL11 5-1,1 DOWN hsa-mir-1202 SEC24B 5-1,3 DOWN hsa-mir-1202 TMEM209 5-1,1 DOWN hsa-mir-1202 TAOK3 5-1,3 DOWN Page 3 of 17
Supplementary Table 3. Independent sample replication subjects. Prefrontal cortex (BA44) brain tissue was obtained in collaboration with the Quebec Coroner s Office from the Quebec Suicide Brain Bank (QSBB). All individuals were matched for age, ph and refrigeration delay. Psychological autopsies were performed postmortem based on DSM-IV criteria. Controls (N=29) and MDD group (N=25) had no history of antidepressant use. The (MDD+A) is composed of depressed individuals with antidepressant history (N=25). We found no significant differences between any of these variables in the experimental groups. Normality was assessed by Shapiro-Wilk normality tests, and statistical differences between groups were analyzed using One-Way ANOVA with post-hoc correction. Controls MDD MDD+A Statistical Test (P value) Sample 29 25 25 N/A Sex 4F/25M 2F/23M 8F/17M N/A Age 52.5±3.8 44.6±2.9 5±2.8 0.20 RD 44.8±5.5 46.3±4.9 44.7±4.9 0.95 Brain ph 6.5±0.1 6.7±0.1 6.7±0.1 0.24 RIN 6.3±0.1 6.6±0.2 6.7±0.2 0.25 Supplementary Table 4. Cell line screening. Expression of mir-1202 and GRM4 across six different human cell lines Cell Line Description mir-1202 GRM4 HEK293 Human Embryonic Kidney N/A High BeC2 Human Neuroblastoma N/A High SK-N-AS Human Neuroblastoma N/A Low SH-SY5Y Human Neuroblastoma N/A Med U-118 MG Human Glioblastoma N/A Low NPCs Human Neural Progenitor Med High Page 4 of 17
Supplementary Table 5. Human Blood Samples. All male and female patients were ascertained at a community outpatient clinic at the Douglas Mental Health University Institute. Subjects were excluded from the study if they had comorbidity with other major psychiatric disorders, if they had positive tests for illicit drugs at any point during the study or if they had general medical illnesses. MDD Subjects were untreated patients (N=32) with a diagnosis of MDD without psychotic features, according to the Statistical Manual of Mental Disordersfourth edition (DSM-IV). Control subjects (N=18) were also excluded if they had a history of antidepressant treatment. All subjects included in the study provided informed consent and the project was approved by the internal review board for the Douglas Mental Health University Institute. We found no significant differences between any of these variables in the experimental groups. Normality was assessed by Shapiro-Wilk normality tests, and statistical differences between groups were analyzed using One-Way ANOVA with post-hoc correction. Controls NRES REM Statistical Test (P value) Sample 18 16 16 N/A Sex 8F/10M 10F/6M 12F/4M N/A Age 37.8±2.2 41.7±2.5 44.1±3.8 0.30 RIN 8.5±0.1 8.4±0.1 8.5±0.1 0.89 Page 5 of 17
Supplementary Table 6. List of primers, references and sequences. Gene Name TaqMan Assay ID SYBR Green hsa-mir-1202 002858 hsa-mir-125 000449 hsa-mir-486 002093 hsa-mir-150 000473 RNU6B 001093 RNU44 001094 RNU48 001006 U6 001093 U47 001223 HTR1A Hs00265014_s1 HTR3A Hs00168375_m1 HTR3B Hs00175775_m1 HTR3C Hs01118162_m1 SERT (SLC6A4) Hs00169010_m1 GRM4 Hs00904641_m1 F: GAC CTG CTG CCT AAC ATC AC R: ACC TCT GTG CCA TCC TTC TC TOX F: CAC AAC TCA CCA TCT CCA CCT R: CGC TTC TCT CCA CCA TTG A MRPS35 F: AGT GAC GAG AAA TGT GAG AAG R: AAC AGA TGG TCC TGA TGA AAC A RRN3 F: TTG AGC GGA TAG TGA TGA GC R: TGA TGG TGT AGC AGA AGA CGA SELT F: CGT GCC CAG CAA GAG ATT A R: CTC CTC AAA CAC CCG CCT AT EIF2S1 F: CCG AAG TGG ATG GAG ATG R:CGC TGC TCT GTG TTC CTT NUMBL F: CAG TGG GCG GCA TTA GAA G R: ACC TGG AGA TGA TGG AGT GG ZFHX4 F: TTC CTT CAC TCT CCG TTC TTG R: CCA GCA GTT GTC CAG TCA TC NTNG1 F: GCA CAA CTG GAC GAT GAG AA R: AAT CCT GAG CCA TTA CAA CGA HTR2A F: ACA CAG GCA GGA GGA CTA TG R: GGC ACC ACA TCA CCA CAA B-Actin 4310881E-1111031 F: AAG ACC TGT ACG CCA ACA CA R: GCA GTG ATC TCC TTC TGC ATC GAPDH 4310884E-1308057 F: TTG TCA AGC TCA TTT CCT GG R: TGT GAG GAG GGG AGA TTC AG Page 6 of 17
Supplementary Table 7. Expression of mir-1202 targets in blood samples. We measured the expression of the additional genes predicted to be targets of mir-1202 in the clinical treatment samples. Three of the predicted genes (ZHX4, HTR2A and NTG1) were not detectable in blood samples. All the remaining genes were expressed at quantifiable levels, using qrt-pcr. We did not find any significant differences in the expression of any of these genes between groups. Normality was assessed by Shapiro-Wilk normality tests, and statistical differences between groups were analyzed using Student s t-test (two-sided). Gene Controls MDD Statistical Test (P value) TOX 0±9 2±6 0.85 MRPS35 0.91±7 3±9 0.38 RRN3 0±7 3±6 0.74 SELT 0±6 7±9 5 EIF2S1 0.92±7 0.84±7 0.42 NUMBL 0±0.1 0.89±8 0.40 Supplementary Table 8. Expression of metabotropic glutamate receptors in HEK293 cells. To experimentally validate our target prediction analysis, we measured the expression of other metabotropic glutamate receptors, also expressed in HEK293 cells, in the mir-1202 mimic assays reported in the manuscript. Consistent with the in silico and prediction target analyses, our results showed that transfection of HEK293 cells with a mir-1202 mimic showed no effects on GRM2, GRM3, GRM5 or GRM8 (One way ANOVA with Bonferroni correction, P>5), the four other metabotropic glutamate receptors endogenously expressed in HEK293 cells. Gene Untransfected Mock mir-scramble mir-1202 Mimic Statistical Test (P value) GRM2 0±0.3 0±0.1 0.97±0.3 0.97±0.2 0.99 GRM3 0.71±0.1 6±3 0.62±0.1 0.77±0.2 0.26 GRM5 0.88±0.2 0.86±0.1 0.68±0.3 0.73±0.3 0.65 GRM8 0±0.1 1.2±0.3 0.83±0.1 2±3 0.10 Page 7 of 17
Supplementary Figure 1A. mir-1202 Conservation. Conservation of mir-1202 across 100 different animal genomes available through the UCSC genome browser hg19 assembly. Highlighted in light blue are the genomes that code for mir-1202. Page 8 of 17
Supplementary Figure 1B. Let-7A1 Conservation. Conservation of Let7a1 across 100 different animal genomes available through the UCSC genome browser hg19 assembly. Highlighted in light blue are the 95 genomes that code for Let-7A1. Page 9 of 17
Qty Mean (mir-1202/rnu6b) Qty Mean (GRM4/Endo Ctrls) GRM4 mrna GRM4 Lopez et al GRM4 - Protein GRM4 2.5 2.5 2.0 2.0 Controls MDD 1 2 3 Protein GRM4 Supplementary Figure 2 Protein expression of GRM4. We measured protein expression levels of GRM4 using western blot analysis in the PCF from the depressed individuals and psychiatrically healthy controls included in the microarray analysis. We observed an increase in GRM4 protein expression levels significantly correlated with the increased GRM4 mrna levels originally reported in the manuscript (P<3, Pearson s r = 0.45, R²=0.2). When comparing GRM4 protein levels between groups, we observed a trend toward increased GRM4 levels among depressed cases (P=8, FC=1.15) mir-1202 GRM4 ** ** ** ** Controls MDD Anxiety + MDD Controls MDD Anxiety + MDD Supplementary Figure 3 Effects of Anxiety on mir-1202 and GRM4. We investigated brain tissue from 16 individuals who at the time of their death met criteria to a major anxiety disorders (OCD, PTSD, panic disorder or GAD) and were of similar age to our depressed cases and controls. However, as the vast majority of these individuals died by suicide (N=12/16), as expected they also met criteria for major depressive disorder or depression NOS. We compared the expression of mir-1202 and GRM4 in PFC brain tissue from these individuals to a depressed group (N: 10) and psychiatrically health controls (N: 15). These results showed a significant difference in the expression of mir-1202 between groups (One way ANOVA with Bonferroni correction, P>01). Consistently, we found a significant increase of GRM4 in the anxiety group (One way ANOVA with Bonferroni correction, P>01) as compared to controls. Page 10 of 17
Supplementary Figure 4 Interaction between GRM4, mir-1202 and target protectors *** *** Supplementary Figure 5 Target Protector Treatment and Controls There were no effects on GRM4 after treatment with any of the two target protectors alone, mir-mimic scramble or vehicle controls. Supplementary Figure 6 Expression of mir-1202 in NPCs across time (0-38 days) Human NPCs show relatively high levels of mir-1202 after one week of differentiation Page 11 of 17
Metabolic Activity Lopez et al 0.8 MTT-Assay 0.6 0.4 0.2 Control L-AP4 (100uM) MSOP (150 um) Supplementary Figure 7 MTT Assay L-AP4 and MSOP showed no toxicity in the MTT cytotoxicity assay. Page 12 of 17
A. B. Supplementary Figure 8 Expression of serotonergic phenotype in Human NPCs (A) Using qrt-pcr we found endogenous expression of the serotonin transporter (SERT) and the serotonin receptors HRT1A, HRT2A, HRT3A and HRT3B. (B) Upper panels present immunocytochemistry performed on NPCs while middle and lower panels display negative and positive controls performed on undifferentiated cells and mouse brain tissue, respectively. In green from left to right: α-tubulin determines the cellular structure of the cells showing the neuron-like Page 13 of 17
shape of the neural progenitors, SERT confirms the presence of the 5-HT transporter in neural progenitors, TpOH the rate limiting enzyme of 5-HT synthesis confirms that the neural progenitors express a serotoninergic phenotype, GRM4 shows the presence of this metabotropic glutamate receptor on the neural progenitors and TH shows that monoaminergic phenotype is expressed by really rare neural progenitors. In blue: nucleus staining with Hoescht stain shows the presence of the cells. These results demonstrate that the vast majority of NPcs express a serotoninergic phenotype, by synthesizing and re-uptaking 5-HT. Moreover, they express mglu4 receptors Supplementary Figure 9 Antidepressant concentrations. Antidepressants were tested at four different concentrations (Citalopram: 0uM, 50uM, 100uM and 200uM, Imipramine: 0uM, 6.25uM, 12.5uM and 25uM) and 3 different time points (24hrs, 48hrs and 1 week) using the MTT cytotoxicity assay. Page 14 of 17
mean gray value (% of control) Lopez et al 120 100 80 60 40 20 0 GRM4 - Protein ** *** *** Control Imipramine Citalopram No Ab Supplementary Figure 10 Expression of GRM4 in human NPCs. Using imaging and immunocytochemistry assays, we measured the expression levels of GRM4 in neural progenitor cells (NPCs) treated with citalopram and imipramine, as previously described in figures 2G and 2H. Consistent with our mrna results, chronic treatment with both citalopram and imipramine significantly reduced GRM4 protein expression levels (One way ANOVA with Bonferroni correction, P< 001). Page 15 of 17
Qty Mean (SERT/GAPDH) Qty Mean (mir-1202/rnu6b) Qty Mean (mir-1202/rnu6b) Qty Mean (SERT/B-Actin & GAPDH) Qty Mean (mir-1202/rnu6b) Qty Mean (GRM4/B-Actin & GAPDH) Qty Mean (SERT/B-Actin & GAPDH) Lopez et al A SERT *** *** Controls Imipramine Citalopram B SERT C mir-1202 D GRM4 Controls Lithium Valproate Control Lithium Valproate Control Lithium Valproate E SERT F 2.5 2.0 Knock-down Cells G 2.5 2.0 RNA Hairpin Control Cells * * *** Untreated Ctrl KD KD Ctrl Control Imipramine Citalopram Controls Imipramine Citalopram Supplementary Figure 11 SERT, mir-1202, GRM4 and antidepressant treatment (A) A one way ANOVA with Bonferroni correction (P<001) shows that there is a significant decrease in the expression of SERT after chronic antidepressant treatment. These results confirm that both citalopram and imipramine alter the expression of SERT in NPCs, and suggest that the effects seen on mir-1202 and GRM4 may be related to the reuptake blockade induced by chronic antidepressant action. (B). NPCs were treated with lithium and valproate for two weeks. Our results showed that chronic treatment with lithium or valproate do not alter the expression of the serotonin transporter (One way ANOVA with Bonferroni correction, P>5). (C-D) We measured the expression of mir-1202 and GRM4 after chronic treatment with lithium and valproate. Our results showed no significant differences in the expression of mir-1202 or GRM4 after chronic treatment as compared to controls (One way ANOVA with Bonferroni correction, P>5). (E) Expression of SERT is significantly reduced in the SERT knocked down cells as compared to untreated controls (P< 001; FC=0.36) and cells treated with a control hairpin RNA (P< 001; FC=0.35). These results confirm a 75% knockdown of SERT in human NPCs. (F-G) SERT knock-down and control cell lines were grown and treated chronically with citalopram and imipramine. Our results showed that the effects of chronic antidepressant treatment on mir- 1202 levels are not present in the SERT knock-down cells, while they are still present in the control cell lines (One way ANOVA with Bonferroni correction, P< 5). Page 16 of 17
A. B. C. D. Supplementary Figure 12 Expression levels of ubiquitously expressed mirnas (mir-125, mir-486, mir-150) and endogenous controls (RNU44, RNU48). (A,C) Chronic (2 weeks) Imipramine treatment. (B,D) Chronic (2 weeks) Citalopram treatment. There were no significant differences in the expression levels of any of these mirnas or endogenous controls as a result of chronic antidepressant treatment (P>5) Page 17 of 17