Fig 1.
ORY-2001 is a covalent inhibitor of KDM1A and MAO-B.
(A) Chemical structure of ORY-2001. (B) A representative dose-response curve of rKDM1A inhibition by ORY-2001 (IC50 KDM1A = 105 nM); N = 1, n = 2, mean ± SD. (C) A representative dose-response curve of MAO-B inhibition by ORY-2001 (IC50 MAO-B = 58 nM); N = 1, n = 2, mean ± SD. (D) Dose curve of induction of the differentiation marker CD11B in THP-1 cells by ORY-2001 (EC50 = 21 nM); N = 1, n = 1; (E) Dose curve of KDM1A target engagement in THP-1 cells treated 24 hr with ORY-2001 (EC50 = 1 nM); N = 1, n = 3, mean ± SD.
Fig 2.
ORY-2001 rescues the memory deficit in SAMP8 mice.
(A) Novel Object Recognition Test (NORT) Discrimination Index (DI) meta-analysis of untreated animals showing cognitive decline in SAMP8 vs. SAMR1 mice (Males F7,224 = 1.970; p = 0.060 Interaction; Females F7,224 = 2.403; p = 0.022 Interaction; n = 15 group/age). (B) ORY-2001 dose response effect on DI in the NORT (F4,77 = 24.74; p < 0.0001; SAMR1 veh N = 25; SAMP8 veh N = 29, ORY-2001 0.11 mg/kg/day N = 6, 0.32 mg/kg/day N = 8, 0.96 mg/kg/day N = 24, 3.20 mg/kg/day N = 15). (C) Dose response of KDM1A target engagement in the brain cortex of SAMP8 mice at end of 2 month treatment with ORY-2001.
Fig 3.
KDM1A inhibition is key for memory rescue by ORY-2001.
(A) Comparison of the effect of ORY-2001 and ORY-LSD1 on the DI in the NORT reflects higher efficacy of ORY-2001 in SAMP8 mice (F4,38 = 10.40; p < 0.0001; Vehicle: N = 11/group; ORY-2001 and ORY-LSD1: N = 8/dose). (B) Comparison of the effect of ORY-2001 and RSG on the DI in the NORT reflects higher efficacy of ORY-2001 in SAMP8 mice (F3,28 = 16.84; p < 0.0001; N = 8/group).
Fig 4.
Symptomatic and disease modifying potential of ORY-2001.
(A) Effect of one week ORY-2001 treatment on NORT DI (t(19) = 5.230; p < 0.0001; N = 9-12/group), assessed 2 hr after training (B) Effect of ORY-2001 on NORT DI in a crossover design; 0: one month treatment with vehicle; I: one month treatment with ORY-2001 (F3,57 = 25.65; p < 0.0001; N = 12-17/group). (C) Effect of ORY-2001 on DI in the NORT of aged SAMP8 mice (t(19) = 6.600; p < 0.0001; N = 9-12/group). Means and SEM are represented. SAMR1 and SAMP8 vehicle groups were compared by Student t-test. Within SAMP8 mice, different drug treatments were compared by t-test or by oneway-ANOVA with Dunnett and SNK post-hoc analysis. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5.
ORY-2001 induces GE changes in the hippocampus of SAMP8 mice.
Microarray analysis of hippocampal tissue from vehicle- and ORY-2001-treated SAM mice: (A) GE changes in SAMR1 (genes with changes > 2-fold are shown) compared to vehicle-treated SAMP8 mice. Genes are ranked according to the highest expression change. Genes on chr4 with higher copy number in SAMP8 compared to SAMR1 mice (Oprs1, Ccl27 and Ccl19) are genotype controls and highlighted in grey. (B) GE changes in SAMP8 mice treated with ORY-2001 compared to vehicle-treated SAMP8 mice (genes with changes > 1.4-fold in ORY-2001 0.96 mg/kg/day samples and > 1.32-fold in ORY-2001 3.2 mg/kg/day samples) are shown. Genes are ranked according to the highest expression change in the SAMP8 0.96 mg/kg/day condition. All values represent Log2(Fold Change). The survey was performed on pooled samples from each group. Veh = Vehicle. qRT-PCR analysis of the effect of ORY-2001 treatment on hippocampal mRNA expression: (C) S100a9 (F2,13 = 9.224; p = 0.0032), (D) Baiap3 (F2,13 = 4.933; p = 0.025), (E) Npw (F2,13 = 34.66; p < 0.0001), (F) Prph F2,13 = 26.47; p < 0.0001) (SAMR1 veh N = 8; SAMP8 veh N = 7, ORY-2001 0.96 mg/kg/day N = 3, 3.20 mg/kg/day N = 6) and (G) Ccl19 (N = 3 group). Data are ΔCp values relative to Gapdh. Means and SEM are represented. SAMR1 and SAMP8 vehicle groups were compared by Student t-test. Within SAMP8 cohorts, different drug treatments were compared by oneway-ANOVA with Dunnett and SNK post-hoc analysis. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 6.
S100A8 and S100A9 are differentially expressed in LOAD.
Re-examination of the expression of the orthologues of SAMP8 biomarkers in human prefrontal cortex in Control and LOAD samples from NCBI GEO GSE44770 [43]: (A) S100A8, (B) S100A9. Control: N = 101 and LOAD: N = 129 subjects. Samples are represented as Log2 (sample/reference sample). Mean ± SD are represented. Fold changes (FC) were calculated as 2^[average Log2(LOAD/Reference values)–average Log2(Control/Reference values)]. Normality was verified by D'Agostino & Pearson omnibus normality test. If data passed the normality test statistical significance was calculated by unpaired t-test, otherwise the Mann-Whitney test was applied. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Double-labeling immunofluorescence and confocal microscopy of paraffin sections with S100A9 (green, left), beta-amyloid (red, middle) and merged with DRAQ5TM stained nuclei (blue, right) in temporal cortex of patients with Alzheimer’s disease. Examples of S100A9-immunoreactive microglia localized in the vicinity of β-amyloid plaques. Bar = 25 μm. (D) S100A8/A9 levels in CSF samples from AD patients (N = 51) and healthy controls (N = 26), as determined by S100A8/A9 ELISA. Data are expressed as Log2 of the S100A8/A9 heterodimer concentration in pg/ml. Each dot represents one CSF donor and mean ± SD of each group are shown. Fold Change was calculated as the ratio of the average S100A8/A9 concentrations of the AD and control sample groups: average ([S100A8/A9 pg/ml]AD SAMPLES)/average([S100A8/A9 pg/ml]CONTROL SAMPLES). Normality was verified by D'Agostino & Pearson omnibus normality test and statistical significance was calculated by unpaired t-test (t(75) = 2.810; p = 0.0063). **p < 0.01.
Fig 7.
ORY-2001 does not have anxiolytic or sedative effects.
Effect of ORY-2001 on SAMP8 mice: (A) Locomotor activity as assessed by total distance travelled in the open field test (N = 8/group), (B) anxiety as assessed by the time spent in the open arms in the elevated plus maze (t(14) = 2.183; p = 0.0465; N = 8/group), Effect of ORY-2001 on rats in the isolation rearing model: (C) Locomotor activity (N = 18/group) and (D) anxiety in the Elevated Plus Maze (N = 18/group); Means and SEM are represented. SAMR1 and SAMP8 vehicle or Non Isolated and Isolated groups were compared by Student’s t-test. Within SAMP8 mice or Isolated groups, different drug treatments were compared by t-test or one way-ANOVA with Dunnett and SNK post-hoc analysis. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 8.
Inhibition of KDM1A with ORY-2001 remedies behavior alterations.
Effect of ORY-2001 on SAMP8 mice: (A) aggressive behavior as assessed by the total number of clinch attacks in the resident intruder test (F2,18 = 16.76; p < 0.0001; N = 5–8 males/ group) and (B) social interest as assessed by the time exploring the new mice in the three chamber test (t(19) = 6.008; p < 0.0001; N = 9–12 females/group). Effect of ORY-2001 on social behavior of rats in the isolation rearing model in the Resident Intruder test reflected by (C) the time spent without social interaction and (D) the number of active avoidances. ORY-2001 reduced both the time without social interaction (t(22) = 2.094; p = 0.048) and the number of social avoidances (F2,33 = 4.941; p = 0.013) (N = 12/group). Means and SEM are represented. SAMR1 and SAMP8 or Non Isolated and Isolated vehicle groups were compared by t-Test. Among the SAMP8 or Isolated groups, different drug treatments were compared by one way-ANOVA with Dunnett and SNK post-hoc analysis. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 9.
ORY-2001 restores the IEG stress response in the SAMP8 prefrontal cortex.
Gene expression changes in SAMP8 mice: (A) Top 5 differentially regulated genes (up and down) in the PFC of SAMP8 vs SAMR1 mice in the basal condition identified by microarray survey. Ccl19, Ccl27 on chr4 amplified in SAMP8 compared to SAMR1 mice are genotype controls and highlighted in grey. (B) Microarray survey performed on PFC tissue of vehicle- and ORY-2001-treated mice shows that ORY-2001 partially restores the GE pattern seen in SAMP8 mice to SAMR1 controls. GE changes in basal conditions following treatment with ORY-2001 (genes with an expression change Log2 > 0.5 or Log2 < -0.5 (1.4 fold up or down) at the dose of 0.96 mg/kg/day in relation to vehicle-treated SAMP8 mice. Genes are ranked according to the highest expression change in the ORY-2001 0.96 mg/kg/day condition. All values represent Log2(Fold Change). The survey was performed on pooled samples from each experimental group (N = 3–5 mice per group). Veh = Vehicle. (C) qRT-PCR analysis of Ccl19 expression performed on cortex tissue was used to verify the genotype of individual mice. Dots: basal; squares: stress cohorts. Data were normalized by GusB and are plotted as ΔCp values. Graphs represent mean ± SEM. Solid line: one-way ANOVA analysis of basal and stress levels using Dunnett’s multiple comparison test relative to the vehicle-treated SAMR1 condition. ***p < 0.001.(D) KDM1A TE analysis used to verify the treatment group of individual mice. Dots: basal; squares: stress cohorts. Graphs represent mean ± SEM. Solid line: one-way ANOVA analysis of basal and stress levels using Tukey's multiple comparison test. Dotted line: two-way ANOVA with Bonferroni post-hoc analysis. *p < 0.05, ***p < 0.001. The effect of ORY-2001 treatment on the expression in the prefrontal cortex, following exposure to stress of (E) Fos (F3,34 = 1.128; p = 0.3514 Interaction), (F) Npas4 (F3,30 = 2.312; p = 0.0961), (G) Calb2 (F3,30 = 7.361; p = 0.0008) and (H) Gng4 (F3,30 = 5.648; p = 0.0034). Basal: N = 3–5 per group; RI test: N = 3–8 per group). Dots: basal; squares: stress cohorts. All qRT-PCR data are plotted as -ΔΔCp values relative to the basal vehicle-treated SAMP8 group. Fold changes (FC) were calculated as 2^[average(stress -ΔΔCp values)—average(basal -ΔΔCp values)]. Fos data were normalized by Gapdh; Npas4, Calb2 and Gng4 by GusB. Graphs represent mean ± SEM. Solid line: two-way ANOVA with Bonferroni post-hoc analysis. Dotted line: one-way ANOVA analysis of basal or stress levels using Dunnett’s multiple comparison test relative to the vehicle-treated SAMP8 condition. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 10.
Biomarkers modulated by ORY-2001 are differentially expressed in LOAD.
Re-examination of the expression of the orthologues of SAMP8 biomarkers in human prefrontal cortex in Control and LOAD samples from NCBI GEO GSE44770 [44] reveals differential expression of immediate early genes (A) ERG1, (B) ERG2, (C) NPAS4 and (D) FOS, (E) CALB2, (F) GAD1, (G) GNG4 and (H) DOC2A. Control: N = 101, average age 62.1 years, male to female ratio = 4.3; LOAD: N = 129 subjects, average age 80.1 years, male to female ratio = 0.9. All samples are represented as Log2 (sample/reference sample) calculated from GSE44770 data. Mean ± SD are represented. Fold changes (FC) were calculated as 2^[average Log2(LOAD/Reference values)–average Log2(Control/Reference values)]. Normality was verified by D'Agostino & Pearson omnibus normality test. If data passed the normality test statistical significance was calculated by unpaired t-test, otherwise the Mann-Whitney test was applied. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 11.
KDM1A is recruited by transcription factors that modulate IEGs.
(A) Schematic representation of KDM1A-interacting proteins detected by mass spectrometry and by ELISA. The figure was generated using STRING. Blue line: interaction detected by ELISA on SH-SY5Y cells; red line: interaction detected by ELISA on human hippocampal tissue; orange line: interaction detected by mass spectrometry; grey line: protein-protein interaction displayed by STRING. (B) Schematic representation of KDM1A interaction ELISAs (left) and chemoprobe interaction ELISA (right). The KDM1A-containing multiprotein complexes are captured either by an antibody directed against one of its components or by the OG-881 chemoprobe; and detected using an antibody directed against KDM1A or another component of the complex. (C) Chemoprobe- interactor ELISAs in SH-SY5Y cells. OG-881 chemoprobe-bound KDM1A complex was captured on streptavidin coated plates and detected using with an HDAC1 or HDAC2 antibody. (D) ELISA on SH-SY5Y cells (N = 1; n = 3 [n = 2 for ZMYM3, ZNF217, REST, SRF, GFI1, HDAC1, HDAC2]). Protein amounts used were 2.5, 25, or 44 μg per well (light, mid, dark blue). (E) ELISA on human post-mortem hippocampal tissue from healthy controls (N = 1; n = 2). Protein amounts were 10, 30, or 80 μg per well (light, mid, dark red). Data in b-d represent Log2 values of the relative luminescence signal (RLU) normalized to the corresponding background. Mean ± SD is shown. The statistical significance was calculated by oneway-ANOVA with Dunnett multiple comparison test relative to the negative control condition (when n = 3) or by Student’s t-test relative to the negative control condition (when n = 2). *p < 0.05, **p < 0.01, ***p < 0.001.