The Melatonin Analog IQM316 May Induce Adult Hippocampal Neurogenesis and Preserve Recognition Memories in Mice

Abstract

Neurogenesis in the adult hippocampus is a unique process in neurobiology that requires functional integration of newly generated neurons, which may disrupt existing hippocampal network connections and consequently loss of established memories. As neurodegenerative diseases characterized by abnormal neurogenesis and memory dysfunctions are increasing, the identification of new anti-aging drugs is required. In adult mice, we found that melatonin, a well-established neurogenic hormone, and the melatonin analog 2-(2-(5-methoxy-1 H-indol-3-yl)ethyl)-5-methyl-1,3,4-oxadiazole (IQM316) were able to induce hippocampal neurogenesis, measured by neuronal nuclei (NeuN) and 5-bromo-2'-deoxyuridine (BrdU) labeling. More importantly, only IQM316 administration was able to induce hippocampal neurogenesis while preserving previously acquired memories, assessed with object recognition tests. In vitro studies with embryonic neural stem cells replicated the finding that both melatonin and IQM316 induce direct differentiation of neural precursors without altering their proliferative activity. Furthermore, IQM316 induces differentiation through a mechanism that is not dependent of melatonergic receptors (MTRs), since the MTR antagonist luzindole could not block the IQM316-induced effects. We also found that IQM316 and melatonin modulate mitochondrial DNA copy number and oxidative phosphorylation proteins, while maintaining mitochondrial function as measured by respiratory assays and enzymatic activity. These results uncover a novel pharmacological agent that may be capable of inducing adult hippocampal neurogenesis at a healthy and sustainable rate that preserves recognition memories.

Keywords: adult neurogenesis; long-term memory; melatonin; neural stem cells.

Fig. 1.

Effect of acute and chronic 2-(2-(5-methoxy-1H-indol-3-yl)ethyl)-5-methyl-1,3,4-oxadiazole (IQM316) or melatonin administration on adult hippocampal neurogenesis. Animals were treated with vehicle (Vhc), IQM316 (IQM), or melatonin (Mel) for 7 (acute) or 28 d (chronic). (A) Quantification of the percentage of 5-bromo-2′-deoxyuridine (BrdU) and neuronal nuclei (NeuN) double-labeling positive cells per dentate gyrus (DG) section upon acute administration. Both IQM316 and melatonin acute administrations induce neurogenesis, with being IQM316 more potent than melatonin. (B) Representative confocal images of BrdU (green) and NeuN (red) immunohistochemistry of the DG quantified in A. BrdU and NeuN double labeling is shown in yellow (merged images). Higher magnification insets of double labeled neurons are displayed on the right. Scale bar is 25 µm. (C) Quantification of the percentage of BrdU and NeuN double-labeling positive cells per DG section upon chronic administration. Both IQM316 and melatonin chronic administrations induce neurogenesis with similar efficacies. Data are mean ± SEM, n = 7 animals per group. **P < 0.01, ***P < 0.001, significantly different from vehicle, Bonferroni post hoc test.

Fig. 2.

Effect of acute and chronic 2-(2-(5-methoxy-1H-indol-3-yl)ethyl)-5-methyl-1,3,4-oxadiazole (IQM316) or melatonin administration on long-term recognition memory. Animals were treated with vehicle (Vhc), IQM316 (IQM), or melatonin (Mel) for 7 (Acute) or 28 d (Chronic). (A) Experimental design of drug administration and novel object recognition test analyzed in B and C. (B) Discrimination index (DI) quantified at day 29. Chronic IQM316 administration increased the DI, indicating that the animals recalled the object, whereas Fig. 2. (continued). melatonin administration did not alter it (C) Ratio of object exploration time as day 29 versus day 1, during the familiarization phase. IQM316-treated mice spent less time exploring at day 29, suggesting that they recalled the object from day 1. Melatonin-treated mice displayed similar exploration times at both days 1 and 29. (D) Experimental design of drug administration and novel object recognition test analyzed in E. (E) DI quantified at day 29, after acute and chronic administration. Neither acute nor chronic administration of IQM316 or melatonin modified short-term recognition memory. Data are mean ± SEM, n = 8 animals per group. *P < 0.05, **P < 0.01, significantly different from vehicle, Bonferroni post hoc test.

Fig. 3.

Effect of acute and chronic 2-(2-(5-methoxy-1H-indol-3-yl)ethyl)-5-methyl-1,3,4-oxadiazole (IQM316) or melatonin administration on hippocampal oxidative phosphorylation (OXPHOS) protein levels. Animals were treated with vehicle (Vhc), IQM316 (IQM), or melatonin (Mel). Quantitative analysis of the effect of acute (A) and chronic (C) administrations on hippocampal OXPHOS protein levels. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalization. Representative Western blots for acute (B) and chronic (D) administrations quantified in A and C, respectively. Acute administration of either IQM316 or melatonin significantly increased subunit B of complex II and complex I subunit (COX I) protein levels but reduced COX IV and ATP-5β. Nonetheless, chronic IQM316 or melatonin administration only increased the protein levels of NDUFB8. Data are mean ± SEM, n = 8 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from vehicle, Bonferroni post hoc test.

Fig. 4.

Effect of acute 2-(2-(5-methoxy-1H-indol-3-yl)ethyl)-5-methyl-1,3,4-oxadiazole (IQM316) or melatonin administration on mitochondrial biogenesis. Animals were treated with vehicle (Vhc) or IQM316 (IQM) or melatonin (Mel). (A) Quantitative analysis of the complex I subunit (COX I)/COX IV protein level ratio upon acute and chronic administrations. Acute administration of either IQM316 or melatonin increased the COXI/COXIV ratio, suggesting activation of mitochondrial DNA replication or translation. Chronic administration had no effect on the ratio. (B) Quantification of the mitochondrial DNA/nuclear DNA ratio upon acute and chronic administrations. Acute administrations increased the ratio, whereas chronic administrations did not. (C) Quantification of relative mRNA expression levels of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), mitochondrial transcription factor A (Tfam), and nuclear respiratory factor 1 (NRF-1) upon acute administration. Expression levels were normalized to hypoxanthine guanine phosphoribosyltransferase and relative to vehicle. Acute administration of either compound did not activate the expression mitochondrial biogenesis genes. (D) Quantitative analysis and representative Western blots of Voltage-dependent anion-selective channel 1 (VDAC1) protein levels upon acute administration. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the normalization control. Acute administration of either compounds did not alter the mitochondrial protein levels, indicating that mitochondrial biogenesis was not affected. Data are mean ± SEM, n = 8 animals per group. *P < 0.05, **P < 0.01, significantly different from vehicle, Bonferroni post hoc test.

Fig. 5.

Effect of 2-(2-(5-methoxy-1H-indol-3-yl)ethyl)-5-methyl-1,3,4-oxadiazole (IQM316) or melatonin on embryonic neural stem cell (NSC) proliferation and differentiation. (A, B) Dose–response effect of IQM316 or melatonin on cell proliferation, showing no effect of either compound. (A) Cell proliferation quantified by XTT assay. (B) Cell proliferation quantified by 4′,6-diamidino-2-phenylindole (DAPI) staining and cell counting. (C, D) Quantification of doublecortin (Dcx) relative mRNA expression levels under differentiation conditions. Expression levels are normalized to hypoxanthine guanine phosphoribosyltransferase and relative to the vehicle. (C) Dose–response effect of IQM316 or melatonin on Dcx relative mRNA expression levels. Both IQM316 and melatonin treatments increase Dcx mRNA levels, indicating activation of neuronal differentiation and therefore neurogenic potential. (D) Cells were differentiated and treated with either IQM316 or melatonin 10⁻³ µM, in the presence or absence of luzindole (0.2 µM). The MT1 and MT2 antagonist luzindole was able to fully block neuronal differentiation induced by melatonin but did not block the effect of IQM316. (E) Representative confocal images of Dcx (red) and DAPI (blue) immunocytochemistry, with higher inset below showing the detailed morphology (Dcx, black) of NSCs differentiated in the presence of vehicle, IQM316 or melatonin 10^–3 µM. Scale bar is 20 µm. (F) Cells under the same conditions as described in D. Quantification of dendritic density. NSCs differentiated in the presence of either IQM316 or melatonin display a higher number of dendritic processes. The effect of melatonin is completely blocked by luzindole, whereas the IQM316-induced effect is not. IQM316 induces neuronal differentiation independently of melatonergic receptors MT1 and MT2. Data are mean ± SEM, n = 3 independent experiments. *P< 0.05, **P < 0.01, ***P < 0.001, significantly different from vehicle, Bonferroni post hoc test.