Results - Ulsan National Institute of Science and Technology

Figure III-1-1. Adult hippocampal neurogenesis was modulated by DSCR1.

(A) Representative image with the BrdU and Ki67 to detect dividing cells in the subgranular zone of the DG. Magnified images of the white squares are displayed in the bottom panels. Double-positive cells (White arrowheads). (B) Quantification analysis of BrdU⁺ and Ki67⁺ cells DG in DSCR1 mutants compared to WT mice. (C) Representative image of immunohistochemistry staining of BrdU and DCX.

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(D) Differentiating cells are quantified by the counting BrdU-DCX double-positive cells (E) Immunofluorescence staining images with BrdU and NeuN (F) Quantification of maturation of progenitor cells (BrdU⁺ and NeuN⁺). A total of 24 sections were collected from a hippocampal area at 40 μm thickness. N = 3 mice for each strain. Data are shown as Mean ± SEM and statistical significance was analyzed by one way ANOVA followed by Bonferroni post hoc test, *P< 0.05, **P< 0.01.

Figure III-1-2. DSCR1 has a specific function in learning and memory

(A) Morris water maze (MWM) test was performed and escape latency was shown during platform trials training days in DSCR1 mutants and WT mice (B) During the 30-second of probe trial, the total target quadrant was recorded and analyzed. (C) Frequency of platform crossings in the probe trial was calculated. (D) Open field was estimated in all mice. N = 10 WT mice, N = 12 DSCR1 KO mice, N = 12 DSCR1 TG mice. Mean ± SEM. One-way ANOVA, Bonferroni post hoc test, *P< 0.01, **P<

0.001.

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Figure III-1-3. DSCR1 controls proliferation of progenitor neurons

(A) Representative bright field images of NSCs from DSCR1 mutants and WT mice. The proliferation and self-renewal capacity of primary (top panels) and secondary (lower panels) were measured. (B-E) DSCR1 mutants showed a decreased number and size of primary and secondary neurospheres. N = 3 mice for each condition. Mean ± SEM. One-way ANOVA, Bonferroni post hoc test, *P< 0.05, **P<

0.01. (F) Differentiation potential of established NSCs was confirmed by the immunoreactivity for Tuj1, GFAP, and Olig2 in DSCR1 mutants and WT mice. Scale bar, 100 μm.

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Ⅲ-1-2. DSCR1 expression during adult hippocampal neurogenesis

Next, different types of SGZ cells were isolated by fluorescence-activated cell sorting (FACS) ⁵², followed by qRT-PCR to characterize the expression pattern of DSCR1 during adult hippocampal neurogenesis (Fig Ⅲ-1-4). We first analyzed proliferating neurons through injection of 5-ethynyl-2’- deoxyuridine (EdU) to label proliferating cells in the hippocampus. We then sorted cells based on the known cell markers with EdU from hippocampi (Fig Ⅲ-1-4A-F). qRT-PCR analysis revealed that the DSCR1 was highly expressed in sorted NeuN expressing mature neurons, whereas we observed low expression of DSCR1 in the DCX expressing neuroblasts compared to that of SOX2 expressing neural progenitor cells (Fig Ⅲ-1-4G).

Figure III-1-4. The expression pattern of DSCR1 is determined by FACS

(A-F) Flow cytometry analysis of single neurons that were isolated from the hippocampus. Each population was gated and isolated by fluorescent labeling by click chemistry with markers for a particular cell type (SOX2-EdU; neural progenitor cells, DCX-EdU; neuroblasts, NeuN-EdU; mature neurons) for RNA analysis, N=5 experiments. (G) DSCR1 mRNA levels were determined by qRT- PCR using sorted neurons as described above flow cytometry gating. Quantitative gene expression data were normalized by GAPDH. N = 4 independent experiments. Mean ± SEM. One-way ANOVA followed by Bonferroni post hoc test, *P< 0.05, **P< 0.01.

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Ⅲ-1-3. DSCR1 controls the expression of miR-124

Previous work has shown that miR-124 is the most abundant microRNA in the adult brain and increased during the process of adult neurogenesis (AN) ⁵⁴. Moreover, adjustment of the expression of miR-124 induced the AN defects in the SVZ, located along the lateral ventricle ^{55, 56}. However, the exact mechanism concerning how miR-124 is regulated during the development of the AN has not been fully investigated. Moreover, DSCR1 also fundamentally functioned in normal AHN (Fig Ⅲ-1-1). Hence, we tested that those two genes would be involved in similar functions or biological processes during adult hippocampal neurogenesis.

We first validated the expression of different forms of miR-124 in the hippocampi, including pri- miRNA and pre-miRNA/mature using qRT-PCR (Fig Ⅲ-1-5A) ⁵⁷. We found that all three forms of miR- 124 were markedly increased in DSCR1 null mice, but upregulation of DSCR1 showed reduced gene expression of those forms. These results demonstrated that DSCR1 is not involved in the processing of miR-124, but DSCR1 can regulate the miRNA-124 transcription. Next, we assessed the impact of DSCR1 on miR-124 promoter activity. We used Neuro2A cells co-transfected with the DSCR1shRNA or DSCR1 transgene and luciferase reporters containing the control of the miR-124 promoter. Our reporter assays revealed that DSCR1 reduction increases luciferase activity, while decreased activity was detected in transfected cells overexpressing DSCR1 (Fig Ⅲ-1-5B). Together, these findings demonstrate that DSCR1 is an important factor for modulating promoter strength of miR-124.

Next, we examined the possibility that DSCR1 could alter the methylation state in the promoter regions of miRNA-124 using bisulfite sequencing. Interestingly, a significant proportion of CpGs sites were completely demethylated in the DSCR1 KO hippocampus, while DSCR1 overexpression increased the number of CpG methylation sites compared to the methylation status of the wild-type littermates (Fig

Ⅲ-1-5C). Furthermore, we evaluated altered methylation level by DSCR1 was directly associated with the promoter activity of miR-124. We induced site-specific mutation at the CpG sites in the normal miR-124 promoter to block methylation. The luciferase activities of the mutated promoter construct were increased, comparable to when DSCR1 was knocked down in Neuro2A cells (Fig Ⅲ-1-5D). We also induced mutations at all methylation sites residing in the hippocampus of DSCR1 TG mice. We found significantly improved miR-124 promoter activity of mutated luciferases compared to when DSCR1 was overexpressed alone (Fig Ⅲ-1-5E). However, we could not rule out the possibility that other CpGs are located in the promoter elsewhere and DNA methylation happened. These findings show that DSCR1 regulates the expression of miR-124 via controlling the DNA methylation level of the miR- 124 promoter.

Figure III-1-5. miR-124 was epigenetically regulated by DSCR1-mediated methylation on the miR-124 promoter.

(A) Effects of DSCR1 on miR-124 expression. N = 3 mice for each strain, *P< 0.01. (B) Neuro2A cells were harvested for luciferase activity, and miR-124 promoter activity was measured with DSCR1 reduction or overexpression. N = 3 all sets of replicates, *P< 0.05, **P< 0.01. (C) Differentially methylated CpG sites are indicated in the two different regions of the miR-124 promoter using bisulfite sequencing. The open and closed circles respectively represent unmethylated and methylated CpG sites.

(D) miR-124 promoter activity with C-to-T transition mutations at methylation sites (31 and 58) displays a similar luciferase activity to that of Neuro2A cells containing DSCR1shRNA. N = 3 all sets of replicates, *P< 0.01. (E) miR-124 promoter at all methylation sites with the directed mutation was increased, whereas overexpressed DSCR1 decreased the luciferase signal intensity. N = 3 all sets of replicates. (A), (B), (D), (E) Mean ± SEM. One-way ANOVA, Bonferroni post hoc test, *P< 0.05, **P<

0.01.

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Ⅲ-1-4. DSCR1 controls TET1 gene expression at the transcriptional level

We then examined how DSCR1 influences the status of methylation of the miR-124 promoter region.

Notably, any conserved domain of specific demethylases or methyltransferases has not been identified in DSCR1. Hence, we tested that DSCR1 could indirectly modulate other enzyme activity that affects DNA methylation level. Since TET1 is a demethylase and it has been recently reported that neuronal TET1 regulates adult hippocampal neurogenesis ¹¹, we questioned whether DSCR1 could control TET1 expression and function. First, we tested whether DSCR1 could modulate TET1 transcription by performing qPCR of hippocampal mRNA. We confirmed that DSCR1 knockout mice significantly increases Tet1 levels, but Tet1 expression substantially decreased in DSCR1 transgenic mice (Fig Ⅲ-1- 6A). However, pre-mRNA levels of TET1 were not changed in both mice (Fig Ⅲ-1-6B). Then, luciferase reporter assay indicated that the TET1 promoter activity did not show any change with different levels of DSCR1 (Fig Ⅲ-1-6C). We lastly investigated DSCR1 regulates TET1 mRNA stability. We applied actinomycin D to N2A cells with reduced or overexpressed DSCR1 to inhibit transcription and monitored the TET1 mRNA decay for 15 hours. We found the decay rate of TET1 transcripts seem to be similar during the same period with irrespective of the expression of DSCR1, suggesting DSCR1 does not impact the TET1 transcripts stability (Fig Ⅲ-1-6D). Collectively, it is therefore possible TET1 mRNA transcription is regulated by DSCR1 but not relevant to the processing or the stability of its transcripts.

Figure III-1-6. DSCR1 modulates TET1 transcription

(A) Quantitative real-time PCR analysis of Tet1 transcript levels in the hippocampi of wild type, DSCR1 mutants. (B) qRT-PCR analysis of pre-mRNA levels of TET1 in WT and DSCR1 mutants. (C) TET1 promoter activities were not attenuated by co-expression with the knockdown or overexpressed DSCR1 in N2A cells. (D) The decay of TET1 mRNA transcripts was monitored by qRT-PCR (qPCR). N=3 for each condition. GAPDH was used for normalization. Mean ± SEM. One-way ANOVA, Bonferroni post

hoc test, *P < 0.05.

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Ⅲ-1-5. DSCR1 mediates the TET1 pre-mRNA splicing

Previous findings suggest that DSCR1 may control the TET1 pre-mRNA splicing. Interestingly, DSCR1 has RNA recognition motif (RRM), but its function has not been properly defined ⁵⁸. First, we evaluated whether DSCR1 could interact with the introns of TET1 directly. TET1 contains 12 introns and 13 exons. We generated biotin-labeled RNAs of TET1 8th exon and 8th and 9th introns randomly chosen using in vitro transcription for this test. Neuro2A cell lysates were prepared and incubated with synthesized biotin-labeled RNAs, then RNAs were affinity-precipitated using streptavidin magnetic beads. We found that DSCR1 interacts with the TET1 intron 8 and 9 but does not bind to TET1 exon 8 (Fig Ⅲ-1-7A). During spliceosome assembly, U1 small nuclear ribonucleoprotein (snRNP) is required for splicing of pre-mRNA, followed by the stable binding of U2 snRNP to form the pre-spliceosome.

We tested whether DSCR1 influences the interaction with 8th intron of TET1 in the presence of the U1/U2 snRNA ⁵⁹. Interestingly, the strength of the interaction between DSCR1 and TET1 intron 8 decreased by increasing levels of U1 and U2 snRNA (Fig Ⅲ-1-7A), suggesting that DSCR1 could affect pre-mRNA splicing of TET1 through the inhibition of spliceosome assembly at the TET1 introns. Next, we tested the ability of the RNA recognition motif (RRM) domain of DSCR1 that interacts with introns of TET1. We transfected DSCR1 containing RRM or ΔRRM domain encoding with a Flag-tag and evaluated its interaction ability to the intron of TET1. We observed the impaired abilities of DSCR1 without the RRM domain to bind to TET1 intron (Fig Ⅲ-1-7B). Since DSCR1 expression controlled the splicing by binding to TET1 introns (Fig Ⅲ-1-7C), we further verified our results using the separated luciferase reporter containing TET1 intron 8 and intron 9 (Fig Ⅲ-1-7D). Notably, we confirmed that knockdown of DSCR1 dramatically enhances luciferase activity, while overexpressed DSCR1 showed decreased signal strength in N2A cells. Also, we designed the intron 3 and 4 of GAPDH integrated into luciferase reporter as control (Fig Ⅲ-1-7D) and identified that those luciferase intensities were not changed depending on the presence of DSCR1. These results suggested that DSCR1 precisely alter the splicing of the mRNAs of TET1. Moreover, IHC results showed that TET1 expression level is increased in DSCR1 knockout mice, whereas a significant reduction was observed in DSCR1 transgenic mice (Fig Ⅲ-1-7E). We next examined whether the direct impact of altering TET1 levels on miR-124 expression. Knockdown of TET1 significantly reduced the expression of miR-124 in Neuro2A cells (Fig Ⅲ-1-8A-C) and methylation levels of the miR-124 promoter were enhanced with the presence of TET1 shRNA, suggesting that downregulation of TET1 directly modulated expression of miR-124 transcript. (Fig S4D, E).

Figure III-1-7. DSCR1 controls the splicing of TET1

(A) Identification of DSCR1 binding to TET1 introns. Western blot demonstrated decreased binding affinity between DSCR1 and the TET1 8th intron, while U1 and U2 snRNA were dose-dependently increased. (B) Deletion of RRM in DSCR1 affected its binding ability with the intron of TET1.

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(C, D) Illustration depicting the intron-containing (Luc-intron) luciferase reporters with TET1 intron 8, 9 and GAPDH intron 3, 4. N2A cells were transfected with those luciferase reporters with DSCR1 reduction or overexpression, and relative luciferase activities were measured. N = 3 independent experiments, *P< 0.05, **P< 0.01. ***P< 0.001. (E) Expression of TET1 was analyzed at the DG of hippocampus in WT and DSCR1 mutant mice. N = 3 mice for each strain, *P< 0.05, **P< 0.01. (C), (D), (E) Mean ± SEM. One-way ANOVA, Bonferroni post hoc test.

Figure III-1-8. TET1 regulates miR-124 expression

(A, B) The silencing efficiency of TET1 was evaluated immunoblotting and qRT-PCR (C) miR‑124 expression was significantly lower with TET1 shRNA. (D) DNA methylation levels of the miR-124 promoter with control shRNA or TET1 shRNA. (E) A graph of % DNA methylation is shown. N = 3 independent experiments. Mean ± SEM. One-way ANOVA followed by Bonferroni post hoc test, *P<

0.05.

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Ⅲ-1-6. TET1 reduction prevents impaired adult neurogenesis of DSCR1 knockout hippocampus As shown that defects of adult hippocampal neurogenesis in DSCR1 KO mice have been linked that expression of TET1 was increased in the hippocampus, we investigated restoration of TET1 expression in the adult hippocampus could rescue the deficits in adult neurogenesis on DSCR1 knockout background. The lentiviral particles encoded by GFP-TET1 shRNA were unilaterally injected at one side of the hippocampal DG, and lentivirus-expressing GFP and random shRNA was injected contralaterally (Fig Ⅲ-1-9A). Three weeks after virus injection, we found significantly larger number of double-positive cells for GFP and Sox2, a well-established marker of neural stem and progenitor cells, in the DG injected with GFP-labeled lentivirus encoding TET1 shRNA than the contralateral DG injected with GFP control (Fig Ⅲ-1-9B, C), indicating that negatively regulated expression of TET1 mitigated AHN dysfunction in the dentate gyrus of DSCR1 null mice. Taken collectively, these results strengthened the idea that DSCR1 mediates the AHN by controlling TET1 expression.

Figure III-1-9. AHN is recovered by TET1 knockdown in DSCR1 null mice

(A) Timeline of experiments and injections of stereotaxic lentiviral GFP-TET1 shRNA or control shRNA are mapped. Two-month-old male DSCR1 KO mice and wild-type mice were used for this experiment. (B) The subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) is analyzed with staining of SOX2⁺ and GFP⁺ cells 3 weeks after viral injection to identify neural progenitor cells (C) Quantification analysis revealed that the percentage of Sox2-GFP cells/total GFP positive cells in GFP- TET1 shRNA injected DSCR1 KO mice is recovered compared to that of GFP-control shRNA infected wild-type cells (Scale bars represent 10μm) N = 3 mice for each strain. Mean ± SEM. One-way ANOVA, Bonferroni post hoc test, *P< 0.05, **P< 0.01.

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Ⅲ-1-7. Normalized DSCR1 in Ts65Dn mouse alleviated deficits of adult neurogenesis and cognition defects.

Previous studies found that defective adult hippocampal neurogenesis in the Ts65Dn mouse model of Down syndrome ^{60, 61}. Also, DNA methylation was globally perturbed, and TET1 expression was changed in Down syndrome patients ^{62, 63}. Those studies and our previous results prompted us to test that normalized DSCR1 expression can rescue adult neurogenesis dysfunction and cognitive defects observed in Ts65Dn mice. First, we crossed Ts65Dn mice with DSCR1 KO mice to normalize DSCR1 expression in these model mice (Fig Ⅲ-1-10A). Next, we assessed AHN and evaluated spatial cognitive abilities in Diploid, Ts65Dn and Ts65Dn/DSCR1^+/– mice (Fig 6B-J). After 1 day of BrdU injection, BrdU and Ki67 cells were analyzed to calculate the number of neural progenitor cells (Fig Ⅲ-1-10B, C). We observed that Ki67⁺BrdU⁺ double-labeled cells were decreased in DS mice. By contrast, about two-fold increased cell numbers were shown in Ts65Dn/DSCR1^+/– mice compared with control mice (Fig Ⅲ-1-10B, C). We concluded that higher DSCR1 levels induce the defects of AHN in DS mice.

Since we identified that DSCR1 is an upstream regulator of the TET1 and miRNA-124 pathway by controlling AHN, we examined altered TET1 and miRNA-124 expression could be recovered in Ts65Dn/DSCR1^+/– mice. Interestingly, we found Ts65Dn/DSCR1^+/– show normal expression levels of miRNA TET1 and miRNA-124 (Fig Ⅲ-1-10D-F). Notably, we also observed enhanced the learning and memory ability in Ts65Dn/DSCR1^+/– mice (Fig Ⅲ-1-10G-J). Collectively, we concluded that altering DSCR1 levels in Ts65Dn alleviates the impaired adult hippocampal neurogenesis and spatial learning ability defects through TET1 and miRNA-124 pathway.

Dalam dokumen Ulsan National Institute of Science and Technology (Halaman 41-81)