WO2020036372A1

WO2020036372A1 - Use of dscr1 regulating adult neurogenesis

Info

Publication number: WO2020036372A1
Application number: PCT/KR2019/010023
Authority: WO
Inventors: 민경태; 최치열
Original assignee: 울산과학기술원
Priority date: 2018-08-13
Filing date: 2019-08-08
Publication date: 2020-02-20
Also published as: KR102076475B1

Abstract

The present invention relates to a use of Down syndrome critical region 1 (DSCR1) regulating adult neurogenesis and, in particular, has discovered that DSCR1 regulates miRNA-124 and TET1 which are known to be involved in the process of adult neurogenesis. It is expected that as DSCR1 regulates the miRNA-124/TET1 regulatory axis, DSCR1 can be usefully employed in treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, in which adult neurogenesis is impaired, and Down syndrome, which is a neurodevelopmental disorder.

Description

Use of DSCR1 to Regulate Adult Neurogenesis

The present invention relates to the use of Down syndrome critical region 1 (DSCR1) to regulate adult neurogenesis.

Adult neurogenesis is the process by which new neurons are made from adult neural precursors, localized in the dentate gyrus of the adult brain hippocampus and in the subventricular zone of the lateral ventricle. Get up. Newly formed neurons play an important role in the formation of the hippocampus's learning and memory, and recently, the extent of adult neurogenesis has been reduced in neurodegenerative diseases such as Alzheimer's and Parkinson's. However, little is known about which physiological and pathological mechanisms regulate adult neurogenesis.

Down syndrome (DS) is a congenital disorder most commonly occurring with a probability of about 1 in about 800 people, and is a complex disorder caused by unbalanced gene mass in part or all of chromosome 21. Down syndrome is caused by the presence of an extra chromosome on chromosome 21. The specific genes that cause intellectual disability in patients with Down syndrome are still unclear. Down syndrome critical region 1 (also called DSCR1, or RCAN1) is located on human chromosome 21 and is highly expressed in the brain and is particularly abundant in hippocampal neurons. DSCR1 belongs to an evolutionarily conserved family of calcineurin (CaN) inhibitors called calcipressins, including nebula in Drosophila, and DSCR1 in mice and humans. It is known that DSCR1 is essential for learning and memory, supporting that DSCR1 may play a major role in intellectual disability in patients with Down syndrome.

MicroRNAs (miRNAs) are small RNAs (~ 22 nucleotides) that do not encode proteins and are involved in many biological processes. miRNAs form incomplete or complete base pairs with complementary sequences in 3 'untranslated regions (UTRs) of target mRNAs, acting as post-transcriptional regulators of gene expression. Each miRNA can target hundreds of thousands of individual target RNAs, indicating that regulation of miRNAs expression is important for regulating several target genes. One of the most microRNAs in the brain is microRNA-124 (miR-124), which is involved in many biological processes such as mRNA splicing, development, synapse formation, and neuronal differentiation. However, how the transcription of miR-124 is regulated is unknown.

DNA methylation by DNA methyltransferase is known to regulate transcription of many genes. In contrast, Ten-eleven translocation methylcytosine dioxygenase (Tet1) enzymes play a major role in the demethylation of methylated DNA through oxidation of 5-methylcytosine (5mC). Recently, Tet1 has been reported to be involved in the regulation of adult hippocampal neurogenesis. Tet1 deficiency inhibits proliferation of neural stem cells (NSCs) and results in DNA hypermethylation of genes involved in proliferation and neuroprotection, including Galanin, Ng2 and Neirglobin (Ngb), which in turn result in nerve Results in reducing outbreaks. In addition, Tet1 KO mice exhibit impaired spatial learning and memory, indicating that Tet1-mediated DNA demethylation is important in regulating learning and memory and is also involved in the regulation of adult neurogenesis.

An object of the present invention is to provide a reagent composition for controlling in vitro miR-124 or TET1 expression comprising Down syndrome critical region 1 (DSCR1) protein as an active ingredient.

It is also an object of the present invention to provide a method of regulating miR-124 or TET1 expression comprising treating neurons with DSCR1 protein in vitro.

The present invention provides a reagent composition for controlling in vitro miR-124 expression comprising Down syndrome critical region 1 (DSCR1) protein as an active ingredient.

The present invention also provides a reagent composition for controlling in vitro TET1 expression comprising the DSCR1 protein as an active ingredient.

The present invention also provides a method for regulating miR-124 expression comprising treating neurons with DSCR1 protein in vitro.

The present invention also provides a method for regulating TET1 expression comprising treating neurons with DSCR1 protein in vitro.

The present invention relates to the use of Down syndrome critical region 1 (DSCR1) for regulating adult neurogenesis, in particular the regulation of miRNA-124 and TET1, which DSCR1 is known to be involved in the adult neurogenesis process. By regulating the miRNA-124-TET1 regulatory axis, DSCR1 may be useful for the treatment of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease and Down's syndrome with neurodevelopmental disorders. It is expected to be.

1 shows adult neurogenesis of damaged hippocampus in the dentate gyrus of mice lacking DSCR1 and overexpressed mice. BrdU and Ki67 double positive cells (n = 3 pairs of mice) in DSCR1 deficient mice and overexpressed mice. Values were expressed as mean ± sem and analyzed by one-way ANOVA. * P <0.05; ** P <0.01.

2 shows that DSCR1 regulates miR-124 expression by inhibiting demethylation of the miR-124 promoter. (A-C) The amounts of pri-, pre- and mature forms of miR-124 in the hippocampus of WT, DSCR1 TG and DSC1 KO mice were analyzed by qRT-PCR (n = 3 pairs of mice). *** P <0.001. ** P <0.01. (D) The results of the luciferase analysis of the luciferase gene linked to the promoter of miR-124 are shown. The results are expressed as mean ± SD (n = 3). *** P <0.001. ** P <0.01. * P <0.05. (E) Promoter methylation in miR-124 promoter of WT, DSCR1 TG, DSCR1 KO mice is confirmed by gene-specific bisulfite sequencing (n = 6 pairs of mice). White or black circles represent unmethylated and methylated CpG sites, respectively.

3 shows that DSCR1 downregulates TET1. (A) The amount of TET1 in the hippocampus of WT, DSCR1 TG and DSC1 KO mice was analyzed by qRT-PCR (n = 3 pairs of mice). ** P <0.01. * P <0.05. (B) The results of counterstaining of DET1 labeled cells with DAPI in hippocampus. (C) Relative TET1 expression is quantified in dentate gyrus (DG) of WT, DSCR1 TG and DSCR1 KO mice (n = 3 pairs of mice). (D) The results of the luciferase analysis of the luciferase gene linked to the promoter of TET1 are shown. The results are expressed as mean ± SD (n = 3). NS, no significance. (E) The results of the luciferase analysis of the luciferase gene linked to the promoter of miR-124 are shown. The results are expressed as mean ± SD (n = 3). * P <0.05.

4 shows that DSCR1 specifically binds to TET1 pre-mRNA and regulates mRNA splicing. (A) Pre-mRNA of TET1 was analyzed by qRT-PCR in hippocampus of WT, DSCR1 TG and DSC1 KO mice (n = 3 pairs of mice). NS, no significance. (B) N2a cells transfected with or without DSCR1 were treated with actinomycin D, and total RNA was extracted at the indicated time and analyzed by qRT-PCR. (C) Biotinylated RNA / protein pull-down assay results are shown. (D) Competitive assay is shown by mixing N2a cell extracts with biotinylated or unbiotinylated TET1 exon1, intron1.

Specifically, the composition can reduce the activity of the miR-124 promoter, downregulating miR-124 expression, and reducing the activity of the miR-124 promoter inhibits demethylation of the CpG site in the miR-124 promoter region. It may be.

Specifically, the composition can downregulate TET1 expression by regulating TET1 pre-mRNA splicing.

Specifically, the method can reduce the activity of the miR-124 promoter, downregulating miR-124 expression, and reducing the activity of the miR-124 promoter inhibits demethylation of the CpG site in the miR-124 promoter region. It may be.

Specifically, the method can downregulate TET1 expression by regulating TET1 pre-mRNA splicing.

In the present invention, the DSCR1 protein may be Gene ID 1827, but is not limited thereto.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<실시예 1> 성체 신경 발생 과정에서의 DSCR1의 역할Example 1 Role of DSCR1 in Adult Neurogenesis

Adult neurogenesis is the proliferation, differentiation, maturation and maturation of adult neuronal stem cells and progenitor cells, and new neurons connect to existing neurons. It involves a process of integration that forms. The present inventors examined the proliferation of dentate hepatic hepatocytes through bromodeoxyuridine (BrdU) injection, and found that the proliferation was reduced in mice lacking DSCR1 (DSCR1 KO mice) and overexpressed mice (DSCR1 TG mice). It was. This shows that normal DSCR1 expression is essential for normal proliferation of neural stem cells (FIG. 1).

<실시예 2> miR-124에 대한 메틸화-의존 침묵을 유발하는 DSCR1Example 2 DSCR1 Induces Methylation-Dependent Silence on miR-124

microRNAs are non-coding RNAs that participate in a variety of biological processes, and several microRNAs are involved in adult neurogenesis. Among them, miR-124 is particularly expressed in all areas of the brain and is known to play a leading role in neurodevelopment. The inventors found that DSCR1 and miR-124 interacted with each other. The inventors confirmed that miR-124 binding site was present at 3′UTR of DSCR1. To confirm that DSCR1 downregulates miR-124, we performed qRT-PCR using hippocampus of DSCR1 TG and DSCR1 KO mice. In DSCR1 TG, pri-miR-124, pre-miR-124 and mature miR-124 expression was shown to be reduced. In contrast, the expression of pri-miR-124, pre-miR-124, and mature miR-124 was increased in DSCR1 KO (FIGS. 2A-2C). This indicates that DSCR1 may be involved in transcriptional regulation without being involved in the miR-124 maturation process. To confirm that DSCR1 regulates the transcription of miR-124, we performed transcriptional analysis in N2A cells using a luciferase construct comprising the miR-124 promoter. Overexpression of DSCR1 significantly reduced the activity of the miR-124 promoter, whereas reduction of DSCR1 enhanced the activity of the miR-124 promoter. These results support that DSCR1 regulates the transcription of miR-124 (FIG. 2D). On the other hand, the present inventors estimated that methylation sites may exist in miR-124, and DSCR1 may regulate it. To confirm this, we have identified epigenetic changes in the miR-124 promoter region. We performed bisulfite sequencing to confirm the methylation status of CpGs in the miR-124 promoter region. Compared with the control, it was confirmed that methylation was significantly increased in CpGs in the miR-124 promoter region of DSCR1 TG. However, most of the cytosine in the DSCR1 KO promoter was demethylated cytosine (FIG. 2E).

<실시예 3> TET1을 음성적으로 조절하는 DSCR1Example 3 DSCR1 Negatively Adjusts TET1

Previous studies using mice lacking TET1 have shown that neural stem cell proliferation was reduced by over-methylation of genes associated with neural stem cell proliferation and neuroprotection, thereby reducing adult neurogenesis. DNA methylation in Down syndrome patients is known to occur differently than normal patients, and this difference seems to cause neurogenesis.

Interestingly, DSCR1 specifically affected TET1 expression. We confirmed changes in TET1 levels in the hippocampus of DSCR1 TG and DSCR1 KO mice via qRT-PCR (FIG. 3A). Next, the inventors confirmed the protein level of TET1 in the dentate gyrus (DG) of DSCR1 TG and DSCR1 KO mice by immunohistochemistry. Consistent with TET mRNA levels, TET1 protein levels were also negatively associated with DSCR1 levels (FIGS. 3B and 3C). However, DSCR1 did not change the promoter activity of TET1 (FIG. 3D). The inventors then confirmed whether TET1 regulates miR-124 expression. Knocking down DSCR1 with TET1 reversed the increased miR-124 promoter activity (FIG. 3E). Taken together, the results support that DSCR1 modulates TET1 to regulate miR-124.

However, we confirmed via qRT-PCR that, in the hippocampus of DSCR1 TG and DSCR1 KO mice, DSCR1 did not affect TET1 pre-mRNA expression levels (FIG. 4A). To confirm how DSCR1 regulates TET1 expression, we first identified the effect of DSCR1 on the stability of TET1 mRNA. We treated 20% of the actinomycin D (act D), a transcription inhibitor, in N2A cells. TET1 mRNAs were reduced to a similar extent as the control (FIG. 4B). Next, it was confirmed that DSCR1 regulates TET1 pre-mRNA splicing. In the end, we produced biotin-labeled TET1 pre-mRNAs. Mibiotinylated or biotinylated TET1 exon1, intron1 and intron2 were treated with N2A cell lysate and pulled down to streptavidin-conjugated beads. DSCR1 binds directly to TET1 intron1 and intron2, but not to TET1 exon1 (FIG. 4C). To determine whether DSCR1 influences binding affinity directly or indirectly in the splicing process of TET1 pre-mRNA, the interaction between DSCR1 and TET1 intron1 in the presence of U1 snRNA and U2 snRNA was observed. Intron1 and DSCR1 binding intensity decreased with increasing U1 snRNA and U2 snRNA (FIG. 4D). Taken together, the results support that DSCR1 influences splicing by directly competing for the splicing process.

Claims

A reagent composition for controlling in vitro miR-124 expression comprising a Down syndrome critical region 1 (DSCR1) protein as an active ingredient.
The reagent composition of claim 1, wherein the composition reduces the activity of the miR-124 promoter to downregulate miR-124 expression.
The reagent composition of claim 2, wherein the decrease in activity of the miR-124 promoter inhibits demethylation of CpG sites in the miR-124 promoter region.
In vitro TET1 expression control reagent composition comprising the DSCR1 protein as an active ingredient.
The reagent composition of claim 4, wherein the composition modulates TET1 pre-mRNA splicing to downregulate TET1 expression.
A method of modulating miR-124 expression comprising treating neurons with an DSCR1 protein in vitro.
The method of claim 6, wherein the method reduces the activity of the miR-124 promoter to downregulate miR-124 expression.
8. The method of claim 7, wherein the reduced activity of the miR-124 promoter inhibits demethylation of CpG sites in the miR-124 promoter region.
A method of modulating TET1 expression comprising treating neurons with an DSCR1 protein in vitro.
The method of claim 9, wherein the method modulates TET1 pre-mRNA splicing to downregulate TET1 expression.