Patent Grant
10544427
The present invention relates to a vector pair for screening tau oligomer formation, a mouse embryo introduced with the vector pair, a transgenic model mouse of neurological disease obtained from the mouse embryo, and a method of screening a tau oligomer formation inhibitor candidate using the transgenic model mouse.
Tau protein is a kind of microtubule-associated protein having a molecular weight of 50,000 to 70,000, which shows remarkable molecular diversity by its phosphorylation. In the human brain, six tau isoforms are generated by insertion of 29 or 58 amino acid residues in the N-terminal region and mRNA alternative splicing of 3 or 4 repeating structures (also referred to as microtubule-associated sites). It was previously thought that tau protein is specific for the central nervous system and found primarily in axons, but it is currently known that tau protein is expressed in astrocytes and oligodendroglia in relatively many tissues, as well as neurons, and is also found in dendrities in addition to axons. Tau contributes to the stability of microtubules, and the excessive aggregation of tau protein by phosphorylation causes various brain neurological diseases. Namely, central nervous diseases, including Alzheimer disease, fontotemporal lobar degeneration (FTLD), Pick's disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP) and the like, are known to involve tau protein aggregates. Because of such pathological features, such neurological diseases are collectively called tauopathies. Tau protein aggregates appearing in tauopathy patients are mainly found in neuronal cell bodies and dendrite, called neurofibrillary tangles (NFT) and neuropil threads. Neurofibrillary tangles are composed of paired helical filaments (PHFs) of aggregated and hyperphosphorylated tau protein, unlike normal tau protein. Although the role of abnormal tau protein aggregation, which appears in tauopathies, in a severe disease stage, has not been clearly known, it is similar to an aggregation phenomenon that appears commonly in neurodegenerative brain diseases, including Huntington's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and the like, and there is a significant correlation between neurofibrillary tangle formation and cognitive impairment. In this respect, it is evident that tau protein plays an important role.
A series of recent studies indicate that various tau oligomers rather than neurofibrillary tangles directly cause neuronal cell toxicity and are also metastasized to other sites of the brain to spread tauopathy. For example, Mirbaha et al. suggested tau trimers as structurally modified bases for intracellular tau, which can be received by cells. On the contrary, Michel et al. suggested that tau monomers are suitable fundamental metastatic forms, based on high-resolution imaging. In 2013, Wu et al. suggested that dimeric or trimeric tau having low molecular weight is a fundamental form that is metastasized between cells. Some studies on the isolation of tau protein and the identification of toxic tau oligomers by a series of biochemical experimental techniques were reported. However, direct observation of tau oligomers in the brain has not yet been reported, and the biggest reason why observation of tau oligomers is difficult is because of the absence of an experimental method that can specifically distinguish tau oligomers in the early stage of aggregation from normal tau protein present in large amounts. Regarding methods for detecting tau aggregates in neurons, immunotherapeutic technology using tau antibodies (U.S. Pat. No. 8,778,343, and US Patent Publication Nos. 2013-0209453 and 2014-0161875), and a tau-related disease cell model based on stem cells (US Patent Publication No. 2014-0011197) were developed. However, the development of technology related to cell models enabling real-time monitoring of tau aggregation mechanisms in living cells is still insufficient.
The present inventors have developed a method for constructing a transgenic model mouse of neurological disease, which displays fluorescence in the brain upon tau oligomer formation. The developed transgenic model mouse is a transgenic model mouse in which tau oligomer formation can be detected by a bimolecular fluorescence complementation (BiFC) technique. Particularly, the present invention provides a transgenic mouse in which tau oligomer formation in living tissue can be detected. Thus, the present invention makes it possible to directly detect tau oligomer formation in tissue, particularly the brain, and to screen a tau oligomer formation inhibitor. Furthermore, the present invention makes it possible to screen a tau oligomer formation inhibitor candidate and access an agent for treating neurological disease such as dementia whose perfect curing method has not yet been developed.
Objects to be solved by the present invention are not limited to the above-mentioned objects, and other objects that are not mentioned herein may be clearly understood by those skilled in the art from the following description.
Hereinafter, various embodiments of the present invention will be described with reference to the accompanying drawings.
The present invention can be embodied in a variety of different forms and may have various embodiments, and exemplary embodiments are illustrated in the accompanying drawings and will be described in detail in the detailed description. However, it should be understood that the present invention is not limited to specific embodiments and encompasses all modifications, equivalents or replacement s that fall within the spirit and technical scope of the present invention. In the following description, the detailed description of related known technology will be omitted when it may obscure the subject matter of the present invention.
Terms used in this specification are used only to describe a specific embodiment and are not intended to limit the scope of the present invention. Singular expressions are intended to include plural expressions unless specified otherwise in the context thereof. In this specification, the terms “comprise”, “have”, etc., are intended to denote the existence of mentioned characteristics, numbers, steps, operations, components, parts, or combinations thereof, but do not preclude the probability of existence or addition of one or more other characteristics, numbers, steps, operations, components, parts, or combinations thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.
A first aspect of the present invention is directed to a vector pair for screening tau oligomer formation, the vector pair comprising: a first vector comprising a first tau gene, a first fluorescence protein gene and a first neuron-specific promoter; and a second vector comprising a second tau gene, a second fluorescence protein gene and a second neuron-specific promoter, wherein a protein expressed from the first fluorescence protein gene and a protein expressed from the second fluorescence protein gene bind to each other to display fluorescence, by association between a protein expressed from the first tau gene and a protein expressed from the second tau gene.
The first tau gene and/or the second tau gene may be a full-length tau, a fragment of the full-length tau, or a variant of the full-length tau. For example, the tau gene may be human full-length tau gene Tau-P301L, but is not limited thereto. In an example of the present invention, a full-length tau gene represented by SEQ ID NO: 1 was used.
Tau protein is a kind of microtubule-associated protein (MAP) having a molecular weight of 50,000 to 70,000, and it is known that abnormal tau aggregation is a primary pathological hallmark in Alzheimer's disease (AD) and multiple other neurodegenerative disorders, collectively called tauopathies (Biochimica. et biophysica. acta. 1739: 331-354). In a healthy neuron, tau stabilizes microtubules by promoting axonal outgrowth and neuronal cell polarization. When pathologically hyperphosphorylated, tau dissociates from microtubules and forms insoluble aggregates (Mol. Neurodegener. 4: 13). A structural framework for tau aggregation has been suggested for many years. Evidences have been suggested that insoluble filaments are formed from 10 soluble monomers, and such filaments associate into higher order structures, called neurofibrillary tangles (NFTs). However, the pathophysiological importance of neurofibrillary tangles in tauopathies, the causes and molecular mechanisms responsible for triggering the process remain largely unknown, and this is because there is no reliable method for monitoring tau aggregation under physiological conditions. Until now, most studies on tau aggregation have been conducted using purified tau or tau fragments under non-physiological conditions. Furthermore, due to its extreme solubility, tau aggregation needs to be induced artificially by adding cofactors such as heparin. For this reason, animal models that can induce and monitor tau oligomer formation in living animals may provide a useful tool to investigate tau pathology and to discover methods capable of preventing and restoring the process.
The first fluorescence protein and/or the second fluorescence protein is one allowing proteins expressed from the first and second first fluorescence proteins to bind to each other to display fluorescence, by association between proteins expressed from the first tau gene and the second tau gene. Namely, when proteins expressed from the first tau gene and the second tau gene interact with each other, first fluorescence protein and the second fluorescence protein bind to each other simultaneously with or subsequently to the interaction to thereby display fluorescence. For example, the fluorescence protein may be a Venus protein. In addition, the first fluorescence protein gene may be represented by SEQ ID NO: 3, and the second fluorescence protein gene may be represented by SEQ ID NO: 5.
The present invention is an application of a method for visualizing protein-protein interactions that is based on a bimolecular fluorescence complementation (BiFC) technique of forming a fluorescence protein complex from non-fluorescent constituents attached to the proteins of interest (Annu. Rev. Biophys. 37: 465-487). Previously, a split green fluorescence protein (GFP) complementation technique was used to quantify tau aggregation. In the assay, tau is fused to a smaller GFP fragment (GFP 11), and co-expressed in cells with a larger GFP fragment (GFP 1-10). When tau exists as a monomer or low degree aggregate, the large GFP fragment is able to access the small GFP fragment fused to tau, leading to the association of the fluorescently active GFP. However, when tau aggregates, the reconstitution of active GFP is inhibited and GFP fluorescence decreases in cells. As a method of quantifying tau aggregation, the split-GFP assay has been highlighted; however, the limited scope and resolution of the assay do not allow the monitoring of tau oligomers that cause neuronal toxicity.
The BiFC technique based on the Venus protein, a kind of yellow fluorescence protein (YFP), is based on the principle according to which, when two different target proteins approach each other for interaction, fluorescence protein fragments linked to the target proteins also approach each other, and as a result, reconstruction between the fluorescence protein fragments occurs to display fluorescence. The use of this technique makes it possible to visually observe that an interaction between two target proteins occurred. Thus, this technique has the advantage of enabling protein-protein interactions to be visually observed in an optimal physical/chemical environment in which protein-protein interactions in cells or tissue may occur and be maintained. This technique makes it possible to determine not only a position where protein-protein interactions in cells or tissue occur, but also information about movement of these proteins.
The protein that is used in the present invention may be the Venus protein. The Venus protein can be effectively used for analysis of proteins such as tau protein, which are difficult to analyze spatially and temporally, because (1) it has fast and efficient maturation, (2) its self-assembly rate is low compared to that of other BiFC pairs, and (3) the fluorescence intensity of Venus-based BiFC is 10 times higher than that of EYFP-based BiFC (Biotechniques 40: 61-66; Biotechniques 49: 793-805).
According to one embodiment of the present invention, the first tau gene and the first fluorescence protein gene may be operably linked to each other, and the second tau gene and the second fluorescence protein gene may be operably linked to each other. Namely, in each of the first vector and the second vector, the tau gene and the fluorescence protein gene are preferably sequentially expressed by a single promoter. For example, the first tau gene and the first fluorescence protein gene may be linked to each other by a first linker, and the second tau gene and the second fluorescence protein gene may be linked to each other by a second linker. In an example of the present invention, the first linker represented by SEQ ID NO: 2 and the second linker represented by SEQ ID NO: 4 were used.
The first neuron-specific promoter and/or the second neuron-specific promoter serves to express the vector pair of the present invention, is not particularly limited, and may comprise any promoter known in the art. For example, the first neuron-specific promoter and/or the second neuron-specific promoter is preferably a Thy1 promoter. Since the Thy1 promoter is expressed specifically in neurons, it can induce expression of the genes inserted in the vector in neurons or neural tissue, particularly brain tissue.
The present invention is characterized in that, when a protein expressed from the first fluorescence protein gene and a protein expressed from the second fluorescence protein gene bind to each other to display fluorescence, by association between a protein expressed from the first tau gene and a protein expressed from the second tau gene. Particularly, the present invention is characterized in that the genes inserted in the vector can be expressed by the neuron-specific promoter in neurons or neural tissue, particularly brain tissue. Accordingly, tau oligomer formation in the mouse brain can be visualized directly by fluorescence, thereby monitoring and quantifying the tau oligomerization process in the brain.
According to one embodiment of the present invention, each of the first vector and the second vector, which comprise the Thy1 promoter, may be a pTSC21K vector comprising the Thy1 promoter, but is not limited thereto.
Furthermore, the first tau gene, the first fluorescence protein gene and the first linker may be inserted into the XhoI site of the pTSC21K vector, and the second tau gene, the second fluorescence protein gene and the second linker may be inserted into the XhoI site of the pTSC21K vector, but the scope of the present invention is not limited thereto.
Moreover, the first vector may comprise a nucleotide sequence of SEQ ID NO: 6, and the second vector may comprise a nucleotide sequence of SEQ ID NO: 7, but the scope of the present invention is not limited thereto.
In addition, the present invention may be directed to a vector pair for screening tau oligomer formation, comprising: a first vector which comprises a Thy1 promoter and wherein a full-length tau gene represented by SEQ ID NO: 1, a first linker represented by SEQ ID NO: 2, and a first fluorescence protein gene represented by SEQ ID NO: 3 are operably linked to one another; and a second vector which comprises a Thy1 promoter and wherein a full-length tau gene represented by SEQ ID NO: 1, a second linker represented by SEQ ID NO: 4, and a second fluorescence protein gene represented by SEQ ID NO: 5 are operably linked to one another.
A second aspect of the present invention is directed to a mouse embryo for screening tau oligomer formation, which has introduced therein the vector pair according to the first embodiment of the present invention.
The mouse and its embryo are not particularly limited. When this mouse embryo is used, a mouse can be obtained in which a protein expressed from the first fluorescence protein gene and a protein expressed from the second fluorescence protein gene bind to each other to display fluorescence, by association between a protein expressed from the first tau gene and a protein expressed from the second tau gene in neuronal tissue.
In according to one embodiment of the present invention, the mouse embryo may be a mouse embryo deposited in the Korean Collection for Type Cultures under accession number KCTC13076BP, but is no limited thereto. The date of the deposit is Aug. 16, 2016, and the address of the depository is Korea Research Institute of Bioscience and Biotechnology (KRIBB) 181, Ipsin-gil, Jeongeup-si, Jeolllabuk-do 56212, Republic of Korea, The present inventors obtained a mouse embryo using the above-described vector pair, and produced a transgenic mouse from the mouse embryo, and confirmed the target gene in the transgenic mouse. In this process, a mouse embryo, showing high gene expression levels and enabling fluorescence to be easily observed, was selected and deposited.
A third aspect of the present invention is directed to a transgenic model mouse of neurological disease, which is obtained from the mouse embryo according to the second aspect of the present invention. For example, the neurological disease is preferably a neurodegenerative brain disease, particularly dementia.
In the transgenic model mouse, human full-length tau proteins and two kinds of Venus proteins (VN173 and VC155) linked thereto, respectively, are expressed by a Thy1 promoter. The Thy promoter operates specifically in neurons, and thus when the tau proteins having VN173 and VC155, respectively, form an oligomer in the brain of the mouse, BiFC fluorescence is displayed by the Venus proteins. According to this principle, whether or not a tau oligomer would be formed in the brain tissue of the mouse and the extent of tau oligomer formation can be visually observed.
According to one embodiment of the present invention, the transgenic mouse may be a mouse wherein fluorescence is displayed by binding between the first fluorescence protein and the second fluorescence protein, when the full-length tau expressed from the first vector and the full-length tau expressed from the second vector form an oligomer. However, the transgenic mouse of the present invention is not limited thereto.
A fourth aspect of the present invention is directed to a method of detecting tau oligomer formation using a transgenic model mouse, the method comprising the steps of: obtaining a brain tissue section from the brain of the transgenic model mouse according to the third aspect of the present invention; subjecting the obtained brain tissue section to imaging analysis to measure fluorescence intensity; and comparing the measured fluorescence intensity with a reference value.
The step of obtaining the brain tissue section may comprises injecting a tau aggregation inducer into the transgenic model mouse and obtaining the brain tissue section from the brain of the transgenic model mouse injected with the tau aggregation inducer.
A fifth aspect of the present invention is directed to a method for screening a tau oligomer formation inhibitor candidate, the method comprising the steps of: inducing tau oligomer formation in the brain of the transgenic model mouse; evaluating the level of tau oligomer formation in the brain of the transgenic model mouse; administering a tau oligomer formation inhibitor candidate to the transgenic model mouse; evaluating the level of tau oligomer formation in the brain of the transgenic model mouse; and comparing the level of tau oligomer formation between before and after administering the tau oligomer formation inhibitor candidate.
For example, the step of evaluating the level of tau oligomer formation may comprise quantifying the fluorescence intensity of the fluorescence protein, that is, the Venus protein, but is not limited thereto.
According to one embodiment of the present invention, the step of inducing tau oligomer formation in the brain of the transgenic model mouse may comprise administering forskolin or okadaic acid to the transgenic model mouse, but is not limited thereto. The forskolin or the okadaic acid may be used to induce hyperphosphorylation of tau protein.
Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention as defined in the appended claims.
1. Construction of Neuron-Specific Vectors for Screening Tau Oligomer Formation
To express tau protein specifically in mouse neurons, two bimolecular fluorescence complementation (BiFC) constructs (tau-VN173 and tau-VC155) were cloned into 323-pTSC21K vectors including a Thy1 promoter.
Particularly, in the present invention, vectors were constructed using a Venus-based BiFC system. To this end, the mammalian expression vector pCMV6-hTau40-GFP was purchased from OriGene Technologies Inc. (Rockville, Md., USA), and the amino acid proline at position 301 was replaced with leucine, thereby constructing pCMV6-hTau40P301 L-GFP. The forward and reverse primer sequences used herein are shown in Table 1 below.
To replace GFP with a Venus fluorescence protein fragment, pBiFC-VN173 and pBiFC-VC155 were purchased from Addgene (Cambridge, Mass.), and then amplified using PCR primers having XhoI/PmeI restriction enzyme sequences. Next, a substituted human full-length tau (441 amino acids) was fused to the N-terminal fragment (1-172, VN173) (first fluorescence protein) and C-terminal fragment (155-238, VC155) (second fluorescence protein) of the fluorescence protein Venus.
pCMV6-TauP301L-GFP and the PCR-amplified insert were digested with XhoI/PmeI and ligated with each other, thereby constructing pCMV6-TauP301L-VN173 and pCMV6-TauP301L-VC155 which are insertion genes. The linker peptide and fluorescence protein sequences used in construction of the insertion genes are shown in Tables 2 and 3 below.
The Thy1 promoter from mouse thy1.2 gene is a promoter that is expressed specifically in mouse brain neurons. Mouse Thy1 gene (mouse Thy-1.2 glycoprotein gene) is 5572 bp in total length and includes three exon regions and three intron regions. TauP301L-VN173 and Tau-P301L-VC155 were inserted into exon 3 of the Thy1 gene.
Each of two tau-BiFC plasmids (i.e., pCMV6-TauP301L-VN173 and pCMV6-TauP301L-VC155) prepared as insertion genes was cloned into the Xho-1 site of a 323-pTSC21K vector.
2. Construction of Transgenic Model Mice
For injection into mouse embryos, the Thy1-TauP301L-VN173 and Thy1-Tau-P301L-VC155 recombinant plasmids were linearized with the restriction enzyme EcoRI.
3. Observation of Tauopathy in Transgenic Mice
The constructed Tau-P301L BiFC transgenic mice display fluorescence when tau protein formed oligomers in the brain. In order to actually confirm whether fluorescence would be observed by tau oligomer formation in the brain tissue of the Tau-P301L BiFC transgenic mice, the brain of 7-month-old mice was purfused, fixed, and extracted, and the extracted brain was sectioned to a thickness of 40 μm. The brain tissue sections of various regions were imaged without immunofluorescence staining, and as a result, BiFC fluorescence was observed. Specifically, BiFC fluorescence could be observed in the hippocampus, cortex, amygdale and the like of tau-expressing animal models known to show tau aggregation.
4. Observation of Induction of Tau Oligomer Formation in Transgenic Mice
Using 4-month-old Tau-P301L BiFC transgenic mice which have not yet shown tau aggregation, whether tau oligomer BiFC fluorescence would be observed following injection of a tau aggregation inducer was examined. The tau aggregation inducing drug forskolin was filled in a drug injection kit, and then the drug was allowed to flow into a ventricle in the brain of about 5-month-old Tau-P301L BiFC transgenic mice (
As described above, according to the present invention, tau oligomer formation occurring in the brain of mice can be visualized directly by fluorescence, thereby monitoring and quantifying the tau oligomerization process in the brain. The use of this technology makes it possible to investigate diseases such as dementia in which tau protein is involved, and to screen a tau oligomer formation inhibitor candidate. Thus, the present invention may be used as a useful tool in development of dementia therapeutic agents.
While the present invention has been described with reference to the particular illustrative embodiments, it will be understood by those skilled in the art to which the present invention pertains that the present invention may be embodied in other specific forms without departing from the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be considered in a descriptive sense only and not for purposes of limitation. For example, each component described in a single form may be carried out in a distributed fashion, and likewise, components described in a distributed form may be carried out in a combined fashion.
Therefore, the scope of the present invention is defined not by the detailed description, but by the claims and their equivalents, and all variations within the scope of the claims and their equivalents are to be construed as being included in the scope of the present invention.
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| Number | Date | Country | |
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| 20180119169 A1 | May 2018 | US |
