Mitochondrial transcriptional regulatory factor Mt-CREB and application thereof in screening and preparing anti-brain aging drugs

Abstract

The invention discloses a mitochondrial transcriptional regulatory factor Mt-CREB and application thereof in screening and preparing a drug for resisting brain aging. According to the present invention, based on the characteristic that the mitochondrial circular DNA has the conservative cAMP reaction element sequence CRE, the CREBWT capable of selectively identifying and combining the CRE or the amino acid RRPSYR mutant CREBDIEDML of the CREBWT at the 130-135th site is adopted as the key element of the gene enhancement regulation, and the CREBWT or the CREBDIEDML is selectively positioned to the mitochondria by using the mitochondrial targeting sequence so as to achieve the detection and the regulation of the mitochondrial gene expression enhancement; on one hand, the invention discloses an amino acid sequence of the Mt-CREBDIEDML, a nucleotide sequence for coding the Mt-CREBDIEDML, an expression vector containing the nucleotide sequence, and an application of the Mt-CREBDIEDML in preparation of a drug for resisting brain aging; on the other hand, the invention discloses a fluorescence and / or chemiluminiscence detection method for screening the anti-brain aging drugs by using the Mt-CREBWT. Through research on biochemical indexes, immunohistochemistry, gene expression level and the like, a new way is provided for preparing and screening anti-brain aging drugs and treating memory decline caused by aging.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an artificially synthesized mitochondrial transcription regulatory factor Mt-CREB and its application in screening and preparing anti-brain aging drugs. Background Art

[0002] As human life expectancy increases and the aging population increases, the normal aging process of the brain has attracted widespread attention. Brain aging refers to the process in which the structure and function of the brain gradually change with age, manifested as a decline in cognitive function, emotional and social function, and the emergence of neurodegenerative changes. This aging process is biologically inevitable, but its speed and extent vary depending on individual differences, lifestyle and other factors. In the nervous system, the function and health of neurons are essential for maintaining brain function. Mitochondria are important organs within neurons. In addition to producing the energy needed by cells, they also affect the release of neurotransmitters, determine the firing pattern of neurons, and provide energy to neurons. With age, mitochondrial function gradually declines, resulting in a slowdown in cellular energy metabolism, which affects the normal function of neurons.

[0003] Abnormal mitochondrial function plays an important role in the aging process of the brain, and the decreased level of mitochondrial-encoded mRNA is a sign of aging. The decrease in mitochondrial mRNA reflects the decline of mitochondrial function, which is a key step in the aging process of the brain. In the brain, the decline of mitochondrial function is particularly significant because the brain has a very high demand for energy, and mitochondria are the main place to provide energy. Abnormal mitochondrial function can lead to insufficient energy supply to neurons, accelerate the aging and death of neurons, and further aggravate the aging process of the brain, leading to problems such as decline in learning and memory ability in ordinary elderly people. This decline in mitochondrial function is closely related to the normal aging process of the brain. Therefore, finding factors that can promote mitochondrial function and maintain the stability of the mitochondrial genome has become one of the important directions of current research.

[0004] Studies have found that calcium / cyclic adenosine monophosphate response element binding protein (cAMP-responsive element binding protein, CREB) is a transcription factor related to learning and memory in the brain, and its wild-type amino acid sequence is shown in SEQ ID NO.1. Although there have been many studies on broad activators of nuclear CREB in the past, abnormal activation of nuclear CREB is closely related to inflammation, tumor development, depression, pain and other diseases, and has many accompanying side effects. At present, there is no research on the role of mitochondrial calcium / cyclic adenosine monophosphate response element binding protein (Mitochondrial cAMP-responsive element binding protein, Mt-CREB) in brain learning and memory, and the coupling of neuronal activity-mitochondrial gene transcription has not been studied in cell models and in vivo models. Summary of the invention

[0005] In view of the above problems, the present invention aims to provide an artificially designed and synthesized mitochondrial transcriptional regulatory factor Mt-CREB and its application in screening and preparing anti-brain aging drugs, wherein CREB includes wild-type CREB WT and mutant CREB DIEDML The mitochondrial transcriptional regulatory factor Mt-CREB is based on the characteristics of the conserved cAMP response element (CRE) sequence in mitochondrial circular DNA, and selectively recognizes CREB bound to CRE. WT CREB WT RRPSYR mutant CREB at amino acids 130-135 DIEDML As a key element of gene enhancement regulation, CREB is targeted by mitochondrial targeting sequence WT CREB DIEDML Selectively localize to mitochondria to achieve detection and regulation of enhanced mitochondrial gene expression.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a transcriptional regulatory factor CREB DIEDML , which is in CREB WT The amino acids 130-135 of RRPSYR mutated to DIEDML, and the CREB WT The amino acid sequence has 95% or more homology to the sequence shown in SEQ ID NO.1.

[0008] The second aspect of the present invention provides a mitochondrial transcriptional regulatory factor Mt-CREB DIEDML, which is involved in the transcriptional regulatory factor CREB DIEDML A mitochondrial targeting sequence is added to the N-terminus of the amino acid sequence of , and the amino acid sequence of the mitochondrial targeting sequence is shown in SEQ ID NO.2.

[0009] In a preferred embodiment, the Mt-CREB DIEDML The amino acid sequence is shown in SEQ ID NO.3.

[0010] The third aspect of the present invention provides a method for encoding the mitochondrial transcriptional regulatory factor Mt-CREB. DIEDML of nucleic acid.

[0011] In a preferred embodiment, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO.4.

[0012] The fourth aspect of the present invention provides a method comprising the above-mentioned coding mitochondrial transcriptional regulatory factor Mt-CREB DIEDML The expression vector is a lentiviral expression vector or an adeno-associated virus expression vector.

[0013] The fifth aspect of the present invention provides a mitochondrial transcription regulatory factor Mt-CREB DIEDML Application in the preparation of anti-brain aging drugs.

[0014] In a preferred embodiment, the active ingredient of the drug includes the above-mentioned mitochondrial transcription regulatory factor Mt-CREB DIEDML , the above encoding mitochondrial transcription factor Mt-CREB DIEDML The nucleic acid encoding the mitochondrial transcriptional regulatory factor Mt-CREB DIEDML One or more combinations of nucleic acid expression vectors.

[0015] In a preferred embodiment, the drug has one or more of the following uses:

[0016] (1) Selectively promote the expression of neuronal mitochondrial genome;

[0017] (2) Slow down the decline in adenosine triphosphate levels caused by aging;

[0018] (3) Improve the brain's spatial memory function.

[0019] The sixth aspect of the present invention provides a method for delivering an anti-aging drug for the brain, comprising the following steps:

[0020] Step S1, using the above-mentioned protein encoding the mitochondrial transcriptional regulatory factor Mt-CREB DIEDML An expression vector for a nucleic acid;

[0021] Step S2, encapsulating the expression vector in step S1 into lentivirus or adeno-associated virus particles;

[0022] Step S3: using the lentivirus or adeno-associated virus particles in step S2 to deliver anti-brain aging drugs, wherein the anti-brain aging drugs are delivered to target cells or living bodies through the infection of the lentivirus or adeno-associated virus particles.

[0023] The seventh aspect of the present invention provides a fluorescence and / or chemiluminescence detection method for screening anti-brain aging drugs, comprising the following steps:

[0024] Step S1, cloning the above mitochondrial targeting sequence into the mitochondrial targeting sequence containing the above CREB WT The fluorescent and / or chemiluminescent vector is recombined to obtain a first biosensor, wherein the first biosensor is used to specifically monitor CREB in mitochondria WT The transcriptional activity of CREB in mitochondria is reflected by changes in fluorescence and / or chemiluminescence WT The binding strength with downstream response elements indicates the expression of mitochondrial genes and neuronal activity;

[0025] Step S2, expressing the first biosensor in step S1 in neurons;

[0026] Step S3, treating the neurons in step S2 with the drug to be screened, and determining the promoting effect of the drug to be screened on mitochondrial gene expression and neuronal activity by detecting the changes in fluorescence and / or chemiluminescence of the first biosensor.

[0027] In a preferred embodiment, the step S1 contains the CREB WT The fluorescent and / or chemiluminescent vector comprises far-red fluorescent protein and / or red fluorescent protein.

[0028] In a preferred embodiment, the far-red fluorescent protein is mMaroon.

[0029] In a preferred embodiment, the red fluorescent protein is mCyRFP2.

[0030] In a preferred embodiment, the step S1 contains the CREB WT The fluorescent and / or chemiluminescent vectors include lentiviral or adeno-associated viral vectors.

[0031] A preferred embodiment, the CREB WT The fluorescent and / or chemiluminescent vector is AAV-hSyn-R CREB sensor pair.

[0032] In a preferred embodiment, the detection method further comprises:

[0033] In the above step S1, it also includes cloning the above mitochondrial targeting sequence into a fluorescent and / or chemiluminescent vector containing calmodulin to recombinantly construct a second biosensor, wherein the second biosensor is used to specifically monitor the calcium ion level in mitochondria, and reflects the binding of calmodulin and calcium ions in mitochondria through changes in fluorescence and / or chemiluminescence, thereby indicating the calcium ion concentration in mitochondria;

[0034] In the above step S2, it also includes expressing the second biosensor described in step S1 in neurons;

[0035] In the above step S3, it also includes determining the promoting effect of the drug to be screened on the calcium ion concentration in mitochondria by detecting the change of fluorescence and / or chemiluminescence of the second biosensor.

[0036] In a preferred embodiment, the fluorescent and / or chemiluminescent carrier containing calmodulin in step S1 comprises green fluorescent protein.

[0037] In a preferred embodiment, the fluorescent and / or chemiluminescent vector containing calmodulin in step S1 comprises a lentiviral or adeno-associated viral vector.

[0038] In a preferred embodiment, the fluorescent and / or chemiluminescent vector containing calmodulin in step S1 is AAV-αCaMKII-GCaMP6s.

[0039] In a preferred embodiment, in step S1, the mitochondrial targeting sequence is cloned before the coding region of the fluorescent and / or chemiluminescent vector.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) The present invention firstly transforms the wild-type transcription factor CREB WT The amino acids RRPSYR at positions 130-135 of the gene were mutated to DIEDML, and a mitochondrial targeting sequence was added to the N-terminus of the mutant sequence, thereby obtaining a new type of mitochondrial transcriptional regulatory factor Mt-CREB with enhanced transcriptional activity and the ability to specifically localize to mitochondria and promote mitochondrial gene expression. DIEDML , avoiding the side effects caused by abnormal activation of CREB transcription factor in the cell nucleus.

[0042] (2) The present invention provides a fluorescence and / or chemiluminescence detection method that can be used to screen anti-aging drugs for the brain, which constructs a fluorescent and / or chemiluminescence detection method that locates CREB expression in mitochondria. WTThe biosensor can be used to determine the expression of CREB by changes in fluorescence and / or chemiluminescence on the biosensor. WT The transcriptional activity of mitochondrial genes can indicate the expression of mitochondrial genes and neuronal activity, creating a new method for screening potential anti-brain aging drugs.

[0043] (3) The present invention is the first to study the coupling of neuronal activity and mitochondrial gene transcription in cell models and in vivo models. Through studies from multiple aspects such as biochemical indicators, immunohistochemistry, and gene expression levels, it was found that the novel mitochondrial transcription regulatory factor Mt-CREB prepared by the present invention DIEDML It alleviates the progression of brain aging and can be used to prepare targeted drugs for the treatment of cognitive dysfunction, providing a new way to treat brain cognitive decline caused by aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 In one embodiment of the present invention, the mitochondrial transcription regulatory factor Mt-CREB DIEDML Schematic diagram of the sequence structure;

[0045] Figure 2 In one embodiment of the present invention, Mt-CREB was overexpressed by lentivirus in two different neuronal models. DIEDML Immunofluorescence staining results of DIEDML, A is the mutation location and action principle of DIEDML, B is Mt-CREB DIEDML (Green) Expression pattern in mitochondria (red) of neuroblastoma SK-N-SH, C is Mt-CREB DIEDML (Green) Expression pattern in mitochondria (red) of primary hippocampal neurons; Scale bar: 20 μm;

[0046] Figure 3 The two-photon imaging result of hippocampal neurons in one embodiment of the present invention is shown in Figure 1. A is a schematic diagram of the construction tool, B is a diagram showing the changes in hippocampal neurons overexpressing two sensors before and after stimulation with bicuculline (BIC), and C is a statistical diagram of B, wherein green is the fluorescence intensity of the sensor Mt-GCaMP6s, which is used to measure the calcium ion content in mitochondria, and red is the fluorescence half-life of the sensor Mt-R-CREB sensor pair, which is used to measure CREB in mitochondria WT Transcriptional activity; Scale bar: 20 μm;

[0047] Figure 4 In one embodiment of the present invention, Mt-CREB is overexpressed by lentivirus in a non-proliferative neuron model. DIEDML Related results of post-biochemical detection, A is overexpression of Mt-CREB DIEDMLFigure 2. Changes in mitochondrial mRNA levels in primary neurons infected with (Mt-DIEDML, dark brown) or control virus (Mt-Ctrl, black) before and after BIC stimulation. BC is overexpression of Mt-CREB DIEDML The effect of Mt-DIEDML (Mt-DIEDML, Zong) or control virus (Mt-Ctrl, black) on mitochondrial membrane potential characterized by CMXROS staining, D is the effect of Mt-CREB overexpression DIEDML The effect of Mt-DIEDML (Mt-DIEDML, dark brown) or control virus (Mt-Ctrl, black) on the intracellular adenosine triphosphate (ATP) content of primary neuronal cells. E is the overexpression of Mt-CREB DIEDML Figure 3. Effects of Mt-DIEDML (green, bottom) or control virus (Mt-Ctrl, green, top) on the aging marker p16 (red) in cholinergic transdifferentiated neurons of the human basal forebrain. F is a statistical graph of E (control virus group: Mt-Ctrl, gray; Mt-CREB DIEDML Group: Mt-DIEDML, brown); *p<0.05, ****p<0.0001, scale bar: 20 μm;

[0048] Figure 5 In one embodiment of the present invention, adeno-associated virus overexpressed Mt-CREB in the hippocampus of aged mice DIEDML Behavioral test results, A is the overexpression of Mt-CREB in the hippocampus of 16-month-old mice DIEDML Figure 2 shows the results of a novel location recognition test on old mice after the induction of Mt-DIEDML (Mt-DIEDML, brown) or its control virus (Mt-Ctrl, gray); B shows the results of a novel location recognition test on old mice after the induction of Mt-CREB (Mt-DIEDML, brown) or its control virus (Mt-Ctrl, gray); DIEDML (Mt-DIEDML, brown) or its control virus (Mt-Ctrl, gray), young mice (black) and old mice were tested for the escape hole; *p<0.05;

[0049] Figure 6 In one embodiment of the present invention, adeno-associated virus overexpressed Mt-CREB in the hippocampus of aged mice DIEDML Related results of post-biochemical detection, A is Mt-CREB DIEDML (Green) Expression pattern in hippocampal CA1, B is overexpression of Mt-CREB in the hippocampus of 16-month-old mice DIEDML Figure 2. β-galactosidase staining signal in Mt-DIEDML (Mt-DIEDML, brown) or its control virus (Mt-Ctrl, gray) and young mice (black). C is the overexpression of Mt-CREB in the hippocampus of 16-month-old mice. DIEDMLFigure 3. Changes in ATP content in the hippocampus of mice with Mt-DIEDML (Mt-DIEDML, brown) or its control virus (Mt-Ctrl, gray) and young mice (black). D is a graph showing changes in ATP content in the hippocampus of 16-month-old mice overexpressing Mt-CREB. DIEDML Immunohistochemical staining of p16 in (Mt-DIEDML, Mt-Ctrl) or its control virus (Mt-Ctrl, gray) and young mice (black). E is the statistical analysis of D (young mouse group: black; old control virus group: Mt-Ctrl, gray; old Mt-CREB DIEDML Group: Mt-DIEDML, brown); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, scale bar: 20 μm. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that the embodiments are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0051] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0052] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels; various processes and methods not described in detail in the embodiments of the present invention are conventional methods known in the art.

[0053] Example 1 Overexpression of Mt-CREB in two different neuronal models DIEDML

[0054] 1. Experimental methods

[0055] (1) Mt-CREB DIEDML Gene synthesis. Full gene synthesis of wild-type calcium / cyclic AMP response element binding protein (CREB WT ), in this embodiment, CREB WT The wild-type calcium / cyclic AMP response element binding protein isoform 1 of the mouse, whose amino acid sequence is shown in SEQ ID NO.1, was used to mutate the amino acids RRPSYR at positions 130-135 to DIEDML by in vitro mutagenesis to form a mutant protein CREB. DIEDML Subsequently, the synthesized mitochondrial targeting sequence TXN2 (Thioredoxin-2, thioredoxin-2, amino acid sequence as shown in SEQ ID NO.2) was added to the N-terminus of the mutant protein sequence, and finally the mitochondrial transcription factor Mt-CREB was synthesized. DIEDMLIts amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4. The correctness of its nucleotide sequence was verified by sequencing (gene synthesis and sequence verification were completed by TingsKe Company).

[0056] It should be noted that CREB WT Transcription factors are highly conserved in humans and mice, and the amino acid sequence homology between humans and mice is greater than 99%. Although this example uses mouse transcription factor (amino acid sequence as shown in SEQ ID NO.1) as the research object, amino acid sequences with 95% or more homology to the sequence shown in SEQ ID NO.1 can also be used as CREB WT The amino acid sequence of human or mouse CREB WT The amino acid sequence is only the preferred research object of this embodiment. In addition, the mitochondrial targeting sequence used in this embodiment is TXN2 with an amino acid sequence such as SEQ ID NO.2, which can also be other available mitochondrial targeting sequences in the prior art. Among them, each sequence is specifically as follows:

[0057] CREB WT Amino Acid Sequence:

[0058] MTMDSGADNQQSGDAAVTEAESQQMTVQAQPQIATLAQVSMPAAHATSSAPTVTLVQLPNGQTVQVHGVIQAAQPSVIQSPQVQTVQSSCKDLKRLFSGTQISTIAESEDSQESVDSVTDSQKRREILSRRPSYRKILNDLSSDAPGVPRIEEEKSEEETSAPAITTVTVPT PIYQTSSGQYIAITQGGAIQLANNGTDGVQGLQTLTMTNAAATQPGTTILQYAQTTDGQQILVPSNQVVVQAASGDVQTYQIRTAPTSTIAPGVVMASSPALPTQPAEEAARKREVRLMKNREAARECRRKKKEYVKCLENRVAVLENQNKTLIEELKALKDLYCHKSD(SEQ ID NO.1)

[0059] Amino acid sequence of mitochondrial targeting sequence TXN2:

[0060] MAQRLLLRRFLASVISRKPSQGQWPPLTSRALQTPQCSPGGLTVTPNPARTIYTTR ISLT(SEQ IDNO.2)

[0061] Mt-CREBDIEDML Amino Acid Sequence:

[0062] MAQRLLLRRFLASVISRKPSQGQWPPLTSRALQTPQCSPGGLTVTPNPARTIYTTRISLTMTMDSGADNQQSGDAAVTEAESQQMTVQAQPQIATLAQVSM PAAHATSSAPVTLVQLPNGQTVQVHGVIQAAQPSVIQSPQVQTVQSSCKDLKRLFSGTQISTIAESEDSQESVDSVTDSQKRREILSDIEDMLKILNDLS SDAPGVPRIEEEKSEEETSAPAITTVTVPTPIYQTSSGQYIAITQGGAIQLANNGTDGVQGLQTLTMTNAAATQPGTTILQYAQTTDGQQILVPSNQVVVQ AASGDVQTYQIRTAPTSTIAPGVVMASSPALPTQPAEEAARKREVRLMKNREAARECRRKKKEYVKCLENRVAVLENQNKTLIEELKALKDLYCHKSD(SEQ ID NO.3)

[0063] Mt-CREB DIEDML Nucleotide sequences (where lowercase letters represent the mitochondrial targeting sequence TXN2, and uppercase letters represent CREB DIEDML sequence):

[0064]

[0065] (2) Mt-CREB DIEDML Gene cloning. Mt-CREB was cloned using the seamless cloning kit (AG11803) from Accurate Biology. DIEDML The nucleotide sequence was cloned into the pCDH (Addgene, 72266) lentiviral vector or AAV-αCaMKII (Addgene, 107790) adeno-associated virus vector to construct pCDH-Mt-CREB DIEDML or AAV-αCaMKII-Mt-CREB DIEDML Recombinant plasmid.

[0066] The constructed recombinant plasmid was transformed into Transgene's Tran5a E. coli competent cells (CD201-01). After transformation, the cells were cultured overnight in a selective LB medium containing ampicillin antibiotics (Biyuntian Company, ST008) to screen out single colonies containing the recombinant plasmid. After selecting a single colony, PCR and enzyme digestion verification were performed, and finally sequencing was performed to ensure the accuracy of the clone.

[0067] (3) Lentivirus packaging. Before transfecting HEK-293T cells (ATCC, CRL-3216), ensure that the cells are in good growth condition and free of contamination. On the day of transfection, adjust the cell density to 70-80% and replace with fresh culture medium before transfection. DIEDML Mixed with lentiviral coating auxiliary plasmids pMD2.G (Addgene, 12259) and psPAX2 (Addgene, 12260) in a ratio of 9:3:1, a total of 4 μg, and transfected using Thermo's Lipo3000 (L3000075) according to the manufacturer's recommended ratios and steps. Fresh culture medium was replaced 8 hours after transfection to promote cell recovery. After 48 hours, the supernatant containing viral particles was collected and purified using a kit (C2901M) from Bio-Tech, and cell debris and untransfected cells were removed by centrifugation at 3500g, 4°C for one hour. The collected viral particles were stored in a lentiviral storage solution containing 0.01M PBS and 10% glycerol to maintain the activity and stability of the viral particles.

[0068] (4) Isolation and culture of primary hippocampal neurons. E18 pregnant mice were dissected in DMEM high-glucose medium (Thermo Company, 11965092) to obtain hippocampal tissue from fetal mouse embryos. The tissue was gently digested with trypsin (Thermo Company, 15050065) to separate single cells. After the digestion process, the digestion reaction was terminated using DMEM high-glucose medium containing 20% ​​serum. Subsequently, the cell suspension was centrifuged at 800g for 4 minutes at room temperature to precipitate the cells. After centrifugation, the supernatant was removed and the cells were resuspended. The cell suspension was filtered using a filter of appropriate pore size to remove undigested tissue fragments and cell clumps. The filtered cell suspension was adjusted to an appropriate cell density, usually 5×10 4 Each well is then planted on a glass slide that has been treated with poly-D-lysine (Sigma, P6407) in advance to promote the adhesion and growth of neurons. After 24 hours, the culture medium is replaced with a special culture medium for primary neurons, such as Brainphys culture medium (Stem Cell, 05792) containing 2% SM1, which provides neurons with suitable growth factors and nutrients. During the culture process, the medium is changed every four and a half days to maintain the cleanliness of the culture environment and supplement the necessary nutrients. Until 14-18 days, the neurons mature and reach the maturity required for the experiment, and subsequent experimental operations can be carried out at this time.

[0069] (5) Overexpression of Mt-CREB using lentivirus DIEDML And immunofluorescence staining. The above coated lentiviral particles (MOI = 5) were added to primary neurons and neuroblastoma SK-N-SH (ATCC, HTB-11), respectively, and immunofluorescence staining was performed after 72-96 hours. The immunofluorescence staining method is as follows:

[0070] The cells were first fixed in a 0.01M PBS fixative containing 4% paraformaldehyde (Sigma-Aldrich, 158127), 3.2mM EGTA, and 90mM sucrose, and fixed at 4°C for 10 minutes to ensure the integrity of the cell structure and the stability of the antigen. Then 0.1% Triton X-100 (Sigma-Aldrich, 9036-19-5) was used for permeation treatment to increase the permeability of the cell membrane and facilitate the entry of antibodies into the cell interior. Next, the sample was blocked in 0.01M PBS with 7.5% normal donkey serum (EMD Millipore, KC S30-100KC) for 30 minutes to reduce nonspecific binding. Afterwards, the corresponding concentration of specific primary antibody (Table 1) was incubated at 4°C for 16 hours to specifically identify the target antigen. The detection of the primary antibody was completed by incubating the corresponding fluorescent secondary antibody (Table 2) in the dark at 20°C for 1 hour to ensure the stability and specificity of the fluorescent label. After incubation, the slides were mounted with anti-fading medium containing or without 4',6-diamidino-2-phenylindole (Yeasen Biotechnology, 36308ES110, 36307ES08) to protect the fluorescent signal and reduce photobleaching. In all steps, after removing the solution, the cells were washed twice with 0.01M PBS to ensure thorough washing.

[0071] Table 1 Primary antibodies required for this example

[0072]

[0073] Table 2 Fluorescent secondary antibodies required for this example

[0074]

[0075] 2. Experimental results

[0076] (1) Mt-CREB constructed in this example DIEDML Sequence structure, mutation location and mechanism of action Figure 1 and Figure 2 As shown in A, Figure 1 The purple part of the N-terminal is the mitochondrial targeting sequence, which is used to guide the transfer of transcription factors to mitochondria. The amino acid mutation position is in the wild-type transcription factor CREB WT The 130-135 site of the transcription factor CREB is mutated to DIEDML, as shown in the red area in the figure. The mutation site is located in the kinase-induced domain (KID domain) of the transcription factor CREB. The mutation modification can increase the transcriptional activity of CREB and promote the expression of the mitochondrial genome. WT Mutational modification of Mt-CREB DIEDMLIt can then be specifically localized in the mitochondria of the cell, thereby activating genome expression only within the mitochondria.

[0077] (2) Figure 2 As shown in B and 2C, Mt-CREB was shown by immunofluorescence staining after lentiviral transfection. DIEDML The mitochondria of neuroblastoma SK-N-SH and primary neurons showed puncta morphology, indicating that the mitochondrial targeting sequence successfully transferred the transcription factor Mt-CREB to the mitochondria. DIEDML It was localized in the mitochondria of cells, indicating its potential role in binding to double-stranded DNA.

[0078] Example 2 Increased neuronal activity activates Mt-CREB WT

[0079] 1. Experimental methods

[0080] (1) Gene cloning. Using the same gene cloning method as in Example 1, the mitochondrial targeting sequence was cloned before the coding region of AAV-hSyn-R CREB sensor pair (Addgene, 137008 and 137009) and AAV-αCaMKII-GCaMP6s (Addgene, 107790), and the first biosensor Mt-R-CREB sensor pair and the second biosensor Mt-GCaMP6s were recombinantly constructed without changing the codons, wherein the Mt-R-CREB sensor pair is used to specifically monitor CREB in mitochondria. WT In order to detect the transcriptional activity of mitochondria, Mt-GCaMP6s were used to specifically monitor the calcium ion level within mitochondria.

[0081] (2) Adeno-associated virus packaging. AAV2 / 9-Mt-GCaMP6s and AAV2 / 9-Mt-R-CREB sensor pair viruses were produced by Shanghai Biobio Biotech Co., Ltd. and used to express biosensors in hippocampal neurons. Hippocampal neurons were imaged in a living cell workstation under living cell conditions.

[0082] 2. Experimental results

[0083] The sequence structure and working principle of the Mt-GCaMP6s and Mt-R-CREB sensor pair biosensors constructed in this example are as follows Figure 3 As shown in A, the N-terminal is a mitochondrial targeting sequence. Calmodulin (CaM) in Mt-GCaMP6s can bind calcium ions (Ca 2+), when the intracellular calcium concentration rises, calmodulin will bind to calcium ions, causing its conformation to change, which in turn causes green fluorescent protein to emit light. The Mt-R-CREB sensor pair reflects CREB through the interaction strength between the far-red fluorescent protein mMaroon and the red fluorescent protein mCyRFP2 (the indicator quantifies the fluorescence half-life of mCyRFP2). WT The binding strength of the protein to the downstream KIX domain is measured to measure mitochondrial CREB WT It is worth noting that since the activation of the Mt-R-CREB sensor pair is S133 site-dependent, the DIEDML mutation is not applicable to this scenario.

[0084] Bicuculline (BIC) is a γ-aminobutyric acid type A receptor (GABA_A) antagonist. It can increase the excitability of neurons by blocking the inhibitory effect of γ-aminobutyric acid (GABA, a major inhibitory neurotransmitter found in the mammalian central nervous system, which reduces the excitability of neurons by binding to specific receptors, usually GABA_A receptors and GABA_B receptors). After the hippocampal neurons were stimulated by 40 micromolar BIC, the changes in the fluorescence intensity or fluorescence half-life of the biosensor were recorded in real time. Figure 3 B-3C shows that mitochondrial calcium concentration is related to mitochondrial CREB WT The activity of the brain will be activated by neuronal activity. Based on this, the above-mentioned biosensor can be used as a tool to screen potential anti-brain aging drugs that promote neuronal activity.

[0085] Example 3 Overexpression of Mt-CREB in a non-proliferative neuronal model DIEDML Promote mitochondrial gene transcription and delay cell aging

[0086] 1. Experimental methods

[0087] (1) Detection of mitochondrial MtCo1 mRNA expression. A total amount of lentivirus with a virus titer of 5 was added to the primary neuron culture system. After 72 to 96 hours, RNA extraction or immunofluorescence staining was performed. The method for detecting mitochondrial MtCo1 mRNA expression is as follows:

[0088] Total RNA was extracted from cultured cells using TRIzol reagent (Sigma-Aldrich, 93289). RNA was transcribed into cDNA using HiScript IIQ Select RT SuperMix for qPCR (with gDNA cleanup agent) (Vazyme, R233-01) for reverse transcription. To evaluate mitochondrial encoded mRNA, cDNA was reverse transcribed using random hexamer primers (42°C for 2 minutes; 50°C for 60 minutes; 85°C for 5 seconds). cDNA was diluted and stored in DNase / RNase-free Tris-EDTA buffer (Beyotime, R0225). Quantitative real-time polymerase chain reaction was performed on a Roche Light Cycler. Evaluation of mouse mitochondrial MtCo1 mRNA levels was performed using a probe primer set purchased from the Accurate Biology commercial database, modified to Fam, and amplified by Pro-Taq HS (Accurate Biology, AG11730) (step 1: 95°C for 30 seconds, 1 cycle; step 2: 95°C for 5 seconds, 60°C for 30 seconds, 40 cycles). The ΔCt method was used to quantify changes in mRNA levels. The average Ct value for each target gene was normalized to the average Ct value of the housekeeping gene 18s rRNA. The fold change was calculated by normalizing each ΔΔCt value to the corresponding control ΔΔCt value.

[0089] (2) The mitochondrial membrane potential in the cells was measured by the staining solution CMXROS (Invitrogen, M46752). Wash three times with PBS to remove serum and protein. Dissolve CMXROS in an appropriate amount of DMF or deionized water according to the manufacturer's guidelines to prepare a working concentration of 50 μM. Preheat the cell culture plate to 37°C and 5% CO2. After adding the CMXROS working solution, incubate at 37°C and 5% CO2 for 30 minutes. After the incubation, wash three times with PBS again to remove unbound probes. Fix and seal the slides with an anti-fluorescence attenuation sealing solution containing DAPI. Observe using a fluorescence microscope or confocal microscope, measure the CMXROS fluorescence intensity to evaluate the cell membrane potential, and high-potential cells show strong fluorescence. After collecting the image, use software to quantitatively analyze the fluorescence intensity and evaluate changes in cell membrane potential.

[0090] (3) The ATP content in cells was measured using the CellTiter-Glo fluorescent cell activity kit (Promega, G7570). 4Neurons were cultured in 48-well plates and cultured to full maturity as described above. After 72-96 hours of lentivirus infection, the solid powder provided by the kit was completely added to the reaction solution to make a working solution. Because the working solution can lyse the monolayer cells, there is no need for an extraction step. The neuron culture solution was aspirated, 150 microliters of working solution was added to each well, and transferred to a white-bottomed 96-well plate (Invitrogen, 136101), and incubated at room temperature in the dark for 10 minutes. ATP (Sigma, A2383) (0, 0.1, 1, 10 micromolar) was used as a standard curve, and ATP bioluminescence was measured at 560 nanometers by a plate reader.

[0091] (4) Transdifferentiation of skin cells from healthy aging patients into human basal forebrain cholinergic transdifferentiated neurons (BFCN) was achieved using a specific transdifferentiation protocol provided by Esai Teng Biotech. The transdifferentiated neurons were cultured in DMEM / F12 medium (Thermo Fisher, 11320033) containing 2% N2A supplement (Stem Cell, 07152) for 30 to 40 days to ensure that the cells were fully mature and exhibited the characteristics of basal forebrain cholinergic neurons. Compared with IPSC cells, the transdifferentiated cells can better retain the age information of the cell source. During the culture process, after the cells reached an appropriate state, a lentivirus with an MOI of 5 was added to the transdifferentiated cells to infect and potentially change the gene expression of the cells. 72 to 96 hours after infection, immunofluorescence staining was performed to evaluate the expression of specific markers of the transdifferentiated cells.

[0092] 2. Experimental results

[0093] (1) Figure 4 As shown in A, after lentivirus transfection of primary neurons, they were stimulated with bicuculline (BIC). Compared with the control group, overexpression of Mt-CREB DIEDML The primary neurons showed higher mitochondrial mRNA transcription levels after excitatory stimulation, proving that this mutation promoted neural activity-induced mitochondrial gene expression in primary hippocampal neurons.

[0094] (2) Figure 4 B and 4C show that Mt-CREB was overexpressed in primary neurons at resting state. DIEDML It did not affect the mitochondrial membrane potential characterized by CMXROS staining, suggesting that overexpression of this transcription factor would not damage mitochondrial quality.

[0095] (3) Figure 4 D shows that overexpression of Mt-CREB DIEDML It can increase the adenosine triphosphate (ATP) content in primary neuronal cells in the resting state.

[0096] (4) Figure 4 As shown in E and 4F, the results of cell immunofluorescence staining showed that Mt-CREB was overexpressed in human basal forebrain cholinergic transdifferentiated neurons (BFCN) DIEDML The level of senescence marker p16 can be improved. p16 is a cell cycle inhibitory protein, and an increase in its level is usually associated with cell senescence. In this example, a decrease in the level of p16 can be observed, indicating an improvement in the cell senescence state.

[0097] Example 4 Overexpression of Mt-CREB DIEDML Effects on spatial cognition in aging mice

[0098] 1. Experimental methods

[0099] (1) Animal model: 30-40 male C57BL / 6 mice, divided into young mice (3 months, >10 mice) and old mice (16-18 months, >10 mice in each group). The experiment was divided into the following groups:

[0100] Experimental group 1 (young control group): no treatment;

[0101] Experimental group 2 (old control group): The hippocampus was specifically injected with the control virus of Mt-mNeonGreen, with an injection volume of 3×10 12 TU per well, and to cover the entire hippocampus, each mouse was injected with four wells;

[0102] Experimental group 3 (old experimental group): the same as experimental group 2, but the injected virus was adeno-associated virus AAV2 / 9-Mt-CREB DIEDML .

[0103] (2) AAV2 / 9-Mt-CREB DIEDMLThe virus was produced by Shanghai Shengbo Biotechnology Co., Ltd. and was used for genetic manipulation of the mouse hippocampal CA1 region. Before the experiment began, the mice were first anesthetized with isoflurane mixed gas (0.5% oxygen / 3% isoflurane) to ensure that the mice maintained an appropriate depth of anesthesia during the operation. Once the mice entered a stable state of anesthesia, the concentration of isoflurane was reduced to (0.5% oxygen / 1.5% isoflurane) to maintain anesthesia. According to standard surgical procedures, a precise hole was punched in the mouse skull with a hole diameter of approximately 0.5 mm to facilitate subsequent virus injection. The precise coordinates of the hippocampal CA1 region were: 2.0-2.5 mm posteriorly and 1.4-1.7 mm laterally from the bregma point of the brain. Using the Nanoject II system, the AAV virus was precisely injected into the mouse hippocampal CA1 region. After the injection was completed, the injection electrode was left at the injection site for 10 minutes to ensure sufficient diffusion of the virus in the target area. Afterwards, the injection electrode was slowly removed to avoid damage to surrounding tissues. After the virus injection, the mice were returned to their cages to recover, and the virus expression period lasted for 4-6 weeks. During this time, specific genes in the CA1 region of the mouse hippocampus will be expressed, preparing for subsequent behavioral experiments.

[0104] (3) After the viral expression period, mice began behavioral training related to spatial memory.

[0105] a. The new location recognition test was conducted in an empty open field box. Before the test, the mice first underwent a 10-minute adaptation period in the field box, and different visual spatial markers were affixed to each boundary of the field box to provide sufficient spatial information. After the adaptation period, the mice were placed in the field box with two identical objects (positions B and C) in the box, and the mice explored the two objects for 10 minutes. After a 2-hour memory retention period, the mice were placed back in the field box again, and at this time a familiar object (original position C) was moved to a new position (position A), while the other object remained in place. The mice compared and explored the new and old positions of the objects and were allowed to explore freely in the box for another 10 minutes. By observing the exploratory behavior of the mice in response to changes in the position of the objects, their spatial memory ability can be evaluated. The mice showed more interest in exploring objects in new positions, indicating that they have good spatial memory ability. The recognition coefficient calculation method is as follows:

[0106]

[0107] b. In the behavioral experiment of Barnes maze, a white PVC circular platform with a diameter of 100 cm was used, which was designed with 20 5 cm holes. The platform was fixed at a height of 100 cm from the ground to ensure the safety of mice when performing the task. In order to provide the necessary visual cues, 4 visual cues were placed around the platform, which helped the mice to locate and remember the space during the experiment. During the adaptation period of the experiment, the mice were first placed in the central starting cylinder (8 cm in diameter and 12.5 cm in height), which provided a familiar and safe environment for the mice. After being placed in the cylinder for 5 seconds, the mice were guided to the escape box through the target hole, which provided a dark and quiet refuge for the mice. The mice stayed in the escape box for 2 minutes, which helped the mice to establish a preliminary memory of the location of the target hole. Entering the acquisition phase, the mice were placed in the starting cylinder again, and after 5 seconds of adaptation, the mice were allowed to explore the platform freely. The goal of the mice was to find and enter the target hole to enter the escape box. The test will end if the mouse successfully enters the escape box or the 3-minute time has expired. Each mouse undergoes 3 trials per day for 4 consecutive days, with a 20-minute interval between each trial. This repeated trial design helps the mice gradually learn and remember the location of the target hole. In the detection test (day 5), both the target hole and the error hole were blocked, and the mouse was placed in the center of the platform and allowed to explore the venue for 3 minutes. The purpose of this stage is to test the mouse's memory of the location of the target hole and its spatial navigation ability. By measuring the number and time that the mouse enters the target hole or target area, the mouse's spatial memory ability, learning ability, and ability to adapt to environmental changes can be evaluated.

[0108] 2. Experimental results

[0109] like Figure 5 As shown, 16-month-old mice received AAV2 / 9-Mt-CREB DIEDML Behavioral tests related to spatial memory were performed 8-12 weeks after virus injection into the hippocampus. Figure 5 A) and Barnes maze ( Figure 5 B) In both behavioral paradigms, aged mice showed impaired spatial memory, whereas overexpression of Mt-CREB DIEDML It can improve the spatial recognition ability of aged mice. Both behavioral results suggest that enhancing mitochondrial gene transcription in the hippocampus can reshape the loss of spatial memory caused by aging.

[0110] Example 5 Overexpression of Mt-CREB DIEDML Effects on neuronal senescence in aged mice

[0111] 1. Experimental methods

[0112] (1) Preparation of brain tissue slices. Mice of the required age were transfected with AAV2 / 9-Mt-CREB DIEDML After virus overexpression treatment, terminal anesthesia was performed and then perfused with PBS to remove blood components in the blood vessels and reduce background interference during subsequent tissue processing. It was then perfused with 4% paraformaldehyde. After perfusion, the mouse brain was removed and fixed in 4% paraformaldehyde for 3 hours to ensure adequate tissue fixation. The fixed brain was dehydrated with 30% sucrose at 4°C for 24 hours. The dehydration effect of sucrose helps prevent the formation of ice crystals and protect tissue structure. The dehydrated brain tissue was sliced ​​at -20°C using a Leica CM1800 cryostat to cut brain slices with a thickness of 35 microns. The cut slices were collected in a cryoprotective buffer consisting of 40% glycerol, 30% 0.1M PBS, and 30% ethylene glycol.

[0113] (2) Brain tissue sections were subjected to immunohistochemical staining. After washing, tissue sections were blocked with 10% normal donkey serum for 1 hour and then incubated at 4°C. Sections were incubated with primary antibodies at 4°C overnight (Table 1). Detection was performed by incubation with appropriate fluorescently labeled Alexa Fluro secondary antibodies (Table 2) at 20°C in the dark for 3 hours. Sections were mounted on gelatin-coated slides and mounted with a medium containing or not containing 4',6-diamidino-2-phenylindole. In all steps, after removing the solution, the sections were washed three times with PBS.

[0114] (3) The brain tissue sections were stained with β-galactosidase to detect tissue aging. This staining uses X-Gal as a substrate and generates a dark blue product under the catalysis of aging-specific β-galactosidase. The color change is observed under an optical microscope to analyze the aging status of the tissue. The SA-β-gal activity of brain tissue sections was incubated with the chromogenic substrate X-Gal in a pH 6.0 buffer (Cell Signaling Technology, 9860). After incubation in a 37°C incubator in the dark for 12 to 16 hours, the sections were washed with PBS, sealed with a neutral resin (Biosharp, BL704A), and observed under a bright field or phase contrast microscope.

[0115] (4) The ATP content in the tissue was measured by CellTiter-Glo fluorescent cell activity kit (Promega, G7570) on a white 96-well plate (Invitrogen, 136101). The mouse tissue was weighed and homogenized in 500 μl of Tris-saturated phenol (Shyuanye, product number R21022). ATP was extracted by 400 μl of chloroform (Sigma, CX1060) and 300 μl of ddH2O. After mixing the reagents, centrifuge at 4°C 13000g for 20 minutes, and the upper aqueous phase contained ATP. The extracted ATP was subjected to luciferase detection. Using known concentrations of ATP (Sigma, A2383) (0, 0.1, 1, 10 μM) as the standard curve, ATP bioluminescence was measured at 560 nm by a plate reader.

[0116] 2. Experimental results

[0117] (1) Figure 6 A shows Mt-CREB DIEDML Expression pattern in mouse hippocampal CA1. Figure 6 B, 6D, and 6E show that Mt-CREB is overexpressed in the hippocampus of 16-month-old mice DIEDML The results showed that the SA-β-gal activity and p16 level in the mouse brain were significantly reduced, indicating that Mt-CREB DIEDML May have potential in vivo effects on aging-related changes.

[0118] (2) Figure 6 C shows young mice, virus control group and Mt-CREB DIEDML ATP levels in the hippocampus of the overexpression group. The results showed that aging reduces ATP levels and overexpression of Mt-CREB DIEDML Afterwards, ATP levels in the brains of aged mice were partially reshaped.

[0119] Through the above cell and animal experiments, it was found that Mt-CREB DIEDML The transcription factor can delay the aging process of the mouse brain, which is manifested by slowing down the decline of adenosine triphosphate caused by aging and improving spatial memory function. This discovery provides a new idea for exploring ways to improve the normal aging process of the brain and provides support for the health and social functions of the elderly. In summary, the present invention deeply explores the potential application of this new factor that promotes mitochondrial genome transcription in delaying the normal aging process of the brain, provides a theoretical and experimental basis for the future development of related drugs or treatment methods, and provides support for the health and social functions of the elderly.

[0120] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A transcriptional regulator CREB DIEDML , characterized in that, In CREB WT The amino acids 130-135 of RRPSYR mutated to DIEDML, and the CREB WT The amino acid sequence has 95% or more homology to the sequence shown in SEQ ID NO.

1.

2. A mitochondrial transcriptional regulator Mt-CREB DIEDML , characterized in that, The transcriptional regulatory factor CREB according to claim 1 DIEDML A mitochondrial targeting sequence is added to the N-terminus of the amino acid sequence of , and the amino acid sequence of the mitochondrial targeting sequence is shown in SEQ ID NO.

2.

3. The mitochondrial transcriptional regulatory factor Mt-CREB according to claim 2 DIEDML , characterized in that, Mt-CREB DIEDML The amino acid sequence is shown in SEQ ID NO.

3.

4. A protein encoding the mitochondrial transcription factor Mt-CREB as claimed in claim 2 or 3 DIEDML of nucleic acid.

5. The nucleic acid according to claim 4, characterized in that The nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

4.

6. An expression vector comprising the nucleic acid according to claim 4, characterized in that The expression vector is a lentiviral expression vector or an adeno-associated virus expression vector.

7. The mitochondrial transcriptional regulatory factor Mt-CREB according to claim 2 or 3 DIEDML Application in the preparation of anti-brain aging drugs.

8. The use according to claim 7, characterized in that: The active ingredient of the drug includes the mitochondrial transcription regulatory factor Mt-CREB according to claim 2 or 3 DIEDML , the nucleic acid according to claim 4 or 5, and the expression vector according to claim 6, or a combination of one or more thereof.

9. The use according to claim 7, characterized in that: The drug has one or more of the following uses: (1) Selectively promote the expression of neuronal mitochondrial genome; (2) Slow down the decline in adenosine triphosphate levels caused by aging; (3) Improve the brain's spatial memory function.

10. A method for delivering an anti-aging drug for the brain, characterized in that: The steps include: Step S1, using an expression vector as claimed in claim 6; Step S2, encapsulating the expression vector in step S1 into lentivirus or adeno-associated virus particles; Step S3: using the lentivirus or adeno-associated virus particles in step S2 to deliver anti-brain aging drugs, wherein the anti-brain aging drugs are delivered to target cells or living bodies through the infection of the lentivirus or adeno-associated virus particles.

11. A fluorescence and / or chemiluminescence detection method for screening anti-brain aging drugs, characterized in that: The steps include: Step S1, cloning the mitochondrial targeting sequence of claim 2 into a cell containing the CREB of claim 1 WT The fluorescent and / or chemiluminescent vector is recombined to obtain a first biosensor, wherein the first biosensor is used to specifically monitor CREB in mitochondria WT The transcriptional activity of CREB in mitochondria is reflected by changes in fluorescence and / or chemiluminescence WT The binding strength with downstream response elements indicates the expression of mitochondrial genes and neuronal activity; Step S2, expressing the first biosensor in step S1 in neurons; Step S3, treating the neurons in step S2 with the drug to be screened, and determining the promoting effect of the drug to be screened on mitochondrial gene expression and neuronal activity by detecting the changes in fluorescence and / or chemiluminescence of the first biosensor.

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