Mouse model construction method for simulating morbidity process of brain premature senility

Abstract

The invention provides a mouse model construction method for simulating the morbidity process of brain premature senility, ABAD knock-down is achieved in the hippocampus of a healthy adult mouse, and the pathological process of brain premature senility morbidity is simulated. According to the mouse model obtained by the invention, the expression quantity of the brain hippocampus ABAD gene is 30% or more lower than that of a wild type mouse, and the mouse model shows pathological characteristics which tend to be consistent with those of a mouse with brain premature senility.

Description

Technical Field

[0001] This invention relates to the field of biomedicine and discloses a mouse model for simulating the pathogenesis of premature brain aging and its construction method. Background Technology

[0002] Premature brain aging refers to the phenomenon where an individual's cognitive function, neural structure, and physiological and biochemical indicators prematurely exhibit signs of aging before reaching the physiological age of old age. This phenomenon is not a natural aging process but is induced or accelerated by a variety of complex factors (such as genetic susceptibility, chronic stress, unhealthy lifestyle, environmental toxins, and specific diseases). The key to recognizing premature brain aging lies in focusing on its core symptoms, which typically manifest as: significant memory decline, especially difficulty retrieving recent memories; inattention, difficulty concentrating on tasks; decreased executive function, manifested as weakened planning, organization, decision-making, and problem-solving abilities; slower information processing speed; difficulty learning new knowledge or skills; increased mood swings, prone to anxiety, depression, or apathy; and the possible occurrence of non-cognitive symptoms such as sleep disturbances and fatigue. These symptoms not only severely impact an individual's quality of life, work performance, and social interactions but also serve as important risk signals for developing serious neurodegenerative diseases in the future (such as Alzheimer's disease and Parkinson's disease).

[0003] Because premature brain aging poses a significant threat and is receiving increasing attention, establishing and researching reliable models of premature brain aging is crucial. The importance of premature aging models is mainly reflected in the following aspects:

[0004] 1. Unveiling Pathological Mechanisms: By simulating key characteristics of premature aging in the human brain (such as neuronal loss, synaptic dysfunction, neuroinflammation, oxidative stress, and protein homeostasis imbalance), the model provides scientists with a controllable experimental platform to delve into the core pathological mechanisms driving premature aging at the molecular, cellular, and circuit levels. This is far more direct and efficient than conducting research in the complex and ever-changing human body.

[0005] 2. Accelerating Drug Development and Screening: Premature aging models are key tools for evaluating the effectiveness and safety of potential interventions (drugs, gene therapy, lifestyle interventions, etc.). Rapidly screening a large number of candidate compounds or therapies on these models can significantly shorten the long cycle from basic research to clinical application, providing strong support for the development of drugs that delay or even reverse premature brain aging.

[0006] 3. Understanding Disease Links and Early Warning: Premature brain aging is often seen as a "prelude" to serious neurodegenerative diseases. Models can be used to study the process of premature aging transforming into more serious diseases, and to identify key biomarkers (such as specific proteins, metabolites, and imaging features), laying the foundation for early diagnosis and risk warning in humans.

[0007] To promote a healthy aging process and prevent age-related health problems, a correct understanding of aging mechanisms and the development of effective and affordable anti-aging intervention strategies are of significant social importance and economic benefit. In this process, animal models of aging are powerful tools for studying the mechanisms of aging. Aging models are generally divided into two categories: natural aging models and accelerated aging models. During the aging process, naturally aging mice exhibit many phenotypes similar to those of normal human aging; however, research in these models is time-consuming, labor-intensive, and expensive, and individual differences are significant compared to accelerated aging models. Therefore, induced accelerated aging models are favored by researchers due to their readily available resources, shorter modeling time, and relatively controllable aging effects.

[0008] Currently, commonly used mouse aging models have many problems:

[0009] 1. Natural Aging Model: Although this model can realistically reflect the progressive aging process, it still has obvious drawbacks: The primary limitation is the excessively long experimental period, which usually requires feeding mice for 18 to 24 months to reach the required old age stage, resulting in low overall research efficiency and slow experimental progress. At the same time, due to the multiple influences of individual genetic background differences, subtle changes in the feeding environment, and individual random factors, different old mice exhibit significant fluctuations in cognitive, physiological, and other aging phenotypes, making it difficult to ensure the stability and consistency between experimental groups. This leads to deviations between experimental results and actual conditions, interfering with the complete execution of the experimental design and data collection.

[0010] 2. D-galactose-induced aging model: The core mechanism involves intraperitoneal injection of D-galactose, which induces aging through oxidative stress, thus mimicking aging. Natural aging, however, involves complex multi-dimensional characteristics such as genomic instability and telomere loss, leading to mechanistic differences between the two. Pathologically, while this model can partially simulate age-related changes such as neuroinflammation, it struggles to reproduce disease-induced neurological damage at the neural level, showing significant differences compared to naturally aging mice.

[0011] 3. The SAMP8 rapid aging model: While widely used in aging and AD research, its pathological manifestations, though capable of simulating cognitive decline and neuronal damage, struggle to reproduce typical pathological features of premature brain aging and AD (such as β-amyloid deposition and neurofibrillary tangles). Furthermore, its cerebral blood flow changes and neuroinflammatory mechanisms are relatively limited. Its application is also restricted, primarily suited for oxidative stress or inflammation research, and insufficient for simulating complex mechanisms (such as hereditary premature aging and multidimensional aging pathways). Therefore, it needs to be combined with other models, such as transgenic models, for complementary validation to improve reliability.

[0012] In summary, the causes and pathological mechanisms of premature brain aging are not yet fully understood, and existing models used to study premature brain aging have limited applicability due to numerous inherent limitations. Therefore, developing methods for constructing mouse models that can accurately simulate the pathogenesis of premature brain aging is of indispensable importance for deeply understanding the core mechanisms of premature brain aging and for providing reliable preclinical animal testing vectors for drug development. Summary of the Invention

[0013] To address the existing technical challenges, this invention provides a method for constructing a mouse model that simulates the pathogenesis of premature brain aging. By knocking down ABAD in the hippocampus of healthy adult mice, the pathological process of premature brain aging can be simulated.

[0014] Specifically, the technical solution of the present invention is as follows:

[0015] The first aspect of this invention provides a method for constructing a mouse model that simulates the pathogenesis of premature brain aging. A minimally invasive method is used to introduce a recombinant knockout vector carrying the ABAD knockout gene into the hippocampus of a mouse brain to knock down the ABAD gene in neurons, thereby achieving "simulation of the pathogenesis of premature brain aging." The mouse model obtained by this method shows that the expression level of the ABAD gene in the hippocampus of the mouse model is more than 30% lower than that of wild-type mice, and its pathological characteristics are consistent with those of mice with premature brain aging.

[0016] A second aspect of the invention provides a vector system for knocking out target genes, including adenovirus, adeno-associated virus, lentivirus, Cre-Lox and CRISPR-Cas systems; preferably, a plasmid and a recombinant knockdown system of AAV9 virus, pAAV9-U6-M-shABAD-EGFP, are selected.

[0017] A third aspect of the present invention provides steps in a model building process, including the following steps:

[0018] (1) Submit the coding region sequence of ABAD to siDirect for siRNA target screening, select the optimal interference target based on the score and specificity index, and synthesize the corresponding shRNA sequence accordingly.

[0019] (2) Insert the fragment synthesized in (1) into the pHBAAV-U6-MCS-CMV-EGFP vector to construct the recombinant target plasmid pAAV9-U6-M-shABAD-EGFP;

[0020] (3) The recombinant plasmid pAAV9-U6-M-shABAD-EGFP and the helper packaging plasmid were co-transfected into cells to package the virus and harvest the target virus particles.

[0021] (4) The bilateral hippocampal regions of the mouse were located using a stereotaxic instrument (location coordinates: -2.3 mm posterior to the anterior fontanelle, ±1.8 mm lateral to the midline, depth -2.2 mm). The viral suspension from (3) was slowly injected into the target brain region through a micro-injection system to achieve stable gene knockdown.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] 1. Compared with drug-induced aging mice, the aging etiology of this model mice is clear, and other toxic side effects caused by drugs or chemical stimulation are excluded. At the same time, the manufacturing process is repeatable and has high reproducibility.

[0024] 2. The vector system used in this method has high efficiency and specificity infectivity, mainly focusing on infecting neurons in the hippocampus, which can effectively eliminate off-target problems;

[0025] 3. This model is directly linked to mitochondrial-related functions and causes aging by regulating neurons, making the model convincing in the theory of aging. Attached Figure Description

[0026] The accompanying drawings, which are included to provide a further understanding of the embodiments of the present invention and form part of this application, are not intended to limit the embodiments of the present invention.

[0027] Figure 1 Schematic diagram of ABAD knockdown plasmid construction, where A is a schematic diagram of siRNA structure; B is a schematic diagram of AAV vector system structure.

[0028] Figure 2 Construction and evaluation of the ABAD knockdown model in SY5Y cells. A and B show the expression and analysis data of ABAD protein in SY5Y cells 48 h after transfection, detected by Western blotting; C and D show the fluorescence effect of mitochondrial membrane potential by flow cytometry and the analysis data; E and F show the fluorescence effect of mitochondrial reactive oxygen species by flow cytometry and the analysis data; G is a visual representation of two sets of data from the mitochondrial respiration test; H shows the basal respiration level of cells in the mitochondrial respiration test; I shows the proton leakage level of cells in the mitochondrial respiration test; J shows the maximum electron transport capacity of cells in the mitochondrial respiration test; K shows the non-mitochondrial respiration level of cells in the mitochondrial respiration test; L shows the ATP level of cells in the mitochondrial respiration test; M shows the reserve respiration capacity of cells in the mitochondrial respiration test. N and O are schematic diagrams and analysis data of cellular senescence-related β-galactosidase staining. Bar = 100 μm. *Data are expressed as mean ± standard error of mean (SEM) (*P<0.05, **P<0.01, ***P<0.001).

[0029] Figure 3Construction and evaluation of an ABAD knockdown model in the hippocampus of normal C57BL / 6 mice. A shows the effect of fluorescent GFP after stereotactic injection into the hippocampus, Bar = 1000 μm; B and C show the expression and analysis of ABAD protein in the mouse hippocampus detected by Western blotting; D and E show the expression and statistical analysis of ABAD protein in the mouse hippocampus detected by immunofluorescence, Bar = 50 μm; F shows a visual representation of mitochondrial respiration test data for two groups; G shows the proton leakage level of complex I; H shows the oxidative phosphorylation capacity of complex I + complex II; I shows the oxidative phosphorylation capacity of complex I; J shows the maximum electron transport capacity of complex II; K shows the non-mitochondrial respiration level; L shows the positive co-localization of neuronal NeuN, nuclear DAPI, and TUNEL apoptosis cells; M shows the statistical graph of co-localized positive cells; N shows the co-localization of neuronal postsynaptic dense protein and synaptic vesicle protein; O shows the statistical graph of co-localization positive analysis, Bar = 50 μm. Data are expressed as mean ± standard error of mean (SEM) (*P<0.05, **P<0.01, ***P<0.001).

[0030] Figure 4 Inflammatory response was induced in normal C57BL / 6 mice by ABAD knockdown in the hippocampus. A is a schematic diagram representing the number of microglia in the hippocampus (Bar = 100 μm); B is a schematic diagram representing the number of microglia in the CA1, CA3, and DG regions of the hippocampus (Bar = 50 μm); C is a statistical graph showing the number of microglia in the CA1, CA3, and DG regions; D is a schematic diagram representing the number of astrocytes in the hippocampus (Bar = 100 μm); E is a schematic diagram representing the number of astrocytes in the CA1, CA3, and DG regions of the hippocampus (Bar = 50 μm); F is a statistical graph showing the number of astrocytes in the CA1, CA3, and DG regions; G is a statistical graph showing the analysis of the pro-inflammatory factor IL-1β in the hippocampus; H is a statistical graph showing the analysis of the pro-inflammatory factor TNF-α in the hippocampus; and I is a statistical graph showing the analysis of the pro-inflammatory factor IL-6 in the hippocampus. Data are expressed as mean ± standard error of mean (SEM) (*P<0.05, **P<0.01, ***P<0.001).

[0031] Figure 5ABAD knockdown in the hippocampus of normal C57BL / 6 mice induced senescence. A and B show the number and analysis of senescence-associated β-galactosidase (SA-β-gal) positive cells in the mouse hippocampus (Bar = 100 μm); C shows the statistical analysis of advanced glycosylation (AGE) levels in the mouse hippocampus; D shows the Sudan Black B staining pattern in the mouse hippocampus (Bar = 100 μm); E and F show the expression and analysis of the senescence marker P21 in the mouse hippocampus; G and H show the number and analysis of DCX (dicortin) positive cells in the subgranular region (SGZ) of the dentate gyrus in the mouse hippocampus (Bar = 50 μm). Data are expressed as mean ± standard error of mean (SEM) (*P < 0.05, **P < 0.01, ***P < 0.001). Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the following description is provided in conjunction with its implementation.

[0035] Examples and accompanying drawings will be used to further illustrate the present invention in detail. The illustrative embodiments and descriptions of the present invention are for explaining the present invention only and are not intended to limit the present invention.

[0036] Example 1: Construction of knockdown plasmid

[0037] 1.1 siRNA Design and Construction

[0038] First, the mRNA sequence was accessed from the NCBI database to obtain the complete coding sequence (CDS) information of mouse ABAD. Second, using the siDirect online tool, the ABAD gene CDS sequence was input to screen for ideal siRNA targets. The following optimal targets and their sequences were ultimately selected: Forward strand sequence: 5'-CCAGCGAGUUCUUGAUGUGAATT-3'; Complementary strand: 5'-UUCACAUCAAGAACUCGCUGGTT-3'. The confirmed sequences were then chemically synthesized by Guangzhou Aiji Biotechnology Co., Ltd., lyophilized, and dissolved in DEPC-treated water to prepare a 20 μM stock solution.

[0039] 1.2 Carrier Construction

[0040] The recombinant plasmid vector pAAV9-U6-M-shABAD-EGFP was constructed by inserting sh-ABAD into the MCS of pHBAAV-U6-MCS-CMV-EGFP. To verify and eliminate the influence of the blank plasmid, the control plasmid pAAV9-U6-M-scramble-EGFP was also constructed. The construction methods of the two plasmids are almost identical, and the specific procedures are as follows:

[0041] First, the target fragments sh-ABAD and sh-scramble were cloned using PCR technology. The primer design is shown in the table below.

[0042]

[0043]

[0044] After purifying the target PCR product, the backbone vectors pHBAAV-U6-MCS-CMV-EGFP and sh-ABAD / sh-scramble were digested with BamHI and EcoRI at 37°C for 3 hours. The digested products were then recovered using a purification kit. The mixtures were then combined at a product:backbone ratio of 3:1, and T4 ligase was added for overnight ligation.

[0045] Take 100 μL of competent Ecoli DH5α cell suspension, add 1 μL of ligation product and transfect on ice for 30 min. Then add 1 mL of LB liquid medium, mix well and at 37 °C, take 100 μL of bacterial culture and spread it on a selection plate containing antibiotics. After single colonies grow, pick single colonies for PCR verification.

[0046] Positive clones obtained by PCR were sent to a sequencing company for sequencing to verify the sequence of the vector contained in the positive clone bacteria. After obtaining positive clones with correct sequences, high-purity recombinant plasmids pAAV9-U6-M-shABAD-EGFP and pAAV9-U6-M-scramble-EGFP were extracted for further experiments. The results of vector construction are shown below. Figure 1 As shown, where Figure 1 A shows a schematic diagram of the si-RNA structure. Figure 1 B is a schematic diagram of the plasmid-virus vector system structure.

[0047] Example 2: Construction of ABAD knockdown cell model and assessment of its mitochondrial function and aging indicators

[0048] 2.1 Construction of SH-SY5Y cell model

[0049] (1) Experimental subject: SH-SY5Y cells;

[0050] (2) Experimental Procedure: First, cell transfection was performed. Cells were seeded into 6-well plates. When the confluence reached 30%-50%, Opti-MEM, Lipofectamine RNAiMAX, and siRNA were mixed and added to the wells for transfection. The siRNA added here was the sequence designed in Example 1, and the transfection method was transient transfection. 300 μL of the mixture was added to each well. After the above treatment was completed, the 6-well plates were placed back in a cell culture incubator and incubated at 37°C and 5% CO2 for 48 h before collecting the cells. Finally, Western blotting was used to verify the expression of ABAD protein in the two groups of cells.

[0051] (3) Experimental results: After cell transfection, Western blot was used to verify the expression of ABAD protein in the two groups of cells. The results showed that the expression level of ABAD protein in the ABAD knockdown experimental group was significantly reduced by more than 30% compared with the control group. Figure 2 A and B, NC are the control group, and siABAD is the ABAD knockdown group, indicating that the cell model was successfully established in the experiment and the intracellular ABAD protein level decreased in the experimental group.

[0052] 2.2 Assess mitochondrial respiratory function

[0053] (1) Experimental subjects: SH-SY5Y cell control group (NC) and ABAD knockdown experimental group (siABAD);

[0054] (2) Experimental Procedure: Mitochondrial respiratory function was assessed using the Oxygraph-2k high-resolution respiratory measurement system. ABAD knockdown cells constructed using the method described in Example 2.1 were collected and counted. The measurement chamber was cleaned and background correction was performed. After adding cells and sealing, the basic respiratory values ​​of intact cells were observed. Then, 1 μL of 5 mM oligomycin was injected, and the proton oxygen consumption of the cells was observed. After the oxygen consumption stabilized, 1 μL of 1 mM CCCP uncoupling agent was injected into the chamber in fractional titrations, and the maximum respiratory rate was observed. Finally, 1 μL of 1 mM rotenone and 1 μL of 5 mM antimycin A were injected into the chamber sequentially. After the oxygen consumption curve stabilized, the residual oxygen consumption of non-mitochondrial respiration was observed. Relevant oxygen consumption data were obtained after the experiment.

[0055] (3) Experimental Results: To investigate the effect of ABAD knockdown on related mitochondrial functions, mitochondrial respiratory function was assessed using the Oxygraph-2k high-resolution respiratory measurement system. The results showed that, compared with the control group, the ABAD knockdown group had a significantly higher maximum electron transport capacity (…). Figure 2 J), respiratory reserve capacity ( Figure 2Both M) were significantly reduced, indicating a severe decline in their ability to cope with the energy crisis and their potential to adapt to increased demand; basal respiratory level ( Figure 2 The H) showed a decreasing trend, indicating a tendency for energy demand to decrease under resting conditions, consistent with the overall functional impairment; in addition, the proton leakage level ( Figure 2 I) and nonmitochondrial respiration ( Figure 2 Both groups showed a significant decrease in cellular ATP (K). Figure 2 The lack of statistically significant difference in ABAD levels may be related to compensatory mechanisms. These findings suggest that intracellular ABAD knockdown leads to impaired mitochondrial respiratory function.

[0056] 2.3 Assess mitochondrial membrane potential and ROS

[0057] (1) Experimental subjects: SH-SY5Y cell control group (NC) and ABAD knockdown experimental group (siABAD);

[0058] (2) Experimental Procedure: Mitochondrial membrane potential and ROS were detected using flow cytometry. ABAD knockdown cells constructed using the method described in Example 2.1 were collected and stained at 37°C for 30 min using the JC-1 mitochondrial assay kit and the ROS assay kit, respectively. After washing with buffer and centrifugation, the cells were resuspended. The cells were then added to a flow cytometer for detection: JC-1 monomeric and polymeric fluorescence signals were acquired using 530 nm and 590 nm filters, respectively; ROS fluorescence signals were acquired using a 530 nm filter. Data were analyzed using FlowJo software.

[0059] (3) Experimental results: Mitochondrial membrane potential and ROS content were assessed by flow cytometry using mitochondrial JC-1 and ROS. The results showed that compared with the control group, the ABAD knockdown group had significantly higher mitochondrial membrane potential impairment indicators. Figure 2 C and D) indicate a decrease in mitochondrial membrane potential; simultaneously, the ROS fluorescence intensity also significantly increased (C and D). Figure 2 E and F) indicate elevated levels of intracellular reactive oxygen species (ROS). These results suggest that intracellular ABAD knockdown not only impairs mitochondrial respiratory function but also leads to impaired mitochondrial membrane potential and elevated ROS, ultimately driving mitochondrial functional deterioration.

[0060] 2.4 Assessment of aging-related β-galactosidase staining

[0061] (1) Experimental subjects: SH-SY5Y cell control group (NC) and ABAD knockdown experimental group (siABAD);

[0062] (2) Experimental Procedure: Cell senescence was assessed using a senescence-associated β-galactosidase (SGA) kit. An ABAD knockdown cell model was constructed as described in Example 2.1. Cells were fixed in fixative at room temperature for 15 min, washed with PBS, and then incubated overnight at 37°C in a CO2-free incubator with SGA staining solution. The next day, the staining solution was removed, and the cells were washed with PBS and mounted. Finally, the cells were observed under an optical microscope.

[0063] (3) Experimental Results: Cellular senescence was assessed using senescence-related β-galactosidase staining. Results showed that, compared to the control group, the ABAD knockdown group exhibited more blue-positive cells (…). Figure 2 N). Statistical analysis shows that ( Figure 2 The proportion of senescence-related β-galactosidase-positive cells in the ABAD knockdown group was significantly higher than that in the control group, indicating a significant cellular senescence phenomenon in the ABAD knockdown group. These results suggest that ABAD knockdown induces mitochondrial dysfunction, thereby promoting cellular senescence.

[0064] Example 3: Construction of ABAD knockdown mouse model and assessment of mitochondrial function, neuronal damage and aging 3.1 Construction of ABAD knockdown mouse model

[0065] (1) Experimental subjects: C57BL / 66-month-old mice (control group, scramble) and ABAD knockdown experimental group (shABAD); (2) Experimental procedure: Six-month-old C57BL / 6 mice were anesthetized by intraperitoneal injection of 1.25% tribromoethanol and fixed on a stereotaxic instrument. The anterior and posterior fontanelles of the mice were fully exposed. The anterior fontanelle Bregma was used as the 0 point. According to the determined position parameters of the injection brain region (anterior fontanelle -2.3mm, lateral ±1.8mm, depth -2.2mm), the cranial drill was moved to open a suitable bone window. The microsyringe (1ul specification) was rinsed with PBS and the patency was checked at the same time. 1μl of diluted virus was drawn up, the microsyringe pump was assembled and placed above the bone window, with the needle tip parallel to the skull (Z=0), and slowly lowered to the target depth. The virus injection was completed within 10 minutes and the needle was left in place for 5 minutes to facilitate virus diffusion. After the operation, the wound was disinfected and sutured, and the mice were placed on a warming pad to recover. The injected virus was a recombinant adeno-associated virus (AAV9) with a shABAD knockout sequence. The viral particles were obtained by transfecting HEK293 cells with the recombinant plasmid pAAV9-U6-M-shABAD-EGFP, maturing, and then isolated and purified.

[0066] Six months after viral injection, mice were sacrificed and their brains were harvested. The right cerebral cortex and hippocampus were cryopreserved at -80°C (for Western blotting), while the left brain was fixed in 4% paraformaldehyde, dehydrated using a sucrose gradient, embedded in OCT, and cut into 30 μm frozen sections (for immunoblotting). Western blotting: Cryopreserved hippocampal tissue was lysed using a lysis buffer containing RIPA, PICT, and PMSF. The supernatant was collected by centrifugation, and protein concentration was normalized using the BCA method. Proteins were then separated by electrophoresis on a 10% SDS gel for 1 hour, transferred to a PVDF membrane, blocked with 10% skim milk for 1 hour, and incubated overnight with primary antibodies such as ABAD antibody and α-TUBULIN. The primary antibody was recovered the next day, and the tissue was incubated with secondary antibody for 1 hour, followed by washing with TBST. Finally, the tissue was exposed using ultrasensitive developer. Immunofluorescence staining: Frozen sections were thawed for 30 minutes, fixed in 4% paraformaldehyde at room temperature for 10 minutes, and washed with PBS. Permeabilization buffer was then applied for 5 minutes, followed by washing again with PBS. After treatment with blocking solution for 1 hour, the cells were incubated overnight at 4°C with ABAD and TUB3 primary antibodies. The next day, the primary antibody was recovered and incubated with the corresponding fluorescent secondary antibody at room temperature for 1 hour. The cells were washed with PBS and finally stained with nuclear staining agent DAPI for 3 minutes.

[0067] (3) Experimental Results: The above cell experiments have confirmed that ABAD knockdown can induce cellular senescence by impairing mitochondrial function. To verify these findings in an in vivo environment closer to physiological conditions and to further explore the effects of ABAD knockdown on the brain at the levels of histopathology and comprehensive aging phenotypes, we constructed a hippocampus-specific ABAD knockdown adult mouse model. We verified hippocampal ABAD protein expression using immunofluorescence and Western blotting. The results showed that the viral expression location conformed to the hippocampal localization (…). Figure 3 A) and decreased ABAD expression in the hippocampus ( Figure 3 D and E), and Western blotting was used to verify the expression of ABAD protein in the hippocampus of the two groups of mice. The results showed that the expression level of ABAD protein in the experimental group was more than 30% lower than that in the control group. Figure 3 In summary, the virus was accurately located, indicating that the ABAD knockdown model in the adult mouse hippocampus was successfully constructed.

[0068] 3.2 Assessment of mitochondrial respiration capacity in ABAD knockdown mouse model

[0069] (1) Experimental subjects: C57BL / 66-month-old mice (control group, scramble) and ABAD knockdown experimental group (shABAD); (2) Experimental procedure: Approximately 5 mg of fresh mouse hippocampal tissue was added to 2.5 mL of respiratory fluid (MIR05-Kit) and homogenized. After background correction, the homogenate was added to the Oxygraph-2k measurement chamber and sealed. After the values ​​stabilized, 10 μL of substrate 2M glutamate and 10 μL of substrate 400 mM malic acid were added. After the system stabilized, the LEAK value of complex I was obtained. 20 μL of 500 mM ADP was added, and the maximum oxidative phosphorylation value of complex I was determined. 20 μL of 1M succinic acid was added, and the maximum oxidative phosphorylation values ​​of complex I and complex II were obtained. 10 μL of 1 mM rotenone was added, and the maximum electron transfer value of complex II was determined. Finally, 10 μL of 5 mM antimycin A was added to obtain the non-mitochondrial respiratory value.

[0070] (3) Experimental Results: Based on previous cell experiments confirming that ABAD knockdown impairs mitochondrial function, mitochondrial respiratory function in mice was assessed using fresh mouse hippocampal tissue homogenate in a successfully constructed in vivo model. The results showed that, compared to the control group, the ABAD knockdown group mice exhibited reduced mitochondrial function in complex I (… Figure 3 I) and the oxidative phosphorylation capacity of complex I + complex II ( Figure 3 H) decreased significantly, while the maximum electron transport capacity of complex II ( Figure 3 J) showed a decreasing trend, but the proton leakage level of complex I ( Figure 3 G) and non-mitochondrial respiratory levels ( Figure 3 There was no significant difference in K), suggesting that ABAD knockdown impairs mitochondrial respiratory function in the hippocampus of mice. This impairment likely stems from significant disruption of electron transport chain complexes I and II, limiting electron transport and being the primary cause of decreased respiratory capacity. Furthermore, this result is consistent with cellular findings, confirming that ABAD knockdown leads to impaired mitochondrial function.

[0071] 3.3 Evaluation of neurons in ABAD knockdown mouse model

[0072] (1) Experimental subjects: C57BL / 66-month-old mice (scramble) as control and ABAD knockdown experimental group (shABAD); (2) Experimental procedure: Immunofluorescence: The steps of section fixation, permeabilization, washing and blocking were the same as in Case 3.1. PSD95 and SYP primary antibody were used for overnight incubation at 4℃. The subsequent steps of secondary antibody incubation, washing and DAPI nuclear staining were the same as in Case 3.1. TUNEL staining: After section fixation, permeabilization, washing and blocking, NeuN primary antibody, corresponding secondary antibody and DAPI were used for incubation. After washing with PBS, TUNEL staining solution was incubated at 37℃ for 1 h. After washing with PBS again, the slides were finally sealed with anti-quenching agent and coverslip. Images were acquired using an Olympus FV3000 confocal microscope and analyzed using ImageJ software to observe neuronal apoptosis and synaptic structure and function.

[0073] (3) Experimental Results: Studies have shown that mitochondrial dysfunction is an important inducing factor for neuronal apoptosis and synaptic damage. Therefore, we hypothesize that ABAD knockdown-induced mitochondrial dysfunction leads to hippocampal neuronal damage. Neuronal function was assessed by TUNEL staining and PSD95 and SYP co-staining. The results showed that compared with normal control mice, the number of TUNEL-positive and neuronal co-identical cells in the ABAD knockdown group was significantly higher than that in the control group. Figure 3 L and M) indicate that neuronal apoptosis is significantly aggravated in the ABAD knockdown group. Simultaneously, compared to the control group, the ABAD knockdown group showed a significant reduction in PSD95 and SYP co-determined positive signals in immunofluorescence analysis. Figure 3 The presence of N and O indicates that the co-localization of key presynaptic and postsynaptic proteins in the neurons of mice knocked down by ABAD is impaired, disrupting both the integrity of synaptic structure and functional connectivity. These results suggest that ABAD knockdown exacerbates apoptosis and synaptic structural and functional damage in hippocampal neurons, and its mechanism is closely related to mitochondrial dysfunction.

[0074] 3.4 Assessment of hippocampal inflammation levels in ABAD knockdown mouse models

[0075] (1) Experimental subjects: C57BL / 66-month-old mice (control group, scramble) and ABAD knockdown experimental group (shABAD); (2) Experimental procedure: Immunofluorescence: The steps of section fixation, permeabilization, washing, and blocking were the same as in Case 3.1. Iba1 and GFAP primary antibodies were used for overnight incubation at 4°C. The subsequent steps of secondary antibody incubation, washing, and DAPI staining were the same as in Case 3.1. ELISA detection: The steps of obtaining hippocampal tissue protein supernatant were the same as in Case 3.1. The IL-1β, TNF-α, and IL-6 kit (Boster) instructions were strictly followed.

[0076] (3) Experimental Results: Neuroinflammation itself is one of the core characteristics of aging and neurodegeneration. Therefore, the level of inflammation in the hippocampus was assessed by observing the number of astrocytes and microglia in the hippocampal subregions (CA1, CA3, DG) and detecting inflammatory factors. The results showed that compared with the control group, the number of microglia in the CA1, CA3, and DG regions of the hippocampus in the ABAD knockdown group mice was significantly higher. Figure 4 AC) and the number of astrocytes ( Figure 4 The significant increase in DF indicates widespread activation of glial cells. Simultaneously, the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 were significantly elevated. Figure 4 These results confirm that ABAD knockdown significantly induces enhanced neuroinflammatory responses in the hippocampus, manifested as glial cell activation and elevated levels of pro-inflammatory factors. They further elucidate that ABAD knockdown-induced mitochondrial dysfunction leads to a neuronal apoptosis cascade, driving the activation of neuroinflammatory activity.

[0077] Validation of aging-related biomarkers in 3.5ABAD knockdown mice

[0078] (1) Experimental subjects: C57BL / 66-month-old mice (scramble) and ABAD knockdown experimental group (shABAD); (2) Experimental procedure: Aging-related β-galactosidase staining: After the sections were warmed, they were treated in fixative for 20 min, washed with PBS, and incubated overnight at 37℃ with aging-related β-galactosidase staining solution. The next day, the staining solution was removed and washed with PBS. Sudan Black B staining: After the sections were warmed, they were rinsed with 70% ethanol and immersed in Sudan Black B staining solution for 1 min. They were rinsed with 70% ethanol and washed with distilled water. They were counterstained with nuclear solid red staining solution for 3 min, rinsed with distilled water, and then mounted. Double corticosteroid (DCX) staining: The steps of section fixation, permeabilization, washing, and blocking were the same as in Case 3.1. The DCX primary antibody was incubated overnight at 4℃. The subsequent steps of secondary antibody incubation, washing, and DAPI nuclear staining were the same as in Case 3.1. Detection of advanced glycation end products (AGEs): Hippocampal tissue protein supernatant was collected (preparation method as before) and processed according to the instructions of the (FineTest) AGEs ELISA kit. Western blotting: Preparation, electrophoresis, transfer, and blocking of mouse hippocampal protein supernatant were performed as before. The mixture was incubated overnight at 4°C with P21 antibody and α-TUBULIN antibody. Subsequent secondary antibody incubation, TBST washing, and ECL chemiluminescence imaging were performed as before.

[0079] (3) Experimental Results: Previous cell experiments have confirmed that ABAD knockdown exacerbates cellular senescence. Therefore, to assess the effect of ABAD knockdown on the aging process in vivo, we examined multiple aging biomarkers in the hippocampus. The results showed that, compared with the control group, the number of senescence-related β-galactosidase-positive cells in the hippocampus of mice in the ABAD knockdown group was significantly increased (…). Figure 5A and B) directly indicate significant cellular senescence in this brain region. Simultaneously, AGEs levels are significantly elevated ( Figure 5 C), and its significantly elevated levels provide important evidence for the aging process. Sudan Black B staining showed a significantly enhanced signal of lipofuscin deposition in the hippocampus of the ABAD knockdown group (C). Figure 5 D), further supporting the presence of aging-related metabolites; the level of p21 protein in the mouse hippocampus showed an increasing trend ( Figure 5 E and F), whose upregulation is one of the key markers of cellular senescence. Furthermore, the number of DCX-positive cells in the subgranular region of the hippocampal dentate gyrus was significantly reduced. Figure 5 (G and H) indicate impaired neurogenesis. These results show that ABAD knockdown significantly exacerbates cellular senescence in the mouse hippocampus, manifested by the abnormal accumulation of multiple aging biomarkers (increased SA-β-gal activity, AGEs accumulation, lipofuscin deposition, upregulated p21 expression, and impaired neurogenesis). The conclusion is that ABAD knockdown significantly accelerates brain aging in mice by impairing mitochondrial function, triggering neuronal damage and neuroinflammation, leading to the abnormal accumulation of multiple aging biomarkers and reduced neurogenesis.

Claims

1. A method for constructing a mouse model simulating the pathogenesis of premature brain aging, characterized in that, A gene knockout recombinant vector carrying the ABAD fragment was introduced into the hippocampus of adult healthy mice, knocking down the ABAD gene in the hippocampus by more than 30%, thereby disrupting neuronal function and realizing the construction of a mouse model that simulates the onset of premature brain aging.

2. The method for constructing a mouse model according to claim 1, comprising the following steps: (1) Submit the coding region sequence of ABAD to siDirect for siRNA target screening, select the optimal interference target based on the score and specificity index, and synthesize the corresponding shRNA sequence accordingly. (2) Insert the fragment synthesized in (1) into the pHBAAV-U6-MCS-CMV-EGFP vector to construct the recombinant target plasmid pAAV9-U6-M-shABAD-EGFP; (3) The recombinant plasmid pAAV9-U6-M-shABAD-EGFP and the helper packaging plasmid were co-transfected into cells to package the virus and harvest the target virus particles. (4) The bilateral hippocampal regions of the mouse were located using a stereotaxic instrument (location coordinates: -2.3 mm posterior to the anterior fontanelle, ±1.8 mm lateral to the midline, depth -2.2 mm). The viral suspension from (3) was slowly injected into the target brain region through a micro-injection system to achieve stable gene knockdown.

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