Application of PKA agonist in preparation of medicine for preventing and treating Alzheimer disease

Abstract

The invention relates to the field of biological medicine, and provides application of a PKA agonist in preparation of a medicine for preventing and treating Alzheimer's disease. The invention particularly relates to application of the PKA protein, a nucleic acid molecule or a recombinant expression vector for coding the protein and a PKA accelerant to preparation of a product for preventing and / or treating AD (Alzheimer's disease). Experimental results show that PKA activation can recover membrane localization and lymphatic-like functions of AQP4, so that AD symptoms are relieved, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and relates to the application of a PKA agonist in the preparation of a drug for preventing and treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease in the world and one of the main causes of dementia. The hallmark neuropathological changes in the brain of AD patients are mainly manifested as abnormal aggregation of amyloid β (Aβ) and abnormal hyperphosphorylation of microtubule-associated protein Tau. The glymphatic pathway is a unique fluid transport system in the brain, which was first identified and named by Maiken Nedergaard's team in 2012. This system is rich in the water channel protein AQP4 through the perivascular space (PVS) formed at the end of astrocytes, and can effectively remove pathological proteins, metabolic waste and inflammatory products, including Aβ and Tau.

[0003] The water channel protein AQP4 is highly polarized and arranged in a square array on the luminal surface of these astrocyte terminals. It plays an important role in water transport between brain tissue and cerebrospinal fluid (CSF) circulation, intracranial pressure regulation, and fluid homeostasis. Therefore, AQP4 is a key node in glymphatic function, and its function depends on the exchange of cerebrospinal fluid and interstitial fluid (CSF-ISF). Autopsy results showed that AD patients had significantly reduced AQP4 polarization in perivascular astrocyte terminals in the frontal lobe compared with non-AD patients of the same age, which is considered to be one of the hallmarks of AD glymphatic dysfunction. Genetic knockout of AQP4 increased the accumulation of Aβ in the hippocampus and cortex, worsened cerebral amyloid angiopathy, and exacerbated cognitive deficits in AD mice. However, the molecular mechanism of AQP4 polarization in AD and its role in regulating glymphatic function are not fully understood.

[0004] The water permeability of AQP4 is regulated by gating and transport. A loss of polarization of AQP4 is clearly observed in AD brains, and membrane-directed transport may be disturbed. AQP4 is believed to be localized to the plasma membrane in a phosphorylation-dependent manner, and the most commonly studied phosphorylation sites are S180 and S276. Studies have shown that phosphorylation of the S180 site by protein kinase C (PKC) leads to increased internalization of AQP4. Protein kinase A (PKA) phosphorylation events occur at the S276 site at the C-terminus, because the S276A mutant fails to transport to the membrane after hypotonic stimulation. In addition, calmodulin kinase II (CaMKII) inhibition can inhibit AQP4 function, but this effect is more through indirect pathways. However, the activity of both PKA and PKC is decreased in AD brains, and which kinase is involved in regulating AQP4 polarization remains to be further elucidated. This study proposes that glymphatic dysfunction is mediated by abnormal membrane distribution of AQP4 in the early stages of AD, and PKA deactivation may be a potential mechanism for the loss of AQP4 polarization. Enhancing glymphatic function by activating PKA may alleviate Aβ deposition and cognitive dysfunction in AD mice. Summary of the invention

[0005] The present invention is based on the above research and studies the application of protein kinase A (PKA) and its activators in alleviating Alzheimer's disease.

[0006] The present invention firstly constructed an Alzheimer's disease (AD) model mouse, and confirmed that the AD model mice and AD patients were both associated with impaired glymphatic system function; then the molecular mechanism was explored, showing that the expression of AQP4 was reduced and the polarity was impaired in the AD mouse model; and further found that there was a correlation between the reduction of PKA and the impaired polarity of AQP4. PKA is a key kinase that regulates the membrane localization of AQP4, and its reduction may be a key factor in the depolarization of AQP4 in AD mice; finally, the therapeutic strategy of restoring AQP4 polarity and improving glymphatic function was explored. After treatment with PKA agonists, AD mice showed significant improvement in cognitive function tests, and the accumulation of Aβ in the brain was reduced, indicating that PKA activation can restore the membrane localization and glymphatic function of AQP4, thereby alleviating the symptoms of AD.

[0007] Based on the above research, the technical solution to be protected by the present invention is as follows:

[0008] The main purpose of the present invention is to provide the use of PKA agonists in alleviating AD, and further provide their use in treating AD or symptoms. The medicine or pharmaceutical composition of the present invention can be used to effectively alleviate the symptoms of AD.

[0009] In a first aspect, the present invention provides the use of a PKA agonist in the preparation of a drug for preventing and treating Alzheimer's disease.

[0010] Preferably, the PKA agonist is selected from exogenous PKA protein, a nucleic acid molecule encoding the protein or a recombinant expression vector thereof, an enzyme activity promoter thereof, and also selected from mRNA, cDNA or precursor of the PKA protein.

[0011] In a preferred embodiment of the present invention, the PKA protein is selected from any of the following situations:

[0012] (a) A polypeptide having an amino acid sequence as shown in SEQ ID NO.1:

[0013] MGNAAAAKKGSEQESVKEFLAKAKEDFLKKWESPAQNTAHLDQFERIKTLGTGSFGRVMLVKHKETGNHYAMKILDKQKVVKLKQIEHTLNEKRILQAVNFPFLVKLEFSFKDNSNLYMVMEYVPGGEMFSHLRRIGRFSEPHARFYAAQIVLTFEYLHSLDLIYRDLKPENLLI DQQGYIQVTDFGFAKRVKGRTWTLCGTPEYLAPEIILSKGYNKAVDWWALGVLIYEMAAGYPPFFADQPIQIYEKIVSGKVRFPSHFSSDLKDLLRNLLQVDLTKRFGNLKNGVNDIKNHKWFATTDWIAIYQRKVEAPFIPKFKGPGDTSNFDDYEEEEIRVSINEKCGKEFSEF

[0014] (b) a substance having homology or sequence identity with the amino acid sequence shown in SEQ ID NO.1 and having the ability to promote PKA function;

[0015] (c), a substance having a PKA-promoting function, wherein one or more amino acids are substituted, deleted or added in the amino acid sequence of (a) or (b), or a protein or polypeptide derived from (a) or (b),

[0016] The nucleic acid molecule encoding the PKA protein is selected from any of the following situations:

[0017] (i) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO.2;

[0018]

[0019] (ii) a molecule that hybridizes to the nucleotide sequence defined in (i) under stringent conditions;

[0020] (iii) a nucleic acid molecule homologous to or having sequence identity with the nucleotide sequence shown in SEQ ID NO.2;

[0021] (iv) A nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of (i), (ii) or (iii).

[0022] In some embodiments, the PKA protein is: a naturally purified protein, a chemically synthesized product, or produced from a prokaryotic or eukaryotic host using recombinant technology. The host is selected from: bacteria, yeast, higher animals and mammalian cells, preferably human PKA.

[0023] Furthermore, the present invention also provides a PKA recombinant expression vector, comprising an expression vector and a nucleic acid molecule encoding a PKA protein inserted into the expression vector, wherein the nucleic acid molecule encoding the PKA protein is as described above.

[0024] Wherein, the expression vector is a conventional vector such as a plasmid vector, a cosmid vector, a phage vector or a viral vector, and the specific type is selected from the prior art according to the actual situation. The "viral vector" includes adeno-associated virus and lentivirus. Suitable viral vectors are well known to those of ordinary skill in the art. The remaining "non-viral vectors" include liposomes or lipid complexes, cationic polymers, chitosan polymers and nanoparticle vectors. Suitable non-viral vectors are well known to those of ordinary skill in the art.

[0025] In other preferred embodiments of the present invention, the PKA enzyme activity promoter is selected from bucladesine.

[0026] The present invention injected bucladesine into AD mice by intraventricular micropump injection. After one month of continuous treatment, the lymphatic infusion and outflow functions of AD mice were restored. In addition, the polarization and membrane localization analysis of AQP4 showed that bucladesine treatment effectively restored the distribution of AQP4 around the blood vessels in the hippocampus of 4XFAD mice. Further analysis showed that the expression level of AQP4 in the membrane protein fractionation of the bucladesine treatment group was significantly higher than that of the saline group.

[0027] The second aspect disclosed in the present invention provides a product for alleviating AD, which is a pharmaceutical composition or a medicine kit, for example, a pharmaceutical composition or a medicine kit in a form suitable for administration by a method selected from the following group: oral administration, injection (for example, direct naked DNA or protein injection, liposome-encapsulated DNA or protein injection), gold-coated gene gun bombardment, reproduction-defective bacteria carrying plasmid DNA, replication-defective adeno-associated virus carrying target DNA, or nasal administration, pulmonary administration, oral administration or transdermal administration of a protein encoded by a target gene.

[0028] Preferably, the pharmaceutical composition or kit of the present invention comprises:

[0029] (A) a therapeutically or preventively effective amount of the PKA, nucleic acid molecules encoding the protein and / or a compound promoter thereof;

[0030] (B) a pharmaceutically or immunologically acceptable carrier or excipient;

[0031] (C) optionally, one or more other active substances for preventing or treating AD and its related disorders and / or symptoms.

[0032] Generally, liquid preparations can be kept stable for at least one year at 2°C-8°C, and freeze-dried preparations can be kept stable for at least six months at 30°C. The preparations can be suspensions, water injections, freeze-dried preparations and other preparations commonly used in the pharmaceutical field.

[0033] When the composition of the present invention is administered to animals including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and administration route. The results of animal experiments and various circumstances can be referred to, and the total dosage cannot exceed a certain range.

[0034] Beneficial protection and effects of the present invention:

[0035] 1. Multimodal imaging technology for comprehensive evaluation of glymphatic system function

[0036] Technical advantages: This study combines DTI-ALPS (diffusion tensor imaging glymphatic system index) with traditional CSF tracing experiments to achieve a comprehensive assessment of glymphatic system function by quantitatively analyzing the clearance of water in the brain. DTI-ALPS can accurately assess the clearance of water and metabolites in mouse models and reveal glymphatic system dysfunction in the early stages of AD.

[0037] Innovation: This technology not only draws on methods from human studies, but also makes appropriate optimization and adjustments to the anatomical structure and imaging technology of the mouse model, thus achieving accurate monitoring of the lymphatic system in small animals.

[0038] 2. Markers for early diagnosis of Alzheimer's disease

[0039] Technical advantages: The study uses functional changes in the glymphatic system as a marker for early diagnosis. In the AD mouse model, damage to the glymphatic system is manifested by a decrease in the DTI-ALPS index, weakened CSF permeability, and reduced polarity and expression of AQP4. These changes can be detected early before AD symptoms appear. This provides a potential technical means for the early diagnosis of AD and can detect the disease before symptoms appear.

[0040] Innovation: Early detection and intervention are the key to the treatment of Alzheimer's disease, and this technology can provide a reliable biomarker for early detection of AD, which may help delay or reduce the occurrence of cognitive decline.

[0041] 3. Therapeutic interventions to restore AQP4 polarity

[0042] Technical advantages: Through the use of the PKA agonist bucladesine, studies have shown that the membrane localization of AQP4 can be restored, thereby improving the function of the glymphatic system. This intervention can significantly improve the cognitive function of mice without significantly affecting other pathological markers of AD mice (such as Aβ accumulation) or inflammatory cells.

[0043] Innovation: This discovery highlights the strategy of restoring AQP4 polarity as a possible treatment approach. Compared with traditional drug interventions, it does not directly target Aβ deposition or neurodegenerative changes, but indirectly alleviates the pathological process by restoring the normal function of the glymphatic system, providing a new treatment idea.

[0044] 4. A new perspective on the lymphatic system as a therapeutic target

[0045] Technical advantages: The glymphatic system is believed to play an important role in the pathogenesis of Alzheimer's disease, especially in the clearance of cerebrospinal fluid and interstitial fluid. This study has opened up a new direction for the study of the glymphatic system as a therapeutic target. By restoring the polarity of AQP4, it may be possible to improve the clearance of metabolic waste in the brain and slow the progression of neurodegenerative diseases.

[0046] Innovation: This treatment strategy is different from traditional interventions targeting Aβ or tau proteins. The glymphatic system, as an early pathological change of AD, provides a new therapeutic target that can slow down or reverse the pathological process of AD by improving waste clearance function.

[0047] 5. Close association between small animal models and human diseases

[0048] Technical advantages: The study simulated the pathological characteristics of human AD in a mouse model, used highly adaptable imaging technology, and established a pathological mechanism similar to that in clinical practice. This research model of translational medicine not only improves the reliability of the research, but also provides a direct reference for the treatment of human AD.

[0049] Innovation: By comparing the changes in glymphatic function in mouse models with those in human AD patients, the study demonstrated the common changes in the glymphatic system in AD, laying the foundation for future clinical applications.

[0050] 6. Behavioral improvement and biomarker linkage

[0051] Technical advantages: By combining behavioral tests (such as cognitive function assessment), immunohistochemical analysis (such as Aβ accumulation, AQP4 localization) and glymphatic system function assessment, this study evaluates the effects of therapeutic intervention from multiple perspectives to ensure the effectiveness of the intervention measures. This comprehensive evaluation method improves the credibility of the data and the rigor of the experimental results.

[0052] Innovation: The combination of functional biomarkers (such as AQP4 polarity, DTI-ALPS index, etc.) with cognitive behavioral outcomes not only reveals the effect of therapeutic intervention, but also further verifies the important role of the glymphatic system in AD.

[0053] Therefore, the present invention provides new technical support for the treatment of Alzheimer's disease and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The present disclosure will be further described below in conjunction with the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present disclosure rather than for limiting the scope of the present disclosure.

[0055] Figure 1 showed an association between impaired glymphatic system function and Alzheimer's disease (AD) model mice.

[0056] Figure 2 showed a reduction in AQP4 polarity and membrane localization in AD mice.

[0057] Figure 3 showed that PKA reduction induced impaired AQP4 polarity in AD mice.

[0058] Figure 4 PKA activation was shown to improve cognitive function by restoring AQP4-associated glymphatic function. DETAILED DESCRIPTION

[0059] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of conventional methods, such as methods for constructing vectors and plasmids, methods for inserting protein-encoding genes into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and are described in many publications.

[0060] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention, and the preferred implementation methods and materials described in the specific implementation methods are for demonstration purposes only.

[0061] Example 1 Association between impaired lymphatic system function and Alzheimer's disease (AD) model mice

[0062] Technical principle: By quantitatively analyzing the function of the glymphatic system, the researchers explored the damage of the glymphatic system in the AD mouse model. This functional impairment is manifested in two aspects:

[0063] (a) Decreased DTI-ALPS index: Diffusion tensor imaging (DTI) and high-frequency MRI-based glymphatic function index (DTI-ALPS) were used to evaluate the ability of mice to clear water and metabolites in the brain. The results showed that the DTI-ALPS index of 4XFAD mice was significantly lower than that of wild-type (WT) controls, suggesting that the function of the glymphatic system was impaired.

[0064] (b) Decreased penetration and efflux of CSF tracers: The reduced penetration of CSF (cerebrospinal fluid) tracers through different brain regions confirmed the impaired function of the glymphatic system in AD model mice, as manifested by reduced exchange efficiency between CSF and interstitial fluid (ISF).

[0065] 1. Animal Experiment Materials

[0066] APP / PS1 transgenic mice (C57BL / 6 background) were purchased from The Jackson Laboratory, while 4XFAD mice (B6 / JGpt-Tg(Thy-APP / Thy-PSEN1)5 / Gpt) were provided by Gempharmatech Co., Ltd. All mice were housed under standard laboratory conditions at 22°C with a 12-h light / dark cycle and provided with unlimited food and water. Animal care and experimental procedures followed the ethical guidelines approved by the local ethics committee. Transgenic mice and their wild-type (WT) littermates were included in the analysis. Female mice at two age time points were used, namely 4XFAD mice (1.5 and 8 months old) and APP / PS1 mice (4 and 12 months old), with at least 4 mice in each group.

[0067] 2. MRI and DTI-ALPS Animal Experiments

[0068] All MRI scans were performed using an 11.7-T BioSpec Avance III small animal magnetic resonance system (BrukerBioSpin, Ettlingen, Germany). Anesthesia was performed using isoflurane (Abbott Animal Health, Abbott Park, IL, USA) at an induction concentration of 3.5%, a maintenance concentration of approximately 1.7%, and an oxygen-to-air mixture ratio of 1:2. To evaluate the DTI-ALPS index of mice, the region of interest (ROI) for each mouse was manually drawn based on the color fractional anisotropy (FA) map and susceptibility-weighted imaging (SWI). The DTI-ALPS index was calculated according to the established protocol. The specific steps are as follows:

[0069] Step 1: DTI data preprocessing

[0070] Image data were preprocessed and analyzed using the functional magnetic resonance imaging library (FSL) software. Diffusion images were corrected for head motion and eddy current distortion using the Eddy Correct method, and non-brain tissue was removed using the Brain Extraction Tool. Afterwards, visual inspection was performed to ensure accurate removal of non-brain tissue. Then, colored fractional anisotropy (FA) maps were generated using the DTIfit algorithm.

[0071] Step 2: Locate the paraventricular medullary vessels

[0072] Locate the paraventricular medullary vessels on SWI images.

[0073] Step 3: Manually draw a 1 mm diameter spherical ROI mask

[0074] A 1 mm diameter spherical ROI mask was drawn next to the medullary vessels, the projection fiber ROI (primarily aligned with the y-axis, blue fibers), and the connecting fiber ROI (primarily aligned with the z-axis, green fibers).

[0075] Step 4: Apply ROI mask to SWI imaging

[0076] Each mask was applied again to the SWI imaging to ensure that it was located near the medullary vessels.

[0077] Step 5: Calculation of diffusivity and DTI-ALPS index

[0078] For each mask, the mean diffusivity was calculated along the x-, y-, and z-axes. The DTI-ALPS index was calculated as the ratio of two sets of diffusion values: the mean x-axis diffusivity of the projection fiber area (Dxxproj) and the mean x-axis diffusivity of the association fiber area (Dxxassoc), to the mean y-axis diffusivity of the projection fiber area (Dyyproj) and the mean z-axis diffusivity of the association fiber area (Dzzassoc).

[0079] The DTI-ALPS index calculated in this way was used to evaluate the glymphatic system function of the mouse brain.

[0080] 3. Results Analysis

[0081] To investigate whether the impaired glymphatic system observed in patients with Alzheimer's disease (AD) is also present in a mouse model of AD, we performed MRI scans on 2-month-old female 4XFAD mice and their wild-type (WT) littermates. In this study, the DTI-ALPS mouse data processing workflow was based on methods used for human samples but was adapted to fit the specific anatomical and imaging limitations of small animal models. Similar to the changes observed in AD patients, a significant decrease in the DTI-ALPS index was observed in 4XFAD mice ( Figure 1 AB).

[0082] Subsequently, we performed a series of CSF infusion and efflux experiments to evaluate the glymphatic system function of 4XFAD mice. Quantitative analysis showed that CSF tracer penetration in the brains of AD mice was decreased by 42% compared with that of WT mice, both in the midbrain and hippocampus ( Figure 1 CE). The magnified image shows that both low molecular weight (3 kDa) and high molecular weight (42 kDa) fluorescent tracers accumulate around the vessel-like structures ( Figure 1 D).

[0083] Next, we evaluated the clearance of molecules from the brain interstitial fluid (ISF) ( Figure 1F). Two hours after the tracer was injected into the hippocampus, the tracer retention in the hippocampus of 4XFAD mice was significantly higher than that in WT controls ( Figure 1 G and 1H). Representative images show penetration of CSF tracer into intracerebral blood vessels and penetration of tracer into plaque-like structures in the brain parenchyma. Of note, the high molecular weight tracer (45 kDa) accumulates significantly around the blood vessel-like structures, while both tracers are present in the plaque-like areas ( Figure 1 H zoom).

[0084] To verify whether the DTI-ALPS index reflects the clearance capacity of the perivascular space, we performed a correlation analysis between the DTI-ALPS index and the fluorescence intensity in the outflow experiment. The results showed that the in vivo DTI-ALPS index was negatively correlated with the fluorescence intensity in the in vitro outflow experiment ( Figure 1 I). These results suggest that the glymphatic system dysfunction in 4XFAD mice is similar to that observed in AD patients, as evidenced by decreased DTI-ALPS index and impaired CSF-ISF clearance, which may lead to pathological accumulation of macromolecules around blood vessels and plaques.

[0085] Example 2: Mechanism of AQP4 expression and polarity changes in Alzheimer's disease (AD) and treatment strategies 1. Overview of research content

[0086] 1. Molecular mechanism of AQP4 expression and polarity changes

[0087] Technical principle: AQP4 (Aquaporin-4) is a water channel protein closely related to the function of the lymphatic system. This study shows that the expression and polarity of AQP4 in the AD mouse model change, mainly reflected in:

[0088] (a) Reduced AQP4 expression: AD mice, especially in the early asymptomatic stage, showed a significant decrease in the number and area of ​​AQP4-positive structures in the hippocampus and thalamus, suggesting that AQP4 expression in the brain was inhibited.

[0089] (b) Impaired polarity of AQP4: The loss of polarity of AQP4 around blood vessels, especially in the early stages of AD, leads to impaired water removal, thus affecting the function of the glymphatic system. The membrane association of AQP4 is significantly reduced in AD mice, further verifying the impact of impaired polarity of AQP4 on glymphatic function.

[0090] 2. Relationship between PKA and AQP4 polarity disorder

[0091] Technical principle: By analyzing the expression of upstream kinases such as PKA (protein kinase A) in AD mice, it was found that there was a correlation between the reduction of PKA and the impairment of AQP4 polarity. PKA is a key kinase that regulates the membrane localization of AQP4, and its reduction may be a key factor in the depolarization of AQP4 in AD mice.

[0092] By treating AD mice with PKA agonists (such as bucladesine), the researchers were able to significantly restore the membrane localization of AQP4, thereby restoring glymphatic function and improving the cognitive function of AD mice.

[0093] 3. Therapeutic strategies to restore AQP4 polarity and improve lymphoid function

[0094] Technical principle: By applying the PKA agonist bucladesine, the researchers restored the polarity of AQP4 in AD mice, and confirmed its improvement on glymphatic function through behavioral tests and CSF tracing experiments. After treatment, AD mice showed significant improvement in cognitive function tests and reduced Aβ accumulation in the brain, indicating that PKA activation can restore the membrane localization and glymphatic function of AQP4, thereby alleviating the symptoms of AD.

[0095] Restoration of glymphatic function: Bucladesine treatment significantly enhanced the diffusion efficiency of CSF tracers, further demonstrating restoration of the glymphatic system. The membrane localization of AQP4 was restored, thereby improving the exchange efficiency between CSF and interstitial fluid and slowing down the accumulation of Aβ.

[0096] 2. Animal Handling and Experimental Design

[0097] A total of 60 female mice, 6 months old, including wild-type (WT) and 4XFAD mice (n=30 per group) were used in this study. Fifteen mice of each genotype (WT and 4XFAD, n=15 per group) received the PKC agonist Bucladesine (at a dose of 25 mg / kg, approximately 0.5-0.75 mg per mouse) via an intracerebral catheter for 8 weeks (from 6 to 8 months of age) using a microfluidic pump. The remaining WT and 4XFAD mice (n=15 per group) received an equal volume of saline injection and were treated on the same schedule. After the treatment period, all mice were subjected to the novel object recognition test and the Y-maze test to assess their cognitive function.

[0098] 1. Glymphatic system assessment

[0099] 1.1 Cerebrospinal fluid (CSF) injection experiment

[0100] For CSF injection experiments, Alexa Fluor 555 solution of 0.5% Dextran, Texas Red (3000MW) and 0.5% ovalbumin was combined and stereotaxically injected in 5 μL artificial cerebrospinal fluid (aCSF) into the cisterna magna at a depth of 1-1.5 mm for 5 minutes. After injection, the syringe was kept in place for 5 minutes. 30 minutes after injection, mice were perfused. The brain was cut into 100 μm thick coronal sections, and five brain sections from each mouse were observed using a fluorescence microscope (5x magnification).

[0101] 1.2 Cerebrospinal fluid reflux experiment

[0102] In the cerebrospinal fluid reflux experiment, AlexaFluor 488 solution combined with 0.5% Dextran, Texas Red (3000MW) and 0.5% ovalbumin was mixed with artificial cerebrospinal fluid (aCSF) and injected into the striatum of mice under stereotaxic conditions with 1 μL of solution. The injection time was 10 minutes, and the coordinates were 0.5 mm anterior, 2.0 mm lateral, and 3.0 mm deep. After injection, the syringe was kept for 5 minutes. The mice were perfused 2 hours later. The brain was cut into 50 μm thick coronal sections, and ten brain sections of each mouse were observed using a fluorescence microscope (5x magnification).

[0103] Quantitative analysis was performed by a blinded researcher using ImageJ software. Regions of interest (ROIs) were defined based on the entire section area, and the mean fluorescence intensity was evaluated.

[0104] 1.3 Immunofluorescence staining

[0105] Mice were anesthetized with 1% sodium pentobarbital, perfused with phosphate buffered saline, and then fixed with 4% paraformaldehyde. After fixation, brain sections were permeabilized and blocked, followed by overnight incubation with the following primary antibodies at 4°C: anti-AQP4 (1:100), anti-CD31 (1:100), anti-Aβ (1:5000), anti-GFAP (1:5000), and anti-IBA1 (1:200). The next day, sections were incubated with secondary antibodies for 1 h at room temperature. Three brain sections were used per mouse, and each section was observed under confocal microscopy and virtual section microscopy in three different fields. Image analysis was performed by a blinded researcher using ImageJ software. AQP4 polarization was quantified by counting the number of AQP4-positive structures and the total AQP4-positive area. The mean fluorescence intensity was evaluated for the entire section and for regions of interest (ROI) of specific brain regions (including cerebral cortex, thalamus, and hippocampus).

[0106] 1.4 Western blotting experiments

[0107] The samples were lysed using a membrane protein and plasma membrane protein extraction kit (Beyotime), and the protein concentration was measured by a diazaindole triacid (BCA) protein assay kit. Protein samples (20 μL per lane) were separated on a 10-12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-AQP4 (1:200), anti-PKA (1:1000), anti-PKC (GFAP; 1:1000), anti-CSNK2A1 (1:200), anti-Actin (1:500), anti-Na-K-ATPaseα1. The next day, the membrane was incubated with a horseradish peroxidase (HRP)-labeled secondary antibody for 1 hour. The color was developed using a chemiluminescent reagent and analyzed by ImageJ software.

[0108] 2. Behavioral testing

[0109] 2.1 New Object Recognition Test

[0110] Mice were grouped by sex and housed in individually ventilated cages for at least two weeks to adapt to the experimental environment (22°C to 24°C, humidity 50% to 60%, 12-hour light-dark cycle). On the test day, mice were placed in a quiet room for 30 minutes of adaptation, and the light was adjusted to 130-180 lux using a light meter. Two days before the test, each mouse was placed in an empty cage for 10 minutes each time for habituation.

[0111] During the test, mice were exposed to two identical objects for 5 minutes, and the exploration time of each mouse on each object was recorded. The mice went through three sample phases, each followed by a 15-minute rest period. After 24 hours, one of the objects in the third sample phase was replaced with a new object, and the mice explored again for 5 minutes, and the exploration time was recorded.

[0112] To eliminate olfactory cues, new cages and objects were used during the experiment, and each mouse was housed individually to prevent potential inhibition effects. The exploration time and number of explorations of each mouse on each object were recorded, and the discrimination index (DI) was calculated using the following formula: DI = (novel object exploration time - familiar object exploration time) / (novel object exploration time + familiar object exploration time).

[0113] 2.2 Y-maze spontaneous alternation test

[0114] Mice were placed individually in the starting arm of the Y-maze, facing the short wall, and began a 5-minute test. The time each mouse exited the starting arm (all four paws left) and the order of entering the maze arm were recorded. Each maze arm was labeled with a letter or number for identification. After each trial, the mice were weighed and male mice were placed in a new cage to prevent conflict or stress with untested mice. During the test, the maze was cleaned with disinfectant and dried to remove olfactory cues. Mice that entered the maze less than five times were excluded from the analysis. The entry pattern was divided into three types: spontaneous alternation (SPA, entering different arms, such as ABC), alternating arm return (AAR, the first and third arms are the same, such as ABA), and same arm return (SAR, the first and second arms are the same, such as AAB). The SAR in each pattern was counted only once, and the number of entries was analyzed in groups of three. The total number of arm entries and the percentages of spontaneous alternation (SPA), alternating arm return (AAR), and same arm return (SAR) were calculated.

[0115] 3. Results and Analysis

[0116] Reduced AQP4 expression and impaired polarity in AD mice

[0117] After confirming the presence of impaired perivascular drainage in 4XFAD mice, the molecular mechanisms underlying this dysfunction were explored. AQP4 is highly concentrated in the foot ends of astrocytes surrounding blood vessels and plays a crucial role in promoting water clearance. To investigate whether the expression of AQP4 and its polarity are altered in 4XFAD mice, we analyzed the level and localization of AQP4. To represent the early, asymptomatic stage without plaque deposition, we selected 4XFAD mice at 4 weeks of age and APP / PS1 mice at 32 weeks of age. For the symptomatic AD stage characterizing cognitive decline, we included 4XFAD mice at 20 weeks of age and APP / PS1 mice at 54 weeks of age. The results showed that although in the early asymptomatic stage, 4XFAD mice ( Figure 2 AD) and APP / PS1 mice ( Figure 2 EF) showed a significant decrease in the number of AQP4-positive structures and the area of ​​AQP4-positive structures in the hippocampus and thalamus, and were significantly different from their wild-type (WT) controls. However, in the cortex, there was no significant change in the number or area of ​​AQP4-positive structures at this early stage. Interestingly, in APP / PS1 mice, there was an increase in the number of AQP4-positive structures, but a decrease in the total area of ​​AQP4-positive areas, suggesting that these structures were fragmented. Importantly, Aβ deposition was not detected at this stage.

[0118] To further quantify the distribution of AQP4 in different cellular substructures, we separated membrane and cytoplasmic proteins from hippocampal tissue and analyzed the AQP4 content in both fractions to verify whether the observed depolarization of AQP4 localization was related to the total AQP4 expression level and the level of AQP4 on the membrane. Both immunoblotting (IB) and Western blotting experiments showed that the total AQP4 expression in 4XFAD and APP / PS1 mice gradually increased with age compared with the WT group ( Figure 2 G, H, J, and L). However, at the early asymptomatic stage (4 and 20 weeks), the proportion of membrane-associated AQP4 in AD mice was significantly lower than that in the WT group ( Figure 2 I and K). Interestingly, this reduction in membrane-associated AQP4 was evident at an early stage of the disease and did not worsen with age, suggesting that membrane localization of AQP4 is reduced early in disease progression. The similar patterns observed in both AD models suggest that reductions in AQP4 polarity and membrane localization may represent a common early feature in Alzheimer's disease pathology.

[0119] 3.2 PKA reduction induces impaired AQP4 polarity in AD mice

[0120] The polarity and membrane localization of AQP4 are regulated by multiple upstream kinases, including PKA, PKC, and CaMKII. To investigate whether the dysfunction of these kinases contributes to the depolarization and reduced membrane localization of AQP4, we examined the expression levels of these kinases in the hippocampus of AD mice. The results showed that the levels of PKA and PKC were significantly decreased in the hippocampus of 4XFAD mice and APP / PS1 mice, and the decrease was more obvious in 4XFAD mice ( Figure 3 AC). To further explore the role of these kinases in regulating AQP4 membrane localization, we treated in vitro cultured hippocampal slices from 4XFAD mice with a PKA agonist (bucladesine) and a PKC agonist (indolactam V) at different concentrations of the agonists. We found that at a concentration of 1 nM, the PKA agonist bucladesine significantly increased the membrane localization ratio of AQP4, while indolactam V exhibited the opposite effect, reducing AQP4 membrane binding in 4XFAD brain slices ( Figure 3 DE).

[0121] Similarly, in cultured hippocampal slices from APP / PS1 mice, 1 nM bucladesine was the only treatment that effectively restored AQP4 membrane localization. In contrast, indolactam V failed to promote AQP4 membrane binding in this model. These findings suggest that PKA activation can enhance AQP4 membrane localization, while PKC activation may hamper this process in AD pathology ( Figure 3FG).

[0122] 3.3PKA activation improves cognitive function by restoring AQP4-related glymphatic function

[0123] To evaluate whether upregulation of PKA in vivo could restore AQP4 polarity and improve glymphatic function in Alzheimer's disease (AD) mice, we injected bucladesine into 7-month-old 4XFAD mice and their littermate controls via intracerebroventricular micropump for one month ( Figure 4 A). After treatment, behavioral tests and immunofluorescence analysis were performed to evaluate the effects of bucladesine on the glymphatic system, which is regulated by AQP4 polarization and plays a key role in AD pathology. Bucladesine treatment significantly improved cognitive function in 4XFAD mice compared with the WT group ( Figure 4 BC), while the Aβ-positive signal in the hippocampus was significantly reduced ( Figure 4 F and H). In addition, GFAP staining showed that bucladesine treatment significantly reduced the proliferation of astrocytes in 4XFAD mice ( Figure 4 G and I). However, no significant changes were observed in the number of IBA1-positive microglia between the bucladesine-treated and untreated groups.

[0124] To further link these improvements to the restoration of glymphatic function, we performed CSF tracer efflux and infusion experiments. The results showed that bucladesine treatment significantly enhanced the diffusion efficiency of CSF tracers in 4XFAD mice, indicating that glymphatic infusion ( Figure 4 D) and outflow ( Figure 4 In addition, the polarization and membrane localization analysis of AQP4 showed that bucladesine treatment effectively restored the distribution of AQP4 around blood vessels in the hippocampus of 4XFAD mice ( Figure 4 J). Further analysis showed that the expression level of AQP4 in membrane protein fractions of the bucladesine-treated group was significantly higher than that of the saline-treated group ( Figure 4 K and L).

[0125] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of PKA agonists in the preparation of drugs for the prevention and treatment of Alzheimer's disease.

2. The use according to claim 1, characterized in that The PKA agonist is selected from exogenous PKA protein, nucleic acid molecules encoding the protein or their recombinant expression vectors, their enzyme activity promoters, and also selected from mRNA, cDNA or precursors of PKA protein.

3. The use according to claim 2, characterized in that: in, The PKA protein is selected from any of the following situations: (a), a polypeptide having an amino acid sequence as shown in SEQ ID NO.1; (b) a substance having homology or sequence identity with the amino acid sequence shown in SEQ ID NO.1 and having the ability to promote PKA function; (c), a substance having the function of promoting PKA in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (a) or (b), or a protein or polypeptide derived from (a) or (b).

4. The use according to claim 2, characterized in that: in, The nucleic acid molecule encoding the PKA protein is selected from any of the following situations: (i) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO.2; (ii) a molecule that hybridizes to the nucleotide sequence defined in (i) under stringent conditions; (iii) a nucleic acid molecule homologous to or having sequence identity with the nucleotide sequence shown in SEQ ID NO.2; (iv) A nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of (i), (ii) or (iii).

5. The use according to claim 2, characterized in that: in, The PKA enzyme activity promoter is selected from bucladesine.

6. A PKA recombinant expression vector, characterized in that: The recombinant vector includes an expression vector and a nucleic acid molecule encoding a PKA protein inserted into the expression vector. The nucleic acid molecule encoding the PKA protein is shown in SEQ ID NO.

2.

7. The PKA recombinant expression vector according to claim 6, characterized in that: in, The expression vector is a plasmid vector, a cosmid vector, a phage vector or a viral vector, and the viral vector is selected from adeno-associated virus or lentivirus.

8. A pharmaceutical composition for preventing and treating Alzheimer's disease, characterized in that: The active component thereof comprises the PKA agonist described in any one of claims 1 to 5.

9. The pharmaceutical composition for preventing and treating Alzheimer's disease according to claim 8, characterized in that: The pharmaceutical composition is used in combination with other drugs for treating Alzheimer's disease.

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