Polypeptide blocking agent for inhibiting transcellular transport of astrocyte GFAP of Alzheimer's disease to neurons and application of polypeptide blocking agent

Abstract

The invention relates to the technical field of polypeptide drugs for treating neurodegenerative diseases, in particular to a polypeptide blocking agent for inhibiting transcellular transport of alzheimer's disease astrocyte GFAP (glial fibrillary acidic protein) to neurons and application of the polypeptide blocking agent. According to the technical scheme, the interaction between the GFAP and the APP (amyloid precursor protein) can be used as an AD (Alzheimer's disease) intervention target, then polypeptide drugs are developed, and the blocking peptide targeting the binding interface between the N terminal of the APP and the GFAP is designed. The medicine can remarkably improve cognitive impairment of AD model mice by inhibiting GFAP endocytosis, repairing mitochondrial functions, relieving neuroinflammation and other mechanisms. According to the technical scheme, the technical problem that polypeptide drugs capable of targeting and interfering GFAP related pathological processes are lacked in the prior art can be solved, a brand new strategy is provided for early intervention of AD, and the technical scheme has important clinical transformation value.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs for treating neurodegenerative diseases, and in particular to a polypeptide blocker for inhibiting the transcellular transport of GFAP (glial fibrillary acidic protein) from Alzheimer's astrocytes to neurons and its application. Background Art

[0002] Alzheimer's disease (AD) is characterized by β-amyloid (Aβ) deposition and Tau protein aggregation. Existing therapies primarily target Aβ clearance, but clinical efficacy is limited. Recent studies have demonstrated that glial fibrillary acidic protein (GFAP), secreted by reactive astrocytes, can enter neurons through unknown mechanisms, leading to mitochondrial damage and synaptic loss. However, the molecular mechanisms of GFAP transcellular trafficking and its pathological role remain unclear.

[0003] Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by the accumulation of amyloid-beta (Aβ) and abnormal aggregation of tau protein. Although existing therapies focus on the clearance or reduction of Aβ, these approaches have had limited success in clinical trials, indicating that the pathological mechanisms of AD are far more complex than a single factor.

[0004] Existing treatments for AD mainly include drug therapy and non-drug interventions, which aim to relieve symptoms, delay disease progression, and improve patients' quality of life. Drug treatments include cholinesterase inhibitors, NMDA receptor antagonists, etc. In some cases, doctors may recommend the use of multiple drugs at the same time in order to achieve better results. Non-drug interventions include lifestyle adjustments, cognitive training, etc. Although there is currently no cure for AD, the above treatments can control symptoms to a certain extent and provide patients with a better quality of life.

[0005] As research into the pathogenesis of AD deepens, it has been discovered that, in addition to Aβ and Tau proteins, glial fibrillary acidic protein (GFAP), secreted by reactive astrocytes, may enter neurons through a yet-to-be-elucidated mechanism, leading to mitochondrial dysfunction and synaptic loss, further exacerbating neuronal damage and cognitive decline. Although GFAP's crucial role in the central nervous system is widely recognized, as a structural protein involved in maintaining the morphological stability of astrocytes, little is known about how GFAP achieves transcellular trafficking and the precise role of this process in the pathological development of AD.

[0006] Given the above challenges, the development of novel therapeutic strategies is particularly urgent. In recent years, peptide drugs have become a research hotspot due to their high specificity, good biocompatibility, and tunable pharmacokinetic properties. In particular, the design of peptide drugs that can target and interfere with GFAP-related pathological processes is expected to provide a new therapeutic approach to alleviate AD symptoms, slow disease progression, and ultimately improve patients' quality of life. However, to achieve this goal, it is necessary to first analyze in detail the specific molecular mechanisms of GFAP transcellular transport and its pathological role in AD. This is not only crucial for understanding the complex pathophysiology of AD, but also lays a theoretical foundation for the development of innovative peptide-based therapies. Summary of the Invention

[0007] The present invention aims to develop a drug for the treatment of Alzheimer's disease, addressing the existing technical problem of a lack of peptide drugs capable of targeting and interfering with GFAP-related pathological processes. This invention reveals for the first time that GFAP mediates endocytosis by binding to the N-terminus of APP and demonstrates its induction of mitochondrial dysfunction and neuroinflammation. Based on this, a GAB1-blocking peptide was developed for the treatment of Alzheimer's disease and other diseases.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A drug for treating Alzheimer's disease, which is a blocking peptide that blocks the binding between GFAP and the N-terminus of APP; the amino acid sequence of the blocking peptide is shown in any one of SEQ ID NOs. 1-7 and their conservative derivatives.

[0010] The above-mentioned technical solution uses a specially designed blocking peptide to prevent the binding of glial fibrillary acidic protein (GFAP) to the N-terminal region of amyloid precursor protein (APP). The blocking peptide can effectively interfere with the interaction between GFAP and APP, thereby preventing the APP-mediated GFAP endocytosis process and avoiding the abnormal transcellular transport and aggregation of GFAP in neurons. The technical means adopted by the present invention can prevent the damage to mitochondrial function and synaptic structure caused by abnormal GFAP aggregation, as well as the related inflammatory response. In particular, the abnormal accumulation of GFAP in neurons observed in AD patients can be effectively alleviated by administering the blocking peptide of the present invention. After application of the above-mentioned blocking peptide in the 5xFAD transgenic mouse model, it can significantly improve GFAP-induced cognitive dysfunction, including but not limited to improved behavioral performance in water maze, Y maze, and novel object recognition tests. In addition, this treatment method has also demonstrated the ability to repair synaptic damage in the hippocampus of 5xFAD mice, while inhibiting the cGAS-STING-related neuroinflammatory pathway, increasing synaptic density, optimizing mitochondrial structure, and reducing neuroinflammation levels, ultimately enhancing neuronal function. The blocking peptide provided by the present invention not only demonstrates great potential as a drug for treating Alzheimer's disease, but also provides a scientific basis and technical support for the development of new therapies for such diseases.

[0011] Furthermore, the amino acid sequence of the blocking peptide is shown in SEQ ID NO.1.

[0012] Furthermore, the N-terminus of the blocking peptide is modified with a lysine residue; preferably, the number of the lysine residues is 6. Furthermore, the blocking peptide is modified with palmitic acid; preferably, the palmitic acid is linked to the 7th K of SEQ ID NO.1.

[0013] Using the above technical solution, the blocking peptide of this solution is designed with a specific amino acid sequence. This blocking peptide can effectively prevent the interaction between GFAP and APP, thereby slowing the pathological progression of Alzheimer's disease. The amino acid sequence of the blocking peptide is shown in any one of SEQ ID NO.1-7, with SEQ ID NO.1 being the preferred sequence. The blocking peptide is modified with C6K to enhance its stability, bioactivity, and cell membrane penetration ability, thereby improving the efficiency of intracellular delivery. The blocking peptide is also modified with palmitic acid to extend the half-life of the peptide and reduce the efficiency of rapid degradation in the body.

[0014] It should be noted that conservative derivatives with ≥80% sequence identity to SEQ ID NOs. 1-7 can also be used to prepare blocking peptides. Peptide derivatives refer to compounds with improved properties or new functions obtained by chemically modifying or altering naturally occurring polypeptide molecules. These derivatives can be prepared in a variety of ways, including but not limited to amino acid substitutions, insertions or deletions, addition of non-natural amino acids, side chain modifications, cyclization, PEGylation, lipidation, and the like. These peptide derivatives are defined as conservative derivatives if they do not adversely affect the biological function of the peptide.

[0015] Furthermore, the drug is used to inhibit the interaction between GFAP and APP.

[0016] Using this technical solution, the drug effectively prevents the abnormal aggregation of GFAP within neurons by specifically blocking the interaction between GFAP and APP. This mechanism of action helps reduce the accumulation of GFAP within neurons, thereby alleviating the disease burden.

[0017] Furthermore, the drug is used to inhibit the transcellular transport of GFAP from astrocytes to neurons, thereby inhibiting the abnormal aggregation of GFAP in neurons.

[0018] Using this technical solution, the drug blocks the transport of GFAP from astrocytes to neurons, effectively preventing its accumulation within neurons. This mechanism of action helps maintain the normal physiological function of neurons and mitigates damage to the nervous system caused by diseases.

[0019] Furthermore, the drug is used to alleviate mitochondrial dysfunction caused by the accumulation of GFAP in neurons; the mitochondrial dysfunction includes loss of mitochondrial membrane potential, decreased ATP synthesis capacity, decreased respiratory chain function and cellular metabolic disorder.

[0020] Using this technology, GFAP accumulation in neurons leads to mitochondrial dysfunction, including loss of mitochondrial membrane potential, decreased ATP synthesis capacity, decreased respiratory chain function, and cellular metabolic disorders. This drug can alleviate these pathological changes, restore normal mitochondrial function, and thus protect the viability and activity of neurons.

[0021] Furthermore, it also includes a pharmaceutically acceptable carrier.

[0022] Using this technology, the drug contains not only the active ingredient, such as the blocking peptide, but also a pharmaceutically acceptable carrier, such as lipid nanoparticles. These carriers play a crucial role in the drug delivery system, significantly improving the drug's targeting and delivery efficiency to brain tissue.

[0023] The carrier can be a liquid, making the composition suitable for parenteral administration, or can be a solid, i.e., a tablet or pill formulated for oral administration. In addition, the carrier can be in the form of an atomizable liquid or solid, making the composition suitable for inhalation. The blocking peptide of the regimen can also be formulated for topical administration, for example in the form of a cream or gel.

[0024] Furthermore, the carrier is a lipid nanoparticle for improving the brain-targeted delivery efficiency of the blocking peptide.

[0025] The above-mentioned technical solution, using lipid nanoparticles (LNPs) as carriers to improve the efficiency of brain-targeted delivery of blocking peptides, offers several advantages. The blood-brain barrier (BBB) is one of the major obstacles restricting the entry of most drugs into the brain. By designing specifically engineered LNPs, receptor-mediated transcytosis or other mechanisms can be exploited to enhance the ability of drugs to cross the BBB, effectively delivering the blocking peptide to the target site. LNPs provide a physical barrier for the encapsulated blocking peptide, preventing enzymatic degradation during blood circulation while also reducing immune recognition and clearance, thereby prolonging the drug's half-life. Because LNPs enable more efficient targeted delivery, they can achieve the same therapeutic effect at lower doses, reducing the likelihood of systemic side effects. Lipid materials are generally biodegradable and biocompatible, making LNP-based delivery systems relatively safe and reducing potential toxicity risks.

[0026] Therefore, using lipid nanoparticles as carriers of blocking peptides can not only significantly improve the efficiency of brain-targeted drug delivery, but also effectively overcome the various challenges faced by traditional drug delivery methods. It is a highly promising option for diseases such as Alzheimer's disease that require efficient brain delivery treatment strategies.

[0027] Furthermore, a blocking peptide for blocking the interaction between GFAP and APP is provided, which is used to replace GFAP and bind to the TTTTTTESVEE fragment of APP protein.

[0028] Furthermore, a blocking peptide for blocking the interaction between GFAP and APP, the sequence of which at least includes KRNIVVKTVEM.

[0029] Using the above technical solution, molecular docking analysis revealed the key amino acid sites for the interaction between GFAP and APP. The binding sites of GFAP and APP N-terminus are at "TTTTTTESVEE" of APP and at "KRNIVVKTVEM" of GFAP. Blocking peptides can be designed based on the above information.

[0030] Furthermore, the amino acid sequence of the blocking peptide is shown in any one of SEQ ID NOs. 1-7.

[0031] Furthermore, the N-terminus of the blocking peptide is modified with a lysine residue; and the blocking peptide is modified with palmitic acid.

[0032] Furthermore, the number of the lysine residues is 6; and the palmitic acid is connected to the K at the 7th position of SEQ ID NO.1.

[0033] This technical solution also provides a use of a blocking peptide for blocking the interaction between GFAP and APP in the preparation of a drug for treating Alzheimer's disease.

[0034] Using this technology, blocking peptides can slow the pathological progression of Alzheimer's disease by specifically blocking the interaction between GFAP (glial fibrillary acidic protein) and APP (amyloid precursor protein). This interaction plays a key role in the development of Alzheimer's disease, so by blocking this interaction, it is expected to slow the progression of the disease.

[0035] The present technical solution also provides a use of a blocking peptide for blocking the interaction between GFAP and APP in the preparation of a drug for treating and alleviating mitochondrial dysfunction in neurons.

[0036] Using this technology, mitochondrial dysfunction is a key feature of many neurodegenerative diseases, including Alzheimer's disease. Blocking peptides can protect neurons from damage through specific therapeutic mechanisms targeting mitochondrial dysfunction. These include regulating mitochondrial homeostasis, promoting mitophagy, and optimizing mitochondrial energy metabolism.

[0037] The present technical solution also provides a use of a blocking peptide for blocking the interaction between GFAP and APP in the preparation of a reagent for blocking GFAP cellular endocytosis mediated by APP as a membrane receptor.

[0038] Using this technical approach, we have discovered that APP not only participates in the formation of amyloid plaques but also acts as a membrane receptor to transmit signals between neurons and glial cells. Blocking peptides can be designed as new reagents to investigate new aspects of the interaction between GFAP and APP. This will help us better understand the mechanism of this interaction and may provide clues for the development of new treatments.

[0039] This technical solution also provides a use of a blocking peptide for blocking the interaction between GFAP and APP in the preparation of a product for detecting APP.

[0040] Using this technical solution, blocking peptides can be used to develop new APP detection reagents. By specifically binding to APP, blocking peptides can be used as probes to detect APP expression levels and activity. This could aid in the diagnosis of Alzheimer's disease and other APP-related diseases.

[0041] The present technical solution also provides a use of a blocking peptide for blocking the interaction between GFAP and APP in the preparation of a drug for enhancing neuronal synaptic plasticity.

[0042] Using this technical solution, synaptic plasticity, which is the foundation of learning and memory and one of the key processes impaired in Alzheimer's disease, can be enhanced by blocking peptides by improving the interaction between GFAP and APP, promoting synaptic plasticity in neurons. This includes regulating synaptic strength and promoting synapse formation and maintenance.

[0043] This technical solution also provides a use of a blocking peptide for blocking the interaction between GFAP and APP in the preparation of a drug for inhibiting cGAS-STING-mediated inflammatory response.

[0044] Using this technical solution, we demonstrate that the cGAS-STING pathway plays a crucial role in inflammatory responses and is closely linked to the development of Alzheimer's disease. Blocking peptides can alleviate neuroinflammation and neurological damage associated with Alzheimer's disease by inhibiting the cGAS-STING-mediated inflammatory response. This includes modulating the production and release of inflammatory factors and inhibiting the activation and migration of inflammatory cells.

[0045] This technical solution also provides the use of GFAP and APP interaction sites in designing drugs for treating Alzheimer's disease.

[0046] Using this technical solution, the discovery of interaction sites between GFAP and APP allows for drug design based on these sites. These sites are crucial for maintaining the interaction between the two and represent key targets for designing drugs targeting this interaction. By precisely targeting these sites, highly effective and specific drugs can be designed to modulate the interaction between APP and GFAP, thereby treating related diseases.

[0047] Furthermore, the amino acid sequence of the N-terminus of APP is shown in SEQ ID NO.8.

[0048] Furthermore, the amino acid sequence of GFAP is shown in SEQ ID NO.9.

[0049] Furthermore, in the APP protein, the interaction sites with GFAP include at least one of D at position 64, K at position 99, T at position 152, E at position 156, T at position 275, T at position 277, T at position 278, T at position 279, E at position 281, V at position 283, E at position 284, E at position 285, E at position 380, Q at position 406, and Q at position 450.

[0050] Furthermore, in the APP protein, the interaction sites with GFAP include T at position 275, T at position 277, T at position 278, T at position 279, E at position 281, V at position 283, E at position 284, and E at position 285.

[0051] Furthermore, in the GFAP protein, the interaction sites with APP include at least one of R at position 124, D at position 128, R at position 136, Q at position 146, K at position 339, R at position 367, K at position 368, K at position 405, R at position 406, N at position 407, V at position 409, K at position 411, V at position 413, and M at position 415.

[0052] Furthermore, in the GFAP protein, the interaction sites with APP include K at position 405, R at position 406, N at position 407, V at position 409, K at position 411, V at position 413, and M at position 415.

[0053] Furthermore, the drug used to treat Alzheimer's disease is a polypeptide.

[0054] Furthermore, the sequence of the polypeptide covers a core sequence; the core sequence is KRNXVXKXVXM, or a sequence having a sequence similarity of ≥80% to KRNXVXKXVXM, wherein X is any amino acid residue.

[0055] Using this technical solution, drug design can use this as a template or reference to design peptides with similar structural features to specifically block the interaction between APP and GFAP. The designed peptide drug covers the aforementioned core sequence and possesses high specificity and biological activity. This means that the peptide drug can precisely target the N-terminus of APP, specifically binding to it and thereby blocking the interaction between GFAP and APP. This high specificity and biological activity make the peptide drug more effective in treatment while also reducing potential side effects on normal cells.

[0056] Furthermore, the polypeptide is used to bind to TTTTTTESVEE at the N-terminus of APP.

[0057] Using this technical solution, the blocking peptide specifically binds to the "TTTTTTESVEE" residue at the N-terminus of APP (as shown in SEQ ID NO. 8), blocking the interaction between GFAP and APP. This binding prevents GFAP from cleaving APP, thereby reducing Aβ production. By reducing Aβ production and deposition, peptide drugs are expected to have a positive impact on the treatment of Alzheimer's disease.

[0058] Furthermore, the binding free energy of the molecular docking between the polypeptide and the N-terminus of APP is less than -420.11 kcal / mol; and the ipTM value of the molecular docking between the polypeptide and the N-terminus of APP is greater than or equal to 0.7.

[0059] Using this technical solution, the binding free energy of the peptide docking with the N-terminus of APP was less than -420.11 kcal / mol, and the ipTM value was ≥0.7, indicating strong binding ability and stability. This means that the peptide drug binds very tightly to the N-terminus of APP, and this binding is stable and not easily affected by the external environment. This stable binding helps ensure the therapeutic effect and duration of the peptide drug in the body.

[0060] In summary, the technical principle of this technical solution is:

[0061] This technical solution is based on in-depth research on new discoveries in the pathogenesis of Alzheimer's disease (AD). Among them, GFAP abnormally aggregates in neurons of AD patients, and this aggregation is associated with mitochondrial damage, suggesting that GFAP may affect mitochondrial function or neuronal homeostasis. GFAP is not only a marker of astrocyte activation, but may also participate in the pathological mechanism of AD through transcellular transport and its abnormal aggregation in neurons, leading to mitochondrial dysfunction, synaptic damage or inflammatory response. GFAP directly interacts with APP to mediate the transcellular transport of GFAP, indicating the important role of APP as a membrane receptor in GFAP endocytosis. The accumulation of GFAP may not only act through Aβ toxicity, but also directly damage mitochondria, causing neuronal mitochondrial dysfunction, further aggravating neuronal energy metabolism disorders and damage. Therefore, the interaction between GFAP and APP can be used as an intervention target for AD.

[0062] Based on the above research results, the development of peptide blockers targeting the interaction between GFAP and APP can be used as a new treatment method to alleviate AD symptoms and delay disease progression. Further research and development of blockers was carried out, and the interaction between GFAP and the N-terminus of APP was analyzed in detail. Through AlphaFold3 prediction and SPR verification, the binding site of the APP N-terminal AcD domain and GFAP was identified (KD = 83.6nM). The high-affinity binding region and key amino acid residues of the APP N-terminus were further identified. Based on the key sites of the interaction between GFAP and APP, a series of peptide blockers were designed and optimized, and the effects of multiple candidate blockers were evaluated. Ultimately, GAB1 was identified as the best blocker, and the peptide was further modified with C6K or palmitic acid to improve its stability and bioavailability, ensuring its effectiveness in vivo.

[0063] Experimental research results show that GAB1 peptide inhibits GFAP-APP binding and reduces cellular endocytosis of GFAP. In animal experiments, C6K-GAB1 was injected into the tail vein, and in vivo imaging of small animals showed that the peptide successfully entered the brain; GAB1 significantly improved the performance of AD model mice in water maze, Y maze, and novel object recognition tasks. GAB1 treatment also improved hippocampal synaptic damage in 5xFAD mice, reduced mitochondrial damage in the course of AD, and inhibited the cGAS-STING-related neuroinflammatory pathway. Immunohistochemistry and transmission electron microscopy analysis showed that GAB1 peptide reduced Aβ deposition, increased synaptic spine and synapse density, and improved mitochondrial ultrastructure. In terms of safety, no liver or kidney damage was observed in long-term toxicity experiments (ALT / AST were normal).

[0064] Through molecular docking technology and a series of experimental verifications, an effective peptide blocker GAB1 was successfully identified and optimized. This blocker can specifically interfere with the interaction between GFAP and APP, thereby preventing GFAP from transcellular transport into neurons, alleviating mitochondrial damage and other pathological changes caused by it, and providing a new strategy for the treatment of Alzheimer's disease.

[0065] The beneficial effects of this technical solution are:

[0066] (1) Blocking GFAP transcellular transport: By specifically targeting the binding site between GFAP and APP, the polypeptide blocker of this technical solution can effectively prevent the abnormal transcellular transport of GFAP, thereby reducing its accumulation in neurons.

[0067] (2) Protecting mitochondrial function: GFAP accumulation can lead to mitochondrial dysfunction, including loss of mitochondrial membrane potential, decreased respiratory chain function, and cellular metabolic disorders. The peptide blocker of this technical solution can reduce the damage of GFAP to mitochondria, thereby protecting the energy metabolism and viability of neurons.

[0068] (3) Reduce synaptic loss: Abnormal distribution and accumulation of GFAP disrupts normal synaptic structure and function, promoting synaptic loss. The peptide blocker of this technical solution can slow the rate of synaptic loss by preventing the transcellular transport of GFAP, thereby delaying the cognitive decline of AD.

[0069] (4) Inhibit inflammatory response: Abnormal distribution of GFAP can promote the release of inflammatory mediators, exacerbating the local inflammatory environment. The polypeptide blocker of this technical solution can reduce the promoting effect of GFAP on the inflammatory response, thereby alleviating the inflammatory response and damage of neurons.

[0070] (5) Providing new therapeutic strategies: This technical solution not only reveals the important role of GFAP in AD pathology and its molecular mechanism, but also provides a new strategy for the treatment of AD. By optimizing the structure and function of the peptide, we can develop more effective, safe and easy-to-use drugs, providing patients with better treatment effects and quality of life.

[0071] (6) Multifunctional application potential: In addition to being used directly as a drug for the treatment of AD, this type of peptide can also be developed to detect the functional status of APP or used in auxiliary treatments to enhance neuronal synaptic plasticity.

[0072] In summary, this technical solution, through in-depth exploration of the transcellular transport mechanism of GFAP and the design of corresponding peptide blockers, provides new ideas and methods for the treatment of Alzheimer's disease. These research results not only help us better understand the pathogenesis of AD, but also provide possibilities for future peptide-based personalized medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1The results of the study on the mechanism of GFAP trans-astrocyte entry into neurons and aggregation in Example 1 (A: Immunoelectron microscopy showed that compared with WT, AD patient-derived neurons showed obvious abnormal aggregation of GFAP indicated by red arrows, accompanied by obvious mitochondrial vacuolation and structural damage; B: WB detection of GFAP expression changes in human induced pluripotent stem cell-derived astrocytes hiPSC-Astro after 24 hours of stimulation with different concentrations of lipopolysaccharide LPS 0, 0.125, 0.25, 0.5, 1, 2 μg / mL; showing that GFAP expression increased in a dose-dependent manner; C: WB detection of hiPSC-Astro stimulated with different concentrations of amyloid protein Aβ oligomers 0, 0.63, 1.25, 2.5, 5, 10 μM Figure 2: Changes in GFAP expression after 24 hours. It was found that GFAP expression increased with increasing Aβ concentration; D: Schematic diagram of the Transwell co-culture system: stimulated hiPSC-Astro cells were placed in the upper layer of the Transwell, and hiPSC-induced neurons (hiPSC-Ns) or trisomy 21-derived neurons DS-hiPSC-Ns were cultured in the lower layer; after stimulation, GFAP was released by astrocytes in the upper chamber and transported into the neurons in the lower layer; E: WB detection results showed that after 24 hours of LPS 1μg / mL stimulation, GFAP released by hiPSC-Astro cells in the upper layer could be transported across cells to hiPSC-Ns and DS-hiPSC-Ns cultured in the lower chamber, confirming the transcellular transport of GFAP).

[0074] Figure 2 The experimental results of the interaction between APP and GFAP and their endocytosis in Example 2 (A: WB detection of GFAP expression in the culture supernatant of 293T / GFAP cells. The GFAP level in the KD-APP group was significantly increased compared with the WT group; B: WB detection of APP and LRP1 expression in 293T / GFAP cell lysate, indicating that APP and LRP1 were successfully knocked down; C: WB detection of GFAP expression in the culture supernatant of SH-SY5Y / GFAP cells. The GFAP level was significantly increased after KD-APP treatment; D: WB detection of SH-SY5Y / GFAP Cell lysate, verifying the successful knockdown of APP protein expression level; E: pHrodo-GFAP treatment of SH-SY5Y and SH-SY5Y / APP695-mCherry cells, live cell imaging to observe the dynamics of GFAP endocytosis, the results showed that SH-SY5Y / APP695 cells significantly enhanced GFAP endocytosis; white arrows indicate the aggregation of endocytosed GFAP; F: WB detection of APP, GFAP and Aβ expression, the results showed that in the SH-SY5Y / APP695 cells GFAP treatment group, Aβ production was significantly increased; G: ELISA detection of Aβ 42Statistical analysis showed that the Aβ production in the GFAP-treated group was significantly increased; *p<0.05, **p<0.01; data are expressed as mean ± SEM, and statistical analysis was performed using a two-tailed t-test).

[0075] Figure 3 The experimental study results of Example 3 on the damage of GFAP aggregation to neuronal mitochondrial function and cell metabolism (A: Schematic diagram of mitochondrial membrane permeability transition pore mPTP opening detection: Calcein displayed by green fluorescence in the closed state is retained by mitochondria, and calcium ions Ca 2+ After loading, the fluorescence signal is stable; when the mPTP opens, Calcein is released and the fluorescence signal weakens; B: Flow cytometry analysis shows that the openness of mPTP in SH-SY5Y / APP695 cells decreases after GFAP treatment; the left figure is a scatter plot, the middle figure is a histogram, and the right figure is a Calcein AM staining image; the mPTP opening rate after GFAP treatment decreased from 94.44% to 83.63%; C: Statistical data showed that GFAP treatment significantly reduced the opening ratio of mPTP; D: Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) curves showed that the mitochondrial respiration OCR of SH-SY5Y / APP695 cells treated with GFAP was significantly decreased; E: GFAP treatment significantly affected the glycolysis-related ECAR of SH-SY5Y / APP695 cells; F: Transmission electron microscopy showed that the mitochondrial structure of SH-SY5Y / APP695 cells treated with GFAP was damaged).

[0076] Figure 4 This is the distribution of key sites for the interaction between GFAP and APP in Example 4.

[0077] Figure 5 These are the experimental results of structural prediction and functional verification of the key sites of interaction between GFAP and APP in Example 4 (A: Schematic diagram of the interaction between APP and GFAP, based on the molecular docking results of AlphaFold3, showing the GFAP-APP binding site with a docking energy of -420.11 kcal / mol; B: SPR analysis results, showing that the binding affinity Kd of the APP N-terminus and the GFAP end at different concentrations of 320 nM, 160 nM, 80 nM, and 40 nM is 83.6 nM, indicating that the two have high-affinity binding; C: Co-IP experiment verifies the binding of APP N-terminus to GFAP; D E: BiFC experiment analyzes the effect of APP domain deletion on GFAP binding. After the acidic region Del-AcD is deleted, the interaction signal between GFAP and APP is significantly decreased; F: WB detection of the expression of APP, APP deletion mutants and GFAP proteins in E; G, H: IF detection of the expression of exogenous GFAP-triggered APP (SiRIs) fluorescence sensing system, indicating that GFAP promotes APP endocytosis).

[0078] Figure 6 The experimental results of Example 5 for proximity labeling to verify the interaction between the APP N-terminus and GFAP protein in human brain organoids (A: Schematic diagram of the experimental design for analyzing APP695 and its interacting proteins using the miniTurboID labeling method; APP-N-terminus is bound to the biotin tag and verified by mass spectrometry analysis; B: WB shows the effect of 0-10mM concentration of biotin on APP N-terminus and miniTurboID protein labeling in hiPSC-induced brain organoids; C: With the increase of biotin concentration, the degree of binding of APP N-terminus to miniTurboID tag in hiPSC-induced brain organoids increases with time, and the detection period is 0-120 minutes; D: Immunoprecipitation experiment IP shows the binding of APP N-terminus and miniTurboID tag, enriching the interaction between APP N-terminus and labeled protein; E: Venn diagram shows APP The overlapping part of the N-terminus and its interacting proteins was used to screen out significantly up-regulated or down-regulated proteins, such as GFAP, ANXA2, MAP1A, etc. The volcano plot on the right further shows the changes in protein expression; foldchange>2 or <0.5; F: Enrichment analysis shows the role of proteins related to the APP N-terminus in different biological processes).

[0079] Figure 7 Schematic diagram of the docking between GAB1 and the N-terminus of APP in Example 6.

[0080] Figure 8 These are the experimental results of Example 6 on the inhibition of GFAP endocytosis at the cellular level by the APP N-terminal blocking peptide (A: in 293T / GFAP cells, after treatment with 0-10 μM concentrations of GAB1 peptide, the GFAP enrichment level in the supernatant increased with increasing peptide concentration; B: after 293T / GFAP cells were treated with 8 μM GAB1 peptide, at time points of 0, 3, 6, 12, and 24 hours, the GFAP enrichment in the supernatant increased with increasing GAB1 peptide treatment time, suggesting that GAB1 peptide may affect the endocytosis process of GFAP).

[0081] Figure 9The experimental results of Example 7 on the improvement of GFAP-induced cognitive dysfunction in 5xFAD mice by the GAB1 blocking peptide (A: Schematic diagram of the experimental design; B: Fluorescence distribution of the brain, liver, and kidney within 3 hours after intravenous injection of CY5.5-C6KGAB1 peptide (0-1 mg / kg) in mice, with the fluorescent signal in the mouse brain becoming more pronounced with increasing concentration; C: Immunohistochemical analysis showing the distribution of CY5.5-C6KGAB1 peptide in mouse brain tissue, detecting that the GAB1 peptide successfully entered the mouse brain tissue; D: Water maze behavioral test showing the time it took for AAV-GFAP to reach the target point in the GFAP overexpression group The time taken to locate the mouse was significantly longer than that of the AAV-Mock control group; the AAV-GFAP / GAB1 treatment group with GAB1 peptide significantly improved the positioning time and was similar to that of the AAV-Mock group, indicating that its cognitive function was improved; E: Y maze test, the frequency of direction changes in the maze in the GFAP overexpression group was significantly lower than that in the control group, while the frequency of direction changes in the GAB1 peptide treatment group was significantly increased, indicating that its cognitive function was improved; F: In the novel object recognition task, the time spent by the GFAP overexpression group on object exploration was significantly less than that in the control group, and the GAB1 peptide treatment group showed a stronger preference for novel objects, indicating that GAB1 peptide improved the memory ability of mice).

[0082] Figure 10The results of the study on GAB1 treatment in Example 7 to improve synaptic damage in the hippocampus of 5xFAD mice and inhibit the cGAS-STING related neuroinflammatory pathway (A: Immunohistochemical analysis of Aβ deposition in the hippocampus of 5xFAD-AAV-GFAP mice showed that C6KGAB1 treatment significantly reduced the number of Aβ senile plaques; B: Transmission electron microscopy observation of the ultrastructure of mitochondria in hippocampal neurons of 5xFAD mice showed that C6KGAB1 treatment can alleviate mitochondrial damage; C: Golgi staining analysis of the morphological changes of dendritic spines in hippocampal neurons of 5xFAD mice; D: Statistical analysis showed that C6KGAB1 treatment can significantly increase the density of dendritic spines, ***p<0.001; E: Transmission electron microscopy observation of the changes in synaptic density of hippocampal neurons in 5xFAD-AAV-GFAP and C6KGAB1 treated mice; F: Statistical analysis showed that C6KGAB1 treatment significantly increased synaptic density and improved 5xFAD D: Synaptic damage in the hippocampus of mice; ***p<0.001; G: Western blot analysis of the expression levels of cGAS, STING, p-STING, TBK1, p-TBK1, IRF3, and p-IRF3 proteins related to the cGAS-STING signaling pathway in hippocampal tissues. The phosphorylation levels of related proteins were reduced under C6KGAB1 treatment; H: qRT-PCR analysis of the mRNA expression levels of CXCL10, IFNB, IL-18, IL-12, IL-23, and IL-1β in hippocampal tissues; statistical analysis showed that C6KGAB1 treatment significantly inhibited the expression of CXCL10 (***p<0.001), IFNB (*p<0.05), IL-18 (*p<0.05), and IL-1β (**p<0.01), while the expression of IL-12 and IL-23 did not change significantly (ns); data are expressed as mean ± SEM, and statistical analysis was performed using a two-tailed t-test). DETAILED DESCRIPTION

[0083] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available.

[0084] The definitions and explanations of terms are as follows:

[0085] GFAP: Glial Fibrillary Acidic Protein, is an intermediate filament protein primarily expressed in astrocytes. Astrocytes are an important component of the central nervous system (CNS) and are involved in a variety of critical brain functions and maintenance activities.

[0086] AD: Alzheimer's Disease (AD), a progressive neurodegenerative disease that primarily affects the elderly.

[0087] APP: Amyloid Precursor Protein. A fragment of APP, β-amyloid (Aβ), is believed to be one of the main components of plaques that appear in the brains of patients with Alzheimer's disease. These plaques are associated with cognitive impairment (e.g., Alzheimer's disease).

[0088] LPS: Lipopolysaccharide.

[0089] Aβ: The full name is β-amyloid protein (Amyloid-beta, Aβ), which is a peptide produced by amyloid precursor protein (Amyloid Precursor Protein, APP) through a series of enzymatic cleavage processes.

[0090] Aβ 42 : Amyloid-beta 42 (Aβ) is a protein composed of 42 amino acids. 42 ), is one of the key molecules in the pathology of Alzheimer's disease (AD). 40 In contrast, although Aβ 42 It accounts for a small proportion of the total Aβ production, but it is more likely to aggregate to form toxic oligomers, fibers and senile plaques. The deposition of these aggregates in the brains of Alzheimer's patients is considered to be one of the important causes of neuronal damage and death.

[0091] DS: Down syndrome is a genetic disease caused by trisomy of chromosome 21.

[0092] Lipid nanoparticles (LNPs): A drug delivery system currently in use, LNPs are widely used in the medical field, particularly in gene therapy and vaccine development. Composed of lipid molecules, they can stably carry and protect drugs or nucleic acids (such as mRNA, siRNA, and proteins) in the body, thereby improving the bioavailability and targeting efficiency of these therapeutic molecules. Their raw materials include ionic or non-ionic lipids, cholesterol, and PEGylated lipids.

[0093] The cGAS-STING pathway is an important innate immune recognition mechanism within cells that detects DNA in the cytoplasm and triggers an inflammatory response to fight infection, injury, and other threats. cGAS is the enzyme cyclic GMP-AMP synthase; STING is the protein that stimulates interferon genes.

[0094] hiPSC: Human induced pluripotent stem cells (hiPSCs) are obtained by reprogramming adult cells (such as skin cells or blood cells) back to a state similar to embryonic stem cells.

[0095] DS-hiPSC neurons: hiPSCs are reprogrammed from mononuclear blood cells isolated from the peripheral blood of individuals with Down syndrome and then further differentiated into neuronal cells.

[0096] SH-SY5Y: neuroblastoma cell line.

[0097] 293T: human embryonic kidney cell line.

[0098] SH-SY5Y / APP695-mCherry cells: SH-SY5Y cell lines stably overexpressing the amyloid precursor protein APP695 isoform and mCherry fluorescent protein, prepared by conventional genetic engineering methods in the prior art.

[0099] SH-SY5Y / APP695 cells: SH-SY5Y cell line stably overexpressing the amyloid precursor protein APP695 isoform prepared by conventional genetic engineering means in the prior art.

[0100] SH-SY5Y / APPswe cells: SH-SY5Y cell lines stably overexpressing the amyloid precursor protein APPswe isoform prepared by conventional genetic engineering methods in the prior art.

[0101] 293T / GFAP cells: A 293T cell line stably overexpressing GFAP protein prepared by conventional genetic engineering methods in the prior art.

[0102] SH-SY5Y / GFAP cells: SH-SY5Y cell line stably overexpressing GFAP protein prepared by conventional genetic engineering methods in the prior art.

[0103] AAV-GFAP overexpression mice: Glial fibrillary acidic protein (GFAP) is overexpressed in mice through adeno-associated virus (AAV) vector-mediated transduction.

[0104] AAV-GFAP virus: A vector based on the adeno-associated virus (AAV), which integrates the GFAP gene into its genome and then packages it into a virus. It can be used to infect experimental animals, causing them to overexpress GFAP. This virus can be prepared using conventional molecular biology methods.

[0105] 5xFAD mice: A transgenic mouse model commonly used in Alzheimer's disease (AD) research. This model is characterized by the introduction of five familial Alzheimer's disease-associated mutations into the mouse genome. These mutations are located in two key genes: the amyloid precursor protein gene (APP) and the presenilin 1 gene (PSEN1).

[0106] Example 1: Study on the mechanism of GFAP translocation from astrocytes to neurons and aggregation

[0107] Immunoelectron microscopy was used to observe GFAP protein expression in neurons derived from healthy subjects and AD patients. HiPSC-derived astrocytes were stimulated with LPS (0, 0.125, 0.25, 0.5, 1, and 2 μg / mL, 24 hours) or Aβ (0, 0.63, 1.25, 2.5, 5, and 10 μM, 72 hours) to induce reactive astrocytes. GFAP protein expression was assessed by Western blotting.

[0108] To examine GFAP transcellular transport, a Transwell co-culture experiment was performed. Activated human iPSC-derived astrocytes were cultured in the upper layer, while hiPSC-derived neurons (hiPSC-Ns) or neurons derived from trisomy 21 (DS-hiPSC-Ns) were cultured in the lower layer. Culture medium and cell lysates from the lower layer were collected regularly, and GFAP content was determined by Western blotting.

[0109] The experimental results are detailed in Figure 1, showing the abnormal aggregation of GFAP in neurons of AD patients and its transcellular transport under LPS / Aβ stimulation. Transmission electron microscopy results showed that the abnormal enrichment of GFAP in neurons of AD patients was associated with mitochondrial damage, suggesting that GFAP may play an important role in the pathological process of AD by affecting mitochondrial function or neuronal homeostasis. In addition, LPS and Aβ stimulation upregulated the expression of GFAP in hiPSC astrocytes and promoted the upregulated GFAP to enter neurons through the transcellular pathway. Since patients with trisomy 21 syndrome (DS) have overexpression of the APP gene, DS-hiPSC neurons were used to study the effect of APP on the transcellular transport and aggregation of GFAP. The results suggest that GFAP is not only a marker of astrocyte activation, but may also affect mitochondrial dysfunction, synaptic damage or inflammatory response through transcellular transport and abnormal aggregation in neurons, thereby participating in the pathological mechanism of AD.

[0110] Example 2: Study on the molecular mechanism of APP as a membrane receptor mediating GFAP endocytosis

[0111] The APP gene or LRP1 in 293T cells was knocked out using conventional means in the prior art (KD-APP group and KD-LRP1 group), and then the GFAP level in the 293T cell culture supernatant was detected by WB, and the expression of APP and LRP1 in the 293T cell lysate was detected by WB. The APP gene in SH-SY5Y cells was knocked out using conventional means in the prior art (KD-APP group), and then the GFAP level in the SH-SY5Y cell culture supernatant was detected by WB, and the expression of APP in the SH-SY5Y cell lysate was detected by WB.

[0112] Using pHrodo TM GFAP was labeled with pHrodo Green fluorescent dye, and SH-SY5Y and SH-SY5Y / APP695-mCherry cells were treated with pHrodo-GFAP. The rate of GFAP entering neurons was recorded using laser confocal live cell imaging.

[0113] SH-SY5Y / APP695 cells and SH-SY5Y / APPswe cells were treated with GFAP to detect the expression of APP and Aβ in the cells. 42 Level ELISA detection.

[0114] For detailed experimental results, see Figure 2, showing the experimental results of the interaction between APP and GFAP and their endocytosis. GFAP directly interacts with APP and mediates the transcellular transport of GFAP. pHrodo-GFAP tracing experiments showed that GFAP can be mediated by APP into neurons, indicating that APP plays an important role as a membrane receptor in GFAP endocytosis. In addition, APP overexpression significantly increases Aβ 42 levels, suggesting that GFAP endocytosis may be potentially coupled with Aβ production. 42 The concentration (about 400 pg / mL) is much lower than the 1-10 μM (4500-45000 pg / mL) required for the classic Aβ-induced mitochondrial damage cell model, and is only 1 / 10 of the 1 μM damage-inducing concentration, indicating that GFAP-mediated Aβ production may not be sufficient to cause mitochondrial damage, but instead directly affects neuronal function through GFAP.

[0115] Example 3: Study on the damage of GFAP aggregation to neuronal mitochondrial function and cell metabolism

[0116] SH-SY5Y / APP695 cells were treated with GFAP to observe the extent of mitochondrial membrane permeability transition pore (mPTP). Seahorse metabolic analysis was used to evaluate the effects of GFAP on mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in SH-SY5Y / APP695 and SH-SY5Y cells. Transmission electron microscopy was used to observe damage to mitochondrial structure in SH-SY5Y / APP695 and SH-SY5Y cells after GFAP treatment.

[0117] The experimental results are detailed in Figure 3 , demonstrating that GFAP accumulation in neurons leads to mitochondrial dysfunction, including loss of mitochondrial membrane potential, decreased respiratory chain function, and cellular metabolic disturbances. In SH-SY5Y / APP695 cells treated with GFAP, oxidative phosphorylation was significantly impaired, and mitochondrial morphology was also disrupted, suggesting that GFAP accumulation may not only act through Aβ toxicity but also directly damage mitochondria, further exacerbating neuronal energy metabolism disorders and damage.

[0118] Example 4: Structural prediction and functional verification of key sites of interaction between GFAP and APP

[0119] AlphaFold3 (Abramson, J et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 2024) predicted the binding region between the APP N-terminus (containing E1, AcD, OX2, E2, and JMR) and the GFAP sequence. RosettaDock screened the top 10 docking conformations and used the AMBER force field and MM / PBSA calculations to identify the lowest-energy and biologically significant conformations. Molecular dynamics (MD) simulations lasting 100-200 ns were then performed on the selected conformations using GROMACS to analyze hydrogen bonds, hydrophobic interactions, salt bridge formation frequency, RMSD, RMSF, SASA, and changes in binding free energy. The high-affinity binding region and key amino acid residues between the APP N-terminus and GFAP were identified. SPR was used to determine the binding affinities of wild-type and mutant APP N-termini with GFAP. Co-IP experiments verified the binding of the APP N-terminus to GFAP. BiFC experiments analyzed the effects of APP domain deletion on GFAP binding. Western blotting was used to detect the expression of APP and GFAP proteins in APP deletion mutants. IF was used to detect the expression of APP (SiRIs) fluorescence sensing system triggered by exogenous GFAP.

[0120] The amino acid sequence of the APP N-terminus (1-625aa) is shown in SEQ ID NO.8:

[0121]

[0122] The amino acid sequence of GFAP (1-432aa) is shown in SEQ ID NO.9:

[0123]

[0124] In the amino acid sequence of GFAP and the amino acid sequence of APP N-terminus, the double underlined and bolded amino acid residues in the two proteins interact with each other. For detailed schematic diagram, please refer to Figure 4 Analysis indicates that the aforementioned amino acid residues are key sites for interaction between GFAP and the N-terminus of APP, and this could be used as a basis for the subsequent design of peptide blockers. The binding site between GFAP and the N-terminus of APP is located at the "TTTTTTESVEE" region of APP.

[0125] AlphaFold3 molecular docking and SPR analysis revealed a high-affinity interaction between the APP N-terminal ACD domain and GFAP. Co-IP and BiFC experiments further confirmed this, and a fluorescence signal was shown in living cells by bimolecular fluorescence complementation experiments. The results confirmed that the AcD domain of GFAP and APP N-terminus interact. Figure 5 , showing the structural prediction and functional verification results of the key sites of interaction between GFAP and APP.

[0126] Example 5: Study on pathological changes of GFAP-APP interaction in human brain organs

[0127] HiPSCs were induced to form brain organoids (45-60 days) using conventional methods. MiniTurboID labeling (proximity labeling) was then used to analyze the presence of a stable interaction between the APP N-terminus and GFAP. Figure 6 Using a proximity labeling method (miniTurboID), researchers screened and confirmed the interaction between the APP N-terminus and GFAP in hiPSC-derived human brain organoids. Functional enrichment analysis of key proteins revealed that the APP / GFAP interaction may contribute to the pathogenesis of neurodegenerative diseases such as AD by influencing mitochondrial autophagy and inflammatory signaling pathways.

[0128] Example 6: Design of a blocker for blocking the interaction between APP and GFAP and in vitro experimental verification

[0129] Based on the research results of the key sites of interaction between GFAP and APP in Example 4, a blocker was designed.

[0130] First, a blocker was designed based on the K, R, N, V, K, V, and M residues at positions 405-407, 409, 411, 413, and 415 of GFAP. The specific sequence is: GHLKRNIVVKTVEMRDG (GAB3, SEQ ID NO. 3, amino acids 402-418 of the GFAP protein). Experimental results showed that upon docking with the APP N-terminus, its binding free energy was -393.39 kcal / mol (higher than the binding free energy of -420.11 kcal / mol for the APP N-terminus and GFAP), and its ipTM value was 0.8 (better than the ipTM value of 0.13 for the APP N-terminus and GFAP, indicating a close approximation to the true interaction structure). Therefore, the binding free energy of GAB3 does not meet the requirements and needs further improvement. The ipTM value is primarily used to assess the confidence of complex structure predictions in molecular docking. Higher values indicate more reliable results, indicating that the relative positions and interactions between the two proteins are closer to the true reality. The magnitude of the binding free energy directly reflects the strength of the interaction between the two molecules. Lower (more negative) binding free energy indicates a tighter bond between the two molecules and a stronger binding capacity. Conversely, higher (closer to zero or more positive) binding free energy indicates weaker binding. Evaluation tool HDOCKserver. Reference: Yan Y, Zhang D, Zhou P, Li B, Huang SY. HDOCK: A web server for protein-protein and protein-DNA / RNA docking based on a hybrid strategy. Nucleic Acids Res. 2017;45:W365-W373.

[0131] Mutation design was performed based on GAB3, specifically: GHLKR R IVK D VEMRDG (GAB2, SEQ ID NO. 2, double underlined mutation sites) was used to enhance the interaction between the blocker and APP. However, the ipTM value of GAB2 was found to be 0.49 (docking with the APP N-terminus), indicating a low confidence level for the interaction model. Therefore, this short peptide was not suitable as a candidate blocker.

[0132] Based on GAB3, we extended it forward, specifically to: VSEGHLKRNIVVKTVEMRDGEV (GAB1, SEQ ID NO.1, amino acids 399-420 of GFAP protein). The results showed that when docked with the APP N-terminus, its binding free energy was -449.48 kcal / mol (better than the binding free energy of -420.11 kcal / mol between the APP N-terminus and GFAP), and its ipTM value was 0.77 (better than the ipTM value of 0.13 between the APP N-terminus and GFAP, which is close to the real interaction structure), making it a preferred blocker. For details, see the schematic diagram of the docking of GAB1 with the APP N-terminus. Figure 7 .

[0133] To further identify more effective blockers, further structural studies were conducted, extending the GAB3 structure to EGHLKRNIVVKTVEMRDGEVIKE (GAB4, SEQ ID NO. 4). Results showed that its binding free energy for docking with the APP N-terminus was -383.23 kcal / mol (less than the -420.11 kcal / mol binding free energy for APP N-terminus and GFAP), and its ipTM value was 0.73 (better than the ipTM value of 0.13 for APP N-terminus and GFAP, indicating a close approximation to the true interaction structure). As a blocker, its effectiveness was inferior to that of GAB1.

[0134] Based on GAB3, we selected peptides, specifically DTKSVSEGHLKRNIVVKTVEMR (GAB5, SEQ ID NO. 5, amino acids 395-416 of GFAP protein). Although GAB5 also contains the aforementioned key binding sites (K, R, N, V, K, V, M at positions 405-407, 409, 411, 413, and 415 of GFAP), its IPTM value when docked with the APP N-terminus is 0.54, indicating low interaction confidence and is not suitable for use as a blocking agent.

[0135] Based on GAB3, the core amino acids were retained and the peptide chain was shortened to KRNIVVKTVEM (GAB6, SEQ ID NO. 6, amino acids 405-413 of GFAP protein). The results showed that when docked with the N-terminus of APP, its ipTM value was 0.54, indicating low interaction reliability and not suitable for use as a blocking agent.

[0136] Based on GAB3, a slightly shorter peptide chain was selected, specifically EGHLKRNIVVKTVEMRDGE (GAB7, SEQ ID NO. 7, amino acids 401-419 of GFAP protein). The results showed that its ipTM value was 0.31 when docked with the APP N-terminus, indicating low interaction reliability and unsuitable for use as a blocking agent.

[0137] The information summary of GAB1-7 is shown in Table 1, which records the sequences of different APP N-terminal blocking peptides (GAB1 to GAB7) and their molecular docking results with the APP N-terminus. The table lists the ipTM value, docking score and binding affinity with the APP N-terminus of each peptide. It can be seen from the above research results that, based on the information of the key sites of interaction between GFAP and APP, how to design the amino acid composition structure of the blocking peptide is also a key problem that needs to be solved in this solution. The inventors tried to design candidate blockers of different lengths and different positions based on the key sites, and tried the method of amino acid residue point mutation. The results showed that the effect of GAB1 as a blocker was far superior to others, and good technical effects were achieved.

[0138] Table 1: Molecular docking study results of APP N-terminal blocking peptides based on AlphaFold3 (“——” indicates that the binding free energy was not measured due to low confidence in the molecular interaction)

[0139] name serial number sequence Docking protein ipTM value Binding free energy GAB1 SEQ ID NO.1 VSEGHLKRNIVVKTVEMRDGEV APP-N 0.77 -449.48 kcal / mol GAB2 SEQ ID NO.2 GHLKRRIVVKDVEMRDG APP-N 0.49 —— GAB3 SEQ ID NO.3 GHLKRNIVVKTVEMRDG APP-N 0.8 -393.39 kcal / mol GAB4 SEQ ID NO.4 EGHLKRNIVVKTVEMRDGEVIKE APP-N 0.73 -383.23 kcal / mol GAB5 SEQ ID NO.5 DTKSVSEGHLKRNIVVKTVEMR APP-N 0.54 —— GAB6 SEQ ID NO.6 KRNIVVKTVEM APP-N 0.54 —— GAB7 SEQ ID NO.7 EGHLKRNIVVKTVEMRDGE APP-N 0.31 ——

[0140] Based on the above results, the effect of GAB1 was experimentally tested (GAB1 was C6K modified and palmitated, KKKKKKVSEGHLK(Pal)RNIVVKTVEMRDGEV). The experimental results are detailed in Figure 8. In 293T / GFAP cells, the enrichment level of GFAP in the supernatant was detected after treatment with different concentrations (0-10μM) of GAB1 peptide for 24 hours. In 293T / GFAP cells, the enrichment level of GFAP in the supernatant was detected at different time points (0, 3, 6, 12, and 24 hours) after treatment with 8μM GAB1 peptide. 293T / GFAP cells are a 293T cell line that stably overexpresses GFAP protein, and the expressed GFAP is secreted into the supernatant in large quantities. Because the cells are treated with GAB1 peptide, GFAP cannot bind to APP on the cell membrane, so GFAP cannot be effectively internalized by the cells. Therefore, treating the cells with higher concentrations of GAB1 peptide, or treating the cells with GAB1 peptide for a longer time, will result in a large amount of GFAP being unable to be internalized by the cells and being enriched in the supernatant. The results show that GAB1 peptide can specifically block GFAP from entering the cell through APP endocytosis. The above experimental results show the experimental verification results of the inhibition of GFAP endocytosis by APP N-terminal blocking peptide at the cellular level. In addition, in 293T / APP695 cells, GFAP in the cell supernatant was detected to increase with the increase of GAB1 treatment time and concentration, indicating that GAB1 can specifically inhibit GFAP endocytosis.

[0141] In summary, molecular docking analysis revealed that the GAB1 peptide had a high affinity for the APP N-terminus (docking score -449.48 kcal / mol) and exhibited a significant inhibitory effect in GFAP endocytosis experiments. GFAP expression levels in 293T cells changed with increasing GAB1 concentrations, and prolonged peptide treatment also affected GFAP accumulation, suggesting that GAB1 may interfere with GFAP endocytosis by inhibiting the interaction between APP and GFAP.

[0142] Example 7: In vivo experimental study of GAB1 blocking peptides

[0143] The GAB1 peptide was synthesized via conventional solid-phase synthesis. C6K modification (addition of six lysine residues to the N-terminus of the amino acid sequence of SEQ ID NO. 1) enhances its interaction with cell membranes and improves its intracellular delivery efficiency. Palmitation (palmitic acid modification at the first K of SEQ ID NO. 1) extends the peptide's half-life and reduces its rapid in vivo degradation. The peptide was purified using high-performance liquid chromatography (HPLC) to a purity of ≥95%. The modified GAB1 sequence is: KKKKKKVSEGHLK(Pal)RNIVVKTVEMRDGEV (also known as C6KGAB1).

[0144] In the in vivo experiment, in order to facilitate tracing, the second K on C6KGAB1 was modified with CY5.5 fluorescent dye (KKKKKKVSEGHLK(Pal)RNIVVK(CY5.5)TVEMRDGEV), namely CY5.5-C6KGAB1. This experiment used 5×FAD Alzheimer's disease transgenic mice as an animal model to explore the therapeutic effect of GAB1 peptide on Alzheimer's disease pathology and cognitive dysfunction. The specific process was: when the mice were 7 weeks old, AAV-GFAP virus was injected into the bilateral hippocampus using a stereotaxic instrument (virus dose was 2μL / side, virus titer was about 1×10 13 vg / mL) to induce GFAP overexpression in the hippocampus (a procedure that accelerates AD progression and shortens the experimental period), while a control group received an equal dose of AAV-mock virus. Subsequently, starting at 11 weeks of age, mice were injected twice weekly via the tail vein with CY5.5 fluorescently labeled C6KGAB1 peptide (at a dose of 2 mg / kg) for continued treatment until 15 weeks of age. Following treatment, the mice were subjected to Morris water maze, Y-shaped maze, and novel object recognition tests to assess improvements in spatial learning, memory, and cognitive function. Brain tissue was also collected for immunohistochemical analysis to determine GFAP expression, Aβ and Tau protein deposition, and neuroinflammatory pathological changes. This ultimately validated the GAB1 peptide's ameliorative effects on AD pathology and cognitive impairment, and its potential therapeutic potential as a treatment for AD. It should be noted that the 5xFAD mouse model is a typical AD mouse model. These mice exhibit not only Aβ deposition but also neuronal loss, typical pathological changes of Alzheimer's disease. As the disease progresses, 5xFAD mice also develop symptoms of cognitive impairment, including decreased learning and memory abilities. In order to accelerate the progression of the disease and the progress of the experiment, and based on the research results of Example 1 (abnormal aggregation of GFAP in neurons of AD patients), this experimental study overexpressed GFAP protein in the bilateral hippocampus of model animals, so that the symptoms of cognitive dysfunction such as decreased learning and memory ability of experimental animals appeared faster and more obviously, and promoted the faster and more obvious appearance of typical pathological changes including Alzheimer's disease, in order to study the effect of the polypeptide developed in this scheme (overexpression of GFAP accelerated the accumulation process of GFAP in nerve cells and accelerated the appearance of disease symptoms).

[0145] Water maze behavioral test, Y maze test and novel object recognition task are three commonly used behavioral experiments, which are mainly used to evaluate the cognitive function of experimental animals, especially learning and memory ability. Figure 9, showing that GAB1 blocking peptide significantly improved GFAP-induced cognitive dysfunction in 5xFAD mice. GAB1 peptide was injected into AAV-GFAP overexpressing mice via the tail vein, significantly improving their performance in water maze, Y maze and novel object recognition tasks. Among them, behavioral tests (water maze) showed that the escape latency was shortened by 40% after tail vein injection of C6K-GAB1 2mg / kg. Behavioral tests showed that GAB1 peptide may improve the cognitive function of mice by regulating the mechanism of action of GFAP. In addition, small animal in vivo imaging and immunohistochemical analysis showed that the GAB1 peptide was effectively distributed in the mouse brain (C6K-GAB1 2mg / kg was injected into the tail vein, and small animal in vivo imaging showed that the peptide successfully entered the brain), providing an experimental basis for its potential clinical transformation.

[0146] The experimental results are detailed in Figure 10 , presenting experimental results demonstrating that C6KGAB1 treatment ameliorates synaptic damage in the hippocampus of 5xFAD mice and inhibits the cGAS-STING-related neuroinflammatory pathway. The GAB1 peptide significantly enhanced neuronal function by improving synaptic density, mitochondrial structure, and neuroinflammation in the hippocampus of 5xFAD mice. Immunohistochemistry and transmission electron microscopy analysis revealed that the GAB1 peptide reduced Aβ deposition, increased synaptic spine and synaptic density, and improved mitochondrial ultrastructure. Western blot and qRT-PCR results demonstrated that the peptide inhibited the cGAS-STING-mediated inflammatory response and reduced the expression of CXCL10, IFNB, IL-18, and IL-1β. Regarding inflammation suppression, Western blot analysis revealed a 50% decrease in cGAS and STING protein expression (p<0.01), while qRT-PCR revealed a significant decrease in CXCL10, IFNB, IL-18, and IL-1β expression. Overall, these results suggest that the GAB1 peptide may improve cognitive function by regulating GFAP-related pathways, enhancing synaptic plasticity, improving mitochondrial function, and inhibiting inflammation. In addition, safety testing was conducted on the peptide drug developed in this protocol, and long-term toxicity testing (90 days) showed no liver or kidney damage (ALT / AST levels were normal). This demonstrates that the drug developed in this protocol is a safe and effective peptide blocker for the treatment of Alzheimer's disease.

[0147] The above is only an embodiment of the present invention, and the common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the structure of the present invention, and these should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A drug for treating Alzheimer's disease, characterized in that: It comprises a blocking peptide that blocks the binding between GFAP and the N-terminus of APP; the amino acid sequence of the blocking peptide is shown in any one of SEQ ID NO. 1-7 and conservative derivatives thereof.

2. The drug for treating Alzheimer's disease according to claim 1, characterized in that: The amino acid sequence of the blocking peptide is shown in SEQ ID NO.

1.

3. The drug for treating Alzheimer's disease according to claim 2, characterized in that: The N-terminus of the blocking peptide is modified with a lysine residue; preferably, the number of the lysine residues is 6; The blocking peptide is modified with palmitic acid; preferably, the palmitic acid is linked to K at the 7th position of SEQ ID NO.

1.

4. A drug for treating Alzheimer's disease according to any one of claims 1 to 3, characterized in that: The drug is used to inhibit the interaction between GFAP and APP; The drug is used to inhibit the transcellular transport of GFAP from astrocytes to neurons, thereby inhibiting the abnormal aggregation of GFAP in neurons; The drug is used to alleviate mitochondrial dysfunction caused by the accumulation of GFAP in neurons; the mitochondrial dysfunction includes loss of mitochondrial membrane potential, decreased ATP synthesis capacity, decreased respiratory chain function and cellular metabolic disorder.

5. The drug for treating Alzheimer's disease according to claim 4, characterized in that: It also includes a pharmaceutically acceptable carrier; preferably, the carrier is a lipid nanoparticle for improving the brain-targeted delivery efficiency of the blocking peptide.

6. A blocking peptide for blocking the interaction between GFAP and APP, characterized in that: It is used to replace GFAP and bind to the TTTTTTESVEE fragment of APP protein; its sequence includes at least KRNIVVKTVEM; Preferably, the amino acid sequence of the blocking peptide is as shown in any one of SEQ ID NO. 1-7; Preferably, the N-terminus of the blocking peptide is modified with a lysine residue; the blocking peptide is modified with palmitic acid; Preferably, the number of the lysine residues is 6; and the palmitic acid is linked to the K at the 7th position of SEQ ID NO.

1.

7. Use of a blocking peptide for blocking the interaction between GFAP and APP according to claim 6 in the preparation of a medicament for treating Alzheimer's disease, or in the preparation of a medicament for treating and alleviating mitochondrial dysfunction in neurons, or in the preparation of an agent for blocking GFAP endocytosis mediated by APP as a membrane receptor, or in the preparation of a product for detecting APP, or in the preparation of a medicament for enhancing neuronal synaptic plasticity, or in the preparation of a medicament for inhibiting cGAS-STING-mediated inflammatory response.

8. Use of the GFAP and APP interaction site in designing a drug for treating Alzheimer's disease, characterized in that: The amino acid sequence of the N-terminus of APP is shown in SEQ ID NO.8; the amino acid sequence of GFAP is shown in SEQ ID NO.9; In the APP protein, the interaction sites with GFAP include at least one of D at position 64, K at position 99, T at position 152, E at position 156, T at position 275, T at position 277, T at position 278, T at position 279, E at position 281, V at position 283, E at position 284, E at position 285, E at position 380, Q at position 406, and Q at position 450; In the GFAP protein, the interaction sites with APP include at least one of R at position 124, D at position 128, R at position 136, Q at position 146, K at position 339, R at position 367, K at position 368, K at position 405, R at position 406, N at position 407, V at position 409, K at position 411, V at position 413, and M at position 415.

9. Use of the GFAP and APP interaction site according to claim 8 in designing a drug for treating Alzheimer's disease, characterized in that: In the APP protein, the interaction sites with GFAP include T at position 275, T at position 277, T at position 278, T at position 279, E at position 281, V at position 283, E at position 284, and E at position 285; In the GFAP protein, the interaction sites with APP include K at position 405, R at position 406, N at position 407, V at position 409, K at position 411, V at position 413, and M at position 415.

10. Use of the GFAP and APP interaction site according to claim 9 in designing a drug for treating Alzheimer's disease, characterized in that: The drug used to treat Alzheimer's disease is a polypeptide; the polypeptide is used to bind to TTTTTTESVEE at the N-terminus of APP; The sequence of the polypeptide covers a core sequence; the core sequence is KRNXVXKXVXM, or a sequence having a sequence similarity of ≥80% to KRNXVXKXVXM, wherein X represents any amino acid residue.

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