Application of pharmaceutical composition in preparation of preparation for treating Parkinson's disease

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a pharmaceutical composition in preparation of a preparation for treating Parkinson's disease. The research shows that the NPAFP1 protein, the NPAFP4 protein and the NPAFP12 protein can improve the pathological characteristics of a PD (Parkinson's Disease) model by increasing the flowability of alpha-synuclein.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a pharmaceutical composition in the preparation of a preparation for treating Parkinson's syndrome. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people. The course of Parkinson's disease progresses slowly. Its pathogenesis is mainly due to the reduction of dopamine synthesis after pathological changes in the substantia nigra cells in the compact part of the midbrain. The reduced dopamine reduces the inhibitory effect of acetylcholine. After the effect of acetylcholine is enhanced, tremor paralysis occurs, so Parkinson's disease is also called tremor paralysis. Typical motor symptoms are resting tremor, muscle rigidity, bradykinesia, abnormal posture and gait, etc., and non-motor symptoms may also be accompanied. According to statistics, the prevalence of Parkinson's disease in the whole population is about 0.3%, and the prevalence in the elderly population aged 65 and over 85 is 1%~2% and 3%~5% respectively (Ascherio A, Schwarzschild M A. The epidemiology of Parkinson's disease: risk factors and prevention. [J]. Lancet Neurology, 2016, 15(12): 1257-1272.). Therefore, it can be seen that the incidence of Parkinson's disease increases with age. There are also differences in the risk of Parkinson's disease between men and women. A survey shows that the relative risk of Parkinson's disease in men is about 1.46 times that of women (Pringsheim T, Jette N, Frolkis A, et al. The prevalence of Parkinson's disease: a systematic review and meta-analysis. [J]. Movement Disorders Official Journal of the Movement Disorder Society, 2014, 29(13): 1583.). According to literature reports, there is no significant difference in the incidence of Parkinson's disease in different regions of the world. The incidence rates of the entire population of Europe, the entire population of North America, and the entire population of Asia are 9-22 / 100,000 person-years, 11-13 / 100,000 person-years, and 1.7-17 / 100,000 person-years, respectively (Elbaz A, Bower JH, Maraganore DM, et al. Risktables for parkinsonism and Parkinson's disease[J]. Journal of Clinical Epidemiology, 2002, 55(1):25-31.). Currently, there are some drugs such as levodopa and dopamine receptor agonists that can relieve symptoms, but these drugs can only improve clinical manifestations and cannot effectively curb the progression of the disease. Long-term use may even lead to drug side effects.Therefore, the development of new therapeutic strategies, especially drugs that can halt the progression of Parkinson's disease, has become one of the key challenges in the current treatment of neurodegenerative diseases.

[0003] The pathogenesis of PD is complex, and its potential molecular pathogenesis includes abnormal aggregation of α-SYN, mitochondrial dysfunction, oxidative stress, impaired calcium homeostasis, impaired axonal transport and neuroinflammation (Gawish R, Martins R, Bohm B, Wimberger T, Sharif0, Lakovits K, et a1. Triggering receptor expressed on myeloid cells-2 fine-tunes inflammatory responses in murine Gram-negative sepsis[J]. FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 2015: 29f4): 1247-57.). The two main pathological features of PD are the degeneration and loss of dopaminergic neurons in the substantia nigra and the appearance of Lewy bodies in the cytoplasm, which are mainly composed of aggregated α-synuclein (α-SYN) (Samaranch L, Lorenzo-Betancor O, Arbelo JM, Ferrer LLorenzo E, Irigoyen J, et al. PINK1-linked parkinsonism is associated with Lewy body pathology [J]. Brain: aiournal of neurology. 2010; 133 (Pt4): 1128-42.). α-synuclein gene is the first gene found to be associated with Parkinson's disease (Goldberg, AL, Protein degradation and protection against misfolded or damaged proteins. Nature, 2003. 426 (6968): p. 895-9.).After a large number of screenings, three common missense mutations of this gene (A53T, A30P and E46K) were found to be closely related to the onset of Parkinson's disease in patients with familial hereditary Parkinson's disease (Mishizen-Eberz, AJ, et al., Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro. J Neurochem, 2003.86(4): p. 836-47.). α-synuclein is a small soluble protein containing 140 amino acids, mainly located in the presynaptic terminals of the central nervous system (Iwata, A., et al., Alpha-synuclein degradation by serine protease neurosin: implication for pathogenesis of synucleinopathies. Hum Mol Genet, 2003. 12(20): p. 2625-35.). Its normal function is speculated to be related to the transmission of dopaminergic neurotransmitters and synaptic plasticity (Kasai, T., et al., Cleavage of normal and pathological forms of alpha-synuclein by neurosin in vitro. Neurosci Lett, 2008. 436(1): p. 52-6.). α-synuclein is believed to have toxic effects on neurons. Although the molecular mechanism causing this effect is still unclear, its protein content is considered to be the determining factor of whether it has neurotoxicity. Under normal physiological conditions, α-synuclein is a monomeric structure and is not cytotoxic. However, environmental or genetic changes can easily cause conformational changes in α-synuclein, resulting in misfolding and the appearance of oligomers. Oligomers can form fibrotic cores, causing the rapid onset of fibrosis. The amount of fibrous protein continues to increase, leading to the formation of insoluble aggregates (Sung, JY, et al., Proteolytic cleavage of extracellular secreted {alpha}-synuclein via matrix metalloproteinases. JBiol Chem, 2005. 280(26): p. 25216-24.).This aggregated α-synuclein is the main component of Lewy bodies in PD patients. However, the aggregation of α-synuclein does not only occur in Parkinson's disease. Studies have confirmed that pathological deposition of α-synuclein exists in a series of neurodegenerative diseases such as Lewy dementia, Alzheimer's disease, and multiple system atrophy (Levin, J., et al., Increased alpha-synuclein aggregation following limited cleavage by certain matrix metalloproteinases. Exp Neurol, 2009. 215(1): p. 201-8.).

[0004] In addition to α-synuclein aggregation, recent studies have found that α-synuclein phase separation (liquid-liquid phase separation, LLPS) may also play an important role in the pathogenesis of Parkinson's disease. Phase separation refers to the spontaneous formation of droplet-like enriched areas in cells according to their physicochemical properties by proteins or RNA molecules. This process is similar to the formation of liquid-liquid phase structures by biological macromolecules. Studies have shown that α-synuclein can form gel-like or droplet-like aggregates through phase separation under certain conditions, and these aggregates may be precursors to its toxic effects. Although these phase separation bodies may be reversible in the early stages, when phase separation is out of control or excessively aggregated, it may lead to a disorder in the intracellular environment, thereby triggering the occurrence of neurodegenerative diseases. Therefore, regulating the aggregation and phase separation of α-synuclein has become an emerging target for the treatment of Parkinson's disease.

[0005] Currently, therapeutic strategies for α-synuclein aggregation are mainly focused on inhibiting its aggregation, promoting its degradation or enhancing its fluidity. However, existing treatments still have significant limitations. For example, although some drugs can partially inhibit the aggregation of α-synuclein, the efficacy of these drugs is often limited and may be accompanied by serious side effects. In addition, most of the existing treatments intervene in the aggregation process of α-synuclein, while there are few studies on the regulation of its fluidity and phase separation mechanism in cells. Given that the fluidity and phase separation of α-synuclein may be important factors in regulating its aggregation and toxicity, it is necessary to develop a therapeutic strategy that can simultaneously regulate the fluidity and phase separation of α-synuclein, which will provide a new idea for the treatment of Parkinson's disease.

[0006] In the antifreeze protein family, Antarctic fish antifreeze proteins have attracted widespread attention due to their structural characteristics and biological activities. Currently, five types of antifreeze proteins have been found in polar fish, including antifreeze glycoproteins (AFGPs) and four antifreeze proteins (AFPI, AFP II, AFP III, and AFP IV). AFPs and AFGPs are proteins found in some organisms that live in extremely low temperature environments. Their main function is to protect cells from low temperature damage by inhibiting the formation of water crystals. Due to their unique structure and biological activity, these proteins also exhibit significant antioxidant and cytoprotective functions under non-freezing conditions, and can effectively reduce cell damage caused by oxidative stress. Therefore, antifreeze proteins and antifreeze glycoproteins may become therapeutic drugs for the prevention and treatment of a variety of neurodegenerative diseases including PD.

[0007] In a previous study (“Evolution of an antifreeze protein by neofunctionalization under escape from adaptive conflict”, Deng et al. PNAS, 2010.), it was found and proved that the Antarctic fish Lycodichthys dearborni evolved an antifreeze protein AFPIII through neofunctionalization in the process of escaping adaptive conflict, and named it LdAFPII. This protein evolved from SAS-B and has multiple repeated functional domains (such as LdAFPIII-1 with one repeated functional domain, LdAFPIII-4 with four repeated functional domains, LdAFPIII-12 with twelve repeated functional domains, etc.), and each AFPIII domain contains 62 amino acids. The sequence of LdAFPII protein was published in a previous study (“Cloning and evolutionary analysis of the gene of the multimeric type III antifreeze protein of Antarctic eel Pout (Lycodichthys dearborni)”, Yu Jing et al., Acta Genetica Sinica, 2005, 32 (8): 789-794.). Summary of the invention

[0008] The present invention finds that NPAFP1, NPAFP4 and NPAFP12 proteins can improve the pathological characteristics of the PD model by increasing the fluidity of α-synuclein, thereby completing the present invention.

[0009] In a first aspect, the present invention provides a pharmaceutical composition for treating Parkinson's syndrome, wherein the pharmaceutical composition comprises one or more NPAFP series proteins.

[0010] Furthermore, the NPAFP protein is selected from one or more of NPAFP1, NPAFP4 and / or NPAFP12 proteins.

[0011] Furthermore, the pharmaceutical composition also contains other active ingredients, which are drugs that are different from the NPAFP protein and can promote improvement of the body and / or treat the symptoms of Parkinson's syndrome.

[0012] Furthermore, the pharmaceutical composition can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

[0013] Furthermore, the administration of the pharmaceutical composition includes oral administration, injection, implantation, external application, spraying or inhalation.

[0014] In a second aspect, the present invention provides a use of a pharmaceutical composition in preparing a preparation for treating Parkinson's syndrome, wherein the pharmaceutical composition comprises one or more of the NPAFP series proteins.

[0015] Furthermore, the NPAFP protein is selected from one or more of NPAFP1, NPAFP4 and / or NPAFP12 proteins.

[0016] Furthermore, the preparation also contains pharmaceutically acceptable excipients.

[0017] Furthermore, the dosage form of the preparation includes, but is not limited to, one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

[0018] Furthermore, the preparation can be taken orally, injected, or the like to prevent and / or treat neurodegenerative diseases.

[0019] Furthermore, the NPAFP protein can reduce the aggregation and phase separation of α-synuclein in patients with Parkinson's syndrome.

[0020] Furthermore, the NPAFP protein can reduce the death of dopamine neurons in patients with Parkinson's syndrome.

[0021] Beneficial Effects The NPAFP protein proposed in the present invention can increase the fluidity of α-synuclein, inhibit the phase separation of α-synuclein, reduce its aggregation and toxicity, improve the survival rate of dopaminergic neurons in the substantia nigra, and improve the pathological characteristics of the PD model. The effect of the NPAFP protein makes it a potential new candidate drug for PD treatment and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1NPAFP protein increases the fluidity of α-synuclein protein: (A) FRAP experiment detects the fluidity of α-synuclein protein; (B) FRAP experiment detects the effect of NPAFP1 on the fluidity of α-synuclein protein; (C) FRAP experiment detects the effect of NPAFP4 on the fluidity of α-synuclein protein; (D) FRAP experiment detects the effect of NPAFP12 on the fluidity of α-synuclein protein.

[0023] Figure 2 NPAFP protein inhibits the phase separation of α-synuclein.

[0024] Figure 3 NPAFP protein reduces the death of dopamine neurons in A53T model mice. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments, but this should not be construed as limiting the scope of the present invention to the following embodiments.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0027] The Parkinson's syndrome involved in the present invention includes "resting tremor", which is often the first symptom, mostly starting from the distal part of one upper limb, and appears or is obvious when the body is in a static position. When the disease occurs, there is a "pill-rolling" action between the thumb and the flexed index finger.

[0028] The EGFP involved in the present invention, namely Enhanced Green Fluorescent Protein (Enhanced Green Fluorescent Protein), is a genetically modified variant of green fluorescent protein (GFP). EGFP has been optimized through genetic engineering while retaining the basic characteristics of GFP, making its fluorescence intensity higher, more stable, and more adaptable to the environment. EGFP has a wide range of applications in biological and medical research. Due to its strong fluorescent signal, EGFP is often used as a reporter gene or marker gene to track and observe the expression of specific proteins, cells or genes in cells, tissues or organisms.

[0029] The FRAP involved in the present invention, which stands for Fluorescence Recovery After Photobleaching, is a microscopy technique used to study the dynamic behavior of intracellular molecules. This technique is based on the diffusion and transport characteristics of fluorescently labeled molecules in cells. By observing and measuring the recovery of fluorescent signals after photobleaching, the mobility, diffusion rate and interaction between molecules in cells can be inferred.

[0030] Example 1 FRAP experiment to detect the fluidity of α-synuclein protein 1.1 Optimization of LdAFPIII-1, LdAFPIII-4, and LdAFPIII-12 Alphafold3 was used to predict and optimize the protein structure. The amino acids at positions 36 and 37 of LdAFPIII-1, LdAFPIII-4 and LdAFPIII-12 were changed from KL to RI, and the amino acid at position 50 was changed from D to E to increase the flexibility of the protein structure. In order to explore their application in neurological diseases, they were named NeuroProtection AFPIII1 (i.e., NPAFP1), NeuroProtection AFPIII4 (i.e., NPAFP4) and NeuroProtection AFPIII12 (i.e., NPAFP12), and their amino acid sequences are shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5, respectively.

[0031] Table 1 Related sequences of NPAFP protein Amino acid sequence of NPAFP1 SEQ ID NO.1 MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE* Nucleotide sequence of NPAFP1 SEQ ID NO.2 ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA Amino acid sequence of NPAFP4 SEQ ID NO.3 MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE* Nucleotide sequence of NPAFP4 SEQ ID NO.4 ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA Amino acid sequence of NPAFP12 SEQ ID NO.5 MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE* Nucleotide sequence of NPAFP12 SEQ ID NO.6 ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA 1.2 Experimental methods One of the pathological characteristics of Parkinson's disease is the abnormal aggregation of α-synuclein in neurons, forming Lewy bodies, which in turn leads to the death of dopaminergic neurons.

[0032] To evaluate the effect of NPAFP protein on the fluidity of α-synuclein, a FRAP experiment was performed. α-synuclein-EGFP fusion protein and NPAFP protein were expressed using a prokaryotic expression system. The expressed α-synuclein-EGFP protein was co-incubated with NPAFP1, NPAFP4 and NPAFP12 in vitro to ensure their full binding. The α-synuclein-EGFP protein not incubated with NPAFP protein was used as a control group. Subsequently, the fluorescence recovery after photobleaching (FRAP) technique was used to photobleach the EGFP protein using an excitation light wavelength of 488 nm. The fluorescence recovery process was recorded by continuous photography, and the fluorescence recovery of α-synuclein-EGFP protein was analyzed to evaluate the effect of NPAFP protein on the fluidity of α-synuclein.

[0033] 1.3 Experimental Results The results showed that both NPAFP proteins significantly increased the fluidity of α-synuclein protein, as evidenced by a significant shortening of the fluorescence recovery time ( Figure 1 ). NPAFP4 showed a stronger effect than NPAFP1 in improving fluidity, while the effect of NPAFP12 was significantly higher than that of NPAFP4. The experimental results show that NPAFP protein can effectively increase the fluidity of α-synuclein, thereby preventing it from aggregating to form pathological structures, and is expected to be used in the prevention and treatment of Parkinson's disease.

[0034] Example 2 Detection of the effect of NPAFP protein on α-synuclein phase separation in HeLa cells 2.1 Experimental methods In order to detect the regulatory effect of NPAFP protein on α-synuclein phase separation (LLPS), HeLa cell line was selected as a model. HeLa cells are widely used to study protein phase separation and its dynamic characteristics. In the experiment, the plasmid expressing C4-α-synuclein and the plasmid expressing NPAFP protein were co-transfected into HeLa cells and then expressed by Fe 3+ Induce oxidative stress in cells and promote phase separation of α-synuclein. Use tetracysteine ​​tag and bisarsenic compound (FlAsH) combined with EDT2 for live cell labeling. Cells were cultured in DMEM medium. After 24 hours of culture, cells were treated with ammonium ferric citrate to induce oxidative stress. Subsequently, 1µM FlAsH and 1µl 25 mM EDT solution were added and incubated. The distribution of α-synuclein in cells after FlAsH staining was observed, and the phase separation behavior of α-synuclein was analyzed.

[0035] 2.2 Experimental Results Figure 2 The results showed that in the control group, α-synuclein showed obvious phase separation and formed concentrated droplets. In contrast, in cells treated with NPAFP1, NPAFP4 and NPAFP12, α-synuclein did not form droplet-like structures, but remained evenly distributed throughout the cells, indicating that NPAFP protein, especially NPAFP12, can significantly inhibit the phase separation of α-synuclein. This result provides a new idea for the treatment of Parkinson's disease, indicating that by regulating the phase separation of α-synuclein, its aggregation and toxicity can be reduced.

[0036] Example 3 Detection of dopamine neurons in A53T mice injected with AAV virus 3.1 Experimental methods In order to evaluate the protective effect of NPAFP protein on dopaminergic neurons in the animal model of Parkinson's disease, A53T mutant mice were used as an experimental model. First, the AdMax adenovirus packaging system was used to construct adenoviruses that can express NPAFP1, NPAFP4, and NPAFP12. The adenovirus of the NPAFP gene was constructed into a shuttle plasmid (pDC316-mCMV-EGFP), and the shuttle plasmid carrying the NPAFP gene and the auxiliary packaging plasmid (pBHGlox(delta)E1,3Cre) carrying most of the adenovirus genome were co-transfected into HEK293 cells. 7-10 days after transfection, the cell supernatant and cells were collected, repeatedly frozen and thawed, centrifuged, and the supernatant was collected and filtered to obtain adenovirus-associated viruses (AAVs) expressing NPAFP1, NPAFP4, and NPAFP12 genes, which were stored at -80℃ for later use.

[0037] A53T mice were injected with adenovirus-associated viruses (AAVs) expressing NPAFP1, NPAFP4, and NPAFP12 genes into the substantia nigra (SN) of mice at 12 weeks of age by stereotaxic injection. AAV vectors carried NPAFP1, NPAFP4, and NPAFP12 genes, respectively, to ensure effective expression of the target protein. At the same time, AAV vectors without NPAFP1, NPAFP4, and NPAFP12 genes were injected into the substantia nigra of mice as a control. Three months after injection, the mouse brain tissue was perfused and fixed with 4% paraformaldehyde (PFA), then dehydrated with 30% sucrose solution, and finally 30µm thick frozen sections were cut for immunofluorescence (IF) detection. Dopaminergic neurons were labeled with anti-tyrosine hydroxylase antibodies and stained with Alexa Fluor647 secondary antibodies. Fluorescence images were taken, image processing was performed, and the number of TH-positive neurons was quantitatively analyzed.

[0038] 3.2 Experimental Results Figure 3 The results showed that the survival rate of dopaminergic neurons in the substantia nigra of A53T mice expressing NPAFP1, NPAFP4 and NPAFP12 was significantly increased, indicating that NPAFP protein has a significant effect in neuroprotection. This result provides strong support for NPAFP protein as a candidate drug for the treatment of Parkinson's disease.

Claims

1. Use of a pharmaceutical composition in preparing a preparation for treating Parkinson's syndrome, the pharmaceutical composition comprising one or more of NPAFP1, NPAFP4 and / or NPAFP12 proteins, the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.3; the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.

5.

2. The use according to claim 1, wherein the dosage form of the preparation includes but is not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

3. The use according to claim 1, wherein the preparation can be taken orally or injected to prevent and / or treat neurodegenerative diseases.

4. The use according to claim 1, wherein the NPAFP1, NPAFP4 and / or NPAFP12 protein can reduce the aggregation and phase separation of α-synuclein in patients with Parkinson's syndrome.

5. The use according to claim 1, wherein the NPAFP1, NPAFP4 and / or NPAFP12 protein can reduce the death of dopamine neurons in patients with Parkinson's syndrome.

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