Use of a pharmaceutical composition in the preparation of a preparation for treating Parkinson's syndrome

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to the application of a pharmaceutical composition in the preparation of a preparation for treating Parkinson's syndrome. The research of the present invention shows that NPAFP1, NPAFP4 and NPAFP12 proteins can improve the pathological characteristics of PD models by increasing the mobility of α-synuclein.

Description

Technical Field

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

[0002] Parkinson's disease (PD) is a common neurodegenerative disorder in middle-aged and elderly people. Its pathogenesis is primarily due to pathological changes in the substantia nigra cells of the midbrain's compacta, which reduces dopamine synthesis. This reduced dopamine reduces the inhibitory effect of acetylcholine, enhancing the effect of acetylcholine and causing paralysis, hence the name "tremor paralysis." Typical motor symptoms include resting tremor, muscle rigidity, bradykinesia, and abnormal posture and gait. Non-motor symptoms may also be present. According to statistics, the prevalence of Parkinson's disease in the general population is approximately 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 showed that the relative risk of Parkinson's disease in men is approximately 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 European population, the North American population, and the Asian population 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, some drugs such as levodopa and dopamine receptor agonists 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, developing 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 al. 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 hallmarks of Parkinson's disease are the degeneration and loss of dopaminergic neurons in the substantia nigra pars compacta and the appearance of Lewy bodies in the cytoplasm, primarily composed of aggregated α-synuclein (α-SYN) (Samaranch L, Lorenzo-Betancor O, Arbelo JM, Ferrer L, Lorenzo E, Irigoyen J, et al. PINK1-linked parkinsonism is associated with Lewy body pathology [J]. Brain: A Journal of Neurology. 2010;133(Pt 4):1128-42). The α-synuclein gene was the first gene discovered 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 extensive screening, three common missense variants of this gene (A53T, A30P, and E46K) were found to be closely associated with the onset of familial 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 consisting of 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 for 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 formation of oligomers. Oligomers can form fibrotic cores, causing the rapid onset of fibrosis. The amount of fibrous protein continues to increase, and then insoluble aggregates are generated (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 Parkinson's disease. However, α-synuclein aggregation is not limited to Parkinson's disease. Studies have confirmed that pathological deposition of α-synuclein is present 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 revealed that α-synuclein phase separation (LLPS) may also play a significant role in the pathogenesis of Parkinson's disease. Phase separation refers to the spontaneous formation of droplet-like aggregates within cells by proteins or RNA molecules based on their physicochemical properties. This process is similar to the formation of liquid-liquid phase structures by biological macromolecules. Studies have shown that under certain conditions, α-synuclein can form gel-like or droplet-like aggregates through phase separation, which may be precursors to its toxic effects. Although these phase separations may be reversible initially, uncontrolled phase separation or excessive aggregation can lead to disrupted intracellular environments, triggering the development of neurodegenerative diseases. Therefore, regulating α-synuclein aggregation and phase separation has become an emerging target for the treatment of Parkinson's disease.

[0005] Currently, therapeutic strategies for α-synuclein aggregation mainly focus 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 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] Within 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 various neurodegenerative diseases, including PD.

[0007] In a previous study (“Evolution of an antifreeze protein byneofunctionalization under escape from adaptive conflict”, Deng et al. PNAS, 2010.), it was discovered and proved that the Antarctic fish Lycodichthys dearborni evolved an antifreeze protein AFPIII through neofunctionalization in the process of escaping adaptive conflict. It was named 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, and LdAFPIII-12 with twelve repeated functional domains). Each AFPIII domain contains 62 amino acids. The sequence of the LdAFPII protein was published in a previous study (“Cloning and evolutionary analysis of the multimeric type III antifreeze protein gene of the 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 has been completed by discovering that NPAFP1, NPAFP4 and NPAFP12 proteins can improve the pathological characteristics of the PD model by increasing the fluidity of α-synuclein.

[0009] In a first aspect, the present invention provides a pharmaceutical composition for treating Parkinson's disease, 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 the improvement of the body and / or treat Parkinson's syndrome symptoms.

[0012] Furthermore, the pharmaceutical composition can be prepared into various 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 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 further comprises 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

[0022] The NPAFP protein proposed in this study can increase the fluidity of α-synuclein, inhibit its phase separation, reduce its aggregation and toxicity, improve the survival rate of dopaminergic neurons in the substantia nigra, and improve the pathological features of the PD model. These effects make NPAFP a potential new drug candidate for PD treatment, with broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 2 NPAFP protein inhibits the phase separation of α-synuclein.

[0025] Figure 3 NPAFP protein reduces the death of dopamine neurons in A53T model mice. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] The Parkinson's disease described herein includes "resting tremor," which is often the first symptom and typically begins in the distal part of one upper limb. Symptoms are often present or pronounced when the body is in a static position. During the onset of the tremor, there is a "pill-rolling" motion between the thumb and flexed index finger.

[0029] The EGFP (Enhanced Green Fluorescent Protein) involved in this invention is a genetically modified variant of green fluorescent protein (GFP). While retaining the essential properties of GFP, EGFP has been optimized through genetic engineering to achieve higher fluorescence intensity, greater stability, and enhanced environmental adaptability. EGFP has a wide range of applications in biological and medical research. Due to its strong fluorescence 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.

[0030] FRAP, or Fluorescence Recovery After Photobleaching (FRAP), is a microscopy technique used to study the dynamic behavior of molecules within cells. This technique, based on the intracellular diffusion and transport characteristics of fluorescently labeled molecules, uses observation and measurement of the post-bleaching recovery of fluorescence signals to infer information such as intracellular molecular motility, diffusion rates, and intermolecular interactions.

[0031] Example 1 FRAP assay to detect the mobility of α-synuclein protein

[0032] 1.1 Optimization of LdAFPIII-1, LdAFPIII-4, and LdAFPIII-12

[0033] 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. To explore their applications in neurological diseases, they were named NeuroProtection AFPIII1 (i.e., NPAFP1), NeuroProtection AFPIII4 (i.e., NPAFP4), and NeuroProtection AFPIII12 (i.e., NPAFP12), respectively. Their amino acid sequences are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, respectively.

[0034] Table 1 Related sequences of NPAFP protein

[0035] Amino acid sequence of NPAFP1 SEQ ID NO.1 MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE* Nucleotide sequence of NPAFP1 SEQ ID NO.2 ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGA TGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA Amino acid sequence of NPAFP4 SEQ ID NO.3 MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLM PDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE* Nucleotide sequence of NPAFP4 SEQ ID NO.4 ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA Amino acid sequence of NPAFP12 SEQ ID NO.5 * Sequence of NPAFP12 SEQ ID NO.6 ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA

[0036] 1.2 Experimental methods

[0037] 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.

[0038] To evaluate the effect of NPAFP on α-synuclein mobility, a FRAP assay was performed. An α-synuclein-EGFP fusion protein and NPAFP protein were expressed using a prokaryotic expression system. The expressed α-synuclein-EGFP protein was incubated in vitro with NPAFP1, NPAFP4, and NPAFP12 to ensure sufficient binding. α-synuclein-EGFP protein not incubated with NPAFP served as a control. Subsequently, the EGFP protein was photobleached using fluorescence recovery after photobleaching (FRAP) using an excitation wavelength of 488 nm. The fluorescence recovery process was recorded by continuous imaging, and the fluorescence recovery of the α-synuclein-EGFP protein was analyzed to assess the effect of NPAFP on α-synuclein mobility.

[0039] 1.3 Experimental Results

[0040] The results showed that both NPAFP proteins significantly increased the mobility of α-synuclein protein, which was manifested by a significant shortening of the fluorescence recovery time ( Figure 1 NPAFP4 showed a stronger effect than NPAFP1 in improving mobility, while NPAFP12 was significantly more effective than NPAFP4. These experimental results suggest that NPAFP protein can effectively increase the mobility of α-synuclein, thereby preventing its aggregation and formation of pathological structures, and may be used for the prevention and treatment of Parkinson's disease.

[0041] Example 2 Detection of the effect of NPAFP protein on α-synuclein phase separation in HeLa cells

[0042] 2.1 Experimental methods

[0043] In order to examine the regulatory effect of NPAFP on α-synuclein phase separation (LLPS), HeLa cells were 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 were co-transfected into HeLa cells and the cells were induced by Fe 3+Inducing oxidative stress in cells promotes phase separation of α-synuclein. Live cell labeling was performed using a tetracysteine ​​tag and a bisarsenic compound (FlAsH) combined with EDT2. Cells were cultured in DMEM for 24 hours and then treated with ferric ammonium citrate to induce oxidative stress. Subsequently, 1µM FlAsH and 1µl of 25 mM EDT solution were added and incubated. The intracellular distribution of α-synuclein after FlAsH staining was observed, and the phase separation behavior of α-synuclein was analyzed.

[0044] 2.2 Experimental Results

[0045] Figure 2 The results showed that in the control group, α-synuclein exhibited significant phase separation, forming 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 cell, indicating that NPAFP proteins, particularly NPAFP12, can significantly inhibit α-synuclein phase separation. This result provides new insights into the treatment of Parkinson's disease, suggesting that regulating α-synuclein phase separation can reduce its aggregation and toxicity.

[0046] Example 3 Detection of dopamine neurons in A53T mice injected with AAV virus

[0047] 3.1 Experimental methods

[0048] To evaluate the protective effect of NPAFP protein on dopaminergic neurons in a Parkinson's disease model, A53T mutant mice were used as an experimental model. First, adenoviruses expressing NPAFP1, NPAFP4, and NPAFP12 were constructed using the AdMax adenoviral packaging system. The NPAFP gene-expressing adenovirus was incorporated into a shuttle plasmid (pDC316-mCMV-EGFP). This shuttle plasmid and a helper packaging plasmid (pBHGlox(delta)E1,3Cre), which carries the majority of the adenoviral genome, were co-transfected into HEK293 cells. Seven to ten days after transfection, the cell supernatant and cells were harvested, repeatedly frozen and thawed, and centrifuged. The supernatant was collected and filtered to obtain adeno-associated viruses (AAVs) expressing NPAFP1, NPAFP4, and NPAFP12 genes, which were then stored at -80°C until further use.

[0049] At 12 weeks of age, A53T mice were injected stereotaxically with adeno-associated viruses (AAVs) carrying the NPAFP1, NPAFP4, and NPAFP12 genes into the substantia nigra (SN). AAV vectors carrying the NPAFP1, NPAFP4, and NPAFP12 genes, respectively, ensured efficient expression of the target proteins. A control AAV vector lacking the NPAFP1, NPAFP4, and NPAFP12 genes was also injected into the SN. Three months after injection, brain tissue was perfusion-fixed with 4% paraformaldehyde (PFA), dehydrated with 30% sucrose solution, and 30µm-thick frozen sections were cut for immunofluorescence (IF). Dopaminergic neurons were labeled with an anti-tyrosine hydroxylase antibody and stained with an Alexa Fluor 647 secondary antibody. Fluorescence images were captured and processed, and the number of TH-positive neurons was quantified.

[0050] 3.2 Experimental Results

[0051] 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 improved, 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 the preparation of a preparation for treating Parkinson's syndrome, wherein the pharmaceutical composition comprises one or more of NPAFP1, NPAFP4 and / or NPAFP12 proteins, and the amino acid sequence of NPAFP1 is shown as SEQ ID NO.1; the amino acid sequence of NPAFP4 is shown as SEQ ID NO.3; the amino acid sequence of NPAFP12 is shown as SEQ ID NO.

5.

2. The use according to claim 1, wherein the preparation can achieve the treatment of Parkinson's syndrome by oral or injection.

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

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

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