Method for regulating lysosomal biogenesis by using RPL10A and application thereof

Abstract

The invention discloses a method for regulating lysosome biogenesis through RPL10A and application of the method, and relates to the technical field of genetic engineering, and the method specifically comprises the steps that after RPL10A is knocked down to cause variable shear change of lysosome related genes, the number of lysosomes is reduced; or the RPL10A is over-expressed, and the number of lysosomes is increased. The invention also provides application of the RPL10A gene or the protein coded by the RPL10A gene as a target spot in screening of drugs for treating or inhibiting neurodegenerative diseases. According to the invention, the molecular mechanism of the RPL10A for regulating the occurrence of the lysosome is defined, and the biogenesis of the lysosome can be specifically regulated; and a new potential target is provided for generation regulation and control of targeted lysosome and related neurodegenerative diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for regulating lysosome biogenesis and an application thereof in preventing and treating diseases. Background Art

[0002] In the 1950s, Christian De Duve won the Nobel Prize for his discovery that established lysosomes as the degradation and metabolic center of cells. Lysosomes are single-membrane organelles whose membranes contain hundreds of integral and peripheral membrane proteins, including different transporters and ion channels. The acidity of the lysosomal lumen is maintained by V-ATPase, and low pH activates a variety of lysosomal hydrolases that can digest macromolecules including proteins, nucleic acids, lipids, and carbohydrates; lysosomes can also use a variety of endocytic pathways to absorb macromolecules from outside the cell through the cell membrane and transport them to lysosomes for degradation; in addition, lysosomes can also capture macromolecules, damaged or misfolded proteins, and even entire organelles in the cell through autophagy and transport them to lysosomes. Therefore, lysosomes have always been considered as the garbage can of cells, and their dysfunction can lead to a variety of human diseases, such as lysosomal storage diseases and neurodegenerative diseases.

[0003] Lysosomes play an important role in many cellular processes. In addition to being the cell's trash can, they are also the cell's signaling center, essential for energy, amino acid sensing, signal transduction, and autophagy regulation. In addition, lysosomes interact with other intracellular organelles, such as mitochondria and endoplasmic reticulum, to regulate each other's homeostasis. Lysosomes are also involved in secretion and plasma membrane repair. When eukaryotic cells are damaged, lysosomes repair the plasma membrane through exocytosis. Lysosomes also play an important role in tumorigenesis, immune response, and many other physiological or pathological processes. Lysosomes play an important role in cellular homeostasis, so lysosome biogenesis is one of the most important mechanisms of lysosomes, which increases the number of lysosomes to meet different cellular needs, such as starvation-induced autophagy and the allocation of lysosomes to daughter cells during cell division.

[0004] The eukaryotic ribosome is composed of 40S and 60S subunits, which combine to form the 80S ribosome with translational activity. RPL10A is a component of the 60S subunit of the ribosome. RPL10A can restore the antioxidant capacity of defective strains that are sensitive to oxidative stress; it acts as a substrate and specific chaperone in the plant's defense response to viruses and as a regulator of the abscisic acid response; some evidence suggests that RPL10A may be involved in cell proliferation, such as promoting ovarian development in Drosophila and regulating thymus development in zebrafish.

[0005] Pre-mRNA splicing is a very important step before gene expression in prokaryotes and eukaryotes. All pre-mRNAs are composed of alternating introns and exons. During the maturation of mRNA, introns are removed to form mature mRNAs with interconnected exons, which are then translated into proteins to perform their functions. Variable splicing increases the diversity of the transcriptome and proteome of eukaryotic cells. Variable splicing is mediated by the spliceosome, which is a dynamic complex composed of five snRNPs (smallnuclear ribonucleoproteins) and hundreds of regulatory proteins. Variable splicing consists of a complex regulatory process involving cis-acting elements and trans-acting splicing factors. Cis-acting elements include exon / intron splicing enhancers and silencers in pre-mRNA; trans-acting factors include SR (serine–arginine-rich) family proteins, U2AF (U2 smallnuclear ribonucleoprotein), hnRNPs (heterogeneous ribonucleoproteins), etc.

[0006] The brain is a very complex organ with multiple cell types working together to achieve homeostasis. Alternative splicing of pre-mRNA is particularly important in the brain. Compared with other tissues, brain cells have a very high proportion of alternatively spliced ​​genes, thus showing the most complex alternative splicing patterns and producing a rich variety of isoforms.

[0007] At present, the main drugs for neurodegenerative diseases, especially Alzheimer's disease, include N-methyl-D-aspartate receptor (NMDAR), cholinesterase inhibitors, acetylcholinesterase inhibitors, and phosphorylcholine synthesis promoters. There are no relevant patent documents or clinical disease reports on the prevention and treatment of Alzheimer's disease by targeting RPL10A and regulating lysosomes. Summary of the invention

[0008] In view of this, one of the objects of the present invention is to provide a method for regulating lysosome biogenesis by RPL10A; the present invention clarifies the molecular mechanism by which RPL10A regulates lysosome biogenesis, and can achieve specific regulation of lysosome biogenesis.

[0009] To achieve the above objectives, the technical effects of the present invention are:

[0010] RPL10A regulates lysosomal biogenesis by:

[0011] By knocking down RPL10A, the number of lysosomes was reduced after the alternative splicing of lysosome-related genes was changed;

[0012] Or overexpressing RPL10A can increase the number of lysosomes.

[0013] Furthermore, knocking down RPL10A is achieved by introducing siRNA to interfere with the expression of RPL10A, wherein the nucleotide sequence of the siRNA includes SEQ ID NO: 1 and SEQ ID NO: 2.

[0014] Furthermore, the lysosome-related genes include MFSD8 and ATP6V0A4. By knocking down RPL10A, the MFSD8 and ATP6V0A4 undergo exon skipping, wherein the transcripts of the MFSD8 undergoing exon skipping are reduced; and the transcripts of the ATP6V0A4 undergoing exon skipping are increased.

[0015] Furthermore, the lysosome-related genes also include CTSF, ASAH1, NPC2, SLC11A2, CD164, NAGPA, GLB1 and LGMN. By knocking down RPL10A, the expression of CTSF, ASAH1, NPC2, SLC11A2, CD164, NAGPA, GLB1 and LGMN is reduced, thereby reducing the number of lysosomes.

[0016] A second object of the present invention is to provide an application of an RPL10A gene or a protein encoded thereby as a target in screening drugs for treating or inhibiting neurodegenerative diseases.

[0017] Furthermore, the drug targets the RPL10A gene or the protein encoded by it, activates or increases the expression of the RPL10A gene, or increases the level of the protein encoded by RPL10A.

[0018] The third object of the present invention is to provide an agent for activating or increasing the expression of the RPL10A gene for use in the preparation of a drug for treating or inhibiting neurodegenerative diseases, wherein the agent includes small molecules, polypeptides, proteins, and gene drugs.

[0019] Furthermore, the neurodegenerative disease includes Alzheimer's disease.

[0020] The present invention also aims to provide a medicine, including an RPL10A agonist, wherein the medicine is indicated for Alzheimer's disease.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The present invention performs RNA sequencing analysis, variable splicing change analysis, GO and KEGG analysis of related signal pathways after interfering with RPL10A, and uses immunofluorescence and protein immunoblotting to detect changes in protein levels of lysosomal marker LAMP1, Golgi marker GM130, endoplasmic reticulum marker Calnexin, and mitochondrial marker Tom20 after interfering with RPL10A, thereby clarifying the molecular mechanism by which RPL10A regulates lysosome biogenesis; specific regulation of lysosome biogenesis can be achieved; and new potential targets are provided for targeted lysosome generation regulation and related neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The localization map and nuclear entry rate of TFEB after interfering with RPL10A provided in the embodiments of the present invention; (A): After interfering with RPL10A, the subcellular localization of TFEB-GFP (green) was detected by immunofluorescence, blue fluorescence (DAPI) was the cell nucleus, and the scale was 20 μm; (B): After interfering with RPL10A, the statistics of TFEB-GFP nuclear entry were shown, **P<0.01; (C): After interfering with RPL10A, the interference efficiency was detected by qPCR, ****P<0.0001.

[0024] Figure 2 This is a differential analysis of the expression levels of the enriched lysosomal genes after interfering with RPL10A provided in an embodiment of the present invention. The horizontal axis is the logarithm of the mRNA expression level of the experimental group compared with the mRNA expression level of the control group; the vertical axis is -log10 (Pvalue); blue dots represent genes with increased mRNA expression levels, red dots represent genes with decreased mRNA expression levels, and gray dots represent genes with no change in mRNA expression levels.

[0025] Figure 3 The embodiment of the present invention provides that the interference with RPL10A specifically affects the lysosome. (A): After interfering with RPL10A, the fluorescence intensity of LAMP1 (cy3 red) was detected by immunofluorescence; blue fluorescence (DAPI) is the cell nucleus, and the scale is 40μm; (B): After interfering with RPL10A, the fluorescence intensity of LAMP1 was statistically analyzed, and the average fluorescence intensity of si-NC was standardized to 1, the ordinate represents the fluorescence intensity / the average fluorescence intensity of si-NC, and the statistical method is t test, *0.01<P≤0.05, **P<0.01; (C): After interfering with RPL10A, the protein level of LAMP1 was detected by protein immunoblotting, and ACTIN was the internal reference protein; (D): Flow analysis diagram, green is the unstained blank group, red is the uninterfered control group, and blue and orange are the RPL10A interference group.

[0026] Figure 4It is a diagram of Golgi morphology and protein level after interfering with RPL10A provided by an embodiment of the present invention. (A): After interfering with RPL10A, the fluorescence intensity of Golgi marker protein GM130 (green) was detected by immunofluorescence; blue fluorescence (DAPI) is the cell nucleus, and the scale is 40μm; (B): After interfering with RPL10A, the fluorescence intensity of GM130 was statistically analyzed, and the average fluorescence intensity of si-NC was standardized to 1, the ordinate represents the fluorescence intensity / the average fluorescence intensity of si-NC, and the statistical method is t test, ns P>0.05; (C): After interfering with RPL10A, the protein level of GM130 was detected by protein immunoblotting, and ACTIN was the internal reference protein.

[0027] Figure 5 It is a diagram of the endoplasmic reticulum morphology and protein level after interfering with RPL10A provided by an embodiment of the present invention. (A): After interfering with RPL10A, the fluorescence intensity of the endoplasmic reticulum marker protein calnexin (red) was detected by immunofluorescence, blue fluorescence (DAPI) is the cell nucleus, and the scale is 40μm; (B): After interfering with RPL10A, the fluorescence intensity of calnexin was statistically analyzed, the average fluorescence intensity of si-NC was standardized to 1, the ordinate represents the fluorescence intensity / si-NC average fluorescence intensity, and the statistical method is t test, ns P>0.05; (C): Interfering with RPL10A and using protein immunoblotting to detect the protein level of the endoplasmic reticulum marker protein calnexin, β-actin is the internal reference protein.

[0028] Figure 6 It is a diagram of mitochondrial morphology and protein levels after interfering with RPL10A provided in an embodiment of the present invention. (A): After interfering with RPL10A, the fluorescence intensity of the mitochondrial marker protein Tom20 (red) was detected by immunofluorescence, blue fluorescence (DAPI) is the cell nucleus, and the scale is 40μm; (B): After interfering with RPL10A, the average branch length of mitochondria was counted, each point represents a cell, ns P>0.05; (C): After interfering with RPL10A, the protein level of Tom20 was detected by protein immunoblotting, and β-actin was used as the internal reference protein.

[0029] Figure 7 The embodiment of the present invention provides a diagram showing that the LAMPI-reduced phenotype is partially elevated after interfering with overexpression of RPL10A. (A): After interfering with RPL10A, HA-RPL10A is overexpressed and the protein level of LAMP1 is detected by western blotting, with ACTIN as the internal reference protein; (B): The protein level of LAMP1 is statistically analyzed, ns P>0.05, **<0.01.

[0030] Figure 8It is a related map of exon skipping of MFSD8 and ATP6V0A4 after interfering with RPL10A provided by the embodiment of the present invention. (A): The ratio of exon skipping of MFSD8 indicated in RNA seq to the wild-type transcript; (B): The ratio of exon skipping of ATP6V0A4 indicated in RNA seq to the wild-type transcript; (C): RT-PCR proves MFSD8 exon skipping, WT315bp, SE 170bp; (D): Exon skipping statistics of three independent experiments of RT-PCR MFSD8, *0.01<P≤0.05, **P<0.01; (E): RT-PCR proves ATP6V0A4 exon skipping, WT 369bp, SE 251bp; (F): Exon skipping statistics of three independent experiments of RT-PCR ATP6V0A4, *0.01<P≤0.05, **P<0.01.

[0031] Fig. 9 This is an immunoblot image of the target protein immunoprecipitated by the RPL10A antibody provided in the embodiment of the present invention.

[0032] Fig.10 The present invention provides an embodiment of the method for annotating genes immunoprecipitated by RPL10A antibody using the human genome.

[0033] Fig.11 This is a gene enrichment diagram related to RNA shearing in the genes immunoprecipitated by RPL10A provided in an embodiment of the present invention. (A): GO analysis is performed on the biological process-related mRNA bound by RPL10A, the horizontal axis represents the count value of the gene, and the color depth of the column represents p.adjust; (B): GO analysis is performed on the cellular component-related mRNA bound by RPL10A, the horizontal axis represents the count value of the gene, and the color depth of the column represents p.adjust; (C): GO analysis is performed on the molecular function-related mRNA bound by RPL10A, the horizontal axis represents the count value of the gene, and the color depth of the column represents p.adjust. (D): KEGG analysis is performed on the mRNA bound by RPL10A, the horizontal axis is the proportion of this type of gene, the size of the point represents the size of the count value, and the color depth of the point represents p.adjust.

[0034] Fig.12 This is a diagram of the binding of RPL10A and PRPF8 exon 23 provided in an embodiment of the present invention. (A): RIP signal of PRPF8 exon 23; (B): RIP signal of SUPT6H exon 6.

[0035] Fig.13 The following is a diagram of the pathological phenotype of the A53T fruit fly after overexpression in the model organism Drosophila provided by the embodiment of the present invention. A): Western blot detection of α-synuclein protein level and oligomerization in 20-day-old Drosophila adults; (B): α-synuclein protein level statistical graph, ****P<0.0001; (C): Climbing behavior test of 15-day-old Drosophila, ***P<0.001, ****P<0.0001; (D): Immunofluorescence detection of the number of DA neurons (green) in 35-day-old Drosophila; scale is 50 μm; (E): 15-day-old Drosophila DA neurons (488 green) statistical graph, *P≤0.05, **P<0.01. DETAILED DESCRIPTION

[0036] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] Example 1 Interference with RPL10A in cells

[0039] The following experimental steps were used to disrupt RPL10A in cells:

[0040] (1) Wipe the equipment required for interference (RNase-free) with 75% alcohol and place it in a clean bench for ultraviolet sterilization for 30 minutes.

[0041] (2) Configure the interference system in the following order

[0042] Table 1 Reagents and volumes required for the preparation of the interference system

[0043]

[0044] Table 2 siRNA sequences

[0045]

[0046] (3) Oscillate, shake, and let stand for 5 min.

[0047] (4) Transfer the opti-MEM containing lip3000 to the EP tube containing siRNA, shake, centrifuge, and let stand for 15 minutes.

[0048] (5) Vertically add 50 μL of the system to the well plate with cells and gently shake to mix.

[0049] Example 2 Subcellular localization of TFEB after interference with RPL10A

[0050] The cells interfered in Example 1 were taken and TFEB in the cells was localized using the following experimental steps.

[0051] (1) Aspirate the culture medium in the well plate, wash twice with sterilized PBS, and then add 4% PFA fixative and incubate for 10-15 minutes.

[0052] (2) Aspirate PFA and wash twice with PBS, 5 min each time; add 0.1% PBST and let stand for 15 min.

[0053] (3) Aspirate PBST and add PBS to wash twice, 5 min each time.

[0054] (4) Use the bent needle to hook the slide from the bottom of the well plate, pick up the slide with tweezers facing up (the side with cells), and place it in a 15 cm dish covered with sealing film.

[0055] (5) Wash twice quickly with PBS, aspirate the PBS, add 50 μL BSA to the slide for blocking, and incubate at room temperature for 1 h.

[0056] (6) Aspirate the BSA, add BSA containing primary antibody, and incubate at room temperature for h.

[0057] (7) Aspirate the primary antibody and wash with PBS six times, 5 min each time.

[0058] (8) Select secondary antibody according to the species of primary antibody, prepare secondary antibody, add 50 μL of secondary antibody, and incubate at room temperature for 1 h.

[0059] (9) Aspirate the secondary antibody and wash with PBS six times, 5 min each time.

[0060] (11) Add 50-60 μL of DAPI and incubate at room temperature for 2-3 minutes to stain the nuclei.

[0061] (12) Aspirate the DAPI and wash with PBS three times, 5 min each time.

[0062] (13) Add 3 μL of mounting medium onto the slide, pick up the slide gently with tweezers, and gently place it down with the side containing cells facing down, and seal the slide with nail polish.

[0063] (14) Perform fluorescence imaging of cell samples.

[0064] like Figure 1As shown, it was found that the subcellular localization of TFEB was changed and lysosomes were damaged after interference with the ribosomal protein RPL10A.

[0065] Example 3 Lysosomal gene detection after interference with RPL10A

[0066] 1. Take the cells after interference in Example 1 and extract RNA from the cells. The extraction steps are as follows:

[0067] (1) Aspirate the culture medium in the well plate and add an appropriate amount of trizol, such as 1 mL for a 6-well plate. Shake to make sure the trizol covers the cells evenly. Let stand for 5 min and pre-cool the RNase-free EP tube.

[0068] (2) Use a tip to scrape the cells from the bottom of the well plate and transfer them to an RNase-free EP tube.

[0069] (3) Add Trizol: chloroform into the EP tube at a ratio of 5:1.

[0070] (4) Clamp the EP tube with two stands, shake vigorously, and centrifuge at 13,000 g at 4°C for 15 min.

[0071] (5) Transfer the supernatant, i.e., RNA, to a new EP tube.

[0072] (6) Add isopropanol at a ratio of 1:1, shake vigorously, let stand for 10 min, and centrifuge at 13,000 g at 4°C for 15 min. The white precipitate is RNA.

[0073] (7) Discard the supernatant and add 75% alcohol to wash the RNA at 13,000 g at 4°C for 5 min.

[0074] (8) Pour off the supernatant, remove the remaining alcohol with a small tip, and repeat the previous step.

[0075] (9) Allow the RNA to dry at room temperature for approximately 10 min.

[0076] (10) Add an appropriate amount of DEPC water to dissolve RNA for about 1 hour.

[0077] (11) Measure RNA concentration.

[0078] 2. Perform reverse transcription on the above samples. The reverse transcription steps are as follows:

[0079] (1) Removal of gDNA from RNA needs to be done on ice. Add the following systems one by one into an RNase-free PCR tube.

[0080] Table 3 Reagents and volumes required for the first step of reverse transcription

[0081] Reagents volume RNA 1 μg <![CDATA[10×ReactionBufferwithMgcl2]]> 1μL DNaseⅠRNase-free 1μL Water, nuclease-free To10μL

[0082] Mix well, incubate at 37℃ for 30min; add 1μL 50mM EDTA at 65℃ for 10min;

[0083] (2) Add the following systems one by one into the RNase-free PCR tube

[0084] Table 4 Reagents and volumes required for the second step of reverse transcription

[0085]

[0086] Mix well, centrifuge, 65℃ for 5min;

[0087] (3) Add the reagents into the PCR tube one by one in the following order.

[0088] Table 5 Reagents and volumes required for the third step of reverse transcription

[0089] Reagents volume 5×ReactionBuffer 4μL Ribolock RNase Inhibitor 1μL 10mM dNTPMix 2μL RevertAidM-MuLRT 1μL Total volume 20μL

[0090] Mix well, centrifuge, and incubate at 42℃ for 60min or 70℃ for 5min. After the reaction is complete, store the cDNA at -20℃. Meanwhile, take the cDNA sample for sequencing.

[0091] like Figure 2 As shown in the figure, the expression levels of lysosomal genes in the control group (uninterrupted) were lower than those in the RPL10A interference group, indicating that the expression of lysosomal genes was affected after interfering with RPL10A.

[0092] Example 4 Interference with RPL10A specifically affects the expression of lysosomal marker proteins

[0093] In order to explore the molecular mechanism by which RPL10A damages lysosomes, this example detected the lysosomal marker LAMP1 by immunofluorescence and western blotting after interfering with RPL10A.

[0094] The immunofluorescence assay steps were the same as those in Example 1. The protein immunoblotting assay steps were as follows:

[0095] (1) Protein extraction: Add protease and phosphatase inhibitors to 2×SDS in proportion to make the final concentration 1×; observe the cells under a microscope, and when they are fully grown, use a pump to remove the culture medium, rinse the cells with 1×PBS, and remove the PBS; add an appropriate amount of 2×SDS lysis buffer according to the cell density and well plate size, shake well, so that the lysis buffer evenly covers the cells, and let it stand for 5 minutes; use a medium tip to scrape the cells from the bottom of the well plate, transfer them to a marked EP tube, and tap; according to the characteristics of the protein, generally denature the protein at 95℃ for 10 minutes; turn on the ultrasonicator, first rinse the probe of the ultrasonicator with ddH2O, and then wipe off the water stains with toilet paper. Ultrasonicate each protein sample three times. At the end of ultrasonication, rinse each sample with ddH2O before ultrasonicating the next sample; transfer to a -80℃ refrigerator to store the protein.

[0096] (2) Protein quantification: Take out the protein sample from the -80℃ freezer and wait for it to melt; prepare the BCA solution in proportion, and apply the standard (8μL) in the order of increasing concentration on the two rows on the left side of the quantitative plate, with each standard applied twice; first apply 6μL ddH2O in the air space of the sample, then apply 2μL protein sample; finally, add 160μL BCA solution to all wells and incubate at 37℃ for 15min; detect the protein concentration with an ELISA instrument, configure the corresponding system according to the amount of protein loaded, add loading, mix well, and centrifuge.

[0097] (3) Transfer: Precool the transfer solution; pour the precooled transfer solution into a rectangular porcelain basin, open the sandwich transfer clamp, and place a piece of filter paper on top of the sponge; stop electrophoresis according to the size of the target band, remove the PAGE gel from the electrophoresis tank, gently pry it open, cut off the upper layer of gel, and then remove the gel from the glass plate; grab the PAGE gel by hand, turn it over several times in the precooled transfer solution, and place it on the filter paper; write a mark on the PVDF membrane with a water-based pen and soak it in methanol to activate it; turn the activated PVDF membrane over several times in the precooled transfer solution, wash off the methanol on the membrane, cover it on the PAGE gel, scrape off the bubbles between the membrane and the PAGE gel with a plate, then gently cover it with filter paper and close the transfer clamp; put the transfer clamp into the transfer tank, pour in the precooled transfer solution, constant current 290mA, 90min, and the transfer time is set according to the size of the target band.

[0098] (4) Blocking: Prepare 5% milk blocking solution; after the transfer is completed, open the transfer tank, take out the PVDF membrane, put it into the membrane box, pour in the blocking solution, and incubate for 1 hour; after the blocking is completed, wash three times with PBST, each time for 10 minutes; pour the antibody into the membrane box and incubate overnight; the next day, wash three times with PBST, each time for 10 minutes; add the secondary antibody of the corresponding species of the antibody and incubate for 30 minutes; wash three times with PBST, each time for 10 minutes, and develop.

[0099] like Figure 3As shown, the average fluorescence intensity of LAMP1 ( Figure 3 -A, 3-B) and protein levels ( Figure 3 -C) was significantly reduced. This example also uses LysoTracker to stain lysosomes, and this example uses LysoTracker to stain lysosomes and then perform flow analysis ( Figure 3 -D), it was found that the fluorescence curve of the RPL10A interference group was significantly shifted to the left compared with the non-interference group, where the horizontal axis represents the fluorescence intensity and the vertical axis represents the number of cells, indicating that the fluorescence intensity decreased after interfering with RPL10A, indicating a decrease in the number of lysosomes.

[0100] To further explore whether RPL10A specifically damages lysosomes, first, this example used immunofluorescence ( Figure 4 -A, B) and Western blotting ( Figure 4 -C) The Golgi marker GM130 was detected and its mean fluorescence intensity and protein level were found to be unchanged. Interference with RPL10A did not affect the morphology and protein level of the Golgi apparatus.

[0101] Secondly, immunofluorescence ( Figure 5 -A, B) and Western blotting ( Figure 5 -C) The endoplasmic reticulum marker Calnexin was detected and its average fluorescence intensity and protein level were found to be unchanged. Interference with RPL10A did not affect the morphology and protein level of the endoplasmic reticulum.

[0102] Finally, immunofluorescence ( Figure 6 -A, B) and Western blotting ( Figure 6 -C) The mitochondrial marker Tom20 was detected and found that the average branch length and protein level of single cells did not change. Interference with RPL10A did not affect the morphology and protein level of mitochondria. The above experimental results show that interference with RPL10A specifically affects lysosomes.

[0103] In addition, after 24 hours of interference with RPL10A, RPL10A was overexpressed by calcium phosphate transfection, and then the level of LAMP1 was detected by Western blotting ( Figure 7 -A, B). The results of immunoblotting showed that the LAMP1 level in the RPL10A interference group was significantly lower than that in the non-interference group, indicating that the interference with RPL10A affected the biogenesis of lysosomes and reduced the number of lysosomes. The LAMP1 level in the non-interference group tended to increase compared with the non-interference overexpression group. The LAMP1 level in the overexpression group after interference tended to increase compared with the RPL10A interference group, indicating that overexpression of RPL10A can increase the number of lysosomes, thereby improving Alzheimer's disease.

[0104] Example 5 Interference with the expression of lysosomal genes MFSD8 and ATP6V0A4 after RPL10A

[0105] After interfering with RPL10A, lysosome-related genes were significantly enriched in the genes whose alternative splicing forms were changed. In this example, the two genes MFSD8 and ATP6V0A4 were selected for subsequent verification.

[0106] The method of Example 3 was used to extract RNA from cells 48 hours after RPL10A interference and reverse transcribe it, and then PCR was performed using corresponding primers, and agarose identification was performed.

[0107] The experimental steps of PCR are as follows:

[0108] (1) Obtain the gene sequence of specific exon skipping transcripts of the target gene in UCSC, design primers on Primer 3, and order primers from a biological company;

[0109] (2) Using cDNA as a template, add various reagents one by one in the following order

[0110] Table 6 Required reagents and volumes for RT-PCR

[0111] Reagents volume 2×RapidTaqMasterMix 25μL F 2μL R 2μL cDNA 1μL ddH2O To50μL

[0112] (3) The upstream primer of MFSD8 is 5'-GATCATATACTGCTGGTGCT-3'; the downstream primer is 5'-CTTTTACACTGTCTTCCTGA-3'. The RT-PCR program is 95℃3min; 95℃15s, 60℃-50℃ (touch down) 15s, 72℃5s, 10 cycles; 95℃15s, 50℃15s, 72℃5s, 35 cycles; 72℃5min; 4℃ storage.

[0113] (4) The upstream primer of AT6V0A4 was 5'-CAGGATCCAAGAAGATGCCA-3', and the downstream primer was 5'-TCCATGATCAGAAGGATGGC-3'. The RT-PCR program was 95°C for 3 min; 95°C for 15 s, 61°C-51°C (touch down) for 15 s, 72°C for 12 s, 10 cycles; 95°C for 15 s, 51°C for 15 s, 72°C for 12 s, 35 cycles; 72°C for 5 min; and stored at 4°C.

[0114] The steps of agarose identification experiment are as follows:

[0115] (1) Select the desired agarose gel concentration according to the size of the DNA band to be identified. Wash the agarose gel plate, comb and conical flask with ddH2O.

[0116] (2) Use a measuring cylinder to transfer the required TAE solution into a conical flask, and then weigh the corresponding amount of agarose into the conical flask.

[0117] (3) Heat in a microwave oven for 3 min until the solution is clear and transparent with no white agarose particles.

[0118] (4) Rinse the conical flask with cold water, add the dye in a ratio of 1:10000 and shake well.

[0119] (5) Insert a comb onto the agarose plate, pour in the prepared agarose solution, and let it stand for 30 minutes to solidify.

[0120] (6) After solidification, remove the comb and align the gel hole with the black first level of the agarose electrophoresis instrument. First, add 2 μL of DNA marker, then add 5 μL of the sample mixed with loading. Keep the constant voltage at 120 V and set the electrophoresis time according to the band size.

[0121] Construction of ATP6V0A4-minigene:

[0122] (1) According to the results of transcript sequencing, the transcripts with exon skipping were downloaded from UCSC, and the downloaded transcripts were input into Primer3. Primers were designed on the sequences 350 bp upstream and downstream of the first, second, and third exons, and then sent to a biological company for synthesis.

[0123] (2) Using normal human gDNA as a template, add each component one by one according to the following steps.

[0124] Table 7 Reagents and volumes required to amplify ATP6V0A4 from gDNA

[0125] Reagents volume 5×PhusionHFbuffer 4μL 10mM dNTPs 0.4μL F 1μL R 1μL DNA polymerase 0.2μL gDNA (175ng) 3.5μL ddH2O To20μL

[0126] Primers for exon 1 were gDNA-ATP-F: 5′-CAGCTGGGCACATTTCTTTT-3′, gDNA-ATP-R: TGAGCCAAGGAGTCCTCAGT-3′;

[0127] Primers for exon 2 were gDNA-ATP-2-F: 5′-CCTGTTTCTTTCCCAACCAG-3′, gDNA-ATP-2-R: 5′-TTGAACTCCTGGGCTCAAAC-3′;

[0128] The primers for exon 3 were gDNA-ATP-3-F: 5'-ACTGGAGCCTGTGAGGTCAA-3', gDNA-ATP-3-R: 5'-AGATGCCACCTTGCCAATAC-3'. The PCR program was: 98°C for 3 min; 98°C for 10 s, 69°C-59°C (touch down) for 20 s, 72°C for 30 s, 10 cycles; 98°C for 10 s, 59°C for 20 s, 72°C for 30 s, 30 cycles; 72°C for 5 min; and stored at 4°C.

[0129] (3) Using the PCR product as a template, add homology arms identical to those of the vector. The following is the system for adding homology arms to the first and second fragments of the PCR.

[0130] Table 8 Reagents and volumes required for adding homology arm PCR to the amplified products of exons 1 and 2

[0131] volume Reagents 5×PhusionHFbuffer 10μL 10mM dNTPs 1μL F 2.5μL R 2.5μL DNA polymerase 0.5μL gDNA (10ng) 1μL ddH2O To50μL

[0132] (4) The primers for the first and second fragments plus homology arms were homologous recombination ATP-F: 5'-CTTAAGCTTGGTACCCAGCTGGGCACATTTCTTTTCT-3', homologous recombination ATP-R: 5'-CTGGTTGGGAAAGAAACAGGTGAGCCAAGGAGTCCTCA-3'; homologous recombination ATP-2-F: 5'-CCTGTTTCTTTCCCAACCAGGGATCCTTTTGCTCCTCAGG-3', homologous recombination ATP-2-R: 5'-TTGACCTCACAGGCTCCAGTTTGAACTCCTGGG-3'. The PCR program was: 95℃10s; 95℃10s, 72℃30s, 30 cycles; 72℃5min; 4℃ storage.

[0133] (5) Use the PCR product as a template, add the same homology arms as the vector, and add each component one by one according to the following steps

[0134] Table 9 Reagents and volumes required for adding homology arm PCR to exon 3 amplification products

[0135]

[0136]

[0137] The primers for the third fragment plus homology arm are homologous recombination ATP-3-F: 5'-ACTGGAGCCTGTGAGGTCAAGGCTGCAATGAGCCATAA-3', homologous recombination ATP-3-R:

[0138] 5'-CCTCTAGACTCGAGCGCTAGATGCCACCTTGCCAATACTT-3'.

[0139] (6) The program for adding the third fragment to the homology arm was as follows: 95°C for 30 s; 95°C for 15 s, 64.5°C for 20 s, 72°C for 30 s, 30 cycles; 72°C for 5 min; and storage at 4°C.

[0140] like Figure 8 As shown in the figure, the agarose identification results showed that the control group had a band at 300bp and 150bp, which were the wild-type transcript and the transcript of exon 6 skipping, respectively; after interfering with RPL10A, the band at 300bp became brighter, and the band at 150bp became weaker. The experimental results showed that the transcript of MFSD8 exon skipping was significantly reduced after interfering with RPL10A ( Figure 8 -C, D), which is consistent with the results suggested by transcriptome sequencing ( Figure 8 -A). The endogenous ATP6V0A4 gene is very weakly expressed, and the bands of exon skipping cannot be identified by RT-PCR and agarose gel electrophoresis. Therefore, an exogenous ATP6V0A4-minigene plasmid was constructed in this embodiment. The constructed ATP6V0A4-minigene and the interference fragment were co-transfected into the cells using Lipfectimin2000. After 48 hours, the RNA was extracted for reverse transcription, and the exon skipping was identified by RT-PCR and agarose electrophoresis. The agarose identification results showed that the control group without overexpression of ATP6V0A4 had only one band; the control group overexpressing ATP6V0A4-minigene had a band at 400bp and 250bp, respectively, which were the wild-type transcript and the exon skipping transcript; after interfering with RPL10A, the exon skipping transcript of ATP6V0A4 increased significantly ( Figure 8 -E, F), which is consistent with the transcriptome sequencing results ( Figure 8 -B).

[0141] Therefore, RPL10A affects the biogenesis and function of lysosomes by changing the alternative splicing pattern of lysosomal genes.

[0142] Example 6 RIP-Seq experiment using RPL10A antibody

[0143] The results of previous experiments showed that interfering with RPL10A would affect the splicing form of lysosomal gene transcripts. RPL10A is a ribosomal protein. This example uses RNA immunoprecipitation technology to study the mRNA bound to RPL10A. This example uses antibodies to precipitate endogenous RPL10A, and a portion is separated for immunoblotting to detect whether RPL10A is precipitated by the antibody. Then, the RNA bound to RPL10A is separated and purified, reverse transcribed into cDNA, and then a cDNA library is constructed, and finally sequenced. The specific experimental steps are as follows:

[0144] (1) Immunoprecipitation

[0145] Expand the HeLa cells to 4 15 cm dishes. When the cells are fully grown, take one of the dishes and digest the cells with 3 mL of trypsin and 6 mL of culture medium to terminate the digestion.

[0146] Take 10 μL of cell suspension and mix it with 10 μL of trypan blue in a ratio of 1:1, and then spot it into a counting plate for cell counting (2.5×107).

[0147] Add 1.8 mL of formaldehyde solution to the culture medium to a final concentration of 1% and incubate at room temperature for 10 min.

[0148] Add 1 / 10 volume of 2.66 M glycine to terminate the cross-linking reaction, incubate at room temperature for 5 min, place on ice to cool, and then transfer to a cold storage for incubation for 10 min.

[0149] Pour off the culture medium, wash the cells twice with 3 mL of ice-cold 1× PBS, then scrape the cells from the bottom of the dish with a cell scraper, wash the 15 cm dish once with 3 mL of PBS, transfer the cell suspension to a 15 mL centrifuge tube, and centrifuge at 1000 g at 4°C for 5 min.

[0150] The supernatant was removed, and the cells were resuspended in 500 μL LB1, incubated at 4°C for 10 min, and then centrifuged at 1400 g for 5 min at 4°C.

[0151] The supernatant was removed, and the cells were resuspended in 500 μL LB2, incubated at 4°C for 10 min, and centrifuged at 4°C and 1400 g for 5 min.

[0152] Remove the supernatant, resuspend the cells in 350 μL LB33, incubate at 4°C for 10 min, and centrifuge at 4°C and 1400 g for 5 min (1 mM DTT, 200 units / mL RNase out, protease and phosphatase inhibitors need to be added to LB1, LB2, and LB3 solutions).

[0153] Ultrasonication for 20 min (80% energy, ultrasonication for 10 s, stop for 30 s).

[0154] After sonication, the cells were centrifuged at 17,000 g for 10 min at 4°C, and the supernatant was transferred to an RNase-free EP tube for BCA quantification.

[0155] Take 50 μL as protein input and 20 μL as RNA input.

[0156] Add 50 μL of protein to 50 μL of 4×SDS and denature at 95°C.

[0157] (2) Antibody-beads incubation:

[0158] Vortex the original beads tube thoroughly, take 75 μL of beads into each of two EP tubes, and mark the EP tubes.

[0159] Resuspend the beads with 1 mL of freshly prepared Nuclei resuspension buffer, spin off, place the EP tube on the magnetic rack and remove the supernatant. Repeat this step 6 times, and resuspend the beads with 100 μL for the last time.

[0160] According to the protein concentration, 8 μL RPL10A antibody and 1.6 μL IgG were added to the EP tubes respectively and incubated at room temperature for 1 h.

[0161] The beads were washed 6 times with 1 mL Nuclei resuspension buffer and finally the protein was added and incubated at 4°C overnight.

[0162] After overnight incubation, place the EP tube on a magnetic stand and remove the supernatant.

[0163] Add 1 mL of ice-cold IP buffer (with protease and phosphatase inhibitors) to each EP tube, vortex the sample vigorously, centrifuge, place on a magnetic rack to remove the supernatant, repeat this step 5 times, and resuspend for the last time to take 50 μL of beads-protein mixture for immunoblotting to detect whether the protein has been IPed.

[0164] (3) RNA purification:

[0165] Resuspend the RNA-beads precipitate in 150 μL of Protein K solution; add 102 μL NT-2, 15 μL 10% SDS, and 18 μL Proteinase K to 9.75 μL RNA input; incubate the sample at 55°C for 30 min and shake to digest the protein.

[0166] After incubation for 30 min, the EP tube was placed on a magnetic rack, the supernatant was transferred to an RNase-free EP tube, and 250 μL NT-2 was added to each tube.

[0167] A phenol-chloroform mixed solution was prepared at a ratio of 125:24, 400 μL of the mixed solution was added to each EP tube, vortexed for 15 s, and centrifuged at 17000 g for 10 min at room temperature.

[0168] Transfer 350 μL of supernatant to a new EP tube, add 400 μL of chloroform, vortex for 15 s, and centrifuge at 17000 g for 10 min at room temperature.

[0169] Transfer 300 μL of supernatant to a new EP tube, add 50 μL of 5 M ammonium acetate, 15 μL of 7.5 M Licl, 1.25 μL of glycogen, and 850 μL of anhydrous ethanol to each tube; incubate at -80°C overnight.

[0170] The EP tube was centrifuged at 17000 g for 30 min at 4°C and the supernatant was removed.

[0171] Add 500 μL of ice-cold 80% ethanol and centrifuge at 17,000 g for 15 min at 4°C.

[0172] The RNA was air-dried in a clean bench and 20 μL of DEPC water was added to dissolve the RNA at 4°C.

[0173] Measure RNA concentration, remove gDNA, and reverse transcribe into cDNA.

[0174] (4) cDNA library construction: 1. DNA end repair

[0175] Table 10 Reagents and volumes required for DNA end repair

[0176] Reagents volume NEBNextUltraⅡDNALibraryPrepReactionBuffer 3.5μL NEBNextUltraⅡDNALibraryPrepEnzymeMix 1.5μL cDNA 20μL ddH2O To30μL

[0177] Use a gun to blow and mix, and then perform PCR. The PCR program is as follows:

[0178] 20℃ 30min 65℃ 30min 4℃ Forever

[0179] 2. Add Adaptor

[0180] Add the components to the PCR tube one by one in the following order.

[0181] Table 11 Reagents and volumes required for adding Adaptor

[0182]

[0183]

[0184] Use a gun to blow the system to mix, tap, and place the system in a PCR instrument at 20°C for 15 minutes, with a lid temperature of 30°C. Add 2 μL of USER Enzyme to the system, mix, tap, and place in a PCR instrument at 37°C for 15 minutes, with a lid temperature of 47°C.

[0185] 3. Product purification

[0186] Transfer 49 μL of the PCR system to a new EP tube, add 45 μL of AMPure XP beads restored to room temperature to the EP tube, mix well by pipetting, and let stand at room temperature for 5 minutes.

[0187] Place the EP tube on the magnetic rack and let it stand at room temperature for 5 minutes. When the beads are fully adsorbed on the magnetic rack, aspirate the supernatant.

[0188] Add freshly prepared 80% ethanol to the EP tube for washing. Invert the EP tube and the magnetic stand twice. After standing for 30 seconds, aspirate the supernatant and repeat this step twice.

[0189] After absorbing the ethanol, the EP tube is still placed on the magnetic rack, and the lid of the EP tube is opened to allow the ethanol to evaporate.

[0190] After the magnetic bead-DNA mixture in the EP tube is fully dried, add 17 μL of water, remove the EP tube, gently tap the EP tube with your fingers to mix it, and let it stand at room temperature for 5 minutes.

[0191] Place the fully dissolved water-magnetic beads-DNA mixture on a magnetic rack and let it stand at room temperature for 5 minutes; after the magnetic beads are adsorbed to the magnetic rack, aspirate 15 μL of supernatant into a new PCR tube for subsequent use.

[0192] 4. PCR amplification after purification

[0193] Add each component one by one in the following order

[0194] Table 12 Reagents and volumes required for PCR amplification after purification

[0195] Reagents volume DNA purified in the previous step 15μL NEBNextUltraⅡQ5MasterMix 25μL Indexprimer 5μL NEBNextUniversalPCRprimer 5μL

[0196] Mix well by pipetting, centrifuge, and then perform PCR. The PCR program is: 98℃ for 30s; 98℃ for 10s, 65℃ for 75s, this step is 10 cycles; 65℃ for 5min; 4℃ forever. Repeat step (4), measure the OD value of the product, and store at -20℃.

[0197] (5) Analysis of sequencing results:

[0198] Upload the sequencing results in fastq format to the X-shell7 server, use fastQC for quality control to obtain fastqc files; use trim_galore to trim and filter the raw data to obtain trim-cleandata;

[0199] Use Hisat2 to compare the sequencing results with the human genome to obtain the sam file; use samtools to convert the sam file to a bam file, sort the bam file, and finally create an index; use picard to remove duplicates; use RIPSeeker to analyze the sequencing results after removing duplicates to obtain the gff3 file; download the gff3 file to the computer and change its suffix to txt; copy the txt file to an excel spreadsheet, remove the header, delete useless information, add a column of peak values, and paste the content into the txt document; change the suffix of the txt document to bed and upload it to the X-shell7 server to annotate the genes; obtain the annotated document, upload it to R studio, and perform enrichment analysis using GO and KEGG. The enrichment function is: GO<-enrichGO(df$ENTREZ.ID,OrgDb=org.Hs.eg.db,keyType="ENTREZID",ont="ALL",#(ont is ALL, so it includes three parts: Biological Process, Cellular Component, and Molecular Function)pvalueCutoff=0.05,qvalueCutoff=0.05,readable=T)KEGG<-enrichKEGG(gene$ENTREZID,organism=KEGG_database,pvalueCutoff=0.05,qvalueCutoff=0.05).

[0200] like Fig. 9 As shown, the Western blot image shows that the endogenous RPL10A can be precipitated by the antibody. Then, the RNA bound to RPL10A is isolated and purified, reverse transcribed into cDNA, and then a cDNA library is constructed, and finally sequenced.

[0201] After the raw data were obtained by sequencing, the data were first quality controlled, trimmed and unnecessary data were filtered, and then the data were analyzed using RIPSeeker. Finally, the obtained genes were annotated using the human genome. The results of gene annotation showed that 38.11% of the genes bound by RPL10A were introns, 32.22% were intergenic intervals, 11.75% were exons, and 5.2% were 3'UTRs ( Fig.10), among which the intron has the highest binding ratio with RPL10A. As we all know, intron is the part with the highest proportion in genes, and the annotation result is in line with expectations.

[0202] The annotated results were then imported into R studio, and clusterProfiler was used to perform GO and KEGG analysis ( Fig.11 -A, B, C, D), the results show that the genes related to RNA shearing are significantly enriched. In this example, the enriched RNA shearing-related genes are sorted according to the log2FC size (Table 9).

[0203] Table 13 Expression levels of RNA splicing-related gene mRNA in the RPL10A interference group compared with the control group

[0204]

[0205]

[0206] Match the sequenced fragments to the reference genome. The greater the probability of protein binding at a certain position in the genome, the higher the RNA fragment stacking detected at that position. If there is no protein binding, there will be no RNA fragment stacking at that position. In this example, RIPSeeker is used for peak calling and then the bed file is output. After GO and KEGG analysis is performed, the file is enriched for RNA splicing-related genes, which are then imported into IGV to obtain a peak graph. Looking at the peak graph of the enriched RNA splicing-related genes, the peaks at the 23rd exon of the PRPF8 gene and the 6th exon of SUPT6H co-precipitated by RPL10A are significantly increased, indicating that RPL10A binds to the gene here ( Fig.12 -A, B).

[0207] Example 7 Overexpression of RPL10A can restore the pathological phenotype of α-synuclein A53T Drosophila

[0208] The above cytological experiments prove that RPL10A regulates lysosomal biogenesis by affecting the alternative splicing of lysosomal related genes, and lysosomal abnormalities are closely related to neurodegenerative diseases. The α-synucleinA53T mutation is closely related to the occurrence of various neurodegenerative diseases, including Parkinson's disease. Existing studies have shown that abnormal aggregation of α-synuclein is related to neuronal damage and death. The present invention explores its potential role in improving pathological phenotypes by overexpressing RPL10A in α-synucleinA53T mutant fruit flies.

[0209] The purpose of the present invention is to provide a method for restoring the pathological phenotype of α-synucleinA53T mutant fruit flies by overexpressing the RPL10A gene, which specifically includes reducing the level and oligomerization of α-synuclein protein, improving the motor ability of fruit flies and protecting dopaminergic neurons.

[0210] The immunofluorescence assay steps are the same as those in Example 4. The Drosophila crawling assay steps are as follows:

[0211] (1) 20-day-old α-synucleinA53T mutant fruit flies were selected and divided into RPL10A overexpression group, control group and wild-type group W118. Each group contained at least 50 fruit flies to ensure the statistical significance of the experimental results;

[0212] (2) Gently remove the fruit flies from the culture bottle to avoid mechanical damage to the fruit flies. Place the fruit flies in a temperature-controlled room at 25°C for acclimatization within 2 hours before the experiment;

[0213] (3) After anesthesia, 30 female flies of the same genotype were placed in three tubes with scale lines (10 flies in each tube) in each group. After they recovered to normal, all the flies were knocked to the bottom. Then, the number of fruit flies that climbed 7 cm within 10 s was counted to calculate their climbing ability.

[0214] The steps for immunofluorescence staining of DA neurons in brain tissue are as follows:

[0215] (1) Dissecting the head: Prepare some pre-cooled 4% PFA drops on a silicone dissecting plate, remove the fruit fly head with forceps, and remove the mouthparts to facilitate the penetration of the fixative;

[0216] (2) Pre-fixation: The dissected head was transferred to a PCR tube containing pre-cooled 4% PFA, placed on a rotating mixer, and fixed at 4°C for 1 h;

[0217] (3) Brain dissection: The fixed fruit fly head is transferred to a silicone dissection plate for further dissection, the head shell is removed, and the brain tissue is obtained;

[0218] (4) Re-fixation: The dissected Drosophila brain tissue was transferred again into a PCR tube containing pre-cooled 4% PFA, placed on a rotating mixer, and fixed at room temperature for 20 min;

[0219] (5) Permeabilization and blocking: aspirate the fixative, add 5% NGS, and permeabilize and block for 1.5 h;

[0220] (6) Incubate with primary antibody: dilute the primary antibody in 5% NGS in proportion. Aspirate the blocking solution, add the prepared primary antibody, place on a rotating mixer, and incubate at 4°C overnight;

[0221] (7) Washing the primary antibody: Recover the primary antibody and store at 4°C. Rinse twice with 0.3% PBST, 15 min each time;

[0222] (8) Incubation with secondary antibody: Add fluorescent secondary antibody to 5% NGS at a ratio of 1:200, place on a rotating mixer, and incubate at room temperature for 1 h;

[0223] (9) Washing the secondary antibody: remove the secondary antibody and wash twice with 0.3% PBST at room temperature, 15 min each time;

[0224] (10) Sealing: Place a new coverslip on a clean slide, use a tip with the tip cut off to absorb the Drosophila brain tissue and transfer it to the coverslip, place them face up, absorb all the liquid, and quickly drop a little Fluoromount on it, and immediately adsorb it on the new slide. Place it at room temperature away from light for 15 minutes, and use clear nail polish to seal the periphery of the coverslip. Store at 4°C away from light.

[0225] like Fig.13 As shown, the expression of α-synucleinA53T protein in Drosophila mutants increased significantly and oligomerized, which interfered with the overexpression of RPL10A gene in Drosophila mutants. It was found that after overexpressing the RPL10A gene, the α-synuclein protein level and TritonX-100 insoluble protein in Drosophila were significantly reduced; at the same time, the Drosophila motor ability was significantly improved and the dopaminergic neurons of Drosophila mutants were protected.

[0226] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0227] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for regulating lysosomal biogenesis by RPL10A, characterized in that include: By knocking down RPL10A, the number of lysosomes was reduced after the alternative splicing of lysosome-related genes was changed; Or overexpressing RPL10A can increase the number of lysosomes.

2. The method according to claim 1, characterized in that: Knockdown of RPL10A is achieved by introducing siRNA to interfere with the expression of RPL10A, wherein the nucleotide sequence of the siRNA includes SEQ ID NO: 1 and SEQ ID NO:

2.

3. The method according to claim 1, characterized in that The lysosome-related genes include MFSD8 and ATP6V0A4. By knocking down RPL10A, exon skipping occurs in MFSD8 and ATP6V0A4, wherein the transcript of MFSD8 undergoing exon skipping is reduced; and the transcript of ATP6V0A4 undergoing exon skipping is increased.

4. The method according to claim 1, characterized in that The lysosome-related genes also include CTSF, ASAH1, NPC2, SLC11A2, CD164, NAGPA, GLB1 and LGMN. By knocking down RPL10A, the expression of CTSF, ASAH1, NPC2, SLC11A2, CD164, NAGPA, GLB1 and LGMN is reduced, thereby reducing the number of lysosomes.

5. Use of the RPL10A gene or the protein it encodes as a target in screening drugs for treating or inhibiting neurodegenerative diseases.

6. The use according to claim 5, characterized in that: The drug targets the RPL10A gene or the protein encoded by it, activates or increases the expression of the RPL10A gene, or increases the level of the protein encoded by RPL10A.

7. Use of an agent for activating or increasing the expression of the RPL10A gene in the preparation of a drug for treating or inhibiting neurodegenerative diseases, wherein the agent comprises a small molecule, a polypeptide, a protein, or a gene drug.

8. The use according to any one of claims 5 to 7, characterized in that: The neurodegenerative diseases include Alzheimer's disease.

9. A drug, characterized in that Including RPL10A agonists, the drug is indicated for Alzheimer's disease.

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