CN111718939B - Use of ferroportin in the treatment of parkinson's disease - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to a target protein of Parkinson's Disease (PD), a coding gene and application thereof, and especially relates to preparation of a gene-targeted drug aiming at the key gene. The invention finds that the target genes ZIP13 and transferrin (Tsf) of the Parkinson disease are provided, and the Tsf is interfered by overexpression of ZIP13 or gene silencing to obviously relieve or delay the PD condition. According to the invention, the target protein of human PD comprises amino acid sequences shown as SEQ ID No.5 and SEQ ID No.6, and the gene comprises nucleotide sequences shown as SEQ ID No.7 and SEQ ID No. 8. According to the target gene, an RNAi vector carrying the target gene is constructed, and the RNAi vector can be used for improving the motor ability of a PD patient and relieving or preventing PD symptoms caused by the knocked-down PINK1 through gene silencing or gene overexpression. The invention is expected to provide a new treatment strategy and a key target for treating hereditary PD.

Description

Use of ferroportin in the treatment of parkinson's disease

Technical Field

The invention relates to the field of genetic engineering, in particular to a target protein of Parkinson's Disease (PD), a coding gene and application thereof, and especially relates to preparation of a gene-targeted drug aiming at the key gene.

Background

Parkinson's Disease (PD) is a neurodegenerative Disease affecting human health. The typical motor symptoms of PD have been considered as an important component of the disease since the initial description of James Parkinson in the 19 th century, and have been subsequently re-labeled by Jean-Martin Charcot. These patients often present with muscular tremor, muscular rigidity, slow movement, impaired writing, unstable gait, general weakness, stiff facial expression and low mood, which makes the patients difficult to take care of themselves, and brings heavy psychological and economic burden to families, seriously affecting social stability and harmony. PD is currently considered to be a complex neurological disease involving multiple systems, is mostly seen in the elderly, and gradually presents a trend of younger age. The pathogenesis of the disease is complex, no effective treatment means exists, and a new treatment strategy is urgently researched.

The causative factors of PD mainly include environmental induction and genetic action. The environmental factors mainly include pesticide exposure (such as rotenone, paraquat, maneb and the like), head injury, life style and the like. In addition to the external environmental induction of PD, more and more studies have found that PD patients have many genetic defects, thus confirming that genetic factors play an important role in the development of PD. To date, scientists have been able to associate mutations in nearly twenty different genes with PD, among which the genes that mediate autosomal dominant inheritance of PD include mainly SNCA, LRRK2, VPS35, EIF4G1, DNAJC13 and chchhd 2, and in addition, the genes associated with autosomal recessive genotype PD are mainly Parkin, PINK1 and DJ-1. PINK1 contains an N-terminal mitochondrial target sequence, a transmembrane domain (a highly conserved serine/threonine kinase domain), and a C-terminal autoregulatory domain. Once mitochondria are damaged, PINK1 can be gathered on a mitochondrial membrane, so that damaged mitochondria can be cleared in time through a plurality of regulation modes such as starting mitophagy, promoting mitochondrion and the like, and once PINK1 is down-regulated, the protection mechanism is damaged, and PD can be induced to occur. Studies have shown that PINK1 deficiency is an important factor in PD, mutations are common in early-onset PD patients and familial genetic cases, and PINK1 deficient patients often exhibit psychiatric symptoms (depression, anxiety and psychosis) in addition to somatic symptoms. Dan MF van Aalten et al believe that the alteration of PINK1 affects the sustained productivity of the cells, and if there is insufficient energy to drive the biochemical process, the affected cells eventually die. From the therapeutic point of view, obtaining a new risk gene of the Parkinson disease, establishing the causal connection of the risk gene and preparing a gene-targeted drug on the basis of the causal connection is a key research and development direction.

There is increasing evidence that endogenous metal ion imbalances are strongly linked to the pathological processes of PD. The literature shows that the black iron of PD patients is increased, striatum iron is unchanged, and pallidoluous iron is reduced; in addition to changes in iron levels in the brain, researchers have also found that PD patients have reduced iron levels in their hair and serum. Therefore, the PD patient has obvious iron metabolism abnormality, and the two are closely related, so that the potential for preventing or improving PD symptoms is provided by regulating the body level of iron ions.

Although proteins involved in iron regulation in human body have been identified to include DMT1, Ferritin, ZIP13, transferrin (Tsf), Mitoferrin1(Mfrn1), etc., the distribution of iron ions in different cells of human body and the regulation of iron ion balance are not clear at present, and especially the association of genes such as ZIP13, Tsf with Parkinson's disease has never been established.

RNA interference can regulate the expression of target genes, thereby changing the expression level of target proteins. RNAi drugs are designed aiming at specific targets of PD, so that specific, lasting and efficient regulation and control effects are achieved, disease progress is slowed down, and the application prospect is very wide. Although some in vitro experiments indicate that iron ion may be involved in the onset of PD, there has been no more detailed, convincing in vivo evidence. Therefore, it is necessary to provide a protein and a gene sequence thereof that can treat PD.

Disclosure of Invention

Drosophila melanogaster is widely applied to human neurodegenerative diseases and metal metabolism regulation and control research, so that the application of iron ion transporters (ZIP 13 positioned on Golgi bodies and cell secretory protein Tsf) in treating PD can be explored by taking Drosophila melanogaster as a model organism. Knocking down the expression of PINK1 in drosophila muscle can reproduce the main symptoms of human PD, including dyskinesia, muscle atrophy, mitochondrial dysfunction and the like. Through a gene overexpression technology and an RNAi gene silencing technology, dZIP13 (the drosophila homologous gene of human ZIP 13) is selectively overexpressed in PD drosophila muscle, and Tsf1 (the drosophila homologous gene of human Tsf) is selectively silenced, so that the PD process of drosophila can be remarkably relieved. The invention provides two key gene target sites for treating PD diseases, and is expected to become a new PD treatment strategy by regulating the expression of the gene target sites.

The amino acid sequence of the target protein dZIP13 of the human PD drosophila model is shown as SEQ ID No.1

The amino acid sequence of the human PD drosophila model target protein Tsf1 is shown as SEQ ID No.2

The Drosophila model target gene dZIP13 for treating PD disorders according to the present invention encodes the above-mentioned dZIP13 protein, and its nucleotide sequence is shown in SEQ ID No.3, for example.

The drosophila model target gene Tsf1 for the treatment of PD disorders according to the invention, which encodes the above-mentioned Tsf1 protein, e.g. the nucleotide sequence of which is shown in SEQ ID No. 4.

The target protein of human PD according to the invention includes the sequence of amino acid ZIP13 as shown in SEQ ID No. 5.

The target protein of human PD according to the invention comprises the sequence of amino acids Tsf as shown in SEQ ID No.6,

the target gene of human PD according to the present invention encodes the above-mentioned ZIP13 protein, and the gene includes the nucleotide sequence shown in SEQ ID No. 7.

The target gene of human PD according to the present invention encodes the above-mentioned Tsf protein, and the gene includes a nucleotide sequence shown as SEQ ID No. 8.

The vector according to the present invention including the target genes ZIP13, Tsf may be a sense vector of ZIP13, Tsf or an RNAi interference vector.

The above-mentioned vector according to the present invention may be any vector constructed to interfere with or suppress expression using the Tsf nucleotide sequence; all over-expression vectors constructed using the ZIP13 nucleotide sequence.

The target protein for PD according to the invention is used in a medicament for treating PD.

The target genes ZIP13 and Tsf of PD are used for preparing the medicine for treating PD.

The above-mentioned carrier according to the present invention may be used for the preparation of a medicament for the treatment of PD.

The target gene according to the present invention is used for improving the motor ability of PD patients and alleviating or inhibiting mitochondrial dysfunction through RNAi interference.

The experimental results show that:

1) dZIP13 and Tsf1 were able to modulate PINK1 deficient Drosophila PD-like phenotype. A PiNK 1-knocked-down drosophila PD model is constructed by utilizing a UAS/Gal4 system, and dZIP13 is overexpressed or Tsf1 is silenced at the muscle part of drosophila by utilizing the system, so that the behavioral influence on the drosophila is observed. Experiments show that the drosophila muscle dZIP13OE or Tsf1 RNAi can relieve the wing abnormality, the reduction of jumping times and the muscle damage (drosophila back depression) caused by PINK1 RNAi. The trunk muscle of the drosophila is dissected, and observation by a laser confocal microscope shows that PINK1 RNAi causes muscle atrophy and rupture, and dZIP13OE or Tsf1 RNAi at the muscle can reduce the muscle damage degree.

2) The PINK1 RNAi-induced respiratory dysfunction can be improved by regulating the expression levels of dZIP13 and Tsf 1. Overexpression of dZIP13 or silencing of Tsf1 increased levels of ATP for PINK1 RNAi. The Seahorse XF-24 energy metabolism analyzer is applied to detecting mitochondrial respiration, and results show that compared with a PINK1 RNAi group, the mitochondrial respiration capacity can be obviously improved by over-expressing dZIP13 or silencing Tsf 1.

3) Modulation of expression levels of dZIP13 and Tsf1 ameliorated mitochondrial complex activity impairment caused by PINK1 RNAi. Muscle PINK1 knockdown resulted in decreased mitochondrial complex I, II, III and IV activity, and our experimental results showed that increased mitochondrial complex I, II, III and IV activity at the muscle site was increased by PD drosophila overexpressing increased expression of dZIP13 and by PD drosophila decreasing expression of Tsf1 by RNAi.

4) Modulation of the expression level of Mfrn may affect the modulation of PD by dZIP13 and Tsf 1. Mfrn OE increases mitochondrial iron content. Compared with PINK 1-deficient drosophila, PD drosophila with muscle Mfrn OE reduced abnormal wing shape, trunk concavity and increased ATP levels in drosophila, which directly indicates that mitochondrial iron protects against injury from PINK1 RNAi. The experimental results show that Mfrn RNAi can weaken the protective effect of dZIP13OE and Tsf1 RNAi on PD caused by PINK1 RNAi. Therefore, the invention simultaneously provides a technical scheme that Mfrn and dZIP13/Tsf1 are intervened together to realize accurate fine adjustment of disease symptoms

Technical effects

Specific overexpression dZIP13 in drosophila muscle tissues, RNAi interference Tsf1 expression, PD drosophila mitochondrial respiration function and weakened mitochondrial complex activity can be obviously improved, so that drosophila dyskinesia and muscle injury are obviously improved. Reduction of mitochondrial iron levels by Mfrn RNAi can attenuate the rescue effect of dZIP13OE and Tsf1 RNAi. The above findings indicate that the regulation and control of the expression of dZIP13 and Tsf1 iron transporters can change the iron level of the body, and finally realize the effect of improving the PD process. The invention establishes the regulation and control functions of the ferroportin ZIP13 and Tsf1 on the Parkinson disease for the first time, so that the genes are identified as key risk genes, RNAi vectors carrying the genes are designed, and the industrial applicability is realized. The invention provides a novel gene therapy target of the Parkinson disease, and simultaneously, the adopted suppression and overexpression vector taking the nucleotide sequence as the target constitutes a substantial molecular element of the corresponding gene targeted drug. The invention is expected to improve new targets and new ideas for treating PD.

Drawings

FIG. 1: phylogenetic tree analysis drosophila dZIP13 has high homology with human ZIP 13.

FIG. 2: phylogenetic tree analysis drosophila Tsf1 shares high homology with human Tsf.

FIG. 3: behavioral manifestations of Drosophila parkinsonii. (A) Counting abnormal wing shapes of drosophila melanogaster, wherein the abnormal wing shapes caused by muscle PINK1 RNAi can be rescued by Mhc-Gal4 > dZIP13OE and Mhc-Gal4 > Tsf1 RNAi; (B) statistics is carried out on fruit fly jumping behaviors, and the injury of jumping ability caused by muscle PINK1 RNAi can be rescued by Mhc-Gal4 > dZIP13OE and Mhc-Gal4 > Tsf1 RNAi; (C) the fruit fly trunk depression is observed, the muscle of the depression is marked by a white arrow in the figure, and dZIP13OE and Tsf1 RNAi have a rescue effect on the trunk injury caused by the muscle PINK1 RNAi; (D) counting the proportion of the injured trunk, compared with the normal group, the muscle PINK1 RNAi significantly increases the trunk pit proportion, and Mhc-Gal4 > dZIP13OE and Mhc-Gal4 > Tsf1 RNAi can improve the injury; (E, F) Drosophila muscle tissue dissection observation, observation of Drosophila muscle at 20X and 63X using confocal laser microscopy, respectively, compared to the normal group, the PINK1 RNAi group exhibited muscle disruption and disorganization of myofibrils, and the muscles dZIP13OE and Tsf1 RNAi were able to alleviate this muscle injury. The muscle in the figure is stained red with rhodamine-labeled phalloidin for microscopic observation. Data were counted as mean ± SEM, t-test, × P < 0.01, × P < 0.001, scale bar, 200 μm (e), 20 μm (f).

FIG. 4: the regulation and control of the iron transporter can save the mitochondrial respiration of the drosophila parkinsonii. (A) Mhc-Gal4 > PINK1 RNAi Drosophila's reduced ATP levels can be elevated by dZIP13OE and Tsf1 RNAi; (B) detecting respiration of a drosophila trunk mitochondrial complex III, wherein sn-glycerol-3-phosphate is used as a substrate, and antimycin A is used as an inhibitor; (C) and (3) detecting the respiration of the mitochondrial complex IV of the trunk line of the drosophila, wherein TMPD/ascorbic acid is used as a substrate, and sodium azide is used as an inhibitor. The OCR value represents the magnitude of the breathing capacity. Compared with Mhc-Gal4 > PINK1 RNAi fruit flies, Mhc-Gal4 > dZIP13OE and Mhc-Gal4 > Tsf1 RNAi fruit flies have obviously improved trunk respiration. Data were counted as mean ± SEM, t-test, × P < 0.05, × P < 0.01, × P < 0.001.

FIG. 5: iron transporters are involved in the regulation of mitochondrial function. NADH dehydrogenase activity (A), succinate dehydrogenase activity (B), cytochrome C reductase activity (C), and cytochrome C oxidase activity (D) were examined. PINK1 RNAi causes the activity of four mitochondrial enzymes to be reduced, and dZIP13OE and Tsf1 RNAi can obviously increase the activity of mitochondrial respiratory chain enzyme. Data were counted as mean ± SEM, t-test, × P < 0.05, × P < 0.001.

FIG. 6: mitochondrial iron transporters regulate drosophila parkinsonian processes. (A) Detecting mitochondrial iron content, wherein the muscle Mfrn RNAi reduces the muscle mitochondrial content, the muscle Mfrn OE can increase the muscle mitochondrial content, and the increase effect of the dZIP13OE and the Tsf1 RNAi on the mitochondrial iron is reduced after the Mfrn RNAi; (B) abnormal wing type caused by PINK1 RNAi can be rescued by Mhc-Gal4 > Mfrn OE, and the regulation effect of muscle dZIP13OE and Tsf1 RNAi is inhibited by Mfrn RNAi; (C) trunk damage caused by PINK1 RNAi can be rescued by Mhc-Gal4 > Mfrn OE, and the rescue effect of muscle dZIP13OE and Tsf1 RNAi is inhibited by Mfrn RNAi; (D) measuring muscle ATP levels, Mfrn RNAi inhibited the effect of dZIP13OE and Tsf1 RNAi on ATP increase. Data were counted as mean ± SEM, t-test, × P < 0.05, × P < 0.01, × P < 0.001.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified.

Example 1: evolutionary tree analysis

Phylogenetic tree analysis was performed on human genes ZIP13 and Tsf, drosophila genes dZIP13 and Tsf1 using MEGA software, indicating that drosophila dZIP13 and human ZIP13, and drosophila Tsf1 and human Tsf belong to orthologous genes and have the same or similar functions (fig. 1-2).

Example 2: tsf1 RNAi and acquisition of dZIP13OE transgenic Drosophila strains

Firstly, using wild drosophila w1118 strain genome DNA as a template, separating and cloning Tsf1 and dZIP13 by PCR, and then carrying out enzyme digestion, ligation and transformation to introduce the clones into escherichia coli competent cells. And obtaining positive clones through colony PCR, plasmid enzyme digestion identification and sequencing identification.

Secondly, transforming the constructed plasmid into a new egg by using a p-element mediated method by using a wild fruit fly w1118 as a receptor and using a microinjection technology, and hybridizing the number of each fruit fly with the w1118 after the egg develops into an adult, so that the red eye of the fruit fly of the filial generation shows that the construction is successful.

Example 3: effect of Drosophila dZIP13 and Tsf1 genes on PINK 1-deficient Drosophila PD-like behaviours

Effect of dZIP13OE and Tsf1 RNAi on PD locomotor Activity

In order to construct a genetic PD model, a PINK1 RNAi fruit fly is hybridized with a Mhc-Gal4 fruit fly, the filial generation is the PD fruit fly with the muscle part PINK1 reduced, in addition, in order to change the level of the iron transporter in muscle tissue, a dZIP13OE and a Tsf1 RNAi fruit fly are respectively hybridized with the Mhc-Gal4, and the filial generation is the fruit fly with the muscle part dZIP13OE or Tsf1 RNAi. Collecting offspring drosophila melanogaster, namely Mhc-Gal 4- +/+ normal group drosophila melanogaster, Mhc-Gal4 > PINK1 RNAi model group drosophila melanogaster, Mhc-Gal4 > PINK1 RNAi; dZIP13OE Drosophila and Mhc-Gal4 > PINK1 RNAi; tsf1 RNAi Drosophila. And counting the number of abnormal wings of each group, comparing the number with the total number of the drosophila, and calculating the abnormal wing proportion. And counting the number of jumps when each pipe is the same. The results show (FIGS. 3A-B): in muscle tissues, specific dZIP13OE (Mhc-Gal4 > PINK1 RNAi; dZIP13 OE) and Tsf1 RNAi (Mhc-Gal4 > PINK1 RNAi; Tsf1 RNAi) can improve dyskinesia of PINK1 RNAi (Mhc-Gal4 > PINK1 RNAi).

Second, in muscle dZIP13OE and Tsf1 RNAi effects on muscle injury

Separately collecting groups of drosophila, including Mhc-Gal 4- + normal group drosophila, Mhc-Gal4 > PINK1 RNAi model group drosophila, Mhc-Gal4 > PINK1 RNAi; dZIP13OE Drosophila and Mhc-Gal4 > PINK1 RNAi; and (4) observing the back depression condition under a Tsf1 RNAi fruit fly body type microscope, and counting the proportion of the number of depressed fruit flies in the total number of fruit flies. Meanwhile, the trunk of the fruit fly is separated under a body microscope, a cut is cut along the median line of the back by using a pair of tweezers, the trunk is divided into two parts, and the two parts are placed into PBS buffer solution for temporary storage. After the separation of all groups is finished, absorbing PBS, adding 4% paraformaldehyde for fixing for ten minutes, washing with 0.3% PBST for three times, adding phalloidin to stain muscles, washing with 0.3% PBST, finally sealing the stained tissues, and observing the tissue morphology under 20 Xand 63 Xoil microscope by using a laser confocal microscope. The results show (FIGS. 3C-F): compared with the Mhc-Gal4 & gtPINK 1 RNAi model group drosophila melanogaster, the muscle tissue specific dZIP13OE (Mhc-Gal4 & gtPINK 1 RNAi; dZIP13 OE) and Tsf1 RNAi (Mhc-Gal4 & gtPINK 1 RNAi; Tsf1 RNAi) can obviously reduce the depressed ratio of the muscle back and effectively relieve the disorganization and breakage degree of muscle fibers.

Example 4: the specific regulation of the expression levels of dZIP13 and Tsf1 in Drosophila muscle tissue can significantly affect mitochondrial respiration

The experiment shows that Mhc-Gal 4- +/+ normal group drosophila, Mhc-Gal4 > PINK1 RNAi model group drosophila, Mhc-Gal4 > PINK1 RNAi; dZIP13OE Drosophila and Mhc-Gal4 > PINK1 RNAi; tsf1 RNAi drosophila trunks are separated, the supernatant is obtained by homogenizing and centrifuging, and the ATP level of the supernatant is detected by using a Promega ATP detection kit. Detecting the respiratory function of mitochondria at muscles by utilizing seahorse XF-24, dissecting the trunk of fruit fly, adding a mitochondria extracting solution, evenly crushing tissues by using a glass homogenizer, and centrifuging by adopting a gradient centrifugation method to obtain a mitochondria precipitate. Mitochondrial concentration was measured with the Bradford kit and mitochondria were added to seahorse24 well plates. To detect mitochondrial complex III respiration, 4mM sn-glycerol-3-phosphate was used as the oxidation substrate and 4 μ M antimycin a was used as the inhibitor. To detect mitochondrial complex IV activity, 200 μ M of N, N' -tetramethylp-phenylenediamine/10 mM ascorbic acid was used as the oxidation substrate and 20mM sodium azide was used as the inhibitor. The results show (fig. 4): overexpression of dZIP13 or silencing of Tsf1 increases ATP levels and improves mitochondrial respiratory function.

Example 5: the specific regulation of the expression levels of dZIP13 and Tsf1 in drosophila muscle tissue can significantly affect the mitochondrial complex activity

For the detection of mitochondrial complex I activity, 0.5M potassium phosphate buffer, 50mg/ml bovine serum albumin, 10mM potassium cyanide, 10mM NADH were added as reaction substrates, and immediately after the addition of 10mM coenzyme Q1, absorbance was measured at 340nm, and the activity levels of the respective groups were compared. To detect the mitochondrial complex II activity, 0.5M potassium phosphate buffer, 50mg/ml bovine serum albumin, 10mM potassium cyanide, 400mM succinic acid, 0.015% DCPIP were added, and finally 12.5M DUB was added and absorbance was measured at 600nm for three minutes, and the activity of each group was compared. To detect mitochondrial complex III activity, 0.5M potassium phosphate buffer, 1mM oxidized cytochrome C, 10mM potassium cyanide, 5mM EDTA, 2.5% Tween 20, and 10mM DUB ethanol solution were added, and absorbance was measured at 550nm to compare the activity of each group. To detect the mitochondrial complex IV activity, the sample was reacted well with 100mM potassium phosphate buffer, 1mM reduced form of cytochrome C, and absorbance was measured at 550nm to compare the activities of the respective groups. The results show (fig. 5): muscle tissue-specific modulation of dZIP13OE and Tsf1 RNAi can increase PINK1 RNAi muscle-damaged mitochondrial complex activity.

Example 6: specific regulation of Mfrn expression levels in Drosophila muscle influences the regulatory role of dZIP13 and Tsf1

The experiment divides the drosophila into the following groups: Mhc-Gal4 > +/+ Normal group Drosophila, Mhc-Gal4 > PINK1 RNAi model group Drosophila, Mhc-Gal4 > PINK1 RNAi; dZIP13OE Drosophila, Mhc-Gal4 > PINK1 RNAi; dZIP13OE + Mfrn RNAi Drosophila, Mhc-Gal4 > PINK1 RNAi; tsf1 RNAi Drosophila melanogaster, Mhc-Gal4 > PINK1 RNAi; tsf1 RNAi + Mfrn RNAi Drosophila, Mhc-Gal4 > PINK1 RNAi; mfrn RNAi Drosophila, Mhc-Gal4 > PINK1 RNAi; mfrn OE Drosophila melanogaster. In the experiment, the ferroso-called felazine method is used for detecting the iron content of mitochondria of each group. Extracting drosophila trunk mitochondria, homogenizing, centrifuging, boiling the supernatant with hydrochloric acid for twenty minutes, centrifuging again, reacting the supernatant with ascorbic acid completely, adding felazine and saturated ammonium acetate successively, reacting completely, detecting absorbance value at 562nm, wherein the size is in positive correlation with iron content. The experiment is similar to the method, and the abnormal wing shape proportion, trunk sinking proportion and ATP level of each group of drosophila are counted. The results show (fig. 6): compared with the normal group, PINK1 RNAi had no significant effect on mitochondrial iron content, and Mfrn OE localized on the mitochondrial membrane increased mitochondrial iron content, and further, muscle dZIP13OE and Tsf1 RNAi also increased mitochondrial iron content, and the increased effect of both changes was attenuated by Mfrn RNAi (fig. 6A). For drosophila abnormal wing and trunk pit ratio experiments, statistical results show that muscle PINK1 RNAi increased the abnormal wing and pit ratio compared to the normal group, Mfrn OE mitigated this change, and Mfrn RNAi attenuated the rescue effect of dZIP13OE and Tsf1 RNAi on both phenotypes (fig. 6B-C). When ATP water of each group is detected, compared with a normal group, the ATP of the PINK1 RNAi group is obviously reduced; in comparison with the PINK1 RNAi model group, Mfrn OE increased ATP levels based on this, whereas Mfrn RNAi attenuated the effect of dZIP13OE and Tsf1 RNAi in increasing ATP levels (fig. 6D).

In addition, other modifications within the spirit of the invention will occur to those skilled in the art, and it is understood that such modifications are included within the scope of the invention as claimed.

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gagaaaatgg ctaaggaggg atacaaagat cctgcagatt caaaactcct gcgagtgctt 240

ctgagtttcg cggtcggcgg tctgctgggc gatgtgttcc ttcacctgtt gccagaagcc 300

tgggagggcg ataatcaaga tccttctagt cacccatccc tgcgctcggg cctttgggta 360

ctttccggca tactgatctt cacgatcgtg gagaaaatct tttccggata tgccagcgcg 420

gacgaggaga accctcagcc caagtgcgtg gagatagcca actgcctgtt gcgtcgacat 480

ggcggccaac taccagaggg cgaaacctct gagagttgtg gcggcgcctg cgacatcgaa 540

gatgtaggta aagtctgttt cctacgcgag caggaacaga agtcaaagga aaggaaggaa 600

cagccgaaga aggtggctgg ttatctgaac ctcttggcca actcaattga caatttcaca 660

cacggtctag ccgtggctgg atcctttttg gtgtccttca gacacggcat tctagccact 720

tttgccatat tgcttcatga aattccgcac gaggtggggg atttcgcaat cctgcttcga 780

tccggattca gtcgctggga cgccgcgcgt gcgcaactac tcacggcggg agctggcctg 840

ctcggtgctc tggtggccat cggaggctcc ggcgtaacgt cggccatgga ggcacgtact 900

tcgtggatta tgccgttcac cgccggcggc tttctgcaca ttgctctggt cacagtatta 960

cctgatctct tgaaggagga ggagcgcaag gagtccatta agcagctgct agcactggta 1020

tttggcattg cgttaatggc cgtgatgacc atgctattcg aacactag 1068

<210> 4

<211> 1926

<212> DNA

<213> Drosophila melanogaster (Drosophila melanogaster)

<400> 4

atgatgtcgc cgcataagac ccacacctgg ctgccactgg cggtggccgc cctcctgctg 60

atccttggac cgcagtcctc cctggcggag gaacccattt atcgcctgtg tgtgccgcaa 120

atctatttgg ccgagtgcca gcagctgctg gccgatccct cggaggcggg catccgcatg 180

gagtgcgtgg ctggacggga tcgagtggac tgcctggagc tgatcgagca gcgcaaggcc 240

gatgtgctgg ccaccgagcc ggaggacatg tacatcgcct atcatcgcaa gaacgaggac 300

tatcgcgtga tctctgagat ccgaacgcag caggacaagg atgccgcctt ccgttacgag 360

ggcattatcc tggtgaagaa ggactccccc attcgcaccc tgcagcagct gcgtggcgcc 420

aagtcctgcc acactggctt cggccgcaac gtcggctaca agatccccat caccaagctg 480

aagaacacgc acgtcctgaa ggtgtccgcc gatccgcaga tctccgctac ggagcgcgaa 540

ctgaagtcgc tgtccgagtt cttcacgcag tcgtgcctgg tgggcaccta ctccacgcat 600

ccggaaacgg atcgcctgct gaagaagaag tacgccaatc tgtgcgctct gtgcgagaag 660

ccggagcagt gcaactatcc ggacaagttc agtggctatg atggcgccat acgctgcctg 720

gacaagggtc agggcgaggt ggccttctcc aaggtgcagt acatcaaaaa gtactttggt 780

ctgccgggtg ccggcccaga tgcgccgcca gcggagggca atccggagaa tttcgaatat 840

ctgtgcgagg atggcacccg gcgcccggtc accggacccg cctgctcctg ggcccagcgc 900

ccctggagcg gctacatctc caacgagcag gccgttcaca actcggagca gctgcaccaa 960

ttgcagtcgc gtctggagcg cttcttcgcc aatggactgc aggcgcagaa caaggacgcc 1020

gccgcccatc tgctcatcca gccgaatgcc gtgtaccaca gcaaggatgc tgccatcgat 1080

cccaaggtct atttggagcg tgccggctac aaggatgtga tcgagcgtga tggcagtgcc 1140

atcaggaaga tccgcttgtg cgcccagaac gacgacgagt tcgccaaatg ccaggcgctg 1200

caccaggctg cctacgcccg cgacgctcgt ccggaactcg agtgcgttca gtccaccgat 1260

tgtgtggtgg ctctgaccaa gaaggaggcg gatttgacca ttgttcgcgc aactggctac 1320

gcggatgccc gtagcaacca gctgcagcca atcgtctacg agcagagggc tcaggatgat 1380

gtccttgtgg cggtcgcagc acccggcgtt acacgggagg ctctccagaa ggccagcatc 1440

aaattcaatg agaattgcga acgatcccgt gctgctgccg ccttgttgaa caagcgacgt 1500

ggcctggacg cttgtcgtgt ctcatccagc gacgatggag aggtgcagat cgtgcccgcc 1560

tccgagctgg agaagcacaa ggacgcgcag ctggtgtgcc ccagtctgga gcggcgaccg 1620

gtcaccgact tccgtgactg caacgtggac gtacagctgc cccgtgccat cttcatccga 1680

tcggacacca ccagcgtgga gcaggagaca gtgaagcatc tgttctcgct gatctcggac 1740

aagtttggtg cccgcggcaa gctggtggat gttttcgctc tgttcggcga attccagaag 1800

ggcaagaaga atgtgtattt caacgacaaa gccgttcagc tcaccacgga gctcaaaaac 1860

gaaatccaga acgagcagat ctacacagat ctccagtgca atgctaacaa gattgccaag 1920

cagtga 1926

<210> 5

<211> 371

<212> PRT

<213> Drosophila melanogaster (Drosophila melanogaster)

<400> 5

Met Pro Gly Cys Pro Cys Pro Gly Cys Gly Met Ala Gly Pro Arg Leu

1 5 10 15

Leu Phe Leu Thr Ala Leu Ala Leu Glu Leu Leu Glu Arg Ala Gly Gly

20 25 30

Ser Gln Pro Ala Leu Arg Ser Arg Gly Thr Ala Thr Ala Cys Arg Leu

35 40 45

Asp Asn Lys Glu Ser Glu Ser Trp Gly Ala Leu Leu Ser Gly Glu Arg

50 55 60

Leu Asp Thr Trp Ile Cys Ser Leu Leu Gly Ser Leu Met Val Gly Leu

65 70 75 80

Ser Gly Val Phe Pro Leu Leu Val Ile Pro Leu Glu Met Gly Thr Met

85 90 95

Leu Arg Ser Glu Ala Gly Ala Trp Arg Leu Lys Gln Leu Leu Ser Phe

100 105 110

Ala Leu Gly Gly Leu Leu Gly Asn Val Phe Leu His Leu Leu Pro Glu

115 120 125

Ala Trp Ala Tyr Thr Cys Ser Ala Ser Pro Gly Gly Glu Gly Gln Ser

130 135 140

Leu Gln Gln Gln Gln Gln Leu Gly Leu Trp Val Ile Ala Gly Ile Leu

145 150 155 160

Thr Phe Leu Ala Leu Glu Lys Met Phe Leu Asp Ser Lys Glu Glu Gly

165 170 175

Thr Ser Gln Ala Pro Asn Lys Asp Pro Thr Ala Ala Ala Ala Ala Leu

180 185 190

Asn Gly Gly His Cys Leu Ala Gln Pro Ala Ala Glu Pro Gly Leu Gly

195 200 205

Ala Val Val Arg Ser Ile Lys Val Ser Gly Tyr Leu Asn Leu Leu Ala

210 215 220

Asn Thr Ile Asp Asn Phe Thr His Gly Leu Ala Val Ala Ala Ser Phe

225 230 235 240

Leu Val Ser Lys Lys Ile Gly Leu Leu Thr Thr Met Ala Ile Leu Leu

245 250 255

His Glu Ile Pro His Glu Val Gly Asp Phe Ala Ile Leu Leu Arg Ala

260 265 270

Gly Phe Asp Arg Trp Ser Ala Ala Lys Leu Gln Leu Ser Thr Ala Leu

275 280 285

Gly Gly Leu Leu Gly Ala Gly Phe Ala Ile Cys Thr Gln Ser Pro Lys

290 295 300

Gly Val Val Gly Cys Ser Pro Ala Ala Glu Glu Thr Ala Ala Trp Val

305 310 315 320

Leu Pro Phe Thr Ser Gly Gly Phe Leu Tyr Ile Ala Leu Val Asn Val

325 330 335

Leu Pro Asp Leu Leu Glu Glu Glu Asp Pro Trp Arg Ser Leu Gln Gln

340 345 350

Leu Leu Leu Leu Cys Ala Gly Ile Val Val Met Val Leu Phe Ser Leu

355 360 365

Phe Val Asp

370

<210> 6

<211> 698

<212> PRT

<213> Drosophila melanogaster (Drosophila melanogaster)

<400> 6

Met Arg Leu Ala Val Gly Ala Leu Leu Val Cys Ala Val Leu Gly Leu

1 5 10 15

Cys Leu Ala Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu

20 25 30

His Glu Ala Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser Val

35 40 45

Ile Pro Ser Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr

50 55 60

Leu Asp Cys Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr

65 70 75 80

Leu Asp Ala Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu

85 90 95

Lys Pro Val Val Ala Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr

100 105 110

Phe Tyr Tyr Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met

115 120 125

Asn Gln Leu Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser

130 135 140

Ala Gly Trp Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu

145 150 155 160

Pro Arg Lys Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser

165 170 175

Cys Ala Pro Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu

180 185 190

Cys Pro Gly Cys Gly Cys Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser

195 200 205

Gly Ala Phe Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala Phe Val

210 215 220

Lys His Ser Thr Ile Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp

225 230 235 240

Gln Tyr Glu Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu

245 250 255

Tyr Lys Asp Cys His Leu Ala Gln Val Pro Ser His Thr Val Val Ala

260 265 270

Arg Ser Ile Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln

275 280 285

Ala Gln Glu His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe

290 295 300

Ser Ser Pro His Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala His Gly

305 310 315 320

Phe Leu Lys Val Pro Pro Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr

325 330 335

Glu Tyr Val Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu

340 345 350

Ala Pro Thr Asp Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His

355 360 365

His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys

370 375 380

Ile Glu Cys Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile

385 390 395 400

Met Asn Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val Tyr

405 410 415

Ile Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Asn

420 425 430

Lys Ser Asp Asn Cys Glu Asp Thr Pro Gly Ala Gly Tyr Phe Ala Val

435 440 445

Ala Val Val Lys Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys

450 455 460

Gly Lys Lys Ser Cys His Thr Ala Val Gly Arg Thr Ala Gly Trp Asn

465 470 475 480

Ile Pro Met Gly Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp

485 490 495

Glu Phe Phe Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser

500 505 510

Leu Cys Lys Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn

515 520 525

Asn Lys Glu Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val

530 535 540

Glu Lys Gly Asp Val Ala Phe Val Lys His Gln Thr Val Pro Gln Asn

545 550 555 560

Thr Gly Gly Lys Asn Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys

565 570 575

Asp Tyr Glu Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu

580 585 590

Tyr Ala Asn Cys His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr

595 600 605

Arg Lys Asp Lys Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln

610 615 620

His Leu Phe Gly Ser Asn Val Thr Asp Cys Ser Gly Asn Phe Cys Leu

625 630 635 640

Phe Arg Ser Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys

645 650 655

Leu Ala Lys Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu

660 665 670

Glu Tyr Val Lys Ala Val Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser

675 680 685

Leu Leu Glu Ala Cys Thr Phe Arg Arg Pro

690 695

<210> 7

<211> 1116

<212> DNA

<213> Drosophila melanogaster (Drosophila melanogaster)

<400> 7

atgcctggat gtccctgccc tggctgtggc atggcgggcc caaggctcct cttcctcact 60

gcccttgccc tggagctctt ggaaagggct gggggttccc agccggccct ccggagccgg 120

gggactgcga cggcctgtcg cctggacaac aaggaaagcg agtcctgggg ggctctgctg 180

agcggagagc ggctggacac ctggatctgc tccctcctgg gttccctcat ggtggggctc 240

agtggggtct tcccgttgct tgtcattccc ctagagatgg ggaccatgct gcgctcagaa 300

gctggggcct ggcgcctgaa gcagctgctc agcttcgccc tggggggact cttgggcaat 360

gtgtttctgc atctgctgcc cgaagcctgg gcctacacgt gcagcgccag ccctggtggt 420

gaggggcaga gcctgcagca gcagcaacag ctggggctgt gggtcattgc tggcatcctg 480

accttcctgg cgttggagaa gatgttcctg gacagcaagg aggaggggac cagccaggcc 540

cccaacaaag accccactgc tgctgccgcc gcgctcaatg gaggccactg tctggcccag 600

ccggctgcag agcccggcct cggtgccgtg gtccggagca tcaaagtcag cggctacctc 660

aacctgctgg ccaacaccat cgataacttc acccacgggc tggctgtggc tgccagcttc 720

cttgtgagca agaagatcgg gctcctgaca accatggcca tcctcctgca tgagatcccc 780

catgaggtgg gcgactttgc catcctgctc cgggccggct ttgaccgatg gagcgcagcc 840

aagctgcaac tctcaacagc gctggggggc ctactgggcg ctggcttcgc catctgtacc 900

cagtccccca agggagtagt tgggtgttct cccgctgcag aggagacggc agcctgggtc 960

ctgcccttca cctctggcgg ctttctctac atcgccttgg tgaacgtgct ccctgacctc 1020

ttggaagaag aggacccgtg gcgctccctg cagcagctgc ttctgctctg tgcgggcatc 1080

gtggtaatgg tgctgttctc gctcttcgtg gattaa 1116

<210> 8

<211> 2097

<212> DNA

<213> Drosophila melanogaster (Drosophila melanogaster)

<400> 8

atgaggctcg ccgtgggagc cctgctggtc tgcgccgtcc tggggctgtg tctggctgtc 60

cctgataaaa ctgtgagatg gtgtgcagtg tcggagcatg aggccactaa gtgccagagt 120

ttccgcgacc atatgaaaag cgtcattcca tccgatggtc ccagtgttgc ttgtgtgaag 180

aaagcctcct accttgattg catcagggcc attgcggcaa acgaagcgga tgctgtgaca 240

ctggatgcag gtttggtgta tgatgcttac ctggctccca ataacctgaa gcctgtggtg 300

gcagagttct atgggtcaaa agaggatcca cagactttct attatgctgt tgctgtggtg 360

aagaaggata gtggcttcca gatgaaccag cttcgaggca agaagtcctg ccacacgggt 420

ctaggcaggt ccgctgggtg gaacatcccc ataggcttac tttactgtga cttacctgag 480

ccacgtaaac ctcttgagaa agcagtggcc aatttcttct cgggcagctg tgccccttgt 540

gcggatggga cggacttccc ccagctgtgt caactgtgtc cagggtgtgg ctgctccacc 600

cttaaccaat acttcggcta ctcgggagcc ttcaagtgtc tgaaggatgg tgctggggat 660

gtggcctttg tcaagcactc gactatattt gagaacttgg caaacaaggc tgacagggac 720

cagtatgagc tgctttgcct ggacaacacc cggaagccgg tagatgaata caaggactgc 780

cacttggccc aggtcccttc tcataccgtc gtggcccgaa gtatgggcgg caaggaggac 840

ttgatctggg agcttctcaa ccaggcccag gaacattttg gcaaagacaa atcaaaagaa 900

ttccaactat tcagctctcc tcatgggaag gacctgctgt ttaaggactc tgcccacggg 960

tttttaaaag tcccccccag gatggatgcc aagatgtacc tgggctatga gtatgtcact 1020

gccatccgga atctacggga aggcacatgc ccagaagccc caacagatga atgcaagcct 1080

gtgaagtggt gtgcgctgag ccaccacgag aggctcaagt gtgatgagtg gagtgttaac 1140

agtgtaggga aaatagagtg tgtatcagca gagaccaccg aagactgcat cgccaagatc 1200

atgaatggag aagctgatgc catgagcttg gatggagggt ttgtctacat agcgggcaag 1260

tgtggtctgg tgcctgtctt ggcagaaaac tacaataaga gcgataattg tgaggataca 1320

ccagaggcag ggtattttgc tatagcagtg gtgaagaaat cagcttctga cctcacctgg 1380

gacaatctga aaggcaagaa gtcctgccat acggcagttg gcagaaccgc tggctggaac 1440

atccccatgg gcctgctcta caataagatc aaccactgca gatttgatga atttttcagt 1500

gaaggttgtg cccctgggtc taagaaagac tccagtctct gtaagctgtg tatgggctca 1560

ggcctaaacc tgtgtgaacc caacaacaaa gagggatact acggctacac aggcgctttc 1620

aggtgtctgg ttgagaaggg agatgtggcc tttgtgaaac accagactgt cccacagaac 1680

actgggggaa aaaaccctga tccatgggct aagaatctga atgaaaaaga ctatgagttg 1740

ctgtgccttg atggtaccag gaaacctgtg gaggagtatg cgaactgcca cctggccaga 1800

gccccgaatc acgctgtggt cacacggaaa gataaggaag cttgcgtcca caagatatta 1860

cgtcaacagc agcacctatt tggaagcaac gtaactgact gctcgggcaa cttttgtttg 1920

ttccggtcgg aaaccaagga ccttctgttc agagatgaca cagtatgttt ggccaaactt 1980

catgacagaa acacatatga aaaatactta ggagaagaat atgtcaaggc tgttggtaac 2040

ctgagaaaat gctccacctc atcactcctg gaagcctgca ctttccgtag accttaa 2097

Claims (2)

1. The application of the substance for improving the expression of Mitoferrin1 and the vector for inhibiting the expression of Transferrin in the preparation of the drug for treating Parkinson's disease is characterized in that the drug improves the motor capacity of Parkinson's disease patients, improves muscle injury, and slows down or inhibits mitochondrial dysfunction.

2. The application of the substance for improving the expression of Mitoferrin1 and the vector for improving the expression of ZIP13 in preparing the medicine for treating Parkinson's disease is characterized in that the medicine improves the motor ability of a patient with Parkinson's disease, improves muscle injury, and slows down or inhibits mitochondrial dysfunction.

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