KR102040893B1 - Use of ARL6IP1 for treatment of Hereditary Spastic Paraplegia - Google Patents

Abstract

The present invention comprises a protein of ARL6IP1 (ADP ribosylation factor like GTPase 6 interacting protein 1) or a gene encoding the same as an active ingredient, the pharmaceutical composition for treatment of Hereditary Spastic Paraplegia (HSP) and the gene The present invention provides a method for screening a therapeutic agent for hereditary tonic paraplegia by measuring expression levels.

Description

Use of ARL6IP1 for treatment of Hereditary Spastic Paraplegia}

The present invention comprises a protein of ARL6IP1 (ADP ribosylation factor like GTPase 6 interacting protein 1) or a gene encoding the same as an active ingredient, a therapeutic pharmaceutical composition of Hereditary Spastic Paraplegia (HSP) and the gene Method for Screening the Therapeutic Agents for Hereditary Stiff Paraplegia by Measuring Expression Levels will be.

Hereditary spastic paraplegia (HSP) is a hereditary nervous system disorder in which the muscles of the legs gradually weaken and become paralyzed, muscle tension increases and stiffness occurs. More than 70 genes associated with hereditary stiff paraplegia (SPG) are known, and dozens of related mutants have been reported, but the pathogenesis is not clear (Lo Giudice et al, Experimental neurology 261: 518). ˜539). The symptoms of hereditary tonic paraplegia are presumed to be due to the loss of function caused by mutations in the SPG genes leading to gradual degeneration of the spinal cord into the cortical spinal cord. The genetic pattern of hereditary stiff paraplegia is classified into autosomal dominant (AD type), autosomal recessive (AR type) or X-chromosome liked type.

Recently, a variety of genetic mutations have been found in families with AR type (recessive) hereditary tonic paraplegia (Novarino G). et al, Science 343: 506-511, 2014). However, although various genes are known to have their expression levels and / or mutations in patients with hereditary stiff paraplegia, such genes have not been studied as a material for treating diseases of hereditary stiff paraplegia.

There is no fundamental treatment for hereditary stiff paraplegia, and symptomatic therapy to help patients with muscle stiffness is an alternative. In the treatment method using the gene, the trait of the mutation is dominant in the AD type, so it is difficult to expect a therapeutic effect even if it is replaced with the wild type gene, but in the case of the AR type, the therapeutic effect can be expected by delivering the wild type gene into the body. have. Accordingly, there is a need for research on new mechanisms or fundamental therapeutic agents that can treat hereditary stiff paraplegia.

Based on this background, the present inventors have made efforts to develop a gene therapy agent for hereditary stiff paraplegia, and as a result of the functional study of ARL6IP1 in neurons, confirming a new mechanism by which neuronal survival and death are regulated by overexpression of ARL6IP1. In the case of a material targeting this, it was confirmed that the therapeutic effect on hereditary stiff paraplegia was completed, thereby completing the present invention.

One object of the present invention is a pharmaceutical composition for the treatment of Hereditary Spastic Paraplegia (HSP), which comprises a protein of ARL6IP1 (ADP ribosylation factor like GTPase 6 interacting protein 1) or a gene encoding the same as an active ingredient. To provide.

It is another object of the present invention to provide a method for treating hereditary ankylosing paraplegia, comprising administering the pharmaceutical composition to a suspected hereditary spastic paraplegia (HSP).

Another object of the present invention is to: (a) treating a candidate substance for the treatment of hereditary spastic paraplegia with a hereditary spontaneous paraplegia (HSP) induced neuronal cell; And (b) measuring the protein level of ARL6IP1 (ADP ribosylation factor like GTPase 6 interacting protein 1) or its activity in the neurons treated with the candidate substance, providing a method of screening for a treatment for hereditary tonic paraplegia will be.

To achieve the above object, one embodiment of the present invention includes a protein of ARL6IP1 (ADP ribosylation factor like GTPase 6 interacting protein 1) or a gene encoding the same as an active ingredient, Hereditary Spastic Paraplegia (HSP) It provides a pharmaceutical composition for the treatment of).

Specifically, in the present invention, the treatment restores the function of ARL6IP1 in neuronal cells in which ARL6IP1 dysfunction has occurred (1) a decrease in protein expression of p53, (2) an increase in protein expression of MDM2, (3) LC3B-II It may be a pharmaceutical composition, which is carried out by any one of an increase in protein expression and (4) an increase in the formation of ATG5-ATG12 conjugate, but is not particularly limited thereto.

"ARP6IP1 (ADP ribosylation factor like GTPase 6 interacting protein 1)" in the present invention belongs to the ARL6IP family and encodes a transmembrane protein mainly in the cytoplasmic membrane. For the purposes of the present invention, a new mechanism of regulating the survival and death of neurons according to the expression of ARL6IP1 in neurons was identified.

ARL6IP1 is called AIP1, apoptotic regulator in the membrane of the endoplasmic reticulum (ARMER), SPG61, and so on. In addition, in the present invention, the ARL6IP1 may be used in the same sense as the ARMER, and they may be used interchangeably. Genetic information thereof may be obtained from a known database. Examples thereof include GenBank of NCBI, but are not limited thereto.

The ARL6IP1 protein is about 23.2 kDa and encoded by the gene of ARL6IP1. The ARL6IP1 protein of the present invention or the gene encoding the ARL6IP1 protein derived from various mammals including humans has a function of inhibiting p53, promoting MDM2 activity, promoting LC3B-II activity by binding, and forming ATG5-ATG12 conjugates. The concept includes a gene encoding the same or a homologous protein functionally equivalent thereto.

The ARL6IP1 protein of the present invention is not only a protein having its wild-type amino acid sequence, but also its homologous protein is also included in the scope of the present invention. The ARL6IP1 protein or homolog thereof can be extracted from nature or prepared by genetic recombination methods based on synthetic or DNA sequences. In addition, the gene encoding the ARL6IP1 protein in the present invention means a nucleic acid molecule encoding the ARL6IP1 protein, these may be isolated or artificially modified in nature.

On the other hand, in the pharmaceutical composition of the present invention, when using the protein as an active ingredient, the pharmaceutical composition may include the protein itself as an active ingredient, it is possible to more easily express ARL6IP1 or its homologous protein in the body of the individual. Thus, the nucleic acid encoding the protein may be included in the pharmaceutical composition in the form contained in the expression vector for gene therapy. In this case, the expression vector is not particularly limited, but may be a vector capable of replicating and / or expressing the protein in mammalian cells (eg, human, monkey, rabbit, rat, hamster, mouse cells, etc.). In particular, the protein may be operably linked to an appropriate promoter to express the protein in a host cell, and may be a vector including at least one selection marker, and more specifically, phage, plasmid, cosmid, mini- Nucleic acid encoding the protein may be introduced into a chromosome, an adenovirus vector, an adenovirus vector, a baculovirus vector, a retrovirus vector, or the like.

The term "gene therapy" of the present invention, in order to treat a variety of genetic diseases caused by gene abnormalities, the gene associated with the disease is directly injected into the patient's body and the gene is expressed in cells To normalize its function.

The expression vector according to the present invention can be introduced into cells using methods known in the art. For example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns and other known methods for introducing nucleic acids into cells It can be introduced into the cell by the method of.

In the present invention, it was confirmed that increasing the expression of ARL6IP1 in neurons can inhibit cell death. Therefore, the material overexpressing ARL6IP1 of the present invention can be used as a pharmaceutical composition for treating hereditary tonic paraplegia.

According to one embodiment of the present invention, ATG6-ATG12 which regulates the autophagy signal of neurons by reducing the apoptosis by inhibiting the expression of p53 in neurons by ARL6IP1 overexpression (Figs. 4c and 4d). An increase in conjugate and LC3B-II was observed (FIG. 6). The present inventors have developed the present invention by judging that ARL6IP1 can be overexpressed to treat hereditary tonic paraplegia. This corresponds to what is not known in the art of conventional hereditary stiff paraplegia.

The term "p53" of the present invention is a protein associated with apoptosis and is encoded by the TP53 gene. More specifically, at the end of the G1 phase, proteins produced by the p53 gene are examined for damage to the cell's DNA. If the DNA is healthy, p53 will allow cell division to progress, and if damaged DNA is found, it will induce other proteins to activate and repair the DNA. If DNA damage is too fatal to repair, the p53 protein causes cells to destroy themselves. This is called apoptosis. Apoptosis prevents the mutated cells from continuing to grow. Other healthy cells promote cell division to replace damaged cells.

When ARL6IP1 is overexpressed in the neuronal cells of the present invention, the expression of p53 is reduced. Thus, substances that increase the protein expression level of ARL6IP1 of the present invention can be used as a therapeutic agent for hereditary tonic paraplegia.

According to one embodiment of the present invention, when confirming the expression level of p53 protein that controls apoptosis in order to confirm the reason why apoptosis is suppressed when overexpression of ARL6IP1, it was confirmed that p53 protein is reduced by overexpression of ARL6IP1 ( 4d). This suggests that overexpression of ARL6IP1 may be a target for the treatment of hereditary tonic paraplegia and development of new therapeutic agents.

As used herein, the term "MDM2" refers to a protein obtained as a result of expression of the mdm2 gene. Within the meaning of this term, MDM2 includes all proteins encoded by mdm2, variants thereof, alternative fragment proteins thereof, and phosphorylated proteins thereof. In addition, “MDM2” includes MDM2 analogs, such as MDMX, also known as MDM4, and analogs of MDM2 homologs and other animals, eg, human homologous HDM2 or human analog HDMX.

MDM2 is the key negative regulator of the p53 function. It binds to the amino terminal transcriptional activation region of p53 to form a negative autoregulatory loop, thus MDM2 inhibits p53's ability to activate transcription and p53 is for protein degradation purposes.

In the present invention, the MDM2 protein is an E3 ubiquitin ligase, which is used to reduce proteome-dependent degradation by inducing proteasome dependent degradation by conjugating a multimeric ubiquitin to the substrate protein p53.

According to one embodiment of the present invention, it was confirmed by the in vivo MDM2 ubiquitination assay (Fig. 5d) whether the enzyme activity of the MDM2 increased by the increase in the expression of ARL6IP1 in the cell at the time of overexpression of ARL6IP1. In the case of overexpression of ARL6IP1 in neurons, it was confirmed that the expression of p53 was decreased by increasing the enzyme activity of MDM2. This suggests that overexpression of ARL6IP1 may be a target for the development of therapeutic agents for hereditary tonic paraplegia.

As used herein, the term "LC3B-II" is an active form of LC3B (light chain 3β) as a protein involved in autophagy. LC3B protein is a protein encoded by the LC3B gene. LC3 is an important protein in the progeny pathway and is widely used as a marker for autophagosomes. Child care in neurons is important for the turn-over of intracellular organelles and plays an important part in survival.

LC3-II is a lipid modified form of LC3, involved in the expansion and fusion of autophagosome membranes. For the purposes of the present invention, overexpression of ARL6IP1 activates progeny resulting in an increase in LC3B-II. This means that the intracellular activity is activated. Therefore, a substance that increases the mRNA or protein activity of ARL6IP1 of the present invention can be used as an agent for the treatment of hereditary tonic paraplegia.

As used herein, the term "ATG5" is a protein encoded by the human ATG5 gene as a progeny protein. It is also an E3 ubiquitin ligase required for offspring acting as an autophagosome extension. It is activated by ATG7 and forms a conjugate with ATG12.

As used herein, the term "ATG12" is a protein encoded by the human ATG12 gene as a progeny related protein. Progeny is the process of mass proteolysis, including cellular organelles, cellular components, and double membrane structures called autophagosomes. Progeny requires covalent binding of protein ATG12 to ATG5 via a ubiquitin-like conjugation system. ATG5-ATG12 conjugates promote the binding of ATG8 to lipid phosphatidyl. The progeny of the cell proceeds by sequential interactivation of ATG proteins, and finally the progeny is completed by formation of the endoplasmic reticulum by LC3-II.

For the purposes of the present invention, the overexpression of ARL6IP1 activates the progeny resulting in an increase in the ATG5-ATG12 conjugate. Therefore, the substance which increases the mRNA of ARL6IP1 or its protein activity of this invention can be used as a therapeutic agent for hereditary tonic paraplegia.

According to one embodiment of the present invention, when confirming the expression level of p53 protein that controls apoptosis in order to confirm the reason why apoptosis is suppressed when overexpression of ARL6IP1, it was confirmed that p53 protein is reduced by overexpression of ARL6IP1 ( 4d).

Through this, it can be seen that the overexpression of ARL6IP1 can be screened for therapeutic agents by measuring the decrease in p53 protein or the increase in Mdm2 activity.

According to one embodiment of the present invention, it was confirmed whether ARL6IP1 can regulate the autophagy signal of neurons. As a result, the overexpression of ARL6IP1 increased the ATG5-ATG12 conjugate and increased the expression of LC3B-II (FIG. 6).

Through this, the overexpression of ARL6IP1 of the present invention, the increase in the protein expression of LC3B-II or the formation of ATG5-ATG12 conjugate was measured, it can be seen that the agent for the treatment of hereditary tonic paraplegia can be screened.

"Hereditary Spastic Paraplegia (HSP)" in the present invention is a hereditary nervous system disease in which the muscles of the legs gradually weaken and become paralyzed, muscle tension increases, and stiffness appears. Hereditary stiff paraplegia is divided into simple hereditary stiff paraplegia (Uncomplicated HSP) and complex hereditary stiff paraplegia (Complicated HSP). Simple hereditary stiff paraplegia may cause progressive stiff paraplegia with initial findings, muscle stiffness, and difficulty controlling bladder, but complex hereditary stiff paraplegia may show additional neurological abnormalities. , Hearing impairment, mental retardation, voluntary impairment of motor control (ataxia), and the like may appear, but is not limited thereto.

As used herein, the term "treatment" refers to any action in which the symptoms of hereditary tonic paraplegia are improved or beneficially altered upon administration of the pharmaceutical composition.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients or diluents commonly used in the manufacture of pharmaceutical compositions. Such carriers, excipients or diluents may be unnaturally occurring.

Specifically, the pharmaceutical compositions are formulated in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as solutions, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, respectively, according to conventional methods. Can be used. In the present invention, carriers, excipients and diluents that can be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose, and the like in the pharmaceutical composition. Or it is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are simple diluents commonly used in suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, twin (twin) 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The content of the ARL6IP1 protein or a gene encoding the same contained in the pharmaceutical composition of the present invention is not limited as long as the pharmaceutical composition has a therapeutic effect on hereditary stiff paraplegia, but is 0.0001 to 99.9 weight based on the total weight of the final composition. %, More specifically, may be included in an amount of 0.01 to 80% by weight.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective dose level is the severity of the disease, the activity of the drug, the age, weight, health, sex of the patient, the sensitivity to the drug of the patient, the time of administration of the composition of the invention used, the route of administration and the rate of release, the duration of treatment, the pattern used. And factors well known in the art and other ingredients, including drugs used in combination or coincidental with the compositions of the invention. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.

Another aspect of the invention provides a method of treating hereditary ankylosing paraplegia, comprising administering the pharmaceutical composition to a Hereditary Spastic Paraplegia (HSP) suspect subject.

The "pharmaceutical composition" "genetic stiff paraplegia" and "treatment" are as described above.

The term "individual" of the present invention refers to humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs that the disease may invent or develop. Means all animals, including. If the pharmaceutical composition of the present invention can be effectively treated by administering to the subject, the type of subject is included without limitation.

The term "administration" of the present invention means providing a predetermined substance to a subject by any suitable method, and the route of administration of the composition of the present invention may be administered via any general route as long as it can reach the target tissue. have. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. In addition, the pharmaceutical compositions of the present invention may be administered by any device in which the active substance may migrate to target cells. Specific modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, drip and the like.

In addition, the dosage of the compound according to the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and generally based on an adult patient having a weight of 70 kg. 0.01 mg to 5000 mg per day, depending on the judgment of the doctor or pharmacist may be divided into once or several times a day at regular intervals, but is not limited thereto.

Another embodiment of the present invention comprises the steps of: (a) treating a hereditary anterior paraplegia (HSP) -induced neuronal cells with hereditary anterior paraplegia; And (b) measuring the protein level of ARL6IP1 (ADP ribosylation factor like GTPase 6 interacting protein 1) or its activity in the candidate cells treated neurons. .

Specifically, the present invention (c) the protein level of ARL6IP1 measured in step (b); Or if its activity is increased relative to the untreated neurons of the candidate substance, the method may further comprise determining a treatment for hereditary tonic paraplegia.

Specifically, the present invention further comprises the steps of (b) measuring any one of (1) protein expression of p53, (2) protein expression of LC3B-II, and (3) formation of ATG5-ATG12 conjugates. Can be.

Specifically, the present invention provides a method for increasing the protein expression of ARL6IP1 to which the candidate substance measured in step (b) is not treated, (1) decreasing protein expression of p53, (2) increasing protein expression of LC3B-II, and (3) if any of the increased formation of ATG5-ATG12 conjugates, may further comprise the step of determining a treatment for hereditary tonic paraplegia.

"ARL6IP1", "p53", "LC3B-II", "ATG5-ATG12" and "genetic stiff paraplegia" are as described above.

As used herein, the term "test agent" includes any substance, molecule, element, compound, entity, or combination thereof. Examples include, but are not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, a synthetic compound or a combination of two or more substances.

As used herein, the term "expression level of ARL6IP1" is a concept including both the expression level of ARL6IP1 mRNA or the expression level of ARL6IP1 protein, and in the present invention, "measurement of expression level of ARL6IP1" is used to measure the expression level of the ARL6IP1 protein. Can be performed through.

In a specific embodiment of the present invention, to confirm the expression level of ARL6IP1, it was confirmed that ARL6IP1 expression was increased during differentiation in ReNcell neuronal cell lines (FIG. 8 b), and that apoptosis was decreased as ARL6IP1 expression was increased. (FIG. 4C).

In a specific embodiment of the present invention, cell death and neuronal differentiation induced by AR-type ARL6IP1 knock-down condition of hereditary stiff limb paralysis are inhibited by neuronal cell death and neuronal differentiation when ARL6IP1 overexpression is induced. Induced was confirmed (Fig. 9, 10).

In a specific embodiment of the present invention, it was confirmed that the lower extremity stiff paralysis appears in the mouse knocked down the ARL6IP1 gene (FIG. 11).

Through this, it was found that the gene can be targeted as an agent for inhibiting apoptosis by using the overexpression of ARL6IP1 of the present invention or as a gene therapy material for hereditary anterior paraplegic disease.

As used herein, the term "measurement of protein expression level" refers to a process of determining the expression level of ARL6IP1, which affects collagen accumulation in hereditary tonic paraplegic disease. Specifically, the antibody or aptamer specifically binds to a protein expressed from the ARL6IP1 gene, but is not limited thereto. If such antibodies are polyclonal antibodies, monoclonal antibodies or antigen binding, fragments of these antibodies are also included in the antibodies of the invention. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies, human antibodies and the like, and may include antibodies already known in the art in addition to novel antibodies. Such antibodies include functional fragments of antibody molecules, as well as complete forms having the full length of two heavy and two light chains, as long as they have the property of binding to specifically recognize proteins expressed from the ARL6IP1 gene. The functional fragment of the molecule of the antibody means at least a fragment having an antigen binding function, and includes, but is not limited to, Fab, F (ab '), F (ab') ₂ and Fv.

For the purpose of the present invention, the protein expression level is measured by protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, Surface Enhanced Laser Desorption / SELDI-TOF Ionization Time of Flight Mass Spectrometry, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid phase chromatography-mass spectrometry liquid chromatography-mass spectrometry (LCMS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), western blot, and enzyme linked immunosorbentassay (ELISA), but are not limited thereto.

According to the present invention, a protein containing ARL6IP1 (ADP ribosylation factor like GTPase 6 interacting protein 1) or a gene encoding the same as an active ingredient can be used as a pharmaceutical composition for treatment of Hereditary Spastic Paraplegia (HSP). In addition, it can be used for screening for the treatment of hereditary tonic paraplegia by measuring the activity of the ARL6IP1.

Figure 1a shows the result of confirming the nucleotide sequence by preparing an AR type ARL6IP1 mutant plasmid.
FIG. 1B shows the expression level of mRNA between ARL6IP1 mutant plasmid (HSP-MT) and wild type (WT) prepared in FIG. 1A using HEK293 cell line, by RT-PCR.
Figure 1c is confirmed by Western blot protein expression in the conditions of Figure 1b.
Figure 2a is a result of observing the cell phenotype through a microscope three days after treatment with neuronal differentiation conditions retinoic acid (retinoic acid, RA) after the knock-down of the ARL6IP1 gene using adenovirus in the ReNcell neuronal cell line.
FIG. 2B is a result of confirming Western blot protein expression levels of ARL6IP1 and neuronal differentiation marker MAP2 at each time point after retinoic acid treatment, which is a neuronal differentiation condition under the conditions of FIG. 2A.
Figure 2c is a result of confirming the distribution of dead cells using flow cytometry after staining cells with the apoptosis marker Annexin V after harvesting cells 3 days retinoic acid treatment in the conditions of Figure 2a.
Figure 3a is a result of fluorescence staining using calnexin ARL6IP1 and ER membrane markers in the U2OS cell line.
Figure 3b is a result showing the intracellular organelle position where ARL6IP1 is expressed in a ReNcell neuronal cell line ARL6IP1 expression after induction of ARL6IP1 overexpression using adenovirus vector at 2.5 to 25% iodixanol concentration gradient conditions.
Figure 4a is confirmed by Western blot protein expression of ER stress marker according to the overexpression of ARL6IP1 and whether tunicamycin (TM), which is an ER stress inducer.
Figure 4b is confirmed by RT-PCR mRNA expression of the target gene of ATF4, an ER stress marker and transcriptional regulator according to the overexpression of ARL6IP1 and the treatment of ER stress inducer tappsigagin (TG).
Figure 4c is treated with ER stress inducer after ARL6IP1 overexpression in the neuronal cells to investigate apoptosis according to the overexpression and TG treatment of ARL6IP1 and stained with Annexin V, an apoptosis marker, using a flow cytometer It is confirmed.
Figure 4d is confirmed by Western blot that the apoptosis pathway p53 and caspase activity is inhibited according to the expression level of ARL6IP1.
5a shows the mRNA expression level of p53 according to the expression level of ARL6IP1 by RT-PCR.
Figure 5b is confirmed by Western blot after treatment with proteasome inhibitor MG132 to confirm the p53 protein regulatory pathway according to the expression level of ARL6IP1.
Figure 5c is confirmed by Western blot after in vitro MDM2-ubiquitination analysis to confirm whether the protein regulatory pathway of p53 by ARL6IP1 is due to the enzymatic activity of MDM2, the E3 ubiquitin ligase of p53.
Figure 5d is confirmed by Western blot after performing in vivo MDM2-ubiquitination assay in HEK293 cell line to confirm the change in MDM2 enzyme activity in the cell according to the expression of ARL6IP1.
Figure 6a is confirmed by Western blot the effect of overexpression of ARL6IP1 on the regulation of autophagy, one of the important signals that control the life of neurons.
Figure 6b is confirmed by Western blot protein binding between LC3B and ARL6IP1 to form an autophagosome (autophagosome).
Figure 6c is confirmed by Western blot after pull-down assay whether the protein binding of the LC3 family and ARL6IP1 similar to LC3B.
Figure 6d is a result of observing the formation of autophagosome (autophagosome) after overexpression of ARL6IP1 using HeLa-GFP-LC3B cell line to continuously express GFP-LC3B through fluorescence microscopy.
FIG. 6E is a result of quantitative and graphical representation of fluorescence expression of autophagosome formation of FIG. 6D using imageJ software.
Figure 7a is a result of observing the formation of autophagocyte after ARL6IP1 gene knock-down using adenovirus in HeLa-GFP-LC3B cell line through a fluorescence microscope.
FIG. 7B is a result of quantitative and graphical representation of fluorescence expression of autophagosome formation of FIG. 7A using imageJ software.
Figure 7c shows the results of Western blot analysis of gene regulation 48 hours after gene knock-down of ARL6IP1 using adenovirus in HeLa-GFP-LC3B cell line.
Figure 8a is a result of observing the phenotype of the cell line under a microscope for 10 days under neuronal differentiation conditions using ReNcell neuron line.
Figure 8b is the result of confirming the expression change of the protein under the conditions of Figure 8a by Western blot.
Figure 8c shows the expression level of ARL6IP1, calnexin (marker of ER vesicles), LC3B at 2.5% to 25% iodixanol concentration gradient after cells were treated with retinoic acid, a neuronal differentiation condition, in ReNcell neuronal cell line for 3 days. The expression position was confirmed by the Western blot.
Figure 9a shows that adenovirus Ad-sh-ARL6IP1 with 200 MOI titer in SH-SY5Y neuronal cell line and knocked down the ARL6IP1 gene, and then treated with adenovirus Ad-ARL6IP1 with 100 MOI titer to induce overexpression. Given the differentiation conditions, the phenotype of the cells was observed under a microscope.
Figure 9b is a result confirmed by Western blot whether the expression control of the gene in the condition of Figure 9a.
Figure 9c is a result of confirming the distribution of killed cells through a flow cytometer after staining with Annexin V, apoptosis marker in the conditions of Figure 9a.
10A shows the results of observing dendrite arborization of neurons by fluorescence microscopy to induce overexpression of ARL6IP1 after ARL6IP1 knock-down using let hippocampal neuronal cells.
FIG. 10B is a result obtained by counting primary dendrites of neurons under the conditions of FIG.
FIG. 10C is a result of counting secondary dendrites of neurons under the conditions of FIG.
11A is a schematic diagram of a method for producing ARL6IP1 knock-out (KO) mouse.
Figure 11b is the result of confirming the mRNA expression by RT-PCR compared to the normal tissue in the brain tissue of the constructed ARL6IP1 KO mouse.
Figure 11c is the result of confirming the protein expression in Western blot compared to the normal tissue in the brain tissue of the constructed ARL6IP1 KO mouse.
FIG. 11D is a photographic result of comparing ARL6IP1 KO mice with normal mice to identify lower extremity stiff paralysis, an HSP disease phenotype.
Figure 11e is the result of confirming the abduction angle of the foot between normal mice and ARL6IP1 KO mice to confirm the HSP disease phenotype.
Figure 11f is a quantitative graph showing the degree of abduction angle of the foot compared to the normal mouse.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example  One: ARL6IP1  Confirmation of Mutation of Protein Expression in Mutants

Hereditary stiff paraplegia is known to occur in autosomal dominant (AD type) and chromosomal recessive (AR type) diseases. Recently, a variety of genetic mutations have been found in families with AR type (recessive) hereditary tonic paraplegia (Novarino G). et al, Science 343: 506-511, 2014). Based on the known literature, AR-type ARL6IP1 mutant recombinant plasmids were prepared and compared with wild-type to determine whether the mutations were dysfunctional and possible as a gene therapy material.

As a result of sequencing by constructing the ARL6IP1-HSP-MT recombinant plasmid in the pFlag-CMV2 vector, the mutant was deleted from four bases at positions 576-579 of chromosome 16, resulting in a frameshift mutation at amino acid sequence lysine 193. mutation) occurred and it was confirmed that 36 amino acids were added and expressed compared to the wild type (FIG. 1A).

In order to compare the expression of mRNA and protein at the AR-type ARL6IP1 mutant recombinant plasmid of the AR type, wild-type Flag tagged ARL6IP1 (WT) and HSP mutant (HSP-MT) plasmids 5, 15 Cells were harvested by incubation for 24 hours after ug transduction. Total RNA from harvested cells was isolated and purified (easy-spin ™ (DNA free) Total RNA Extraction Kit, Intron), and mRNA was synthesized with cDNA (SuperScript® III Reverse Transcriptase, Invitrogen) and identical using β-actin primers. PCR confirmed whether positive cDNA was synthesized. Thereafter, PCR was performed by constructing the ARL6IP1 gene specific sequence with a primer (Table 1) (FIG. 1B).

In addition, the cells were harvested under the same conditions as in FIG. 1B to confirm protein levels, followed by lysis buffer (RIPA, 50 mM Tris-cl, pH8.0, 150 mM NaCl, 0.1% NP-40, 0.1% SDS, 0.5% sodium). deoxycholate) was dissolved and centrifuged (13000 rpm, 10 min, 4 ° C.) to obtain a protein sample. Thereafter, 12% SDS-PAGE gels were used to separate the proteins according to mass differences and transfer them to PVDF membranes. The membrane was reacted with blocking buffer (1 × PBS, 0.05% Tween-20, 5% skim milk) at room temperature for 1 hour and washed with PBST. To confirm the expression change of the protein, ARL6IP1 (1: 3000) and β-actin (1: 5000, sigma-aldrich) were treated with PBST as a primary antibody and then treated at 4 ° C. for 12 hours. The ARL6IP1 antibody was directly prepared through mouse immunization using a protein isolated and purified from Escherichia coli . After the first antibody treatment, washed with PBST and then HRP (horseradish peroxidase) bound secondary antibody (1: 10000, santa-cruz) was diluted with PBST and reacted at room temperature for 1 hour, and the unbound antibody was washed. . After treatment with luminol (Luminol) was confirmed through the film development through the color development (Fig. 1c).

As a result, it was confirmed that the expression of the AR type ARL6IP1 mutation was inhibited at the translation stage of the gene when compared with the wild type.

Example  2: Of ARL6IP1  Inhibiting expression sh - ARL6IP1 is  Induces death of nerve cells

Preparation of sh-ARL6IP1 to determine the effect on neuronal differentiation when inducing knock-down of the ARL6IP1 gene in neurons is a well-known method ( Fengjie 　 Guo et al. Mol Biol Rep 37: 3819-3825, 2010) and homologates the plasmid containing the adenovirus genome and the plasmid cloned with sh-ARL6IP1 at the pSuper vector BglII / HindIII site for knock-down of ARL6IP1. Adenovirus vectors were produced by recombination. The produced adenovirus vector was transfected into the HEK293 cell line to produce and concentrate the adenovirus. Adenoviruses were produced to improve the low transduction rate of neurons. Adenovirus (Ad-sh-ARL6IP1) prepared in ReNcell neuron lines was treated with 100 and 200 MOI titers for 24 hours in neuronal differentiation medium (DMEM / F12, 1% FBS, penicillin, streptomycin and 10 uM retinoic acid). Phenotype of differentiated neurons by culturing for 3 days was observed under a microscope (FIG. 2A).

ReNcell neuronal cell lines were treated with adenoviruses (Ad-sh-ARL6IP1, Ad-sh-Ctrl) at 100 MOI titers for 24 hours and then treated with retinoic acid, a neuronal differentiation condition, to induce neuronal differentiation. Was confirmed by Western blot.

Protein samples collected at each time point were separated using 12% SDS-PAGE gel and treated with ARL6IP1, a neuronal differentiation marker MAP2 antibody as primary antibody, at 4 ° C. for 12 hours. After the primary antibody treatment, after washing with PBST, the HRP labeled secondary antibody was treated at room temperature for 1 hour. Unbound secondary antibody was confirmed by washing and treating the luminol and film development through color development (Fig. 2b).

It was confirmed that knock-down of the ARL6IP1 gene inhibited the differentiation of neurons, and that adenoviruses (Ad-sh-ARL6IP1, Ad-sh-Ctrl) were reversed to 100 MOI to confirm cell death regulation under the same conditions. Cells treated with retinoic acid were harvested for 3 days after 24 hours transverse staining, stained with annexin V, an apoptosis marker, and confirmed the degree of apoptosis distribution using a flow cytometer.

As a result, it was confirmed that knock-down of ARL6IP1 gene induced 41.85% cell death compared to the control group (Ad-sh-Ctrl) (FIG. 2C).

Example 3: Confirmation of the expression of ARL6IP1 in the ER (ER)

To identify intracellular organelles where ARL6IP1 (ADP ribosylation factor like GTPase 6 interacting protein 1) is expressed, U2OS cell lines were placed in 6 well plates at 1 × 10 ⁵ cells / well. After seeding, the cells were incubated for 24 hours. Thereafter, the cells were fixed with 4% para-formaldehyde at room temperature for at least 12 hours, fixed with cells, washed with PBS and treated with 0.5% Triton X-100 for 5 minutes, followed by blocking buffer (1 x PBS, 0.05% Tween 20, 0.5% Triton X-100, 5% BSA) for 1 hour at room temperature. The primary antibody was diluted with ARL6IP1 (1: 1000) and the ER membrane marker calnexin (1: 1000, AbFrontier) antibody with PBST (1 x PBS, 0.05% Tween 20) and treated for 16 hours at 4 ° C. Remaining primary antibody was washed using PBST. For reading using a fluorescence microscope, ARL6IP1 used a secondary antibody (1: 500, sigma-aldrich) labeled with a rodamin fluorescent dye, and calnexin was used as a secondary antibody labeled with FITC fluorescent dye (1: 500, sigma-aldrich) and the secondary antibody remaining unbound using PBST after 1 hour at room temperature was washed. Using a dye labeled through a fluorescence microscope, the expression and location of specific proteins in cultured cells were confirmed (FIG. 3A).

In order to confirm the expression of ARL6IP1 in neurons and the location of intracellular organelles, the ARL6IP1 gene was cloned at the XhoI / HindIII position in the pShuttle-CMV vector, and adenovirus vectors were prepared by homologous recombination with Adeasy vectors. The produced adenovirus vector was transfected into the HEK293 cell line to produce and concentrate the adenovirus.

After treatment with 100 MOI titer using ARL6IP1 containing ARL6IP1 in ReNcell neuron line, cells were harvested and cultured for 24 hours after lysis buffer (0.25 M sucrose, 1 mM EDTA, 10 mM HEPES-NaOH, After the addition of pH7.4), the cells were crushed using a sonicator, and only the supernatant (lysate) was collected by centrifugation (5000 rpm, 5 min, 4 ° C.). Thereafter, 2.5 to 25% iodixanol concentration gradient was applied, and then the prepared supernatant was raised and centrifuged (35000 rpm, 3 h, 4 ° C.), and 500 ul of samples were collected from the tube top, and the western blot was collected. Confirmed.

Western blots were separated by protein mass differences using a 12% SDS-PAGE gel and then transferred to PVDF membranes (millipore), after which the membranes were blocked with buffer (1 x PBS, 0.05% Tween 20, 5% skim milk). After treatment for 1 hour at room temperature using PBST (1XPBS, 0.05% Tween 20) was washed. The primary antibody was diluted with PBST using ARL6IP1 (1: 3000) and calnexin (1: 3000) to react at 4 ° C for 16 hours. Each membrane was washed with PBST and HRP bound secondary antibody (1: 10000, santa-cruz) was diluted with PBST. After 1 hour reaction at room temperature, the unbound secondary antibody was washed. The degree of expression was confirmed using luminol (ECL solution, Intron) by the chemiluminescence method (Fig. 3b).

As a result of confirming the location of the intracellular organelles of ARL6IP1 through the two experiments, it was confirmed that the fractional samples of the ER membrane markers Kalnexin and ARL6IP1 were identical, and the expression of Kalnexin also increased when ARL6IP1 expression was increased by adenovirus. Confirmed. This suggests that ARL6IP1 is located in the endoplasmic reticulum (ER) of intracellular organelles.

Example  4: in neurons Of ARL6IP1  Overexpression inhibits apoptosis in ER stress-induced conditions

As described above, it was confirmed that ARL6IP1 is present in the ER membrane and plays an important function in the shape of the ER. To confirm the function in ER when ARL6IP1 is expressed as one of the ER membrane proteins, expression regulation of ER stress markers was confirmed after ER stress induction. Flag tagged ARL6IP1 plasmid (Flag-ARL6IP1) and transfection reagent (Promega) were mixed in the U2OS cell line to make DNA / liposome complexes, and then treated with cells and incubated for 24 hours. After 4 hours of treatment with ER stress inducer tunicamycin (tunicamycin, TM, sigma-aldrich) at a concentration of 10 μg / ml to determine the degree of protein expression of ER stress markers when ER stress was induced under ARL6IP1 Cells were harvested and centrifuged at 4 ° C. for 10 min using lysis buffer (RIPA, 50 mM Tris-cl, pH8.0, 150 mM NaCl, 0.1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate). (13000 rpm, 10 min, 4 ° C.) to prepare a total supernatant. It was then separated according to protein mass using a 12% SDS PAGE gel. After moving to the PVDF membrane, it was reacted for 1 hour at room temperature using a blocking buffer.

Flag (1: 5000, sigma-aldrich) and ARL6IP1 (1: 3000) antibodies were used to confirm overexpression of ARL6IP1, and ATF4 (1: 2000, cell signaling) and CHOP were used to confirm the expression of ER stress markers. (1: 2000, cell signaling), XBP-1 (1: 2000, cell signaling), and ATF6α (1: 2000, cell signaling) were used as primary antibodies and diluted for 12 hours at 4 ° C. after dilution with PSBT. HRP-bound secondary antibody (1: 10000, santa-cruz) using a luminol treatment after 1 hour reaction at room temperature to determine the expression through color development. As a result, ATF4-CHOP is known to activate apoptosis signal among ER stress markers, but overexpression of ARL6IP1 was confirmed to induce stronger XBP-1 and ATF6α signals as well as ATF4-CHOP signal (Fig. 4a), it induces the expression of chaperon, which is one of the target genes of XBP-1, ATF6α, etc., and it is confirmed that survival / growth rather than cell death is activated.

Among ER stress markers induced after overexpression of ARL6IP1, ATF4 is a transcriptional regulator and regulates the expression of various target genes. To determine whether ATF4 induced by ARL6IP1 can function as a transcriptional regulator, Flag-ARL6IP1 plasmid was used in the U2OS cell line after transfection, to induce ER stress inducer (thapsigargin, TG, sigma-aldrich). ) Was treated for 4 hours at a concentration of 10 uM to induce ER stress. Cells were then collected and mRNA was isolated (easy-spin ™ (DNA free) Total RNA Extraction Kits, Intron), and then the specific sequences of the target genes of each ATF4 were prepared as primers (Table 1) and confirmed by PCR. Various target genes of ATF4 were confirmed to be increased in the mRNA level (Fig. 4b). Primer sequences used in the experiments are shown in Table 1 below.

RT- PCR primer  order designation Primer Sequence SEQ ID NO: ARL6IP1
Forward direction 5’-GACTGCAAGTCTGGAAGAAC-3 ’ One Reverse 5’-GAACAAGGTAGTCAGCCAAG-3 ’ 2 ATF4
Forward direction 5’-CTGACCACGTTGGATGACAC-3 ’ 3 Reverse 5’-GGGCTCATACAGATGCCACT-3 ’ 4 E-selectin
Forward direction 5’-TGTTTCCAAAACCCTGGAAG-3 ’ 5 Reverse 5’-AACTGGGATTTGCTGTGTCC-3 ’ 6 ASNS
Forward direction 5’-CTCTGAGGAAGGCATTCAGG-3 ’ 7 Reverse 5’-CATTTCTGGTGGCAGAGACA-3 ’ 8 CHOP
Forward direction 5’-CTTGGCTGACTGAGGAGGAG-3 ’ 9 Reverse 5’-TCACCATTCGGTCAATCAGA-3 ’ 10 VEGF
Forward direction 5’-GGAGTACCCTGATGAGATCG-3 ’ 11 Reverse 5’-TGCAGGAACATTTACACGTC-3 ’ 12 p53
Forward direction 5’-CGTGTGGAGTATTTGGATGA-3 ’ 13 Reverse 5’-TTCTCCATCCAGTGGTTTCT-3 ’ 14 MAP2
Forward direction 5’-GAACCAGCAGAGATTCAGAG-3 ’ 15 Reverse 5’-GCTGCCTCTTCTACTTCAAA-3 ’ 16 β-actin
Forward direction 5’-AGGAGAAGCTGTGCTACGTC-3 ’ 17 Reverse 5’-CTCAGGAGGAGCAATGATCT-3 ’ 18

According to the expression level of these genes, it was confirmed whether overexpression of ARL6IP1 affects the growth and death of cells. In order to confirm the change in cell cycle due to ER stress induced after overexpression of ARL6IP1, Flag-tagged ARL6IP1 plasmid was incubated for 24 hours after transduction into Neuro-2a cell line.

Cells were collected and fixed with 70% ethanol at 4 ° C. for at least 12 hours, and then stained with annexin V, an apoptosis marker, to confirm apoptosis. The control group showed 75.88% cell death compared to ARL6IP1. When overexpressed, apoptosis was found to be markedly low at 45.06% even though ER stress was induced. It was found that overexpression of ARL6IP1 can inhibit apoptosis under stress conditions (FIG. 4C).

In order to identify the reason why apoptosis was inhibited when ER stress was induced after ARL6IP1 overexpression, ARL6IP1 overexpression was induced by incubation for 24 hours after transduction (5, 15ug) of a tagged TRL plasmid in U2OS cell line. Cells were harvested after 4 hours treatment of ER stress inducer tunicamycin (TM) at a concentration of 10 ug / ml in the ARL6IP1 overexpressed cell line. Protein samples were prepared using RIPA lysis buffer, and proteins were separated by mass with 12% SDS-PAGE gel. Apoptosis pathways p53 (1: 5000, santa-cruz), BAX (1: 3000, santa-cruz), caspase-9 (1: 2000, cell signaling), caspase-3 (1: 2000, cell signaling ) And cleavage caspase-3 (1: 2000, cell signaling) were used as primary antibodies for 12 hours at 4 ° C. The HRP-bound secondary antibody was treated by luminol treatment after treatment for 1 hour at room temperature, and confirmed by developing the film with a film. As a result, when the expression level of p53 protein that regulates apoptosis was confirmed, it was confirmed that p53 protein was decreased by overexpression of ARL6IP1, and the activities of BAX, caspase-9, and caspase-3, which are sub-paths of p53, were decreased It could be confirmed (Figure 4d).

Example  5: ARL6IP1 is Of MDM2  E3 ligase enzyme activity Activate  regulates the protein of p53

To determine whether p53 protein reduction following ARL6IP1 overexpression is regulated at the transcriptional or posttranslational stage, cells were harvested after induction of overexpression using the Flag-ARL6IP1 plasmid in the U2OS cell line under the same conditions as in FIG. 4. Total RNA was isolated and cDNA synthesis was confirmed by PCR using ARL6IP1, p53 specific primer (Table 1). β-actin was used as a control indicating that PCR was performed at the same concentration of cDNA.

As a result, it was confirmed that there was no change in the mRNA level of p53 after ARL6IP1 overexpression. This suggests that p53 by ARL6IP1 is regulated at post-translational levels (FIG. 5A).

The protein of p53 is known to be ubiquitinated by EDM ubiquitin ligase, MDM2, to be proteasome dependently degraded. Based on this, to determine whether the reduction of p53 protein by ARL6IP1 overexpression is due to proteasome-dependent degradation by ubiquitination, the U2OS cell line was transfected with Flag-ARL6IP1 plasmid (5, 15 ug) for 24 hours. After inducing overexpression of ARL6IP1, cells were harvested after 12 hours of proteasome inhibitor MG132 at a concentration of 10 uM. The harvested cells prepared protein samples using RIPA lysis buffer and proteins were separated by 12% SDS-PAGE gel.

In addition, Flag (1: 5000) and ARL6IP1 (1: 3000) were used as primary antibodies to determine whether ARL6IP1 was overexpressed in a concentration-dependent manner, and p53 (1: 5000, santa-) was treated with proteasome inhibitor treatment. cruz), MDM2 (1: 2000, BD pharmingen) was confirmed. The primary antibody was diluted with PBST and treated at 4 ° C. for 12 hours, and the HRP-bound secondary antibody was treated with luminol after treatment for 1 hour at room temperature (FIG. 5b).

As a result, it was confirmed that overexpression of ARL6IP1 decreased the p53 protein level, and the proteasome inhibitor did not show a decrease in p53. In addition, the expression of p53 E3 ubiquitin ligase MDM2 did not show a change in expression due to the overexpression of ARL6IP1. This suggests that a decrease in p53 by ARL6IP1 is associated with ubiquitination.

p53 is known to modulate ubiquitination by MDM2 (Mouse double minute 2 homolog), an E3 ubiquitin ligase.MDM2 is a substrate protein that is used as a substrate as well as p53 to connect multimeric ubiquitin to substrate protein. It is known that theasome can be dependent on the degradation of matrix proteins.

In the present invention, it was confirmed that p53 protein by ARL6IP1 is reduced by ubiquitination. However, the increase in the protein of MDM2 was not confirmed by the increase in the expression of ARL6IP1 in the cell (Fig. 5b). Based on these results, EDM enzymatic activity of MDM2 by ARL6IP1 was confirmed by in vitro or in vivo MDM2 ubiquitination assay by self-ubiquitination.

In vitro MDM2-ubiquitination assay was performed to determine whether the protein regulatory pathway of p53 by ARL6IP1 is due to the enzymatic activity of MDM2, the E3 ubiquitin ligase of p53. In vitro MDM2-ubiquitination assays were performed using His-E1; 0.5 μg, His-UBC5Hc; 0.2 μg, GST-MDM2; 0.2, 0.5, 1 μg, His-ARL6IP1; 0.2, 0.5, 1 μg, Flag-ubiquitin (Sigma-Adrich); 1.25 μg of reaction buffer (25 mM Tris-HCl pH7.5, 1 mM MgCl ₂ , 2.5 mM DTT, 5 mM ATP, and ATP regeneration system 1 mM creatine phosphate (Sigma-Adrich), 1 mM creatine kinase) (Sigma-Adrich), 0.5μg / ml ubiquitin aldehyde (Boston Biohem, MA, USA)] and after 1 hour reaction at 37 ° C, it was confirmed by Western blot.

As a result, there was no change in MDM2 enzymatic activity depending on the protein concentration of ARL6IP1, which confirmed that ARL6IP1 does not directly regulate the enzymatic activity of MDM2 (FIG. 5C).

In vivo MDM2 ubiquitination assay was performed to confirm the change of MDM2 enzymatic activity in cells according to the expression of ARL6IP1. HEK293T cell lines were incubated with GST tagged MDM2 (5 ug), Flag tagged ARL6IP1 (5 ug) and HA tagged ubiquitin (5 ug) plasmids for 24 hours after transfection. After 12 hours after treatment with proteasome inhibitor MG132 (10 uM), cells were harvested using lysis buffer (50 mM Tris-cl, pH7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA). GST resin (Amersharm) was added to react for 4 to 2 hours to dissolve and to pull down GST-MDM2. Subsequently, unbound proteins were washed with PBST except GST-MDM2 bound to GST resin by centrifugation. Resin was resuspended with 2 X SDS sample buffer (0.125 M Tris pH 6.8, 4% SDS, 20% glycerol, 0.2 M DTT, 0.02% bromophenol blue) and then separated with 10% SDS PAGE gel and then HA (1: 2000, AbFrontier) was used as the primary antibody and treated at 4 ° C. for 12 hours. The ubiquitination of MDM2 was confirmed using an HRP bound secondary antibody.

As a result, the overexpression of ARL6IP1 resulted in a sharp increase in the ubiquitination of MDM2 compared to the control group. Through this, ARL6IP1 does not directly affect the enzymatic activity of MDM2, but it is a co-factor that regulates the enzymatic activity of MDM2 in cells. It can be seen that by increasing the enzyme activity of MDM2 was increased (Fig. 5d).

Example  6: ARL6IP1 is  Child action autophagy Activation of cells to regulate cell lifespan

The association of ARL6IP1 gene with autophagy, one of the important pathways that regulate the lifespan, survival and death of ER organelles, has been identified. Based on the results of Example 3, where ARL6IP1 is located in the membrane of the ER organelle, it was confirmed whether this ARL6IP1 can regulate the autophagy signal of neurons. Flag-ARL6IP1 plasmid was transduced (5, 15 ug) in a U2OS cell line and incubated for 24 hours to induce overexpression of ARL6IP1 and treated with ER stress inducer tunicamycin (TM) at a concentration of 10 ug / ml for 4 hours. Cells were then harvested and protein samples were prepared using RIPA buffer. LC3B-, the active form of the ATG5-ATG12 conjugate (1: 2000, santa-cruz) and LC3B (light chain 3β, 1: 5000, sigma-aldrich) involved in progeny signaling after protein separation using a 12% SDS-PAGE gel Treatment was performed at 4 ° C. for 12 hours using a primary antibody capable of confirming the amount of protein expression of II. Using the HRP-bound secondary antibody was confirmed the degree of protein expression through the film phenomenon after the reaction (Fig. 6a).

As a result, the expression of ATG5-ATG12 conjugate and LC3B-II increased as the expression of ARL6IP1 increased, indicating that a child signal was induced.

In order to confirm whether ARL6IP1 directly induces a child signal, protein-to-protein binding with LC3B protein forming an autophagosome was confirmed. ARL6IP1 amplified by PCR in the pET28a (His tagged) vector, an E. coli expression vector, was prepared using a BamHI / NotI restriction enzyme. Recombinant plasmids were constructed. Thereafter, each protein was isolated and purified from E. coli . The purified His-ARL6IP1 (0.5 ug) and GST-LC3B (0.5, 1 ug) were reacted in PBS buffer at 4 ° C for 2 hours and then pulled down by adding GST resin. Pulled-down samples were identified using His (1: 2000, santa-cruz) as the primary antibody after separation through 12% SDS-PAGE gel to identify His-ARL6IP1 bound to GST-LC3B ( 6b). As a result, it was confirmed that ARL6IP1 binds directly to LC3B.

In addition, the binding between the LC3B family protein and ARL6IP1 protein, which perform a function similar to LC3B capable of forming autophagosomes, was confirmed. GST-ARL6IP1 and His tagged LC3B family were isolated and purified from E. coli , respectively . After that, GST-ARL6IP1 (1 ug) and His-LC3 family (1 ug) were reacted in PBS buffer for 2 hours at 4 ° C. GST resin was added and pulled down. Pulled-down samples were identified using His (1: 2000, santa-cruz) as the primary antibody after separation through 12% SDS-PAGE gel (Fig. 6c). GST protein was used as a negative control.

In order to confirm that ARL6IP1 and LC3B directly bind intracellularly to form autophagosomes and induce a child signal, a GFP-LC3B point formation assay was performed. A stable cell line continuously expressing GFP-LC3B in the HeLa cell line was prepared, and the adenovirus vector containing ARL6IP1 was treated with 100 MOI titer and incubated for 24 hours. Subsequently, HBSS (induction of fertility signals), wortmannin (inhibitor of autophagocytosis), and bafilomycin A1 (lysosome inhibitor) were incubated at 37 ° C. for 4 hours. After 4 hours-paraformaldehyde (para-formaldehyde) was left at room temperature for at least 12 hours to fix the cells and washed with PBS. GFP-LC3B puncta formation was observed through fluorescence microscopy (FIG. 6D).

As a result, when overexpression of ARL6IP1 was induced, it was confirmed that a strong LC3B point (puncta) was formed compared to the control group, and this resulted in a strong induction signal by ARL6IP1. On the other hand, even when overexpression of ARL6IP1 was induced when autophagy inhibitors were treated, the LC3B point (puncta) was not observed, indicating that the increase in expression of ARL6IP1 was directly involved in autophagy formation of LC3B. LC3B dot formation of FIG. 6D was quantified using image J software (FIG. 6E).

Example  7: ARL6IP1  Knock-down of genes inhibits child interaction

Induction of offspring was confirmed by overexpression of ARL6IP1. In order to confirm whether these results are specifically observed in ARL6IP1 gene expression, we utilized a cell line that expresses GFP-LC3B continuously in HeLa cell line and adenovirus (Ad-sh-ARL6IP1) was used to achieve a 100 MOI titer of 48. Treatment for hours induced knock-down of the ARL6IP1 gene.

Subsequently, after treatment for 4 hours with HBSS, a condition for inducing child action signal, autophagy formation was observed through a fluorescence microscope (FIG. 7A). In addition, the degree of fluorescence expression was quantified and plotted using image J software (FIG. 7B).

Cells were collected after treatment with adenovirus at 100 MOI titer for 48 hours to determine the protein expression level of LC3B-II, which can confirm the regulation of ARL6IP1 gene by Adenovirus (Ad-sh-ARL6IP1) and control of child action. Confirmed by Western blot (Fig. 7c). Ad-sh-Ctrl virus was used as a control for adenovirus.

As a result, ARL6IP1 gene expression was suppressed after adenovirus treatment and LC3B-II expression was also reduced under the same conditions.

Example  8: in the differentiation of neurons Of ARL6IP1  Manifestation Will increase  Child action is induced

The aim of this study was to determine the expression of ARL6IP1 and control of offspring activity during differentiation of neurons. ReNcell neuronal cells were cultured in neuronal differentiation medium (DMEM / F12, 1% FBS, penicillin, streptomycin and 10 uM retinoic acid) for up to 10 days to confirm the expression regulation of ARL6IP1 gene under neuronal differentiation conditions. Microscopic observation was performed to confirm the change in the phenotype of each cell (FIG. 8A). The cells were harvested at each time point to obtain protein samples using RIPA lysis buffer. This was followed by Western blot using ARL6IP1, LC3B and β-actin as the primary antibody after protein separation using a 12% SDS-PAGE gel (FIG. 8B).

As a result, neuronal phenotypic changes were observed after neuronal differentiation conditions, and the expression of ARL6IP1 protein was maximized at 1 to 3 days after retinoic acid treatment, which is an autophagosome. An increase in protein of LC3B-II forming was observed. MAP2 protein, which is used as a differentiation marker of neurons, was found to continuously increase expression under retinoic acid treatment conditions, indicating that neuronal differentiation was induced. During the differentiation process, neurons were able to activate the progeny pathway for survival.

Three days after the ReNcell treatment with Retinoic acid, cells were harvested and centrifuged under 2.5-25% iodixanol concentration gradient conditions. Protein samples were collected and confirmed by Western blot. Through the above experiments, it was confirmed that the expression of ARL6IP1 and LC3B-II was increased in the retinoic acid treated group, which is a neurodifferentiation condition, compared with the control group, and that the expression of ARL6IP1, the ER membrane markers Kalnexin and LC3B were expressed at the same fraction sample. . It was confirmed that ARL6IP1 is present in the intracellular organelle ER and induces a child under neurodifferentiation conditions (FIG. 8C).

Example  9: Of ARL6IP1  Nerve cell death due to inhibition of expression Of ARL6IP1  Overexpression suppressed

To confirm the potential of ARL6IP1 as a therapeutic agent for ALS6IP1 as a gene therapy for hereditary stiff paraplegia, we tried to determine whether apoptosis was inhibited by overexpression of ARL6IP1 when apoptosis was induced after knocking down ARL6IP1 using sh-ARL6IP1. .

SH-SY5Y cell line, which is a neuronal cell line, was treated with 200 MOI titer using adenovirus Ad-sh-ARL6IP1 to induce knock-down of ARL6IP1 gene, and then 200 MOI using adenovirus Ad-ARL6IP1 / Ad-LacZ. Incubation for 24 hours with titer induced overexpression of ARL6IP1. After treatment with retinoic acid, which induces differentiation of neurons, the phenotype of neurons differentiated after 3 days of culturing was observed through a microscope (FIG. 9A).

As a result, it was confirmed that when ARL6IP1 overexpression was induced in a condition in which apoptosis was induced after ARL6IP1 knock-down and neuronal differentiation was not induced, neuronal differentiation proceeded similarly to the untreated condition.

In addition, each adenovirus (Ad-sh Ctrl, Ad-sh-ARL6IP1 and Ad-ARL6IP1) was treated with 200 MOI titers in neuro-2a neuronal lines to confirm protein expression regulation of the ARL6IP1 gene by adenoviruses. Cells were harvested after time incubation. The harvested cells obtained protein samples using RIPA lysis buffer. This was followed by Western blot using ARL6IP1, ATF4 and β-actin as the primary antibody after protein separation using a 12% SDS-PAGE gel (FIG. 9B).

In order to determine whether apoptosis can be controlled when ARL6IP1 overexpression is induced in apoptosis conditions induced by knock-down of the ARL6IP1 gene, each adenovirus (Ad-sh Ctrl, Ad) -sh-ARL6IP1 and Ad-ARL6IP1) 200 MOI titers and cells were harvested after 24 hours incubation. The harvested cells were confirmed by flow cytometry after staining using Annexin V, a cell death marker (FIG. 9C).

These results indicate that neuronal cell death and differentiation are inhibited by the knock-down of AR type ARL6IP1, a hereditary stiff paraplegic disease, and that overexpression of ARL6IP1 is induced under this condition, which inhibits neuronal cell death and increases differentiation. The results suggest that there is potential as a gene therapy material for hereditary tonic paraplegia disease caused by mutations in the gene.

Example  10: Let hippocampal  In neurons ARL6IP1  After gene knock-down ARL6IP1  Validation of treatment effect due to overexpression

Lett hippocampal neuron primary cells were isolated from 18 days of embryonic brain tissue and cultured on glass coverslips. 5 days after primary cell culture, the cells were transduced with calcium phosphate. After 9 days, the cells were fixed and stained and observed under a microscope (FIG. 10a).

When the primary and secondary dendrites of neurons were counted to determine the degree of neuronal dendrite arborization after ARL6IP1 knock-down, both primary and secondary dendrite of neurons were significantly reduced by ARL6IP1 knock-down. . In addition, the primary dendrite decreased when the overexpression of ARL6IP1 at the same time showed a significant increase effect similar to the control group, the secondary dendrite did not recover to the control level, but a significant increase was confirmed (Fig. 10b).

These results indicate that knock-down of ARL6IP1 significantly inhibited dendrite arborization of neurons, and when ARL6IP1 overexpressed, dendrite arborization increased to a control level, suggesting that ARL6IP1 could be used as a gene therapy material.

Example  11: ARL6IP1  Mouse by gene knock-out HSP  Establish disease model

A knock-out mouse of ARL6IP1 was constructed to verify whether HSP disease phenotype is identified by knock-down of the ARL6IP1 gene based on the results of confirming the possibility of ARL6IP1 as a gene therapy agent for hereditary stiff paraplegia (FIG. 11A).

The expression of ARL6IP1 in the tissues of ARL6IP1 KO mice, a constructed HSP disease model, was confirmed to be reduced in mRNA and protein levels of ARL6IP1 in brain tissues. (FIGS. 11B and 11C)

ARL6IP1 KO mice showed muscle stiffness in the lower extremity due to neurodegeneration at 10 months (FIG. 11D). In addition, as a result of confirming the abduction angle of the mouse foot by the neurodegenerative phenotypic analysis, the abduction angle of the foot was more than 25 degrees compared to the normal mouse in 10 months of ARL6IP1 KO mice (FIGS.

These results indicate that inhibition of ARL6IP1 expression causes autosomal recessive HSP disease and HSP disease-specific phenotypes have been identified in ARL6IP1 KO mice, suggesting that they can be used to validate therapeutic efficacy as mouse HSP disease models.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> Use of ARL6IP1 for treatment of Hereditary Spastic Paraplegia <130> KPA161072-KR-P1 <150> KR 10-2016-0109406 <151> 2016-08-26 <160> 19 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer F1 <400> 1 gactgcaagt ctggaagaac 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer R1 <400> 2 gaacaaggta gtcagccaag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer F2 <400> 3 ctgaccacgt tggatgacac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer R2 <400> 4 gggctcatac agatgccact 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer F3 <400> 5 tgtttccaaa accctggaag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer R3 <400> 6 aactgggatt tgctgtgtcc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer F4 <400> 7 ctctgaggaa ggcattcagg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer R4 <400> 8 catttctggt ggcagagaca 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer F5 <400> 9 cttggctgac tgaggaggag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer R5 <400> 10 tcaccattcg gtcaatcaga 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer F6 <400> 11 ggagtaccct gatgagatcg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer R6 <400> 12 tgcaggaaca tttacacgtc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer F7 <400> 13 cgtgtggagt atttggatga 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer R7 <400> 14 ttctccatcc agtggtttct 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer F8 <400> 15 gaaccagcag agattcagag 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer R8 <400> 16 ctcaggagga gcaatgatct 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer F9 <400> 17 aggagaagct gtgctacgtc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer R9 <400> 18 ctcaggagga gcaatgatct 20 <210> 19 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> ARL6IP1-shRNA <400> 19 gatgctgatg gctgacaaa 19

Claims (9)

delete delete delete delete (a) treating a candidate candidate for treating hereditary spontaneous paraplegia with isolated neuronal cells induced with Hereditary Spastic Paraplegia (HSP); And
(b) measuring the protein expression level or activity of ARL6IP1 (ADP ribosylation factor like GTPase 6 interacting protein 1) in isolated neurons treated with the candidate substance,
Step (b) further comprises the step of measuring any one of the following (1) to (3), the method of screening for the treatment of hereditary tonic paraplegia
(1) protein expression of p53;
(2) protein expression of LC3B-II; And
(3) Formation of ATG5-ATG12 conjugates.
The method of claim 5,
The method comprises (c) the protein level of ARL6IP1 measured in step (b); Or if its activity is increased relative to the isolated neuron, which has not been treated with the candidate substance, determining the treatment for hereditary tonic paraplegia.
delete The method of claim 5,
When the candidate substance measured in step (b) is increased in the protein activity of ARL6IP1 compared to the untreated isolated neurons, and if any one of the following (1) to (3), as a treatment for hereditary tonic paraplegia The screening method further comprises the step of determining:
(1) reduced protein expression of p53;
(2) increased protein expression of LC3B-II; And
(3) Formation of ATG5-ATG12 conjugates is increased.
The method of claim 5,
The expression level of the protein is measured by an antibody that specifically binds to the protein.

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