Intracellular domain of AXL or fragment derived therefrom, and use for regulating autophagy using same

Abstract

The present invention relates to an intracellular domain of AXL or a fragment derived therefrom, and a use for regulating autophagy using same, wherein the intracellular domain of AXL or the fragment derived therefrom can regulate a selective autophagy pathway in response to an intracellular amyloid beta signal.

Description

Intracellular domain of AXL or fragment derived therefrom and use thereof for regulating autophagy

[0001] The present invention relates to an intracellular domain of AXL or a fragment derived therefrom and a use thereof for regulating autophagy.

[0002] Autophagy is a natural survival mechanism of cells that degrades and recycles cellular components, such as unnecessary or damaged proteins and organelles. It has been recognized as a key cellular mechanism for eliminating pathological proteins in various diseases. However, existing autophagy inducers suffer from various problems, including nonspecific effects, side effects, limitations in signaling pathways, non-selective activation, and difficulties in clinical application. For example, rapamycin is a representative drug that induces autophagy by inhibiting the mTOR signaling pathway. However, because mTOR is involved in various physiological functions, including cell growth, metabolism, and survival, this nonspecific effect has a wide-ranging effect on the entire cell and can cause cytotoxicity with chronic administration. These problems can be particularly detrimental in organs such as the brain, where homeostasis and cellular composition are meticulously regulated.

[0003] Meanwhile, astrocytes, one of the brain's most resilient cells, can transform into reactive astrocytes in response to toxic proteins such as amyloid beta (Aβ) in conditions such as Alzheimer's disease. However, the non-cell-autonomous neuropathological mechanisms mediated by reactive astrocytes remain unclear.

[0004] These researchers aimed to develop a precisely regulated autophagy control platform that could overcome the toxicity and nonspecificity of existing autophagy inducers for the treatment of neurodegenerative diseases. As a result, they discovered that in astrocytes, a non-neuronal cell, the intracellular domain (ICD) of the AXL receptor is cleaved and translocated to the nucleus, where it interacts with transcription factors to regulate autophagy-inducing genes, thereby completing the present invention.

[0005] One aspect is to provide an isolated peptide comprising the amino acid sequence of sequence number 1.

[0006] Another aspect is to provide a polynucleotide encoding the above peptide.

[0007] Another aspect is to provide a carrier comprising the peptide or the polynucleotide.

[0008] Another aspect provides a method of inhibiting autophagy in a subject comprising administering to the subject an effective amount of the peptide, the polynucleotide or the carrier.

[0009] Another aspect provides a method of inhibiting autophagy comprising administering to a cell an effective amount of the peptide, the polynucleotide or the carrier.

[0010] Another aspect provides the use of the peptide for inhibition of autophagy.

[0011] Another aspect provides a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising as an active ingredient an intracellular domain of AXL; a polynucleotide encoding the same; or a transporter comprising the intracellular domain of AXL or the polynucleotide.

[0012] Another aspect provides a method for preventing, ameliorating or treating a neurodegenerative disease comprising administering to a subject in need thereof an effective amount of an intracellular domain of AXL, a polynucleotide encoding the same, or a carrier comprising the intracellular domain of AXL or the polynucleotide.

[0013] Another aspect provides the use of an intracellular domain of AXL, a polynucleotide encoding the same, or a delivery system comprising the intracellular domain of AXL or the polynucleotide for the prevention, amelioration or treatment of a neurodegenerative disease.

[0014] Another aspect provides the use of an intracellular domain of AXL, a polynucleotide encoding the same, or a carrier comprising the intracellular domain of AXL or the polynucleotide for use in the manufacture of a pharmaceutical preparation for preventing, ameliorating or treating a neurodegenerative disease.

[0015] Another aspect is to provide a method for screening drugs for the prevention or treatment of neurodegenerative diseases.

[0016] Another aspect is to provide a method for screening autophagy regulators.

[0017] One aspect provides an isolated peptide comprising the amino acid sequence of SEQ ID NO: 1. The isolated peptide may be used for the purpose of regulating amyloid beta-selective autophagy in neurons.

[0018] In one specific embodiment, the peptide may further comprise an addition of no more than 20 amino acids at the N-terminus, the C-terminus, or both. The peptide may further comprise an addition of no more than 20, no more than 19, no more than 18, no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid, but is not limited thereto. It should be understood that peptides having (or comprising) an amino acid sequence in which some sequences are modified or added are also included in the present invention, provided that they have the same or corresponding activity as the isolated peptide.

[0019] Specifically, the peptide may further comprise an addition of no more than 10 amino acids at the N-terminus or no more than 10 amino acids at the C-terminus. More specifically, the peptide may further comprise an addition of no more than 6 amino acids at the N-terminus.

[0020] The peptide may be a fragment of the intracellular domain of AXL. The peptide may further comprise an addition of a continuous amino acid sequence derived from the intracellular domain of AXL.

[0021] The term "AXL" in this specification refers to one of the individual receptors belonging to the TAM receptor family. The main ligand is Gas6, and binding to Gas6 induces autophosphorylation of AXL, which activates downstream signaling pathways. "TAM receptor" is an acronym for the first letters of the names of three receptor tyrosine kinases (RTKs), Tyro3, ​​AXL, and MerTK, and refers to a family of receptors that play an important role in maintaining immune homeostasis, efferocytosis, and regulation of inflammatory responses.

[0022] In one specific example, the peptide may further comprise 20 or fewer amino acids in the N-terminal direction from the 459th amino acid of the AXL protein of SEQ ID NO: 7 at the N-terminus. Specifically, the peptide may further comprise 20 or fewer, 15 or fewer, 10 or fewer, or 6 or fewer amino acids in the N-terminal direction from the 459th amino acid of the AXL protein of SEQ ID NO: 7 at the N-terminus.

[0023] In one specific example, the peptide may further comprise 20 or fewer amino acids in a C-terminal direction from the 467th amino acid of the AXL protein of SEQ ID NO: 7 at the C-terminus. Specifically, the peptide may further comprise 20 or fewer, 15 or fewer, 10 or fewer, or 6 or fewer amino acids in a C-terminal direction from the 467th amino acid of the AXL protein of SEQ ID NO: 7 at the C-terminus.

[0024] In one specific example, the peptide may comprise the amino acid sequence of SEQ ID NO: 2.

[0025] In one specific example, the peptide may be an isolated peptide consisting of an amino acid sequence of SEQ ID NO: 1 or 2.

[0026] In one specific embodiment, the peptide may further comprise a cell penetrating peptide (CPP) at the N-terminus, the C-terminus, or both.

[0027] The above cell penetrating peptide may be TAT peptide, Penetratin, SynB1, RVG29, Angiopep-2, Pep-1, Transportan, MAP (model amphipathic peptide), KALA, MPG, CADY, Buforin II, or R9(RRRRRRRRR), but any peptide known to those skilled in the art as a cell penetrating peptide may be used in the present invention regardless of its amino acid sequence, origin, or length.

[0028] Specifically, the cell penetrating peptide may comprise the amino acid sequence of SEQ ID NO: 3 or may consist of the amino acid sequence of SEQ ID NO: 3.

[0029] In one specific example, the peptide may comprise an addition of a continuous sequence derived from the AXL protein as described above to one or more ends of the amino acid sequence of SEQ ID NO: 1, and may further comprise an addition of the cell penetrating peptide.

[0030] The above peptide may be one in which the C-terminus of the amino acid of SEQ ID NO. 3 is linked to the N-terminus of the amino acid of SEQ ID NO. 1, or the N-terminus of the amino acid of SEQ ID NO. 3 is linked to the C-terminus of the amino acid of SEQ ID NO. 1, or the amino acid of SEQ ID NO. 3 is linked to both.

[0031] The above peptide may be one in which the C-terminus of the amino acid of SEQ ID NO. 3 is linked to the N-terminus of the amino acid of SEQ ID NO. 2, or the N-terminus of the amino acid of SEQ ID NO. 3 is linked to the C-terminus of the amino acid of SEQ ID NO. 2, or the amino acid of SEQ ID NO. 3 is linked to both.

[0032] In one specific example, the peptide may comprise an amino acid sequence of SEQ ID NO: 4, 5, or 6.

[0033] As used herein, the terms "amino acid" and "amino acid residue" refer to a natural amino acid, an unnatural amino acid, or a modified amino acid. Unless otherwise stated, all references to amino acids, either generically or by name, specifically include references to both the D and L stereoisomers (where the structure permits such stereoisomeric forms). Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Non-natural amino acids include modified amino acid residues that are chemically modified, or reversibly or irreversibly chemically blocked, at the N-terminal amino group or side chain, such as N-methylated D and L amino acids or residues in which the side chain functionality is chemically modified with another functional group.

[0034] In the case where it has the same or corresponding activity as the peptide of the present invention, in addition to the mutation of the present invention, it does not exclude meaningless sequence addition before and after the amino acid sequence of the corresponding sequence number, mutation that can occur naturally, or silent mutation thereof, and it is obvious that it falls within the scope of the present invention even if it has a deletion, modification, substitution (e.g., conservative substitution) of a part of the sequence, or an amino acid sequence added. The conservative substitution means replacing an amino acid residue with an amino acid residue having a similar side chain without causing a loss of the biological or biochemical function of the polypeptide or protein. Classes of amino acid residues having similar side chains are defined in the relevant technical field and are well known. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0035]

[0036] Another aspect provides a polynucleotide encoding the peptide.

[0037] Another aspect provides a carrier comprising the peptide or the polynucleotide.

[0038] The polynucleotide may be in the form of a construct for delivery to a host cell either in vitro, in vivo, or ex vivo.

[0039] The above polynucleotide may further comprise a promoter operably linked thereto for expression thereof.

[0040] In one specific example, the polynucleotide may comprise the base sequence of SEQ ID NO: 13, 14, or 15.

[0041] The above vector may be a viral vector or a non-viral vector. The vector may be used without limitation in type as long as it can stably express the peptide or polynucleotide within the host cell by inducing transduction or transfection by infecting or introducing the host cell.

[0042] The above viral vector may be a retrovirus, lentivirus, adenovirus, adeno-associated virus, or vaccinia virus vector, and in addition to these, various viral delivery means known in the art may be used without limitation.

[0043] The non-viral vector may include lipid nanoparticles (LNPs), polymeric nanoparticles, liposomes, peptide-based nanoparticles, electroporation, the calcium phosphate method, nanocomplexes, anionic or cationic polymers (polyplexes or lipoplexes), physical delivery methods (e.g., microneedles, gene guns), etc. In addition to these, various non-viral delivery means known in the art may be used without limitation.

[0044] Another aspect provides a method of inhibiting autophagy in a subject comprising administering to the subject an effective amount of the peptide, the polynucleotide or the carrier.

[0045] Another aspect provides a method of inhibiting autophagy comprising administering to a cell an effective amount of the peptide, the polynucleotide or the carrier.

[0046] In one specific example, the step of treating the cell with the peptide may be, but is not limited to, a method of directly treating the cell by adding the purified peptide to a cell culture medium, introducing a vector containing a gene that causes the peptide to be expressed into the cell, using a cell-penetrating peptide, delivering the peptide by encapsulating it in a liposome, nanoparticle, or other drug delivery system, or introducing the peptide into the cell by using electroporation.

[0047] Another aspect provides a use of the peptide for autophagy inhibition.

[0048]

[0049] Another aspect provides a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising as an active ingredient an intracellular domain of AXL, a polynucleotide encoding the same, or a transporter comprising the intracellular domain of AXL or the polynucleotide.

[0050] In one specific example, the intracellular domain of AXL may include an amino acid sequence from a γ-secretase cleavage site to the C-terminus of the AXL protein. Specifically, the intracellular domain of AXL may include a sequence in which the C-terminus is cleaved by α-secretase and the N-terminus is cleaved by γ-secretase. The γ-secretase cleavage site of the AXL protein may refer to 452YVLLGAVV459 within the transmembrane domain, as previously reported in the art [Lu Y, et al. "Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells". FASEB J. 2017 Apr;31(4):1382-1397. doi: 10.1096 / fj.201600702R.].

[0051] In one specific example, the intracellular domain of AXL may comprise the 473rd to 894th amino acid sequence, the 470th to 894th amino acid sequence, the 466th to 894th amino acid sequence, the 460th to 894th amino acid sequence and / or the 454th to 894th amino acid sequence from the N-terminus of the AXL protein. Preferably, the intracellular domain of AXL may comprise the 460th to 894th amino acid sequence from the N-terminus of the AXL protein.

[0052] In one specific example, the intracellular domain of AXL may include the 473rd to 894th amino acid sequence, the 470th to 894th amino acid sequence, the 466th to 894th amino acid sequence, the 460th to 894th amino acid sequence and / or the 454th to 894th amino acid sequence from the N-terminus of the full-length AXL protein sequence represented by SEQ ID NO: 7. Preferably, the intracellular domain of AXL may include the 460th to 894th amino acid sequence from the N-terminus of the full-length AXL protein sequence represented by SEQ ID NO: 7.

[0053] In one specific embodiment, the intracellular domain of AXL may comprise an amino acid sequence represented by SEQ ID NO: 9, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0054] In one specific example, the polynucleotide sequence encoding the intracellular domain of AXL may comprise a gene sequence represented by SEQ ID NO: 15, a portion thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0055] Even if the present application describes "comprising a gene sequence / amino acid sequence of a specific sequence number" or "having a gene sequence / amino acid sequence of a specific sequence number", it is obvious that a gene sequence / amino acid sequence in which some sequences are deleted, modified, substituted or added can also be used in the present application, as long as it has the same or corresponding function as that composed of the gene sequence / amino acid sequence of the corresponding sequence number. In other words, the variant of the present invention may also be included within the scope of the present invention. Specifically, the variant of the present invention may include deletion, modification, substitution (e.g., conservative substitution) or addition of some sequences that have a minimal effect on the characteristics and secondary structure of the polypeptide, and if it has the same or corresponding activity as the variant of the present invention, it does not exclude meaningless sequence additions before and after the amino acid sequence of the corresponding sequence, mutations that may occur naturally, or silent mutations thereof. Additionally, the N-terminus of the variant may be conjugated with a signal (or leader) sequence that is involved in translocation of the protein co-translationally or post-translationally, and may be conjugated to other sequences to enable identification, purification, or synthesis.

[0056] Homology and identity refer to the degree to which two given sequences are related, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0057] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering the composition of the present invention to a subject. For preventive purposes, the composition may be administered to a subject at risk of developing a specific disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0058] As used herein, the term "treatment" refers to any action that improves the symptoms of a disease or provides benefit by administering the composition of the present invention to a subject. As used herein, the terms "treatment," "palliation," and "improvement" may be used interchangeably. A therapeutic benefit refers to any therapeutically significant improvement or effect on one or more diseases, conditions, or symptoms under treatment.

[0059] In one specific example, the composition may regulate intracellular amyloid beta selective autophagy.

[0060] The term "autophagy" as used herein refers to the cellular self-phagy that degrades and recycles damaged organelles, proteins, or other intracellular components.

[0061] As used herein, the term "selective autophagy" refers to autophagy that targets and selectively degrades specific damaged organelles or proteins within a cell. For the purposes of the present invention, "selective autophagy" refers to amyloid-beta selective autophagy, which specifically recognizes and removes amyloid-beta protein.

[0062] The composition can induce amyloid beta-selective autophagy. The AXL intracellular domain can act as an amyloid beta receptor and can induce intracellular autophagy in response to amyloid beta signals. When intracellular amyloid beta accumulates, the intracellular domain of AXL is cleaved by gamma-secretase upon receiving the amyloid beta signal, and the cleaved intracellular domain of AXL can translocate into the nucleus and activate the expression of genes that regulate the autophagy pathway.

[0063] The composition can regulate the expression of proteins and / or genes that regulate the autophagy pathway in astrocytes. For example, the composition can regulate the expression of autophagy-related proteins such as LC3B, p62, ATG7, or GABARAPL1.

[0064] The composition may regulate intracellular amyloid beta to an appropriate level. When intracellular amyloid beta is excessively accumulated, the composition may induce intracellular amyloid beta-selective autophagy to remove amyloid beta.

[0065] The neurodegenerative disease may be at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Lewy body dementia, progressive supranuclear palsy, frontotemporal dementia, tauopathies, cerebral amyloid angiopathy, and Dutch-type hereditary cerebral amyloid angiopathy, but is not limited thereto, and may be used for preventing, improving, or treating a disease associated with amyloid beta pathology.

[0066] The above neurodegenerative disease may be a neurodegenerative disease associated with amyloid beta pathology.

[0067] The composition is present in an amount of 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, 0.00001 wt% to 10 wt%, 0.00001 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 The intracellular domain of AXL, the polynucleotide, or the delivery system may be comprised in an amount of from 0.1 wt% to 30 wt%, from 0.1 wt% to 20 wt%, from 0.1 wt% to 10 wt%, or from 0.1 wt% to 5 wt%.

[0068] The above composition “comprising” the protein or the polynucleotide means that it is added to the composition to an extent that it can exhibit the above-mentioned effect, and includes formulation in various forms by adding various components as auxiliary components for drug delivery and stabilization, etc.

[0069] The content of the protein or the polynucleotide in the pharmaceutical composition of the present invention can be appropriately adjusted depending on the purpose of use of the pharmaceutical composition, the form of the formulation, etc.

[0070] The pharmaceutical composition of the present invention may be in any form suitable for the intended method of administration. In the pharmaceutical composition of the present invention, "administration" means introducing a predetermined substance into a patient by any suitable method, and the route of administration of the pharmaceutical composition may be administered through any common route as long as the drug can reach the target tissue. Administration may be by a method known in the art, and examples thereof include, but are not limited to, topical ocular administration (e.g., periocular (e.g., subTenon's), subconjunctival, intraocular, intravitreal, intracameral, subretinal, suprachoroidal, and retrobulbar administration), intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. In addition, the active ingredient may be administered by any device capable of transporting to target cells, and the route of administration is preferably determined depending on the type of disease to which it is applied.

[0071] The above administration is 0.00001 mg to 1,000 mg of the composition according to one specific example per subject per day, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, Alternatively, it may be administered in doses of 10 mg to 25 mg.

[0072] However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The frequency of administration may be once a day or twice or more within the range of clinically acceptable side effects, and the administration may be done in one or more sites, and the total number of administration days may be from 1 to 30 days per treatment, daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after an appropriate period. For animals other than humans, the same dosage as for humans per kg may be used, or the above dosage may be converted into an amount based on the volume ratio (e.g., average value) of the organs (e.g., heart) of the target animal and the human.

[0073] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., formulated according to conventional methods, or parenteral formulations such as suspensions, emulsions, lyophilized preparations, external preparations, suppositories, sterile injection solutions, and implantable preparations. The pharmaceutical composition may further comprise, in addition to the active ingredient, a pharmaceutically acceptable excipient that can be used in formulation.

[0074] The above excipients include carriers, vehicles, diluents, solvents, for example, monohydric alcohols, for example, ethanol, isopropanol, and polyhydric alcohols, for example, glycerol, and edible oils, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, oily esters, for example, ethyl oleate, isopropyl myristate; It may include at least one selected from the group consisting of binders, adjuvants, solubilizers, thickeners, stabilizers, disintegrants, glidants, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweeteners, coloring agents, flavoring agents, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starches, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low melting point waxes, ion exchange resins, etc., but is not limited thereto.

[0075] The carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0076] The pharmaceutical composition of the present invention may be formulated in the form of an oral administration dosage form, for example, tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, etc. These oral administration dosage forms may, in addition to the active ingredient according to the typical composition of each dosage form, contain pharmaceutically acceptable carriers, such as diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine, or lubricants such as silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol.

[0077] If the oral dosage form is a tablet, it may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethyl cellulose and / or polyvinylpyrrolidine, and in some cases, a disintegrating agent such as starch, agar, alginic acid or its sodium salt, an effervescent mixture and / or an absorbent, a coloring agent, a flavoring agent or a sweetening agent.

[0078] The pharmaceutical composition of the present invention being formulated in the form of a parenteral administration dosage form may mean that it is administered by a method such as subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. In this case, in order to formulate the pharmaceutical composition in the parenteral administration dosage form, the active ingredient is mixed with a stabilizer or buffer in water to prepare a solution or suspension, and this solution or suspension can be prepared in a unit dosage form of an ampoule or vial.

[0079] In addition, the pharmaceutical composition may be sterilized or may further contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifying agents, salts for osmotic pressure control and / or buffers, and may further contain other therapeutically useful substances, and may be formulated according to conventional methods of mixing, granulating or coating.

[0080] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and may be adjusted according to factors including the type of the patient's disease, the severity of the disease, the type of active ingredient administered, the type of formulation, the patient's age, sex, weight, health condition, diet, sensitivity, the time and method of drug administration, the combination of the composition or concurrently used drugs, and other factors well known in the medical field.

[0081] The pharmaceutical composition of the present invention can prevent or treat a disease in a subject, including a step of administering to the subject an amount effective to prevent or treat the disease.

[0082] The dosage of the pharmaceutical composition for the prevention or treatment of diseases according to the present invention may range from 0.01 ug / kg to 10 g / kg per day, specifically from 0.01 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once daily or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0083] The above object may be a mammal. The mammal may be a human, a dog, a cat, a cow, a goat, or a pig.

[0084] In one specific example, the composition may further comprise a SIRT2 protein.

[0085] In one specific example, the composition may be administered in combination with SIRT2 protein.

[0086] The term "SIRT2 protein" in this specification refers to one of the Sirtuin (SIRT) family proteins, also called NAD +-It is a deacetylase enzyme. It corresponds to the second isoform among the seven types (SIRT1-SIRT7) and is known to be involved in cell cycle, inflammation, metabolism, and neuroprotection.

[0087] The above AXL intracellular domain can interact with the SIRT2 protein. The above AXL intracellular domain can bind to the SIRT2 protein and have the above effect.

[0088] The above combination administration may mean administering the AXL intracellular domain and the SIRT2 protein simultaneously, sequentially, separately, or in any order.

[0089] Specifically, the combination administration may be by administering the AXL intracellular domain and the SIRT2 protein simultaneously, or by administering one of the AXL intracellular domain and the SIRT2 protein followed by the other. The combination therapy according to the present invention may be defined as providing a synergistic effect if the efficacy, as measured by, for example, the degree of response, the rate of response, the time until disease progression, or the duration of survival, is therapeutically superior to the efficacy that can be obtained by administering one or the other of the components of the combination therapy at a usual dose. For example, the efficacy of the combination therapy is synergistic if the efficacy is therapeutically superior to the efficacy obtained by using each of the above alone. In particular, a synergistic effect is considered to exist if the usual dose of the proteins can be reduced without compromising one or more of the degree of response, the rate of response, the time until disease progression, and the duration of the response, and in particular without compromising the duration of the response, while reducing and / or reducing problematic side effects compared to when each component is used at a usual dose.

[0090] Another aspect provides a method for preventing, ameliorating or treating a neurodegenerative disease comprising administering to a subject in need thereof an effective amount of an intracellular domain of AXL, a polynucleotide encoding the same, or a carrier comprising the intracellular domain of AXL or the polynucleotide.

[0091] Another aspect provides the use of an intracellular domain of AXL, a polynucleotide encoding the same, or a delivery system comprising the intracellular domain of AXL or the polynucleotide for the prevention, amelioration or treatment of a neurodegenerative disease.

[0092] Another aspect provides the use of an intracellular domain of AXL, a polynucleotide encoding the same, or a carrier comprising the intracellular domain of AXL or the polynucleotide for use in the manufacture of a pharmaceutical preparation for preventing, ameliorating or treating a neurodegenerative disease.

[0093]

[0094] Another aspect provides a method for screening drugs for the prevention or treatment of neurodegenerative diseases. To achieve this goal, the present invention provides a screening method comprising the following steps:

[0095] (a) a step of treating a candidate substance to a cell;

[0096] (b) a step of measuring the interaction of AXL protein and a protein binding to AXL protein in cells treated with the candidate substance or the expression of the intracellular domain of AXL protein; and

[0097] (c) A step of selecting a candidate substance that changes the interaction between the AXL protein and a protein binding to the AXL protein or the expression of the intracellular domain of the AXL protein compared to the untreated control group.

[0098] In one specific example, the protein binding to the AXL protein may include at least one selected from the group consisting of SIRT2, RUVBL1, RUVBL2, ERLIN1, ERLIN2, WDR5, and ATAD3A.

[0099] In one specific example, the measurement of the interaction between the AXL protein and the protein binding to the AXL protein can be confirmed using immunoprecipitation (IPP), surface plasmon resonance (SPR) method, fluorescence resonance energy transfer (FRET) system, immunofluorescence staining, GST-Pull down, yeast two-hybrid system (Y2H), bimolecular fluorescence complementation (BiFC) technique, or TAP-tag (Tandem affinity purification) method, but is not limited thereto, and any method capable of measuring protein-protein interaction known in the art can be used.

[0100] In one specific example, the measurement of the expression of the intracellular domain of the AXL protein may be performed by labeling with a label that generates a detectable signal (the label is, for example, chemically (e.g., covalently or non-covalently), recombinantly, or physically bound) or by labeling in a form in which a tag to which the label can be bound is attached, and then measuring the signal generated from the label through conventional enzymatic reaction, fluorescence, luminescence, and / or radiological detection. The measurement of the signal may be measured by any signal detection means conventionally used to detect or measure it (e.g., conventional fluorescence microscope, fluorescence camera, fluorescence intensity measurement (quantitation) device, etc.).

[0101] In one specific example, the selection step may further include a step of determining the candidate substance as a drug for preventing or treating a neurodegenerative disease when the interaction between the AXL protein and a protein binding to the AXL protein is increased compared to an untreated control group.

[0102] In one specific example, the selection step may further include a step of determining the candidate substance as a drug for preventing or treating a neurodegenerative disease if the expression of the intracellular domain of the AXL protein is increased compared to an untreated control group.

[0103]

[0104] Another aspect provides a method for screening autophagy regulators. To achieve this objective, the present invention provides a screening method comprising the following steps:

[0105] (a) a step of treating a candidate substance to a cell;

[0106] (b) a step of measuring the interaction of AXL protein and a protein binding to AXL protein in cells treated with the candidate substance or the expression of the intracellular domain of AXL protein; and

[0107] (c) A step of selecting a candidate substance that changes the interaction between the AXL protein and a protein binding to the AXL protein or the expression of the intracellular domain of the AXL protein compared to the untreated control group.

[0108] In one specific example, the measurement of the interaction between the AXL protein and the protein binding to the AXL protein can be confirmed using immunoprecipitation (IPP), surface plasmon resonance (SPR) method, fluorescence resonance energy transfer (FRET) system, immunofluorescence staining, GST-Pull down, yeast two-hybrid system (Y2H), bimolecular fluorescence complementation (BiFC) technique, or TAP-tag (Tandem affinity purification) method, but is not limited thereto, and any method capable of measuring protein-protein interaction known in the art can be used.

[0109] In one specific example, the measurement of the expression of the intracellular domain of the AXL protein may be performed by labeling with a label that generates a detectable signal (the label is, for example, chemically (e.g., covalently or non-covalently), recombinantly, or physically bound) or by labeling in a form in which a tag to which the label can be bound is attached, and then measuring the signal generated from the label through conventional enzymatic reaction, fluorescence, luminescence, and / or radiological detection. The measurement of the signal may be measured by any signal detection means conventionally used to detect or measure it (e.g., conventional fluorescence microscope, fluorescence camera, fluorescence intensity measurement (quantitation) device, etc.).

[0110] In one specific example, the selection step may further include a step of determining the candidate substance as an autophagy regulator when the interaction between the AXL protein and a protein binding to the AXL protein increases compared to an untreated control group.

[0111] In one specific example, the selection step may further include a step of determining the candidate substance as an autophagy regulator if the expression of the intracellular domain of the AXL protein is increased compared to an untreated control group.

[0112] In one specific example, the autophagy regulator may be an amyloid beta selective autophagy regulator.

[0113]

[0114] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0115] According to one aspect, the intracellular domain of AXL or a fragment derived therefrom can regulate the expression or activity of genes that regulate the autophagy pathway in response to intracellular amyloid beta signals. Accordingly, the intracellular domain of AXL or a fragment derived therefrom can be used for the prevention or treatment of neurodegenerative diseases, and also has a useful effect in screening for neurodegenerative disease therapeutic agents or autophagy regulators.

[0116] Figure 1 is a schematic diagram of the mechanism of action of Aβ-selective autophagy induced by AXL-ICD.

[0117] Figure 2 is a schematic diagram of Aβ oligomer treatment and immunoblot analysis in human astrocyte culture system.

[0118] Figure 3 is an image of immunoblot analysis of autophagy-related proteins after Aβ treatment in human astrocytes.

[0119] Figure 4 is an image of immunoblot analysis of LC3B protein expression in human astrocytes with knockdown of TAM receptors (Tyro3, ​​AXL, and Mertk).

[0120] Figure 5 is publicly available data analyzing TAM receptor RNA expression in various types of cells in the brain. (brainrnaseq.org / )

[0121] Figure 6 shows an image of LC3B protein observed by immunofluorescence in human astrocytes knocked down with AXL, a graph quantifying signal intensity, and the results of qRT-PCR analysis normalizing LC3B mRNA expression to GAPDH.

[0122] Figure 7 is an immunoblot analysis image analyzing autophagy flow after Bafilomycin A1 treatment in human astrocytes with AXL knocked down.

[0123] Figure 8 is an immunoblot analysis image of LC3B protein expression after Aβ treatment in human astrocytes with AXL knocked down and a quantitative graph of fold change in LC3-I levels normalized to GAPDH.

[0124] Figure 9 shows immunoblot images of AXL and LC3B proteins in human astrocytes (left) or U87MG cells (right) treated with Aβ oligomers in the presence or absence of GAS6. GAPDH was used as a loading control, and the arrow indicates the AXL intracellular domain.

[0125] Figure 10 shows immunofluorescence images of human astrocytes expressing full-length AXL after treatment with Aβ and GAS6, and immunostaining images observed using antibodies that recognize only the N-terminus of AXL or antibodies that recognize both the N- and C-termini.

[0126] Figure 11 is an image of an immunoblot analysis of AXL in cytoplasmic and nuclear extracts fractionated from human astrocytes treated with various concentrations of Aβ.

[0127] Figure 12 shows the results of immunoblot analysis for LC3B protein in human astrocytes treated with Aβ in the presence or absence of DAPT, a γ-secretase inhibitor.

[0128] Figure 13 is a schematic diagram of the amino acid sequence surrounding the potential γ-secretase cleavage site (Y452-V459) within the transmembrane domain (Y452-V472) of AXL and an AXL truncation mutant with GFP fused to the C-terminus.

[0129] Figure 14 is a representative immunofluorescence image obtained after treating human astrocytes expressing AXL truncation mutants with DMSO or Leptomycin B (LMB).

[0130] Figure 15 is a representative immunofluorescence image obtained after treatment with DMSO or Leptomycin B (LMB) in U87MB cells expressing AXL truncation mutants.

[0131] Figure 16 is an image showing the consensus sequence of the classical nuclear export signal (NES) sequence. NES has a general consensus pattern Φ-X 1- ₃-Φ-X 1- It is characterized by a leucine-rich motif following the sequence ₃-Φ-X-Φ, where Φ represents a hydrophobic residue such as leucine (L), isoleucine (I), valine (V), phenylalanine (F), or methionine (M), and X represents any amino acid.

[0132] Figure 17 is a graph showing the quantitative analysis of the nuclear / cytoplasmic GFP fluorescence intensity ratio and the number and intensity of intranuclear puncta in AXL truncation mutant human astrocytes.

[0133] Figure 18 shows in silico analysis and immunoblot analysis results supporting the possibility that AXL truncates form dimers or multimers.

[0134] Figure 19 is an image comparing immunoblot analysis of LC3B and p62 proteins in U87MG cells expressing AXL truncations of various lengths.

[0135] Figure 20 is an image comparing the cutting positions of AXL and APP.

[0136] Figure 21 is an image of quantitative RT-PCR analysis for LC3B and p62 mRNA levels in U87MG cells expressing AXL-ICD (Δ460-894).

[0137] Figure 22 is an image of LC3B-GFP analysis of AXL-ICD overexpressed cells.

[0138] Figure 23 shows the results of immunoblot analysis and quantitative analysis of autophagy-related proteins in U87MG cells expressing AXL-ICD (Δ460-894).

[0139] Figure 24 is a volcano plot showing differentially expressed genes (DEGs) between AXL-ICD overexpressing cells and the mock control group.

[0140] Figure 25 is a heatmap image showing the relative expression (Z-score) of selected genes.

[0141] Figure 26 is an image showing the results of KEGG pathway analysis for DEGs.

[0142] Figure 27 shows the results of GO biological process analysis.

[0143] Figure 28 shows the results of GSEA analysis.

[0144] Figure 29 shows the results of ATAC-seq analysis.

[0145] Figure 30 is a graph showing representative immunofluorescence images and quantitative analysis of SIRT2 intensity in the nucleus per cell obtained after Aβ treatment in U87MG cells with AXL knockdown applied.

[0146] Figure 31 is an image showing SIRT2 nuclear translocation observed after Aβ treatment in mouse astrocytes.

[0147] Figure 32 is an immunofluorescence image showing the distribution of SIRT2 in U87MG cells overexpressing AXL-ICD, a graph showing quantitative analysis of SIRT2 intensity, and an immunoblot analysis image showing nuclear enrichment of SIRT2 induced by AXL-ICD.

[0148] Figure 33 is an image of immunoblot analysis using kinase-dead mutant (KD).

[0149] Figure 34 is an image of the immunoco-precipitation analysis between HA-tagged AXL-ICD and FLAG-tagged Sirtuin family proteins (SIRT1-SIRT7) in HEK293T cells.

[0150] Figure 35 shows the results of proximity ligation assay (PLA) between AXL-ICD-HA and endogenous SIRT2 observed in U87MG cells.

[0151] Figure 36 is an immunoco-precipitation analysis image showing the binding sites between FLAG-tagged SIRT2 constructs and HA-tagged AXL-ICD in HEK293T cells.

[0152] Figure 37 shows the results of protein structure simulation analysis predicting the detailed binding site between AXL-ICD and SIRT2, and images of immunoblotting performed with anti-HA and anti-SIRT2 antibodies after immunoprecipitation with HA antibody in HEK293T cells.

[0153] Figure 38 is an immunofluorescence image observing the expression of AXL in astrocytes of brain tissue of an Alzheimer's patient and a graph showing its quantification.

[0154] Figure 39 is an immunofluorescence image and a graph showing the expression and nuclear location of SIRT2 in astrocytes of brain tissue of an Alzheimer's patient and its quantification.

[0155] Figure 40 shows the results of proximity ligation assay (PLA) confirming colocalization of AXL-SIRT2 in brain tissue of Alzheimer's patients.

[0156] Figure 41 shows the results of immunoblot analysis of autophagy-related proteins induced by AXL-ICD overexpression in U87MG cells with SIRT2 knockdown.

[0157] Figure 42 shows the results of immunoblot analysis of autophagy-related proteins induced by AXL-ICD overexpression in U87MG cells after treatment with the SIRT2 inhibitor AGK2.

[0158] Figure 43 is an immunofluorescence image and a quantification graph analyzing the nuclear condensate of AXL in U87MG cells with SIRT2 knockdown.

[0159] Figure 44 shows the peptide sequence information used in the experiment and the results of immunoblot analysis of LC3B protein after treatment with the peptide in U87MG cells, and images of immunoprecipitation with anti-HA antibody and immunoblot analysis performed with anti-HA and anti-SIRT2 antibodies.

[0160] Figure 45 is an image analyzing autophagy flux under glucose starvation conditions after AICD-TAT peptide treatment in U87MG cells.

[0161] Figure 46 shows the results of immunoblot analysis of autophagy-related proteins after treatment with Aβ and AICD-TAT peptides in U87MG cells.

[0162] Figures 47 and 48 are a schematic diagram of proteomic analysis to identify transcriptional cofactors interacting with AXL-ICD, and graphs of the results of gene ontology (GO) analysis and Volcano plot analysis.

[0163] Figure 49 is an image of immunoblotting performed after HA immunoprecipitation in astrocytes and U87MG cells expressing AXL-ICD-HA to identify proteins interacting with AXL-ICD identified through proteomic analysis.

[0164] Figure 50 is an image showing PAQosome components interacting with AXL-ICD identified through proteomic analysis.

[0165] Figure 51 shows the results of immunostaining and Western blot analysis for RPB1 protein in U87MG cells overexpressing AXL-ICD.

[0166] Figure 52 shows the results of immunostaining and Western blot analysis for RPB1 protein in AXL knockdown U87MG cells.

[0167] Figure 53 shows the immunoblotting results of RUVBL2 and RPB1 proteins.

[0168] Figure 54 is a diagram comparing histone modifications by Aβ and AXL-ICD.

[0169] Figure 55 is a fluorescence microscope image and FACS result image observing autophagosome induction by AXL-ICD.

[0170] Figure 56 is a graph showing a fluorescence microscope image confirming the removal of Aβ plaques by autophagosomes induced by AXL-ICD and its quantitative analysis.

[0171] The following examples are provided for more detailed description. However, these examples are provided solely to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0172]

[0173] Experimental Example 1. Confirmation of AXL's regulation of gene expression related to amyloid beta (Aβ)-selective autophagy.

[0174] 1.1 Confirmation of Aβ induction of autophagy gene expression

[0175] First, we examined whether Aβ treatment induces autophagy gene expression in human astrocytes. Specifically, human astrocyte cultures were treated with 4 mM Aβ oligomers for 24 hours, and immunoblot analysis of autophagy-related proteins was performed.

[0176] As a result, it was confirmed that when human astrocytes were treated with Aβ, the expression of autophagy-related proteins such as LC3B, p62, and ATG7 significantly increased (Fig. 2 and Fig. 3).

[0177]

[0178] 1.2 Confirmation of TAM (Tyro3, ​​AXL, and Mertk) receptor knockdown and autophagy gene expression

[0179] Recently, it has been shown that TAM receptors act as Aβ receptors [Huang Y, et al., “Microglia use TAM receptors to detect and engulf amyloid β plaques” Nat Immunol. 2021 May;22(5):586-594. doi: 10.1038 / s41590-021-00913-5] and are highly expressed in astrocytes (Fig. 5, https: / brainrnaseq.org / ), so we investigated the role of Tyro3, ​​AXL, and Mertk in linking Aβ and autophagy gene activity.

[0180] Specifically, LC3B expression was observed after knockdown of each receptor. Specifically, AXL knockdown resulted in a decrease in LC3B expression, whereas Mertk and Tyro3 knockdown did not induce significant changes. Furthermore, the decrease in LC3B expression in AXL knockdown cells appeared to be due to a decrease in mRNA levels (Figures 4 and 6).

[0181]

[0182] Additionally, autophagy flow was analyzed in AXL knockdown cells treated with 100 nM Bafilomycin A1 for 12 hours, and the results are shown in Fig. 7, and immunoblot analysis of LC3B expression in AXL knockdown cells treated with 4 mM Aβ oligomers for 24 hours is shown in Fig. 8.

[0183] As a result, in the autophagy flow analysis using Bafilomycin A1, it was confirmed that AXL knockdown reduced autophagy flow, and LC3B gene expression was not induced in the AXL knockdown state despite treatment with Aβ.

[0184]

[0185] Taken together, these results imply that AXL is a key mediator of Aβ-induced autophagy gene expression in astrocytes.

[0186]

[0187] Experimental Example 2. Confirmation of AXL fragmentation and nuclear localization.

[0188] 2.1 Confirmation of AXL fragmentation of Aβ

[0189] First, we investigated the effect of Aβ on AXL expression. Specifically, immunoblot analysis for AXL and LC3B was performed in human astrocytes treated with Aβ oligomers in the presence or absence of 250 ng / ml GAS6 for 24 h.

[0190] As a result, we observed a marked increase in AXL fragments measuring approximately 50 kDa, although total AXL expression remained unchanged. This fragmentation was further enhanced in the presence of GAS6, a ligand for AXL (Fig. 9, left). A similar phenomenon was observed in U87MG, a glioma cell line derived from astrocytes (Fig. 9, right).

[0191]

[0192] 2.2 Confirmation of nuclear localization of AXL fragments

[0193] Like many receptor tyrosine kinases (RTKs), AXL undergoes cleavage processing, with the C-terminal fragment formed by α-secretase, which is then cleaved again by γ-secretase to release the AXL intracellular domain (ICD) into the cytoplasm. Although it has been previously reported that the intracellular domain of AXL translocates to the nucleus, its functional role was unknown. To elucidate this, we analyzed immunofluorescence images of human astrocytes expressing full-length AXL fused to the C-terminus with GFP, treated with DMSO or 4 mM Aβ and 250 ng / ml GAS6 for 24 h. We also analyzed immunostaining images using antibodies that recognize only the N-terminus of AXL or antibodies that recognize both the N- and C-termini. Additionally, immunoblot analysis of AXL was performed on cytoplasmic and nuclear extracts fractionated from human astrocytes treated with various concentrations of Aβ, using Lamin B1 and β-actin as nuclear and cytoplasmic markers, respectively.

[0194] Consistent with previous results, treatment of cells expressing full-length AXL fused to the C-terminus with Aβ and GAS6 resulted in translocation of the GFP signal to the nucleus. Immunostaining with an AXL antibody that recognizes the C-terminus but not the N-terminus confirmed the presence of truncated C-terminal AXL in the nucleus (Fig. 10).

[0195] In cell fractionation analysis, the level of AXL intracellular domain in the nuclear fraction increased in a concentration-dependent manner following Aβ treatment (Fig. 11).

[0196]

[0197] Additionally, immunoblot analysis for LC3B was performed in human astrocytes treated with 4 mM Aβ oligomers for 24 h in the presence or absence of DAPT (10 mM), a γ-secretase inhibitor.

[0198] When treated with DAPT, a γ-secretase inhibitor, the increase in LC3B expression induced by Aβ was suppressed (Fig. 12).

[0199]

[0200] Taken together, these results suggest that Aβ cleaves AXL via γ-secretase to generate the intracellular domain of AXL, which likely increases LC3B mRNA expression in the nucleus.

[0201]

[0202] Experimental Example 3. Identification of a functional sequence in the AXL intracellular domain.

[0203] To identify γ-secretase-truncation AXL mutants that translocate to the nucleus in response to Aβ, various AXL truncation mutants with GFP fused to the C-terminus were constructed as shown in Table 1. The amino acid sequence from position 477 to position 894 is omitted.

[0204]

[0205] Production mutant amino acid sequences SEQ ID NO: Δ454-894-GFPLLGAVVAAACVLILALFLVHRRKK … SEQ ID NO: 8 Δ460-894-GFPAAACVLILALFLVHRRKK … SEQ ID NO: 9 Δ466-894-GFPILALFLVHRRKK … SEQ ID NO: 10 Δ470-894-GFPFLVHRRKK … SEQ ID NO: 11 Δ473-894-GFPHRRKK … SEQ ID NO: 12

[0206] Representative immunofluorescence images were analyzed after human astrocytes expressing AXL truncation mutants were treated with DMSO or the nuclear export inhibitor leptomycin B (LMB) for 16 h. Nuclei were visualized by DAPI staining (Fig. 14).

[0207] In addition, the ratio of nuclear / cytoplasmic GFP fluorescence intensity was quantified in each construct (Fig. 17, top), and the number and intensity of intranuclear puncta were quantified and analyzed in LMB-treated AXL(Δ454-894), AXL(Δ460-894), and AXL(Δ466-894)-expressing cells (Fig. 17, bottom). All quantitative analyses were performed using ImageJ, and data are expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical significance was analyzed by one-way analysis of variance (ANOVA) and Tukey's post hoc test, and is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0208] As a result, AXL(Δ454-894), which had the N-terminus deleted, did not exhibit any nuclear GFP signal, and no nuclear fluorescence was observed even after treatment with leptomycin B (LMB), which inhibits nuclear export. This may be because the construct contained most of the transmembrane domain and was therefore anchored to the membrane. AXL(Δ460-894) exhibited a stronger nuclear GFP signal than Δ454-894, and some fluorescence still remained in the cytoplasm. Notably, LMB treatment formed nuclear condensates similar to those observed in Aβ-treated cells. AXL(Δ466-894) exhibited a stronger nuclear GFP signal, but the number and intensity of nuclear condensates after LMB treatment were significantly reduced compared to Δ460-894.

[0209] This suggests that the 460-466 region of AXL plays an important role in the formation of condensates within the nucleus.

[0210]

[0211] Additional truncations, such as Δ470-894 and Δ473-894, exhibited strong nuclear signals even without LMB treatment, likely due to the presence of a nuclear localization signal (NLS) in the 473-477 (HRRKK) region. This nuclear localization change was similarly observed in U87MG cells (Fig. 15 ).

[0212] Interestingly, despite containing an NLS, Δ466-894 showed a significantly weaker nuclear signal compared to Δ473-894, suggesting the presence of a nuclear export signal (NES) between 466 and 473 (Fig. 13). Supporting this hypothesis, the “465LILALFLV472” sequence contains numerous hydrophobic amino acid residues and shares features consistent with the consensus sequence of classical NES sequences (Fig. 16). This suggests that this sequence may function as a functional NES.

[0213]

[0214] Additionally, a molecular docking model was generated using Schrodinger software to gain structural insight into AXL (Δ466-894).

[0215] As a result, it was found that the N-terminus and C-terminus of AXL (Δ460-894) bind in opposite directions to form a reverse dimer. The binding sites are calculated and expressed as amino acid positions of AXL-ICD, which are R476-E826, E495-R746, R499-Y866, and K542-E815 (Fig. 18).

[0216] To determine whether AXL (Δ460-894) forms a dimer within cells, constructs in which different tags, FLAG tag or HA tag, were fused to the C-terminus of AXL (Δ460-894) were expressed in HEK293T cells, and immunoprecipitation analysis was performed to determine whether they bind to each other. After immunoprecipitation with anti-HA antibody, immunoblotting was performed with anti-FLAG and anti-HA antibodies, and the protein input amount was presented as a loading control.

[0217] As a result, FLAG-tagged AXL (Δ460-894) and HA-tagged AXL (Δ460-894) were found to bind to each other, which means that at least AXL (Δ460-894) forms a dimer. In addition, it was observed that Δ460-854, which is a truncated version of the C-terminus of AXL (Δ460-894), showed a greater binding affinity than Δ460-894, suggesting that the amino acids between 854 and 894 are the sites that interfere with dimer formation (Fig. 18).

[0218] Next, to observe whether dimer formation is related to the autophagy activation of AXL-ICD, immunoblot analysis and quantitative analysis of autophagy-related proteins were performed in U87MG cells expressing Δ460-894 and Δ460-854, respectively. Quantification was normalized to GAPDH.

[0219] As a result, it was observed that Δ460-854 increased the amount of LC3B more than Δ460-894, suggesting that dimer formation is directly related to autophagy activity.

[0220]

[0221] Experimental Example 4. Confirmation of induction of transcriptional expression of autophagy-related genes in AXL-ICD.

[0222] 4.1 Comparison of the ability of AXL intracellular domains to induce autophagy-related gene expression

[0223] Based on the analysis of nuclear entry of the AXL (Δ460-894) construct, various AXL truncations near residue 460 were designed and introduced into cells, and their ability to induce expression of autophagy-related genes such as LC3B and p62 was evaluated.

[0224] First, immunoblot analysis of LC3B and p62 was performed in U87MG cells expressing AXL truncates fused to the C-terminus with an HA tag. HA blots indicate expression of each construct, and GAPDH served as a loading control.

[0225] As a result, the Δ460-894 construct strongly induced the expression of LC3B and p62, but progressive N-terminal truncation gradually weakened this effect, and the Δ464-894 construct had no inducing activity at all. Conversely, Δ456-894 and Δ458-894, which had only a few amino acids extended from the N-terminus of Δ460-894, showed significantly reduced autophagy gene induction abilities compared to Δ460-894 (Fig. 19).

[0226] These results suggest that Δ460-894 is the most potent transcriptionally active form of AXL produced by γ-secretase and is particularly effective in inducing autophagy genes. Interestingly, the cleavage site at position 460 of AXL shares sequence similarity with the γ-secretase cleavage site (position 40) of APP (Fig. 20).

[0227]

[0228] 4.2 Confirmation of induction of autophagy-related gene expression in AXL-ICD

[0229] To elucidate the function of AXL-ICD, we analyzed whether it could induce autophagy-related gene expression. Various AXL truncations were designed and introduced into cells, and changes in LC3B and p62 expression were observed. Among them, AXL (Δ460-894) most significantly increased LC3B and p62 expression (Fig. 19). Therefore, AXL (Δ460-894) was designated AXL-ICD and used in subsequent experiments.

[0230] Overexpression of AXL-ICD induced a marked increase in LC3B and p62 mRNA levels (Fig. 21). Gene expression was normalized to GAPDH and expressed as relative expression compared to the control group. Data are the means ± SEM from three independent experiments, and statistical significance was analyzed using a Student's t-test between the two groups (*p < 0.05, **p < 0.01). Furthermore, this was accompanied by autophagosome formation (Fig. 22).

[0231]

[0232] We also performed immunoblot analysis and quantitative analysis of autophagy-related proteins in U87MG cells expressing AXL-ICD (Δ460-894) fused to the C-terminus with an HA tag. Quantification was normalized to GAPDH, and data are expressed as the mean ± SEM from at least three independent experiments. Statistical analysis was performed using Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

[0233] As a result, AXL-ICD increased the protein expression of p62, ATG7, and LC3B, while leaving ULK1 unchanged (Fig. 23). This expression pattern was very similar to that observed in Aβ-treated cells (Fig. 2).

[0234] These results suggest that Aβ-induced autophagy gene activation is mediated through AXL-ICD generation and nuclear translocation.

[0235]

[0236] 4.3 RNA-seq analysis

[0237] To understand the overall transcriptional effects of AXL-ICD, RNA-seq analysis was performed between U87MG cells overexpressing AXL-ICD and the control group.

[0238] Volcano plot analysis revealed that numerous genes involved in autophagy, including LC3B, SQSTM1, GABARAPL1, ATG3, and RAB39B, were significantly upregulated (Fig. 24). Heatmap clustering also showed that autophagy-related gene signatures were extensively induced in AXL-ICD-expressing cells (Fig. 25). KEGG pathway analysis revealed that inflammatory pathways, including autophagy, NF-κB, TNF, and IL-17, were upregulated, while cell cycle and DNA replication-related pathways were downregulated (Fig. 26).

[0239] GO biological process analysis also showed a significant enrichment of terms related to macroautophagy and its regulation (Fig. 27). GSEA analysis also showed a marked enrichment of gene sets related to autophagosome structure and membrane components (Fig. 28). In addition, ATAC-seq analysis was performed to determine whether AXL-ICD transcriptionally activates gene expression and is accompanied by changes in chromatin structure. As a result, chromatin accessibility was confirmed to be increased in the promoters of key autophagy genes such as LC3B, SQSTM1, and GABARAPL1 in AXL-ICD-expressing cells (Fig. 29).

[0240]

[0241] Taken together, these results suggest that AXL-ICD not only induces transcriptional activation of autophagy genes but also reorganizes chromatin structure to promote their expression.

[0242]

[0243] Experimental Example 5. Confirmation of the interaction between AXL-ICD and SIRT2.

[0244] 5.1 Confirmation of Aβ treatment and changes in SIRT2 expression

[0245] First, we investigated whether SIRT2, a major histone deacetylase (HDAC), acts as a key regulator of Aβ stimulation in mice as well as in human astrocytes, and its expression was increased by Aβ treatment. Specifically, immunofluorescence images were analyzed after applying a nonspecific control (NS) or AXL knockdown in human astrocytes and then treating them with DPBS or 4 μM Aβ for 24 h. Nuclei were stained with DAPI, and the scale bar is 10 μm. The quantitative data of SIRT2 intensity in the nucleus per cell are expressed as the mean ± SEM, and statistical analysis was performed using a two-way ANOVA and Tukey's post-hoc test (p < 0.0001 for all pairwise comparisons).

[0246] As a result, SIRT2 nuclear intensity increased upon Aβ treatment, and this increase was suppressed in AXL knockdown cells. In other words, we observed that Aβ stimulation induced the nuclear localization of SIRT2 in mouse astrocytes as well (Figs. 31 and 30). In particular, when AXL was knocked down, the nuclear translocation of SIRT2 induced by Aβ was significantly reduced.

[0247] This suggests that AXL plays a key role in regulating SIRT2 localization in response to Aβ.

[0248]

[0249] 5.2 Confirmation of SIRT2 nuclear translocation mediation in AXL-ICD

[0250] To confirm whether AXL-ICD directly mediates the nuclear translocation of SIRT2, after forcibly expressing AXL-ICD, the distribution of SIRT2 was confirmed by immunofluorescence imaging and SIRT2 intensity was quantitatively analyzed. Nuclei were stained with DAPI, and the scale bar is 10 μm. Quantitative analysis is the mean ± SEM from three independent experiments, and statistical analysis was performed by two-way ANOVA and Tukey's post hoc test (p < 0.0001). In addition, to analyze the expression of SIRT2 and AXL-ICD-HA in the cytoplasmic and nuclear fractions, GAPDH and Lamin B1 were used as cytoplasmic and nuclear markers, respectively, and nuclear enrichment of SIRT2 was confirmed by immunoblot analysis.

[0251] As a result, the nuclear intensity of SIRT2 increased in response to AXL-ICD expression. This confirms that AXL-ICD is sufficient to induce nuclear translocation of SIRT2 even in the absence of Aβ (Fig. 32, top). Further supporting this, in a doxycycline (Dox)-inducible AXL-ICD expression system, the nuclear translocation of SIRT2 was also found to increase in proportion to the level of AXL-ICD (Fig. 32, bottom).

[0252]

[0253] 5.3 Confirmation of the combination of AXL-ICD and SIRT2

[0254] We confirmed that AXL-ICD selectively interacts with SIRT2 among sirtuin family proteins.

[0255] Specifically, we performed an immunoco-precipitation assay between HA-tagged AXL-ICD and FLAG-tagged Sirtuin family proteins (SIRT1-SIRT7) in HEK293T cells. After immunoprecipitation with an anti-HA antibody, immunoblotting was performed with anti-HA and anti-FLAG antibodies. An empty vector (Mock) was used as a negative control. In addition, a proximity ligation assay (PLA) was performed to detect the proximity between AXL-ICD-HA and endogenous SIRT2. The number of PLA puncta per cell was quantified as the mean ± SEM from three independent experiments, and statistical analysis was performed using an unpaired two-tailed Student's t-test (p < 0.001).

[0256] As a result, we confirmed that AXL-ICD selectively interacts with SIRT2 (Fig. 34). We also confirmed that AXL-ICD and SIRT2 coexist in both the cytoplasm and nucleus (Fig. 35). We confirmed that this interaction is independent of the enzymatic activity of AXL, as the binding to SIRT2 is maintained even in the absence of AXL kinase activity (Fig. 33).

[0257]

[0258] 5.4 Identification of binding sites of AXL-ICD and SIRT2

[0259] Mutational analysis was performed to identify the binding site involved in the interaction. Immunoprecipitation analysis was performed between FLAG-tagged SIRT2 constructs and HA-tagged AXL-ICD in HEK293T cells for full-length (WT, aa 1-373), N-terminal deletion (ΔN, aa 89-389), C-terminal deletion (ΔC, aa 1-356), and a construct containing only the catalytic domain (aa 89-356). Immunoprecipitation was performed with anti-FLAG antibody, followed by immunoblotting with anti-HA and anti-FLAG antibodies. The protein input amount was presented as a loading control.

[0260] As a result, we revealed that the catalytic domain of SIRT2 (Fig. 36, left) and a four-amino acid sequence (460AAAC463) on AXL-ICD are essential for binding (Fig. 36, right). Interestingly, this AXL-ICD sequence exactly matches the region identified as crucial for the induction of autophagy genes in Fig. 19.

[0261] This suggests that AXL-ICD / SIRT2 interaction is a central mechanism for AXL-ICD-mediated autophagy induction.

[0262]

[0263] Additionally, molecular docking models were generated using Schrodinger software to gain structural insights into this interaction.

[0264] As a result, the AXL-ICD peptide (amino acids 460-467; AAACVLI) was found to bind to the groove on the surface of SIRT2 and contact residues E163, Q164, and E165 within the SIRT2 catalytic domain (Fig. 37, left).

[0265] To verify the functional importance of this region, a mutant was created in which the 163EQE165 sequence of SIRT2 was substituted with 163AQA165, and its binding ability to AXL-ICD was evaluated.

[0266] Co-immunoprecipitation analysis revealed that the AQA mutant exhibited altered interaction with AXL-ICD compared to the wild type (Fig. 37, right), suggesting that the EQE motif of SIRT2 regulates AXL-ICD binding and constitutes part of the direct interaction surface.

[0267] The above results suggest that AXL-ICD specifically binds to the catalytic domain of SIRT2, thereby inducing nuclear translocation of SIRT2 and mediating autophagy gene expression.

[0268]

[0269] Experimental Example 6. Confirmation of the expression and interaction of AXL and SIRT2 in patient brain tissue.

[0270] The expression of AXL and SIRT2 in astrocyte tissues of normal subjects and Alzheimer's patients was confirmed through immunostaining and quantified, and the binding of the two proteins was confirmed through PLA.

[0271]

[0272] As a result, it was confirmed that the expression of AXL in astrocytes of the brain tissue of Alzheimer's patients increased and was located in the nucleus (Fig. 38), the expression of SIRT2 also increased and was located in the nucleus (Fig. 39), and it was observed that AXL and SIRT2 bind and that the binding increased in Alzheimer's patients (Fig. 40).

[0273]

[0274] Experimental Example 7. Confirmation of Aβ-selective autophagy activation through AXL-ICD and SIRT2 interactions.

[0275]

[0276] 7.1 Confirmation of the role of SIRT2 in AXL-ICD-induced autophagy gene expression

[0277] First, we confirmed the role of SIRT2 in autophagy gene expression induced by AXL-ICD.

[0278] Specifically, the effects of SIRT2 knockdown using shRNA (Figure 41) or the SIRT2 inhibitor AGK2 (Figure 42) on autophagy-related gene expression induced by AXL-ICD overexpression in U87MG cells were analyzed by immunoblot. GAPDH was used as a loading control, and data are presented as the mean ± SEM of five and three independent experiments, respectively. Statistical analysis was performed using two-way ANOVA and Tukey's post-hoc test, and significance was shown at the **p < 0.01 and ***p < 0.001 levels.

[0279] As a result, the increase in LC3B expression induced by AXL-ICD was effectively suppressed, indicating that SIRT2 is essential for AXL-ICD-mediated LC3B induction (Figure 41). In contrast, AGK2, a drug that inhibits the enzymatic activity of SIRT2, did not affect the increase in LC3B expression (Figure 42). Furthermore, SIRT2 knockdown also suppressed AXL nuclear condensate formation (Figure 43).

[0280] This suggests that the interaction itself, rather than the catalytic activity of SIRT2, is important in this process.

[0281]

[0282] Additionally, we verified whether direct binding between AXL-ICD and SIRT2 is essential for autophagy induction. To this end, we designed a competitive inhibitory peptide containing the SIRT2-binding domain of AXL-ICD (460AAACVLI466) and fused it to the TAT peptide (TAT-AICD) to facilitate intracellular uptake. The effect of this inhibitory peptide on LC3B expression induced by AXL-ICD overexpression was analyzed by immunoblot. HA indicates AXL-ICD expression, and GAPDH served as a loading control.

[0283]

[0284] Furthermore, the effect of inhibitory peptides on the interaction between HA-tagged AXL-ICD and SIRT2 was evaluated using immunoprecipitation analysis. HA-tagged AXL-ICD was overexpressed in U87MG cells for 24 h, followed by treatment with inhibitory peptides for 24 h. Cell lysates were then immunoprecipitated with anti-HA antibodies, and immunoblot analysis was performed with anti-HA and anti-SIRT2 antibodies. Input SIRT2 served as a loading control.

[0285] Analysis of the effects of peptides on LC3B expression revealed that the TAT fusion site was critical for inhibitory activity. Specifically, the C-terminal TAT-bound form (AICD-TAT) inhibited the AXL-ICD-induced increase in LC3B expression even at a concentration of 5 μM, whereas the N-terminal fusion form (TAT-AICD) exhibited a similar effect at a higher concentration of 50 μM (Figure 44, top).

[0286] This suggests that proper exposure of the SIRT2 binding site is important for effective interaction with SIRT2 and signaling inhibition.

[0287] In addition, AICD-TAT effectively inhibited the interaction between AXL-ICD and SIRT2 in a concentration-dependent manner (Fig. 44, bottom).

[0288]

[0289] 7.2 Analysis of autophagy flux

[0290] Autophagy flux was analyzed under glucose starvation conditions after treatment with inhibitory peptides or TAT control in U87MG cells. Cells were cultured for 18 h with glucose deprivation simultaneously with peptide treatment, and then treated with 100 nM bafilomycin A1 for an additional 6 h.

[0291] As a result, the amount of LC3-II accumulation measured after treatment with Bafilomycin A1 under glucose deprivation conditions did not differ between the TAT treatment group and the AICD-TAT treatment group (Fig. 45), indicating that the peptide did not affect autophagy induced by nutrient deprivation.

[0292] This suggests that the autophagy pathway induced by AXL-ICD / SIRT2 is a distinct mechanism from the canonical autophagy pathway induced by nutritional deprivation.

[0293]

[0294] Next, we confirmed through immunoblot analysis whether the AXL-ICD / SIRT2 interaction is a critical mechanism for Aβ-induced autophagy. Protein expression was quantified using ImageJ, and data are presented as the mean ± SEM from three independent experiments. Statistical analysis was performed using a two-way ANOVA followed by Tukey's post hoc test, and significance was indicated at the *p < 0.05 and **p < 0.01 levels.

[0295] As a result, AICD-TAT significantly inhibited the increase in LC3B and p62 expression induced by Aβ treatment (Fig. 46).

[0296] This demonstrates that the AXL-ICD / SIRT2 interaction plays a key role in regulating the Aβ-specific atypical autophagy pathway.

[0297] In conclusion, we confirmed that the interaction between AXL-ICD and SIRT2 acts as an important molecular switch in the regulation of autophagy induced by Aβ stimulation.

[0298]

[0299] Experimental Example 8. Confirmation of the interaction between AXL-ICD and transcriptional cofactors.

[0300] 8.1 Gene Ontology (GO) Analysis and Volcano Plot Analysis

[0301] Because AXL-ICD does not have a DNA-binding domain, we hypothesized that its transcriptional regulatory function is achieved through interactions with transcriptional cofactors. To verify this, we performed immunoprecipitation followed by mass spectrometry (IP-MS) in human astrocytes and U87MG cells expressing AXL-ICD-HA. Specifically, human astrocytes or U87MG cells were infected with lentiviruses expressing mock or AXL-ICD-HA. After collecting cell lysates, immunoprecipitation using an anti-HA antibody and mass spectrometry (MS)-based proteomics analysis were performed to identify AXL-ICD interacting proteins. Gene ontology (GO) analysis and Volcano plot analysis were performed on the identified interacting proteins.

[0302] Gene ontology (GO) analysis revealed a significant enrichment of biological processes related to protein metabolism regulation in both cell types, suggesting that AXL-ICD interacts with conserved cellular machinery (Fig. 47). Volcano plot analysis highlighted high-confidence AXL-ICD interacting proteins, including transcriptional regulators WDR5, RUVBL1, and RUVBL2, as well as proteins such as ERLIN1 / 2 and ATAD3A, which were consistently enriched in both astrocytes and U87MG cells (Fig. 48).

[0303]

[0304] 8.2 Immunoblot analysis results

[0305] To verify the AXL-ICD interacting proteins, HA immunoprecipitation and immunoblotting were performed in astrocytes and U87MG cells expressing AXL-ICD-HA. As a result, specific interacting proteins were detected in the HA-IP samples (Fig. 49, left).

[0306]

[0307] Additionally, the effects of knockdown of RUVBL2, SIRT2, and INO80 on AXL-ICD-induced LC3B expression were analyzed by immunoblotting. The results confirmed that RUVBL2, SIRT2, and INO80 interact with various transcriptional regulators (Fig. 49, middle and right).

[0308]

[0309] Experimental Example 9. Confirmation of assembly and stabilization of the RNA polymerase II (RNAP II) complex of AXL-ICD.

[0310] Proteomic analysis identified several PAQosome components as proteins that interact with AXL-ICD (Fig. 50). The PAQosome-mediated RNAP II assembly mechanism involves the R2TP subunit, the URI1 prefoldin subunit, RNAP II subunits, and Hsp90.

[0311]

[0312] AXL-ICD likely stabilizes RNAP II by increasing the interaction between RNAP II and the PAQosome. As shown in immunohistochemistry and Western blot analysis, overexpression of AXL-ICD increased the expression of RPB1, a subunit of RNAPII, in U87MG cells (Fig. 51), whereas knockdown of AXL decreased RPB1 protein levels in U87MG cells (Fig. 52). Expression of DKDNFFJ and AXL-ICD increased the interaction between RUVBL2, a component of the PAQosome, and RPB1 (Fig. 53).

[0313]

[0314] Furthermore, histone modifications altered by Aβ treatment and AXL-ICD expression are associated with RNAP II (Fig. 54). Amyloid-β (Aβ) treatment alters specific histone methylation sites, including histone H3 lysine K4 dimethylation (H3K4me2), H3K4 trimethylation (H3K4me3), H3K36 trimethylation (H3K36me3), and H3K79 trimethylation (H3K79me3), in human astrocytes. Expression of AXL-ICD alters the same histone modification sites altered by Aβ in U87MG cells.

[0315]

[0316] In summary, AXL-ICD binds to SIRT2 to form the AXL-ICD / SIRT2 complex, which interacts with downstream proteins, R2TP and the INO80 family. This binding promotes chromatin remodeling and the recruitment of RNA polymerase II, ultimately inducing the expression of autophagy genes.

[0317]

[0318] Experimental Example 10. Confirmation of autophagosome induction by AXL-ICD.

[0319] We further analyzed whether AXL-ICD induces autophagosome formation using Fyco1-mRFP, a probe capable of visualizing endogenous LC3A and LC3B, and ATG4B(T), a probe capable of visualizing endogenous GABARAP and GABARAPL1. Mock or AXL-ICD was expressed in U87MG cells expressing each probe, and then observed under a fluorescence microscope.

[0320] As a result, we confirmed that the formation of autophagosomes containing LC3 and GABARAP was induced in AXL-ICD-expressing cells (Fig. 55, top). Furthermore, using the Cyto-ID Autophagy Detection Kit (Enzo Life Sciences), fluorescence-activated cell sorting (FACS) was performed, and we observed that autophagosome formation increased in AXL-ICD-expressing cells (Fig. 55, bottom).

[0321]

[0322] Additionally, we evaluated whether AXL-ICD-induced autophagosomes were effective in clearing amyloid-β plaques. U87MG cells were infected with lentiviruses expressing Mock or AXL-ICD-HA, treated with the same concentration of Aβ 24 h later, and fixed and stained with an Aβ-recognizing antibody 72 h later. Fluorescence microscopy images were acquired at seven randomly selected locations and quantitatively analyzed.

[0323] As a result, it was confirmed that the amount of Aβ was significantly reduced in cells expressing AXL-ICD (Fig. 56).

[0324]

[0325] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. An isolated peptide comprising the amino acid sequence of sequence number 1.

2. A peptide according to claim 1, wherein the peptide further comprises an addition of 20 or fewer amino acids at the N-terminus, the C-terminus, or both.

3. In claim 1, the peptide further comprises 20 or fewer amino acids in the N-terminal direction from the 459th amino acid of the AXL protein of SEQ ID NO: 7; or A peptide comprising an additional 20 or fewer amino acids in a C-terminal direction from the 467th amino acid of the AXL protein of sequence number 7 at the C-terminus.

4. A peptide according to claim 1, wherein the peptide further comprises a cell penetrating peptide (CPP) at the N-terminus, the C-terminus, or both.

5. In claim 1, the peptide comprises an amino acid sequence of any one or more of SEQ ID NOs: 2, 4, 5, and 6.

6. A peptide according to claim 1, wherein the peptide inhibits intracellular amyloid beta selective autophagy.

7. A carrier comprising a peptide according to any one of claims 1 to 6, or a polynucleotide encoding the same.

8. A pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising as an active ingredient an intracellular domain of AXL; a polynucleotide encoding the same; or a transporter comprising the intracellular domain or the polynucleotide.

9. A pharmaceutical composition according to claim 8, wherein the intracellular domain of AXL comprises the amino acid sequence of SEQ ID NO:

9.

10. A pharmaceutical composition according to claim 8, further comprising a SIRT2 protein.

11. A pharmaceutical composition according to claim 8, which is administered in combination with SIRT2 protein.

12. A pharmaceutical composition according to claim 1, wherein the neurodegenerative disease is at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Lewy body dementia, progressive supranuclear palsy, frontotemporal dementia, tauopathies, cerebral amyloid angiopathy, and Dutch-type hereditary cerebral amyloid angiopathy.

13. Step of treating the candidate material to cells; A step of measuring the interaction of AXL protein and a protein binding to AXL protein in cells treated with the candidate substance or the expression of the intracellular domain of AXL protein; and A step of selecting a candidate substance that changes the interaction between the AXL protein and a protein binding to the AXL protein or the expression of the intracellular domain of the AXL protein compared to an untreated control group. A method for screening a drug for the prevention or treatment of a neurodegenerative disease including:

14. Step of treating the candidate material to cells; A step of measuring the interaction of AXL protein and a protein binding to AXL protein in cells treated with the candidate substance or the expression of the intracellular domain of AXL protein; and A step of selecting a candidate substance that changes the interaction between the AXL protein and a protein binding to the AXL protein or the expression of the intracellular domain of the AXL protein compared to an untreated control group. A method for screening an autophagy regulator comprising:

15. A method for screening an autophagy regulator according to claim 14, wherein the autophagy regulator is an amyloid beta-selective autophagy regulator.

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