WO2022262830A1 - 治疗神经元蜡样质脂褐质沉积症的方法和药物 - Google Patents

治疗神经元蜡样质脂褐质沉积症的方法和药物 Download PDF

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WO2022262830A1
WO2022262830A1 PCT/CN2022/099308 CN2022099308W WO2022262830A1 WO 2022262830 A1 WO2022262830 A1 WO 2022262830A1 CN 2022099308 W CN2022099308 W CN 2022099308W WO 2022262830 A1 WO2022262830 A1 WO 2022262830A1
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cln3
disease
compound
keap1
neurons
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French (fr)
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支赟
丁胜
马天骅
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清华大学
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Publication of WO2022262830A1 publication Critical patent/WO2022262830A1/zh

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Definitions

  • the invention relates to a method for treating neurodegenerative diseases such as neuron ceroid lipofuscinosis, especially juvenile neuron ceroid lipofuscinosis (JNCL) and a medicine used for the treatment.
  • neurodegenerative diseases such as neuron ceroid lipofuscinosis, especially juvenile neuron ceroid lipofuscinosis (JNCL) and a medicine used for the treatment.
  • JNCL juvenile neuron ceroid lipofuscinosis
  • NCL Neuronal ceroid lipofuscinosis
  • LSDs lysosomal storage diseases
  • Symptoms of NCLs mostly appear in infancy and childhood, and most patients will show a progressive neurodegenerative clinical process.
  • JNCL juvenile neuronal ceroid lipofuscinosis
  • Batten disease juvenile neuronal ceroid lipofuscinosis
  • the cells of patients with JNCL will show the characteristics of obvious lysosomal dysfunction, and many substances such as intracellular mitochondrial ATP synthase c subunit (SCMAS), lipoproteins and glycoproteins cannot be degraded normally, so that in lysosomes Gradually build up.
  • Neurons are one of the cell types most affected by CLN3 mutations due to their high dependence on lysosomes for material metabolism. Patients will show severe visual impairment around the age of 2-10, followed by epilepsy, progressive movement disorders, and cognitive decline, and usually die of illness between the ages of 20-30.
  • the lysosome is an organelle that plays a key role in the degradation and recycling of extracellular and extracellular materials (Luzio et al., 2009; Mizushima and Komatsu, 2011).
  • a network of lysosomes, endosomes, autophagosomes, and other cellular components function to degrade both extracellular and extracellular materials (Saftig and Klumperman, 2009).
  • Changes in lysosomal acidity can affect the activities of various enzymes in the lumen, thereby disrupting the clearance process of their substrates, leading to the occurrence of various neurodegenerative diseases including NCL (Song et al., 2020).
  • One aspect of the present invention relates to a method for treating diseases related to defects in autophagy function and/or lysosomal function, the method comprising inhibiting the activity of Kelch-like epichlorohydrin-associated protein 1 (KEAP1), and/or A step of enhancing the activity of the transcription factor E2-related factor 2 (NRF2) and/or its downstream proteins.
  • KEAP1 Kelch-like epichlorohydrin-associated protein 1
  • NEF2 transcription factor E2-related factor 2
  • the disease related to autophagy function defect and/or lysosome function defect is a neurodegenerative disease.
  • the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's dementia, Alexander disease, Alper's disease (Alper'sdisease), a total of Ataxic disorders-telangiectasia, bovine spongiform encephalopathy (BSE), Canavan disease, Cockaynesyndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington's disease, HIV-associated dementia, Kennedy disease, Krabbe Krabbedisease, dementia with Lewy bodies, Machado-Joseph disease (spinocerebellar ataxia type 3), multiple sclerosis, multiple system atrophy, neuroborreliosis, Parkinson's disease, Pey-Mey disease , Pick's disease, primary lateral sclerosis, Prion's disease, Refsum's disease, Sandhoff disease, Hilder's disease, schizophrenia, Spielmeyer-Vogt-Sjo
  • ALS amyotrophic
  • the neurodegenerative disease is selected from Parkinson's disease, Alzheimer's disease, Huntington's disease, Kennedy's disease, amyotrophic lateral sclerosis, primary lateral sclerosis, multiple sclerosis, Frontotemporal dementia or neuronal ceroid lipofuscinosis (NCL).
  • Parkinson's disease Alzheimer's disease, Huntington's disease, Kennedy's disease, amyotrophic lateral sclerosis, primary lateral sclerosis, multiple sclerosis, Frontotemporal dementia or neuronal ceroid lipofuscinosis (NCL).
  • the neurodegenerative disease is preferably neuronal ceroid lipofuscinosis, more preferably juvenile neuronal ceroid lipofuscinosis (JNCL).
  • JNCL juvenile neuronal ceroid lipofuscinosis
  • the disease is caused by a mutation of the CLN3 gene, for example, the deletion mutation CLN3 ⁇ ex7/8 of exons 7 and 8 of the CLN3 gene, wherein the deletion mutation is about 1.02kb.
  • siRNA, sgRNA or a vector constructed with shRNA is used to silence or knock down the expression of KEAP1 gene, thereby enhancing the activity of NRF2 and/or its downstream proteins.
  • KEAP1 inhibitors and/or NRF2 activators are used to enhance the expression of NRF2 and/or its downstream genes, thereby enhancing the activity of NRF2 and/or its downstream proteins.
  • the KEAP1 inhibitor and/or NRF2 activator are selected from cysteine residues capable of interacting with cysteine residues including cysteine 151, 272 and/or 288 on KEAP1 Reagents for covalent reactions.
  • the KEAP1 inhibitor and/or NRF2 activator is selected from Carvedilol (CAS: 72956-09-3), Ketoconazole (CAS: 65277-42-1), GANT61, CAS (500579 -04-4), Protriptyline hydrochloride(CAS:1225-55-4), LP 44(CAS:824958-12-5), Doxepin HCl(CAS:1229-29-4), Dimethyl Fumarate (DMF ), acetyl-11-carbonyl- ⁇ -boswellic acid (AKBA), isothiocyanates, bardoxolone (CDDO), bardoxolone methyl (CDDO-Me), and derivatives or analogs of these compounds , or one or more selected from ⁇ , ⁇ -unsaturated carbonyl compounds, phenols and polyphenols.
  • Carvedilol CAS: 72956-09-3
  • Ketoconazole CAS: 65277-42-1
  • GANT61 CAS (500579 -04-4
  • the KEAP1 inhibitor is compound G, namely (Z)-guggulsterone, its structural formula is:
  • Another aspect of the present invention relates to the preparation of KEAP1 inhibitors and/or NRF2 activators for inhibiting the activity of KEAP1, and/or enhancing the activity of transcription factor E2-related factor 2 (NRF2) and/or its downstream proteins to treat cells related to Use in medicine for diseases related to autophagy function defect and/or lysosome function defect.
  • NRF2 transcription factor E2-related factor 2
  • the disease related to autophagy function defect and/or lysosome function defect is a neurodegenerative disease.
  • said neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's dementia, Alexander disease, Alper's disease (Alper'sdisease), total Ataxic disorders-telangiectasia, bovine spongiform encephalopathy (BSE), Canavan disease, Cockaynesyndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington's disease, HIV-associated dementia, Kennedy disease, Krabbe Krabbedisease, dementia with Lewy bodies, Machado-Joseph disease (spinocerebellar ataxia type 3), multiple sclerosis, multiple system atrophy, neuroborreliosis, Parkinson's disease, Pey-Mey disease , Pick's disease, primary lateral sclerosis, Prion's disease, Refsum's disease, Sandhoff disease, Hilder's disease, schizophrenia, Spielmeyer-Vogt-Sjogren - Batten disease, spino
  • the neurodegenerative disease is preferably neuronal ceroid lipofuscinosis, more preferably juvenile neuronal ceroid lipofuscinosis (JNCL).
  • JNCL juvenile neuronal ceroid lipofuscinosis
  • the disease is caused by a mutation of the CLN3 gene, for example, a deletion mutation CLN3 ⁇ ex7/8 of exons 7 and 8 of the CLN3 gene, wherein the deletion mutation is about 1.02kb.
  • siRNA, sgRNA or a vector constructed with shRNA is used to silence or knock down the expression of the KEAP1 gene, thereby enhancing the activity of NRF2 and/or its downstream proteins.
  • KEAP1 inhibitors and/or NRF2 activators are used to enhance the expression of NRF2 and/or its downstream genes, thereby enhancing the activity of NRF2 and/or its downstream proteins.
  • the KEAP1 inhibitor and/or NRF2 activator is selected from the group that can interact with cysteine residues including cysteine 151, 272 and/or 288 on KEAP1 Reagents for covalent reactions.
  • the KEAP1 inhibitor and/or NRF2 activator is selected from one or more of the following compounds: Carvedilol (CAS: 72956-09-3), Ketoconazole (CAS: 65277-42 -1), GANT61, CAS (500579-04-4), Protriptyline hydrochloride (CAS: 1225-55-4), LP 44 (CAS: 824958-12-5), Doxepin HCl (CAS: 1229-29-4) , Dimethyl Fumarate (DMF), Acetyl-11-Carbonyl- ⁇ -Boswellic Acid (AKBA), Isothiocyanate, Bardoxolone (CDDO), Bardoxolone Methyl (CDDO-Me) And derivatives or analogs of these compounds, or selected from ⁇ , ⁇ -unsaturated carbonyl compounds, phenols and polyphenols.
  • Carvedilol CAS: 72956-09-3
  • Ketoconazole CAS: 65277-42 -1
  • the KEAP1 inhibitor is compound G, namely (Z)-guggulsterone, whose structural formula is:
  • Another aspect of the present invention relates to a pharmaceutical composition for treating diseases related to autophagy function defect and/or lysosomal function defect, which comprises a therapeutically effective amount of one or more KEAP1 inhibitors and/or or an NRF2 activator, and a pharmaceutically acceptable carrier and/or excipient.
  • the KEAP1 inhibitor and/or NRF2 activator is selected from the group that can interact with cysteine residues on KEAP1 including cysteine 151, 272 and/or 288 Reagents for covalent reactions.
  • the KEAP1 inhibitor and/or NRF2 activator is selected from one or more of the following compounds: Carvedilol (CAS: 72956-09-3), Ketoconazole (CAS: 65277 -42-1), GANT61, CAS (500579-04-4), Protriptyline hydrochloride (CAS: 1225-55-4), LP 44 (CAS: 824958-12-5), Doxepin HCl (CAS: 1229-29- 4), dimethyl fumarate (DMF), acetyl-11-carbonyl- ⁇ -boswellic acid (AKBA), isothiocyanate, bardoxolone (CDDO), bardoxolone methyl (CDDO- Me) and derivatives or analogs of these compounds, or selected from ⁇ , ⁇ -unsaturated carbonyl compounds, phenols and polyphenols.
  • Carvedilol CAS: 72956-09-3
  • Ketoconazole CAS: 65277 -42-1
  • GANT61 CAS (5005
  • the KEAP1 inhibitor is compound G, namely (Z)-guggulsterone, its structural formula is:
  • kits for treating diseases related to autophagy function defect and/or lysosome function defect which comprises a reagent for inhibiting KEAP1 gene expression.
  • the kit according to the present invention comprises siRNA, sgRNA or a vector constructed with shRNA for silencing or knocking down the KEAP1 gene so as to inhibit the expression of the KEAP1 gene.
  • Another aspect of the present invention relates to a method for alleviating or eliminating the differentiation defect of neural stem cells into neurons in a subject, the method comprising administering to a subject in need inhibiting KEAP1 activity and/or enhancing NRF2 and/or or a drug for its downstream protein activity, wherein the subject suffers from a disease related to autophagy function defect and/or lysosome function defect.
  • the disease is caused by a mutation of the CLN3 gene, such as a deletion mutation of exons 7 and 8 of the CLN3 gene
  • the disease is preferably JNCL.
  • the drug is selected from one or more of the following drugs: Compound G (Z)-guggulsterone, Carvedilol (CAS: 72956-09-3), Ketoconazole (CAS: 65277 -42-1), GANT61, CAS (500579-04-4), Protriptyline hydrochloride (CAS: 1225-55-4), LP 44 (CAS: 824958-12-5), Doxepin HCl (CAS: 1229-29- 4), dimethyl fumarate (DMF), acetyl-11-carbonyl- ⁇ -boswellic acid (AKBA), isothiocyanate, bardoxolone (CDDO), bardoxolone methyl (CDDO- Me) and derivatives or analogs of these compounds, or selected from other ⁇ , ⁇ -unsaturated carbonyl compounds, phenols and polyphenols.
  • drugs selected from one or more of the following drugs: Compound G (Z)-guggulsterone, Carvedilol (CAS: 72956-09-3
  • the drug is preferably compound G (Z)-guggulsterone
  • Another aspect of the present invention relates to a system for screening substances that can activate cell autophagy flow and/or enhance lysosome function, which is characterized in that the system comprises a double fluorescent-labeled Tandem LC3 reporter system.
  • the autophagosome where the double fluorescently labeled LC3 is located in the cytoplasm exhibits, for example, red and green double fluorescence, and when the autophagosome fuses with the lysosome to form an autolysosome, then Only present as red single fluorescence.
  • the screening system of the present invention comprises an NRK cell line stably expressing tandem LC3.
  • Another aspect of the present invention relates to a method for screening substances that can activate cell autophagy flow and/or enhance lysosome function, characterized in that the aforementioned screening system is used, if the substance to be screened makes the dual fluorescence and If the number of single fluorescently labeled autophagosomes and autolysosomes are both increased, then the substance is the target substance that can activate the autophagic flow of cells.
  • Another aspect of the present invention relates to an in vitro model based on neural stem cells or neurons, wherein the model has deletion mutations of exons 7 and 8 of the CLN3 gene, namely CLN3 ⁇ ex7/8 .
  • Another aspect of the present invention relates to the use of the in vitro model to screen compounds that induce autophagy and/or verify the effect of the compounds.
  • the present invention directly or indirectly activates or enhances the expression of NRF2 and/or its downstream genes by inhibiting the expression of KEAP1 gene or activating the expression of NRF2, thereby improving the lysosome acidic environment of neurons and the enzymatic activity of cathepsin in neurons , reduce the abnormal accumulation of protein in neurons, protect the homeostasis of mitochondria in neurons and improve the ability of mitochondria to produce ATP, so as to prevent or treat neurodegeneration related to autophagy function defects and/or lysosome function defects effect of the disease.
  • the terms "subject” and “patient” are used interchangeably and refer to mammals in need of treatment, such as pets (e.g., dogs, cats, etc.), livestock (e.g., cows, pigs, horses, sheep, goats, etc.) etc.) and experimental animals (such as rats, mice, guinea pigs, etc.).
  • the subject is a human being in need of treatment.
  • treatment refers to obtaining a desired pharmacological and/or physiological effect.
  • the effects may be therapeutic and include partially or substantially achieving one or more of the following: partially or completely reducing the severity of the disease, disorder or syndrome; alleviating or improving clinical symptoms or indicators associated with the disorder; and delaying, inhibiting or reducing the likelihood of progression of a disease, disorder or syndrome.
  • the compounds of the invention are useful in the treatment of neurodegenerative disorders.
  • neurodegenerative disorders include, but are not limited to: Amyotrophic Lateral Sclerosis (ALS), Alzheimer's Dementia, Alexander's Disease, Alper's disease, Ataxia-Telangiectasia , Bovine spongiform encephalopathy (BSE), Canavan disease, Cockaynesyndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington's disease, HIV-related dementia, Kennedy disease, Krabbedisease, Lewy bodies Dementia, Machado-Joseph disease (spinocerebellar ataxia type 3), multiple sclerosis, multiple system atrophy, neuroborreliosis, Parkinson's disease, Pey-May's disease, Pick's disease, protozoa Lateral sclerosis, Prion disease, Refsum's disease, Sandhoff disease, Hilder's disease, schizophrenia, Spielmeyer-Vogt-Sjogren
  • the small molecule substance of the present invention can be administered to the subject by itself or as a part of a pharmaceutical composition.
  • a pharmaceutical composition refers to a preparation of one or more active ingredients described herein together with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate the administration of a compound to an organism.
  • a compound described herein, or a pharmaceutically acceptable salt thereof, or other form thereof is administered to a subject using any suitable method of delivery, including topical, enteral, parenteral, transdermal, transmucosal, via A compound described herein, or a pharmaceutically acceptable salt thereof, is administered to a subject by inhalation, intracisternal, epidural, intravaginal, intravenous, intramuscular, subcutaneous, intradermal, and intravitreal.
  • Administration of a compound described herein, or a pharmaceutically acceptable salt thereof, or other forms thereof, to a subject also includes topical, enteral, parenteral, transdermal, transmucosal, via inhalation, intracisternal, dura mater, Intravaginal, intravenous, intramuscular, subcutaneous, intradermal, and intravitreal administration to a subject in or on the surface of his body, metabolizable to a compound described herein, or a pharmaceutically acceptable salt thereof, or other form of the compound .
  • the pharmaceutical composition of the present invention can be produced by methods known in the art, such as conventional mixing, dissolving, granulating, sugar-coating, levigating, emulsifying, encapsulating, entrapping or freeze-drying.
  • a compound described herein, or a pharmaceutically acceptable salt thereof can be administered systemically (eg, orally) in combination with a pharmaceutically acceptable carrier, such as an inert diluent or an assimilable edible carrier. It can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or can be incorporated directly into the patient's meal.
  • a pharmaceutically acceptable carrier such as an inert diluent or an assimilable edible carrier. It can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or can be incorporated directly into the patient's meal.
  • the compounds, substances, or pharmaceutically acceptable salts thereof, or other forms described herein may be combined with one or more excipients and presented as ingestible tablets, buccal tablets , buccal tablets, capsules, elixirs, suspensions, syrups or wafers and other forms of administration.
  • the tablets, lozenges, pills, capsules, etc. may include the following: binders such as tragacanth, acacia, cornstarch and gelatin; excipients such as dicalcium phosphate; disintegrants such as cornstarch, Potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; sweeteners, such as sucrose, fructose, lactose, and aspartame; and flavoring agents.
  • binders such as tragacanth, acacia, cornstarch and gelatin
  • excipients such as dicalcium phosphate
  • disintegrants such as cornstarch, Potato starch, alginic acid, etc.
  • lubricants such as magnesium stearate
  • sweeteners such as sucrose, fructose, lactose, and aspartame
  • flavoring agents such as sucrose, fructose, lactose, and aspartame.
  • Exemplary pharmaceutical dosage forms for intravenous, intramuscular, subcutaneous, intradermal etc. injection or infusion include sterile aqueous solutions or dispersions containing the active ingredient and sterile powders suitable for sterile injectable or Extemporaneous preparation of infusible solutions or dispersions.
  • the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent with various other ingredients enumerated above and below as required, followed by filtered sterilization.
  • preferred methods of preparation may be vacuum drying and freeze-drying techniques which yield the active ingredient plus any additional desired ingredients present in a sterile-filtered solution.
  • Ingredient powder may be vacuum drying and freeze-drying techniques which yield the active ingredient plus any additional desired ingredients present in a sterile-filtered solution.
  • compositions and preparations should contain at least about 0.1% active ingredient.
  • the percentages of such compositions and formulations may of course vary and may conveniently be from about 2 to 60% by weight of a given unit dosage form.
  • the amount of the active ingredient in the therapeutic pharmaceutical composition is to obtain an effective dosage level.
  • the amount of a compound described herein, or a pharmaceutically acceptable salt thereof, or other form thereof, required for treatment may vary with the particular form selected and may vary with the route of administration, the nature of the condition being treated, and the age and age of the patient. Conditions vary and are ultimately at the discretion of the attending physician or clinician.
  • the dose of a compound provided herein, or a pharmaceutically acceptable salt thereof, or other forms thereof administered to a subject may be 10 ⁇ g to 5,000 mg; 10 ⁇ g to 1 mg; 1 to 500 mg; or 500 to 5,000 mg, etc.
  • it is conveniently administered in unit dosage form.
  • each unit dosage form contains 0.01-10 mg or 0.05-1 mg of active ingredient.
  • the dose is 5 mg/kg or less.
  • the required dose may be presented in a single dose or in divided doses administered at appropriate intervals.
  • Useful dosages of compounds described herein, or pharmaceutically acceptable salts thereof, or other forms, can be determined by comparing their in vitro and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” are used interchangeably to mean a carrier or diluent that does not cause significant irritation to the organism and does not abrogate the biological activity and properties of the administered compound , including adjuvants.
  • Exemplary solid carriers can include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like.
  • Suitable liquid carriers include water, alcohols, glycols and water-alcohol/glycol blends.
  • a compound described herein, or a pharmaceutically acceptable salt thereof, or other form, can be dissolved or dispersed at effective levels, optionally with the aid of a nontoxic surfactant.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and the like.
  • the present invention uses CRISPR/Cas9 gene editing technology to introduce the most common homozygous deletion mutations of exon 7 and 8 of CLN3 gene in JNCL patients into hESC, and induce its differentiation to neural lineage, and construct human neuropathy for JNCL disease. metamodel. This model recapitulates multiple JNCL disease-associated phenotypes, including defective lysosomal and autophagic function, and dysregulation of mitochondrial homeostasis. Since the normal function of autophagy and lysosome is crucial to maintain the homeostasis of neurons, a high-throughput screening system aimed at discovering novel autophagy-promoting small molecules was established.
  • guggulsterone enhanced autophagy and lysosome function by inhibiting KEAP1 protein, and alleviated JNCL-related disease phenotypes.
  • DMEM/F-12 medium Advanced DMEM/F-12 medium, DMEM/F12 medium, DMEM basal medium, DPBS buffer, Opti-MEM medium, Fetal Bovine Serum (FBS), GlutaMAXTM (100 ⁇ ), N2 Supplement (100 ⁇ ), B27 Supplement (50 ⁇ ), Penicillin/Streptomycin (Penicillin/Streptomycin, 100 ⁇ ), Trypsin-EDTA, StemPro Accutase were all purchased from Gibco Company; mTeSRTM1 medium was purchased from STEMCELL Company; boric acid Salt buffer was purchased from Thermo Fisher Scientific; Matrigel was purchased from BD; Poly-D-lysine (PDL) was purchased from Sigma Aldrich.
  • FBS Fetal Bovine Serum
  • GlutaMAXTM 100 ⁇
  • N2 Supplement 100 ⁇
  • B27 Supplement 50 ⁇
  • Penicillin/Streptomycin Penicillin/Streptomycin, 100 ⁇
  • Trypsin-EDTA StemPro Accutase were all purchased from Gibco
  • CHIR99021 (S1263), SB431542 (S1067), rapamycin (S1039), Bafilomycin A1 (S1413), Y27632 2HCl (S1049) were purchased from Selleck Company;
  • cAMP (A9501), L-ascorbic acid (A4403) were purchased from Sigma Aldrich Company;
  • Compound G ((Z)-Guggulsterone) was purchased from Tocris;
  • Human GDNF 450-10-500), Human/Murine/Rat BDNF (450-02-500) were purchased from PeproTech;
  • Recombinant Mouse EGF Protein (2028-EG), Recombinant Mouse FGF basic (3139-FB) was purchased from R&D Company;
  • Leukemia Inhibitory Factor Human (hLIF, LIF1010) was purchased from Millipore Company.
  • Liposome 3000 ( 3000 Reagent), Hochest 33342, ECL chromogenic solution and substrate luminescence solution (Super SignalTM West Dura Extended Duration Substrate), PageRuler TM Prestained Protein Ladder were purchased from Thermo Fisher Scientific; DMSO, paraformaldehyde, absolute ethanol, isopropyl Alcohol, ammonium persulfate (AP), 30% Acr/Bis gel solution, tetramethylethylenediamine, sodium lauryl sulfate, and iodoacetamide (IAA) were purchased from Sigma Aldrich; HiFi DNA Assembly kit was purchased from NEB Company; proteinase K, Taq DNA polymerase, 6 ⁇ DNA Loading Buffer, 100bp DNA Ladder, DNA Marker, PerfectStart TM Green qPCR SuperMix, One-Step gDNA Removal and cDNA Synthesis SuperMix and competent cells were purchased from Quanshijin Company; Tris, glycine and sucrose were purchased from Am
  • p62 antibody (PM045) was purchased from MBL; LC3 antibody (2775) and ⁇ 3-Tubulin antibody (5568P) were purchased from Cell Signaling Technology; ⁇ -Actin (C4) antibody (sc-47778) was from Santa Cruz; recombinant Anti- ATP synthase C antibody (ab181243), Pax6 antibody (ab195045) were purchased from Abcam Company; Sox1 antibody (AF3369) was purchased from R&D Company; ⁇ -actin antibody (sc-47778) was purchased from Santa Cruz Company; Nestin antibody (MAB353, MAB5326) , Sox2 antibody (AB5603) was purchased from Millipore; MAP2 antibody (M9942) was purchased from Sigma Aldrich; Oct4 antibody (MA5-14845) was purchased from Thermo Fisher Scientific.
  • Table 1 qPCR primer information for detecting gene expression
  • pMXS-IP GFP-OMP25 (#38249), pBABE-puro-mCherry-EGFP-LC3B (#22418), pSpCas9(BB)-2A-Puro (PX459, #48139) were purchased from Addgene; ATG5 shRNA plasmid, KEAP1 shRNA plasmid , CLN3 shRNA plasmid selected from shRNA plasmid library (MERCK); human p62-GFP plasmid (a plasmid for overexpressing p62 protein in mammalian cells driven by ef1a promoter), Flag plasmid and Flag-KEAP1 plasmid (respectively for overexpression of p62 protein in mammalian cells Plasmids expressing FLAG and FLAG-KEAP1 proteins, both driven by CMV promoters).
  • Cln3 knockout mice B6.129S6-Cln3tm1Nbm/J (029471) were purchased from Jackson Laboratory and bred on the C57BL/6 background; wild-type C57BL/6 mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Animals were housed in individually ventilated cages, with a maximum of 6 mice per cage. The mice were housed with a 12/12 hour light/dark cycle and the ambient temperature was maintained at 22-26°C. Mice had free access to sterile pelleted food and drinking water. All animal experiments were approved by the Institutional Animal Use and Care Committee (IACUC).
  • IACUC Institutional Animal Use and Care Committee
  • HEK293T/17 cell culture medium DMEM medium + 10% FBS + 1% Penicillin/Streptomycin
  • DMEM medium + 10% FBS + 1% Penicillin/Streptomycin DMEM medium + 10% FBS + 1% Penicillin/Streptomycin
  • aspirate the medium wash the cells once with dPBS, and then digest them with 0.25% Trypsin-EDTA at room temperature for 1 minute to single cells, add 5 times the volume of HEK293T/17 cell culture medium to neutralize Trypsin-EDTA, and centrifuge After resuspension, the cells were subcultured at a ratio of 1:5.
  • Rat kidney NRK cells Rat kidney NRK cells:
  • NRK cell culture medium NRK rat kidney cell culture medium: DMEM medium + 10% FBS + 1% Penicillin/Streptomycin
  • DMEM medium + 10% FBS + 1% Penicillin/Streptomycin NRK rat kidney cell culture medium
  • H9 human embryonic stem cells were cultured on cell culture plates coated with 2% matrigel in advance. Change medium daily with fresh mTeSRTM1 medium. Subculture when the cell clone density reaches 70%-80%. When subculture, aspirate the medium, wash the cells once with dPBS, then add Versene, and digest in a 37°C cell culture incubator for 5-7min. Aspirate the Versene when the cells are round but not floating, use mTeSRTM1 medium with 10 ⁇ M Y27632 to blow the cells into single cells, and transfer them to 2% matrigel pre-coated cells according to the ratio of 1:10-1:20 culture plate.
  • the medium on the day of subculture was mTeSRTM1 medium with 10 ⁇ M Y27632, and Y27632 was removed after 24 hours.
  • Human embryonic stem cell (hESC) medium mTeSRTM1 medium + 1% Penicillin/Streptomycin.
  • the culture medium on the day of subculture is the mTeSRTM1 medium with 10 ⁇ M Y27632, and after 24 hours, replace with human neural stem cell induction medium (human neural stem cell induction medium : Advanced DMEM/F12 medium: Medium (1:1)+3 ⁇ M CHIR99021+2 ⁇ M SB431542+0.1 ⁇ M Compound E+5 ⁇ g/mL BSA+10ng/mL hLIF+1 ⁇ N2+1 ⁇ B27+1% GlutaMAXTM+1% Penicillin/Streptomycin), and continuously Cultured for 7 days to induce the formation of human neural stem cells, during which the medium was changed every day.
  • human neural stem cell induction medium Advanced DMEM/F12 medium: Medium (1:1)+3 ⁇ M CHIR99021+2 ⁇ M SB431542+0.1 ⁇ M Compound E+5 ⁇ g/mL BSA+10ng/mL hLIF+1 ⁇ N2+1 ⁇ B27+1% GlutaMAXTM+1% Penicillin/Streptomycin
  • human neural stem cell maintenance medium Advanced DMEM/F12 medium: Medium (1:1)+3 ⁇ M CHIR99021+2 ⁇ M SB431542+5 ⁇ g/mL BSA+10ng/mL hLIF+1 ⁇ N2+1 ⁇ B27+1% GlutaMAXTM+1% Penicillin/Streptomycin) to blow the cells into single cells, According to the ratio of 1:3, it was transferred to the cell culture plate coated with 2% matrigel in advance.
  • Y27632 was withdrawn after 24 hours. Afterwards, the cells were cultured in human neural stem cell maintenance medium for a long time, and when the cell density reached 85%-95%, the cells were subcultured according to the above method. Human neural stem cells can be maintained in vitro for more than 10 passages, and can also be stored in liquid nitrogen for later use.
  • passaging is carried out according to the above method at 1:20-1:50.
  • the culture medium on the day of passaging was the human neural stem cell maintenance medium with 10 ⁇ M Y27632, and Y27632 was removed after 24 hours, and the culture was continued in the human neural stem cell maintenance medium for 48 hours.
  • human neuron differentiation medium human neuron medium : DMEM/F12 medium+10ng/ml hBDNF+10ng/ml hGDNF+300ng/mL cAMP+0.2mM vitamin C+1 ⁇ N2+1 ⁇ B27+1% Penicillin/Streptomycin
  • human neuron medium DMEM/F12 medium+10ng/ml hBDNF+10ng/ml hGDNF+300ng/mL cAMP+0.2mM vitamin C+1 ⁇ N2+1 ⁇ B27+1% Penicillin/Streptomycin
  • flow cytometry was used: aspirate the cell culture medium, wash once with dPBS, and digest into single cells by adding digestive enzymes corresponding to the cell type ; Centrifuge to remove the supernatant, resuspend the cells with 1 mL of fresh medium; filter the cell suspension with a 40 ⁇ m filter membrane to collect the single-cell filtrate; transfer the single-cell filtrate to a flow tube, and use BD FACS Aria III for analysis or Sorting; during analysis, analyze the fluorescence intensity of 1 ⁇ 104 cells per sample to obtain the mean fluorescence intensity (MFI); during sorting, use a 96-well plate with medium to receive cells, and each well receives one cells to establish cell lines.
  • MFI mean fluorescence intensity
  • Amaxa TM P3 primary cell 4D-Nucleofector TM electrotransfection kit for transfection, taking a 12-well cell culture plate as an example: Amaxa TM P3 Primary Cell 4D-NucleofectorTM electrotransfection reagent The box was equilibrated to room temperature; when the human embryonic stem cells grew to a density of about 70%, they were digested with versene at 37°C for 10 minutes; the cells were blown into single cells with 1 mL of mTeSRTM1 medium containing 10 ⁇ M Y27632; Centrifuge for 5 minutes, and remove the supernatant as much as possible; mix 82 ⁇ L Nucleofector TM Solution and 18 ⁇ L Supplement included in the kit, and add PX459 plasmid, 1 ⁇ g each of the two sgRNA plasmids targeting CLN3, and mix by pipetting.
  • HEK293T/17 cells used for co-immunoprecipitation were mainly transfected by Lipofectamine 3000 kit. Take a 10cm cell culture dish as an example: culture HEK293T/17 cells to 40-50% density; first add 10 ⁇ g plasmid and 20 ⁇ L P3000 reagent to 500 ⁇ L opti-MEM medium, shake and mix; add 30 ⁇ L Liposome 3000 to another In a tube of 500 ⁇ L opti-MEM medium, mix it upside down; add liposome 3000 to the opti-MEM medium containing the plasmid above, mix upside down; leave it at room temperature for 15 minutes, add it dropwise to the HEK293T/17 cell plate . Eight hours after transfection, the cell culture medium was aspirated, and fresh HEK293T/17 cell culture medium was added to complete the transfection; 36 hours after transfection, the cells could be collected for co-immunoprecipitation experiments.
  • Lentiviral packaging was carried out in HEK293T/17 cells: spread HEK293T/17 cells in cell culture plates and grow to a density of 40%-60%; the lentiviral plasmid and two viral packaging plasmids psPAX2 and pMD2.G according to 5 : 3:2 ratio mixed for transfection, transfection steps refer to liposome 3000 cell transfection steps; 8 hours after transfection, absorb liposome 3000, replace with fresh HEK293T/17 cell culture medium; transfection After 32-56 hours, collect the culture medium of HEK293T/17 cells, and filter out the cell debris with a 0.45 ⁇ m filter membrane; After nitrogen quick-freezing, store at -80°C for subsequent use, avoid repeated freezing and thawing; after 8 hours of infection, replace with fresh medium of target cells, and the infection is complete.
  • Sample preparation For adherent cultured cells, take cells cultured in six-well cell plates as an example, suck off the cell culture medium, wash once with dPBS, add about 100 ⁇ L of RIPA containing protease inhibitors and phosphatase inhibitors to each well to lyse solution (50mM Tris-HCl, pH 7.5, 150mM sodium chloride, 0.25% sodium deoxycholate, 0.1% NP-40, 0.1% Triton X-100), all the cell lysate was scraped off with a cell scraper, and collected to 1.5 mL EP tubes and placed on ice for 30 minutes.
  • lyse solution 50mM Tris-HCl, pH 7.5, 150mM sodium chloride, 0.25% sodium deoxycholate, 0.1% NP-40, 0.1% Triton X-100
  • For animal tissue weigh the animal tissue, add 10 times the volume of RIPA lysate containing protease inhibitors and phosphatase inhibitors, use a tissue grinder to grind the tissue on ice until there are no obvious tissue pieces, and collect 1.5mL EP Tubes were placed on ice and lysed for 30 minutes.
  • Fix remove the cell culture medium, wash once with dPBS, then add 4% paraformaldehyde solution to fix the cells, incubate at room temperature for 20 minutes, wash with dPBS three times, each time for 5 minutes; block and permeabilize: add blocking solution (5% BSA and 0.3% Trinton-X100 dissolved in dPBS), incubate at room temperature for 1 hour, wash 3 times with dPBS, 5 minutes each time; primary antibody incubation: dilute the antibody with dPBS containing 5% BSA according to the product instructions, 4 Incubate overnight at °C or at room temperature for 2 hours, wash 3 times with dPBS, 5 minutes each time; secondary antibody incubation: Dilute the secondary antibody of the corresponding species and fluorophore with dPBS containing 5% BSA at a ratio of 1:1000, Incubate at room temperature for 1 hour, then wash 3 times with dPBS, 5 minutes each; nuclear staining: dilute DAPI to 100ng/mL with
  • Blocking and permeabilization add blocking solution (5% BSA and 0.3% Trinton-X100 dissolved in dPBS), incubate at room temperature for 1 hour, wash 3 times with dPBS, 5 minutes each time; primary antibody incubation: use 5% BSA containing dPBS Dilute the antibody according to the product instructions, incubate overnight at 4°C, and then wash 3 times with dPBS, 5 minutes each time; Secondary antibody incubation: Use dPBS containing 5% BSA with the secondary antibody of the corresponding species and fluorophore at a ratio of 1:1000 Dilute according to the ratio, incubate at room temperature for 1 hour, then wash 3 times with dPBS, 5 minutes each time; cell nucleus staining: dilute DAPI to 100ng/mL with dPBS, incubate at room temperature for 5 minutes, then wash 3 times with dPBS, 5 minutes each time; Spread the brain tissue on a glass slide, drop the anti-fade reagent, cover the cover
  • the cells are counted using a hemocytometer. Then the cell suspension was diluted to the same cell concentration (10 5 -10 6 /mL). Take out 100 ⁇ L of cell suspension to a white 96-well assay plate, add 100 ⁇ L of CellTiter-Lumi TM Steady Plus reagent equilibrated to room temperature, shake to mix, and incubate at room temperature for 15 minutes. Then read the chemiluminescence intensity of the sample with a multi-functional microplate reader to indicate the relative ATP content in the cells.
  • mtMinArc-R AGAGCTCCCGTGAGTGGTTA;
  • b2M-R CCATGTACTAACAAATGTCTAAAATGGT.
  • RNA extraction and real-time fluorescent quantitative PCR qRT-PCR
  • the activity of the intracellular protease cathepsin D was completed using Abcam’s Cathepsin D Activity Assay kit. Each sample was prepared with more than 3 replicates. The specific steps were as follows: absorb the cell culture medium, wash it once with dPBS, and then digest it into single cells.
  • the mouse brain tissue tumor before grinding add the IS methanol solution in step 4) into the ground brain tissue according to the volume of 5 mL per gram of brain tissue, shake and mix well; Collect the supernatant; mix 100 ⁇ L of supernatant and 100 ⁇ L of 10% aqueous methanol solution, and transfer to an HPLC sample vial; mix the supernatant of blank tissue with 100 ⁇ L of 10% aqueous methanol solution, and add the standard substance of compound G to the following concentration, Transfer to HPLC vials as a standard curve: 1000ng/mL, 500ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 20ng/mL, 10ng/mL, 5ng/mL, 2ng/mL, 1ng/mL , 0.5ng/mL, 0.2ng/mL, 0.1ng/mL; use UPLC-MS/MS to identify the peak area of compound G in the sample.
  • the chromatographic conditions are as follows: the chromatographic column is Waters XSelect HSS T3 column (100 ⁇ 2.1mm, 1.8 ⁇ m); the mobile phase A is 0.1% formic acid methanol, and the mobile phase B is 0.1% formic acid water; gradient elution: 0-3 minutes 40 % mobile phase A to 100% mobile phase A, maintain for 2 minutes, then return to the initial 40% mobile phase A in 5.1 minutes, and end a run in 8 minutes; the flow rate is 0.2mL/min; the injection volume is 10 ⁇ L; the column temperature is 30°C.
  • the mass spectrometry conditions are as follows: electrospray ionization source (ESI), using positive ion mode, multiple reaction monitoring (MRM), ion source temperature is 500 °C, curtain gas 30psi, collision gas is medium, ion voltage is 5500V, spray gas and auxiliary heating The gas is 50psi; use the software AB SCIEX Analyst 1.6.3 Software to calculate the peak area of compound G, and use the standard to make a standard curve to calculate the drug concentration in the sample.
  • ESI electrospray ionization source
  • MRM multiple reaction monitoring
  • ion source temperature 500 °C
  • curtain gas 30psi collision gas is medium
  • ion voltage is 5500V
  • the gas is 50psi
  • Mark * is quantitative fragment ion
  • the neural stem cells were cultured in a 10 cm cell culture dish to a cell density of 90%. Neural stem cells in a 10cm cell culture dish can be lysed to obtain 1-2mg protein; aspirate the supernatant, add fresh medium containing 30 ⁇ M compound G, or medium containing an equal volume of solvent DMSO, and incubate in a cell culture incubator at 37°C for 2 hours; remove the cell culture medium, wash once with 10mL dPBS, and aspirate as much as possible.
  • mice born 21 days after birth were isolated from the breeding cage, and 1 mg of /mL Compound G for treatment.
  • the control group was Cln3 KO mice whose drinking water contained the same amount of solvent.
  • the body weight changes of the mice were recorded during the administration period.
  • the mice were 2 months old, the mice were euthanized and immunohistochemical staining was performed on the brain tissue to observe the abnormal accumulation of SCMAS protein in the brain of the mice.
  • mice were 4-5 months old, using Morris water maze test, rolling wheel test and mine field test to compare the differences in learning and memory ability and motor ability between Cln3 KO mice and wild-type mice, and the effect of compound G on Cln3 Treatment effect in KO mice.
  • a Morris water maze experiment was carried out. The specific steps are as follows: place the mice to be tested in a single cage Raise for more than 1 week to adapt to the environment; the maze used in this study is a circular pool with a diameter of 120 cm and a depth of 40 cm; the pool is divided into northeast (northeast, NE ), northwest (northwest, NW), southwest (southwest, SW) and southeast (southeast, SE) four quadrants.
  • mice Remove the mouse from the platform, wipe it clean with a dry towel and put it back into the cage.
  • Each mouse was trained 4 times a day with a 30-minute interval between each training session for a total of 6 days. Mice that could not swim were excluded; on the 7th day, the platform in the water was removed, and the mice were taken from the NE position. Gently put it into the water, and record the movement of the mouse within 60 seconds with video equipment, use Noldus software to analyze and count the time when the mouse finds the platform for the first time, and the time when the mouse stays in the SW quadrant. In addition, the experimenter manually calculates The number of times the mouse crossed the original platform location within 60 seconds.
  • Table 5 The position information of mice placed in the water maze
  • the shRNA knockdown experiments of genes such as CLN3 and KEAP1 were carried out.
  • the specific steps are as follows: According to the experimental requirements, the neural stem cells were planted in the required cell culture plate, to about 50% of the cells Density, or differentiate neural stem cells into human neurons in cell culture plates; shRNA lentiviral plasmids for genes such as CLN3 and KEAP1, and control lentiviral plasmids expressing no target sequences were selected from the MISSIOH human shRNA library of Sigma Aldrich, and the sequences are listed in the table 6.
  • pack the lentivirus in a 10cm cell culture plate and collect the supernatant containing the virus by filtration; mix the supernatant with the lentivirus concentrate (40% PEG-8000 and 1.2M chloride Dissolve sodium in dPBS solution (pH ⁇ 7.0) and mix according to the volume ratio of 1:3, slowly rotate at 4°C, and incubate overnight; centrifuge at 4°C for 30 minutes with a centrifugal force of 13,000g, discard the supernatant, and use 1mL of fresh cells Resuspend the pellet in the medium and add it to the target cells; 8 hours after infection, aspirate the virus and add fresh cell culture medium; cells for subsequent experiments.
  • the lentivirus concentrate 50% PEG-8000 and 1.2M chloride Dissolve sodium in dPBS solution (pH ⁇ 7.0) and mix according to the volume ratio of 1:3, slowly rotate at 4°C, and incubate overnight
  • transcriptome sequencing was performed on the neural stem cells treated with compound G, and the specific steps were as follows: CLN3 ⁇ ex7/8 human neural stem cells were planted in 2% Matrigel-coated 6-well cell plates, to 80% cell density; add fresh cell culture medium containing 6 ⁇ M compound G or an equal volume of DMSO, each treatment condition contains two biological replicates; place the neural stem cells in a 37°C cell culture incubator for 6 hours; aspirate The cell culture medium was washed once with dPBS, and then the intracellular RNA was extracted using the Qiagen RNeazy Mini Plus Kit; the extracted RNA was sent to Annoroad, and the quality of the RNA was first tested.
  • the Aligent 2100 RNA Nano 6000 Assay kit and qPCR were used for quality detection; the Hiseq PE Cluster kit of Illumina was used for clustering, and the Hiseq 2500 sequencing platform was used for clustering. Double-end sequencing was performed to obtain 150bp paired-end sequencing reads; FastQC software was used to evaluate the quality of the raw data obtained by sequencing, and then TrimGalore was used to remove adapter sequences and low-quality sequences to obtain filtered data for subsequent analysis.
  • HISAT2 Use HISAT2 to compare the filtered data with the human reference genome hg38, and obtain the Bam file after comparison, use FeatureCount to count the number of counts compared to each gene, and use the gtf (GRCh38.92) file provided by Ensembl for annotation. Ensembl IDs were converted to gene names using the R package Biomart. Differentially expressed genes were analyzed using DESeq2 software, and genes with a fold change > 2 and corrected p ⁇ 0.05 were selected as differential genes.
  • the data were statistically analyzed using GraphPad Prism 7 software.
  • the data shown in the charts are mean ⁇ variance or mean ⁇ standard deviation, see the labeling of each experiment icon for details. * stands for p ⁇ 0.05; ** stands for p ⁇ 0.01; *** stands for p ⁇ 0.001; ns stands for no significant difference.
  • the gray value of western blot images was analyzed using ImageJ software to indicate protein abundance.
  • FIG. 1 Introduction of JNCL patient mutations in human embryonic stem cells using CRISPR/Cas9 technology.
  • Figure 2 Flowchart of the differentiation of hESC cell lines into neural lineages.
  • FIG. 3 Lysosomal function impairment exists in CLN3 ⁇ ex7/8 neural stem cells.
  • Lysotracker Red staining was carried out in neural stem cells, and the intracellular red fluorescence intensity distribution was measured by flow cytometry;
  • the average fluorescence intensity of Lysotracker staining in WT and CLN3 ⁇ ex7/8 neural stem cells (n 3 );
  • (c) Comparison of cathepsin D enzyme activity in lysosomes of WT and CLN3 ⁇ ex7/8 neural stem cells (n 4);
  • SCMAS Western blot measurement of abnormally accumulated ATP in WT and CLN3 ⁇ ex7/8 neural stem cells Enzyme c subunit
  • Figure 4 Lysosomal defects in CLN3 ⁇ ex7/8 neurons.
  • Lysotracker Red dye was used to stain neurons, and the distribution of red fluorescence intensity in cells was measured by flow cytometry.
  • Figure 5 Abnormal accumulation of SCMAS protein in CLN3 ⁇ ex7/8 neurons.
  • (c) Quantification of gray value of SCMAS bands in Western blot experiments (n 4).
  • Figure 6 Lysosomal function deficits in CLN3 knockdown neurons.
  • hNSCs infected with the same amount of control shRNA and CLN3 shRNA were differentiated into neurons, and the expressions of neuronal lineage proteins MAP2 and TUJ1 were detected by immunofluorescence staining after 17 days of differentiation. Scale bar is 200 ⁇ m.
  • Lysotracker dye staining In CLN3 knockdown neurons, Lysotracker dye staining, flow cytometry measurement of intracellular Lysotracker fluorescence intensity.
  • c Western blot assay to measure the protein levels of CLN3 and SCMAS in neurons infected with control shRNA and CLN3 shRNA.
  • Figure 7 Defective autophagic flux in CLN3 ⁇ ex7/8 neural stem cells.
  • Neural stem cells were infected with tandem LC3 lentivirus, autophagosomes were labeled, and imaged by high-content confocal laser microscopy. White arrows point to autophagosomes that are single-positive for red fluorescence. Scale bar is 50 ⁇ m.
  • Neural stem cells were treated with autophagy inhibitor Baf-A1 for 12 hours, and the number of autophagosomes with red fluorescence in the cells were counted before and after Baf-A1 treatment. The picture shows 64 fields of view of Opera Phenix data analysis workstation statistical results.
  • FIG. 8 Mitochondrial damage is present in CLN3 ⁇ ex7/8 neurons.
  • (a) Mitochondrial morphology in green fluorescent fused OMP25-labeled human neurons. White arrows are damaged punctate mitochondria. Scale bar is 20 ⁇ m.
  • (b) Cell Titer Glo was used to detect the ATP content in the same number of wild-type and CLN3 ⁇ ex7/8 neurons (n 4).
  • Figure 9 Screening of small molecule compounds that enhance lysosomal acidity. Hits is a representative image of small molecule compounds that enhance lysosome staining with Lysotracker Red. Arrowheads are autophagosomes colocalized with lysosomes after compound treatment. Scale bar is 100 ⁇ m.
  • Figure 10 Small molecule compounds enhance lysosomal acidity.
  • Figure 11 Compound G promotes the differentiation of CLN3 ⁇ ex7/8 neurons. Take the same number of neural stem cells and start the induction of neurons.
  • (c) Relative cell number of differentiated neurons (n 4).
  • Figure 12 Compound G does not affect cell fate of CLN3 ⁇ ex7/8 hNSCs.
  • Figure 13 Compound G enhances CLN3 ⁇ ex7/8 hNSC lysosomal acidity.
  • Compound G treated CLN3 ⁇ ex7/8 hNSC for 48 hours, stained with Lysotracker dye, and imaged with confocal laser confocal high-content imaging microscope. Shown is a mosaic of nine near-field images. Scale bar is 200 ⁇ m.
  • Figure 14 Compound G alleviates lysosomal defects in CLN3 ⁇ ex7/8 human neurons.
  • Figure 15 Compound G reduces SCMAS protein accumulation in CLN3 ⁇ ex7/8 neurons.
  • (c) Quantitative analysis of the gray value of SCMAS bands in Western blot experiments (n 4).
  • FIG. 16 DMF reduces SCMAS protein accumulation in CLN3 ⁇ ex7/8 neurons.
  • Figure 17 Compound G enhances autophagic flux in CLN3 ⁇ ex7/8 human neural stem cells.
  • White arrows point to autophagosomes that are single-positive for red fluorescence. Scale bar is 50 ⁇ m.
  • Figure 18 Compound G enhances autophagic flux in CLN3 ⁇ ex7/8 human neurons.
  • (a) is a representative p62 western blot picture;
  • Figure 19 Compound G protects mitochondrial homeostasis in CLN3 ⁇ ex7/8 neurons.
  • (b) Count the same number of neurons, add Cell Titer Glo reagent, and measure the ATP level in the cells with a microplate reader (n 3).
  • Figure 21 Effect of compound G on body weight and brain tissue mass of Cln3 KO mice.
  • (b) When the mice were 6 months old, they were euthanized, and the brain tissues were taken out and weighed (n 5).
  • Figure 22 Compound G reduces SCMAS protein accumulation in the brain of Cln3 KO mice.
  • Figure 23 Learning and memory ability of Cln3 KO mice measured by Morris water maze test.
  • FIG 24 Rolling wheel test and mine field test to test the motor ability of mice.
  • the mice were trained for 3 days, and the training time was 300 seconds, and the time when the mice fell from the roller for the first time was recorded.
  • mice were tested and the time when the mice first fell off the roller was recorded.
  • Figure 26 Compound G does not affect the transcription of autophagy and lysosomal genes in mouse neural stem cells and neurons.
  • Compound G treated the primary (a) neural stem cells and (b) neurons of Cln3 KO mice for 6 hours, extracted the mRNA in the cells, and reverse-transcribed it into cDNA.
  • Figure 28 Compound G increases transcription of autophagy genes in a FXR-dependent manner.
  • Figure 29 The effect of compound G on the expression profile of CLN3 ⁇ ex7/8 human neural stem cells.
  • (b) is a distance map.
  • (c) is a PCA plot.
  • RNA-seq shows that compound G does not affect the expression of autophagy and lysosomal genes in neural stem cells.
  • Figure 31 RNA-seq data showing that Compound G activates the expression of NRF2 downstream genes.
  • Figure 32 Compound G activates the expression of NRF2 downstream genes in CLN3 ⁇ ex7/8 human neural stem cells.
  • Figure 33 Quantitative thiol reactivity analysis in CLN3 ⁇ ex7/8 human neural stem cells.
  • Figure 34 KEAP1 knockdown in CLN3 ⁇ ex7/8 human neural stem cells.
  • Figure 35 Effect of knockdown of KEAP1 on lysosomal acidity of CLN3 ⁇ ex7/8 human neurons.
  • Figure 36 Effect of knockdown of KEAP1 on cathepsin activity in CLN3 ⁇ ex7/8 cells.
  • Figure 37 KEAP1 knockdown reduces SCMAS protein accumulation in CLN3 ⁇ ex7/8 neurons.
  • (b) Statistics of SCMAS fluorescence area (n 4).
  • Figure 38 The effect of knockdown of KEAP1 on the autophagic flow of CLN3 ⁇ ex7/8 human neural stem cells.
  • (a) White arrows point to autophagosomes that are single-positive for red fluorescence. Scale bar is 50 ⁇ m.
  • (b) The figure shows the statistical results of the number of autophagosomes in 64 fields of view by the Opera Phenix data analysis workstation. The number of red fluorescent single-positive autophagosomes (the number of mCherry-positive autophagosomes - the number of EGFP-positive autophagosomes).
  • Figure 39 KEAP1 knockdown reduces mitochondrial damage in CLN3 ⁇ ex7/8 human neurons. White arrows point to damaged punctate mitochondria. Scale bar is 20 ⁇ m.
  • Figure 41 Compound G increases intracellular p62 protein levels.
  • (a) After human neurons were treated with compound G for 24 hours or KEAP1 was knocked down, the transcription level of p62 gene was detected by qRT-PCR (n 3).
  • (b) After human neurons were treated with compound G for 24 hours or KEAP1 was knocked down, the intracellular p62 protein level was detected by Western blotting, and the band grayscale statistics were performed (n 3).
  • Figure 42 Overexpression of p62 protein increases lysosomal acidity.
  • Figure 43 Overexpression of p62 protein reduces SCMAS protein accumulation and cell death in CLN3 ⁇ ex7/8 neurons. Using DOX to overexpress p62 protein in CLN3 ⁇ ex7/8 human neural stem cells and differentiate them into human neurons.
  • Figure 44 Compound G reduces A[beta] protein accumulation and secretion levels of toxic A[beta]42 in APP mutant neurons.
  • Embodiment 2 Construction of the human neuron model of JNCL disease
  • hNSCs human neural stem cells
  • hNSCs human neural stem cells
  • Example 3 CLN3 ⁇ ex7/8 human neural stem cells and neurons recapitulate disease-associated phenotypes in vitro
  • Lysotracker dye which is enriched in acidic lysosomes and emits red fluorescence, and then the fluorescence intensity of stained cells was analyzed by flow cytometry.
  • CLN3 gene mutations can cause JNCL-related lysosomal dysfunction phenotypes such as disordered acidity regulation in the lysosomal cavity, decreased protease activity, and abnormal protein storage.
  • Lysosome and autophagy functions were further detected in CLN3 ⁇ ex7/8 neurons.
  • the neurons were first stained with Lysotracker Red dye, and the fluorescence intensity of the dye in the wild-type and CLN3 ⁇ ex7/8 neurons were detected by flow cytometry. Consistent with the results observed in neural stem cells, CLN3 ⁇ ex7/8 neurons had a phenotype of decreased lysosomal acidity compared with wild-type neurons (Fig. 4(a,b)); and CLN3 ⁇ ex7/8 neurons The enzymatic activity of cathepsin D in metalysosomes decreased significantly (Fig. 4(c)). Cell immunofluorescence experiments and Western blot experiments jointly proved that there was a large amount of abnormal accumulation of SCMAS protein in CLN3 ⁇ ex7/8 human neurons ( FIG. 5 ).
  • short hairpin RNA (shRNA) targeting CLN3 transcripts was performed in wild-type neural stem cells.
  • the CLN3 gene was knocked down, and wild-type neural stem cells infected with no target shRNA (Ctrl shRNA) were used as a control, and then differentiated into human neurons.
  • the changes of CLN3 protein level in neurons expressing CLN3 shRNA were detected by Western blot. The results showed that, compared with the control shRNA, the protein level of CLN3 in neurons could be significantly reduced by using CLN3 shRNA (Fig. 6(c)).
  • Lysotracker dye was used to detect the effect of CLN3 gene knockdown on lysosome acidity in human neurons. The results showed that after CLN3 gene knockdown, the Lysotracker staining intensity in neurons was weakened, indicating that the lysosome acidity decreased (Fig. 6(b )). It was observed by Western blot that knockdown of CLN3 gene would cause abnormal accumulation of SCMAS protein in neurons (Fig. 6(c)).
  • the number of autophagosomes in the cells was counted before and after treatment with the autophagy inhibitor bafilomycin A1 (Baf-A1).
  • the results showed that the changes in the number of autophagosomes in wild-type neural stem cells (with Baf The number of autophagosomes after -A1-the number of autophagosomes without Baf-A1) was significantly higher than that in CLN3 ⁇ ex7/8 NSCs (Fig. 7(b)), confirming the autophagosomes in CLN3 ⁇ ex7/8 NSCs Phagocytosis is flawed.
  • Example 4 The compound of the present invention is an autophagy flux activator capable of enhancing lysosome acidity
  • the inventors constructed a normal rat kidney (NRK) cell line stably expressing tandem LC3.
  • the tandem LC3 system is a LC3 reporter system that uses red fluorescence and green fluorescence to simultaneously label.
  • the autophagosomes When the autophagosomes where the double-fluorescence-labeled LC3 is located are located in the cytoplasm, the autophagosomes simultaneously display red fluorescence and green fluorescence ( yellow).
  • red fluorescence and green fluorescence yellow.
  • the acidic environment in lysosomes will quench the green fluorescence on tandem LC3, so autolysosomes only show red fluorescence (Shunsuke Kimura et al. ,2007).
  • the system is able to distinguish autophagic flux activators from lysosomal inhibitors.
  • the inventors have obtained 7 small molecular compounds, which can simultaneously increase the number of red fluorescent and green fluorescent double-positive autophagosomes in cells by two times or more when tested with the above-mentioned tandem LC3 system, and maintain The number of autophagosomes with single positive red fluorescence did not decrease, indicating that they were all activators of autophagy flux rather than lysosome inhibitors. Moreover, after treating NRK cells stably expressing BFP-LC3 with these 7 small molecular compounds for 18 hours, staining with Lysotracker dyes, it can be observed that these 7 small molecular compounds can significantly increase the intracellular Lysotracker activity compared with the control cells. (Fig.
  • the 7 small molecular compounds are respectively:
  • Example 5 Compound G attenuates the disease phenotype in the JNCL human neuronal model
  • the inventors took compound G as an example to test the potential effects of the aforementioned small molecule compounds on JNCL.
  • Compound G alleviates the differentiation defect of CLN3 ⁇ ex7/8 neural stem cells
  • compound G was found to effectively alleviate the differentiation defect of CLN3 ⁇ ex7/8 neural stem cells into neurons.
  • CLN3 ⁇ ex7/8 neural stem cells treated with compound G can generate more MAP2 and TUJ1 positive neurons, which is close to the neuron density produced by differentiation of wild-type neural stem cells (Figure 11).
  • Compound G alleviates lysosomal and autophagy defects in CLN3 ⁇ ex7/8 neurons
  • the treatment of the screened compound G can effectively alleviate the imbalance of lysosomal acidity in CLN3 ⁇ ex7/8 human neurons, increase the activity of proteases in the lysosome, and reduce the abnormal accumulation of intracellular proteins, that is, compound G Can partially alleviate JNCL disease-associated phenotypes in the CLN3 ⁇ ex7/8 human neuron model.
  • the inventors also tested some other potentially useful compounds and found that, similar to compound G, they can also relieve JNCL disease-related phenotypes in the CLN3 ⁇ ex7/8 human neuron model to a certain extent.
  • DMF dimethyl fumarate
  • Lysotracker staining indicates the acidity of lysosomes in cells
  • flow cytometry to analyze the mean fluorescence intensity of the staining
  • the Lysotracker average fluorescence intensity of ⁇ ex7/8 human neurons was significantly enhanced, indicating that DMF can increase the lysosomal acidity of CLN3 ⁇ ex7/ 8 human neurons; test for the effect of DMF on SCMAS protein accumulation in CLN3 ⁇ ex7/8 human neurons It was shown that DMF treatment significantly reduced the accumulation of SCMAS protein in CLN3 ⁇ ex7/8 human neurons ( FIG. 16 ).
  • Compound G increases the level of autophagy and mitophagy in CLN3 ⁇ ex7/8 neurons and maintains mitochondrial function and steady state
  • the autophagosomes in neural stem cells were labeled with tandem LC3, and the number of autophagosomes with green fluorescence and the number of autolysosomes with only red fluorescence were observed by high-content laser confocal microscopy.
  • CLN3 ⁇ ex7/8 human neural stem cells the number of red fluorescent single-positive autophagosomes decreased, and the treatment of compound G could increase the number of red fluorescent single-positive autophagosomes in cells ( Figure 17), indicating that the compound G treatment rescues the defect in autophagic flux in CLN3 ⁇ ex7/8 human neural stem cells.
  • the protein level of p62 in cells was detected.
  • the p62 protein expressed by p62 is an autophagy-related protein responsible for connecting the autophagy substrate to the LC3 protein located on the autophagosome membrane. Since p62 can be transported to lysosomes for degradation along with autophagosomes, by comparing the changes in the amount of p62 protein in cells after treatment with lysosomal inhibitor Baf-A1 (p62 (Baf-A1 treatment group) - p62 (untreated group) Baf-A1 treatment group)), can indicate the intensity of intracellular autophagic flux.
  • Damaged mitochondria in cells can be cleared through an autophagic process called mitophagy.
  • Functional defects in autophagy affect the clearance of damaged mitochondria, thereby disrupting mitochondrial homeostasis in neurons.
  • the mitochondrial uncoupler CCCP was used to induce mitochondrial damage in wild-type and CLN3 ⁇ ex7/8 neurons, and then Baf-A1 was used to inhibit the clearance of damaged mitochondria, the number of intracellular mitochondria (mtDNA/nDNA) decreased after Baf-A1 treatment The increase of can represent the level of intracellular mitophagy.
  • compound G significantly increased the level of autophagy and mitophagy in CLN3 ⁇ ex7/8 neurons, and maintained the function and homeostasis of mitochondria.
  • Example 6 Compound G improves the disease phenotype of JNCL mice
  • Cln3 knockout (Cln3 KO) JNCL mouse model was purchased from Jackson Lab.
  • Compound G improves disease-associated phenotypes in JNCL mouse models
  • Compound G can pass through the blood-brain barrier
  • Compound G can reduce the abnormal accumulation of protein in the brain of JNCL mouse model
  • compound G can improve the learning and memory ability of JNCL mice
  • mice were trained to learn the water maze for 6 days, and the time required to find the underwater platform was recorded.
  • the underwater platform was removed, and the mice were tested for 60 seconds, and the time it took for the mice to reach the original platform location for the first time, the number of shuttles at the original platform location and the quadrant of the original platform were recorded.
  • the time of staying is indicative of whether it has acquired the location of the underwater platform through the training of the previous 6 days.
  • mice were tested for learning and memory ability. To test, the mouse was gently lowered into the water from a completely new position farthest from the original platform. The results showed that, compared with wild-type mice, the Cln3 KO mice took significantly longer to reach the original platform for the first time (Fig. 23(b)), even longer than the time spent on the 6th day (Fig. 23 (a)), this may be due to the fact that mice started to find the platform from a completely new position on day 7, which was different from the familiar starting position during training. In contrast, the treatment of compound G can effectively shorten the time it takes for Cln3 KO mice to reach the position of the original platform for the first time (Fig. 23(b)), indicating that the mice treated with compound G can better learn and remember water levels Where the lower platform is located.
  • Cln3 KO mice stayed in the quadrant of the original platform significantly shorter than wild-type mice ( Figure 23(c)), further indicating that their ability to remember the location of the original platform was not as good as that of wild-type mice, and after compound G treatment, Cln3 KO mice prolonged their residence time in the quadrant of the original platform to a certain extent.
  • Cln3 KO mice shuttled at the original platform location tended to decrease, while the number of shuttles at the original platform location of the Cln3 KO mice was closer to that of wild-type mice after treatment with compound G (Fig. 23 (d)).
  • Compound G can improve the exercise capacity of JNCL mice
  • Example 7 Compound G inhibits KEAP1 and enhances autophagy-lysosome function
  • Compound G's known target is barely expressed in neurons
  • TFEB transcription factor EB
  • FXR farnesoid X receptor
  • mouse TTF was infected with lentivirus packaged with control shRNA or FXR shRNA-2, and after compound G was treated, mRNA was extracted for detection of gene transcription. It was found that compound G could significantly increase the expression of autophagy-related genes in control shRNA-infected cells. However, in TTF infected with FXR shRNA-2, the treatment of compound G could not increase the expression of these genes (Fig. 28(b)). The data showed that the knockdown of FXR itself could significantly increase the expression levels of Tfeb and autophagy-related genes (Fig. 28(b)).
  • Compound G activates the expression of NRF2 downstream genes
  • transcriptome sequencing experiments were performed on wild-type human neural stem cells, CLN3 ⁇ ex7/8 human neural stem cells, and CLN3 ⁇ ex7 / 8 human neural stem cells treated with compound G for 6 hours, and Differentially expressed genes were analyzed.
  • PCA analysis showed that in CLN3 ⁇ ex7/8 human neural stem cells, compound G treatment for 6 hours did not have a significant effect on its gene expression profile, and the compound G-treated and untreated CLN3 ⁇ ex7/8 human neural stem cells clustered in One place (Fig. 29(b, c)).
  • Compound G inhibits KEAP1 protein activity
  • NRF2 protein is mainly regulated by KEAP1 protein.
  • Multiple electrophiles or electrophile precursor compounds can increase NRF2 activity by inhibiting KEAP1, such as isothiocyanates, ⁇ , ⁇ -unsaturated carbonyl compounds, phenols and polyphenols, etc.
  • the basic mode of action involves a covalent reaction with cysteine residues on KEAP1, mainly including cysteine residues 151, 272, and 288, resulting in changes in the binding mode of KEAP1 and NRF2, thereby activating NRF2 (Wells, 2015 ).
  • Compound G contains an ⁇ , ⁇ -unsaturated carbonyl group, so a quantitative thiol reactivity assay was performed to determine whether compound G could covalently bind to the cysteine on KEAP1.
  • CLN3 ⁇ ex7/8 human neural stem cells were treated with compound G for 2 hours, the cells were lysed, and the thiol reactivity of cysteine residues on KEAP1 in the lysate was analyzed by mass spectrometry. The results showed that the treatment of compound G could reduce the thiol reactivity of cysteine residue at position 288 of KEAP1 protein ( FIG. 33 ). It indicated that compound G may inhibit the function of KEAP1 by covalently binding to the 288-position cysteine residue of KEAP1 protein, thereby activating the expression of NRF2 and its downstream genes.
  • KEAP1 protein was knocked down in the CLN3 ⁇ ex7/8 human neuron model using shRNA targeting KEAP1.
  • shRNA targeting KEAP1 Five shRNA lentiviral plasmids targeting KEAP1 were selected from the human shRNA library, and then packaged into lentiviruses using HEK293T cells and infected with human neural stem cells.
  • shRNA was extracted, and the relative abundance of KEAP1 mRNA in cells was detected by qRT-PCR. The results showed that KEAP1 shRNA-3 had the highest knockdown efficiency ( FIG. 34 ), and then this shRNA was used to conduct subsequent experiments to detect the effect of KEAP1 knockdown on CLN3 ⁇ ex7/8 human neurons.
  • Lysotracker staining was used to indicate the acidity of lysosomes in cells, and the mean fluorescence intensity of staining was analyzed by flow cytometry.
  • the average fluorescence intensity of Lysotracker in KEAP1-knockdown CLN3 ⁇ ex7/ 8 human neurons was significantly enhanced, but the addition of Compound G in KEAP1-knockdown CLN3 ⁇ ex7/8 human neurons could not further increase the average fluorescence intensity of Lysotracker ( Figure 35).
  • tandem LC3 protein was overexpressed in human neural stem cells to label intracellular autophagosomes, and imaged using high-content laser confocal microscopy.
  • knockdown of KEAP1 could significantly increase the number of red fluorescent single-positive autophagosomes in CLN3 ⁇ ex7/8 human neural stem cells.
  • KEAP1 knockdown CLN3 ⁇ ex7/8 human neurons were infected with OMP25-GFP lentiviral plasmid to mark the morphology of mitochondria in the cells. The cells were then imaged with a confocal laser microscope. There are a large number of damaged punctate mitochondria in CLN3 ⁇ ex7/8 human neurons, and the treatment of compound G or knockdown of KEAP1 can significantly reduce the appearance of damaged mitochondria in CLN3 ⁇ ex7/8 human neurons ( FIG. 39 ).
  • knockdown of KEAP1 can mimic the effects of compound G on lysosomal function, autophagic flux intensity, and mitochondrial homeostasis in the CLN3 ⁇ ex7/8 human cell model.
  • Compound G exerts the above functions in a KEAP1-dependent manner.
  • Compound G enhances lysosomal acidity through the p62-KEAP1 pathway
  • the present invention detects the effect of compound G treatment or KEAP1 knockdown on intracellular p62 protein level.
  • Compound G was added to human neurons, and the cell lysate was extracted after incubation for 24 hours, and the transcription level of p62 gene was detected by qRT-PCR.
  • the results showed that the treatment of compound G or the knockdown of KEAP1 could significantly increase the transcription level of p62 in human neurons ( FIG. 41( a )).
  • treatment of Compound G or KEAP1 knockdown also increased p62 protein levels ( FIG. 41( b )).
  • compound G can still significantly increase the protein level of p62 in cells under the condition of adding Baf-A1, which indicates that the regulation of p62 protein by compound G is not by inhibiting the function of lysosome (Fig. 22(a, b)).
  • DMF dimethyl fumarate
  • KEAP1 inhibitor Treatment with dimethyl fumarate (DMF), another known KEAP1 inhibitor, also significantly increased the acidity of intracellular lysosomes (Fig. 42(a)).
  • a similar phenomenon was observed after overexpressing GFP-p62 protein in human neural stem cells.
  • the overexpression of p62 protein can significantly increase the lysosomal acidity in neural stem cells ( Figure 42(b)), and alleviate the expression of CLN3 ⁇ ex7 Abnormal accumulation of toxic proteins and neuronal death in /8 human neurons (FIG. 43).
  • Example 8 Compound G reduces the accumulation of toxic proteins in a human neuronal model of Alzheimer's dementia
  • the present invention examines the therapeutic effect of compound G treatment on Alzheimer's dementia.
  • the APP gene mutant human neurons treated with compound G showed a significant reduction in the deposition of A ⁇ plaques, as well as a significant reduction in the level of toxic A ⁇ 42 secreted by the cells into the medium ( FIG. 44 ).
  • the present invention reveals that small molecule compounds, including Compound G, enhance the autophagy flux and lysosome function in human neurons by inhibiting KEAP1, thereby benefiting the treatment of JNCL, and at the same time prove that knocking down the KEAP1 gene can also achieve the same effect .
  • small molecule compounds including Compound G
  • defects in lysosomal function and reduced autophagy are also involved in the pathogenesis of a variety of NCL and other late-onset neurodegenerative diseases, and it is reasonable to infer that inhibition of KEAP1 activity has a positive effect on other autophagy and lysosomal defects.
  • Neurodegenerative diseases also have a certain therapeutic effect, and the means of inhibiting KEAP1 include the use of small molecule compounds identified in the present invention including compound G or knocking down the KEAP1 gene.

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Abstract

一种通过抑制KEAP1活性和/或增强转录因子E2相关因子2(NRF2)和/或其下游基因的表达以治疗与细胞自噬功能缺陷和/或溶酶体功能缺陷相关疾病如神经退行性疾病的方法，其中采用KEAP1抑制剂、敲减KEAP1基因、和/或NRF2活化剂来增强NRF2和/或其下游基因的表达。所述方法和产品能够提高神经元的溶酶体酸性环境，提高组织蛋白酶的酶活，减少蛋白质的异常贮积，保护神经元中线粒体稳态，提高线粒体产生ATP能力。

Description

治疗神经元蜡样质脂褐质沉积症的方法和药物 技术领域
本发明涉及治疗神经退行性疾病如神经元蜡样质脂褐质沉积症尤其是青少年神经元蜡样质脂褐质沉积症(JNCL)的方法及治疗所用的药物。
背景技术
神经元蜡样脂褐质沉积症(Neuronal ceroid lipofuscinosis,NCL)是一类罕见的遗传性神经系统疾病，属于溶酶体贮积疾病(Lysosomal storage diseases,LSD)家族，其中大多数是常染色体隐性遗传病。NCLs症状多出现于婴儿和儿童时期，大多数患者会表现出一个渐进的神经退行性临床过程。作为一种典型的NCL疾病，青少年型神经元蜡样脂褐质沉积症(Juvenile neuronal ceroid lipofuscinosis,JNCL)，又称巴顿病(Batten disease)，是最常见的遗传性青少年神经系统疾病之一，由CLN3基因突变引起。JNCL患者的细胞会表现出明显的溶酶体功能缺陷的特征，胞内线粒体ATP合成酶c亚基(SCMAS)、脂蛋白和糖蛋白等多种物质无法被正常降解，从而在溶酶体中逐渐聚积。神经元由于高度依赖溶酶体进行物质代谢，因而是受CLN3突变影响最大的细胞类型之一。患者在2-10岁左右会表现出严重的视力障碍，随后出现癫痫、进行性运动障碍和认知能力下降，通常在20-30岁之间因病去世。目前，JNCL尚无根治方法，临床上所有治疗都是对症和姑息性的。
溶酶体是一种在细胞内外物质的降解和循环利用中起着关键作用的细胞器(Luzio et al.,2009；Mizushima and Komatsu,2011)。为了维持细胞稳态，一个由溶酶体、内体、自噬小体和其他细胞成分组成的网络，行使对细胞内外的物质进行降解的功能(Saftig and Klumperman,2009)。溶酶体酸度的变化会影响腔内多种酶的活性，从而破坏其底物的清除过程，导致包括NCL在内的多种神经退行性疾病的发生(Song et al.,2020)。
已有的研究显示，CLN3蛋白主要定位于细胞溶酶体膜上，其在细胞内行使的功能至今尚不明确。对体外和体内JNCL疾病模型的研究显示，其具备溶酶体酸度失调、溶酶体内蛋白酶活性降低和自噬缺陷等溶酶体受损表型。自噬和溶酶体功能异常是导致衰老和神经退行性疾病的重要原因之一，但自噬和溶酶体在衰老和神经退行性疾病发病机制中扮演的角色仍是需研究和探索的内容。
研究显示，通过促进自噬起始来激活细胞自噬对于CLN3突变的细胞并不产生治疗作用。目前，最经典的诱导自噬的化合物是靶向mTORC1信号通路的小分子，如雷帕霉素 (Rapamycin)。利用mTORC1抑制剂处理Cln3 Δex7/8细胞会增加自噬小体的形成，但是形成的自噬小体并不能被溶酶体有效降解，并在细胞内聚积(Centa et al.,2020)，疾病表型无法得到有效缓解。
因此，仍然需要有效治疗JNCL等相关神经退行性疾病的手段。
发明简述
本发明的一个方面涉及一种治疗与细胞自噬功能缺陷和/或溶酶体功能缺陷相关疾病的方法，该方法包括抑制Kelch样环氧氯丙烷相关蛋白1(KEAP1)的活性，和/或增强转录因子E2相关因子2(NRF2)和/或其下游蛋白的活性的步骤。
根据本发明所述的治疗方法，所述与细胞自噬功能缺陷和/或溶酶体功能缺陷相关的疾病为神经退行性疾病。
根据本发明所述的治疗方法，所述神经退行性疾病为选自肌萎缩性侧索硬化症(ALS)、阿尔茨海默氏痴呆、亚历山大病、阿尔珀斯病(Alper’sdisease)、共济失调-毛细血管扩张症、牛海绵状脑病(BSE)、Canavan病、科凯恩综合征(Cockaynesyndrome)、皮质基底节变性、克-雅病、亨廷顿病、HIV相关痴呆、肯尼迪病、克拉伯病(Krabbedisease)、路易体痴呆、马查多-约瑟夫病(脊髓小脑共济失调3型)、多发性硬化症、多系统萎缩、神经疏螺旋体病(Neuroborre1iosis)、帕金森病、佩-梅病、皮克氏病、原发性侧索硬化、Prion病、雷夫叙姆病(Refsum’s disease)、桑德霍夫病(Sandhoff disease)、希尔德病、精神分裂症、Spielmeyer-Vogt-Sjogren-Batten病、脊髓小脑共济失调、脊髓性肌肉萎缩症、或神经元蜡样脂褐质沉积症(NCL)。
在一些具体实施方案中，所述神经退行性疾病选自帕金森病、阿尔茨海默症、亨廷顿病、肯尼迪病、肌萎缩性侧索硬化症、原发性侧索硬化、多发性硬化、额颞叶痴呆或神经元蜡样脂褐质沉积症(NCL)。
在一些优选的实施方案中，所述神经退行性疾病优选为神经元蜡样脂褐质沉积症，更优选青少年神经元蜡样脂褐质沉积症(JNCL)。
根据本发明所述的治疗方法，所述疾病由CLN3基因突变所致，例如由CLN3基因外显子7和8的缺失突变CLN3 Δex7/8所致，其中所述缺失突变约1.02kb。
根据本发明所述的治疗方法，其中采用siRNA、sgRNA或构建有shRNA的载体沉默或敲减KEAP1基因的表达，从而增强NRF2和/或其下游蛋白的活性。
根据本发明所述的治疗方法，其中采用KEAP1抑制剂和/或NRF2活化剂增强NRF2和/或其下游基因的表达，从而增强NRF2和/或其下游蛋白的活性。
根据本发明所述的治疗方法，其中所述KEAP1抑制剂和/或NRF2活化剂选自能够与KEAP1上包括第151、272和/或288位半胱氨酸在内的半胱氨酸残基发生共价反应的试剂。
根据本发明所述的治疗方法，其中所述KEAP1抑制剂和/或NRF2活化剂选自Carvedilol(CAS:72956-09-3)、Ketoconazole(CAS:65277-42-1)、GANT61,CAS(500579-04-4)、Protriptyline hydrochloride(CAS:1225-55-4)、LP 44(CAS:824958-12-5)、Doxepin HCl(CAS:1229-29-4)、富马酸二甲酯(DMF)、乙酰-11-羰基-β-乳香酸(AKBA)、异硫氰酸酯、巴多索隆(CDDO)、甲基巴多索隆(CDDO-Me)以及这些化合物的衍生物或类似物，或者选自α,β-不饱和羰基化合物、酚类和多酚类化合物中的一种或多种。
根据本发明所述的治疗方法，其中所述的KEAP1抑制剂为化合物G，即(Z)-guggulsterone，其结构式为：
Figure PCTCN2022099308-appb-000001
本发明的另一个方面涉及KEAP1抑制剂和/或NRF2活化剂在制备用于抑制KEAP1的活性，和/或增强转录因子E2相关因子2(NRF2)和/或其下游蛋白的活性以治疗与细胞自噬功能缺陷和/或溶酶体功能缺陷相关疾病的药物中的用途。
根据本发明所述的用途，其中所述与细胞自噬功能缺陷和/或溶酶体功能缺陷相关的疾病为神经退行性疾病。
根据本发明所述的用途，其中所述神经退行性疾病为选自肌萎缩性侧索硬化症(ALS)、阿尔茨海默氏痴呆、亚历山大病、阿尔珀斯病(Alper’sdisease)、共济失调-毛细血管扩张症、牛海绵状脑病(BSE)、Canavan病、科凯恩综合征(Cockaynesyndrome)、皮质基底节变性、克-雅病、亨廷顿病、HIV相关痴呆、肯尼迪病、克拉伯病(Krabbedisease)、路易体痴呆、马查多-约瑟夫病(脊髓小脑共济失调3型)、多发性硬化症、多系统萎缩、神经疏螺旋体病(Neuroborre1iosis)、帕金森病、佩-梅病、皮克氏病、原发性侧索硬化、Prion病、雷夫叙姆病(Refsum’s disease)、桑德霍夫病(Sandhoff disease)、希尔德病、精神分裂症、 Spielmeyer-Vogt-Sjogren-Batten病、脊髓小脑共济失调、脊髓性肌肉萎缩症、或神经元蜡样脂褐质沉积症(NCL)。
在一些优选的实施方案中，其中所述神经退行性疾病优选为神经元蜡样脂褐质沉积症，更优选青少年神经元蜡样脂褐质沉积症(JNCL)。
根据本发明所述的用途，其中所述疾病由CLN3基因突变所致，例如由CLN3基因外显子7和8的缺失突变CLN3 Δex7/8所致，其中所述缺失突变约1.02kb。
根据本发明所述的用途，其中采用siRNA、sgRNA或构建有shRNA的载体沉默或敲减KEAP1基因的表达，从而增强NRF2和/或其下游蛋白的活性。
根据本发明所述的用途，其中采用KEAP1抑制剂和/或NRF2活化剂增强NRF2和/或其下游的基因的表达，从而增强NRF2和/或其下游蛋白的活性。
根据本发明所述的用途，其中所述KEAP1抑制剂和/或NRF2活化剂选自能够与KEAP1上包括第151、272和/或288位半胱氨酸在内的半胱氨酸残基发生共价反应的试剂。
根据本发明所述的用途，其中所述KEAP1抑制剂和/或NRF2活化剂选自以下化合物中的一种或多种：Carvedilol(CAS:72956-09-3)、Ketoconazole(CAS:65277-42-1)、GANT61,CAS(500579-04-4)、Protriptyline hydrochloride(CAS:1225-55-4)、LP 44(CAS:824958-12-5)、Doxepin HCl(CAS:1229-29-4)、富马酸二甲酯(DMF)、乙酰-11-羰基-β-乳香酸(AKBA)、异硫氰酸酯、巴多索隆(CDDO)、甲基巴多索隆(CDDO-Me)以及这些化合物的衍生物或类似物，或者选自α,β-不饱和羰基化合物、酚类和多酚类化合物。
根据本发明所述的用途，其中所述的KEAP1抑制剂为化合物G，即(Z)-guggulsterone，其结构式为：
Figure PCTCN2022099308-appb-000002
本发明的又一个方面涉及一种用于治疗与细胞自噬功能缺陷和/或溶酶体功能缺陷相关疾病的药物组合物，其包含治疗上有效量的一种或多种KEAP1抑制剂和/或NRF2活化 剂，和药物上可接受的载体和/或赋形剂。
根据本发明所述的药物组合物，其中所述KEAP1抑制剂和/或NRF2活化剂选自能够与KEAP1上包括第151、272和/或288位半胱氨酸在内的半胱氨酸残基发生共价反应的试剂。
根据本发明所述的药物组合物，其中所述KEAP1抑制剂和/或NRF2活化剂选自以下化合物中的一种或多种：Carvedilol(CAS:72956-09-3)、Ketoconazole(CAS:65277-42-1)、GANT61,CAS(500579-04-4)、Protriptyline hydrochloride(CAS:1225-55-4)、LP 44(CAS:824958-12-5)、Doxepin HCl(CAS:1229-29-4)、富马酸二甲酯(DMF)、乙酰-11-羰基-β-乳香酸(AKBA)、异硫氰酸酯、巴多索隆(CDDO)、甲基巴多索隆(CDDO-Me)以及这些化合物的衍生物或类似物，或者选自α,β-不饱和羰基化合物、酚类和多酚类化合物。
根据本发明所述的药物组合物，其中所述的KEAP1抑制剂为化合物G，即(Z)-guggulsterone，其结构式为：
Figure PCTCN2022099308-appb-000003
本发明的再一个方面涉及一种用于治疗与细胞自噬功能缺陷和/或溶酶体功能缺陷相关疾病的试剂盒，其包含抑制KEAP1基因表达的试剂。
根据本发明所述的试剂盒，其包含siRNA、sgRNA或构建有shRNA的载体，用于沉默或敲减KEAP1基因，以抑制KEAP1基因的表达。
本发明的另一个方面涉及一种用于缓解或消除受试者神经干细胞向神经元的分化缺陷的方法，所述方法包括向有需要的受试者施用抑制KEAP1活性和/或增强NRF2和/或其下游蛋白活性的药物，其中所述受试者患有与细胞自噬功能缺陷和/或溶酶体功能缺陷相关的疾病。
根据本发明所述的方法，其中所述疾病由CLN3基因突变所致，例如由CLN3基因外显子7和8的缺失突变所致，所述疾病优选JNCL。
根据本发明所述的方法，其中所述的药物选自以下药物中的一种或多种：化合物G即(Z)-guggulsterone、Carvedilol(CAS:72956-09-3)、Ketoconazole(CAS:65277-42-1)、GANT61,CAS(500579-04-4)、Protriptyline hydrochloride(CAS:1225-55-4)、LP 44(CAS: 824958-12-5)、Doxepin HCl(CAS:1229-29-4)、富马酸二甲酯(DMF)、乙酰-11-羰基-β-乳香酸(AKBA)、异硫氰酸酯、巴多索隆(CDDO)、甲基巴多索隆(CDDO-Me)以及这些化合物的衍生物或类似物，或者选自其它α,β-不饱和羰基化合物、酚类和多酚类化合物。
根据本发明的方法，其中所述药物优选为化合物G即(Z)-guggulsterone
Figure PCTCN2022099308-appb-000004
本发明的另一个方面涉及一种用于筛选可激活细胞自噬流和/或增强溶酶体功能的物质的系统，其特征在于该系统包含双荧光标记的Tandem LC3报告系统。
根据本发明所述的筛选系统，其中所述双荧光标记的LC3所在的自噬小体位于细胞质时呈现例如红色和绿色双荧光，自噬小体与溶酶体融合形成自溶酶体时则只呈现如红色单荧光。
根据本发明的筛选系统，其包括稳定表达tandem LC3的NRK细胞系。
本发明的又一个方面涉及一种筛选可激活细胞自噬流和/或增强溶酶体功能的物质的方法，其特征在于使用前述的筛选系统，如果待筛选物质使得所述系统中双荧光和单荧光标记的自噬小体数量和自溶酶体数量均增强，则该物质为能够激活细胞自噬流的目标物质。
本发明的再一个方面涉及基于神经干细胞或神经元的体外模型，其中该模型存在CLN3基因外显子7和8的缺失突变即CLN3 Δex7/8。
本发明的另一个方面涉及利用所述体外模型筛选诱导自噬的化合物和/或验证所述化合物的效果的用途。
本发明通过抑制KEAP1基因的表达或活化NRF2的表达，直接或间接地激活或增强NRF2和/或其下游基因的表达，提高了神经元的溶酶体酸性环境和神经元中组织蛋白酶的酶活，减少了神经元中蛋白质的异常贮积，保护神经元中线粒体稳态并提高线粒体产生ATP能力，从而达到预防或治疗与细胞自噬功能缺陷和/或溶酶体功能缺陷相关的神经退行性疾病的效果。
发明详述
定义
为了使本文更容易理解，首先定义某些术语。在整个发明详述中还阐述了其他的定义。
如本文所用，术语“受试者”与“患者”可互换使用，且指需要治疗的哺乳动物，如宠物(例如，狗、猫等)、牲畜(例如牛、猪、马、绵羊、山羊等)及实验动物(例如大鼠、小鼠、豚鼠等)。典型地，受试者为需要治疗的人类。
术语“治疗”是指获得所需的药理学和/或生理学效应。所述效应可为治疗性的，其包括部分或实质实现以下的一或多个：部分地或完全地降低疾病、病症或综合征的严重程度；缓解或改善与病症有关的临床症状或指标；及延迟、抑制或降低疾病、病症或综合征的进展可能性。
本发明的化合物可用来治疗神经退行性病症。神经退行性病症的实例包括但不限于：肌萎缩性侧索硬化症(ALS)、阿尔茨海默氏痴呆、亚历山大病、阿尔珀斯病(Alper’sdisease)、共济失调-毛细血管扩张症、牛海绵状脑病(BSE)、Canavan病、科凯恩综合征(Cockaynesyndrome)、皮质基底节变性、克-雅病、亨廷顿病、HIV相关痴呆、肯尼迪病、克拉伯病(Krabbedisease)、路易体痴呆、马查多-约瑟夫病(脊髓小脑共济失调3型)、多发性硬化症、多系统萎缩、神经疏螺旋体病(Neuroborre1iosis)、帕金森病、佩-梅病、皮克氏病、原发性侧索硬化、Prion病、雷夫叙姆病(Refsum’s disease)、桑德霍夫病(Sandhoff disease)、希尔德病、精神分裂症、Spielmeyer-Vogt-Sjogren-Batten病、脊髓小脑共济失调、脊髓性肌肉萎缩症、神经元蜡样脂褐质沉积症(NCL)、巴顿病(Batten disease)。
本发明小分子物质可以本身或者作为药物组合物的一部分送给受治疗者。本文所用的“药物组合物”是指一种或多种本文所述的活性成分与其他化学组分例如生理学上适合的载体和赋形剂的制品。药物组合物的目的是促进化合物施用至生物体。
将本文所述的化合物或其药学上可接受的盐，或其他形式，施用于受试者，可使用任何合适的递送方法，包括表面、经肠、胃肠外、经皮、经黏膜、经由吸入、脑池内、硬膜、阴道内、静脉内、肌肉内、皮下、皮内及玻璃体内将本文所述的化合物或其药学上可接受的盐施用于受试者。将本文所述的化合物或其药学上可接受的盐，或其他形式，施用于受试者还包括表面、经肠、胃肠外、经皮、经黏膜、经由吸入、脑池内、硬膜、阴道内、静脉内、肌肉内、皮下、皮内及玻璃体内向受试者施用在其身体表面内或上，可代谢为本文所述的化合物或其药学上可接受的盐，或其他形式的化合物。
本发明的药物组合物可以通过本领域公知的方法制造，例如通过常规的混合、溶解、造粒、糖衣片制作、磨细、乳化、装胶囊、包埋或冷冻干燥。
本文所述的化合物或其药学上可接受的盐可与药学上可接受的载体(如惰性稀释剂或可同化食用的载体)组合全身性(例如经口)施用。其可被封入硬壳或软壳明胶胶囊中，可被压制为片剂，或可直接与患者的膳食合并。关于经口治疗性施用，本文所述的化合物、物质，或其药学上可接受的盐，或其他形式，可与一种或多种赋形剂组合，且以可摄取片剂、颊片剂、口含锭、胶囊、酏剂、混悬液、糖浆或糯米纸等形式施用。
所述片剂、口含锭、丸剂、胶囊等可包括以下：黏合剂，如黄蓍胶、阿拉伯胶、玉米淀粉和明胶；赋形剂，如磷酸二钙；崩解剂，如玉米淀粉、马铃薯淀粉、褐藻酸等；润滑剂，如硬脂酸镁；甜味剂，如蔗糖、果糖、乳糖和阿斯巴甜糖；及调味剂。
用于静脉内、肌肉内、皮下、皮内等注射或输注的示例药物剂型包括，含有活性成分的无菌水溶液或分散液及无菌粉末，所述无菌粉末适用于无菌可注射或可输注溶液或分散液的临时制备。最终剂型均应在制造和储存条件下为无菌、流体且稳定的。
无菌可注射溶液可通过将所需量的活性成分与所需的上下文列举的多种其他成分一起并入适当溶剂中，随后过滤灭菌来制备。在制备无菌可注射溶液的无菌粉末的情况下，优选制备方法可为真空干燥和冷冻干燥技术，所述技术可产生活性成分加上存在于经无菌过滤的溶液中的任何额外所需成分的粉末。
所述组合物和制剂应含有至少约0.1％的活性成分。所述组合物和制剂的百分比当然可变化且可便利地在给定单位剂型重量的约2～60％。所述可用于治疗的药物组合物中的活性成分的用量为可获得有效剂量水平。
用于治疗所需的本文所述的化合物，或其药学上可接受的盐，或其他形式的用量可随所选择的特定形式变化，且可随施用途径、所治疗的病状性质及患者的年龄和状况而变化，且最终可由主治医师或临床医师判断。
施用于受试者的本文所提供的化合物，或其药学上可接受的盐，或其他形式的剂量可为10μg～5,000mg；10μg～1mg；1～500mg；或500～5,000mg等。优选地，可便利地以单位剂型施用。例如每单位剂型含有0.01～10mg或0.05～1mg活性成分。在一些实施方案中，5mg/kg或更少剂量。所需剂量可以单次剂量提供或在适当时间间隔下以分次剂量施用。
本文所述的化合物，或其药学上可接受的盐，或其他形式的适用剂量可通过在动物模型中比较其体外活性与体内活性来测定。用于将小鼠及其他动物中的有效剂量外推至人类 的方法为本领域中已知的。
可互换使用的短语“生理学上可接受的载体”和“药学上可接受的载体”是指不引起对生物体的显著刺激并且不消除所施用的化合物的生物活性和性质的载体或稀释剂，包括佐剂。
示例性的固体载体可包括精细分散的固体，如滑石、黏土、微晶纤维素、硅石、氧化铝等。适用的液体载体包括水、醇、二醇及水-醇/二醇掺混物。本文所述的化合物，或其药学上可接受的盐，或其他形式，可以有效水平溶解或分散，任选地借助于无毒表面活性剂。
本文术语“赋形剂”是指被加入到药物组合物中进一步促进活性成分的施用的惰性物质。赋形剂的非限制性实例包括碳酸钙、磷酸钙、各种糖和各种类型的淀粉、纤维素衍生物、明胶、植物油和聚乙二醇等。
本发明利用CRISPR/Cas9基因编辑技术，在hESC中引入了JNCL患者中最常见的CLN3基因外显子7和8的纯合缺失突变，并诱导其向神经谱系分化，构建了JNCL疾病的人神经元模型。该模型重现了溶酶体和自噬功能缺陷以及线粒体稳态失调等多个JNCL疾病相关表型。由于自噬和溶酶体的正常功能对于维持神经元的稳态至关重要，建立了旨在发现新型促自噬小分子的高通量筛选体系。
通过筛选，得到了一系列激活细胞自噬和溶酶体功能的小分子化合物，并在构建好的JNCL疾病人神经元模型中检测了化合物缓解疾病表型的效果。结果显示，有一个小分子化合物G(guggulsterone)可在体外有效缓解JNCL疾病相关的自噬和溶酶体功能缺陷，并保护线粒体稳态。在JNCL小鼠模型中，guggulsterone被证明可以穿透血脑屏障，并部分缓解小鼠表现出的学习记忆缺陷和运动能力缺陷。
结合RNA测序和定量硫醇反应性分析，进一步探索了guggulsterone的作用机制，发现guggulsterone通过抑制KEAP1蛋白，增强了细胞自噬和溶酶体功能，缓解JNCL相关疾病表型。
实验材料
细胞培养试剂：
Figure PCTCN2022099308-appb-000005
培养基、Advanced DMEM/F-12培养基、DMEM/F12培养基、DMEM基础培养基、DPBS缓冲液、Opti-MEM培养基、胎牛血清(Fetal Bovine Serum，FBS)、GlutaMAXTM(100×)、N2 Supplement(100×)、B27 Supplement(50×)、青霉素/链霉素 (Penicillin/Streptomycin，100×)、Trypsin-EDTA、StemPro Accutase均购自Gibco公司；mTeSRTM1培养基购自STEMCELL公司；硼酸盐缓冲液购于Thermo Fisher Scientific公司；Matrigel购自BD公司；多聚赖氨酸(Poly-D-lysine，PDL)购自Sigma Aldrich公司。
小分子及细胞因子：
CHIR99021(S1263)、SB431542(S1067)、rapamycin(S1039)、Bafilomycin A1(S1413)、Y27632 2HCl(S1049)购自Selleck公司；cAMP(A9501)、L-抗坏血酸(A4403)购自Sigma Aldrich公司；化合物G((Z)-Guggulsterone)购自Tocris公司；Human GDNF(450-10-500)、Human/Murine/Rat BDNF(450-02-500)购自PeproTech公司；Recombinant Mouse EGF Protein(2028-EG)、Recombinant Mouse FGF basic(3139-FB)购自R&D公司；Leukemia Inhibitory Factor Human(hLIF，LIF1010)购自Millipore公司。
分子生化实验试剂：
脂质体3000(
Figure PCTCN2022099308-appb-000006
3000 Reagent)、Hochest 33342、ECL显色液与底物发光液(Super SignalTM West Dura Extended Duration Substrate)、PageRuler TM Prestained Protein Ladder购自Thermo Fisher Scientific公司；DMSO、多聚甲醛、无水乙醇、异丙醇、过硫酸铵(AP)、30％Acr/Bis制胶溶液、四甲基乙二胺、十二烷基硫酸钠、碘乙酰胺(IAA)购自Sigma Aldrich公司；
Figure PCTCN2022099308-appb-000007
HiFi DNA Assembly kit购自NEB公司；蛋白酶K、Taq DNA聚合酶、6×DNA Loading Buffer、100bp DNA Ladder、
Figure PCTCN2022099308-appb-000008
DNA Marker、PerfectStart TM Green qPCR SuperMix、
Figure PCTCN2022099308-appb-000009
One-Step gDNA Removal and cDNA Synthesis SuperMix、感受态细胞购自全式金公司；Tris、甘氨酸、蔗糖购自Amresco公司；无水甲醇购自北京通广精细化工；蛋白酶抑制剂、磷酸酶抑制剂购自Roche公司；硝酸纤维膜购自Bio-Rad公司；
Figure PCTCN2022099308-appb-000010
Max DNA聚合酶购自Takara公司；QuickBlock TM Western封闭液、QuickBlock TM Western一抗稀释液、CellTiter-Lumi TM Steady Plus发光法细胞活力检测试剂盒、BCA蛋白浓度测定试剂盒(P0010)购自碧云天公司；DTT(A620058-0025)购自BBI Life Sciences；Light Az-UV-biotin(EVU102)、Heavy Az-UV-biotin(EVU151)购自KeraFast；
Figure PCTCN2022099308-appb-000011
Blood&Tissue Kit(69506)、
Figure PCTCN2022099308-appb-000012
Gel Extraction Kit(28706)、RNeazy Mini Plus Kit(74136)、HiSpeed Plasmid Maxi Kit(12663)购自QIAGEN公司；AxyPrep Total RNA Mini Prep Kit(AP-MN-MS-RNA)购自Axygen公司；无内毒素小提中量试剂盒(DP118-02)购自Tiangen公司；Pierce TM Protein A/G Magnetic Beads(88802)购自Thermo Fisher Scientific公司；Amaxa TM P3 Primary Cell 4D-Nucleofector TM电转试剂盒(V4XP-3024)购自Lonza公司；组织蛋白酶D活力试剂盒(ab65302)来自Abcam公司；组织蛋白酶B活力试剂盒(937)购自Immunochemistry技术公司。
抗体：
p62抗体(PM045)购自MBL公司；LC3抗体(2775)、β3-Tubulin抗体(5568P)购自Cell Signaling Technology公司；β-Actin(C4)抗体(sc-47778)来自Santa Cruz公司；重组Anti-ATP synthase C抗体(ab181243)、Pax6抗体(ab195045)购自Abcam公司；Sox1抗体(AF3369)购自R&D公司；β-actin抗体(sc-47778)购自Santa Cruz公司；Nestin抗体(MAB353，MAB5326)、Sox2抗体(AB5603)购自Millipore公司；MAP2抗体(M9942)购自Sigma Aldrich公司；Oct4抗体(MA5-14845)购自Thermo Fisher Scientific公司。
引物序列：
如下表1所示。
表1：检测基因表达情况的qPCR引物信息
Figure PCTCN2022099308-appb-000013
质粒：
pMXS-IP GFP-OMP25(#38249)、pBABE-puro-mCherry-EGFP-LC3B(#22418)、pSpCas9(BB)-2A-Puro(PX459，#48139)购自Addgene；ATG5 shRNA质粒、KEAP1 shRNA质粒、CLN3 shRNA质粒选自
Figure PCTCN2022099308-appb-000014
shRNA质粒文库(MERCK)；人p62-GFP质粒 (一种由ef1a启动子驱动的在哺乳动物细胞中过表达p62蛋白的质粒)、Flag质粒和Flag-KEAP1质粒(分别为在哺乳动物细胞中过表达FLAG和FLAG-KEAP1蛋白的质粒，均由CMV启动子驱动)。
实验动物：
Cln3敲除小鼠B6.129S6-Cln3tm1Nbm/J(029471)购自Jackson Laboratory，在C57BL/6背景繁育；野生型C57BL/6小鼠购自北京维通利华实验动物技术有限公司。动物饲养在独立通气笼具中，每笼最多饲养小鼠6只。小鼠饲养环境具有12/12小时的光照/黑暗循环，环境温度维持在22-26℃。小鼠可以自由获取无菌颗粒食物和饮水。所有动物实验获得实验动物使用和管理委员会(IACUC)批准。
细胞培养和分化
人胚肾细胞HEK293T/17细胞：
在细胞培养皿或细胞培养板中用HEK293T/17细胞培养基(DMEM培养基+10％FBS+1％Penicillin/Streptomycin)贴壁培养，每天换液，至95％-100％细胞密度时传代。传代时，吸去培养基，用dPBS清洗1次细胞，而后用0.25％的Trypsin-EDTA在室温消化1分钟至单细胞，加入5倍体积的HEK293T/17细胞培养基中和Trypsin-EDTA，离心重悬后按照1:5的比例进行传代。
大鼠肾细胞NRK细胞：
在细胞培养皿或细胞培养板中用NRK细胞培养基(NRK大鼠肾细胞培养基：DMEM培养基+10％FBS+1％Penicillin/Streptomycin)贴壁培养，每天换液，至95％-100％细胞密度时传代。传代时，吸去培养基，用dPBS清洗1次细胞，而后用0.25％的Trypsin-EDTA在室温消化3分钟至单细胞，加入5倍体积的NRK细胞培养基中和Trypsin-EDTA，离心重悬后按照1:10的比例进行传代。
人胚胎干细胞：
H9人胚胎干细胞培养在2％matrigel提前包被的细胞培养板上。每天利用新鲜的mTeSRTM1培养基换液。待细胞克隆密度达到70％-80％时传代。传代时，吸去培养基，用dPBS清洗1次细胞，而后加入Versene，在37℃细胞培养箱消化5-7min。待细胞变圆但还未漂浮时吸去Versene，用带有10μM Y27632的mTeSRTM1培养基将细胞吹打成单细胞，按照1:10-1:20的比例传到2％matrigel提前包被的细胞培养板上。 传代当天的培养基为带有10μM Y27632的mTeSRTM1培养基，24小时后撤去Y27632。其中人类胚胎干细胞(hESC)培养基：mTeSRTM1培养基+1％Penicillin/Streptomycin。
人类神经干细胞的分化和维持：
当人胚胎干细胞克隆密度达到80％时，吸去培养基，用dPBS清洗1次细胞，而后加入Versene，在37℃细胞培养箱消化5-7min。待细胞变圆但还未漂浮时吸去Versene，用带有10μM Y27632的mTeSRTM1培养基将细胞吹打成单细胞，按照1:5的比例传到2％matrigel提前包被的细胞培养板上。传代当天的培养基为带有10μM Y27632的mTeSRTM1培养基，24小时后换成人神经干细胞诱导培养基( 人神经干 细胞诱导培养基：Advanced DMEM/F12培养基:
Figure PCTCN2022099308-appb-000015
培养基(1:1)+3μM CHIR99021+2μM SB431542+0.1μM Compound E+5μg/mL BSA+10ng/mL hLIF+1×N2+1×B27+1％GlutaMAXTM+1％Penicillin/Streptomycin)，并连续培养7天，诱导形成人神经干细胞，期间每天换液。7天后，吸去培养基，用dPBS清洗1次细胞，而后加入Accutase，在37℃细胞培养箱消化10min，待细胞变圆但还未漂浮时吸去Accutase，用带有10μM Y27632的人神经干细胞维持培养基( 人神经干细胞维持 培养基：Advanced DMEM/F12培养基:
Figure PCTCN2022099308-appb-000016
培养基(1:1)+3μM CHIR99021+2μM SB431542+5μg/mL BSA+10ng/mL hLIF+1×N2+1×B27+1％GlutaMAXTM+1％Penicillin/Streptomycin)将细胞吹打成单细胞，按照1：3的比例传到2％matrigel提前包被的细胞培养板上。24小时后撤去Y27632。之后，细胞长期培养在人神经干细胞维持培养基中，当细胞密度到达85％-95％时，按上述方法进行传代。人神经干细胞可在体外维持10代以上，也可存在液氮中待用。
人神经元的分化和维持：
人诱导神经干细胞的密度到达85％-95％时，按照上述方法1:20-1:50进行传代。传代当天(记为分化第1天)的培养基为带有10μM Y27632的人神经干细胞维持培养基，24小时后撤去Y27632，并继续在人神经干细胞维持培养基中培养48小时。分化第4天，将培养基换成人神经元分化培养基( 人神经元培养基：DMEM/F12培养基+10ng/ml hBDNF+10ng/ml hGDNF+300ng/mL cAMP+0.2mM vitamin C+1×N2+1×B27+1％Penicillin/Streptomycin)，隔天半换液。分化第17天可以得到高表达MAP2和TUJ1蛋白的人神经元。之后，人神经元可被继续培养在人神经元分化培养基中，隔天半换液。
流式细胞分析和分选
在分析神经干细胞和神经元染色的平均荧光强度或NRK细胞单细胞建系时，使用了流式细胞术：吸去细胞培养基，用dPBS清洗一次，加入细胞类型对应的消化酶消化成单细胞；离心去上清，用1mL新鲜的培养基重悬细胞；用40μm滤膜过滤细胞悬液，收集单细胞滤液；将单细胞滤液转移至流式管中，并利用BD FACS Aria III进行分析或分选；分析时，每个样品分析1×10 4个细胞的荧光强度，得出平均荧光强度(MFI)；分选时，用带有培养基的96孔板接收细胞，每个孔接收一个细胞以建立细胞系。
细胞转染
细胞电转染：
对人胚胎干细胞进行CRISPR/Cas基因编辑时利用Amaxa TM P3 primary cell 4D Nucleofector TM电转染试剂盒进行转染，以12孔细胞培养板为例：提前将Amaxa TM P3 Primary Cell 4D-NucleofectorTM电转试剂盒平衡到室温；待人胚胎干细胞长至密度为70％左右，利用versene在37℃消化10分钟；用1mL含有10μM Y27632的mTeSRTM1培养基将细胞吹打成单细胞；利用台式离心机以500g的转速离心5分钟，尽量将上清去除干净；将试剂盒包含的82μL Nucleofector TM Solution和18μL Supplement混匀，并在其中加入PX459质粒，靶向CLN3的两条sgRNA质粒各1μg，吹打混匀。利用得到的溶液重悬所述人胚胎干细胞，并将其转移至电转杯中，转移过程中避免气泡的产生；利用Lonza电转仪进行电转，电转程序为CB-150；将电转后的细胞转移至5mL新鲜的含有10μM Y27632的mTeSRTM1培养基中，并种植到2％Matrigel提前包被的细胞培养板中；电转24小时后，撤去Y27632，转成新鲜的mTeSRTM1培养基，电转完成。
脂质体3000细胞转染：
用于免疫共沉淀的HEK293T/17细胞主要通过脂质体3000试剂盒进行转染。以10cm细胞培养皿为例：将HEK293T/17细胞培养至40-50％密度；首先将10μg质粒和20μL P3000试剂加入500μL opti-MEM培养基中，震荡混匀；将30μL脂质体3000加入另一管500μL opti-MEM培养基中，颠倒混匀；将脂质体3000加入上述含有质粒的opti-MEM培养基中，颠倒混匀；室温放置15分钟后，逐滴加入HEK293T/17细胞板中。转染8小时后，将细胞培养基吸去，加入新鲜的HEK293T/17细胞培养基， 完成转染；转染36小时后，可以收集细胞进行免疫共沉淀实验。
慢病毒包装和感染
慢病毒包装在HEK293T/17细胞中进行：将HEK293T/17细胞铺在细胞培养板中，长至40％-60％的密度；将慢病毒质粒和两个病毒包装质粒psPAX2和pMD2.G按照5:3:2的比例混合进行转染，转染步骤参照脂质体3000细胞转染步骤；转染8小时后，吸去脂质体3000，换成新鲜的HEK293T/17细胞培养基；转染32-56小时后，收集HEK293T/17细胞培养基，用0.45μm滤膜滤去细胞碎片；按照所需的病毒量，将过滤好的病毒液加入目标细胞的培养基中，剩余的病毒可以液氮速冻后保存在-80℃供后续使用，避免反复冻融；感染8小时后换成目标细胞的新鲜培养基，感染完成。
蛋白免疫印迹
制样：对于贴壁培养的细胞，以六孔细胞板培养的细胞为例，吸去细胞的培养基，用dPBS清洗一次，每孔加入约100μL含有蛋白酶抑制剂和磷酸酶抑制剂的RIPA裂解液(50mM Tris-HCl，PH 7.5，150mM氯化钠，0.25％脱氧胆酸钠，0.1％NP-40，0.1％Triton X-100)，用细胞刮刀将细胞裂解液全部刮下，收集到1.5mL EP管中，置于冰上继续裂解30分钟。对于动物组织，称量动物组织的重量，加入10倍体积的含有蛋白酶抑制剂和磷酸酶抑制剂的RIPA裂解液，利用组织研磨器在冰上研磨组织至无明显组织块，收集到1.5mL EP管中，置于冰上继续裂解30分钟。
以13000rpm的转速在4℃离心细胞或组织裂解液5分钟，并收集上清；利用BCA蛋白浓度测定试剂盒测定裂解液中的蛋白浓度，并将样品稀释到目标浓度；将4倍体积的裂解液上清和1倍体积的5×上样缓冲液(0.3125M Tris-HCl，PH 6.8，10％SDS，50％甘油，0.25％溴酚蓝，0.5M DTT)混合，98℃加热10分钟；聚丙烯酰胺凝胶电泳：利用SDS-PAGE对蛋白样品进行电泳(p62、CLN3使用7.5％浓缩胶+10％分离胶；LC3、SCMAS使用7.5％浓缩胶+15％分离胶)；电泳缓冲液成分为25mM Tris-Base，0.2M甘氨酸，1％SDS，pH 8.3；电泳条件为80V 30分钟，120V 1小时；转膜：利用湿转法将蛋白从分离胶转移至PVDF膜(p62、CLN3使用0.45μm膜；LC3、SCMAS使用0.22μm膜)；转膜液配方为25mM Tris-Base，0.2M甘氨酸，20％甲醇，预冷到4℃；转膜条件为150mA 1-2小时；封闭：将转上蛋白的PVDF膜放置于QuickBlock TM Western封闭液中，在摇床室温封闭1小时，PBST洗涤3次，每次5分钟；一抗孵育：将抗体按照说明书指示的比例稀释到QuickBlock TM Western一抗稀释液，而后加 到封闭完的PVDF膜上，在摇床上4℃孵育过夜或室温孵育2小时，用PBST洗涤3次，每次5分钟；二抗孵育：将对应物种的二抗使用PBST按照1:5000的比例进行稀释，而后在摇床上室温孵育1小时，用PBST洗涤3次，每次5分钟；显色：将ECL显色液底物和发光液1:1混合，而后滴到PVDF膜上，室温反应5分钟后利用化学发光成像仪显色；条带灰度计算：利用ImageJ软件对目的蛋白的灰度进行定量分析。
免疫荧光染色
固定：吸去细胞培养基，用dPBS清洗1次，而后加入4％的多聚甲醛溶液固定细胞，室温孵育20分钟，用dPBS洗涤3次，每次5分钟；封闭和通透：加入封闭液(5％BSA和0.3％Trinton-X100溶于dPBS中)，室温孵育1小时，用dPBS洗涤3次，每次5分钟；一抗孵育：用含有5％BSA的dPBS按照产品说明稀释抗体，4℃孵育过夜或室温孵育2小时，用dPBS洗涤3次，每次5分钟；二抗孵育：将对应物种和荧光基团的二抗使用含有5％BSA的dPBS按照1:1000的比例进行稀释，室温孵育1小时，之后使用dPBS洗涤3次，每次5分钟；细胞核染色：用dPBS稀释DAPI至100ng/mL，室温孵育5分钟，用dPBS洗涤3次，每次5分钟；成像：使用Olympus倒置荧光显微镜IX83或GE高内涵显微镜Opera Phenix成像。
免疫组化染色
对小鼠脑组织进行免疫组化染色：腹腔注射Avertin麻醉剂麻醉小鼠，打开胸腔，心脏灌流20mL预冷的dPBS，冲去小鼠体内血液，而后心脏灌流20mL预冷的4％多聚甲醛溶液，对组织进行预固定；解剖并取出完整的小鼠脑组织，置于含有4％多聚甲醛溶液的50mL离心管中，4℃固定过夜；弃去4％多聚甲醛溶液，dPBS洗涤3次；加入含有15％蔗糖的dPBS溶液，4℃脱水12个小时，至脑组织沉至50mL离心管管底；弃去15％蔗糖的dPBS溶液，加入含有30％蔗糖的dPBS溶液4℃脱水12到24个小时，至脑组织沉至50mL离心管管底；将脑组织取出，用吸水纸吸干组织表面水分后，利用OCT包埋剂包裹脑组织，置于-20℃冷冻；利用冷冻切片机切片，脑组织切片厚度为30μm，将切下的脑片置入dPBS中洗涤3次；加入4％的多聚甲醛溶液固定细胞，室温孵育20分钟，之后使用dPBS洗涤3次，每次5分钟。
封闭和通透：加入封闭液(5％BSA和0.3％Trinton-X100溶于dPBS中)，室温孵育1小时，用dPBS洗涤3次，每次5分钟；一抗孵育：用含有5％BSA的dPBS 按照产品说明稀释抗体，4℃孵育过夜，之后使用dPBS洗涤3次，每次5分钟；二抗孵育：将对应物种和荧光基团的二抗使用含有5％BSA的dPBS按照1:1000的比例进行稀释，室温孵育1小时，之后使用dPBS洗涤3次，每次5分钟；细胞核染色：用dPBS稀释DAPI至100ng/mL，室温孵育5分钟，之后使用dPBS洗涤3次，每次5分钟；将脑组织平铺在载玻片上，滴上防淬灭剂，而后盖上盖玻片，用透明指甲油封闭盖玻片四周，室温避光风干；成像：使用Olympus倒置荧光显微镜IX83或Zeiss780激光共聚焦显微镜进行成像。
细胞内相对ATP含量测定
将神经干细胞或神经元消化成单细胞后，利用血球计数板进行细胞计数。而后将细胞悬液稀释成同样的细胞浓度(10 5～10 6/mL)。取出100μL细胞悬浊液至白色96孔分析板中，加入100μL平衡到室温的CellTiter-Lumi TM Steady Plus试剂，震荡混匀，并在室温孵育15分钟。然后用多功能微孔板读板机读取样品的化学发光强度，以指征细胞内相对ATP含量。
细胞内相对线粒体拷贝数测定
吸去细胞培养板中的培养基，用dPBS清洗一次后，将细胞消化下来。利用细胞培养基终止消化后，离心去除上清，并用200μL dPBS重悬细胞。
Figure PCTCN2022099308-appb-000017
Blood&Tissue Kit被用来提取细胞内的DNA。后参照表2和表3的qRT-PCR体系和反应程序，用提取的DNA替代cDNA进行qRT-PCR反应。数据分析在Prism 7软件中进行，使用2 -ΔΔCt法，以基因组基因b2M为内参，对线粒体序列MinArc的数量进行相对定量。测定细胞内线粒体数量的引物如下：
mtMinArc-F:CTAAATAGCCCACACGTTCCC；
mtMinArc-R：AGAGCTCCCGTGAGTGGTTA；
测定细胞内基因组数量的引物如下：
b2M-F:GCTGGGTAGCTCTAAACAATGTATTCA；
b2M-R:CCATGTACTAACAAATGTCTAAAATGGT。
RNA提取和实时荧光定量PCR(qRT-PCR)
收取细胞培养皿中的细胞，使用AxyPrep Total RNA Mini Prep Kit或是Qiagen AxyPrep Total RNA Mini Prep Kit进行提取，使用Nanodrop测定提取的RNA浓度； 利用全式金的
Figure PCTCN2022099308-appb-000018
One-Step gDNA Removal and cDNA Synthesis SuperMix将提取的mRNA反转录为cDNA，反转录所用引物为Oligo dT引物；逆转录完成后，利用PerfectStart TM Green qPCR SuperMix进行qRT-PCR反应，检测目的基因的表达情况；qRT-PCR反应在Bio-Rad CFX96 Real-Time PCR系统进行；20μL qRT-PCR反应体系和反应程序分别见表2和表3；数据分析在Prism 7软件中进行，使用2 -ΔΔCt法，以GAPDH作为内参，对目的基因的表达量进行相对定量。
表2：qRT-PCR反应体系
Figure PCTCN2022099308-appb-000019
表3：qRT-PCR反应程序
Figure PCTCN2022099308-appb-000020
细胞内组织蛋白酶D活性测定
细胞内的蛋白酶cathepsin D的活性是使用Abcam的Cathepsin D Activity Assay试剂盒完成，每个样品准备3个以上重复，具体步骤如下：吸去细胞培养基，利用dPBS清洗一次，而后消化成单细胞，并重悬在新鲜的细胞培养基中；利用血球计数板进行细胞计数，每个样品计取10 6个细胞，离心去上清后用100μL dPBS清洗一次，4℃离心去上清；加入200μL试剂盒中含有的CD细胞裂解液，冰上孵育10分钟；以13000rpm的转速在4℃离心5分钟，收集上清至新的1.5mL EP管中；将50μL反应液和2μL底物混合，加入40μL CD裂解液和步骤4)中的10μL细胞上清，转移至黑壁透明底的细胞培养板中，在37℃避光孵育1-2小时；利用多功能微孔板读板机读取样品的荧光强度，激发光和发射光波长 分别为328nm和460nm；实验组和对照组的荧光强度的比值即为实验组细胞中组织蛋白酶D酶活性的相对倍数变化。
组织内药物浓度测定
为测定化合物G是否能通过血脑屏障及其在小鼠脑组织中的药物浓度，进行以下实验：将化合物G以1mg/mL的浓度溶解在饮水中喂食小鼠，对照小鼠饮水中加入溶剂；在8个不同的随机时间点取出化合物G处理的小鼠，用Avertin麻醉后，心脏灌流20mL dPBS，以冲洗小鼠体内血液；另外用同样方法灌流未给药的小鼠，作为空白组织；取出完整的小鼠脑组织，称重后，液氮速冻并用混合冷冻研磨仪(RETSCH MM400)进行研磨，震动频率26次/秒，时间5分钟；提前准备和化合物G结构性质类似的小分子作为内参(记为IS)，将IS溶解于80％的甲醇水溶液中至100ng/mL。
根据研磨前小鼠脑组织肿瘤，按照每克脑组织5mL的体积将步骤4)中的IS甲醇溶液加入研磨后的脑组织中，充分震荡混匀；以13000g的转速在4℃离心8分钟，收集上清；将100μL上清和100μL 10％的甲醇水溶液混合后，转入HPLC样品瓶中；将空白组织的上清和100μL 10％的甲醇水溶液混合后，加入化合物G的标准品，至以下浓度，转入HPLC样品瓶中，作为标准曲线：1000ng/mL、500ng/mL、200ng/mL、100ng/mL、50ng/mL、20ng/mL、10ng/mL、5ng/mL、2ng/mL、1ng/mL、0.5ng/mL、0.2ng/mL、0.1ng/mL；利用UPLC-MS/MS鉴定样品中化合物G的峰面积。
色谱条件如下：色谱柱为Waters XSelect HSS T3色谱柱(100×2.1mm,1.8μm)；流动相A为0.1％甲酸甲醇，流动相B为0.1％甲酸水；梯度洗脱：0-3分钟40％流动相A到100％流动相A，维持2分钟，然后5.1分钟回到40％流动相A初始，并在8分钟结束一次运行；流速为0.2mL/min；进样量为10μL；柱温为30℃。
质谱条件如下：电喷雾离子源(ESI)，采用正离子模式，多反应监测(MRM)，离子源温度为500℃，气帘气30psi，碰撞气为中等，离子电压为5500V，喷雾气和辅助加热气为50psi；利用软件AB SCIEX Analyst 1.6.3 Software计算化合物G的峰面积，并利用标准品做出标准曲线，以计算样品中的药物浓度。
表4：质谱参数
Figure PCTCN2022099308-appb-000021
Figure PCTCN2022099308-appb-000022
标记*为定量碎片离子
定量硫醇反应性分析
为测定和化合物G共价结合的蛋白质，进行了化合物G结合的蛋白质组学分析，具体步骤如下：将神经干细胞培养在10cm细胞培养皿中，至90％的细胞密度。一个10cm细胞培养皿的神经干细胞裂解后能得到1-2mg蛋白质；吸去上清，加入含有30μM化合物G的新鲜培养基，或含有等体积溶剂DMSO的培养基，在细胞培养箱37℃孵育2小时；吸去细胞培养基，用10mL dPBS清洗一次，并尽量吸干净。
将细胞培养皿置于冰上，并加入500μL带有蛋白酶抑制剂和磷酸酶抑制剂的RIPA细胞裂解液(配方见流式细胞分析和分选部分)；在冰上，使用细胞刮刀刮下全部细胞，收集到1.5mL EP管中；将样品置于冰上超声破碎，超声功率为20％，每次超声3秒，超声5次；在每个样品中加入终浓度100μM的ENE探针，避光旋转孵育1小时。ENE能够与蛋白质半胱氨酸残基中的巯基反应，产生稳定的硫醚键。
在每个样品中加入终浓度8mM现配的DTT终止反应，避光旋转孵育1小时；在每个样品中加入终浓度32mM现配的碘乙酰胺(iodoacetamide，IAA)，避光旋转孵育0.5小时，以烷基化还原状态的半胱氨酸；在每个样品中加入500μL预冷的无水甲醇和125μL预冷的氯仿，颠倒混匀后，在4℃以1700g的离心力离心30分钟，去上清；在每个样品中加入500μL预冷的无水甲醇和500μL预冷的氯仿，颠倒混匀后，在4℃以11000g的离心力离心10分钟，去上清；重复一次。
打开EP管盖，将样品在37℃金属浴略微加热以蒸干残余液体；加入200μL含有1M尿素的胰蛋白酶消化缓冲液(4mg/ml的碳酸氢铵水溶液，pH＝8)，在冰上超声破碎蛋白质沉淀；加入200μL不含尿素的胰蛋白酶消化缓冲液，以20％的功率超声溶解蛋白，每次超声10秒，超声5次，直至没有肉眼可见的蛋白质颗粒存在；使用BCA蛋白浓度测定试剂盒进行蛋白定量；使用胰蛋白酶消化缓冲液将蛋白质浓度稀释到1mg/mL，转移1mL稀释好的蛋白到一个新的1.5mL EP管中；加入20μg测序级胰蛋白酶原液(Promega，V5113)，并在37℃在热搅拌器中孵育过夜；在室温以14000g离心力离心5分钟，收集 上清液到一个新的1.5mL EP管中；通过抽真空离心浓缩样品至约200μL；选择使用HLB SPE滤芯进行脱盐处理，它可以在中性条件下有效地分离多肽和无机盐。
在HLB SPE滤芯加入1mL的HPLC级乙腈，使其通过重力流过滤芯；加入1mL HPLC级别的水平衡滤芯；重复1次。将约200μL样品全部加入滤芯的吸附床；加入1mL HPLC级别的水清洗滤芯；加1mL HLB溶剂A(配方为：80％乙腈、10％甲醇和10％HPLC级水)到滤芯，并收集含有肽段的洗脱液。利用真空离心机在45℃离心约2小时以蒸干收集的肽段；在蒸干的肽段中加入20μL HPLC级别水和10μL乙腈，震荡复溶；依次加入终浓度为1mM L Az-UV-biotin(化合物G处理的样品)或H Az-UV-biotin(DMSO处理的对照)、4mM抗坏血酸钠、1.25mM TBTA和4mM硫酸铜，轻轻震荡以混匀，室温避光孵育2小时；加入120μL链霉菌素结合缓冲液(50mM NaOAc,pH 4.5)稀释反应混合物，立即涡旋30秒以混匀；在室温用14000g的离心力离心五分钟，取出等量的化合物G处理的样品和对照组样品上清，在一个新的1.5mL EP管内混匀；转移200μL链霉菌素珠到15mL管中，并用10mL链霉菌素结合缓冲液洗两次；加入10mL链霉菌素结合缓冲液以重悬链霉菌素珠，并加入步骤29中混匀的样品，避光室温孵育2小时；在室温用1700g的离心力离心3分钟，去除上清；依次使用链霉菌素结合缓冲液、链霉菌素洗涤缓冲液(50mM NaOAc,2M NaCl,pH 4.5)和HPLC级别的水各洗链霉菌素珠两次；用1.2mL的光解缓冲液(25mM NH 4HCO 3)重悬链霉菌素珠，并将它们转移到一个带有磁力搅拌棒的薄壁玻璃瓶中；将玻璃瓶放在磁力搅拌器上，并用手持式紫外线照射灯在室温下用365nm紫外线照射2小时；将链霉菌素珠悬浮液转移到一个新的2mL EP管中，并在室温下以2000g的离心力离心4分钟。收集上清液，并转移到一个新的1.5mL EP管中；而后用真空离心机浓缩至约200μL。使用C18 StageTips滤芯进行最终脱盐处理。在滤芯加入1mL的HPLC级乙腈，使其通过重力流过滤芯；加入1mL HPLC级别的水平衡滤芯；重复1次。将上述的浓缩液加入滤芯的吸附床，而后使用1mL HPLC级别的水进行清洗；用750μL HLB溶剂A和250μL HLB溶剂B洗脱肽段，并将洗脱液混合在一个新的EP管中。
在45℃环境下使用真空离心机干燥洗脱的肽样品，大约需2小时；加入12μL的LC-MS/MS采样缓冲液(0.1％甲酸和5％乙腈的水溶液)重悬肽段；在室温下以20000g的离心力离心10分钟，将10μL的上清液转移到带插管的采样瓶中；将采样瓶放入预冷至4℃的LC自动进样器中，设置进样程序并开始运行。对于每次分析，将每个样品的一半(5μL)注入到LC-MS/MS中，流速设置为600nL/分钟；利用pFind 3对LC-MS/MS的结果进行搜寻和分析。
免疫共沉淀
利用脂质体3000，将Flag质粒和Flag-KEAP1质粒分别转染进入细胞；转染36-48小时后，去除细胞培养基，用预冷的dPBS洗涤细胞一次；以10cm细胞培养皿为例，吸去PBS，加入1ml预冷的含有蛋白酶抑制剂和磷酸酶抑制剂的非变性细胞裂解液(2mM Tris·HCl(pH 7.4),150mM NaCl,1％NP-40,1mM EDTA,5％glycerol)，冰上孵育5分钟；用细胞刮刀刮下细胞，并转移至1.5mL EP管中，置于冰上；在冰上可对样品进行3次超声粉碎，每次5秒；以14000g的离心力在4℃下离心10分钟，然后将上清液转移到新试管中并置于冰上；上清可在-80℃长期保存；利用BCA蛋白浓度测定试剂盒进行蛋白定量，并稀释到1mg/ml；轻微涡旋protein A/G磁珠以重悬，转移20μL珠浆至1.5mL EP管中，将EP管放在磁分离架上10-15秒；待溶液澄清，小心吸去溶液；加入500μL非变性细胞裂解液，洗涤两次。
将200μL稀释好的细胞裂解液加入磁珠，室温旋转孵育20分钟；利用磁力架分离细胞裂解液和磁珠，并将细胞裂解液转移到新的1.5mL EP管中；添加2.5μg Flag抗体或同物种的IgG同型对照至细胞裂解液中，4℃旋转孵育过夜；洗涤磁珠；将细胞裂解液和抗体转移到洗涤好的磁珠中，室温旋转孵育20分钟；利用磁力架分离细胞裂解液和磁珠，并吸去澄清溶液；使用含有蛋白酶抑制剂和磷酸酶抑制剂的非变性细胞裂解液洗涤磁珠5次，洗涤期间将样品置于冰上；利用磁力架分离细胞裂解液和磁珠，并尽量吸去上清，而后加入32μL非变性细胞裂解液和8μL 5×上样缓冲液，98℃加热10分钟，将目的蛋白从磁珠洗脱；按照上文所述方法进行蛋白免疫印迹。
示例性化合物G对Cln3 KO小鼠的治疗
为比较Cln3 KO小鼠和野生型小鼠的差异，以及示例性化合物G对于Cln3 KO小鼠的治疗效果，将出生后21天的小鼠从繁殖笼中分离出来，在其饮水中持续添加1mg/mL化合物G进行治疗。对照组为饮水中含有等量溶剂的Cln3 KO小鼠。给药期间记录小鼠体重变化。在小鼠2月龄时，将小鼠安乐死后进行脑组织的免疫组化染色，观察小鼠脑中SCMAS蛋白异常贮积情况。在小鼠4-5月龄时，利用Morris水迷宫实验、滚轮实验和矿场实验等手段，比较Cln3 KO小鼠和野生型小鼠的学习记忆能力和运动能力的差异，以及化合物G对于Cln3 KO小鼠的治疗效果。
Morris水迷宫实验
为测试Cln3 KO小鼠和野生型小鼠学习记忆能力的差异，以及化合物G对于Cln3 KO小鼠学习记忆能力的影响，进行了Morris水迷宫实验，具体步骤如下：将需要测试的小鼠单笼饲养1周以上，以适应环境；本研究所使用的迷宫为直径120cm，深度40cm的圆形水池；在水池壁上分别使用圆形、正方形、三角形和叉号将水池划分为东北(northeast，NE)、西北(northwest，NW)、西南(southwest，SW)和东南(southeast，SE)四个象限。在SW象限的固定位置放置一个直径11cm，高度18cm的站台；在水池中加入含有钛白粉的乳白色水，加水量以刚没过站台为宜；将水温调节至22℃左右；将小鼠从表5所示的指定位置轻柔放入水中，并通过录像设备记录小鼠在60秒内的运动情况。如果小鼠在60秒内找到平台，则记录其找到平台的时间；如果小鼠在60秒内无法找到平台，则将其引导到平台上，并保持15秒。
将小鼠从平台取下，用干燥的毛巾将其擦拭干净后放回饲养笼中。每只小鼠每天训练4次，每次训练间隔30分钟，共训练6天，不会游泳的小鼠将被排除在外；在第7天，将水中的平台取出，然后从NE方位将小鼠轻柔地放入水中，并用录像设备记录小鼠60秒内的运动情况，用Noldus软件分析和统计小鼠第一次找到平台的时间，以及小鼠停留在SW象限的时间，此外实验人员人工计算小鼠在60秒内穿越原平台所在位置的次数。
表5：小鼠放入水迷宫的位置信息
Figure PCTCN2022099308-appb-000023
滚轮实验
为测试Cln3 KO小鼠和野生型小鼠运动能力的差异，以及化合物G对于Cln3 KO小鼠运动能力的影响，进行了矿场实验，具体步骤如下：将需要测试的小鼠单笼饲养1周以上，以适应环境；将转轮的转速设置为10rpm，并将小鼠轻柔放置在设备上适应1分钟； 在300秒内将转速不断升高至30rpm，如果期间小鼠从滚轮掉落，设备感应并记录小鼠第一次掉落时间，而后，将掉落小鼠放回设备继续进行训练；如果期间小鼠没有掉落，则记为300秒；如果小鼠在300秒内掉落3次以上，则将其放回饲养笼；小鼠每天训练3次，每次间隔30分钟，训练共持续4天；第4天中，3次训练结果获得的平均值可以作为衡量小鼠运动能力的指标。
矿场实验
使用大小为50cm×50cm×40cm的蓝色箱子对小鼠进行了矿场实验。具体步骤如下：将需要测试的小鼠单笼饲养1周以上，以适应环境；将箱子中心25cm×25cm区域划分为中央区域。将小鼠背朝实验人员轻轻放入中央区域，开启录像设备记录小鼠10分钟内的运动轨迹；用75％的乙醇彻底擦拭箱子两次，以消除小鼠气味，而后放入下一只小鼠进行10分钟的运动轨迹记录；使用Noldus软件分析小鼠的运动轨迹，分析指标包括小鼠的运动速度，运动总距离。小鼠在10分钟内后腿直立的次数由实验人员人工统计。
慢病毒介导的目标蛋白敲减
在诱导得到的神经干细胞和神经元中进行了CLN3和KEAP1等基因的shRNA敲低实验，具体步骤如下：根据实验需求，将神经干细胞种植到所需的细胞培养板中，至约50％的细胞密度，或在细胞培养板中将神经干细胞分化为人神经元；CLN3和KEAP1等基因的shRNA慢病毒质粒以及表达无靶标序列的对照慢病毒质粒选自Sigma Aldrich公司的MISSIOH人类shRNA库，序列见表6；按照前文中方法所述，在10cm细胞培养板中包装慢病毒，并过滤收集含有病毒的上清液；将上清液和慢病毒浓缩剂(40％PEG-8000和1.2M的氯化钠溶解在dPBS溶液中，pH～7.0)按照1:3的体积比混合，4℃缓慢旋转，孵育过夜；用13000g的离心力在4℃离心30分钟，弃去上清液，用1mL新鲜的细胞培养基重悬沉淀，并加到目的细胞中；感染8小时后，吸去病毒，加上新鲜的细胞培养基；感染48小时后，加入1.5μg/mL嘌呤霉素筛选72小时，利用存活的细胞进行后续的实验。
表6：基因敲减所用shRNA序列
Figure PCTCN2022099308-appb-000024
转录组测序实验
为检测化合物G处理后对细胞的影响，对化合物G处理的神经干细胞进行了转录组测序，具体步骤如下：将CLN3 Δex7/8人类神经干细胞种植到2％Matrigel包被的6孔细胞板中，至80％的细胞密度；加入含有6μM的化合物G或等体积的DMSO的新鲜细胞培养基，每种处理条件包含两个生物重复；将神经干细胞置于37℃细胞培养箱孵育6小时；吸去细胞培养基，用dPBS清洗一次，而后利用Qiagen RNeazy Mini Plus Kit提取细胞内RNA；将提取的RNA寄送到安诺优达公司，首先对RNA进行质量检测。RNA的纯度使用Nanophotometer分光光度计进行检测；RNA的浓度使用Qubit 3.0 Flurometer进行检测；RNA的完整性使用Aligent 2100 RNA Nano 6000 Assay试剂盒进行检测；从每个检验合格的样品中取出2μg RNA进行建库，并利用Qubit 3.0 Flurometer进行定量。
将建库后的样品稀释到1ng/μL后，利用Aligent 2100 RNA Nano 6000 Assay试剂盒和qPCR等手段进行质量检测；使用Illumina公司的Hiseq PE Cluster试剂盒进行成簇，并使用Hiseq 2500测序平台进行双端测序，得到150bp的双端测序读数；用FastQC软件对测序得到的原始数据进行质量评估，而后使用TrimGalore去除接头序列和低质量序列，获得过滤后的数据用于后续分析。
利用HISAT2将过滤后的数据与人类参考基因组hg38进行比对，比对后得到Bam文件，用FeatureCount统计比对到每个基因上的counts数，注释使用Ensembl提供的gtf(GRCh38.92)文件。Ensembl ID转换为基因名使用R包Biomart完成。差异表达基因分析使用DESeq2软件，筛选倍数变化>2并且矫正后p<0.05的基因作为差异基因。
数据的处理和分析
数据使用GraphPad Prism 7软件进行统计分析。图表中显示的数据为平均值±方差或平均值±标准差，具体细节见各实验图标标注。*代表p＜0.05；**代表p＜0.01；***代表p＜0.001；ns代表无显著性差异。蛋白质印迹图片的灰度值使用ImageJ软件进行分析，以指征蛋白丰度。
附图说明
以下参考附图仅示例性描述本发明。需强调的是，所示细节仅作为示例且说明性地表示本发明的优选实施方案，用以描述本发明的原理和概念。本领域技术人员容易知晓如何参考附图描述以本发明的各种形式进行实践。
图1：利用CRISPR/Cas9技术在人胚胎干细胞中引入JNCL患者突变。
图2：hESC细胞系向神经谱系分化流程图。
图3：CLN3 Δex7/8神经干细胞中存在溶酶体功能损伤。其中(a)在神经干细胞中进行Lysotracker Red的染色，并使用流式细胞术测量细胞内红色荧光强度分布；(b)WT和CLN3 Δex7/8神经干细胞中Lysotracker染色的平均荧光强度(n＝3)；(c)WT和CLN3 Δex7/8神经干细胞溶酶体内组织蛋白酶D的酶活比较(n＝4)；(d)蛋白印迹实验测量WT和CLN3 Δex7/8神经干细胞内异常贮积的ATP酶c亚基(SCMAS)。
图4：CLN3 Δex7/8神经元中存在溶酶体缺陷。(a)在神经元中利用Lysotracker Red染料染色，流式细胞术测量细胞内红色荧光强度的分布。(b)WT和CLN3 Δex7/8神经元中Lysotracker染色的平均荧光强度(n＝3)。(c)WT和CLN3 Δex7/8神经元溶酶体内组织蛋白酶D的相对酶活(n＝4)。
图5：CLN3 Δex7/8神经元中存在SCMAS蛋白的异常贮积。(a)免疫荧光染色显示CLN3 Δex7/8神经元内存在异常贮积的SCMAS蛋白。标尺为20μm。(b)蛋白印迹实验检测WT和CLN3 Δex7/8神经元内SCMAS蛋白量。(c)对蛋白印迹实验中SCMAS条带的灰度值进行定量(n＝4)。
图6：CLN3敲减的神经元中存在溶酶体功能缺陷。(a)将同等数量的对照shRNA和CLN3 shRNA侵染后的hNSCs分化为神经元，分化17天后利用免疫荧光染色检测神经元谱系蛋白MAP2和TUJ1的表达情况。标尺为200μm。(b)在CLN3敲减神经元中，Lysotracker染料染色，流式细胞术测量细胞内Lysotracker荧光强度。(c)蛋白印迹实验测量对照shRNA和CLN3 shRNA侵染后的神经元内CLN3和SCMAS的蛋白量。
图7：CLN3 Δex7/8神经干细胞中存在自噬流缺陷。(a)tandem LC3慢病毒侵染神经干细胞，标记细胞内的自噬小体，高内涵激光共聚焦显微镜成像。白色箭头指向红色荧光单阳性的自噬小体。标尺为50μm。(d)自噬抑制剂Baf-A1处理神经干细胞 12小时，在Baf-A1处理前后分别对细胞内带有红色荧光的自噬小体数量进行统计，图为Opera Phenix数据分析工作站对64个视野的统计结果。
图8：CLN3 Δex7/8神经元中存在线粒体损伤。(a)绿色荧光融合的OMP25标记人神经元中的线粒体形态。白色箭头是损伤的点状线粒体。标尺为20μm。(b)Cell Titer Glo检测同等数量的野生型和CLN3 Δex7/8神经元中ATP的含量(n＝4)。
图9：筛选增强溶酶体酸度的小分子化合物。Hits为提高Lysotracker Red溶酶体着色的小分子化合物的代表性图片。箭头的是化合物处理后，和溶酶体共定位的自噬小体。标尺为100μm。
图10：小分子化合物增强溶酶体酸度。
图11：化合物G促进CLN3 Δex7/8神经元的分化。记取同等数量的神经干细胞，开始神经元的诱导分化。(a)分化17天后的明场图片。标尺为200μm。(b)将分化17天的神经元固定，神经元谱系标记物MAP2和TUJ1的免疫荧光染色。标尺为100μm。(c)分化得到的神经元相对细胞数量(n＝4)。
图12：化合物G不影响CLN3 Δex7/8 hNSC的细胞命运。(a)化合物G处理48后的CLN3 Δex7/8 hNSC的明场照片。标尺为200μm。(b)将hNSC固定，进行神经干细胞谱系蛋白PAX6、SOX1和NESTIN的免疫荧光染色。标尺为100μm。
图13：化合物G增强CLN3 Δex7/8 hNSC溶酶体酸度。(a)化合物G处理CLN3 Δex7/8 hNSC 48小时后，Lysotracker染料进行染色，激光共聚焦高内涵成像显微镜进行成像。图中所示为9个临近视野的拼接图片。标尺为200μm。(b)化合物G处理CLN3 Δex7/8 hNSC 48小时后，AnnexinV/PI细胞凋亡染料染色，流式细胞术分析。
图14：化合物G缓解CLN3 Δex7/8人神经元中的溶酶体缺陷。(a)在神经元中Lysotracker进行染色，流式细胞术测量细胞的平均荧光强度(n＝3)。(b)在WT和CLN3 Δex7/8神经元裂解液中加入组织蛋白酶D的底物，酶标仪测量底物被组织蛋白酶D切割后发出的红色荧光强度(n＝4)。
图15：化合物G减少CLN3 Δex7/8神经元中SCMAS蛋白贮积。(a)免疫荧光染色检测化合物G处理后CLN3 Δex7/8神经元内SCMAS蛋白的异常贮积情况。标尺为20μm。(b)蛋白印迹实验测量化合物G对于SCMAS蛋白量的影响。(c)对蛋白印迹实验中SCMAS条带的灰度值进行定量分析(n＝4)。
图16：DMF减少CLN3 Δex7/8神经元中SCMAS蛋白贮积。
图17：化合物G增强CLN3 Δex7/8人神经干细胞中的自噬流。白色箭头指向红色荧光单阳性的自噬小体。标尺为50μm。
图18：化合物G增强CLN3 Δex7/8人神经元中的自噬流。(a)为具有代表性的p62蛋白印迹图片；(b)为p62蛋白水平的统计图(n＝3)。(c)细胞内线粒体自噬流的水平(n＝6)。
图19：化合物G保护CLN3 Δex7/8神经元中的线粒体稳态。(a)白色箭头指向损伤的点状线粒体。标尺为20μm。(b)计取同等数量的神经元，加入Cell Titer Glo试剂，并用酶标仪测量细胞内的ATP水平(n＝3)。
图20：化合物G在小鼠脑组织中的浓度(n＝8)。
图21：化合物G对于Cln3 KO小鼠体重和脑组织质量的影响。(a)小鼠2月龄和6月龄时，对其体重进行记录(n＝5)。(b)小鼠6月龄时，将其安乐死，并取出脑组织进行称重(n＝5)。
图22：化合物G减少Cln3 KO小鼠脑中SCMAS蛋白贮积。(a)将2月龄的野生型小鼠和Cln3 KO小鼠安乐死，取出脑组织进行冰冻切片和免疫组化染色。标尺为50μm。(b)对SCMAS荧光面积进行统计(WT:n＝4；Cln3 KO:n＝4；Cln3 KO+G:n＝6)。
图23：Morris水迷宫实验测Cln3 KO小鼠的学习记忆能力。(a)对5月龄的小鼠进行为期6天的水迷宫训练，并记录其每天找到平台所需的时间。第7天将水下平台撤去后进行测试。(b)小鼠在第7天测试时，第一次到达原平台所在位置所需的时间。(c)小鼠在第7天测试时，在原平台所在象限SW停留的时间。(d)小鼠在第7天测试时，在原平台所在位置穿越的次数。(e)小鼠在第7天测试时，在水迷宫中运动的速度(WT:n＝8；Cln3 KO:n＝13；Cln3 KO+G:n＝10)。
图24：滚轮实验和矿场实验测试小鼠的运动能力。(a)对小鼠进行了为期3天的训练，训练时长为300秒，并记录小鼠第一次从滚轮跌落的时间。(b)第4天，对小鼠进行了测试，并记录了小鼠第一次从滚轮跌落的时间。(c、d)将小鼠轻轻放入矿场，让其自由运动10分钟，记录并分析了小鼠在10分钟内的运动轨迹长度和运动速度(WT:n＝10；Cln3 KO:n＝10；Cln3 KO+G:n＝12)。
图25：化合物G在小鼠TTF细胞中提高自噬和溶酶体基因的转录。qRT-PCR检测了(a)自噬相关基因和(b)溶酶体相关基因的转录水平(n＝3)。
图26：化合物G在小鼠神经干细胞和神经元中不影响自噬和溶酶体基因的转录。化合物G对Cln3 KO小鼠的原代(a)神经干细胞和(b)神经元进行6小时的处理，提取了细胞中的mRNA，逆转录成为cDNA，qRT-PCR检测了自噬相关基因和溶酶体相关基因的转录水平(n＝3)。
图27：化合物G在人神经干细胞和神经元中不影响自噬和溶酶体基因的转录。化合物G对(a)人神经干细胞和(b)神经元进行了6小时的处理之后，提取细胞中的mRNA，逆转录成为cDNA，并利用qRT-PCR检测了自噬相关基因和溶酶体相关基因的转录水平(n＝3)
图28：化合物G以依赖于FXR的方式增加自噬基因的转录。(a)提取小鼠TTF细胞的mRNA，qRT-PCR检测细胞中FXR的表达水平(n＝3)。(b)化合物G处理表达对照shRNA或FXR shRNA-2的小鼠TTF细胞6小时，qRT-PCR检测细胞中Tfeb和自噬相关基因的表达水平(n＝3)。
图29：化合物G对CLN3 Δex7/8人神经干细胞表达谱的影响。化合物G处理CLN3 Δex7/8人神经干细胞6小时，提取细胞的mRNA，进行建库和RNA-seq实验(n＝2)。(a)为差异表达基因的聚类热图。(b)为distance图。(c)为PCA图。
图30：RNA-seq显示化合物G不影响神经干细胞中自噬和溶酶体基因的表达。化合物G处理CLN3 Δex7/8人神经干细胞6小时，进行RNA-seq，对测序数据中溶酶体基因和自噬相关基因的表达量进行分析(n＝2)。
图31：RNA-seq数据显示化合物G激活NRF2下游基因的表达。化合物G处理CLN3 Δex7/8人神经干细胞6小时，进行RNA-seq，对测序数据中NRF2下游基因的表达量进行分析(n＝2)。
图32：化合物G在CLN3Δex7/8人神经干细胞中激活NRF2下游基因的表达。化合物G处理CLN3 Δex7/8人神经干细胞6小时，进行qRT-PCR实验，检测NRF2下游基因的表达量(n＝3)。
图33：CLN3 Δex7/8人神经干细胞中的定量硫醇反应性分析。
图34：在CLN3 Δex7/8人神经干细胞中进行KEAP1的敲减。
图35：KEAP1的敲减对CLN3 Δex7/8人神经元溶酶体酸度的影响。
图36：KEAP1的敲减对CLN3 Δex7/8细胞中组织蛋白酶活性的影响。(a)利用激光共聚焦高内涵成像显微镜进行细胞成像，观察胞内红色荧光的强度。标尺为100μm。(b)利用Opera Phenix数据分析工作站对细胞内红色荧光强度进行统计，图为528个视野的统计结果。
图37：KEAP1的敲减减少CLN3 Δex7/8神经元中SCMAS蛋白贮积。(a)免疫荧光染色显示KEAP1敲减对于CLN3 Δex7/8神经元内异常贮积的SCMAS蛋白的影响。标尺为20μm。(b)对SCMAS荧光面积进行统计(n＝4)。
图38：KEAP1的敲减对CLN3 Δex7/8人神经干细胞自噬流的影响。(a)白色箭头指向红色荧光单阳性的自噬小体。标尺为50μm。(b)图为Opera Phenix数据分析工作站对64个视野中自噬小体数量的统计结果。红色荧光单阳性自噬小体数量＝(mCherry阳性自噬小体数量－EGFP阳性自噬小体数量)。
图39：KEAP1的敲减减少CLN3 Δex7/8人神经元中线粒体的损伤。白色箭头指向损伤的点状线粒体。标尺为20μm。
图40：KEAP1的敲减增加CLN3 Δex7/8人神经元中ATP含量。血球计数板计取同等数量的神经元，加入Cell Titer Glo试剂，并用酶标仪测量细胞内的ATP水平(n＝6)。
图41：化合物G增加细胞内p62蛋白水平。(a)利用化合物G处理人神经元24小时后或KEAP1敲减后，利用qRT-PCR检测p62基因的转录水平(n＝3)。(b)利用化合物G处理人神经元24小时后或KEAP1敲减后，通过蛋白质免疫印迹手段检测细胞内p62蛋白水平，并进行条带灰度统计(n＝3)。
图42：过表达p62蛋白增加溶酶体酸度。(a)在NRK细胞中过表达GFP-p62蛋白和对照GFP蛋白，48小时后，用Lysotracker染料进行染色，流式细胞术检测绿色荧光阳性细胞内的红色荧光强度(n＝3)。(b)在人神经干细胞中过表达GFP-p62蛋白和对照GFP蛋白，48小时后，用Lysotracker染料进行染色，流式细胞术检测绿色荧光阳性细胞内红色荧光强度(n＝3)。
图43：过表达p62蛋白减少CLN3 Δex7/8神经元中SCMAS蛋白贮积和细胞死亡。利用DOX在CLN3 Δex7/8人神经干细胞中过表达p62蛋白，并将其分化为人神经元。(a)免疫荧光染色显示p62过表达对于CLN3 Δex7/8神经元内异常贮积的SCMAS蛋白的影响。标尺为20μm。(b)免疫荧光染色显示p62过表达对于CLN3 Δex7/8神经元凋亡 的影响。标尺为10μm。
图44：化合物G减少APP突变神经元中Aβ蛋白贮积以及毒性Aβ42的分泌水平。(a)免疫荧光染色检测化合物G处理后APP突变神经元内Aβ蛋白的异常贮积情况以及(b)Aβ斑块面积的统计，标尺为50μm。(c)ELISA实验测量化合物G对于APP突变神经元分泌毒性Aβ42的水平的影响。
具体实施方式
下面结合实施例对本发明作进一步的详细描述，该实施例仅用于说明本发明，而不应视为限定本发明的范围。在不偏离本发明基础上所做的修改或改进，均属于本发明的保护范围。如无特殊说明，下述实施例所用的实验方法，均为常规方法；所用的试剂、材料均可从商业途径得到。
实施例1：建立CLN3 Δex7/8细胞系
为建立体外JNCL人神经元模型，参照已公开的方法(Ran et al.,2013)，利用CRISPR/Cas9基因编辑技术在H9人胚胎干细胞中引入了JNCL患者中最常见的CLN3外显子7和8的纯合缺失(CLN3 Δex7/8)(图1)。过程中所用构建引物参见表7，其它步骤同Ran et al.。
表7：CLN3 Δex7/8细胞系构建引物
Figure PCTCN2022099308-appb-000025
实施例2：构建JNCL疾病的人神经元模型
本研究证实，受CLN3基因突变影响最大的是患者的神经系统，尤其是神经元的稳态和存活。体外诱导CLN3 Δex7/8 hESC向神经谱系定向分化，构建JNCL疾病的人 神经元模型。该模型重现了溶酶体和自噬功能缺陷以及线粒体稳态失调等多个JNCL疾病相关表型。
利用如图2所示的hESC向神经干细胞以及神经元高效分化的方法。首先利用CHIR99021、SB431542、hLIF和Compound E诱导hESC向神经谱系分化；诱导7天后，野生型和CLN3 Δex7/8 hESC均能被高效诱导成为PAX6、SOX1以及NESTIN阳性的人神经干细胞(hNSC)。利用CHIR99021、SB431542和hLIF处理hNSC 3天，而后换成含有hBDNF、hGDNF、cAMP和维生素C的培养基，继续诱导14天后，可以得到MAP2和TUJ1阳性的人神经元。
免疫荧光染色结果显示，经过7天的分化，野生型和CLN3 Δex7/8 hESC均能被高效诱导成为PAX6、SOX1以及NESTIN阳性的人神经干细胞。谱系基因表达检测结果进一步证明，和hESC相比，野生型以及CLN3 Δex7/8神经干细胞都高表达神经干细胞谱系基因PAX6、SOX1和NOGGIN，而胚胎干细胞多能性基因OCT4的表达水平显著下降。
实施例3：CLN3 Δex7/8人神经干细胞和神经元在体外重现疾病相关表型
在分化得到的细胞中，从溶酶体酸度、蛋白酶活性、细胞内蛋白贮积、自噬流和线粒体损伤等角度出发，检测了CLN3基因外显子7和8的纯合缺失对人神经干细胞和神经元细胞稳态的影响。
CLN3 Δex7/8人神经干细胞中存在溶酶体缺陷
利用在酸性溶酶体中富集并发出红色荧光的Lysotracker染料对细胞进行染色，然后利用流式细胞术对染色后的细胞荧光强度进行分析。
结果显示(图3)，比起野生型人神经干细胞，CLN3 Δex7/8细胞中Lysotracker的平均荧光强度显著下降，即细胞溶酶体酸度整体降低，说明CLN3蛋白突变导致神经干细胞的溶酶体pH调节紊乱。利用蛋白免疫印迹实验对神经干细胞中SCMAS的蛋白量进行了检测。结果显示，CLN3 Δex7/8神经干细胞中存在SCMAS的异常贮积，表明其无法在溶酶体内被顺利降解。
综上，在分化得到的人神经干细胞中，CLN3基因突变会引起溶酶体腔内酸度调节紊乱、蛋白酶活性降低以及蛋白质异常贮积等JNCL相关溶酶体功能缺陷表型。
CLN3 Δex7/8人神经元中存在溶酶体缺陷
进一步在CLN3 Δex7/8神经元中进行溶酶体和自噬功能的检测。将野生型和CLN3 Δex7/8人神经干细胞分化成为神经元后，首先利用Lysotracker Red染料对神经元进行染色，流式细胞术分别检测野生型和CLN3 Δex7/8神经元中染料的荧光强度。与在神经干细胞中观察到的结果一致，比起野生型的神经元，CLN3 Δex7/8神经元具有溶酶体酸度下降的表型(图4(a，b))；且CLN3 Δex7/8神经元溶酶体中组织蛋白酶D的酶活显著下降(图4(c))。细胞免疫荧光实验和蛋白质印迹实验共同证明，CLN3 Δex7/8人神经元中存在大量SCMAS蛋白的异常贮积(图5)。
以上结果表明，与人神经干细胞类似，CLN3 Δex7/8人神经元也表现出溶酶体酸度失调、组织蛋白酶活性降低、未能降解的蛋白质异常贮积等溶酶体功能缺陷表型。
为进一步确定上述在CLN3 Δex7/8人神经元中观察到的溶酶体缺陷是由于CLN3蛋白功能缺失导致的，在野生型神经干细胞中利用靶向CLN3转录本的短发夹RNA(shRNA)进行了CLN3基因敲减，并用无靶标的shRNA(Ctrl shRNA)侵染的野生型神经干细胞作为对照，而后将其分化成为人神经元。利用蛋白印迹实验检测了表达CLN3 shRNA的神经元内CLN3蛋白水平的变化。结果显示，和对照shRNA相比，使用CLN3 shRNA可以显著降低神经元内CLN3的蛋白水平(图6(c))。
此外，将同等数量的表达CLN3 shRNA和对照shRNA的神经干细胞种植到新的细胞培养板，并按照之前所述的方法诱导向人神经元分化。分化17天后，免疫荧光染色结果显示，CLN3基因敲减后的神经干细胞可以被成功分化成为MAP2和TUJ1阳性的神经元(图6(a))。并且和在CLN3 Δex7/8细胞中观察到的结果一致，从相同数量的细胞出发，CLN3敲减的神经干细胞最终分化得到的神经元的密度显著降低(图6(a))。
用Lysotracker染料检测了CLN3基因敲减对人神经元中溶酶体酸度的影响，结果显示，CLN3基因敲减后，神经元中Lysotracker染色强度减弱，表明其溶酶体酸度降低(图6(b))。利用蛋白印迹实验观察到，CLN3基因敲减会引起神经元内SCMAS蛋白的异常贮积(图6(c))。
这些结果共同说明，在人神经元中CLN3蛋白功能缺失可以引起溶酶体酸度失调和蛋白质异常贮积等溶酶体功能缺陷表型，影响细胞稳态。
CLN3 Δex7/8人神经元中存在自噬缺陷
检测到CLN3 Δex7/8神经干细胞和神经元中存在溶酶体功能缺陷后，进一步对细胞 中的自噬流进行了检测。利用等量的包装有tandem LC3质粒的慢病毒感染野生型和CLN3 Δex7/8细胞，标记细胞内的自噬小体。和野生型神经干细胞相比，CLN3 Δex7/8人神经干细胞中红色荧光单阳性的自噬小体数量有所下降(图7(a)中箭头所示),表明细胞中可能存在自噬流的阻滞。
在自噬阻滞剂巴佛洛霉素A1(Baf-A1)处理细胞前后分别对细胞内自噬小体数量进行统计，结果显示，野生型神经干细胞中自噬小体数量的变化(加Baf-A1后的自噬小体数量-不加Baf-A1的自噬小体数量)明显高于CLN3 Δex7/8神经干细胞(图7(b))，证实CLN3 Δex7/8人神经干细胞中的自噬流存在缺陷。
已有的研究显示，健康的线粒体形态呈短棍状，线粒体膜两侧存在较大的电势差，能够高效地产生维持细胞正常功能的ATP。损伤的线粒体会经过线粒体裂变(fission)的过程，分裂成点状形态，被自噬小体包裹，运输进入溶酶体降解。为检测CLN3 Δex7/8人神经元中是否存在线粒体形态异常，构建了绿色荧光蛋白标记的OMP25慢病毒质粒，并侵染野生型和CLN3 Δex7/8人神经元。
结果显示，野生型的人神经元中的线粒体多为健康的短棍状。相比之下，CLN3 Δex7/8人神经元中有大量点状线粒体(图8(a))，表明CLN3 Δex7/8人神经元中存在严重的线粒体损伤。进一步利用Cell Titer Glo试剂检测了CLN3 Δex7/8人神经元中ATP的产生能力。利用酶标仪进行定量检测后发现，同等数量的野生型和CLN3 Δex7/8人神经元中，自噬流受损的CLN3 Δex7/8人神经元内含有ATP的水平明显降低(图8(b))。
以上数据共同表明，由CLN3 Δex7/8人胚胎干细胞体外分化得到的神经干细胞和神经元中存在溶酶体和自噬功能的缺陷，并影响了细胞中线粒体的稳态和功能。
实施例4：本发明的化合物为能够增强溶酶体酸度的自噬流激活剂
发明人构建了稳定表达tandem LC3的正常大鼠肾(NRK)细胞系。tandem LC3系统是一种使用了红色荧光和绿色荧光同时标记的LC3报告系统，当双荧光标记的LC3所在的自噬小体位于细胞质中时，自噬小体同时呈现出红色荧光和绿色荧光(黄色)。随着自噬小体和溶酶体融合形成自溶酶体，溶酶体中的酸性环境会淬灭tandem LC3上的绿色荧光，因此自溶酶体只呈现出红色荧光(Shunsuke Kimura et al.,2007)。由此，该系统能够区分自噬流激活剂和溶酶体抑制剂。
发明人获得了7个小分子化合物，这7个小分子化合物在用上述tandem LC3系 统测试时能够同时将细胞中红色荧光和绿色荧光双阳性的自噬小体数量增加两倍及以上，以及保持红色荧光单阳性的自噬小体数量不下降，表明其均为自噬流激活剂而非溶酶体抑制剂。而且，用这7个小分子化合物处理BFP-LC3稳定表达的NRK细胞18h后，进行Lysotracker染料的染色，可观察到，和对照组细胞相比，这7个小分子化合物可以显著提高细胞内Lysotracker的着色(图9、10)，说明其增强了细胞内溶酶体的酸度。作为对照，Baf-A1的处理使得溶酶体酸度显著下降，嗜酸性的Lysotracker染料无法正确定位于溶酶体，在胞质中呈现弥散状态(图9)。
所述7个小分子化合物分别是：
Figure PCTCN2022099308-appb-000026
Figure PCTCN2022099308-appb-000027
依据上述试验结果可知，这些增加细胞自噬流的小分子化合物均是潜在的可用于治疗JNCL疾病的化合物。接下来以化合物G为例予以验证。
实施例5：化合物G缓解JNCL人神经元模型中的疾病表型
发明人以化合物G为例测试了前述小分子化合物对JNCL的潜在影响。
化合物G缓解CLN3 Δex7/8神经干细胞分化缺陷
从同等数量的人神经干细胞起始诱导分化为人神经元，CLN3 Δex7/8神经干细胞最终产生的神经元数量要低于野生型细胞。在神经元的分化过程中，将上述得到的7个小分子化合物分别加入到培养基中，并观察其对于CLN3 Δex7/8神经干细胞向神经元分化过程的影响。
分化17天后，发现化合物G可有效缓解CLN3 Δex7/8神经干细胞向神经元的分化缺陷。从同等数量的细胞出发，化合物G处理后的CLN3 Δex7/8神经干细胞能够生成更多MAP2和TUJ1阳性的神经元，和野生型神经干细胞分化产生的神经元密度接近(图11)。
化合物G：(Z)-gugglsterone，结构式如下：
Figure PCTCN2022099308-appb-000028
进一步检测化合物G对于CLN3 Δex7/8神经元的溶酶体功能、自噬功能和细胞稳态的影响。
用化合物G处理CLN3 Δex7/8神经干细胞48h后，针对神经干细胞谱系标记物SOX1、PAX6和NESTIN进行了免疫荧光染色。结果显示，化合物G处理后的CLN3 Δex7/8神经干细胞保持了SOX1、PAX6和NESTIN的阳性特征(图12)，表明化合物G的处理不会改变CLN3 Δex7/8神经干细胞的细胞命运。
而后检测了化合物G是否在神经干细胞中引起细胞毒性。利用膜联蛋白V(Annexin V)和碘化丙啶(PI)染色的方法检测化合物G处理后细胞的凋亡情况，并用流式细胞术进行分析。Annexin V阳性和PI阴性代表了早期凋亡细胞，而Annexin V阳性和PI阳性代表了晚期凋亡细胞。结果显示，CLN3 Δex7/8神经干细胞中早期凋亡细胞和晚期凋亡细胞的本底比例不高(图13(b))，而化合物G处理48小时后，不会显著改变CLN3 Δex7/8神经干细胞中凋亡细胞的比例。说明化合物G的处理对于CLN3 Δex7/8神经干细胞没有明显的毒性。
在CLN3 Δex7/8神经干细胞中进行Lysotracker染料的染色后，可看到化合物G可 以显著提高神经干细胞中Lysotracker的着色(图13(a))，说明化合物G在人神经干细胞中也可以显著提高细胞内溶酶体的酸性环境。
化合物G缓解CLN3 Δex7/8神经元中溶酶体和自噬缺陷
上文研究表明，CLN3 Δex7/8人神经元具有溶酶体酸度失调的特点。为研究化合物G对于CLN3 Δex7/8神经元溶酶体酸度的影响，利用Lysotracker染料进行染色，并通过流式细胞术分析细胞中Lysotracker染料的荧光强度。结果显示，化合物G处理之后，CLN3 Δex7/8人神经元中Lysotracker荧光强度明显上升(图14(a，b))，和野生型神经元更为接近，这表明化合物G的处理可以提高CLN3 Δex7/8神经元的溶酶体酸度。
检测了经化合物G处理后，CLN3 Δex7/8神经元中组织蛋白酶D的活性。结果显示，化合物G处理后CLN3 Δex7/8神经元中荧光强度显著升高，更接近野生型神经元中的荧光强度，表明组织蛋白酶D的活性有所增强(图14(b))。免疫荧光染色和蛋白印迹实验结果表明，化合物G的处理使CLN3 Δex7/8神经元中异常贮积的SCMAS蛋白显著减少(图15)。
由此可知，筛选得到的化合物G的处理能够有效缓解CLN3 Δex7/8人神经元中溶酶体酸度的失调，提高溶酶体内蛋白酶的活性，并减少细胞内蛋白质的异常贮积，即化合物G可在一定程度上缓解CLN3 Δex7/8人神经元模型中JNCL疾病相关表型。
发明人还测试了其它一些潜在有用的化合物，发现其与化合物G类似，同样能够在一定程度上缓解CLN3 Δex7/8人神经元模型中JNCL疾病相关表型。例如，我们对富马酸二甲酯(DMF)进行了测试：利用Lysotracker染色来指征细胞中溶酶体的酸度，并利用流式细胞术分析染色的平均荧光强度，发现DMF处理后的CLN3 Δex7/8人神经元的Lysotracker平均荧光强度显著增强，表明DMF可以提高CLN3 Δex7/8人神经元的溶酶体酸度；针对DMF对于CLN3 Δex7/8人神经元中SCMAS蛋白贮积的影响的测试表明，DMF的处理显著减少了CLN3 Δex7/8人神经元中SCMAS蛋白的贮积(图16)。
化合物G提高CLN3 Δex7/8神经元中的自噬水平和线粒体自噬水平，维护了线粒体 的功能和稳态
利用tandem LC3标记神经干细胞中的自噬小体，并利用高内涵激光共聚焦显微镜观察细胞内带有绿色荧光的自噬小体数量，以及只带有红色荧光的自溶酶体的数量。在CLN3 Δex7/8人神经干细胞中，红色荧光单阳性的自噬小体数量有所降低，而化合物G的处理可以增加细胞内红色荧光单阳性的自噬小体数量(图17)，说明化合 物G的处理挽救了CLN3 Δex7/8人神经干细胞中自噬流的缺陷。
此外，检测了细胞内p62的蛋白水平。作为NRF2基因的下游基因，p62表达的p62蛋白是一种细胞自噬相关蛋白，负责将自噬底物连接到位于自噬小体膜上的LC3蛋白上。由于p62可以随着自噬小体被运输到溶酶体降解，通过对比溶酶体抑制剂Baf-A1处理后细胞内p62蛋白量的变化(p62(Baf-A1处理组)-p62(未被Baf-A1处理组))，可以指征细胞内自噬流的强度。通过p62蛋白印迹的方法，检测了化合物G处理后CLN3 Δex7/8人神经元内自噬流的变化。发现Baf-A1处理之后，CLN3 Δex7/8人神经元中p62蛋白的增加量少于野生型神经元(图18(a、b))；而用化合物G处理之后再加入Baf-A1，可以使CLN3 Δex7/8人神经元中p62蛋白的增加量变多，达到和野生型神经元中相似的增加量(图18(a、b))。这些数据进一步表明，化合物G的处理可以增加神经元内的自噬流。
细胞内受损伤的线粒体可通过名为线粒体自噬的自噬过程被清除。自噬的功能缺陷会影响损伤线粒体的清除，进而破坏神经元中的线粒体稳态。当利用线粒体解偶联剂CCCP诱导野生型和CLN3 Δex7/8神经元中的线粒体损伤，再利用Baf-A1抑制损伤线粒体的清除后，细胞内线粒体数量(mtDNA/nDNA)在Baf-A1处理后的增加量可以代表细胞内线粒体自噬的水平。实验结果显示，在野生型人神经元中，Baf-A1的处理可以显著增加细胞内线粒体的数量，而在CLN3 Δex7/8神经元中，Baf-A1的处理对线粒体数量几乎没有影响(图18(c))。而当用化合物G处理CLN3 Δex7/8神经元后，Baf-A1可以显著增加线粒体数量(图18(c))，这表明化合物G可以挽救线粒体自噬，增加CLN3 Δex7/8神经元中损伤线粒体的清除效率。
为了检测化合物G对于CLN3 Δex7/8神经元中线粒体稳态的影响，在神经元中过表达荧光标记的线粒体外膜蛋白OMP25-GFP，并通过激光共聚焦显微镜观察线粒体形态的变化。结果显示，CLN3 Δex7/8神经元中存在大量受损伤的点状线粒体，化合物G可以显著减少损伤的点状线粒体的出现(图19(a))。利用Cell Titer Glo试剂，测试了相同数量神经元中产生ATP的量。结果显示，CLN3 Δex7/8神经元产生ATP的能力显著下降，而化合物G的处理可显著提高CLN3 Δex7/8神经元产生ATP的能力(图19(b))。
综合以上数据可知，化合物G显著提高了CLN3 Δex7/8神经元中的自噬水平和线粒体自噬水平，并维护了线粒体的功能和稳态。
实施例6：化合物G改善JNCL小鼠疾病表型
Cln3敲除(Cln3 KO)的JNCL小鼠模型购买于Jackson Lab。
化合物G改善JNCL小鼠模型的疾病相关表型
A、化合物G可透过血脑屏障
先检测了化合物G是否可以透过小鼠血脑屏障：将其加入Cln3 KO小鼠的饮水中，并在不同的时间点麻醉小鼠，利用dPBS将全身血液灌流干净后，安乐死小鼠并取出其脑组织，测量化合物G在小鼠脑组织中的药物浓度。结果显示，在不同时间点取得的小鼠脑组织中均能检测到化合物G的存在，其在小鼠脑中的平均浓度为814.74±834.19ng/g脑组织(图20)。即化合物G可以穿透血脑屏障。
B、化合物G可减少JNCL小鼠模型脑内蛋白的异常贮积
从小鼠21天脱乳开始，将化合物G或者溶剂加入Cln3 KO小鼠的饮水中，并记录小鼠的体重变化。发现在小鼠2月龄或6月龄时，Cln3 KO小鼠的体重和野生型小鼠没有显著区别，同时，化合物G对于小鼠的体重也没有显著的影响(图21(a))。在小鼠达到六月龄时，将其安乐死，取出脑组织称重。数据显示6月龄时，Cln3 KO小鼠的脑组织质量和野生型小鼠没有显著区别，同时，化合物G对于小鼠脑组织的质量也没有显著的影响(图21(b))。这表明，Cln3 KO小鼠在体重和脑组织的大小方面和同年龄的野生型小鼠没有显著的区别。同时，在所用的浓度下，使用化合物G对Cln3 KO小鼠进行长期持续的治疗不会产生显著的毒性。
Cln3 KO小鼠脑组织中存在大量异常贮积的物质。按上述方法灌流并安乐死了2月龄的小鼠，取出脑组织进行冰冻切片以及SCMAS的免疫组化染色。发现和野生型小鼠相比，2月龄的Cln3 KO小鼠脑组织中已经出现了大量SCMAS蛋白的贮积(图22)。在Cln3 KO小鼠21日龄脱乳后开始用化合物G处理，直至小鼠到达2月龄时。发现化合物G的处理能够一定程度减轻Cln3 KO小鼠脑中异常贮积物质的数量(图22)。这证明化合物G能够在Cln3 KO小鼠体内改善JNCL疾病相关的病理表型。
C、化合物G可以改善JNCL小鼠的学习记忆能力
从Cln3 KO小鼠21天脱乳开始持续给予化合物G的处理。在小鼠到达5月龄时，取出同龄的野生型小鼠、未给药的Cln3 KO小鼠以及化合物G处理的Cln3 KO小鼠，开展Morris水迷宫实验。
首先对小鼠进行为期6天的水迷宫学习训练，并记录其找到水下平台所需的时间。在第7天时，将水下平台撤去，并对小鼠进行了60秒的测试，记录了小鼠第一次到达原平台所在位置花费的时间，在原平台所在位置穿梭的次数以及在原平台所在象限停留的时间，表征其通过前6天的训练是否习得水下平台所在的位置。
实验结果显示，野生型小鼠、Cln3 KO小鼠以及化合物G处理的Cln3 KO小鼠在训练第1天时表现类似，几乎不能在训练的60秒时长内找到水下平台(图23(a))。随着学习的进展，野生型小鼠找到水下平台的时间逐渐缩短。相比之下，Cln3 KO小鼠找到水下平台所需的时间较长，而化合物G处理的Cln3 KO小鼠的表现介于两者之间(图23(a))。
训练第7天时，对小鼠进行了学习记忆能力的测试。测试时，从一个全新的离原平台最远的位置将小鼠轻轻放入水中。结果显示，和野生型的小鼠相比，Cln3 KO小鼠第一次到达原平台所在位置的时间明显更长(图23(b))，甚至比第6天花费的时间更长(图23(a))，这可能是由于第7天小鼠是从一个全新的位置开始寻找平台，和训练中熟悉的起始位置不同。相比之下，化合物G的处理可以有效缩短Cln3 KO小鼠第一次到达原平台所在位置花费的时间(图23(b))，表明化合物G处理之后的小鼠能够更好地学习记忆水下平台所在的位置。
Cln3 KO小鼠在原平台所在象限停留的时间显著短于野生型小鼠(图23(c))，进一步表明其对于原平台所在位置的记忆能力不如野生型小鼠，而经化合物G处理后，Cln3 KO小鼠在一定程度上延长了其在原平台所在象限的停留时间。此外，在第7天测试时，Cln3 KO小鼠在原平台所在位置穿梭的次数有减少的趋势，而化合物G处理后能够使其在原平台所在位置穿梭的次数和野生型小鼠更接近(图23(d))。
为排除Cln3 KO小鼠在水中存在游泳能力缺陷，而非学习记忆能力的缺陷，统计了野生型小鼠和Cln3 KO小鼠在水中的运动速度，发现它们没有显著的区别。同时，化合物G的处理对于小鼠在水中的运动速度也没有显著影响(图23(e))。
以上数据共同表明，和野生型小鼠相比，5月龄的Cln3 KO小鼠存在学习记忆能力的缺陷，而化合物G的处理能够在一定程度上改善Cln3 KO小鼠的学习记忆能力。
D、化合物G可以改善JNCL小鼠的运动能力
在临床上，运动能力的丧失也是JNCL患者常见的疾病表型。已有的研究显示，在Cln3基因突变的JNCL小鼠模型中，能够观察到患者所表现出的运动能力的缺陷。 因此，利用矿场实验和滚轮实验对Cln3 KO小鼠的运动能力进行了测试，并检测了化合物G对于Cln3 KO小鼠运动能力的影响。
首先进行了滚轮实验。对21天脱乳之后的小鼠持续给与了化合物G的处理。在野生型小鼠、Cln3 KO小鼠以及化合物G处理的Cln3 KO小鼠达到4-5月龄时，对其进行了为期3天的滚轮训练，并在第4天对小鼠在滚轮上的运动能力进行了测试。
实验数据显示，在滚轮实验训练的第1天，Cln3 KO小鼠第一次从滚轮跌落的时间就早于野生型小鼠，表明Cln3 KO小鼠的运动能力可能存在缺陷，而化合物G处理Cln3 KO小鼠后，其跌落时间处于两者之间(图24(a))。经过3天的训练，野生型小鼠、Cln3 KO小鼠以及化合物G处理的Cln3 KO小鼠从滚轮跌落的时间都逐渐延长(图24(a))。在第4天测试时，Cln3 KO小鼠从滚轮跌落的时间显著早于野生型小鼠，而化合物G的处理显著延长了Cln3 KO小鼠从滚轮跌落的时间，并达到与野生型小鼠接近的水平(图24(b))。这说明化合物G可以缓解Cln3 KO小鼠在滚轮实验中的运动缺陷。
对4-5月龄的野生型小鼠、Cln3 KO小鼠以及化合物G处理的Cln3 KO小鼠进行了矿场实验。将小鼠轻轻置于矿场中，让其自由运动，并记录其在10分钟内运动的路线、路程和速度。数据显示，和野生型小鼠相比，Cln3 KO小鼠在矿场中运动的路程和速度有一个显著的下降。而化合物G的处理可以在一定程度上提高Cln3 KO小鼠在矿场中运动的路程和速度(图24(c、d))。这说明化合物G可以在一定程度缓解Cln3 KO小鼠在矿场实验中的运动缺陷。
实施例7：化合物G抑制KEAP1并增强自噬溶酶体功能
化合物G的已知靶点在神经细胞中几乎不表达
已有的关于化合物G的研究显示，其可通过抑制已知靶点法尼醇X受体(FXR)的活性，提高转录因子EB(TFEB)以及自噬相关基因和溶酶体基因的转录水平。在小鼠尾尖成纤维细胞(TTF)中加入化合物G，孵育6小时后，提取mRNA，并检测了细胞中自噬相关基因和溶酶体基因的转录水平。结果显示，化合物G的处理可以显著提高TTF中自噬相关基因和溶酶体基因的转录水平(图25(a、b))。
然而在小鼠神经干细胞中，化合物G处理6小时后，细胞自噬和溶酶体相关基因的表达水平没有显著的变化(图26(a))。在小鼠神经元中，化合物G也没有显著提高自噬相关基因或溶酶体基因的转录水平(图26(b))。由此猜想化合物G的 已知靶点FXR在神经谱系细胞中表达量低或不表达。对于野生型和CLN3 Δex7/8人神经干细胞的转录组测序数据证实：FXR在人神经干细胞中的RPKM数为0.496±0.573，几乎不表达。
进一步在诱导产生的CLN3 Δex7/8人神经干细胞和神经元中检测了化合物G对于自噬相关基因和溶酶体基因转录水平的影响。结果显示，在CLN3 Δex7/8人神经干细胞中，化合物G的处理没有显著提高自噬相关基因或溶酶体基因的转录水平(图27(a))。同样的，在CLN3 Δex7/8人神经元中，化合物G的处理也没有在整体上显著提高自噬相关基因或溶酶体基因的表达(图27(b))。
为验证在小鼠TTF中，化合物G对于自噬相关基因和溶酶体基因的转录激活是否依赖于FXR，利用shRNA在TTF中敲减了FXR的水平。对shRNA敲减FXR的效率进行测试后，选择了敲低效率最好的shRNA-2进行后续的实验(图28(a))。
首先利用包装了对照shRNA或FXR shRNA-2的慢病毒感染小鼠TTF，在化合物G进行处理后，提取mRNA进行基因转录的检测。发现在对照shRNA感染的细胞中，化合物G可以显著提高自噬相关基因的表达。而在FXR shRNA-2感染的TTF中，化合物G的处理并不能增加这些基因的表达量(图28(b))。数据显示，FXR的敲减本身可以显著提高Tfeb和自噬相关基因的表达水平(图28(b))。
以上结果共同表明，化合物G对于自噬相关基因的转录激活是依赖于FXR。
化合物G激活NRF2下游基因的表达
前述实验的结果表明，在人神经干细胞和神经元中，化合物G可以显著提高细胞的自噬流，并增强溶酶体的功能。由此猜测在人神经干细胞和人神经元中，化合物G通过全新的不依赖于FXR的机制，增强了细胞的自噬流和溶酶体功能。
为了探索化合物G在人神经干细胞中的作用机制，对野生型人神经干细胞、CLN3 Δex7/8人神经干细胞以及化合物G处理6小时后的CLN3 Δex7/8人神经干细胞进行了转录组测序实验，并分析了差异表达基因。PCA分析显示，在CLN3 Δex7/8人神经干细胞中，化合物G处理6小时不会对其基因表达谱产生显著的影响，经化合物G处理和未经处理的CLN3 Δex7/8人神经干细胞聚类在一处(图29(b、c))。当把表达量倍数变化>2并且矫正后p<0.05的基因作为差异基因时，发现化合物G处理6小时后只产生了3个差异基因(图29(a))，分别为NQO1、LPCAT1和HLA-E，其表达量都有所增加。针对RNA测序结果，还分析了化合物G对TFEB、自噬相关基因 以及溶酶体基因转录水平的影响，和之前的实验结果一致，化合物G在CLN3 Δex7/8人神经干细胞中对TFEB以及大部分自噬相关基因和溶酶体基因的转录水平没有显著的影响(图30)。
对RNA测序数据中NRF2蛋白下游基因的转录水平进行分析。结果显示，化合物G处理之后，CLN3 Δex7/8人神经干细胞中多个NRF2下游基因的表达有所上升(图31)。除了NQO1，其它基因的差异表达倍数小于2。
进一步设计引物，对上述NRF2下游被激活的基因进行了qRT-PCR的验证。用化合物G处理CLN3 Δex7/8人神经干细胞6小时后，收集细胞并提取mRNA，用于检测基因的表达水平。结果显示，在CLN3 Δex7/8人神经干细胞中，化合物G的处理可以显著提高大部分NRF2下游基因的表达水平(图32)。
以上数据证明，在CLN3 Δex7/8人神经干细胞中，化合物G的短时间处理可以激活NRF2下游基因的表达。由此可以确定化合物G直接激活了NRF2。
化合物G抑制KEAP1蛋白活性
已有的研究表明，NRF2蛋白的活性主要受KEAP1蛋白的调控。多个亲电试剂或亲电试剂前体化合物，能够通过抑制KEAP1，增加NRF2活性，例如异硫氰酸酯、α,β-不饱和羰基化合物、酚类和多酚类化合物等。基本作用模式涉及和KEAP1上半胱氨酸残基的共价反应，主要包括第151、272和288位半胱氨酸残基，导致KEAP1和NRF2的结合模式改变，进而激活NRF2(Wells,2015)。
化合物G含有一个α,β-不饱和羰基，由此进行了定量硫醇反应性分析，测定化合物G是否可和KEAP1上的半胱氨酸共价结合。用化合物G处理CLN3 Δex7/8人神经干细胞2小时后，裂解细胞，利用质谱分析裂解液中KEAP1上半胱氨酸残基的硫醇反应性。结果显示，化合物G的处理可以减少KEAP1蛋白第288位半胱氨酸残基的硫醇反应性(图33)。表明化合物G可能通过和KEAP1蛋白288位半胱氨酸残基的共价结合，抑制KEAP1的功能，从而激活NRF2及其下游基因的表达。
为验证KEAP1的抑制是否可以缓解JNCL疾病表型，利用靶向KEAP1的shRNA，在CLN3 Δex7/8人神经元模型中敲减了KEAP1蛋白。从人shRNA文库中选取了5条靶向KEAP1的shRNA慢病毒质粒，而后利用HEK293T细胞将其包装成慢病毒，并侵染人神经干细胞。为检测shRNA的敲减效率，提取mRNA，利用qRT-PCR检测细胞内KEAP1 mRNA的相对丰度。结果显示，KEAP1 shRNA-3具有最高的敲减效率(图 34)，接着利用该shRNA进行了后续的实验，检测KEAP1敲减对于CLN3 Δex7/8人神经元的影响。
首先检测KEAP1的敲减是否可以达到类似的效果。利用Lysotracker染色来指征细胞中溶酶体的酸度，并利用流式细胞术分析染色的平均荧光强度。KEAP1敲减的CLN3 Δex7/8人神经元的Lysotracker平均荧光强度显著增强，而在KEAP1敲减的CLN3 Δex7/8人神经元中添加化合物G则不能进一步提高Lysotracker的平均荧光强度(图35)。该数据说明，KEAP1的敲减可以提高CLN3 Δex7/8人神经元的溶酶体酸度，而化合物G提高溶酶体酸度的能力依赖于KEAP1。
接着测试了KEAP1的敲减对于CLN3 Δex7/8人神经干细胞溶酶体内蛋白酶活性的影响。在细胞中加入了组织蛋白酶B的底物，该底物被组织蛋白酶B剪切后可以发出红色荧光。利用Opera高内涵激光共聚焦显微镜进行了细胞成像。结果显示，和野生型神经干细胞相比，组织蛋白酶B剪切底物后发出的红色荧光强度在CLN3 Δex7/8细胞中显著降低，而KEAP1的敲低可以显著提高细胞内的红色荧光强度(图36(a、b))。以上数据说明，KEAP1的敲低可以提高CLN3 Δex7/8细胞溶酶体中的蛋白酶活性。KEAP1敲减的CLN3 Δex7/8人神经元中SCMAS蛋白的贮积也显著减少(图37)。
为进一步检测KEAP1敲减对于细胞内自噬流的影响，在人神经干细胞中过表达了tandem LC3蛋白，以标记细胞内的自噬小体，并利用高内涵激光共聚焦显微镜进行成像。结果显示，和化合物G类似，KEAP1敲减可以显著增加CLN3 Δex7/8人神经干细胞中红色荧光单阳性的自噬小体数量。该结果提示，在CLN3 Δex7/8人神经干细胞中，KEAP1敲减可以有效缓解细胞自噬流的缺陷(图38(a、b))。
接着检测了KEAP1敲减对于CLN3 Δex7/8人神经元稳态的影响。利用OMP25-GFP慢病毒质粒侵染KEAP1敲低的CLN3 Δex7/8人神经元，以标记细胞中线粒体的形态。而后用激光共聚焦显微镜进行细胞成像。CLN3 Δex7/8人神经元中存在大量损伤的点状线粒体，而化合物G的处理或KEAP1的敲减都可以显著减少CLN3 Δex7/8人神经元中损伤线粒体的出现(图39)。
还检测了KEAP1敲减对于CLN3 Δex7/8人神经元中ATP产生能力的影响。结果显示，KEAP1的敲减可以显著增加CLN3 Δex7/8人神经元产生ATP的能力(图40)。上述结果证明，和化合物G的处理效果一致，KEAP1的敲减可以帮助维持CLN3 Δex7/8人神经元中线粒体的稳态和功能。
综上，在CLN3 Δex7/8人类细胞模型中，KEAP1的敲减可以模拟化合物G对于溶酶体功能、自噬流强度和线粒体稳态的作用。化合物G以KEAP1依赖的方式发挥上述功能。
化合物G通过p62-KEAP1通路增强溶酶体酸度
本发明检测了化合物G的处理或KEAP1的敲低对于细胞内p62蛋白水平的影响。在人神经元中加入化合物G，孵育24小时后提取细胞裂解液，利用qRT-PCR检测p62基因的转录水平。结果显示，化合物G的处理或KEAP1的敲低可以显著增加人神经元中p62的转录水平(图41(a))。在人神经元中，化合物G的处理或KEAP1的敲低同样可以增加p62的蛋白水平(图41(b))。前文已经证明，在加入Baf-A1的条件下，化合物G仍然可以显著增加胞内p62的蛋白水平，这表明化合物G对p62蛋白量的调控并非通过抑制溶酶体的功能(图22(a、b))。
检测了p62蛋白水平增加对于细胞溶酶体功能的影响：在NRK细胞中过表达了GFP-p62蛋白，以及对照组GFP蛋白，并利用Lysotracker染料检测了GFP阳性细胞内溶酶体的酸度。流式细胞术分析结果显示，和化合物G类似，p62蛋白的过表达可以显著增加细胞内Lysotracker染料的荧光强度(图42(a))，表明p62蛋白过表达可以提高细胞内溶酶体酸度。
另一个已知的KEAP1抑制剂富马酸二甲酯(DMF)的处理也可以显著提高细胞内溶酶体的酸度(图42(a))。同时，在人神经干细胞中过表达了GFP-p62蛋白后也观察到了类似的现象，p62蛋白的过表达可以显著提高神经干细胞内的溶酶体酸度(图42(b)),并且缓解CLN3 Δex7/8人神经元中毒性蛋白的异常聚积以及神经元死亡(图43)。
以上结果共同表明，KEAP1的抑制通过增加细胞内NRF2下游蛋白如p62的蛋白水平，提高溶酶体的酸度。
实施例8：化合物G降低阿尔茨海默氏痴呆人神经元模型中毒性蛋白的聚积
本发明检测了化合物G的处理对于阿尔茨海默氏痴呆的治疗作用。我们将带有APP基因突变的人胚胎干细胞体外定向分化为人神经元，构建了体外阿尔茨海默氏痴呆的人神经元模型。该模型展现出了细胞外Aβ斑块的沉积，以及细胞分泌到培养基中的毒性Aβ42的水平增加。化合物G处理后的APP基因突变人神经元则表现出了Aβ斑块的沉积的显著减少，以及细胞分泌到培养基中的毒性Aβ42的水平的显著 降低(图44)。
结论
本发明揭示了包括化合物G在内的小分子化合物通过抑制KEAP1增强人神经元中的自噬流和溶酶体功能，从而有利于JNCL的治疗，同时证明敲减KEAP1基因也可达到同样的效果。除了JNCL，溶酶体功能缺陷和自噬功能降低也参与多种NCL和其他晚发性神经退行性疾病的发病过程，合理推断，抑制KEAP1的活性对于其他自噬和溶酶体功能缺陷相关的神经退行性疾病也具有一定的治疗作用，抑制KEAP1的手段包括使用本发明所鉴定的包括化合物G在内的小分子化合物或者敲低KEAP1基因。
尽管已结合本发明的具体实施方式描述了本发明，但显然许多替代、修饰和变化对本领域的技术人员是显而易见的。相应地，本发明包括了落在所附权利要求的精神和范围之内的所有这类替代、修饰和变化。在本说明书中提及的所有公开文件、专利和专利申请均通过引用至本说明书中的方式全文并入本文。

Claims (21)

  1. 一种治疗与细胞自噬功能缺陷和/或溶酶体功能缺陷相关疾病的方法，该方法包括抑制Kelch样环氧氯丙烷相关蛋白1(KEAP1)的活性，和/或增强转录因子E2相关因子2(NRF2)和/或其下游蛋白的活性的步骤。
  2. 根据权利要求1的方法，其特征在于所述与细胞自噬功能缺陷和/或溶酶体功能缺陷相关的疾病为神经退行性疾病。
  3. 根据权利要求2的方法，其特征在于所述神经退行性疾病为选自肌萎缩性侧索硬化症(ALS)、阿尔茨海默氏痴呆、亚历山大病、阿尔珀斯病(Alper’sdisease)、共济失调-毛细血管扩张症、牛海绵状脑病(BSE)、Canavan病、科凯恩综合征(Cockaynesyndrome)、皮质基底节变性、克-雅病、亨廷顿病、HIV相关痴呆、肯尼迪病、克拉伯病(Krabbedisease)、路易体痴呆、马查多-约瑟夫病(脊髓小脑共济失调3型)、多发性硬化症、多系统萎缩、神经疏螺旋体病(Neuroborre1iosis)、帕金森病、佩-梅病、皮克氏病、原发性侧索硬化、Prion病、雷夫叙姆病(Refsum’s disease)、桑德霍夫病(Sandhoff disease)、希尔德病、精神分裂症、Spielmeyer-Vogt-Sjogren-Batten病、脊髓小脑共济失调、脊髓性肌肉萎缩症、或神经元蜡样脂褐质沉积症(NCL)，其中优选所述神经元蜡样脂褐质沉积症为青少年神经元蜡样脂褐质沉积症(JNCL)。
  4. 根据权利要求3的方法，其特征在于所述疾病由CLN3基因突变所致，例如由CLN3基因外显子7和8的缺失突变所致。
  5. 根据权利要求1-4任一项所述的方法，其特征在于采用siRNA、sgRNA或构建有shRNA的载体沉默或敲减KEAP1基因的表达，从而增强NRF2和/或其下游蛋白的活性。
  6. 根据权利要求1-4任一项所述的方法，其特征在于采用KEAP1抑制剂和/或NRF2活化剂增强NRF2和/或其下游基因的表达，从而增强NRF2和/或其下游蛋白的活性。
  7. 根据权利要求6所述的方法，其特征在于所述KEAP1抑制剂和/或NRF2活化剂选自能够与KEAP1上包括第151、272和/或288位半胱氨酸在内的半胱氨酸残基发生共价反应的试剂。
  8. 根据权利要求6所述的方法，其特征在于所述KEAP1抑制剂和/或NRF2活化剂选自Carvedilol(CAS:72956-09-3)、Ketoconazole(CAS:65277-42-1)、GANT61,CAS(500579-04-4)、Protriptyline hydrochloride(CAS:1225-55-4)、LP 44(CAS:824958-12-5)、Doxepin HCl(CAS:1229-29-4)、富马酸二甲酯(DMF)、乙酰-11-羰基-β-乳香酸(AKBA)、异硫氰酸酯、巴多索隆(CDDO)、甲基巴多索隆(CDDO-Me)以及这些化合物的衍生物或类似物，或者选自α,β-不饱和羰基化合物、酚类和多酚类化合物的一种或多种。
  9. 根据权利要求6所述的方法，其中所述的KEAP1抑制剂为化合物G，即(Z)-guggulsterone，其结构式为：
    Figure PCTCN2022099308-appb-100001
  10. 一种用于治疗与细胞自噬功能缺陷和/或溶酶体功能缺陷相关疾病的药物组合物，其包含治疗上有效量的一种或多种KEAP1抑制剂和/或NRF2活化剂，和药物上可接受的载体和/或赋形剂。
  11. 根据权利要求10所述的药物组合物，其特征在于所述KEAP1抑制剂和/或NRF2活化剂选自能够与KEAP1上包括第151、272和/或288位半胱氨酸在内的半胱氨酸残基发生共价反应的试剂。
  12. 根据权利要求10所述的药物组合物，其特征在于所述KEAP1抑制剂和/或NRF2活化剂选自Carvedilol(CAS:72956-09-3)、Ketoconazole(CAS:65277-42-1)、GANT61,CAS(500579-04-4)、Protriptyline hydrochloride(CAS:1225-55-4)、LP 44(CAS:824958-12-5)、Doxepin HCl(CAS:1229-29-4)、富马酸二甲酯(DMF)、乙酰-11-羰基-β-乳香酸(AKBA)、异硫氰酸酯、巴多索隆(CDDO)、甲基巴多索隆(CDDO-Me)以及这些化合物的衍生物或类似物，或者选自α,β-不饱和羰基化合物、酚类和多酚类化合物。
  13. 根据权利要求10所述的药物组合物，其中所述的KEAP1抑制剂为化合物G，即(Z)-guggulsterone，其结构式为：
    Figure PCTCN2022099308-appb-100002
  14. 一种用于治疗与细胞自噬功能缺陷和/或溶酶体功能缺陷相关疾病的试剂盒，其包含抑制KEAP1基因表达的试剂。
  15. 根据权利要求14所述的试剂盒，其特征在于所述试剂盒包含siRNA、sgRNA或构建有shRNA的载体。
  16. 一种用于缓解或消除受试者神经干细胞向神经元的分化缺陷的方法，所述方法包括向有需要的受试者施用抑制KEAP1活性和/或增强NRF2和/或其下游蛋白活性的药物，其中所述受试者患有与细胞自噬功能缺陷和/或溶酶体功能缺陷相关的疾病。
  17. 根据权利要求16所述的方法，其中所述疾病由CLN3基因突变所致，例如由CLN3基因外显子7和8的缺失突变所致。
  18. 根据权利要求16或17的方法，其中所述药物选自以下药物中的一种或多种：化合物G即(Z)-guggulsterone、Carvedilol(CAS:72956-09-3)、Ketoconazole(CAS:65277-42-1)、GANT61,CAS(500579-04-4)、Protriptyline hydrochloride(CAS:1225-55-4)、LP 44(CAS:824958-12-5)、Doxepin HCl(CAS:1229-29-4)、富马酸二甲酯(DMF)、乙酰-11-羰基-β-乳香酸(AKBA)、异硫氰酸酯、巴多索隆(CDDO)、甲基巴多索隆(CDDO-Me)以及这些化合物的衍生物或类似物，或者选自其它α,β-不饱和羰基化合物、酚类和多酚类化合物，优选为化合物G即(Z)-guggulsterone。
  19. 一种筛选可激活细胞自噬流和/或增强溶酶体功能的物质的方法，其特征在于使用一种包含双荧光标记的Tandem LC3报告系统，该系统中所述双荧光标记的LC3所在的自噬小体位于细胞质时呈现双荧光，自噬小体与溶酶体融合形成自溶酶体时则只呈现单荧光；如果待筛选物质使得该报告系统中双荧光和单荧光标记的自噬小体数量和自溶酶体数量均增加，则该物质为能够激活细胞自噬流和/或增强溶酶体功能的目标物质。
  20. 一种基于神经干细胞或神经元的体外模型，其存在CLN3基因外显子7和8的缺失突变即CLN3 Δex7/8。
  21. 化合物G即(Z)-guggulsterone在制备治疗CLN3基因突变引起的疾病或病症中的用途，优选地，所述CLN3基因突变是指外显子7和8的缺失突变。
PCT/CN2022/099308 2021-06-17 2022-06-17 治疗神经元蜡样质脂褐质沉积症的方法和药物 WO2022262830A1 (zh)

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