WO2018171616A1 - Aldh1a及其激动剂、催化产物和抑制剂的用途 - Google Patents

Aldh1a及其激动剂、催化产物和抑制剂的用途 Download PDF

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WO2018171616A1
WO2018171616A1 PCT/CN2018/079758 CN2018079758W WO2018171616A1 WO 2018171616 A1 WO2018171616 A1 WO 2018171616A1 CN 2018079758 W CN2018079758 W CN 2018079758W WO 2018171616 A1 WO2018171616 A1 WO 2018171616A1
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aldh1a
ube3a
group
autism
mice
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PCT/CN2018/079758
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English (en)
French (fr)
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胡荣贵
徐星星
高晓博
李传银
郝子健
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中国科学院上海生命科学研究院
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Priority to US16/495,863 priority Critical patent/US20200268855A1/en
Priority to JP2019552513A priority patent/JP7068334B2/ja
Priority to EP18772528.8A priority patent/EP3622950A4/en
Publication of WO2018171616A1 publication Critical patent/WO2018171616A1/zh

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Definitions

  • the present invention is in the field of biotechnology, and in particular, the invention relates to the use of ALDH1A and its agonists, catalytic products and inhibitors.
  • autism Autism spectrum disorder, referred to as “autism”, also known as “autism”, its core symptoms are social communication and communication barriers, repeated stereotypes, is a serious neurodevelopmental disorder.
  • chromosome 15q11-q13 copy number amplification (CNV) accounts for 1-3%; this CNV can cause excessive activation of UBE3A E3 ubiquitin ligase, but its specific pathogenesis is not clear .
  • Autism spectrum disorder is a kind of extensive neurodevelopmental disorder with a variety of pathogenic causes.
  • the core symptoms are mainly reflected in social communication, communication skills, limited repetitive interests, behaviors or activities.
  • genetic factors that cause autism include point mutations and chromosome copy number variations, and their mutations encompass a variety of involved genes, involving different signaling pathways.
  • autistic patients show similar core phenotypic symptoms, as well as synaptic homeostasis, suggesting that there may be autism in autism Common pathogenesis. Therefore, it can be clarified that the molecular mechanism between genetic factors and synaptic homeostasis in autism is particularly important and critical.
  • the ALDH1A agonist is not an inhibitor of UBE3A.
  • the "inhibitor that is not UBE3A” means that the expression or activity of UBE3A has no or substantially no effect on the expression or activity of the ALDH1A agonist (eg, expression of UBE3A in the experimental group in which the ALDH1A agonist is present) E1 or activity A1, the ratio of expression E0 or activity A0 of UBE3A in the blank control group (when the ALDH1A agonist is absent) (ie, E1/E0 or A1/A0 is 0.7-1.3, preferably 0.8- 1.2, better 0.9-1.1)).
  • the ALDH1A agonist eg, expression of UBE3A in the experimental group in which the ALDH1A agonist is present
  • E1 or activity A1 the ratio of expression E0 or activity A0 of UBE3A in the blank control group (when the ALDH1A agonist is absent)
  • E1/E0 or A1/A0 is 0.7-1.3, preferably 0.8- 1.2, better 0.9-1.1
  • the autism is autism in the human population.
  • the population is a population having chromosome 15q11-q13 copy number amplification
  • the population is a population having UBE3A overexpressed or overactivated.
  • the population is a population of T508A mutations with UBE3A.
  • the ALDH1A is selected from the group consisting of ALDH1A1, ALDH1A2, ALDH1A3, or a combination thereof.
  • the agonist comprises an expression promoter, a protein degradation inhibitor.
  • the ALDH1A agonist is selected from the group consisting of N-(1,3-benzodioxolan-5-methylene)-2,6-dichlorobenzamide (N- (1,3-Benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide), N-(1,3-benzodioxolan-5-methylene)-2,6-dichlorobenzamide ( N-(1,3-Benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide), 6-methyl-2-azophenyl-3-hydroxypyridine (6-methyl-2-(phenylazo)-3- Pyridinol), 2-(benzo[d][1.3]dioxocyclopenten-5-yl)-N-(5,6-dihydro-4H-cyclopentadiene[c]isoxazole-3 -(Benzyl)acetate (2-(benzo[d][1,3]dioxol-5-
  • a retinoic acid or a retinoic acid analog, or a solvate thereof, or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition or formulation, the pharmaceutical composition Or a formulation for the treatment and/or prevention of autism.
  • the retinoic acid analog is selected from the group consisting of retinol, retinal, all-trans retinoic acid, 13-cis retinoic acid, 9-cis retinoic acid, acitretin , Avi A, adapalene, bexarotene, tazarotene, or a combination thereof.
  • a retinoic acid synthesis starting compound or a retinoic acid degrading enzyme inhibitor, for the preparation of a pharmaceutical composition or formulation for use in therapy and/or Prevent autism.
  • the retinoic acid degrading enzyme inhibitor is selected from the group consisting of liarozole, ketoconazole, talarozole, or a combination thereof.
  • an ALDH1A inhibitor for the preparation of a formulation for establishing an autistic animal model.
  • the ALDH1A inhibitor is selected from the group consisting of a small molecule compound, an antibody, a nucleic acid, or a combination thereof.
  • the ALDH1A inhibitor is a UBE3A agonist.
  • the nucleic acid is selected from the group consisting of miRNA, siRNA, sgRNA/Cas9 complex, or a combination thereof.
  • the ALDH1A inhibitor is selected from the group consisting of disulfiram, 4-(N,N-diethyl)aminobenzaldehyde (DEAB), WIN-18446, A37 (CM037) , NCT-501 hydrochloride, CVT-10216, or a combination thereof.
  • a method of assisting diagnosis and/or prognosis for autism comprising the steps of:
  • sample to be tested the sample being selected from the group consisting of blood and body fluid;
  • the marker is selected from the group consisting of retinoic acid, ALDH1A, or a combination thereof.
  • step (3) when the retinoic acid concentration in the sample is lower than the retinoic acid concentration of the normal donor sample (8-20 nM), the patient is at high risk of developing autism. In the normal population or poor prognosis; and / or
  • the indicator to be tested is selected from the group consisting of: the level or concentration of retinoic acid, the amount or activity of ALDH1A expression, or a combination thereof;
  • the measurement result T is significantly lower than the measurement result C, it indicates that the test substance has a risk of causing side effects of the pregnant woman and/or the infant.
  • the substance to be tested is selected from the group consisting of a drug, a daily chemical, or a combination thereof.
  • the side effects include causing autism in the present, autism in the offspring, or a combination thereof.
  • said "significantly lower” means that the T/C ratio is less than 0.7, and a statistically significant difference P ⁇ 0.05 occurs.
  • step (b) the assay is carried out in a model animal or culture system.
  • the method further comprises verifying the substance to be tested which has a side effect in step (c), further verified by animal experiments.
  • a pharmaceutical combination or a kit comprising the pharmaceutical composition, the pharmaceutical combination comprising:
  • a first pharmaceutical composition comprising a pharmaceutically acceptable carrier and a first active ingredient selected from the group consisting of clotrimazole, montelukast, montelukast sodium;
  • a second pharmaceutical composition comprising a pharmaceutically acceptable carrier and a second active ingredient selected from the group consisting of ALDH1A, an ALDH1A agonist, an ALDH1A catalytic product, an ALDH1A catalytic substrate, or Its combination.
  • the second pharmaceutical composition is for reducing the risk of side effects of the first pharmaceutical composition, the risk of side effects being a risk of inducing or triggering autism.
  • a pharmaceutical composition comprising:
  • a first active ingredient selected from the group consisting of clotrimazole, montelukast, and montelukast sodium;
  • a second active ingredient selected from the group consisting of ALDH1A, an ALDH1A agonist, an ALDH1A catalytic product, an ALDH1A catalytic substrate, or a combination thereof.
  • the second active ingredient is used to reduce the risk of side effects of the first active ingredient, which is a risk of inducing or triggering autism.
  • the second pharmaceutically active ingredient is selected from the group consisting of retinoic acid and analogs thereof, retinoic acid degrading enzyme inhibitors, or a combination thereof.
  • a method of screening for a drug candidate (or potential therapeutic agent) for treating or preventing autism comprising the steps of:
  • test index is selected from the group consisting of: the level or concentration of retinoic acid, the amount or activity of ALDH1A expression, or a combination thereof;
  • the candidate substance to be tested can be used as a drug candidate for treating or preventing autism.
  • said "significantly higher" means that the T/C ratio is greater than 1.2, and a statistically significant difference P ⁇ 0.05 occurs.
  • step (b) the assay is carried out in a model animal or culture system.
  • the method further comprises, for step (c), suggesting a candidate substance that can be used as a drug candidate for treating or preventing autism, further verified by animal experiments.
  • a polypeptide or antibody or compound capable of blocking the binding of UBE3A and an ALDH1A family protein for the preparation of a pharmaceutical composition or formulation for use in therapy and / or prevent autism.
  • polypeptide is selected from the group consisting of peptide-1, peptide-2, peptide-3.
  • Figure 1 shows the copy number amplification of chromosome 15q11.2-14 in a sample of autistic patients.
  • FIG. 2 UBE3A binds to members of the ALDH1A family.
  • FIG. 3 shows that UBE3A binds a polyubiquitin chain to ALDH1A2 in a recombinant ubiquitination system.
  • UBE3A links the polyubiquitin chain to ALDH1A2 in a recombinant ubiquitination system.
  • UBE3A In vitro UBE3A promotes ubiquitination of ALDH1A2. The ubiquitinated protein was enriched with HA antibody and immunoblotted with His antibody.
  • Figure 4 Human UBE3A ubiquitinates ALDH1A2 by non-protein degraded Ub linkage.
  • the HA or Flag tagged ALDH1A2 protein is enriched in cells and detected with HA or Flag antibodies.
  • His6-Ub, Myc-UBE3A, and HA-ALDH1A2 containing the designated site KR were co-transduced in H1299 cells.
  • ALDH1A2-HA was ubiquitinated after enrichment with HA antibody.
  • ALDH1A2-HA is mutated to R at K269, K370, K515.
  • (g) The crystal structure of the human ALDH1A2 protein monomer, in which three major ubiquitination sites (marked in red) are located near the active center (Cys320, highlighted in yellow) and the coenzyme NAD + purple. The crystal structure was extracted from the PDB database (DOI: 10.2210/pdb4x2q/pdb).
  • FIG. 5 UBI3A ubiquitination inhibits ALDH1A2 retinal dehydrogenase activity.
  • ALDH1A2-Flag ubiquitination level detection The designated plasmid was co-transformed in HEK-293FT cells, and the ALDH1A2-Flag protein was precipitated with Flag antibody, eluted with Flag short peptide, and treated with USP2cc overnight at 4 °C.
  • b-c The polyubiquitin chain bound by UBE3A on ALDH1A2 reduced its dehydrogenase activity against retinal.
  • the ALDH1A2-Flag protein is enriched in 293FT cells overexpressing Myc-UBE3A, His6-Ub and ALDH1A2-Flag (b), and the polyubiquitin chain (c) is removed by treatment with USP2cc enzyme or without treatment.
  • the enriched ALDH1A2 protein was analyzed for dehydrogenase activity using all-trans retinal as a substrate.
  • ALDH1A2-Flag whether ubiquitination analysis of propionaldehyde dehydrogenase activity.
  • ASD patient-derived immortalized lymphocytes had lower total ALDH1A activity than normal controls, and were analyzed by AldeFluor (h) or RARE-luciferase (i). The data is presented as mean and variance. *, P ⁇ 0.05, **, P ⁇ 0.01, ***, P ⁇ 0.001; (b, c) two-tailed t-test, one-way ANOVA Dunnett" s post-hoc test (h, i).
  • Blocking of neural activity does not alter the level of transcription of a particular gene in primary PFC cells.
  • Neural activity blockade significantly upregulates GluR1 on the surface of primary PFC cell membranes, rather than the protein level of GluR2.
  • GluR1 and PSD-95 were stained on the surface of nerve cell membrane after introduction of no-load, T508E or UBE3A.
  • Figure 7 Excess UBE3A protein down-regulates the homeostasis of RA and induces a similar phenotype of ASD in mice.
  • the scale, 50 ⁇ m was injected at a time of self-combing of EGFP, UEB3A, UBE3A-T508E mice carried by AAV virus.
  • (f) Compare the communication time of the three groups of mice with the unfamiliar mouse I and the strange mouse II.
  • Figure 8 Oral ATRA is able to reverse the autism phenotype that occurs in mice overexpressing UBE3A.
  • communication time (e) Compare the residence time of oral solvents or ATRA mice in the center of the open field.
  • (f) Compare the ratio of the distance traveled to the total distance of the oral solvent or the central region of the ATRA field in mice injected with UBE3A AAV virus.
  • (g) Compare the duration of injection of UBE3A AAV virus oral solvent or ATRA before dropping in the rotating rod test.
  • (h) Compare wild-type mice with oral solvent or ATRA for self-carding time.
  • (i-j) Comparison of time of wild type mouse oral solvent or ATRA to explore unfamiliar mouse I and article (i) or strange mouse I and strange mouse II (j).
  • (k-1) Compare the residence time (k) of the oral solvent or ATRA in the wild type mice in the open field or the walking distance (l) in the central area.
  • (m) Comparison of the duration before dropping in the wild type mouse oral solvent or ATRA rotating rod test. The data is presented as mean and variance.
  • Figure 9 shows the appearance of a similar phenotype of ASD induced by oral administration of the ALDH1A inhibitor DSF to mice.
  • (a) After oral administration of the solvent or different doses of DSF (0.1 or 0.3 mg/g) for 6 weeks, the relative ATRA content was determined by HPLC-MS/MS method and corrected by the control group.
  • (b) There were no significant differences in body weight between the different groups of mice.
  • (c) Compare the time of three groups of mice for self-combing.
  • (d) Compare the residence time of the three groups of mice in unfamiliar mice, in the middle, and in the chamber of the article.
  • the inventors have unexpectedly discovered a new pathway associated with autism for the first time through extensive and in-depth research. For the first time, the inventors have a significant correlation with the occurrence of autism in the pathway core protein, the synthetase ALDH1A. By supplementing the synthetase ALDH1A, or an agonist thereof, or a synthetic product thereof, or a catalytic substrate thereof, it is helpful to prevent and ameliorate autism and symptoms associated with autism.
  • the present invention has been completed on this basis.
  • Retinoic acid and its derivatives are roughly divided into three generations: the first generation includes: retinol, retinal, tretinoin (all-trans-retinoic acid), isotretinoin (13-cis-retinoic acid), alitretinoin (9-cis-retinoic acid);
  • the second generation includes: etretinate, acitretin;
  • the third generation includes: adapalene, bexarotene, tazarotene.
  • Derivatives also include ester derivatives of the individual compounds which are hydrolyzed to the acid or salt form in vivo. Some of the compounds have the following structural formula:
  • ADH1A refers to the Aldehyde dehydrogenase family 1 member A.
  • ALDH1A includes homologous proteins from different mammals, such as ALDH1A from human and non-human mammals such as rodents (eg, mice, rats, cows, sheep, dogs, etc.).
  • rodents eg, mice, rats, cows, sheep, dogs, etc.
  • the term includes not only wild-type ALDH1A but also mutant ALDH1A (the mutant ALDH1A has similar or similar activity compared to wild-type ALDH1A).
  • representative ALDH1A includes, but is not limited to, ALDH1A1, ALDH1A2, ALDH1A3, or a combination thereof.
  • the term "agonist of ALDH1A protein” is a substance that increases the expression and/or activity of ALDH1A. For example, it has a high affinity with the ALDH1A protein and can bind to ALDH1A to produce a substance that enhances the ALDH1A effect.
  • the agonist of the ALDH1A protein is a compound selected from the group consisting of N-(1,3-benzodioxolan-5-methylene) -2,6-dichlorobenzamide (N-(1,3-Benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide, abbreviation ALDA-1), N-(1,3-benzodioxolane) -5-methylene)-2,6-dichlorobenzamide, 2-(benzo[d][1.3]dioxol-5-yl)-N-(5,6-dihydro -4H-cyclopentadienyl[c]isoxazol-3-yl)acetamide, 6-methyl-2-azophenyl-3-hydroxypyridine.
  • N-(1,3-benzodioxolan-5-methylene) -2,6-dichlorobenzamide N-(1,3-Benzodioxol-5-ylmethyl)-2,6-
  • ALDH1A inhibitors are substances that block or reduce the rate of chemical reaction of ALDH1A and its family proteins in biochemical reactions.
  • the term also includes inhibitors that reduce the expression or activity of ALDH1A, such as antisense RNA, miRNA, or antibodies.
  • the ALDH1A and its family inhibitor are compounds selected from the group consisting of:
  • the RA-degrading enzyme CYP26A1 inhibitor refers to a substance that blocks or reduces the rate of degradation of retinoic acid by the CYP26A1 protein in a biochemical reaction.
  • the RA degrading enzyme CYP26A1 inhibitor is a compound selected from the group consisting of:
  • a drug candidate or therapeutic agent refers to a substance which is known to have a certain pharmacological activity or is being tested and which may have a certain pharmacological activity, including but not limited to nucleic acid, protein, chemically synthesized small molecule or large Molecular compounds, cells, and the like.
  • the drug or therapeutic agent can be administered orally, intravenously, intraperitoneally, subcutaneously, or via a spinal canal.
  • the present invention provides a novel therapeutic target for autism, and therefore, according to the present invention, a drug for treating autism can be screened.
  • the present invention can also be used to screen for the potential risk of autism in the offspring caused by the side effects of taking drugs and daily chemicals for pregnant women (as well as the side effects of taking drugs and daily chemicals in infants and young children). To develop an effective clinical assessment diagnostic tool.
  • the copy number variation (male, age: 3-6 years) of 3 Han patients was performed using the Human660W-Quad microarray chip (Illumina). Chip analysis was performed using GenomeStudio v2011.1 (Illumina), according to previous reports (Nava et al., 2013). The copying of the copy number variation was performed using the human reference genome hg18 as a reference.
  • Blood cells were transformed with EBV virus according to a standardized method (Anderson et al., 1984) to obtain immortalized lymphocytes, and cultured in RPMI-1640 (Gibco) medium containing 10% fetal calf serum (Biochrom).
  • HEK-293FT Life Technologies
  • HEK-293 ATCC
  • SH-SY5Y ATCC
  • H1299 ATCC
  • A549 ATCC
  • HEK-293FT cells were transfected with polyethylenimine (Sigma), and SH-SY5Y and H1299 were transfected with Lipofectamine 2000 (Life Technologies), operating according to the manufacturer's instructions.
  • the UBE3A single allele and the biallelic knockout cell line based on H1299 cells were screened by genome editing by the CRISPR/Cas9 system.
  • the sgRNA primer design of the UBE3A gene has been reported according to previous reports (Hsu et al., 2013), and the sequences are listed below (target sequences are underlined):
  • the plasmids in this subject include restriction endonuclease digestion and ligation (NEB), both according to conventional cloning methods. Plasmids for yeast double hybrid screening were obtained using Gateway LR Cloning Enzyme (ThermoFisher) according to the manufacturer's protocol. Point mutations of related plasmids were introduced using site-directed mutagenesis methods. In addition, plasmids containing multiple elements were constructed using the Gibson assembly method. In this work, UBE3A fusion expression tags are placed at the N-end.
  • UBE3A pDEST32-UBE3A (ThermoFisher) and a human cDNA library based on the pDEST22 backbone (ThermoFisher) were co-transformed into the yeast strain Mav203 (ThermoFisher). Positive clones were able to survive in a medium lacking both uracil, histidine, leucine and tryptophan (Clontech), and also in the presence of X-Gal (Sigma).
  • GST, GST-UBE3A and the truncated GST-UBE3A protein were induced to express in BL21 competent (NEB) and purified using glutathione agarose (GE Healthcare).
  • the ALDH1A1-His6, ALDH1A2-His6 and ALDH1A3-His6 proteins were purified using Ni-NTA agarose (Qiagen).
  • the purified ALDH1A2-His6 and GST-UBE3A proteins were incubated in pul l-down buffer (50 mM Tris-Cl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM DTT, 10 mM MgCl2) at 4 °C. hour.
  • the beads were washed 4 times with pull-down buffer and analyzed by immunoblotting.
  • the pull-down analysis of other proteins follows the same steps.
  • HA-UB, UBCH7 and Uba1 are inserted into the first multiple cloning site, ligated to the T7 promoter/lactose operon and the ribose binding site, and each member is separated by the Shine-Dalgarno (SD) sequence to form a poly-shun
  • SD Shine-Dalgarno
  • BL21 competent cells were co-transformed with pACYC and pET22b-ALDH1A2-His6 plasmid (Novagen) by electroporation and screened with chloramphenicol and ampicillin (Sigma). E. coli was induced to culture for 16 hours at 18 ° C with 0.25 mM isopropyl- ⁇ -D-1-thiogalactoside (IPTG, Sigma) when the OD600 absorbance reached 0.8.
  • IPTG isopropyl- ⁇ -D-1-thiogalactoside
  • the cells were harvested and resuspended in RIPA buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.4, 1% NP-40, 0.1% SDS), then the cells were sonicated with a Vibra-Cell processor (SONICS) and the pellet was removed by centrifugation. . The supernatant was purified using Ni-NTA agarose. Purified proteins bound to Ni-NTA beads and Usp2cc enzyme were incubated overnight at 4 °C in the Usp2cc treatment group. The level of ubiquitination of the ALDH1A2 protein was analyzed by immunoblotting.
  • RIPA buffer 150 mM NaCl, 50 mM Tris-Cl, pH 7.4, 1% NP-40, 0.1% SDS
  • SONICS Vibra-Cell processor
  • the supernatant was purified using Ni-NTA agarose. Purified proteins bound to Ni-NTA beads and Usp2cc enzyme were incubated overnight at 4 °C in
  • IP buffer 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol
  • protease inhibitor cocktail (Roche).
  • Vibra-Cell processor for ultrasound. After centrifugation, the cell debris was removed, and the supernatant was added with a specific antibody and protein G agarose beads (Merck Millipore) at 4 ° C overnight.
  • the primary antibodies used were as follows: normal rabbit IgG antibody (sc-2027, Santa Cruz), Flag antibody (F1804, Sigma), ALDH1A2 antibody (sc-367527, Santa Cruz), HA antibody (H6908, Sigma). After immunoprecipitation, 2 x SDS-PAGE loading buffer was added and denatured at 95 ° C for 10 minutes. The initial protein, immunoprecipitated enriched protein and other cell lysate samples were separated by SDS-PAGE gel and transferred to a PVDF membrane (Bio-Rad).
  • Membranes were immunoblotted with specific antibodies: UBE3A antibody (sc-166689, Santa Cruz, 1:500 dilution), Flag tag antibody (F1804, Sigma, 1:8000 dilution), HA-tag antibody (H6908, Sigma, 1:4000) Dilution), ALDH1A2 antibody (sc-367527, Santa Cruz, 1:500 antibody), ALDH1A1 antibody (15910-1-AP, Proteintech, 1:500 antibody), ALDH1A3 antibody (25167-1-AP, Proteintech, 1:500 Diluted), His-tag antibody (H1029, Sigma, 1:4000 dilution), GST-tagged antibody (66001-1-lg, Proteintech, 1:5000 dilution), Myc-tagged antibody (sc-40, Santa Cruz, 1:1000 dilution) ), actin antibody (A2228, Sigma, 1:8000 dilution), GAPDH antibody (sc-32233, Santa Cruz, 1:4000 dilution).
  • UBE3A antibody s
  • SH-SY5Y cells After transfecting the specific plasmid, SH-SY5Y cells (ATCC) were cultured for another 24 hours and then fixed with 4% paraformaldehyde (Sigma). After rupturing the cells, primary antibody (Flag tag antibody, F1804, Sigma) was added overnight at 4 ° C, and then incubated with Alexa Fluor 488-conjugated fluorescent secondary antibody (A11029, ThermoFisher) for 1 hour at room temperature. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, ThermoFisher).
  • DAPI 4,6-diamidino-2-phenylindole
  • Rat primary neuronal cells were transfected into specific plasmids by calcium phosphate transfection at day 10 in vitro, with DMSO (Sigma) or 1 ⁇ M tetrodotoxin (TTX, Shanghai Aladdin) and 100 ⁇ M D-(-)- at DIV12. 2-Amino-5-phosphate valeric acid (D-APV, Tocris) was treated for 24 hours. After fixing the cells, the primary anti-GluR1 antibody (sc-55509, Santa Cruz), PSD95 antibody (ab18258, Abcam) and Alexa Fluor 488 (A11029, ThermoFisher), Alexa Fluor 647 (A21245, ThermoFisher) were combined according to the previous protocol. The secondary antibody is stained.
  • the brains taken from the mice injected with the virus at a fixed point were fixed overnight with PBS containing 4% paraformaldehyde at 4 ° C, and then immersed in PBS containing 30% sucrose (Sigma). Coronal sections of the brain were cut at 40 ⁇ M thickness using a Leica CM3050S ice cutter (Leica Biosystems) and sections were treated with PBST (0.3% Triton X-100) for 15 minutes at room temperature. Brain sections were then blocked with 3% normal goat serum (PhD) and incubated with Flag tag antibody (14793, Cell Signaling, 1:800 dilution) overnight at 4 °C.
  • Sections were incubated with Cy3 binding secondary antibody (111-165-045, Jackson ImmunoResearch) for 1 hour at room temperature, and the nuclei were counterstained with DAPI and mounted with a pad of Mowiol (Sigma). Fluorescent images were acquired using an Olympus FV1200 confocal microscope.
  • Exogenously expressed ALDH1A2-Flag protein was enriched in Flag-conjugated agarose column (Sigma) in HEK-293FT cells transfected or untransfected with UBE3A, and then formulated in ALDH1A enzyme activity assay buffer (0.1M).
  • the Flag short peptide in sodium pyrophosphate, pH 8.0, 1.0 mM EDTA, 2.0 mM DTT was eluted.
  • USP2cc enzyme or bovine serum albumin (BSA) was used to incubate with the eluted protein at 4 °C overnight.
  • the enzyme reaction system consisted of 2.5 mM NAD + , 20 mM DTT and 100 ⁇ M propionaldehyde (Sigma) dissolved in the enzyme activity detection buffer, and the dehydrogenase activity was detected on a BioTek Synergy Neo spectrophotometer at a wavelength of 340 nm at room temperature. Test every 3 minutes. The reaction was terminated when the absorbance of all samples reached the plateau. Using NADH as a standard, the ALDH1A2 dehydrogenase activity was calculated as: (production of NADH (nmol) in total reaction time ⁇ dilution factor of sample) / (reaction time x reaction volume).
  • an acetaldehyde dehydrogenase activity assay kit (Cayman) is required. It is subjected to detection analysis by a method based on fluorescence quantification. Briefly, in the presence of 100 ⁇ M all-trans retinal, the enzyme activity minimizes the interference of retinal on the absorbance by detecting the parameters of the fluorophore at the absorption wavelength of 530-540 nm and the emission wavelength of 585-595 nm.
  • Aldefluor detection was performed using the Aldefluor kit (STEMCELL Technologies) according to the manufacturer's steps. Briefly, 1 x 10 6 immortalized lymphocytes and the fluorescent substrate BODIPY-aminoacetaldehyde-diethylacetate (BAAA-DA) (1.5 ⁇ M) were incubated at 37 ° C for 30 minutes. Each cell is divided into two halves: half for fluorescence analysis and half for pretreatment with the ALDH inhibitor diethylamine-benzaldehyde (DEAB) provided in the kit as a negative in flow cytometry Control.
  • BAAA-DA fluorescent substrate BODIPY-aminoacetaldehyde-diethylacetate
  • the construction of the pGL4.22-RARE-TK-luciferase plasmid included the cloning of 3X RARE (RA-responsive element) in the pGL4.22 vector (Promega).
  • the RA sensor cell line construction method involves transfecting the pGL4.22-RARE-TK-luciferase plasmid into H1299 cells, followed by screening for stable cell lines with puromycin (1 ⁇ g/mL, Sigma).
  • the RA sensor cells and the immortalized lymphocytes as the RA donor were co-cultured in a VP-SFM (virus production serum-free medium, Gibco) medium at a cell number of 1:1. After 8 hours of treatment with 1 ⁇ M all-trans retinal (Sigma), the luciferase activity of the cultured cells was examined.
  • the patch clamp inner solution includes (unit mM): 20KCl, 5MgCl2, 20HEPES, 110K-gluconate, 0.6EGTA, 2Na2-ATP, 0.2Na3-GTP, pH 7.3, 290mOsm, and the internal liquid resistance is about 3-6M ⁇ .
  • the cultured cells were placed in an external solution in which the external liquid component (unit: mM): 129 NaCl, 5 KCl, 1 MgCl 2 , 25 HEPES, 2 CaCl 2 , 30 glucose, pH 7.3, 310 mOsm.
  • the extracellular fluid also contained 1 ⁇ M TTX and 100 ⁇ M picrotoxin (Tocris), and mEPSC was recorded at a voltage of -70 mV.
  • the results were analyzed using Mini Analysis software (Synaptosoft).
  • RNAsimple total RNA kit (Tiangen).
  • a cDNA sample was obtained by reverse transcription using a ReverTra Ace qPCR RT Master Mix (Toyobo).
  • qRT-PCR detection was performed using a SYBR Green Master Mix (Toyobo) on a CFX96 real-time PCR instrument (Bio-Rad). The relative amount of transcription of the designated gene was corrected by the ⁇ Ct method using Gapdh as an internal reference.
  • the QPCR primer sequences in this work are as follows.
  • Biotin-labeled membrane surface protein analysis was performed following previously reported methods (Aoto et al., 2008). Briefly, after 24 hours of treatment with DMSO or 1 ⁇ M TTX and 100 ⁇ M D-APV, primary PFC neurons were washed with PBS and then with biotin solution (1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin, Pierce) Incubate for 2 hours at 4 °C. The reaction was stopped by the addition of 0.1 M glycine, and then washed 3 times with PBS.
  • the biotin-labeled cells were in a cell lysate (containing 25 mM MgCl2, 1% NP-40, 1% Triton X-100, 10% glycerol and protease inhibitors). Lysis in PBS). After removing the cell debris by centrifugation, the supernatant and UltraLink Streptavidin resin (Pierce) were incubated overnight at 4 °C. Biotinylated proteins were collected by centrifugation and washed 3 times with cell lysis buffer.
  • Proteins were denatured in 2X SDS-PAGE loading buffer for 30 min at 75 °C and immunoblotted with GluR1 antibody (13185, Cell Signaling, 1:500 dilution) and GluR2 antibody (13607, Cell Signaling, 1:500 dilution). .
  • mice were housed in 3-5 animals per cage in a standard 12-hour light/12-hour dark cycle to provide autonomous diet water. All behavioral experiments were performed under 12 hours of light. All zoological studies are carried out in strict accordance with the regulations of the Animal Care and Use Committee (IACUC) of the Institute of Biochemical Cell Research of the Chinese Academy of Sciences. The mice in all experiments were C57BL/6 background male mice (SLAC, China).
  • IACUC Animal Care and Use Committee
  • AAV adeno-associated virus
  • AAV2/9 which is packaged by Shanghai Woyuan Biotechnology Co., Ltd. (Obio).
  • the AAV-SynI-Flag-UBE3A and AAV-SynI-Flag-UBE3A-T508E virus titers are approximately 1.5 ⁇ 10 13 copies/mL, and the AAV-SynI-Flag-EGFP virus titer is approximately 9.5 ⁇ 10 12 copies/ mL.
  • mice Three-week-old mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg) and fixed on a locator (Rivend, China). 1 ⁇ L of PBS-diluted AAV virus was injected at a rate of 0.2 ⁇ L/min on both sides of the PFC region inside the mouse brain using a syringe pump (Stoelting). The location of the spot injection (relative to the front door): AP, +2 mm; ML, ⁇ 0.5 mm; DV, -1.3 mm.
  • the amount of virus injection per injection point was: AAV-SynI-Flag-UBE3A and its mutant T508E was 3 ⁇ 10 9 , and AAV-SynI-Flag-EGFP was 1.5 ⁇ 10 9 .
  • the needle was held in place for another 3 minutes to prevent virus reflux.
  • the mice were placed on a 37 ° C electric blanket until they recovered completely from the anesthesia.
  • mice were intraperitoneally injected with 0.5 mg/ml meloxicam (Sigma, 2 mg/kg) to help the mice relieve pain.
  • mice were orally administered ATRA (Sigma, dissolved in olive oil, 3 mg/kg) or olive oil for five consecutive days. Behavioral testing was performed 4 weeks after administration.
  • mice Four-week-old mice were given a low dose (0.1 mg/g) or a high dose (0.3 mg/g) DSF (Sigma, dissolved in olive oil) every other day for 6 weeks.
  • the control group was only given the solvent olive oil (Aladdin).
  • the body weight of the mice was weighed weekly. After 6 weeks of gavage, the mouse behavioral test was started. The brains of the control and DSF mice were dissected, and after liquid nitrogen freezing, tissue homogenization was performed, and ATRA was quantified by the HPLC-MS/MS method described later.
  • the oil phase containing RA and polar retinoids was extracted with 7 mL of n-hexane added again.
  • the extract was evaporated under a nitrogen atmosphere and the dried extract was resuspended with 50 ⁇ L of acetonitrile.
  • the samples were analyzed by HPLC on a 2.1 x 100 mm Supelcosil ABZ + PLUS column (3 ⁇ m, Sigma) column.
  • the mobile phases were: A, water containing 0.1% formic acid; B, acetonitrile containing 0.1% formic acid.
  • the instrumentation was an AB Sciex 4000QTRAP LC-MS/MS system, and the ATRA component was quantified in the APCI cation mode, and the ATRA content in each sample was corrected using a standard ATRA curve.
  • mice used in the self-carding experiments were acclimated for 10 minutes in advance in a cage box filled with a filler having a thickness of about 0.5-1 cm. Then, within 10 minutes, the time the mice were used for self-combing was recorded by a double-blind experimenter through a stopwatch.
  • a three-compartment social experiment was performed according to previously reported procedures (Sztainberg et al., 2015). Briefly, a transparent acrylic box for social experiments was divided into three chambers of uniform size with removable gates mounted on the partitioned partitions. And put an inverted iron mesh cup in each of the left and right chambers. Two days before the experiment, C57BL/6 mice, which were matched with the test mice and matched with the test mice, were placed in a non-category for two hours each day. Test mice were randomized and adapted to the test room for 1 hour before starting the test. Each test mouse was placed in the intermediate chamber for 10 minutes after self-exploration, and the chamber door was closed.
  • a strange mouse is randomly placed in the left or right cup (avoiding positional bias), and the inanimate object is placed in the other side of the cup.
  • Each test mouse was explored for 10 minutes in an open three-chamber, and the communication time between the test mice and the strange mice and the objects was manually recorded.
  • the test mice were kept in the cavity of the strange mouse for 5 minutes, and then another strange mouse was placed in the cup before the object was placed. The test mice were further explored for 10 minutes, and the communication time between the test mice and the familiar animals and the new animals was manually recorded. The observer did not know the group of mice tested.
  • mice were randomized and placed in a field instrument (Med Associates) for 30 minutes.
  • the Ethovision automatic recording software (Noldus) was used to record the time spent by the mouse in the central region (1/3 to 2/3 length on each side of the region) and to calculate the ratio of the distance traveled in the central region to the total distance.
  • mice were placed in a closed arm of the Med Associates and began recording for 5 minutes. Image recording was performed using a dangling camera, and the number of times the mouse entered the open arm and the closed arm within 5 minutes was recorded and analyzed using ANY-maze software (Stoelting).
  • mice After randomization of mice, the motor coordination ability of the mice was recorded using a Rotamex rotarod instrument (Columnbus Instruments). On the same day, the test mice were tested 3 times, each experiment lasted 5 minutes, and the rotating rod was accelerated from 4 revolutions per second to 40 revolutions per second. At least 30 minutes between each experiment. The time before the test mice dropped the rods was automatically recorded by an infrared detection system, and the results of the last test were finally compared.
  • Rotamex rotarod instrument Coldbus Instruments
  • RA retinol dehydrogenase
  • RH10 retinol dehydrogenase
  • ALDH1A1, 2 , 3 families retinal dehydrogenase
  • endogenous or exogenously expressed UBE3A and ALDH1A2 proteins form a complex in HEK-293FT cells (Fig. 2b-c).
  • Flag and RFP-tagged UBE3A and ALDH1A2 proteins were co-localized in the cytoplasm of glioma cell SH-SY5Y (Fig. 2d).
  • the GST pull-down experiment further confirmed that the recombinant proteins UBE3A and ALDH1A2 could bind directly in vitro (Fig. 2e) and bind through the N-terminus (1-280 amino acids) of UBE3A (Fig. 2f). Since the ALDH1A2 protein has greater sequence similarity to other family members ALDH1A1 and 3, it was further verified that UBE3A can bind directly to ALDH1A1 and 3, respectively, in vitro and in vivo (Fig. 2g-j).
  • Peptide-1 (peptide-1): ASRMKRAAAKHLIERYYHQLTEGCG (Seq ID No 9)
  • Peptide-2 NNAAAIKALELYKINAKLCDPH (Seq ID No 10)
  • Peptide-3 (peptide-3): AEALVQSFRKVKQHTKEELKSLQAKDEDKD (Seq ID No 11).
  • the Urease 508 site (T508) of UBE3A has been reported to be phosphorylated by protein kinase A (PKA) and its ubiquitin ligase activity is inhibited.
  • PKA protein kinase A
  • the ubiquitin ligase activity appears to be over-activated due to the inability of phosphorylation of the T508A mutant (Yi et al., 2015).
  • the UBE3A phosphorylation mimetic mutant T508E was found to completely lose E3 ubiquitin ligase activity and was therefore used herein as a ligase inactivating mutant.
  • UBE3A is capable of ubiquitinating the unique retinal dehydrogenase family ALDH1A of the RA synthesis family. Therefore, it is also concluded that UBE3A should affect the anabolism of RA in cells.
  • Aldh1a2 protein was also unchanged in mouse MEF cells containing different Ube3a protein levels (Fig. 4b).
  • the ALDH1A2 protein levels were also consistent in immortalized lymphocyte lines, including autistic patient sources and healthy volunteer-derived cells (Fig. 3i). These results reflect that ubiquitination of ALDH1A2 by UBE3A does not promote substrate degradation as well as other substrates.
  • ubiquitin includes seven lysine (Lys) residues
  • each lysine residue along with the alpha-amino group on the N-terminal methionine, may become under the process of forming a ubiquitin chain.
  • a ubiquitin molecule covalently binds to a site, ultimately forming a polyubiquitinated chain (poly-Ub) containing different lysine linkages.
  • poly-Ub polyubiquitinated chain
  • K-to-R a method that retains lysine at a specific position on the ubiquitin protein and mutates all other lysines into arginine
  • the polyubiquitinated strand forms attached to the ALDH1A2 protein are primarily Lys29 and Lys63.
  • the lysine on K29 and K63 was mutated to arginine, respectively, and the polyubiquitination chain bound to ALDH1A2 was significantly reduced (Fig. 4d-e).
  • the ALDH1A2 protein enriched from bacteria was analyzed by protein profiling technology to find out the ubiquitination site of UBE3A catalyzed ligation in ALDH1A2 protein; K269, K370 and K415 were identified as possible catalytic modification sites. point.
  • the ubiquitination levels on the ALDH1A2 mutant protein were significantly reduced when the K269, K370 and K415 sites on ALDH1A2 were simultaneously mutated to arginine (Fig. 4f).
  • Fig. 4g protein structure analysis that these three sites were distributed near the active center of the dehydrogenase
  • Fig. 4h the UBE3A ubiquitination-modified ALDH1A protein family mainly occurs near the site near the dehydrogenase activity center and does not promote substrate degradation.
  • Flow cytometry-based Aldefluor detection methods are very common when studying cellular ALDH activity (Storms et al., 1999). Its working principle is that intracellular ALDH can oxidize BAAA (BODIPY-aminoacetaldehyde) into BAA (BODIPY-aminoacetate), while BAA as a negative charge product will remain in the cell, thus the cell ALDH dehydrogenase Activity can be quantified by detecting the fluorescence intensity of cells containing BAA. As shown in Figure 5h, the proportion of ALDH positive cells in autistic lymphocytes was reduced by approximately 20% to 50% compared to normal control lymphocytes. This also suggests that a high dose of UBE3A associated with autism may result in down-regulation of ALDH enzyme activity in cells.
  • a co-cultured cell system was established to further verify the ALDH1A dehydrogenase activity in autistic cells.
  • the luciferase plasmid carrying the RA response element RARE was introduced into H1299 cells as RA-responsive cells; the immortalized lymphocyte strain was used as RA-producing cells; the two were cultured in serum-free ratio at a ratio of 1:1.
  • Co-culture was carried out in VP-SFM, and luciferase activity was detected after 8 hours of treatment with retinal.
  • H1299 luciferase activity co-cultured with autism lymphocytes was down-regulated by 60% compared to normal control lymphocytes.
  • RA has been gradually found to play an important role in homeostasis synaptic plasticity (Chen et al., 2014).
  • synaptic calcium levels rapidly decrease when synaptic transmission is blocked, and RA is activated by the synthesis of ALDH1A dehydrogenase (Chen et al., 2014; Aoto et al., 2008).
  • RA binds to the protein translation process originally blocked by RAR ⁇ by binding to the RAR ⁇ protein localized in the synapse, where the regulated protein includes the AMPA receptor, thereby upregulating synaptic transmission (Aoto et al., 2008).
  • the anatomical structure of the PFC region and its associated structural abnormalities with other brain regions have been found to be common in the brains of autistic patients, and this suggests that dysfunction of PFC is closely related to the etiology of autism (Stoner et Al., 2014; Chow et al., 2012).
  • adeno-associated virus (AAV) packaged with EGFP, UBE3A and T508E, respectively was injected into the PFC region of the mouse brain by site-directed injection (Fig. 7a-b).
  • Immunofluorescence results after frozen sections of the brain showed that UBE3A and T508E have similar protein expression levels in the PFC region (Fig. 7c).
  • mice After four weeks of brain injection, the mice were tested for behavioral experiments. As shown in Figure 7d, in mice self-carding experiments, mice overexpressing UBE3A spent twice as much time on self-combing behavior compared to mice expressing EGFP; whereas mice expressing T508E flower The time is only 30% more. This suggests that mice overexpressing UBE3A exhibit repetitive stereotypic behavior.
  • three-chamber social behavioral experiments were used to record and compare the time of interaction between mice and social animals or non-social objects. In mice expressing EGFP and T508E, the time spent interacting with social mice (50 seconds) was approximately double that of the time of interaction with the object (23 seconds); however, in mice expressing UBE3A, Social mice interacted with objects for almost 30 seconds ( Figure 7e).
  • mice overexpressing UBE3A have severe social interaction disorders.
  • the mice were also recorded and compared in the time of interaction with familiar social mice and unfamiliar social mice.
  • mice expressing EGFP and T508E they showed greater interest in unfamiliar social mice than familiar social mice, and spent more time interacting with them (about 60-100 increased). %).
  • mice overexpressing UBE3A took as long as the time spent interacting with familiar and unfamiliar social mice (Fig. 7f). This suggests that overexpression of the UBE3A protein inhibits its perception of social novelty in mice.
  • mice expressing these three proteins showed the same level of tropism (Fig. 7g-h).
  • the results of the rotating rod experiment showed that overexpression of UBE3A did not affect the activity of the mouse compared with the control group (Fig. 7i).
  • Example 8 Compensation for RA attenuates the autism phenotype of mice caused by overactivation of UBE3A
  • mice injected with UAV3A-bearing AAV virus into PFC are orally administered with oral solvents (olive oil) or ATRA (3 mg/kg).
  • oral solvents olive oil
  • ATRA 3 mg/kg
  • the drug was administered for five weeks in a week for a total of four weeks, and then the mice were subjected to behavioral experiments (Fig. 8a).
  • the time spent on self-combing behavior was significantly reduced in mice given oral ATRA compared to the oral solvent group (Fig. 8b).
  • Example 9 Compounds Lead to Steady State Deregulation of RA Inducing the Production of Autistic Phenotypes in Mice
  • mice subjected to DSF administration for six weeks were subjected to a double-blind behavioral test.
  • DSF-administered mice were significantly doubled (30 seconds, 0.1 mg/g dose) and twice (42 seconds, 0.3) compared to the solvent group (15 seconds). The time of the mg/g dose) was self-combed.
  • the mice in the solvent group had significantly more time to interact with the social mice than the non-social subjects; however, there was no such obvious trend in the 0.1 mg/g DSF-administered mice.
  • Figure 9d-e the mice subjected to DSF administration for six weeks were subjected to a double-blind behavioral test.
  • UBE3A binds and ubiquitinates to modify ALDH1A2, thereby inhibiting the dehydrogenase activity of ALDH1A2.
  • ALDH1A1 and ALDH1A3 have also been shown to be substrates for UBE3A.
  • Autism-associated UBE3A overactivation can down-regulate RA levels in cells, thereby affecting RA signaling pathways, particularly its role in regulating synaptic scaling in neuronal cells.
  • DSF a compound inhibitor of ALDH1A
  • the present invention can also be used to screen for the risk of autism in the offspring caused by the side effects of taking drugs and daily chemicals for pregnant women (as well as taking drugs and daily chemicals for infants and young children). Side effects), it is expected to develop an effective clinical evaluation diagnostic tool.
  • a cell model for real-time monitoring of retinoic acid levels was also established.
  • High-throughput screening technology was used to screen FDA-approved commercial drugs, and several drugs capable of interfering with retinoic acid levels were identified and advanced. Verification, the verification results are exactly as expected. Animal experiments are currently underway to verify whether these drugs will experimentally induce autism.
  • the present invention discloses for the first time that UBE3A can bind to and modify ALDH1A family proteins by non-proteasome degradation, thereby inhibiting the synthesis of RA and down-regulating the homeostasis of intracellular RA.
  • blockade of neuronal activity causes rapid generation of RA, which regulates the process of synaptic scaling of neurons.
  • excessive activation of UBE3A interferes with RA by interfering with the formation of RA. The adjustment of the steady state of the touch. It has also been found in mouse behavioral studies that overexpression of UBE3A or disulfiram (DSF) administration of ALDH1A inhibitors can reshape the autism phenotype.
  • DSF disulfiram
  • UBE3A has a wide range of physiological effects, it is not appropriate to directly inhibit UBE3A, which may cause other side effects.
  • targets, drugs, and therapeutic means for preventing and/or treating autism without intervention or intervention of UBE3A are provided.

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Abstract

本发明公开了ALDH1A及其激动剂、催化产物和抑制剂的用途。具体地本发明公开了一种ALDH1A或其激动剂、或其催化产物的用途，用于制备药物组合物或制剂，所述药物组合物或制剂用于治疗和/或预防自闭症。

Description

ALDH1A及其激动剂、催化产物和抑制剂的用途 技术领域
本发明属于生物技术领域，具体地，本发明涉及ALDH1A及其激动剂、催化产物和抑制剂的用途。
背景技术
自闭症谱系障碍，简称“自闭症”，又名“孤独症”，其核心症状表现为社会性交流和沟通的障碍，重复刻板行为，是一种严重的神经发育障碍性疾病。其中在自闭症病例中，染色体15q11-q13拷贝数扩增(CNV)占比1-3％；该CNV能够引起UBE3A E3泛素连接酶的过度激活，但其具体的致病机理并不清楚。
自闭症谱系障碍是一种致病原因多样化的广泛性神经发育障碍型疾病，其核心症状主要体现在社会交往、沟通能力的缺陷，局限重复性兴趣、行为或活动等方面。迄今为止，引起自闭症的遗传因素包括点突变和染色体拷贝数变异，它们的突变涵括多种受累基因，涉及到不同的信号通路。尽管引起自闭症的遗传因素非常复杂和多样化，但自闭症患者表现出相似的核心表型症状，同时都有突触稳态的紊乱出现，这提示在自闭症疾病中可能存在着共同的致病机理。因此，能够阐明在自闭症中遗传因素与突触稳态紊乱间的分子机理显得尤为重要和关键。
在自闭症病例中，母本染色体15q11-q13拷贝数的扩增大约占到1-3％。其中在对多人群病例的研究中发现，E3泛素连接酶UBE3A在15q11-q13CNV症状中扮演主要角色。并且在多种转基因小鼠模型中，UBE3A的过表达同样能够引起小鼠出现自闭症表型。这些证明了UBE3A的过度激活是自闭症亚型中阐述最为清楚的致病因素。在最近的报道中，又发现了与自闭症高度关联的UBE3A点突变，该点突变阻断了蛋白激酶A(PKA)对UBE3A的磷酸化，从而导致了UBE3A的过度激活，以及突触形成的增加(Yi et al.,2015)。现在已经报道了E3泛素连接酶UBE3A的多种底物，但仍不能用来阐述自闭症的发生机制，因此可能存在其它未知的在自闭症患者大脑中功能失调的底物蛋白。
因此现在迫切需要通过系统性的对UBE3A底物进行筛选，开发ALDH1A及其激动剂、催化产物和抑制剂的相关用途。
发明内容
本发明的目的是提供ALDH1A及其激动剂、催化产物和抑制剂的用途。
在本发明的第一方面，提供了一种ALDH1A、或其激动剂、或其催化产物、和/或其催化底物的用途，用于制备药物组合物或制剂，所述药物组合物或制剂用于治疗和/或预防自闭症。
在另一优选例中，所述的ALDH1A激动剂不是UBE3A的抑制剂。
在另一优选例中，所述“不是UBE3A的抑制剂”指对于ALDH1A激动剂对于UBE3A的表达或活性没有影响或基本上没有影响(例如，存在所述ALDH1A激动剂的实验组的UBE3A的表达E1或活性A1，与空白对照组(不存在所述所述ALDH1A激动剂时)的UBE3A的表达E0或活性A0之比(即E1/E0或A1/A0为0.7-1.3，较佳地0.8-1.2，更佳地0.9-1.1))。
在另一优选例中，所述自闭症为人群中自闭症。
在另一优选例中，所述人群为具有染色体15q11-q13拷贝数扩增的人群
在另一优选例中，所述人群为具有UBE3A过度表达或过度活化的人群。
在另一优选例中，所述人群为具有UBE3A的T508A突变的人群。
在另一优选例中，所述ALDH1A选自下组：ALDH1A1、ALDH1A2、ALDH1A3、或其组合。
在另一优选例中，所述的激动剂包括表达促进剂、蛋白降解抑制剂。
在另一优选例中，所述ALDH1A激动剂选自下组：N-(1,3-苯并二氧戊环-5-亚甲基)-2,6-二氯苯甲酰胺(N-(1,3-Benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide)、N-(1,3-苯并二氧戊环-5-亚甲基)-2,6-二氯苯甲酰胺(N-(1,3-Benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide)、6-甲基-2-偶氮苯基-3-羟基吡啶(6-methyl-2-(phenylazo)-3-pyridinol)、2-(苯并[d][1.3]间二氧环戊烯-5-基)-N-(5,6-二氢-4H-环戊二烯[c]异恶唑-3-基)乙酰胺(2-(benzo[d][1,3]dioxol-5-yl)-N-(5,6-dihydro-4H-cyclopenta[c]isoxazol-3-yl)acetamide)及其各衍生物。
在本发明的第二方面，提供了一种维甲酸或维甲酸类似物，或其溶剂化物、或其药学上可接受的盐的用途，用于制备药物组合物或制剂，所述药物组合物 或制剂用于治疗和/或预防自闭症。
在另一优选例中，所述维甲酸类似物选自下组：视黄醇、视黄醛、全反式维甲酸、13-顺式维甲酸、9-顺式维甲酸、阿维A酯、阿维A、阿达帕林、贝沙罗汀、他扎罗汀、或其组合。
在本发明的第三方面，提供了一种维甲酸合成原料化合物、或维甲酸降解酶抑制剂的用途，用于制备药物组合物或制剂，所述药物组合物或制剂用于治疗和/或预防自闭症。
在另一优选例中，所述维甲酸降解酶抑制剂选自下组：利阿唑(liarozole)，酮康唑(ketoconazole)，他拉罗唑(talarozole)，或其组合。
在本发明的第四方面，提供了一种ALDH1A抑制剂的用途，用于制备一制剂，所述制剂用于建立自闭症动物模型。
在另一优选例中，所述的ALDH1A抑制剂选自下组：小分子化合物、抗体、核酸、或其组合。
在另一优选例中，所述的ALDH1A抑制剂为UBE3A激动剂。
在另一优选例中，所述核酸选自下组：miRNA、siRNA、sgRNA/Cas9复合物、或其组合。
在另一优选例中，所述ALDH1A抑制剂选自下组：双硫仑(disulfiram)、4-(N,N-二乙基)氨基苯甲醛(DEAB)，WIN-18446，A37(CM037)，NCT-501盐酸化物，CVT-10216，或其组合。
在本发明的第五方面，提供了一种对自闭症的辅助诊断和/或预后的方法，所述方法包括步骤：
(1)提供一待测样本，所述样本选自下组：血液、体液；
(2)检测标志物浓度、含量、和/或活性；
(3)与标准值或标准曲线比较，从而进行辅助诊断和/或预后；
其中，所述标志物选自下组：维甲酸、ALDH1A、或其组合。
在另一优选例中，在步骤(3)中，当所述样本中的维甲酸浓度低于正常供体样本的维甲酸浓度(8-20nM)时，则表明该患者患自闭症风险高于正常人群或预后不良；和/或
当所述样本中ALDH1A的活性低于正常供体样本时，则表明该患者患自闭症风险高于正常人群或预后不良。
在本发明的第六方面，提供了一种评估待测物质的副作用风险的方法，所述的副作用风险为诱发或引发自闭症的风险，并且所述方法包括步骤：
(a)提供一待测物质；
(b)在施用所述的待测物质的测试组中，测定所述待测物质中对于待测指标的影响；并在未施用所述待测物质的对照组中，测定同一待测指标的数据；其中，所述的待测指标选自下组：维甲酸的水平或浓度、ALDH1A表达量或活性、或其组合；
(c)将测试组的所述待测指标的测定结果T与对照组中所述待测指标的测定结果C进行比较；
如果测定结果T显著低于测定结果C，则提示待测物质具有导致孕妇和/或婴幼儿副作用的风险。
在另一优选例中，所述待测物质选自下组：药物、日用化学品、或其组合。
在另一优选例中，所述的副作用包括导致当代发生自闭症、后代发生自闭症、或其组合。
在另一优选例中，所述的“显著低于”指T/C比值小于0.7，出现统计学显著性差异P<0.05。
在另一优选例中，步骤(b)中，在模型动物、或培养体系中进行测定。
在另一优选例中，所述方法还包括对于步骤(c)中提示有副作用的待测物质，进一步通过动物实验验证。
在本发明的第七方面，提供了一种药物组合或含所述药物组合的药盒，所述的药物组合包括：
(a)第一药物组合物，所述第一药物组合物含有药学上可接受的载体和选自下组的第一活性成分：克霉唑、孟鲁司特、孟鲁司特钠；和
(b)第二药物组合物，所述第二药物组合物含有药学上可接受的载体和选自下组的第二活性成分：ALDH1A、ALDH1A激动剂、ALDH1A催化产物、ALDH1A催化底物、或其组合。
在另一优选例中，所述的第二药物组合物用于降低第一药物组合物的副作 用风险，所述副作用风险为诱发或引发自闭症的风险。
在本发明的第八方面，提供了一种药物组合物，所述的药物组合物含有：
(a)第一活性成分，其选自下组：克霉唑、孟鲁司特、孟鲁司特钠；和
(b)第二活性成分，其选自下组：ALDH1A、ALDH1A激动剂、ALDH1A催化产物、ALDH1A催化底物、或其组合。
在另一优选例中，所述的第二活性成分用于降低第一活性成分的副作用风险，所述副作用风险为诱发或引发自闭症的风险。
在另一优选例中，所述的第二药物活性成分选自下组：维甲酸及其类似物、维甲酸降解酶抑制剂、或其组合。
在本发明的第九方面，提供了一种筛选治疗或预防自闭症的候选药物(或潜在治疗剂)的方法，包括步骤：
(a)提供一待测试的候选物质；
(b)在施用所述的待测试的候选物质的测试组中，测定所述待测试的候选物质对于待测指标的影响；并在未施用所述待测试的候选物质的对照组中，测定同一待测指标的数据；其中，所述的待测指标选自下组：维甲酸的水平或浓度、ALDH1A表达量或活性、或其组合；
(c)将测试组的所述待测指标的测定结果T与对照组中所述待测指标的测定结果C进行比较；
如果测定结果T显著高于测定结果C，则提示所述待测试的候选物质可作为治疗或预防自闭症的候选药物。
在另一优选例中，所述的“显著高于”指T/C比值大于1.2，出现统计学显著性差异P<0.05。
在另一优选例中，步骤(b)中，在模型动物、或培养体系中进行测定。
在另一优选例中，所述方法还包括对于步骤(c)中提示可作为治疗或预防自闭症的候选药物的候选物质，进一步通过动物实验验证。
在本发明的第十方面，提供了一种能够阻断UBE3A和ALDH1A家族蛋白结合的多肽或抗体或化合物的用途，用于制备药物组合物或制剂，所述药物组合物或制剂用于治疗和/或预防自闭症。
在另一优选例中，所述多肽选自下组：肽-1，肽-2，肽-3。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1自闭症病人样本中发现染色体15q11.2-14的拷贝数扩增。(a)在3个ASD病人中包含UBE3A区域的拷贝数变异。(b)3个ASD病人中重复的DNA区域(蓝色)用UCSC映射到hg18人类基因组中，15q11.2-q14红色框标出。已知的基因在底部标出，UBE3A基因用红色标记。(c)人类UBE3A蛋白结构域示意图。(d)UBE3A基因剂量过多ASD病人和正常对照的永生化淋巴细胞中静态UBE3A蛋白水平。
图2UBE3A结合ALDH1A家族成员。(a)酵母双杂分析中人UBE3A蛋白和ALDH1A2的相互作用，阳性克隆既可以在SD-4培养板中存活，也可以在X-gal上显蓝色。(b-c)293-FT细胞中Flag-UBE3A和ALDH1A2-HA或者内源蛋白存在相互作用。细胞表达标签蛋白用特定抗体进行Co-IP分析(b)；用IgG或ALDH1A2抗体进行内原蛋白Co-IP，用UBE3A或ALDH1A2抗体进行免疫印迹(d)。(d)在SH-SY5Y细胞中对Flag-UBE3A和ALDH1A2-RFP进行免疫荧光共定位实验，细胞核用DAPI染色。刻度条代表30μm。(e)体外UBE3A和ALDH1A2蛋白间相互作用。(f)鉴定ALDH1A2和UBE3A的结合区段。(g-h)体外UBE3A和ALDH1A家族其他蛋白相互作用。(i-j)体内人UBE3A和ALDH1A其他家族成员存在相互作用。(k)在GST拉下实验中，加入三个针对UBE3A和ALDH1A2结合区域的不同肽段(肽-1，肽-2，肽-3)，可以不同程度的竞争性阻断GST-UBE3A和ALDH1A2蛋白间的相互结合。
图3在重组泛素化体系中UBE3A把多聚泛素链连接到ALDH1A2上。(a)在重组泛素化体系中UBE3A把多聚泛素链连接到ALDH1A2上。(b)体外UBE3A促进ALDH1A2泛素化。泛素化蛋白用HA抗体富集，并用His抗体免疫印迹分析。(c)检测不同细胞系中内源ALDH1A1,2,3蛋白静态含量。(d)H1299细胞中CRISPR靶向UBE3A基因位序列。上部：UBE3A中敲除后的基因序列，下部：UBE3A -/-细胞测序峰图。红色箭头指示移码起始点(缺失4bp)。(e)与野生型H1299细胞相比，IB分析确认H1299UBE3A -/-细胞中UEB3A基因敲除。(f)H1299细胞不 同UBE3A(+/+,+/-,和和-/-)基因型，ALDH1A2泛素化随着UBE3A缺失而减少。(g-h)MEF细胞或HEK-293FT细胞中，内源Aldh1a2泛素化水平与Ube3a蛋白水平正相关。(i)在ASD病人来源淋巴细胞中，内源ALDH1A2蛋白的泛素化水平随UBE3A蛋白水平增加而增加。(j)UBE3A磷酸化状态影响其对ALDH1A2的E3连接酶活性。(k)重组泛素化系统中UBE3A泛素化ALDH1A家族其他成员。(l-m)体内UBE3A蛋白泛素化ALDH1A家族其他成员。
图4人UBE3A以非蛋白降解Ub连接方式泛素化修饰ALDH1A2。(a-b)HEK-293FT细胞中外源表达增加剂量的Flag-UBE3A蛋白或在MEF细胞中野生型Ube3a，缺失或敲除(Ube3a +/+,+/-,-/-)中内源ALDH1A2的蛋白水平。(c-e)UBE3A以K29和K63多聚泛素链连接方式调节ALDH1A2的泛素化。H1299细胞中共转Myc-UBE3A,ALDH1A2-HA(或-Flag)，和在特定位置只保留一个Lys的His-Ub突变体(c)，或者只在K29位(d)或63位(e)把K突变为R的突变体。HA或Flag标签的ALDH1A2蛋白从细胞中富集，并用HA或者Flag抗体检测。(f)H1299细胞中共转His6-Ub,Myc-UBE3A,和含有指定位点K-R的HA-ALDH1A2。ALDH1A2-HA用HA抗体富集后检测泛素化。3R:ALDH1A2-HA在K269，K370，K515位点K突变成R。(g)人ALDH1A2蛋白单体的晶体结构，其中三个主要泛素化位点(红色标出)在活性中心(Cys320，黄色高亮标出)附近，辅酶NAD +紫色标出。晶体结构从PDB数据库中提取(DOI:10.2210/pdb4x2q/pdb)。(h)人ALDH1A家族蛋白泛素化位点附近区域肽段序列比对，泛素化位点橙色标出。
图5UBE3A泛素化抑制了ALDH1A2视黄醛脱氢酶活性。(a)ALDH1A2-Flag泛素化水平检测。HEK-293FT细胞中共转指定质粒，ALDH1A2-Flag蛋白用Flag抗体沉淀后，用Flag短肽洗脱，并在4℃过夜用USP2cc处理。(b-c)UBE3A在ALDH1A2上结合的多聚泛素链降低了其对视黄醛的脱氢酶活性。ALDH1A2-Flag蛋白从过表达Myc-UBE3A,His6-Ub和ALDH1A2-Flag的293FT细胞中富集(b)，经过USP2cc酶处理或不处理移除多聚泛素链(c)。富集后的ALDH1A2蛋白将全反式视黄醛作为底物做脱氢酶活性分析。泛素化ALDH1A2蛋白酶活与对照组相比进行校正(b:n＝4；c:n＝3)。(d)ALDH1A2-Flag，是否泛素化对丙醛脱氢酶活性分析。***,P<0.001,双尾t-test,n＝4。(e)ALDH1A2-Flag蛋白，是否USP2cc处理，对丙醛脱氢酶活性分析。**,P<0.01,双尾t-test,n＝3.(f)野生型ALDH1A2蛋白或3KR蛋白对丙醛脱氢酶活性分析。酶活表示为校正后NADH产量(左)和相对酶活性(右)。***,P<0.0001,双 尾t-test,n＝3.(g)不同物种中三个主要泛素化位点(K269,K370,K415)附近氨基酸序列比对。(h-i)ASD病人来源永生化淋巴细胞总ALDH1A活性低于正常对照，以AldeFluor分析(h)或者RARE-luciferase报告分析中(i)。数据呈现为平均值和方差。*,P<0.05,**,P<0.01,***,P<0.001；(b,c)双尾t-test,one-way ANOVA Dunnett”s post-hoc test(h,i)。
图6过高UBE3A活性破坏了突触传递的可塑性。(a)神经活动用1μM和100μM TTX阻断后，原代PFC神经细胞中RARE-hrGFP亮度显著增强。代表图在上部(标尺，50μm)，下部，荧光强度以DMSO组矫正。DMSO,n＝8；APV/TTX,n＝11。(b)在原代神经细胞1μM和100μMTTX处理后，与空载或者T508E突变体相比，导入UBE3A显著降低了RARE-hrGFP信号。相对荧光强度代表图在右侧显示。Vector,n＝11；UBE3A,n＝14；T508E,n＝12.标尺:40μm。(c)1μM和100μMTTX的原代神经细胞，电生理试验中mEPSC幅度和频率都有显著性增加。上部，代表mEPSC轨迹；下部，幅度和频率定量(DMSO,n＝15；APV/TTX,n＝12)。(d)相比于空载和T508E组，1μM APV和100μM TTX刺激后，神经细胞中导入UBE3A降低了mEPSC频率。(vector,n＝15；UBE3A,n＝17；T508E,n＝18)。(e)DMSO或APV/TTX处理24h后，PFC神经细胞膜表面GluR1和PSD-95染色(DMSO,n＝31；APV/TTX,n＝33)。定量与PSD-95共定位的GluR1的强度。(f)神经活动阻断不改变特定基因在原代PFC细胞中的转录水平。(g)神经活动阻断显著上调原代PFC细胞膜表面GluR1,而不是GluR2的蛋白水平。(h)APV/TTX处理24h后,导入空载、T508E或者UBE3A后，对神经细胞膜表面GluR1和PSD-95进行染色。在turboRFP阳性的树突中对和PSD-95共定位的GluR1定量分析(vector,n＝21；UBE3A,n＝28；T508E,n＝24)。数据呈现为平均值和方差。*,P<0.05,**,P<0.01,***,P<0.001；(a,c,e)双尾t-test,(b,d,h)one-way ANOVA Bonferroni post-hoc test。
图7过量的UBE3A蛋白下调RA的内稳态，并在小鼠中诱导出现ASD类似表型。(a)PFC区定点注射的脑部示意图(左)，hSynI-启动子驱动表达Flag-EGFP,Flag-UBE3A or Flag-T508E的结构图(右)。(b)AAV-SynI-Flag-EGFP注射入PFC区的小鼠脑部冠状切片代表图。标尺，100μm。(c)PFC区中显示Flag标签的EGFP，UBE3A和T508E蛋白。细胞核用DAPI复染。标尺，50μm(d)定点注射AAV病毒携带的EGFP，UEB3A,UBE3A-T508E组小鼠自我梳理的时间。EGFP(n＝11),UBE3A(n＝14),UBE3A-T508E(n＝9)。(e)比较三组小鼠和陌 生小鼠及物品交流时间。EGFP,n＝12；UBE3A,n＝13；T508E,n＝11.(f)比较三组小鼠和陌生小鼠I及陌生小鼠II交流时间。EGFP(n＝10),UBE3A(n＝14)T508E(n＝11)。*,P<0.05,**,P<0.01,***,P<0.001。(g)注射EGFP，UBE3A或T508E AAV病毒小鼠在旷场中心区停留时间。(h)注射EGFP，UBE3A或T508E AAV病毒小鼠在旷场中心区走的距离与总距离的比值。(i)比较注射不同AAV病毒小鼠在转棒试验中掉落前持续时间。
图8口服ATRA能够逆转过表达UBE3A的小鼠中出现的自闭症表型。(a)定点注射AAV过程及小鼠模型中ATRA回补实验示意图。(b)测试AAV注射小鼠注射溶剂或ATRA后用于自我梳理时间。(olive oil,n＝9)，ATRA(n＝10)。(c-d)比较AAV小鼠注射溶剂或者ATRA后和陌生小鼠I及物体(c)交流时间或陌生小鼠I及陌生小鼠II(d)交流时间。(e)比较口服溶剂或ATRA小鼠在旷场中心停留时间。(f)比较注射UBE3A AAV病毒小鼠口服溶剂或ATRA旷场中心区走的距离与总距离的比值。(g)比较注射UBE3A AAV病毒口服溶剂或ATRA在转棒试验中掉落前持续时间。(h)比较野生型小鼠口服溶剂或ATRA用于自我梳理时间。(i-j)比较野生型小鼠口服溶剂或ATRA探索陌生小鼠I和物品(i)或陌生小鼠I和陌生小鼠II(j)的时间。(k-l)比较野生型小鼠口服溶剂或ATRA在旷场中中心停留时间(k)或在中心区域行走距离(l)百分数。(m)比较野生型小鼠口服溶剂或ATRA转棒试验中落下前持续时间。数据呈现为平均值和方差。
图9给小鼠口服ALDH1A抑制剂DSF诱导ASD类似表型的出现。(a)小鼠口服溶剂或不同剂量DSF(0.1or 0.3mg/g)6周后，HPLC-MS/MS方法测定相对ATRA含量，用对照组矫正。(b)不同组小鼠体重没有显著性差异。溶剂组(n＝10),DSF(0.1mg/g,n＝9),DSF(0.3mg/g,n＝12)。(c)比较三组小鼠用于自我梳理的时间。(d)比较三组小鼠在陌生小鼠、中间、物品腔室中的停留时间。(e)比较三组小鼠和陌生小鼠及物品交流时间。(f)比较小鼠停留在陌生小鼠I、中间、陌生小鼠II腔室中的时间。(g)比较三组小鼠和陌生小鼠I及陌生小鼠II交流时间。(h-i)比较小鼠灌胃溶剂或0.1/0.3mg/g在旷场中心停留时间及中心与活动距离比例。(j)比较不同组在高架十字中进入开放臂的次数比例。(k)比较不同组小鼠在转棒试验中落下前停留时间。数据呈现为平均值和方差。*,P<0.05,**,P<0.01,***,P<0.001,NS,无显著性差异；(e,g)双尾t-test,(a-d,f)one-way ANOVA Bonferroni post-hoc test.c-g,vehicle group(n＝15),DSF(0.1mg/g,n＝17),and DSF(0.3mg/g, n＝19)。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现了一个与自闭症相关的新通路。本发明人首次，该通路核心蛋白，即合成酶ALDH1A与自闭症的发生存在显著的相关性。通过补充所述合成酶ALDH1A、或其激动剂、或其合成产物、或其催化底物，均有助于预防和改善自闭症以及与自闭症相关的症状。在此基础上完成了本发明。
具体地，实验表明，维甲酸(RA)的限速合成酶(ALDH1A2)能够被UBE3A以泛素化修饰依赖的方式进行负调控。自闭症相关的UBE3A蛋白高剂量或过度激活都能够破坏RA介导的神经元突触稳态。在小鼠动物模型中，UBE3A蛋白在小鼠大脑前额叶皮质区(PFC)的过表达和ALDH1A抑制剂的给药都能够导致小鼠自闭症类似症状的出现。通过RA的补充，能够显著的改善UBE3A过表达所引起的小鼠自闭症的症状。这些结果提示，RA通路的干扰在UBE3A过度激活与自闭症表型间关联的潜在机理。
维甲酸及其衍生物
维甲酸及其衍生物大致分为三代：第一代包括：retinol,retinal,tretinoin(all-trans-retinoic acid),isotretinoin(13-cis-retinoic acid),alitretinoin(9-cis-retinoic acid)；第二代包括：etretinate,acitretin；第三代包括：adapalene,bexarotene,tazarotene。(视黄醇，视黄醛，全反式维甲酸，13-顺式维甲酸，9-顺式维甲酸，阿维A酯，阿维A，阿达帕林，贝沙罗汀，他扎罗汀)
衍生物还包括各个化合物的酯类衍生物，其在体内均会被水解为酸或盐的形式。其中部分化合物结构式如下：
Figure PCTCN2018079758-appb-000001
ALDH1A蛋白及其激动剂
如本文所用，术语“ALDH1A”指视黄醛脱氢酶家族1成员A(Aldehyde dehydrogenase family 1 member A)。
在本发明中，ALDH1A包括来自不同哺乳动物的同类蛋白，例如来自人和非人哺乳动物(如啮齿动物(如小鼠、大鼠)、牛、羊、狗等)的ALDH1A。在本发明中，该术语不仅包括野生型ALDH1A，还包括突变型ALDH1A(与野生型ALDH1A相比，该突变型ALDH1A具有类似的或相近的活性)。
在本发明中，代表性的ALDH1A包括(但并不限于)：ALDH1A1、ALDH1A2、ALDH1A3、或其组合。
如本文所用，术语“ALDH1A蛋白的激动剂”是提高ALDH1A表达和/或活性的物质。例如，能够与ALDH1A蛋白间具有高亲和力，能与ALDH1A结合后产生提高ALDH1A效应的物质。
在本发明的一个优选的实施例中，ALDH1A蛋白的激动剂为选自下组的化合物及其对应的衍生物：N-(1,3-苯并二氧戊环-5-亚甲基)-2,6-二氯苯甲酰胺 (N-(1,3-Benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide，简称ALDA-1)、N-(1,3-苯并二氧戊环-5-亚甲基)-2,6-二氯苯甲酰胺、2-(苯并[d][1.3]间二氧环戊烯-5-基)-N-(5,6-二氢-4H-环戊二烯[c]异恶唑-3-基)乙酰胺、6-甲基-2-偶氮苯基-3-羟基吡啶。
Figure PCTCN2018079758-appb-000002
ALDH1A抑制剂
ALDH1A抑制剂(或ALDH1A家族抑制剂)是指生物化学反应中能阻滞或降低ALDH1A及其家族蛋白化学反应速度的物质。此外，该术语还包括降低ALDH1A表达或活性的抑制剂，如反义RNA、miRNA、或抗体。
在本发明的一个优选的实施例中，ALDH1A及其家族抑制剂为选自下组的化合物及其对应的衍生物：
Figure PCTCN2018079758-appb-000003
Figure PCTCN2018079758-appb-000004
RA降解酶CYP26A1抑制剂
RA降解酶CYP26A1抑制剂是指生物化学反应中能阻滞或降低CYP26A1蛋白降解维甲酸速度的物质。
在本发明的一个优选的实施例中，RA降解酶CYP26A1抑制剂为选自下组的化合物及其对应的衍生物：
Figure PCTCN2018079758-appb-000005
候选药物或治疗剂
在本发明中，还提供了一种筛选治疗或预防自闭症的候选药物(或潜在治疗剂)的方法。
在本发明中，候选药物或治疗剂是指已知具有某种药理学活性或正在被检测的可能具有某种药理学活性的物质，包括但不限于核酸、蛋白、化学合成的小分子或大分子化合物、细胞等。候选药物或治疗剂的给药方式可以是口服、静脉注射、腹腔注射、皮下注射、或椎管给药。
本发明的主要优点在于：
(1)本发明提供了新颖的自闭症治疗靶点，故依据本发明可以筛选开发出自闭症治疗的药物。
(2)本发明还可用于筛查潜在的对孕妇服用药物及日用化学品副作用所导致的后代中出现自闭症的风险(以及婴幼儿服用药物及日用化学品等的副作用)，有望于开发出有效地临床评估诊断工具。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。本发明中所涉及的实验材料如无特殊说明均可从市售渠道获得。
材料和方法
1.受试者及样本
所用受试者的招募和评估都是根据中南大学生命科学院，医学遗传学国家重点实验室审查委员会的章程，并且严格坚守赫尔辛基宣言的原则。受试者招募和诊断的细节都遵照先前报道的进行(Xia et al.,2013；Wang et al.,2016)。所有血样样本的采集都收到的书面知情同意书。
2.拷贝数变异(CNV)序列分析
3个汉族病人拷贝数变异(男性，年龄：3-6岁)使用Human660W-Quad微阵列芯片进行(Illumina)。芯片分析利用GenomeStudio v2011.1(Illumina)进行，根据以前报道(Nava et al.,2013)。用人类参考基因组hg18作为参考进行拷贝数变异的绘制。
3.细胞培养和转染
根据标准化方法(Anderson et al.,1984)，用EBV病毒转化血细胞得到永生化的淋巴细胞，并在含有10％胎牛血清(Biochrom)的RPMI-1640(Gibco)培养基中培养。HEK-293FT(Life Technologies),HEK-293(ATCC),SH-SY5Y (ATCC),H1299(ATCC)和A549(ATCC)细胞都在含有10％胎牛血清和青霉素/链霉素(Life Technologies)的DMEM(Corning)培养基中培养。根据已有报道方法准备和建立小鼠成纤维细胞(Xu et al.,2005)。原代神经元细胞从怀孕18天的大鼠(Sprague Dawley)胎鼠大脑的前额叶皮质区分离，并在含有10％胎牛血清的DMEM/F12培养基中培养。第二天，将细胞培养基换为含有B-27补充物(Gibco)和GlutaMax(Gibco)的无血清神经基础培养基(Gibco)中培养。细胞在含有5％CO2饱和湿度培养箱中培养。所有细胞系均定期进行支原体检测。
HEK-293FT细胞用聚乙烯亚胺(Sigma)转染相关质粒，SH-SY5Y和H1299用Lipofectamine 2000(Life Technologies)转染，均依照生产厂商的指示进行操作。基于H1299细胞的UBE3A单等位基因和双等位基因敲除细胞系通过CRISPR/Cas9系统进行基因组编辑后筛选得到。UBE3A基因的sgRNA引物设计依照已有报道(Hsu et al.,2013)，序列如下列出(靶序列用下划线标出)：
(1)5”-CACCG AGCACAAAACTCATTCGTGC-3”(Seq ID No 1)
(2)5”-AAAC GCACGAATGAGTTTTGTGCTC-3”(Seq ID No 2)
原代神经元细胞用磷酸钙转染试剂(碧云天)根据生产厂商的流程转染相关质粒。
4.质粒的构建
本课题中的质粒包括限制性内切酶消化和连接反应(NEB)，均根据传统克隆方法进行。用于酵母双杂筛选的质粒利用Gateway LR克隆酶(ThermoFisher)按照生产厂商的流程反应得到。相关质粒的点突变利用定点突变方法引入。另外，含有多个元件的质粒利用Gibson组装方法进行构建。在本工作中，UBE3A融合表达标签都放在N端。
5.酵母双杂交
利用UBE3A作为诱饵蛋白，将pDEST32-UBE3A(ThermoFisher)和基于pDEST22骨架的人源cDNA文库(ThermoFisher)共转入酵母菌株Mav203(ThermoFisher)中。阳性克隆能够在同时缺乏尿嘧啶、组氨酸、亮氨酸和色氨酸四种成分的培养基(Clontech)中生存，也能够在X-Gal(Sigma)存在条件下显出蓝色。
6.GST拉下实验
GST，GST-UBE3A和截短的GST-UBE3A蛋白在BL21感受态(NEB)中诱导表达，利用谷胱甘肽琼脂糖(GE Healthcare)进行纯化。ALDH1A1-His6,ALDH1A2-His6和ALDH1A3-His6蛋白利用Ni-NTA琼脂糖(Qiagen)进行纯化。纯化后的ALDH1A2-His6和GST-UBE3A蛋白在pul l-down缓冲液(50mM Tris-Cl,pH 8.0,200mM NaCl,1mM EDTA,1％NP-40,1mM DTT,10mM MgCl2)中4℃孵育2小时。珠子用pull-down缓冲液洗4次，用免疫印迹方法分析。其他蛋白的pull-down分析遵守相同步骤。
7.细菌泛素化系统的重建
泛素化系统的全部成分利用传统克隆方法放在在双表达系统载体骨架pACYCDuet-1(Novagen)中。HA-UB,UBCH7和Uba1插入第一个多克隆位点，连接在T7启动子/乳糖操纵子和核糖结合位点后面，每个成员间都由Shine-Dalgarno(SD)序列分开从而形成多顺反子原件。UBE3A插入第二个多克隆酶切位点，生成pACYC-HA-UB-UBCH7-Uba1-UBE3A质粒。BL21感受态细胞用电转方法共转化pACYC和pET22b-ALDH1A2-His6质粒(Novagen)，并用氯霉素和氨苄青霉素(Sigma)进行筛选。大肠杆菌在OD600吸光值到达0.8时，用0.25mM异丙基-β-D-1-硫代半乳糖苷(IPTG，Sigma)在18℃条件下诱导培养16小时。细胞收集并重悬在RIPA缓冲液中(150mM NaCl,50mM Tris-Cl,pH7.4,1％NP-40,0.1％SDS)，然后细胞用Vibra-Cell处理器(SONICS)超声，通过离心去除沉淀。上清用Ni-NTA琼脂糖进行纯化。在Usp2cc处理组，在Ni-NTA珠子上结合的纯化蛋白和Usp2cc酶在4℃孵育过夜。ALDH1A2蛋白的泛素化水平用免疫印迹方法分析。
8.免疫沉淀和免疫印迹实验
表达内源或者外源蛋白的细胞在加有蛋白酶抑制剂混合物(Roche)的IP缓冲液(50mM Tris-Cl,pH 7.5,150mM NaCl,1mM EDTA,1％NP-40,10％glycerol)中裂解，并用Vibra-Cell处理器超声。离心后去除细胞碎片，上清加入特异性抗体和蛋白G琼脂糖珠子(Merck Millipore)4℃过夜孵育。使用的一抗如下列出：正常兔IgG抗体(sc-2027,Santa Cruz),Flag抗体(F1804,Sigma),ALDH1A2抗体(sc-367527,Santa Cruz),HA抗体(H6908,Sigma)。免疫沉淀 富集后加入2×SDS-PAGE上样缓冲液，95℃变性10分钟。初始蛋白，免疫沉淀富集蛋白和其他细胞裂解样品用SDS-PAGE胶进行分离，转至PVDF膜(Bio-Rad)。膜用特异的抗体进行免疫印迹：UBE3A抗体(sc-166689,Santa Cruz,1:500稀释)，Flag标签抗体(F1804,Sigma,1:8000稀释),HA标签抗体(H6908,Sigma,1:4000稀释)，ALDH1A2抗体(sc-367527,Santa Cruz,1:500抗体)，ALDH1A1抗体(15910-1-AP,Proteintech,1:500抗体)，ALDH1A3抗体(25167-1-AP,Proteintech,1:500稀释)，His标签抗体(H1029,Sigma,1:4000稀释)，GST标签抗体(66001-1-lg,Proteintech,1:5000稀释)，Myc标签抗体(sc-40,Santa Cruz,1:1000稀释)，actin抗体(A2228,Sigma,1:8000稀释)，GAPDH抗体(sc-32233,Santa Cruz,1:4000稀释)。
9.免疫荧光
SH-SY5Y细胞(ATCC)经转染特定质粒后，再培养24小时，然后用4％多聚甲醛(Sigma)固定。将细胞破膜后，加入一抗(Flag标签抗体，F1804，Sigma)4℃过夜孵育，然后用Alexa Fluor 488结合的荧光二抗(A11029，ThermoFisher)室温孵育1小时。细胞核用4,6-双脒基-2-苯基吲哚(DAPI，ThermoFisher)复染。大鼠原代神经元细胞在体外第10天时用磷酸钙转染方法转入特定质粒，在DIV12时用DMSO(Sigma)或者1μM河豚毒素(TTX，上海Aladdin公司)和100μM D-(-)-2-氨基-5-磷酸戊酸(D-APV，Tocris)处理24小时。固定细胞后，遵照前面的流程分别用一抗GluR1抗体(sc-55509,Santa Cruz)，PSD95抗体(ab18258,Abcam)和Alexa Fluor 488(A11029,ThermoFisher)，Alexa Fluor 647(A21245,ThermoFisher)结合的二抗进行染色。用Olympus FV1200共聚焦显微镜采集图片，采集分辨率为1024×1024像素。在同一实验中共聚焦设定参数保持一致。荧光强度分析定量使用Image-Pro plus(Media Cybernetics)软件。
10.免疫组化
将从定点注射过病毒的小鼠取出的大脑用含有4％多聚甲醛的PBS 4℃固定过夜，再放入含有30％蔗糖(Sigma)的PBS中沉浸。用Leica CM3050S冰切机(Leica Biosystems)切取40μM厚度的大脑冠状切片，切片用PBST(0.3％Triton X-100)室温处理15分钟。然后脑部切片用3％正常山羊血清(博士德) 封闭，用Flag标签抗体(14793,Cell Signaling,1:800稀释)4℃过夜孵育。切片用Cy3结合二抗(111-165-045,Jackson ImmunoResearch)室温孵育1小时后，用DAPI复染细胞核，并用封片剂Mowiol(Sigma)封片。荧光图片用Olympus FV1200共聚焦显微镜进行采集。
11.ALDH1A酶活检测
在转染或者未转染UBE3A的HEK-293FT细胞中，用Flag偶联的琼脂糖柱子(Sigma)富集出外源表达的ALDH1A2-Flag蛋白，然后用配制在ALDH1A酶活检测缓冲液(0.1M焦磷酸钠,pH 8.0,1.0mM EDTA,2.0mM DTT)中的Flag短肽洗脱。对于ALDH1A2酶活去除泛素化与否的研究中，分别用USP2cc酶或者牛血清白蛋白(BSA)与洗脱蛋白4℃过夜孵育。酶活反应体系包括溶解于酶活检测缓冲液的2.5mM NAD +,20mM DTT和100μM丙醛(Sigma)，脱氢酶活性在BioTek Synergy Neo分光光度计上用340nm波长在室温条件下进行检测，每3分钟测一次。当所有样品的吸光值都达到平台期时终止反应。利用NADH作为标准品，ALDH1A2脱氢酶活性计算方法为：(总反应时间内NADH的产量(nmol)×样品稀释倍数)/(反应时间×反应体积)。测量底物全反式视黄醛(Sigma)时，需要采用乙醛脱氢酶活性分析试剂盒(Cayman)。其通过基于荧光定量的方法进行检测分析。简要地，在100μM全反式视黄醛存在时，酶活通过检测荧光基团在吸收波长530-540nm和发射波长585-595nm的参数来最小化视黄醛对吸收值的干扰。
12.Aldefluor检测
Aldefluor检测利用Aldefluor试剂盒(STEMCELL Technologies)按照厂商提供步骤进行。简要地，将1×10 6个永生化淋巴细胞和荧光底物BODIPY-氨基乙醛-二乙基醋酸盐(BAAA-DA)(1.5μM)37℃孵育30分钟。每一份细胞分为两半：一半用于荧光分析，另外一半细胞先用试剂盒中提供的ALDH抑制剂二乙胺-苯甲醛(DEAB)预处理，以作为在流式细胞分析中作为阴性对照。
13.RARE-荧光素酶活性检测
pGL4.22-RARE-TK-luciferase质粒的构建方法包括，将3X RARE(RA-响应元件)克隆在pGL4.22载体(Promega)中。RA感受器细胞系构建方法包括，在 H1299细胞中转染pGL4.22-RARE-TK-luciferase质粒，接着用嘌呤霉素(1μg/mL，Sigma)筛选稳转细胞株。RA感受器细胞和作为RA供体的永生化淋巴细胞以细胞数1:1在VP-SFM(virus production serum-free medium,Gibco)培养基中共培养。加入1μM全反式视黄醛(Sigma)处理8小时后，检测培养细胞的荧光素酶活性。
14.电生理实验
原代神经元细胞和体外培养12-14转染的神经元细胞用1μM TTX(Aladdin)和100μM D-APV(Tocris)处理24小时，室温条件下记录膜片钳数据。其中，膜片钳内液包括(单位mM)：20KCl,5MgCl2,20HEPES,110K-gluconate,0.6EGTA,2Na2-ATP,0.2Na3-GTP,pH 7.3,290mOsm，内液电阻大约3-6MΩ。将培养的细胞置于外液中，其中外液成分(单位mM)：129NaCl,5KCl,1MgCl2,25HEPES,2CaCl2,30glucose,pH 7.3,310mOsm。细胞外液中还含有1μM TTX和100μM picrotoxin(Tocris)，在电压-70mV条件下记录mEPSC。结果用Mini Analysis软件(Synaptosoft)进行分析。
15.定量RT-PCR
原代神经元细胞用指定的化合物(1μM TTX和100μM D-APV处理24小时，或者0.5μM ATRA处理8小时)进行处理，然后用RNAsimple total RNA试剂盒(Tiangen)提取总RNA。用ReverTra Ace qPCR RT Master Mix(Toyobo)逆转录得到cDNA样品。用SYBR Green Master Mix(Toyobo)在CFX96real-time PCR仪器上(Bio-Rad)进行qRT-PCR检测。指定基因的转录相对量用ΔΔCt方法将Gapdh作为内参进行矫正。本工作中QPCR引物序列如下。
Gapdh
5”AGGTCGGTGTGAACGGATTTG3”(Seq ID No 3)，
5”GGGGTCGTTGATGGCAACA3”(Seq ID No 4)，
GluR1
5”AACCACCGAGGAAGGATACC3”(Seq ID No 5),
5”CGTTGAGGCGTTCTGATTCA 3”(Seq ID No 6)，
GluR2
5”TTCTCCTGTTTTATGGGGACTGA3”(Seq ID No 7),
5”CCCTACCCGAAATGCACTGT3”(Seq ID No 8)。
16.生物素标记细胞膜表面蛋白分析
生物素标记膜表面蛋白分析遵照先前报道方法进行(Aoto et al.,2008)。简要地，在DMSO或1μM TTX和100μM D-APV处理24小时后，原代PFC神经元细胞用PBS洗，再与生物素溶液(1mg/ml EZ-Link Sulfo-NHS-SS-Biotin,Pierce)4℃孵育2小时。加入0.1M甘氨酸终止反应，然后用PBS洗3遍，生物素标记后的细胞在细胞裂解液(含有25mM MgCl2,1％NP-40,1％Triton X-100,10％甘油和蛋白酶抑制剂的PBS)中裂解。离心除去细胞碎片后，上清和UltraLink Streptavidin树脂(Pierce)在4℃旋转孵育过夜。生物素标记的蛋白通过离心收集，并用细胞裂解缓冲液洗3遍。蛋白在2×SDS-PAGE上样缓冲液中75℃变性30分钟，并用GluR1抗体(13185,Cell Signaling,1:500稀释)和GluR2抗体(13607,Cell Signaling,1:500稀释)进行免疫印迹分析。
17.动物饲养
小鼠在标准12小时光照/12小时黑暗循环中，每笼3-5只饲养，提供自主饮食喝水。所有行为学实验都在12小时光照条件下进行。所有动物学研究都严格遵照中国科学院生化细胞所动物维护和使用委员会(IACUC)章程进行。所有实验中的小鼠都是C57BL/6背景的雄性小鼠(SLAC，中国)。
18.病毒的制备和脑定位注射
所有指定基因都由Synapsin I(SynI)启动子驱动，并且在N端融合了Flag标签。腺相关病毒(AAV)的包被蛋白血清型为AAV2/9，由上海禾元生物技术公司(Obio)包装。AAV-SynI-Flag-UBE3A和AAV-SynI-Flag-UBE3A-T508E病毒滴度大约在1.5×10 13拷贝数/mL，AAV-SynI-Flag-EGFP病毒滴度大约在9.5×10 12拷贝数/mL。
3周龄小鼠腹腔注射戊巴比妥(50mg/kg)进行麻醉后，固定在定位仪上(瑞沃德，中国)。在小鼠大脑内侧PFC区域两侧用定量注射泵(Stoelting)以0.2 μL/min速度注射1μL PBS稀释的AAV病毒。定点注射的位置(相对于前囱门)：AP,+2mm；ML,±0.5mm；DV,-1.3mm。每个注射点病毒注射的量分别为：AAV-SynI-Flag-UBE3A及其突变体T508E为3×10 9,AAV-SynI-Flag-EGFP为1.5×10 9。注射针在原位再保持3分钟，以防止病毒回流。将小鼠放置于37℃电热毯上，直至其从麻醉状态中完全恢复。手术后1-2天，给小鼠腹腔注射0.5mg/ml美洛昔康(Sigma，2mg/kg)帮助小鼠缓解疼痛。手术后4周，对小鼠进行行为学测试。在ATRA补偿实验中，注射AAV-SynI-Flag-UBE3A病毒1周后，小鼠每周连续五天灌胃ATRA(Sigma，溶于橄榄油，3mg/kg)或者橄榄油。给药4周后进行行为学测试。
19.Disulfiram(DSF)给药
4周龄小鼠连续6周隔天灌胃低剂量(0.1mg/g),或高剂量(0.3mg/g)DSF(Sigma，溶于橄榄油)。对照组只灌胃溶剂橄榄油(Aladdin)。每周称量小鼠体重。灌胃6周后，开始小鼠行为学测试。解剖取对照组和DSF组小鼠的大脑，液氮速冻后，进行组织匀浆，用后面描述的HPLC-MS/MS方法对ATRA进行定量。
20.HPLC-MS/MS定量检测RA实验
按照之前报道流程进行样品制备、类维生素A萃取和后续HPLC-MS/MS分析(Kane et al.,2010)。在红光条件下，将速冻小鼠的大脑在冰上用2mL 0.9％生理盐水进行匀浆。把13-cis-retinoic acid-d5(20ng/mL,TRC)加入组织匀浆中作为内参。然后在匀浆中加入1.5mL溶于乙醇的0.025M氢氧化钾，再加入7mL正己烷后，混匀并萃取水相，然后用120μL 4M盐酸中和。然后，将含有RA和极性类维生素A的油相用再次加入的7mL正己烷萃取。将萃取物在氮气环境下蒸发，干燥的萃取物用50μL乙腈重悬。将样品用2.1×100mm Supelcosil ABZ+PLUS column(3μm,Sigma)柱子进行HPLC分析，流动相分别为：A,含有0.1％甲酸的水；B,含有0.1％甲酸的乙腈。检测仪器为AB Sciex4000QTRAP LC-MS/MS系统，APCI阳离子模式对ATRA组分进行定量，每个样品中ATRA的含量用标准ATRA曲线进行校正。
21.自梳理实验
用于自梳理实验的小鼠在一个放有厚度约0.5-1cm填料的笼盒中提前适应10分钟。然后，在10分钟内，将小鼠用于自梳理的时间由双盲的实验人员通过秒表进行记录。
22.三室社交实验
根据以前报道的流程进行三室社交实验(Sztainberg et al.,2015)。简要地，进行社交实验的透明丙烯酸盒，分为三个大小一致的腔室，分区的隔板上安装可移动的小门。并在左右腔室中各放入一个倒扣的铁质网孔杯子。在实验前两天，两只杯子中各放入一只非同窝，年龄性别与测试小鼠相匹配的C57BL/6小鼠作为陌生小鼠，每天适应1小时。将测试小鼠随机分组，并在开始测试前在测试房间中提前适应1小时。每一只测试小鼠放入中间腔室自主探索10分钟后，关闭腔室门。第一阶段，将一个陌生小鼠随机放入左边或右边杯子内(避免有位置偏向性)，另一边杯子中放入无生命的物体。每一只测试小鼠在开放的三室中探索10分钟，手动记录测试小鼠分别和陌生小鼠以及物体间的交流时间。第二阶段，将受试小鼠在陌生小鼠腔室内继续放置5分钟，然后把另一只陌生小鼠放入之前放物体的杯子内。测试小鼠继续探索10分钟，手动记录测试小鼠分别和熟悉动物以及新动物间的交流时间。观测者并不知道受试小鼠的组别。
23.旷场行为学检测
小鼠经随机分组后放在旷场仪器(Med Associates)中探索30分钟。用Ethovision自动记录软件(Noldus)记录小鼠在中心区域(区域的每一边1/3to2/3长度)所花费的时间，以及计算出其在中心区域走的距离占总距离的比例。
24.高架十字迷宫实验
小鼠放置在高架十字迷宫(Med Associates)的封闭臂中，开始记录，共5分钟。利用悬空摄像机进行摄像记录，用ANY-maze软件(Stoelting)记录并分析小鼠5分钟内进入开放臂和封闭臂的次数。
25.转棒实验
小鼠随机分组后，用Rotamex rotarod仪器(Columnbus Instruments)记录小鼠的运动协调能力。同一天内，受试小鼠测试3次，每次实验持续5分钟，转棒从4转每秒加速到40转每秒。每次实验间隔至少30分钟。受试小鼠落下转棒前的时间用红外检测系统自动记录，最后一次测试的结果进行最终比较。
26.统计学分析
所有数据分析都用GraphPad Prism软件进行。数据显著性分析分别用Student”s two-tailed t-test,one-way ANOVA(Bonferroni post-hoc test或Dunnett”s post-hoc test)。所有数据都是平均值和方差表示。每一个数据统计的细节都在图注中标出。所有数据采集来自最少3次独立重复试验。没有预先估计样本量。所有小鼠均随机分配至不同组别。
实施例1 自闭症病人样本中发现染色体15q11.2-14的拷贝数扩增
前期收集到三名临床上诊断为自闭症表型的患者，通过对他们外周血进行基因组DNA的抽提和BeadChip芯片的检测，发现了染色体15q11.2-14的扩增(图1a-b)。在该扩增区域内涵括了一系列基因，而它们与自闭症间的关联并不清楚，除了并普遍证实的在15q扩增综合症里扮演关键角色的UBE3A基因(图1b-c)。应用EBV病毒感染的方法将这些病人的外周血淋巴细胞建成永生化细胞系，以便于后续的功能研究。其中，与来自健康志愿者的对照细胞相比，这三个自闭症细胞的UBE3A蛋白表达水平明显增加(图1d)。
实施例2 筛选出UBE3A的新底物ALDH1A蛋白家族
由于迄今为止并没有UBE3A的已知底物能够从机制角度解释ASD的发病与UBE3A过度激活间的关联(Glessner et al.,2009)，首先利用人UBE3A蛋白作为诱饵，通过酵母双杂交的方法来筛选未知的底物蛋白。其中，许多阳性克隆均被发现携带编码RA合成的限速酶ALDH1A2基因的cDNA(图2a)。维甲酸RA作为维生素A(视黄醇retinol)的活性代谢物，对于包括人在内的高等动物的发育和生长均为所需。其中RA的合成由两个步骤完成，包括催化视黄醇至视黄醛的视黄醇脱氢酶(RDH10)，和催化视黄醛到视黄酸的视黄醛脱氢酶(ALDH1A1,2,3家族)。通过免疫共沉淀实验验证了内源性或是外源表达的UBE3A和ALDH1A2蛋 白在HEK-293FT细胞中形成复合物(图2b-c)。在免疫荧光实验中，分别加上Flag和RFP标签的UBE3A和ALDH1A2蛋白能够在神经胶质瘤细胞SH-SY5Y的细胞质内共定位(图2d)。GST拉下实验进一步的验证了重组蛋白UBE3A和ALDH1A2能够在体外直接结合(图2e)，并且通过UBE3A的N端(1-280位氨基酸)进行结合(图2f)。由于ALDH1A2蛋白与其它家族成员ALDH1A1和3具有较大的序列相似度，因此也进一步验证UBE3A能够分别在体外和体内直接和ALDH1A1和3结合(图2g-j)。在GST拉下实验中，加入三个针对UBE3A和ALDH1A2结合区域的不同肽段(肽-1，肽-2，肽-3)，可以不同程度的竞争性阻断GST-UBE3A和ALDH1A2蛋白间的相互结合(图2k)，其中肽-1，肽-2，肽-3的序列如下：
肽-1(peptide-1)：ASRMKRAAAKHLIERYYHQLTEGCG(Seq ID No 9)
肽-2(peptide-2)：NNAAAIKALELYKINAKLCDPH(Seq ID No 10)
肽-3(peptide-3)：AEALVQSFRKVKQHTKEELKSLQAKDEDKD(Seq ID No 11)。
实施例3 UBE3A对ALDH1A2进行泛素化修饰
下一步，继续验证UBE3A是否能够对ALDH1A2进行泛素化修饰。首先，在大肠杆菌中建立了泛素化系统(具体细节见方法部分)(Keren-Kaplan et al.,2012)，将ALDH1A2和泛素化反应所需的成员共转化进细菌内，利用IPTG来诱导蛋白的表达。如图3a所示，当UBE3A蛋白存在的条件下，ALDH1A2蛋白被高度泛素化，并且该泛素化条带能够被去泛素化酶USP2的催化核心部分USP2cc蛋白去除，这提示ALDH1A2被UBE3A进行了泛素共价修饰。在HEK-293FT细胞中，过表达UBE3A蛋白同样也能够促进ALDH1A2的泛素化(图3b)。
在进一步对ALDH1A2的研究中，为了去除内源性蛋白的干扰，对各种细胞系中ALDH1A家族蛋白的表达情况进行了筛查。结果发现，在H1299细胞系中，ALDH1A家族蛋白的内源表达情况几乎完全检测不到(图3c)。利用Crispr/Cas9方法，将H1299细胞中的UBE3A基因进行了剔除，结果发现了回转进入的ALDH1A2蛋白泛素化水平的明显降低(图3d-f)。同样的，与对照正常组MEF细胞相比，在来自UBE3a敲除小鼠的MEF细胞内，随着Ube3a蛋白的减少，Aldh1a2蛋白的泛素化水平也相应的减低(图3g)。由于在人UBE3A -/-和小鼠Ube3a -/-细胞内ALDH1A2蛋白仍然存在一定程度的泛素化修饰，因此可能还存在其它的能够泛素化修饰ALDH1A2蛋白的未知E3泛素连接酶。总之，这些结果清楚的证明了包括ALDH1A2在内的ALDH1A蛋白家族均是E3泛素连接酶UBE3A的底物。
由于UBE3A的过度激活与自闭症密切相关，试图进一步验证UBE3A的高活性能够在体内增加ALDH1A2的泛素化修饰。在HEK-293FT细胞中，ALDH1A2的泛素化水平呈现UBE3A剂量依赖性的显著性增加(图3h)。并且，在永生化自闭症淋巴细胞株中的内源性ALDH1A2蛋白泛素化水平与正常对照组相比明显上升(图3i)，这反映了与自闭症关联的高剂量UBE3A蛋白确实能够催化更多的ALDH1A2泛素化水平。
在以前的研究中，UBE3A的苏氨酸508位点(T508)已被报道能够被蛋白激酶A(PKA)磷酸化修饰进而自身的泛素连接酶活性受抑，然而在自闭症病例中发现的T508A突变体由于不能够被磷酸化修饰，因此泛素连接酶活性表现为过度活化(Yi et al.,2015)。同时，UBE3A磷酸化模拟突变体T508E被发现完全丢失E3泛素连接酶活性，因此在本文中被用作连接酶失活的突变体。通过对从HEK-293FT细胞中富集的Flag-ALDH1A2蛋白检测发现，过表达UBE3A蛋白能够明显增加ALDH1A2的泛素化，T508A突变体则增加得更为显著；但T508E突变体的过表达完全不会增加ALDH1A2的泛素化水平(图3j)。这些结果验证了UBE3A T508E至少在催化ALDH1A2泛素化角度上，确实完全丢失了E3泛素连接酶活性。总之，在检测的几个细胞系中均发现，ALDH1A2泛素化水平与UBE3A蛋白的剂量密切相关。
同样考虑到ALDH1A蛋白家族的高度相似性以及前面提及的各成员均与UBE3A直接结合，进一步验证UBE3A是否能够泛素化修饰ALDH1A1和ALDH1A3。结果发现，在原核细胞和哺乳动物细胞中，ALDH1A1和3均能够被UBE3A泛素化修饰(图3k-m)。
因此，UBE3A能够泛素化修饰RA合成家族唯一的视黄醛脱氢酶家族ALDH1A。因此也推断UBE3A应该会影响到细胞中的RA合成代谢。
实施例4 UBE3A对ALDH1A2进行非蛋白酶体依赖的泛素化修饰
在前面报道中，一些UBE3A的底物蛋白比如HHR23A(Kumar et al.,1999)，和RING1B(Zaaroor-Regev et al.,2010)，在缺失人乳头状瘤病毒癌基因E6的条件下，能被泛素化修饰和蛋白酶体依赖的降解。但是p53蛋白在缺失E6时，UBE3A独自并不能够对其进行泛素化修饰和进一步的蛋白降解(Ansari et al.,2012)。然而，在HEK-293FT细胞中，随着UBE3A蛋白表达水平的增加，内源性ALDH1A2的表达水平并没有出现变化(图4a)。同时，在含有不同Ube3a 蛋白水平的小鼠MEF细胞中，Aldh1a2蛋白的表达水平同样没有改变(图4b)。在永生化淋巴细胞系中，包括自闭症病人来源和健康志愿者来源的细胞，ALDH1A2蛋白水平也保持一致(图3i)。这些结果反映了UBE3A对ALDH1A2的泛素化修饰并不会如同其他底物一样，促进底物的降解。
由于泛素包括七个赖氨酸(Lys)残基，在形成泛素链的过程中，每个赖氨酸残基，连同N端甲硫氨酸上的α-氨基，都可能会成为下一个泛素分子共价结合的位点，从而最终形成含有不同赖氨酸联结形式的多泛素化链(poly-Ub)。应用保留泛素蛋白上特定位置的赖氨酸，而将其他所有赖氨酸突变成精氨酸(K-to-R)的方法，能够鉴定出多泛素化链的具体联结形式。在图4c中，连接在ALDH1A2蛋白上的多泛素化链形式主要是Lys29和Lys63。同时，分别将K29和K63上的赖氨酸突变成精氨酸后，结合在ALDH1A2上的多泛素化链显著性的减少(图4d-e)。这些结果反映了UBE3A泛素化修饰ALDH1A2主要以K29和K63的联结形式，而这两种泛素链一般也不会引导催化底物进行蛋白酶体降解。
接下来通过蛋白质谱技术对从细菌中富集出的ALDH1A2蛋白进行分析，以期找出UBE3A在ALDH1A2蛋白催化连接的泛素化修饰位点；找出了K269，K370和K415作为可能的催化修饰位点。在H1299细胞中，当同时将ALDH1A2上的K269，K370和K415三个位点突变成精氨酸后，ALDH1A2突变体蛋白上的泛素化水平明显减少(图4f)。进一步通过蛋白结构分析发现，这三个位点均分布在脱氢酶的活性中心附近(图4g)。并且它们在ALDH1A三个成员间也高度保守(图4h)。因此，UBE3A泛素化修饰ALDH1A蛋白家族主要发生在靠近脱氢酶活性中心附近的位点，并且不会促进底物的降解。
实施例5 UBE3A介导的泛素化修饰抑制了ALDH1A2的脱氢酶活性
进一步需要对UBE3A泛素化修饰后的ALDH1A2的脱氢酶活性进行检测，尤其是其催化视黄醛至视黄酸的活性是否受到影响。结果发现，与对照细胞样品相比，从过表达UBE3A蛋白的HEK-293FT细胞中富集出的高度泛素化的ALDH1A2蛋白的视黄醛脱氢酶活性下降了一半；但该蛋白用USP2cc去泛素化酶体外处理后，活性出现显著性的恢复(图5a-c)。这显示了UBE3A阶段的ALDH1A2泛素化修饰确实显著的下调了视黄醛脱氢酶活性。同样，对ALDH1A2的另一个底物丙醛进行了检测，发现UBE3A同样也会下降ALDH1A2的丙醛脱氢酶的活性，而USP2cc蛋白能够逆转这一现象(图5d-e)。并且，ALDH1A2的K269/K370/K415 三突变体完全丢失了脱氢酶活性(图5f)，这提示这三个赖氨酸残基的完整对于ALDH1A2脱氢酶活性，可能包括整个ALDH1A蛋白家族脱氢酶活性来说都尤为重要(图5g)。
在研究细胞ALDH活性时，基于流式细胞仪的Aldefluor检测方法非常普遍(Storms et al.,1999)。它的工作原理在于细胞内的ALDH能够将BAAA(BODIPY-氨基乙醛)氧化成BAA(BODIPY-氨基乙酸盐)，而BAA作为负电荷产物会滞留在细胞内，从而细胞ALDH的脱氢酶活性可以通过检测含有BAA的细胞荧光强度来定量。如图5h所示，与正常对照组淋巴细胞相比，自闭症淋巴细胞的ALDH阳性细胞比例大约减少了20％到50％。这也提示了自闭症相关的UBE3A高剂量可能会导致细胞中的ALDH酶活性下调。
同时，建立了共培养细胞体系来进一步验证自闭症细胞中的ALDH1A脱氢酶活性情况。首先将带有RA响应元件RARE的荧光素酶质粒引入H1299细胞，作为RA响应细胞；同时将永生化的淋巴细胞株作为RA产生细胞；将两者以1:1的细胞数目比在无血清培养基VP-SFM中进行共培养，加入视黄醛处理8小时后对荧光素酶活性进行检测。如图5i所示，与正常对照淋巴细胞相比，自闭症淋巴细胞所共培养的H1299荧光素酶活性下调了60％。
总之，这些结果提示，与自闭症相关的UBE3A过度激活能够抑制RA的生物合成，从而下调总体的RA内稳态水平。
实施例6 UBE3A过度激活引起突触传递稳态的失调
在成体神经系统中，RA已逐渐被发现在内稳态突触可塑性中扮演重要的角色(Chen et al.,2014)。在神经元细胞中，当突触传递被阻止时，突触钙离子水平会迅速下降，RA通过ALDH1A脱氢酶的合成过程从而被激活(Chen et al.,2014；Aoto et al.,2008)。RA通过结合定位在突触中的RARα蛋白，解除RARα原先阻滞的蛋白翻译过程，其中被调控的蛋白包括AMPA受体，从而上调突触传递(Aoto et al.,2008)。在来自大鼠PFC皮质的原代神经元细胞中，通过D-APV和TTX对突触活性的抑制，细胞中的RA水平上升，从而诱导RARE驱动的人源海肾绿色荧光蛋白(hrGFP)显著性的表达(图6a)。但在神经元细胞中，将UBE3A-IRES-turboRFP和RARE-hrGFP报告质粒共转染后，D-APV和TTX的处理不再诱导hrGFP蛋白的表达(图6b)。然而，将UBE3A连接酶活性缺失的突变体T508E与RARE-hrGFP报告质粒共转染后，D-APV和TTX的处理能够将 hrGFP蛋白诱导表达至与对照组相似的水平(图6b)。这些结果证明了UBE3A的过度激活阻止了突触传递受阻所引起的RA生成。
接下来对UBE3A的过度激活是否会影响到神经元活性受阻时RA所调控的突触内稳态过程。首先，在原代PFC神经元细胞中，APV和TTX的共处理引起了mEPSC(微小兴奋性突触后电流)的幅值和频率的显著性上升(图6c)。这提示在神经元细胞中，兴奋性突触可能通过突触前和突触后的不同机制实现了代偿性的增加。但是，在过表达UBE3A的神经元细胞中，与对照组相比，同样的活性受阻使得mEPSC幅值下降了约30％。但过表达UBE3A T508E的神经元细胞中，mEPSC上升幅值则与对照组相似(图6d)。并且，在过表达UBE3A和UBE3A T508E的细胞中，mEPSC的频率几乎与对照组一致(图6d)。因此，这些结果提示，在神经元活性受阻的条件下，UBE3A的过度激活很可能通过突触后机制，而不是突触前机制来破坏RA所调控的突触内稳态过程。
现在已经知道突触缩放的突触后机制主要通过促进突触受体的翻译来增加兴奋性突触受体的数目(Han et al.,2009)。与前面的报道一致(Aoto et al.,2008)，在神经元活性受阻时，GluR1受体的蛋白水平增加，而GluR2的蛋白水平不变；并且其转录水平不受影响(图6e-g)。然而，在UBE3A表达的神经元细胞中，神经元活性的阻滞并不能增加突触后GluR1的蛋白水平(图6h)。而UBE3A T508E表达的细胞中，神经元活性的阻滞诱导GluR1蛋白水平显著高于UBE3A表达组。这些结果均提示，UBE3A的过度激活所引起的突触内稳态失调，主要通过影响RA上调的突触后蛋白翻译过程。
实施例7 在小鼠中的UBE3A过度激活引起自闭症表型
大脑中PFC区域调控着大脑的各种执行功能和高阶认知过程，包括做出决定、认知移动、社会行为、学习和社会沟通交流等。最近，PFC区的解剖学结构和它与其他脑区的联结结构异常，被发现普遍的在自闭症患者大脑中存在，而这也提示PFC的功能异常与自闭症病因密切相关(Stoner et al.,2014；Chow et al.,2012)。为了验证过度激活的UBE3A能够诱导自闭症行为的产生，将分别包装有EGFP，UBE3A和T508E的腺相关病毒(AAV)通过定点注射的方法打入小鼠大脑内侧PFC区域中(图7a-b)。对大脑冰冻切片后的免疫荧光结果显示，UBE3A和T508E在PFC区域中的蛋白表达水平相仿(图7c)。
大脑注射四周后，对小鼠进行行为学实验检测。如图7d所示，在小鼠自 梳理实验中，与表达EGFP的小鼠相比，过表达UBE3A的小鼠在自梳理行为上所花的时间多了一倍；而表达T508E的小鼠花的时间只多了30％。这提示过表达UBE3A的小鼠显现出重复性的刻板行为。接下来，利用三室社会行为学实验对小鼠在与社会性动物或是非社会性物体间的交往时间进行记录和比较分析。在表达EGFP和T508E的小鼠中，其与社会性小鼠交往的时间(50秒)比与物体交往的时间(23秒)多了约一倍；然而在表达UBE3A的小鼠中，其与社会性小鼠和物体交往的时间几乎一致，均为30秒(图7e)。这提示过表达UBE3A的小鼠出现了严重的社会交往障碍。同时，对小鼠在与熟悉的社会性小鼠和陌生的社会性小鼠交往的时间上也进行记录和比较分析。其中，在表达EGFP和T508E的小鼠中，他们对陌生的社会性小鼠比熟悉的社会性小鼠表现出更大的兴趣，花费了更多的时间与他们交往(约增加了60-100％)。然而，过表达UBE3A的小鼠在与熟悉的和陌生的社会性小鼠交往的时间上所花的时间一样长(图7f)。这提示过表达UBE3A蛋白对于小鼠会抑制其对社交新颖性的认知。这些行为学实验也清楚的显示了，在小鼠PFC脑区中过表达UBE3A蛋白，而不是泛素连接酶失活的突变体T508E，能够显著性的诱导小鼠自闭症表型的出现，包括重复刻板行为，社交的障碍以及社交新颖性的认知缺失。
为了进一步研究过表达UBE3A或是T508E蛋白所引起的潜在表型改变，对小鼠进行了其它的行为学实验检测。实际上，在旷场实验中，表达这三种蛋白的小鼠都表现出相同水平的趋触性(图7g-h)。并且，转棒实验结果显示，与对照组相比，过表达UBE3A没有对小鼠的活动能力产生影响(图7i)。
因此，小鼠PFC脑区中过表达UBE3A蛋白能够特异性的引发小鼠自闭症核心症状的出现。
实施例8 RA的补偿削弱了UBE3A过度激活所引起的小鼠自闭症表型
为了进一步验证UBE3A过度激活引发小鼠自闭症表型是由于RA生成过程的损伤所引起的，对PFC注射携带UBE3A的AAV病毒的小鼠分组口服溶剂(橄榄油)或是ATRA(3mg/kg，每周连续五天给药)共四周时间，然后对小鼠进行行为学实验(图8a)。在小鼠自梳理实验中，与口服溶剂组相比，口服ATRA的小鼠在自梳理行为上所花的时间显著性的减少(图8b)。同时，与溶剂组相比，ATRA给药的小鼠在社交上的障碍也出现了完全的逆转；它们与社会性动物交往的时间比非社会性的物体多了近一倍的时间(图8c)。如图8d所示，这些小鼠对社 会新颖性的认知缺失在ATRA给药后也出现了缓解，它们与陌生小鼠的交往时间多于熟悉小鼠近一倍时间。因此，四周时间的ATRA给药能够大幅的改善UBE3A过表达小鼠的自闭症核心症状。
需要注明的是，在旷场实验中，ATRA给药的小鼠在旷场中间区域逗留的时间和运动的距离要少于溶剂组(图8e-f)，这提示ATRA在该剂量条件下对UBE3A过表达小鼠会引起一定程度的焦虑症状。但是，转棒结果显示，ATAR的给药并不会对小鼠的运动能力产生影响(图8g)。
为了排除ATRA的给药是否会对野生型小鼠也会产生焦虑症状，对野生型小鼠进行了同样的给药方式和行为学检测(图8a)。事实上，溶剂或是ATRA的给药，对野生型小鼠在自梳理实验、三室社会交往实验、旷场实验以及转棒实验都没有引起差异(图8h-m)。这些观察结果表明ATRA的给药只会对UBE3A过表达小鼠产生轻度的焦虑症状，而对于野生型小鼠完全没有影响；这也提示如果在后面期望用ATRA来治疗自闭症的情况下，需要对其潜在的副作用进行考虑和评估。
总之，这些ATRA的补偿实验第一次证明了RA稳态调节在UBE3A过表达所引起的自闭症发生过程中的关键作用。同时，RA的补偿理论上也能够用来缓解UBE3A过度激活所引起的自闭症亚型疾病。
实施例9 化合物导致RA的稳态失调诱导小鼠自闭症表型的产生
为了证明单独ALDH1A活性的受抑是否足够诱导小鼠自闭症表型的发生，给四周龄的野生型C57BL/6小鼠口服Aldh1a的抑制剂disulfiram(DSF)。给药时间共六周，剂量分别为0.1或0.3mg/g，隔天给药。当DSF给药六周后，取出小鼠大脑，利用HPLC-MS/MS方法对其中的ATRA水平进行检测(Kane et al.,2010)。如图9a所示，DSF给药六周的小鼠大脑组织中ATRA的水平与溶剂组相比，分别下降了30％(0.1mg/g剂量)和60％(0.3mg/g剂量)。这表明DSF的给药能够剂量依赖性的抑制小鼠大脑中的ATRA合成。同时通过对小鼠的体重每周进行检测发现，各组间体重并没有出现明显的差异(图9b)。这提示这里的DSF剂量并不会对小鼠引起显著性的毒性作用。
接下来，对DSF给药六周的小鼠进行双盲的行为学实验检测。如图9c所示，在自梳理实验中，与溶剂组(15秒)相比，DSF给药小鼠要明显增加一倍(30秒，0.1mg/g剂量)和两倍(42秒，0.3mg/g剂量)的时间进行自梳理行为。而 在三室社会交往实验中，溶剂组的小鼠与社会性小鼠交往的时间明显多于非社会性物体的时间；但在0.1mg/g DSF给药小鼠中完全没有这样明显的趋势(图9d-e)。并且，在0.3mg/g DSF给药小鼠中，其于社会性小鼠交往的时间和非社会性物体的交往时间完全没有差异(图9d-e)。在社交新颖性角度，0.3mg/g DSF给药组与溶剂组相比，在熟悉的社会性小鼠腔室中逗留的时间增加了0.5倍，而在陌生的社会性小鼠腔室中逗留时间减少了0.3倍(图9f-g)。而在与社会性小鼠交流时间上，DSF给药小鼠也在与熟悉或陌生小鼠的交流时间上完全无差别。总之，但RA的稳态调控被化合物抑制后，小鼠的自闭症表型显著性的表现出来。
同时，也从行为学其它角度对DSF给药对小鼠的影响进行评估。在旷场实验中，三组之间在中间区域逗留的时间和运动的距离均没有差异(图9h-i)。在高架十字迷宫实验中，三组小鼠进入开放臂的次数占所有臂进入次数的比率无明显区别(图9j)。这两个结果清楚的显示了DSF在本研究中的剂量并不会对小鼠的焦虑或是探索性行为产生影响。同时，转棒实验也显示DSF的给药并不会对小鼠的运动能力产生影响(图9k)。通过这些行为学实验也反映了DSF诱导小鼠产生自闭症表型并不是通过其化学毒副作用施加的，这也与前面的小鼠体重结果相符(图9b)。
总之，化合物抑制RA的稳态调节引起了小鼠自闭症表型的出现，也再次强调了UBE3A过度激活导致ALDH1A泛素化，从而下调了RA的水平在自闭症病因中的重要性。
总结和讨论
(1)UBE3A结合并泛素化修饰ALDH1A2，从而抑制了ALDH1A2的脱氢酶活性。同样，ALDH1A的其它家族成员ALDH1A1和ALDH1A3也被证明是UBE3A的底物。
(2)自闭症相关的UBE3A过度激活能够下调细胞内的RA水平，进而影响RA的信号通路，特别是其在神经元细胞中调节突触缩放角度的作用。
(3)UBE3A在小鼠大脑PFC区域的过表达能够引发自闭症表型，并且能够通过RA的补偿来缓解疾病表型。这个可以直接推广至人类自闭症的临床治疗角度上。
(4)ALDH1A的化合物抑制剂DSF能够在小鼠体内引起RA水平的降低，也 能够诱导小鼠自闭症类似表型的发生。
(5)通过本研究可以扩展人类临床上自闭症的诊断范畴，提供了新颖的自闭症诊断和治疗靶点，应该将RA的水平是否改变及其上游调节的因素纳入自闭症诊断的工具中，同时也能够依据此发明筛选开发出自闭症治疗的药物。
(6)同时，也可以将本发明用于筛查潜在的对孕妇服用药物及日用化学品副作用所导致的后代中出现自闭症的风险(以及婴幼儿服用药物及日用化学品等的副作用)上，有望于开发出有效的临床评估诊断工具。
(7)还建立了实时监测维甲酸水平的细胞模型，利用高通量筛选技术，对FDA批准的商品化药物正在进行筛查，找出了若干能够干扰维甲酸水平的药物，并进行了后期验证，验证结果完全符合预期。现正在进行动物实验来验证这些药物是否会实验隔代诱导自闭症的现象。
综上所述，本发明首次揭示了UBE3A能够结合并通过非蛋白酶体降解的形式泛素化修饰ALDH1A家族蛋白，从而抑制RA的合成，下调细胞内RA的内稳态。在培养的原代神经元细胞中，神经元活性阻断会引起RA的快速生成，从而调节神经元的突触缩放过程，然而UBE3A的过度激活通过阻断RA的生成从而干扰RA对神经元突触稳态的调节。在小鼠的行为学研究中也发现，UBE3A的过度表达或者ALDH1A的抑制剂disulfiram(DSF)给药都能够重塑自闭症表型。因此UBE3A过度激活与自闭症表型间存在关联性。然而，由于UBE3A具有广泛的生理作用，因此不宜直接抑制UBE3A，否则会导致其他副作用。基于本发明的研究成果，提供了可以在不干预或不过分干预UBE3A的情况下对自闭症进行预防和/或治疗的靶点、药物和治疗手段。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
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Claims (17)

  1. 一种ALDH1A、或其激动剂、或其催化产物、和/或其催化底物的用途，其特征在于，用于制备药物组合物或制剂，所述药物组合物或制剂用于治疗和/或预防自闭症。
  2. 如权利要求1所述的用途，其特征在于，所述ALDH1A选自下组：ALDH1A1、ALDH1A2、ALDH1A3、或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述ALDH1A激动剂选自下组：N-(1,3-苯并二氧戊环-5-亚甲基)-2,6-二氯苯甲酰胺(N-(1,3-Benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide)、N-(1,3-苯并二氧戊环-5-亚甲基)-2,6-二氯苯甲酰胺(N-(1,3-Benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide)、6-甲基-2-偶氮苯基-3-羟基吡啶(6-methyl-2-(phenylazo)-3-pyridinol)、2-(苯并[d][1.3]间二氧环戊烯-5-基)-N-(5,6-二氢-4H-环戊二烯[c]异恶唑-3-基)乙酰胺(2-(benzo[d][1,3]dioxol-5-yl)-N-(5,6-dihydro-4H-cyclopenta[c]isoxazol-3-yl)acetamide)及其各衍生物。
  4. 如权利要求1所述的用途，其特征在于，所述的激动剂包括表达促进剂、蛋白降解抑制剂。
  5. 一种维甲酸或维甲酸类似物，或其溶剂化物、或其药学上可接受的盐的用途，其特征在于，用于制备药物组合物或制剂，所述药物组合物或制剂用于治疗和/或预防自闭症。
  6. 如权利要求5所述的用途，其特征在于，所述维甲酸类似物选自下组：视黄醇、视黄醛、全反式维甲酸、13-顺式维甲酸、9-顺式维甲酸、阿维A酯、阿维A、阿达帕林、贝沙罗汀、他扎罗汀、或其组合。
  7. 一种维甲酸合成原料化合物、或维甲酸降解酶抑制剂的用途，其特征在于，用于制备药物组合物或制剂，所述药物组合物或制剂用于治疗和/或预防自闭症。
  8. 如权利要求7所述的用途，其特征在于，所述维甲酸降解酶抑制剂选自下组：利阿唑(liarozole)，酮康唑(ketoconazole)，他拉罗唑(talarozole)，或其组合。
  9. 一种ALDH1A抑制剂的用途，其特征在于，用于制备一制剂，所述制剂用于建立自闭症动物模型。
  10. 如权利要求9所述的用途，其特征在于，所述ALDH1A抑制剂选自下组：双硫仑(disulfiram)、4-(N,N-二乙基)氨基苯甲醛(DEAB)，WIN-18446，A37(CM037)，NCT-501盐酸化物，CVT-10216，或其组合。
  11. 一种对自闭症的辅助诊断和/或预后的方法，其特征在于，所述方法包括步骤：
    (1)提供一待测样本，所述样本选自下组：血液、体液；
    (2)检测标志物浓度、含量、和/或活性；
    (3)与标准值或标准曲线比较，从而进行辅助诊断和/或预后；
    其中，所述标志物选自下组：维甲酸、ALDH1A、或其组合。
  12. 一种评估待测物质的副作用风险的方法，其特征在于，所述的副作用风险为诱发或引发自闭症的风险，并且所述方法包括步骤：
    (a)提供一待测物质；
    (b)在施用所述的待测物质的测试组中，测定所述待测物质中对于待测指标的影响；并在未施用所述待测物质的对照组中，测定同一待测指标的数据；其中，所述的待测指标选自下组：维甲酸的水平或浓度、ALDH1A表达量或活性、或其组合；
    (c)将测试组的所述待测指标的测定结果T与对照组中所述待测指标的测定结果C进行比较；
    如果测定结果T显著低于测定结果C，则提示待测物质具有导致孕妇和/或婴幼儿副作用的风险。
  13. 如权利要求12所述的方法，其特征在于，所述待测物质选自下组：药物、日用化学品、或其组合。
  14. 一种药物组合或含所述药物组合的药盒，其特征在于，所述的药物组合包括：
    (a)第一药物组合物，所述第一药物组合物含有药学上可接受的载体和选自下组的第一活性成分：克霉唑、孟鲁司特、孟鲁司特钠；和
    (b)第二药物组合物，所述第二药物组合物含有药学上可接受的载体和选自下组的第二活性成分：ALDH1A、ALDH1A激动剂、ALDH1A催化产物、ALDH1A催化底物、或其组合。
  15. 一种药物组合物，其特征在于，所述的药物组合物含有：
    (a)第一活性成分，其选自下组：克霉唑、孟鲁司特、孟鲁司特钠；
    (b)第二活性成分，其选自下组：ALDH1A、ALDH1A激动剂、ALDH1A催化产物、ALDH1A催化底物、或其组合。
  16. 一种筛选治疗或预防自闭症的候选药物的方法，其特征在于，包括步骤：
    (a)提供一待测试的候选物质；
    (b)在施用所述的待测试的候选物质的测试组中，测定所述待测试的候选物质对于待测指标的影响；并在未施用所述待测试的候选物质的对照组中，测定同一待测指标的数据；其中，所述的待测指标选自下组：维甲酸的水平或浓度、ALDH1A表达量或活性、或其组合；
    (c)将测试组的所述待测指标的测定结果T与对照组中所述待测指标的测定结果C进行比较；
    如果测定结果T显著高于测定结果C，则提示所述待测试的候选物质可作为治疗或预防自闭症的候选药物。
  17. 一种能够阻断UBE3A和ALDH1A家族蛋白结合的多肽或抗体或化合物的用途，其特征在于，用于制备药物组合物或制剂，所述药物组合物或制剂用于治疗和/或预防自闭症。
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