Application of molecular target ptchd1 in synaptic function, consciousness regulation and related diseases

Abstract

This application discloses the application of the molecular target Ptchd1 in neural synaptic function, consciousness regulation, and related diseases. Through genome-wide screening, this application identified Ptchd1 as a key molecular target for consciousness regulation and demonstrated in animal experiments that this target plays a significant regulatory role in neuronal synaptic vesicle release and drug-induced loss of consciousness. Given that Ptchd1 can regulate drug-induced loss of consciousness and neuronal synaptic transmission, this application proposes that Ptchd1-targeted therapies have significant potential value in the following areas: i) application in the preparation of reagents for regulating neural synaptic function and consciousness regulation; ii) application in the preparation of drugs for promoting post-anesthesia recovery; iii) application in the preparation of awakening-promoting therapeutic drugs for consciousness disorders, including vegetative state, minimally conscious state, or coma due to brain injury.

Description

Technical Field

[0001] This application relates to the application of the molecular target Ptchd1 in neural synaptic function, consciousness regulation, and related diseases, and belongs to the field of biomedical technology. Background Technology

[0002] Currently, there are no specific drugs or relevant targets for regulating loss of consciousness induced by general anesthesia. On the other hand, synaptic dysfunction causes a series of neurological diseases (cognitive impairment, epilepsy, autism spectrum disorder, etc.), but treatments targeting the regulation of synaptic vesicle release have not yet been reported.

[0003] Ptchd1 (patched domain containing 1) is a gene located on the X chromosome (Xp22.11). The main function of the Ptchd1 gene is to participate in the regulation of cell growth and differentiation. It affects cell proliferation, apoptosis, and tissue formation by regulating a series of signaling pathways. However, the role of Ptchd1 in regulating abnormal neural synaptic function, consciousness regulation, and related diseases has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide therapeutic targets for diseases related to abnormal synaptic transmission (such as cognitive impairment, epilepsy, autism spectrum disorder, etc.) by intervening in the precise control of general anesthesia resuscitation for the treatment of diseases related to impaired consciousness.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] In the first aspect, this application provides the application of Ptchd1 as a detection target, the application being selected from: i) the application in screening reagents for regulating neural synaptic function and consciousness regulation; ii) the application in screening drugs for precise anesthesia or treatment of diseases or conditions related to disorders of consciousness.

[0007] Secondly, the use of the reagent that inhibits the expression or activity of Ptchd1 in this application is selected from: i) its use in the preparation of reagents for regulating neural synaptic function and consciousness regulation; ii) its use in drugs that promote post-anesthesia recovery; and iii) its use in the preparation of drugs for the treatment of consciousness disorders, including vegetative state (PVS), minimally conscious state (MCS), or coma due to brain injury.

[0008] In some embodiments, the reagents for inhibiting Ptchd1 expression or activity include: i) any one or more of shRNA, siRNA, ASO and delivery vectors of said shRNA, siRNA, ASO and CRISPR / Cas system that specifically inhibit Ptchd1 expression; ii) any one or more of neutralizing antibodies, aptamers and small molecule inhibitors that block the binding of Ptchd1 to its binding protein (Rab3).

[0009] Thirdly, this application provides the use of a reagent that promotes the expression or activity of Ptchd1, the use being in the preparation of a reagent or drug for prolonging anesthesia time.

[0010] In some embodiments, the agents that promote Ptchd1 expression or activity include one or more combinations of small molecule compounds, Ptchd1 gene delivery vectors, and Ptchd1 protein agonists.

[0011] In some embodiments, the delivery vector includes a viral vector or a non-viral vector.

[0012] In some embodiments, the viral vector is selected from one or more combinations of retroviral vectors, adenovirus vectors, and adeno-associated virus vectors; and / or, the non-viral vector includes mRNA liposomes.

[0013] In some embodiments, the drug comprises a pharmacologically active ingredient and pharmaceutically acceptable excipients, wherein the pharmacologically active ingredient includes an agent that inhibits the expression or activity of Ptchd1.

[0014] In some embodiments, the pharmaceutically acceptable excipient is one or more of the following: diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, and buffer.

[0015] In some embodiments, the dosage form of the drug is an oral formulation or an injectable formulation.

[0016] Compared with the prior art, this application has the following beneficial effects:

[0017] This application identified Ptchd1, a molecular target related to consciousness regulation, through whole-genome screening, and demonstrated in animal experiments that this target plays an important regulatory role in the release of neuronal synaptic vesicles. Synaptic transmission is the basic mode of signal communication between neurons, and abnormal synaptic transmission will cause various neurological diseases. This application verified the regulatory role of Ptchd1 in the loss of consciousness induced by general anesthetics. Synaptic function regulation targeting Ptchd1 has great potential value in the treatment of these diseases. Attached Figure Description

[0018] Figure 1 Ptchd1 in the thalamic reticular nucleus (TRN) is crucial for anesthesia-induced loss of consciousness (LOC); among which:

[0019] (A) Ptchd1 knockout treated with propofol (Ptchd1 Y / - Mice and wild-type controls (Ptchd1) Y / + The duration of loss of righting reflex (LORR) in mice;

[0020] (B) Duration of righting reflex loss (LORR) in esketamine-treated Ptchd1 knockout mice and wild-type control mice;

[0021] (C) Schematic diagram of the timeline of intravenous injection of dexmedetomidine (top), and the induction time and duration of righting reflex loss (LORR) in Ptchd1 knockout mice and wild-type control mice (bottom);

[0022] (D) Expression of Ptchd1 mRNA in the thalamic reticular nucleus (TRN), cerebral cortex, hippocampus and thalamic relay nucleus (red, RNA scope technique).

[0023] (EG) Effect of specific knockdown of Ptchd1 in the thalamic reticular nucleus (TRN) on loss of righting reflex (LORR) induced by anesthesia: (E) Duration of LORR induced by propofol; (F) Duration of LORR induced by esketamine; (G) Induction time and duration of LORR induced by dexmedetomidine;

[0024] Figure 2 Ptchd1 is located in presynaptic vesicles of neurons in the thalamic reticular nucleus (TRN); among which:

[0025] (A) Identification of PTR-11 interacting proteins: Using the lysate of PTR-11::GFP transgenic Caenorhabditis elegans, the proteins interacting with PTR-11 were captured by immunoprecipitation with GFP antibody. The proteins were then identified by mass spectrometry analysis, which showed that PTR-11 binds to multiple Rab family proteins.

[0026] (B) Immunoprecipitation experiments of HA-Ptchd1 and Rab3 in mouse thalamic tissue homogenate showed that Ptchd1 and Rab3 bind to each other;

[0027] (C) Schematic diagram of the anatomical structure of the imaging region shown in Figure D: ventral posterior nucleus (VP), posteromedial nucleus (PO), and central thalamus (CT).

[0028] (D) Immunostaining results of Ptchd1 in TRN and thalamic relay nucleus (green); Blue: DAPI-labeled cell nuclei;

[0029] (E) Immunoelectron microscopy analysis of Ptchd1 subcellular localization in TRN neurons (top) and thalamic relay nucleus (bottom); red arrows indicate immunogold particles, and blue arrows indicate excitatory synapses;

[0030] (F) Schematic diagram of the virus injection site and imaging region related to Figure G;

[0031] (G) Representative images of co-localization of HA-Ptchd1 (green) and Rab3 (purple) in thalamic relay nuclei (VP, Po, CT); TRN-derived presynaptic terminals are labeled with synaptic vesicle protein-mCherry (red) mediated by adeno-associated virus (AAV); Scale bar: 2 micrometers (μm); The right-hand chart shows Rab3-positive and synaptic vesicle protein-positive (Rab3) nuclei. + Syn + The proportion of Ptchd1 positive cells in the presynaptic tubercle suggests that Ptchd1 co-localizes with Rab3 in the presynaptic tubercle.

[0032] Figure 3 Ptchd1 deficiency leads to abnormal inhibitory synaptic transmission in the thalamic reticular nucleus (TRN); among which:

[0033] (A) Volcano plot of differentially expressed membrane proteins (DEMPs) in the thalamus tissue of wild-type (WT) mice and Ptchd1 knockout (KO) mice;

[0034] (B) Analysis of genes (GO) that upregulated and downregulated differentially expressed membrane proteins (DEMPs) in the thalamus of Ptchd1 knockout mice revealed downregulated expression of multiple synapse-related proteins;

[0035] (C) Heatmap of expression levels of representative differentially expressed membrane proteins (DEMPs);

[0036] (D) Schematic diagram of the experimental procedure for Correlated Light Microscopy-Sequential Scanning Electron Microscopy (CoLSSEM);

[0037] (E) Representative electron micrographs of TRN-derived inhibitory synapses in the thalamic relay nucleus. Ptchd1 knockout significantly reduces the number of presynaptic release vesicles. Yellow arrows indicate docking vesicles;

[0038] (F) Three-dimensional reconstruction of the presynaptic tubercle using Correlated Light Microscopy-Sequential Scanning Electron Microscopy (CoLSSEM); the three-dimensional structure also shows that the number of presynaptic release vesicles is significantly reduced after Ptchd1 knockout;

[0039] Quantitative analysis of the number of vesicles released per presynaptic tubercle (G), the total number of vesicles (H), and the volume of the presynaptic tubercle (I) in the TRN terminals of wild-type (WT) and Ptchd1 knockout (KO) mice (GI) showed that Ptchd1 knockout significantly reduced the number of vesicles released, the total number of vesicles, and the volume of the presynaptic tubercle.

[0040] (JK) Schematic diagram (J) and representative trajectory (J) of inhibitory postsynaptic current (IPSC) triggered by hyperosmolar sucrose in mouse thalamic neurons, and quantitative analysis (K) of charge transfer in the initial 10 seconds; suggesting that Ptchd1 knockout reduces inhibitory postsynaptic current;

[0041] (LN) Schematic diagram (L), representative trajectory (M), and quantitative analysis (N) of spontaneous inhibitory postsynaptic currents (sIPSCs) in mouse brain slices; the results showed that the frequency of sIPSCs decreased significantly after Ptchd1 knockout, suggesting abnormal presynaptic function;

[0042] (OP) Representative double-pulse stimulation trajectory (O, stimulation interval 20 ms) and quantitative analysis (P); the results showed abnormal synaptic vesicle release after Ptchd1 deletion. Detailed Implementation

[0043] To make the technical solution of this application clearer and easier to understand, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0044] Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0045] Example 1. Ptchd1 plays an important role in the awakening process after loss of consciousness induced by various general anesthetic drugs.

[0046] Experimental methods:

[0047] Construction of genetically modified mice:

[0048] Ptchd1 knockout (KO) and Ptchd1-3×HA-P2A-YFP knock-in (KI) mouse models were constructed by Jicui Pharmaceutical Co., Ltd. using CRISPR-Cas9 technology in a C57BL / 6J background.

[0049] For the Ptchd1 knockout model, Cas9 mRNA and sgRNA targeting exons 2-3 of the Ptchd1 gene were microinjected into fertilized eggs. The resulting first-generation mice were backcrossed three times with wild-type C57BL / 6J mice to establish a pure genetic background. Litters were obtained by mating female heterozygotes with male homozygous knockout mice.

[0050] For the Ptchd1-3×HA-P2A-YFP knock-in model, a donor vector containing 3×HA-P2A-YFP was co-injected with a Cas9 component, inserting the sequence into exon 3. Correctly targeted F0 generation mice were identified by PCR and sequencing. A breeding population was established by mating F1 generation heterozygotes (wt / ki) with hemizygous males (Y / ki).

[0051] Mouse behavioral tests

[0052] Healthy 2-5 month old mice were assessed for loss of righting reflex (LORR) via tail vein injection of the following anesthetics: propofol (Fresenius Kabi, batch number HJ20150655; doses 10, 15, 20, 30, 40 mg / kg), esketamine (Hengrui Medicine Co., Ltd., batch number H20193336; dose 40 mg / kg), or dexmedetomidine hydrochloride (Chenxin Pharmaceutical Co., Ltd., batch number H20130027; dose 0.4 mg / kg). The onset of loss of righting reflex was defined as the time within 10 seconds after the mouse was placed in a supine position when it was unable to right itself, and the duration of loss of righting reflex was defined as the time from the onset of loss to the moment the mouse successfully righted itself. Genotypic blinding was performed on all behavioral assessments.

[0053] Statistical analysis

[0054] All experiments included at least three independent biological replicates, performed in a randomized order. Behavioral testing, presynaptic knot analysis, and electrophysiological recordings were all administered and analyzed using a double-blind method. Statistical analysis was performed using GraphPad Prism 9 software, with detailed descriptions of the specific tests used in each figure caption. Significance was expressed as a corrected p-value. All data are presented as mean ± standard error (mean ± SEM).

[0055] Experimental results:

[0056] Compared to wild-type mice, Ptchd1 knockout mice showed significantly shorter duration of propofol and esketamine anesthesia, earlier awakening time, and increased anesthesia induction time and shortened anesthesia duration in dexmedetomidine-induced loss of consciousness. Figure 1 As shown in A~G.

[0057] The results of this embodiment indicate that Ptchd1 can serve as a target for consciousness modulation, specifically applicable to: 1) Personalized anesthesia, prolonging anesthesia time by promoting Ptchd1 expression or shortening anesthesia time by inhibiting Ptchd1 expression; 2) Development of novel adjuvant anesthesia drugs: developing drugs specifically targeting Ptchd1 or its regulated vesicle release mechanism; 3) Ptchd1 inhibitors for the awakening treatment of disorders of consciousness: treatment of patients in vegetative state (PVS), minimally conscious state (MCS), or coma due to brain injury.

[0058] Example 2. Ptchd1 participates in regulating synaptic vesicle release

[0059] Experimental methods:

[0060] Immunoprecipitation and Western blotting

[0061] To immunoprecipitate HA-tagged PTCHD1, membrane protein extracts from the thalamus of Ptchd1-3×HA-P2A-YFP mice were used. Immunoprecipitation experiments were performed using the Pierce Crosslinked Magnetic Immunoprecipitation / Co-immunoprecipitation Kit (Thermo Fisher Scientific, catalog number 88805). Brief procedure: Using disuccinimide (DSS) from the kit, 25 μL of magnetic beads were crosslinked with 10 μg of rat anti-HA antibody (Roche, catalog number 11867423001); 10 μg of rat IgG antibody (Invitrogen, catalog number 10700) was used as a negative control. Beads containing 5 mouse thalamic proteins from 1 mL of lysis buffer were incubated with the antibody-conjugated beads on a shaker at room temperature for 1 hour. The beads were then washed five times with immunoprecipitation lysis / wash buffer. The bound proteins were eluted with 100 μL of elution buffer. The eluent was mixed with SDS loading buffer and heated at 65°C for 15 minutes before SDS-PAGE electrophoresis and Western blot analysis. The primary antibodies used for detection were as follows: rat anti-HA (1:1000, Roche, catalog number 11867423001), mouse anti-Rab3 (1:2000, Synaptic Systems, catalog number 107111), rabbit anti-Rab10 (1:500, Invitrogen, catalog number PA5-79901), and rabbit anti-Rab11 (1:500, Invitrogen, catalog number PA5-120704). Each experiment was performed independently in triplicate.

[0062] Adeno-associated virus (AAV) injection

[0063] 4-5 week old mice were anesthetized with isoflurane and fixed on a stereotaxic apparatus. 200 nL of mixed adeno-associated virus was injected into two sites in each hemisphere of the bilateral thalamic reticular nucleus (TRN) (coordinates relative to the anterior fontanelle: anteroposterior axis -0.7 mm, medial-lateral axis ±1.65 mm, dorsoventral axis -3.2 mm and -3.55 mm). After 3-4 weeks of recovery, the mice underwent behavioral tests, electrophysiological recordings, immunofluorescence staining, or electron microscopy analysis. The virus was purchased from Wuhan Shumi Brain Science Technology Co., Ltd., and specific information is listed in the supplementary table.

[0064] Immunohistochemistry

[0065] Ptchd1-3×HA-P2A-YFP mice injected with rAAV-hSyn-Synaptophysin-mCherry virus via the thalamic reticular nucleus were perfused with 4% paraformaldehyde via cardiac perfusion. The brains were post-fixed overnight in 4% paraformaldehyde and coronal sections (14 μm thick) were prepared. Sections were blocked for 1 hour (room temperature) in PBS containing 1% bovine serum albumin (BSA) and 0.3% Triton X-100, followed by overnight incubation with primary antibodies (rat anti-HA; mouse anti-Rab3) at 4°C. After washing, sections were incubated with species-matched secondary antibodies at room temperature for 2 hours. Images were acquired using a Nikon TiE-A1 plus laser confocal microscope. To analyze the co-localization of Ptchd1 and Rab3, the percentage of double-positive signals of Ptchd1 and Rab3 was quantified in Rab3-positive synapses labeled with synaptic vesicle proteins (mCherry positive). This experiment used three mice, with five visual fields analyzed for each brain region from each mouse.

[0066] Pre-embedding immunogold labeling and electron microscopy analysis

[0067] For pre-embedding immunogold electron microscopy analysis, Ptchd1-3×HA-P2A-YFP mice were perfused with an ice bath fixative (4% paraformaldehyde, 0.1% glutaraldehyde) via cardiac perfusion. The brain was isolated, post-fixed overnight at 4°C in the same fixative, and coronal sections were prepared. Sections were incubated overnight at 4°C with primary antibody (rat anti-HA), followed by incubation with gold-labeled Fab' fragment goat anti-rat IgG. The gold labeling was then enhanced for 5-10 minutes at room temperature in the dark using a silver enhancement kit. Sections used for electron microscopy analysis were post-fixed with 1% osmium tetroxide, stained with 1% uranium acetate, dehydrated with graded ethanol, cleared with propylene oxide, and finally embedded using an EMBED-812 embedding kit. Ultrathin sections (70-80 nm thick) were prepared from the thalamic reticular nucleus (TRN) and thalamic relay nucleus regions, mounted on 50-mesh copper mesh coated with collodion, and stained with lead citrate. Copper meshes were observed using a transmission electron microscope (JEOL 1230) with an accelerating voltage of 80 kV, and digital photomicrographs were acquired using a Gatan 2048×2048 CCD camera (Orius 830 camera). Three biologically independent mice were used in the experiment.

[0068] Liquid chromatography-mass spectrometry analysis

[0069] To compare the thalamic proteomes of Ptchd1 knockout mice and wild-type control mice, membrane proteins were first enriched from thalamic tissue using a membrane protein extraction kit. Supernatants containing dissolved membrane proteins and membrane-associated proteins were collected and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using a Bruker Daltonics timsTOF Pro2 mass spectrometer. Data were acquired in data-independent acquisition (DIA) mode using the diaPASEF method. Raw data processing, peptide identification, and quantification were performed using Spectronaut 19 software (Biognosys AG). Differential protein expression analysis between genotypes was then performed using the DAVID bioinformatics database. Each group contained three biological replicates.

[0070] Correlated light microscopy-serial scanning electron microscopy (CoLSSEM) analysis

[0071] Four weeks prior to sacrifice, Ptchd1 knockout mice and their littermates were injected with rAAV-hSyn-Synaptophysin-mCherry virus into the thalamic reticular nucleus (TRN) using the method described above. Subsequently, the mice were perfused with an ice-cold fixative solution (4% paraformaldehyde, 0.1% glutaraldehyde, pH 7.4). The brain was isolated, and coronal sections (50 μm thickness) were prepared using a Leica VT1200S vibratory microtome. Floating sections were counterstained with DAPI (Sigma-Aldrich, catalog number D9542) to the cell nuclei, and then imaged under an Olympus FV3000 laser confocal microscope. For electron microscopy processing, samples were osmium-treated using a reduced osmium tetroxide-thiocarbazide (TCH)-osmium tetroxide staining method. The sections were then dehydrated with graded ethanol, cleared with propylene oxide, and embedded in Epon 812 resin. Epon masses were trimmed using a diamond scalpel (DiATome, trimming blade 45°) and serially sectioned (nominal thickness 50 nm) using an ultrathin diamond scalpel (DiATome, ultrathin blade 35°) on a Leica ultramicrotome (UC7). More than 200 serial sections (approximately 400 μm wide and 500 μm long) were collected onto a pre-fabricated silicon wafer. Regions of interest were identified in the sections by observing landmarks such as blood vessels or cell nuclei. Scanning electron microscopy (SEM) imaging was performed using a Zeiss Gemin 300 Sigma field emission scanning electron microscope equipped with a backscattered signal detector. An image series covering the 200 sections was recorded using ATLAS 5 software.

[0072] All acquired image stacks were digitally processed, aligned, and reconstructed using the TrackEM2 plugin in Fiji software (NIH). Presynaptic ultrastructures labeled with synaptophysin-mCherry fluorescence signals were identified and analyzed. To achieve registration of the light microscopy (LM) and scanning electron microscopy (SEM) datasets, manual co-registration of the two 3D reconstructed models was performed in Dragonfly software (Comet Technologies, Canada) by rotation and translation, using the SEM volume as a spatial reference. The light microscopy reconstructed model was adjusted to improve correlation accuracy. This enabled the generation of 3D reconstructed models of the synaptic structures of interest, overlaid with the corresponding fluorescence images.

[0073] primary neuron culture of thalamus

[0074] Primary mouse thalamic neuron cultures were prepared from newborn mice. On the same day, E15.5-E16.5 mice from wild-type and Ptchd1 knockout groups were euthanized by decapitation, and the entire thalamic tissue containing the thalamic reticular nucleus (TRN) was immediately isolated. Cells were isolated and seeded onto coverslips in a minimum essential medium (MEM) supplemented with 10% FetalSelect bovine serum (Gibco, catalog number 10099141C), 0.1 mg / mL transferrin, 2 mM L-glutamine, 0.5% glucose, 0.02% sodium bicarbonate, and 25 μg / mL insulin. The cultures were incubated in a 37°C tissue culture incubator. After approximately 24 hours, the inoculation medium was replaced with growth medium consisting of minimum essential medium (MEM) supplemented with 5% fetal bovine serum, 0.1 mg / mL transferrin, 0.5 mM L-glutamine, 0.5% glucose, 0.02% sodium bicarbonate, and 2% B-27 supplement. On day 3 of in vitro culture, depending on cell growth, 50% or 75% of the medium was replaced with growth medium supplemented with 4 μM cytosine β-D-arabinofuranoside (AraC) to inhibit glial cell growth. Whole-cell patch-clamp recordings were performed on days 16–18 of in vitro culture.

[0075] Preparation of brain slices for patch-clamp recording

[0076] For experiments involving whole-cell patch-clamp manipulation, adult (>8 weeks old) male wild-type or Ptchd1 knockout mice were used. All brain slices were prepared at a coronal angle. Animals were anesthetized with a ketamine compound anesthetic (50 mg / kg body weight) and then decapitated. Brain slices were rapidly separated and immersed in an ice bath of an oxygenated (95% O2 and 5% CO2) sucrose-based slice solution. The chemical composition of this solution was similar to that of artificial cerebrospinal fluid (ACSF), except that sucrose was used instead of sodium chloride. Subsequently, in the ice bath slice solution, the brain slices were cut using a Leica VT1200S vibratory microtome (Germany) to a thickness of 300 μm. The slices were incubated at 34.5°C for 40 minutes and then stored at room temperature. The standard artificial cerebrospinal fluid (ACSF) consists of the following components: 126 mM sodium chloride, 2.5 mM potassium chloride, 2 mM magnesium sulfate, 2 mM calcium chloride, 26 mM sodium bicarbonate, 1.25 mM sodium dihydrogen phosphate, and 25 mM glucose, with an osmotic pressure of 315 mOsm / L and a pH of 7.4. The ACSF is continuously aerated with 95% O2 and 5% CO2 to maintain equilibrium. In IPSC recordings, an additional 1.5 mM kynurenine is added to the solution to block excitatory synaptic transmission.

[0077] Whole-cell patch-clamp recording

[0078] Electrophysiological recordings of cultured thalamic neurons were performed on days 16–18 of in vitro culture, following the previously described method. The resistance of the pulled glass electrodes was 2–5 MΩ. The intracellular fluid composition (for inhibitory postsynaptic current (IPSC) recording) was as follows (mM): 40 CsCl, 90 potassium gluconate, 1.8 NaCl, 1.7 MgCl2, 3.5 KCl, 0.05 EGTA, 2 Mg-ATP, 0.4 Na2-GTP, 10 creatine phosphate, 4 QX314-Cl, 10 HEPES-CsOH (pH 7.2, osmolarity 300 mOsm). Cells were clamped at -70 mV for both inhibitory postsynaptic current (IPSC) and sucrose-induced inhibitory postsynaptic current (IPSC) recordings. Series resistance was monitored during recording and compensated to 3–5 MΩ; cells with uncompensated series resistance exceeding 15 MΩ were discarded. The extracellular fluid composition was as follows (mM): 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES-NaOH (pH 7.4, osmolarity 300 mOsm). All recordings were performed at room temperature (20–24°C). For inhibitory postsynaptic current (IPSC) recordings, D-APV (50 μM, NMDA receptor antagonist) and CNQX (20 μM, AMPA receptor antagonist) were added to the extracellular fluid. Action potentials were induced using a bipolar focusing stimulation electrode made of nichrome wire. For sucrose-induced inhibitory postsynaptic current (IPSC) recordings, TTX (1 μM), CNQX (20 μM), and D-APV (50 μM) were added to the extracellular fluid. The readily releasable pool (RRP) was estimated by injecting 500 mM sucrose solution at a rate of 10 μL / min for 10 seconds using a microsyringe pump. Data were sampled at 5 kHz and low-pass filtered at 2 kHz using an Axon 700B Multiclamp amplifier and digitized using a Digidata 1550B digitizer. All data acquisition and analysis were performed using pClamp10 software.

[0079] For brain slice recording, the slices were placed in a heated recording chamber, and the temperature was maintained at approximately 34.5°C–35.5°C during whole-cell patch-clamp recording. Oxygenated artificial cerebrospinal fluid (ACSF) was continuously aspirated through the slices at a flow rate of 2 mL / min. Neurons in the thalamic reticular nucleus (TRN) were distinguished from other neurons by fluorescence under an upright infrared differential interference contrast (DIC) microscope (BX51WI, Olympus Corporation, Japan). Voltage-clamped or current-clamped recording was performed using a MultiClamp 700B amplifier (Molecular Devices, USA).

[0080] The electrode internal solution used for recording membrane potential responses during current injection consisted of the following components: 140 mM potassium gluconate, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 0.2 mM EGTA, and 2 mM Na2ATP. The pH was adjusted to 7.23 and the osmotic pressure to 315 mOsm / L using KOH. 0.2% biocytin was added to the electrode internal solution for tracking and labeling the recorded cells. The high-chlorine electrode internal solution used for recording inhibitory postsynaptic currents (IPSCs) consisted of the following components: 71 mM KCl, 72 mM potassium gluconate, 2 mM MgCl2, 10 mM HEPES, 0.025 mM BAPTA, 2 mM Na2ATP, and 0.2% biocytin. The pH was adjusted to 7.23 and the osmotic pressure to 315 mOsm / L using CsOH. For double-pulse ratio (PPR) recording, the stimulating electrodes are placed 100 to 200 μm away from the neuron being recorded. The stimulation threshold is defined as the minimum current intensity required to induce an inhibitory postsynaptic current (IPSC). Double-pulse ratio (PPR) is induced with a stimulation intensity twice the threshold, with a stimulation interval of 20 ms. The impedance of the recording electrodes is typically 7–9 MΩ, and the impedance of the stimulating electrodes is 5–7 MΩ. Current and voltage signals are low-pass filtered at 10 kHz and sampled at 25 kHz through a 1401 data acquisition interface and Spike2 software (version 8.0, Cambridge Electronics Design Ltd., UK).

[0081] Electrophysiological Experiment Analysis

[0082] During data collection and analysis, genotype and treatment were blinded. Action potential (AP) waveform analysis included measuring peak amplitude (defined as the voltage difference between the action potential threshold (dV / dt = 20 V / s) and its peak value). The action potential half-width was calculated as the duration at which half the action potential amplitude occurred. Individual inhibitory postsynaptic current (IPSC) events were detected using MiniAnalysis software (RRID: SCR_002184), and analysis of spontaneous inhibitory postsynaptic currents (sIPSCs) was based on a 120-second recording period.

[0083] Statistical analysis

[0084] All experiments included at least three independent biological replicates, performed in a randomized order. Behavioral testing, presynaptic knot analysis, and electrophysiological recordings were all administered and analyzed using a double-blind method. Statistical analysis was performed using GraphPad Prism 9 software, with detailed descriptions of the specific tests used in each figure caption. Significance was expressed as a corrected p-value. All data are presented as mean ± standard error (mean ± SEM).

[0085] Experimental results:

[0086] Ptchd1 is primarily located in presynaptic vesicles of TRN neurons and is enriched in the TRN synaptic terminals of the thalamic relay nuclei (VP, PO, CT), such as... Figure 2 As shown in A~D;

[0087] Immunoelectron microscopy and colocalization experiments confirmed that Ptchd1 colocalizes with the presynaptic vesicle marker Rab3; for example... Figure 2 As shown in E~G;

[0088] In the thalamic tissue of Ptchd1 KO mice, the number of releaseable vesicles at TRN-derived inhibitory presynaptic terminals was significantly reduced, with both the total number of vesicles and the terminal volume decreasing (verified by CoLSSEM 3D reconstruction). Figure 3 Shown by A~I;

[0089] Electrophysiological experiments showed that the frequencies of inhibitory postsynaptic currents (IPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) in the thalamic neurons of KO mice were significantly reduced, while the amplitude and dynamic characteristics remained unchanged, suggesting a decrease in the efficiency of presynaptic vesicle transmission and release. Figure 3 As shown in JP.

[0090] The results of this embodiment indicate that knocking out Ptchd1 leads to abnormal synaptic function. Therefore, promoting Ptchd1 expression or Ptchd1 promoters can be used for: 1) the treatment of sleep disorders; 2) the treatment of neurodevelopmental disorders (ADHD / autism), improving attention and sensory gating problems in patients with ADHD or autism.

[0091] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from this application, and these improvements and additions should also be considered within the scope of protection of this application.

Claims

1. Application of Ptchd1 as a detection target in screening drugs for precision anesthesia.

2. Application of reagents that inhibit Ptchd1 expression or activity in the preparation of drugs for promoting post-anesthesia recovery.

3. Use according to claim 2, characterized in that, The reagents that inhibit Ptchd1 expression or activity include: i) any one or more of shRNA, siRNA, ASO and delivery vectors of said shRNA, siRNA, ASO and CRISPR / Cas system that specifically inhibit Ptchd1 expression; ii) any one or more of neutralizing antibodies, aptamers and small molecule inhibitors that block the binding of Ptchd1 to its binding protein (Rab3).

4. The use of reagents that promote Ptchd1 expression or activity in the preparation of reagents or drugs for prolonging anesthesia time.

5. The use of claim 4, wherein the agent that promotes Ptchdl expression or activity comprises: A combination of one or more of the following: small molecule compounds, Ptchd1 gene delivery vectors, and Ptchd1 protein agonists.

6. Use according to claim 3 or 5, characterized in that, The delivery vector may be a viral vector or a non-viral vector.

7. Use according to claim 6, characterized in that, The viral vector is selected from one or more combinations of retroviral vectors, adenovirus vectors, and adeno-associated virus vectors; and / or, the non-viral vector includes mRNA liposomes.

8. Use according to any one of claims 2 to 5, characterized in that, The drug comprises a pharmacologically active ingredient and pharmaceutically acceptable excipients, wherein the pharmacologically active ingredient contains an agent that inhibits the expression or activity of Ptchd1.

9. Use according to claim 8, characterized in that, The pharmaceutically acceptable excipients are one or more of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, and buffers.

10. Use according to claim 8, characterized in that, The drug is available in oral or injectable form.

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