Neuron toxic compound screening method based on brain organoid model

Abstract

The invention discloses a neuron toxic compound screening method based on a brain organoid model, and belongs to the technical field of chemoinformatics. The method comprises the following steps: constructing stably transferred human embryonic stem cell lines and dividing the stably transferred human embryonic stem cell lines into two groups, namely a first cell line and a second cell line; inserting a first expression element into a genome of a cell of the first cell line by using a gene editing technology, wherein the first expression element expresses neurons for labeling activation; inserting the second expression element into a genome of a cell of a second cell line by using a gene editing technology, wherein the second expression element is used for realizing measurability of neuronal activity; performing differentiation culture on the edited first cell line and the edited second cell line to respectively obtain a first brain organ and a second brain organ; the first brain organoid and the second brain organoid are used as experimental carriers, a neuron toxic compound screening platform is constructed, and the neuron toxic compound screening platform is tested.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical informatics, and in particular to a method for screening neurotoxic compounds based on a brain organoid model. Background Art

[0002] Since brain organoids can better simulate the structure and some functions of the human brain, in order to overcome the shortcomings of existing animal models and 2D cultured cell line models in simulating and studying neurological diseases, researchers created a 3D culture-based brain organoid culture system.

[0003] The existing brain organoid culture method is achieved by culturing human embryonic stem cells / induced pluripotent stem cells into brain organoids, culturing them in neural induction medium, differentiating them into neural ectoderm, and then embedding them in matrix gel, and amplifying neural epithelial buds by shaking culture in neural differentiation medium.

[0004] For example, the patent application with publication number: CN116864027A discloses a method, device and storage medium for screening potential neuron cell toxic chemicals, including: obtaining neuron cell toxicity data; cleaning the neuron cell toxicity data; dividing the neuron cell toxicity data; obtaining a classifier; constructing a weighted integrated model based on the classifier; and screening the neuron cell toxicity data using the weighted integrated model. The present application provides a method, device and storage medium for screening potential neuron cell toxic chemicals. A weighted integrated model NeuTox with a publicly available web server has been developed, which can be used to predict the potential neuron cell toxicity of a compound.

[0005] For example, the patent application with publication number: CN116612833A discloses a method for intelligent screening of active ingredients of traditional Chinese medicine for treating Alzheimer's disease, including: using Olig2 and GSK-3β as targets, through artificial intelligence-assisted precise design, screening out innovative small molecules of traditional Chinese medicine that reduce phosphorylated tau protein and their combination lead compounds, which can not only reduce investment in drug development, save limited experimental resources, and reduce blind waste, but also improve the success rate and efficiency of drug design on the basis of traditional computer-aided drug design, and shorten the drug development cycle; establish a new in vitro cell model of AD patient neurons, conduct in vitro efficacy evaluation, which is closer to the real process of Alzheimer's disease, can reveal major pathological mechanisms hidden in animal models, and help to develop more targeted active compounds of traditional Chinese medicine; establish an AD patient neuronal cell chimeric mouse model App / ps1, conduct efficacy evaluation of traditional Chinese medicine in living animals, and find traditional Chinese medicine lead drugs with significant therapeutic effects.

[0006] However, in the process of implementing the technical solution of the invention in the embodiments of the present application, the present application found that the above technology has at least the following technical problems:

[0007] In the prior art, when screening drugs or testing the toxicity of various compounds to neurons, the screening efficiency of candidate drugs is low, and there is a problem of low efficiency in developing treatment plans. Summary of the invention

[0008] The embodiments of the present application provide a method for screening neurotoxic compounds based on a brain organoid model, thereby solving the problems of low efficiency in screening candidate drugs and low efficiency in developing treatment plans when screening drugs or testing the toxicity of various compounds to neurons in the prior art. The method realizes real-time monitoring of neuronal activity and long-term observation of the dynamic changes of neuronal activity in organoids and the response to drug treatment without damaging the organoids.

[0009] An embodiment of the present application provides a method for screening neurotoxic compounds based on a brain organoid model, comprising the following steps: constructing a stably transformed human embryonic stem cell line and dividing it into two groups, namely a first cell line and a second cell line; using gene editing technology to insert a first expression element into the genome of cells of the first cell line, wherein the first expression element is used to mark activated neurons; using gene editing technology to insert a second expression element into the genome of cells of the second cell line, wherein the second expression element is used to achieve measurable neuronal activity; differentiating and culturing the edited first cell line and the edited second cell line to obtain a first brain organoid and a second brain organoid, respectively; using the first brain organoid and the second brain organoid as experimental vectors to construct a neurotoxic compound screening platform; testing the neurotoxic compound screening platform to obtain verification results and provide feedback.

[0010] Furthermore, the first expression element is used to mark the activated neurons specifically: after the first expression element is expressed, it indicates that the neuron cells have matured and produced fluorescent substances.

[0011] Furthermore, the first expression element is specifically: an E-SARE enhancer element in series with an ArcMini promoter in series with a fluorescent substance element.

[0012] Furthermore, the fluorescent substance element is specifically a fluorescent protein element, and the fluorescent protein element produces a specific color after expression.

[0013] Furthermore, the second expression element is used to achieve measurable measurement of neuron activity as follows: the second expression element is expressed to produce substance one only after the neuron matures, and the second expression element produces substance two during the entire cell growth stage, and the activity of the neuron can be measured by detecting the amount of substance one and substance two.

[0014] Furthermore, the second expression element includes: a promoter associated with neuron activation, a sub-element one expressed after being activated by a promoter associated with neuron activation, a promoter that is continuously and stably expressed in cells, and a sub-element two expressed after being activated by a promoter that is continuously and stably expressed in cells; the sub-element one produces substance one after expression, and the sub-element two produces substance two after expression.

[0015] Furthermore, the sub-element 1 is specifically: E-SARE enhancer element in series with ArcMini promoter in series with Gaussia luciferase.

[0016] Furthermore, the second sub-element is specifically: CAG promoter tandemly connected to secretory luciferase Cypridina luciferase.

[0017] Furthermore, the substance one is specifically: Gaussia luciferase produced by expressing an E-SARE enhancer element in series with an ArcMini promoter in series with Gaussia luciferase; the substance two is specifically: Cypridina luciferase produced by expressing a CAG promoter in series with a secretory luciferase Cypridina luciferase.

[0018] Furthermore, the platform for screening neurotoxic compounds is tested, specifically: for the first cerebral organoid, no neurotrophic factors are added in the early stage of culture, and neurotrophic factors are added when the neurons are mature, and the neuronal activity is verified by comparing the fluorescence expression; for the second cerebral organoid to which neurotrophic factors are added at different culture stages and after the cultured neurons are mature, Gaussia luciferase and Cypridina luciferase are collected, and the values ​​of chemical fluorescence generated by the action of the corresponding substrates are collected on a microplate reader, and then the ratio of the two is analyzed to obtain the changing trend of the ratio of Gaussia protein and Cypridina protein expression, and the activity state of the neurons is judged by comparing the changing trend of the ratio of Gaussia protein and Cypridina protein expression, thereby verifying the reliability of the platform; the neurotrophic factors include: BDNF and GDNF.

[0019] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0020] 1. The present invention provides a method for screening neurotoxic compounds based on a brain organoid model, thereby achieving real-time monitoring of neuronal activity and long-term observation of the dynamic changes of neuronal activity in organoids and responses to drug treatment without damaging the organoids. This allows for effective screening of candidate drugs at an earlier stage, reducing the time spent on failed projects and the overall cost of drug development.

[0021] 2. The present invention constructs a brain organoid model that can visualize and high-throughput screen neuronal activity by utilizing multiple reporter genes such as fluorescence and luciferase, thereby better characterizing the changes in neuronal activity and constructing a high-throughput platform for neuronal activity screening based on brain organoids.

[0022] 3. The present invention connects the E-SARE promoter in series with Gaussia, the CAG promoter in series with Cypridina, and uses Cypridina as an internal reference. The changes in neuronal activity are observed by the changes in the ratio of luciferase fluorescence, thereby achieving real-time monitoring of neuronal activity and dynamic analysis of neuronal activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A flow chart of a method for screening neurotoxic compounds based on a brain organoid model provided in an embodiment of the present application.

[0024] Figure 2 The present application provides an embodiment of a platform for screening neurotoxic compounds based on brain organoids.

[0025] Figure 3 This is the first expression element map provided in the examples of this application.

[0026] Figure 4 This is a second expression element map provided in the examples of this application.

[0027] Figure 5 Validation diagram of the brain organoid neurotoxic compound screening platform provided in the examples of the present application.

[0028] Figure 6 The embodiments of the present application provide a diagram for testing neurological compounds based on a brain organoid model.

[0029] Figure 7 The embodiments of the present application provide a diagram for testing neurological compounds based on a brain organoid model. DETAILED DESCRIPTION

[0030] The embodiments of the present application provide a method for screening neurotoxic compounds based on a brain organoid model, thereby solving the problems in the prior art of low screening efficiency for candidate drugs and low efficiency in developing treatment plans when screening drugs or testing the toxicity of various compounds to neurons. By establishing a screening platform for neurotoxic compounds based on brain organoids, rapid and simple drug screening and testing of the toxicity of various compounds to neurons are achieved, and candidate drugs can be effectively screened at an earlier stage, reducing the time on failed projects and the overall cost of drug development.

[0031] The technical solution in the embodiment of the present application is to solve the above-mentioned problem of low efficiency in screening candidate drugs and low efficiency in developing treatment plans when screening drugs or testing the toxicity of various compounds to neurons. The overall idea is as follows:

[0032] A stably transformed human embryonic stem cell line is constructed and divided into two groups, namely the first cell line and the second cell line; a first expression element is inserted into the genome of cells of the first cell line using gene editing technology, and the first expression element is used to mark activated neurons; a second expression element is inserted into the genome of cells of the second cell line using gene editing technology, and the second expression element is used to achieve measurable neuronal activity; the edited first cell line and the edited second cell line are differentiated and cultured to obtain a first cerebral organoid and a second cerebral organoid, respectively; the first cerebral organoid and the second cerebral organoid are used as experimental vectors to construct a neurotoxic compound screening platform; the neurotoxic compound screening platform is tested to obtain verification results and provide feedback.

[0033] The first expression element is used to mark the activated neurons specifically: after the first expression element is expressed, it indicates that the neuron cells have matured and produced fluorescent substances.

[0034] The first expression element is specifically: an E-SARE enhancer element connected in series with an ArcMini promoter connected in series with a fluorescent substance element.

[0035] The fluorescent substance element is specifically a fluorescent protein element, which produces a specific color after being expressed.

[0036] The second expression element is used to achieve measurable measurement of neuron activity: the second expression element is expressed to produce substance 1 only after the neuron matures, and the second expression element produces substance 2 during the entire cell growth stage, and the activity of the neuron can be measured by detecting the amount of substance 1 and substance 2. Substance 1 and substance 2 can be various detectable luciferases or other similar detectable and labeled substances.

[0037] The second expression element comprises: a promoter associated with neuron activation, a sub-element 1 expressed after being activated by the promoter associated with neuron activation, a promoter stably and continuously expressed in cells, and a sub-element 2 expressed after being activated by the promoter stably and continuously expressed in cells;

[0038] The sub-element 1 produces substance 1 after being expressed, and the sub-element 2 produces substance 2 after being expressed.

[0039] The sub-element 1 is specifically: E-SARE enhancer element in series with ArcMini promoter in series with Gaussia luciferase.

[0040] The second sub-element is specifically: CAG promoter tandem secretory luciferase Cypridina luciferase.

[0041] The substance 1 is specifically: Gaussia luciferase produced by the expression of E-SARE enhancer element in series with ArcMini promoter in series with Gaussia luciferase;

[0042] The second substance is specifically: Cypridina luciferase produced by expressing the secretory luciferase Cypridina luciferase in series with the CAG promoter.

[0043] The neurotoxic compound screening platform is tested as follows:

[0044] For the first cerebral organoid, no neurotrophic factors were added in the early stage of culture. Neurotrophic factors were added when the neurons matured, and the neuronal activity was verified by comparing fluorescence expression;

[0045] For the second cerebral organoids at different culture stages and after the neurons to be cultured mature, add neurotrophic factors, collect Gaussia luciferase and Cypridina luciferase, and collect the values ​​of chemical fluorescence produced by the corresponding substrate on a microplate reader. Then analyze the ratio of the two to obtain the changing trend of the ratio of Gaussia protein and Cypridina protein expression. By comparing the changing trend of the ratio of Gaussia protein and Cypridina protein expression, the activity state of neurons can be judged, thereby verifying the reliability of the platform;

[0046] The neurotrophic factors include: BDNF and GDNF.

[0047] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0048] like Figure 1 As shown, it is a flow chart of a method for screening neurotoxic compounds based on a brain organoid model provided in an embodiment of the present application, the method comprising the following steps: constructing a stably transfected human embryonic stem cell line and dividing it into two groups, namely a first cell line and a second cell line;

[0049] The method for constructing a stable human embryonic stem cell line is as follows: Matrigel is mixed with DMEM / F12 culture medium to prepare a matrix for cell culture, and Matrigel is evenly distributed in the culture wells by refrigeration and mixing; the frozen cells are revived and resuspended in the culture medium, and then the cells are transferred to the culture dish treated with Matrigel and Y27632 is added; EDTA is used to remove the binding of cells to the culture dish wall, and fresh culture medium is used to pass the cells; the remaining cells after passaging are frozen and stored as a human embryonic stem cell line. Matrigel is a basement membrane matrix extracted from Engelbreth-Holm-Sarm (EHS) mouse sarcoma, which is used for a variety of cell cultures; DMEM / F12 is a widely used basal culture medium, especially suitable for the culture of a variety of mammalian cells. When plating, take Matrigel out of a -20°C or lower refrigerator and place it in a 4°C refrigerator to thaw overnight, ensuring that Matrigel thaws slowly and evenly to avoid bubbles and local overheating; when Matrigel is completely thawed, dispense it into sterile centrifuge tubes or cryopreservation tubes according to experimental requirements, and then put Matrigel back into a -20°C refrigerator for storage; take out 12 mL of DMEM / F12 culture medium from the refrigerated environment, pour it into a 15 mL sterile centrifuge tube, prepare two 6-well plates, and aspirate 1 mL of DME in the centrifuge tube. Gently blow M / F12 until Matrigel is thawed and mixed, aspirate the thawed and mixed Matrigel and add it to the remaining DMEM / F12 in the centrifuge tube, and use a 10mL pipette to repeatedly blow and mix again; dispense the mixed Matrigel-DMEM / F12 solution into a 6-well plate, 1mL per well, shake gently to mix so that the solution is evenly distributed in the well, wrap it with sealing film to prevent contamination and water evaporation, and put the entire 6-well plate back into a 4°C refrigerator overnight. This will allow Matrigel to form a uniform gel-like film at the bottom of the well to prepare for subsequent cell inoculation.

[0050] Y27632 is a small molecule inhibitor of Rho-associated protein kinase (ROCK). Its full name can also be Y-27632. Y27632 can prevent stem cell apoptosis and improve cloning efficiency in stem cell research and organoid culture, playing a key role. When the cells are revived, take out the cells from -80℃ or liquid nitrogen and temporarily store them in dry ice. This can keep the cells in a low temperature state and prevent the cells from being damaged during the thawing process before being taken out of the water bath. Then, gently shake them in a 37℃ water bath until they are basically melted. Rapid heating will melt the extracellular crystals quickly, while preventing water from infiltrating into the cells and causing damage to the cells due to recrystallization. Transfer all the liquid to a 50mL centrifuge tube, shake gently and add 3mL of culture medium. The thawed cell suspension can be mixed with the culture medium for subsequent centrifugation and resuspension operations. Place the centrifuge tube in a centrifuge and centrifuge it for 2 minutes at a centrifugal force of 200g. After the centrifugation is completed, take out the centrifuge tube and discard the supernatant. Remove the cells by centrifugation. The freezing solution and possible impurities in the cell suspension were removed, and the cell pellet was collected at the same time; an appropriate amount of culture medium preheated to room temperature was added to the centrifuge tube, and the cell pellet was gently blown with a pipette to completely resuspend it, and then the resuspended cell suspension was transferred to a culture dish treated with Matrigel to promote cell adhesion and growth; according to the cell type and experimental requirements, Y27632 (10mM) was added to the culture medium at a ratio of 1:1000, and the cells were evenly distributed to improve the cell growth state, and the cells were more evenly distributed in the culture dish by cross shaking, and the culture dish was placed in a constant temperature incubator with a preset temperature of 37°C to provide a suitable growth environment for the cells.

[0051] EDTA, the full name of which is ethylenediaminetetraacetic acid, is an important chelating agent widely used in the fields of chemistry, medicine, biology, etc. In biology and cell culture, EDTA is often used as an auxiliary agent for cell separation and digestion. It combines with divalent cations such as calcium ions and magnesium ions on the cell surface, thereby destroying the adhesion between cells and culture dishes, making it easier for cells to fall off the culture dishes. Fresh culture medium refers to culture medium that has been prepared shortly after it has been stored for a long time and maintains its original quality and sterility. In order to maintain the freshness of the culture medium, it should be sterilized immediately after preparation (such as high-pressure sterilization) and stored under appropriate conditions to prevent microbial contamination and loss of nutrients. When performing cell passaging, remove the original culture medium in the culture dish by aspirating the culture medium so as to perform subsequent cell separation operations; according to the size of the culture dish and the density of the cells, add an appropriate amount of 0.5μM EDTA to cover the entire culture dish, ensuring that the entire bottom of the culture dish is covered with EDTA solution; place the culture dish with EDTA in a 37°C constant temperature incubator or water bath, wait for 4 minutes, so that EDTA has enough time to combine with the ions on the cell surface, thereby effectively destroying the adhesion between the cells and the culture dish; after aspirating EDTA, the adhesion between the cells and the culture dish has been destroyed, and then use fresh culture medium to gently blow the cells off the wall of the dish and suspend them in the culture medium, and then pass the separated cells according to experimental requirements and spread them into new culture dishes.

[0052] 2 is a serum-free cell cryopreservation medium that is widely used in the cryopreservation of various animal cell lines and tumor cell lines. 2 Gently pipette down the cells and freeze 200-300 μL per tube.

[0053] Using gene editing technology to insert a first expression element into the safe site AAVS1 of the genome of cells of the first cell line, wherein the first expression element is used to mark activated neurons; using gene editing technology to insert a second expression element into the safe site AAVS1 of the genome of cells of the second cell line, wherein the second expression element is used to achieve measurable neuronal activity;

[0054] The AAVS1 site is a specific genomic safe harbor site located on human chromosome 19. Due to its low mutation rate and genotoxicity, the AAVS1 site is often used as a target for gene editing, especially when stable integration of exogenous genes is required.

[0055] Differentiating and culturing the edited first cell line and the edited second cell line to obtain a first cerebral organoid and a second cerebral organoid, respectively;

[0056] The edited first cell line, the edited second cell line and the gRNA vector expression element are electroporated into the H9 human embryonic stem cell line, and drug screening is performed using Puro. Single clones are selected for subculture, and when the cell fusion degree reaches 80%, the first cerebral organoid and the second cerebral organoid can be obtained.

[0057] The E-SARE, ArcMini, d2GFP and PURO gene sequences were introduced into the expression element, so that the green fluorescent protein was stably expressed after neuron activation, and the first expression element (AAVS1-Puro-E-SARE-ArcMini-d2GFP) was obtained. The map of the first expression element is shown in Figure 3 As shown; Gaussia luciferase is a monomeric enzyme with a length of 185 amino acids, which can be released from the endoplasmic reticulum to the extracellular space. It can catalyze the oxidation reaction of the substrate coelenterazine and emit 480nm fluorescence. Cypridina luciferase is a luciferase composed of 555 amino acids with a molecular weight of 62.171kDa. Its substrate is vargulin, which can oxidize the substrate to produce blue light. By connecting different promoters in series before these two luciferases, quantifiable neuronal activity can be achieved. The second expression element (AAVS1-Puro-E-SARE-ArcMini-Gaussia-CAG-Cypridina) is as shown Figure 4 As shown in the figure, the gRNA vector expression element is usually constructed in the laboratory through molecular cloning technology. It contains the key component of the gene editing system, the gRNA sequence. The gRNA can specifically recognize and bind to the target DNA sequence, which is the key to achieving the accuracy of gene editing. Puro (Puromycin, Chinese name puromycin) is an aminoglycoside antibiotic isolated from the bacteria Streptomyces (such as Streptomyces alboniger).

[0058] E-SARE (Enhanced Synaptic Activity-Responsive Element) is an enhanced synaptic activity response element, which plays a key role in gene expression as a regulatory element (promoter); Arc gene plays an important role in the nervous system, and it is involved in synaptic plasticity, learning and memory consolidation, and addiction. Under normal circumstances, Arc will be activated normally after the neurons mature, but ArcMini inhibits the normal operation of Arc after the neurons mature, and E-SARE can specifically respond to the synaptic activity of neurons, thereby driving the expression of ArcMini gene, so the tandem E-SARE activates the expression of ArcMini; d2GFP is a short-lived green fluorescent protein variant, which retains the fluorescence characteristics of GFP (green fluorescent protein) with a shorter half-life and faster degradation rate, which makes d2GFP an ideal reporter gene for real-time monitoring of gene expression levels and protein dynamic changes. Expression element 1 (E-SARE-ArcMini-d2GFP) refers to the ArcMini gene expression system driven by the E-SARE regulatory element promoter, with the short-lived green fluorescent protein d2GFP connected in series after E-SARE as a reporter gene. The design of this expression element enables activated neurons to express green fluorescence, allowing researchers to track the expression level and dynamic changes of the ArcMini gene in real time by monitoring the fluorescence signal of d2GFP.

[0059] Before electroporation, while providing a suitable growth environment for cell culture, prepare the culture dish as needed. Take a 10 cm dish as an example. Add Matrigel to the culture dish one day in advance to ensure that the entire bottom of the culture dish is evenly covered. Place the culture dish in an incubator and incubate at 37°C and 5% CO2 for at least 1 hour to allow Matrigel to gel and firmly attach to the bottom of the culture dish. Discard Matrigel matrix gel and add 9 mL of mTesR and 1 mL of ClonR to optimize cell culture conditions. Rbuffer can maintain the pH value of the solution stable in the experiment, protect DNA from acid-base changes, enhance the permeability of the cell membrane, and promote the entry of expression element DNA into the cell. mTesR is a serum-free culture medium specifically used for human pluripotent stem cell (hPSC) culture. It is known for its defined composition and serum-free characteristics, providing a stable and reliable environment for cell culture. ClonR is a supplement or culture medium component used to promote cell cloning and growth. Taking a 10 cm dish as an example, to prepare the expression element mixture (i.e., the edited first cell line, the edited second cell line and the gRNA vector expression element mixture), 1-5 μg of the expression element and 110 μL of R buffer should be mixed evenly; Accutase is a trypsin esterase, which is mainly used in cell dissociation and cell culture operations. It contains proteolytic enzyme and collagenase activity, which can effectively decompose the intercellular matrix and extracellular matrix, causing the cells to quickly lose attachment and form a single cell suspension.

[0060] When preparing cells for electroporation, first remove the cell culture medium in the 6-well plate to reduce the effect of the components in the culture medium on the subsequent EDTA and Accutase action, add an appropriate amount of 0.5 μM EDTA, and place the 6-well plate in a 37°C incubator for 4 minutes to allow EDTA to fully act; remove the EDTA solution, add 500 μL Accutase to ensure that the entire culture surface is covered, and place the 6-well plate in a 37°C incubator for another 4 minutes. Add an equal amount of culture medium to neutralize the enzyme activity of Accutase to prevent excessive digestion of cells; use a pipette to remove the EDTA solution. Use a pipette to gently blow the culture surface to ensure that all cells are blown down, transfer the cell suspension to a centrifuge tube, centrifuge at 200g for 2 minutes to allow the cells to settle to the bottom of the tube, aspirate the supernatant and retain the pure cell pellet; resuspend the cells with 1mL of culture medium and count the cells using a cell counter to determine the cell concentration; according to the experimental requirements, if the number of electroporated cells corresponding to a 10cm dish is 1 million, aspirate the cell suspension of the corresponding number of cells from the total cell suspension, centrifuge, and resuspend the cells with the prepared expression element mixture in preparation for electroporation.

[0061] When performing cell electroporation, E2 buffer is a special electrolysis buffer used to protect cells from direct damage by the electric field during electroporation and provide a suitable ionic environment to promote the entry of expression element DNA into cells. Pipette, use 100 μL Tip: Slowly pipette the cell expression component mixture and avoid bubbles during pipetting, as bubbles may affect the effect of electroporation. Pipette vertical insertion Tube, make sure there is no spillage or leakage; Tube Insertion In the electroporator, set the required pulse conditions, pulse voltage 1050V, pulse width 30ms, pulse number 2 times, start the electroporation program, and complete the electroporation process; after completion, add the cells to the pre-prepared culture dish and shake crosswise to evenly distribute the cells for subsequent cell culture and experiments.

[0062] in A pipette is a specialized pipetting tool designed specifically for use with Used in conjunction with the electroporation system to accurately measure and transfer the electrolysis buffer containing the cell expression element mixture; Tip is a micropipette head used to absorb and transfer cell expression component mixture to Tube, which is an important part of the electroporation process, ensuring that the cell expression component mixture is accurately transferred to the electroporation environment; Tube is One of the core components in the electroporation system, it is used to contain the cell expression element mixture and provide the necessary electric field environment during the electroporation process. It is usually made of conductive materials and can withstand the effects of high-voltage electric field pulses.

[0063] Using Puro to screen the electroporated cells for drugs can remove unsuccessfully transfected cells and enrich cells that have successfully integrated exogenous DNA, thereby improving the efficiency of gene editing and cell line construction. Since the expression vector contains the puromycin resistance gene (Puro^R), only cells that have successfully integrated the expression vector can survive in a culture medium containing puromycin. Single cells are picked from the drug-screened cells for culture to form monoclonal cell lines, which will carry the same gene editing and expression elements; when the cell fusion reaches 80%, the cells are collected for genotyping, which usually includes PCR amplification, sequencing verification, and possible Southern blot or FISH analysis. Genotyping is used to confirm whether the exogenous DNA has been correctly integrated into the AAVS1 site and to check whether there are unexpected mutations or integration events, which is a key step in verifying the success of gene editing and cell line construction.

[0064] The process of culturing brain organoids is as follows: first, add 150 μL of 1% PVA (polyvinyl alcohol) to each well of the 96-well plate and leave it at room temperature for about 30 minutes. PVA is an anti-adhesion agent used to prevent cells from growing on the bottom of the well plate, thereby prompting cells to aggregate into clusters to form brain organoids. By coating PVA, an environment that is conducive to cell aggregation rather than adhesion can be created; calculate the required culture medium volume with 150 μL per well, add RC reagent at a ratio of 1:100, and add it to a 15mL centrifuge tube for later use. RC reagent contains factors that have an important effect on cell growth, differentiation or aggregation. The presence of these factors in the culture medium can optimize cell culture conditions and promote the formation and development of brain organoids; then aspirate the cell culture medium in the 6-well plate and add an appropriate amount of 0.5 μM EDTA weakens the connection between cells. Incubate at 37°C for 4 minutes, then remove EDTA and add 500μL Accutase (a mild cell dissociation enzyme) to further dissociate the cells. Incubate at 37°C for 4 minutes, then add an equal amount of culture medium to neutralize Accutase, gently blow down the cells to make them completely dispersed, put them in a centrifuge tube, centrifuge at 200g for 2 minutes, remove the supernatant, resuspend the cells with 1mL culture medium, and count the cells. The combined use of EDTA and Accutase can effectively dissociate cells from the culture dish while maintaining the activity and integrity of the cells. The centrifugation and resuspension steps are to remove residual enzymes and impurities in the culture medium, and at the same time adjust the cell density to prepare for subsequent cell counting and distribution; calculate the total number of cells required with 9000 cells per well, aspirate the required cells and add them to the prepared centrifuge tube to mix well. By accurately distributing the cell suspension, it can be ensured that the number of cells in each well is the same, which is conducive to the formation of brain organoids of uniform size; finally, adjust the range of the discharge gun to 150μL, pour the prepared cell suspension into the sample loading groove, remove the substance or coating (Anti-adherence) used to prevent cell adhesion in the 96-well plate, add 150μL of cell suspension to each well, seal the sealing film, and centrifuge at 100g centrifugal force for 5 minutes to redistribute the cells at the bottom of the well plate and aggregate together. Due to the anti-adhesion effect of PVA, the cells will not adhere to the wall but form clumps, which is conducive to the rapid formation and stable development of brain organoids; remove the sealing film and place the well plate in a cell culture incubator for culture.

[0065] Before using Matrigel, put the Matrigel in an ice box in advance and thaw it overnight in a 4°C refrigerator. Cut the sealing film into small pieces and expose it to UV light for later use to keep the Matrigel in liquid state and provide a sterile and easy-to-operate platform. Use a 200μL pipette tip with a small hole to aspirate the brain organoids in the 96-well plate one by one and place them on the prepared sealing film. Use the gun to aspirate the culture medium around the brain organoids in turn to reduce interference with subsequent Matrigel embedding. Then use the pipette tip to take the dissolved Matrigel. Each brain organoid is about 20 μL. In this step, be sure to keep the gun tip clean and avoid letting the brain organoid dry out. Use the gun tip to gently move the brain organoid so that it is in the middle of the Matrigel droplet to ensure that the brain organoid is evenly embedded in the Matrigel, which is beneficial for its subsequent growth and development. Place the culture dish at 37°C for 20 minutes. When the Matrigel is completely solidified, use 20 mL of Imp-A culture medium to blow the Matrigel clot off the sealing film, remove the sealing film with tweezers, and place it in a cell culture incubator for culture.

[0066] On the third day after the brain organoids were made, mTeSR medium was replaced. mTeSR medium is a serum-free medium designed to support the growth of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs); then Neural Induction medium was replaced on the 5th, 7th and 11th days respectively. Neural Induction medium is used to induce stem cells to differentiate into neural lineages. By continuously replacing this medium, the cells in EBs can be gradually guided to differentiate into neural progenitor cells, thereby forming a neuroepithelial-like structure; on the 13th day, Matrigel matrix glue was used for embedding, and the medium was replaced with Imp-A medium. Imp-A medium is a medium that supports neural differentiation. It contains specific growth factors and nutrients that help promote further differentiation of neural progenitor cells and the formation of neural networks; Imp-A medium was replaced on the 16th day. By regularly replacing Imp-A medium, the stability and consistency of the neural differentiation environment can be ensured, which helps Promote the continuous differentiation of neural precursor cells and the further development of neural tissue; replace Imp+A medium on the 21st day. Imp+A medium may contain more ingredients that promote the maturation and function of neural tissue. Using it for culture can further promote the formation of neural networks and the improvement of their functions. Transfer the culture medium to a shaker for culture, which can simulate the microenvironment in the body, such as mechanical stimulation and fluid shear force. These stimuli contribute to the further maturation and function of neural tissue. After that, replace Imp+A medium once a week. Regular replacement of Imp+A medium can remove metabolites and waste, while adding new nutrients and growth factors, which helps maintain cell health and activity.

[0067] The first and second cerebral organoids were used as experimental carriers to construct a screening platform for neurotoxic compounds;

[0068] like Figure 5 The figure shows the validation of the neurotoxic compound screening platform for brain organoids, where A is a representative image of the bright field (BF) and green fluorescence field (GFP) of d2GFP brain organoids on the 20th, 40th, 60th, and 70th days of culture, and B is the immunofluorescence staining of MAP2, GFP, and PSD95 of d2GFP brain organoids on the 70th day, where MAP2: neuronal marker, PSD95: synaptic marker. Scale bar: 50μm, C is the change in the fluorescence ratio of Gaussia / Cypridina in dual-luciferase brain organoids at different time points. n=8. D is the immunoblot of Gaussia and Cypridina proteins in dual-luciferase brain organoids at different time points, and GAPDH is the internal reference protein.

[0069] like Figure 5 As shown in A, no green fluorescence was observed in the early stage of culture (the first 60 days), indicating that without external stimulation or the action of specific factors, the neuronal activity was at a relatively low level. After adding BDNF and GDNF neurotrophic factors on the 60th day, strong green fluorescence expression was observed in the organoid within 8 hours, indicating that BDNF and GDNF can rapidly and significantly enhance the activity of neurons or promote the expression of molecules related to neuronal activity. Figure 5 As shown in B, immunofluorescence staining of d2GFP organoids on day 70 showed that GFP was clearly co-localized with the neuronal marker protein MAP2 and the synaptic marker protein PSD95. The value of bioluminescence produced by the oxidation of the respective substrates catalyzed by luciferase was detected using an ELISA instrument. The magnitude of the fluorescence value indicated the expression level of the two enzymes, as shown in Figure 5 After adding BDNF and GDNF, the fluorescence value increased significantly, and the activity of neurons was significantly enhanced. In addition, WB analysis was performed on organoids at different culture stages, such as Figure 5 The changing trends of Gaussia protein and Cypridina protein expression shown in D are consistent with the results of the microplate reader test. In summary, genetically engineered brain organoids can quickly and sensitively display neuronal activity. If the fluorescence expression is weak in the experiment, the fluorescence signal can be enhanced by using signal amplification technology, such as tyramide signal amplification (TSA) technology. This technology can significantly increase the intensity of the fluorescence signal without increasing the antibody concentration.

[0070] Test the neurotoxic compound screening platform, obtain validation results and provide feedback.

[0071] Among them, the testing of the neurotoxic compound screening platform includes the neurotoxic compound screening platform KCl gradient test and the neurotoxic compound screening platform compound test.

[0072] The steps of the KCl gradient test of the neurotoxic compound screening platform include: adding different concentrations of KCl to the culture medium to increase the extracellular K+ concentration; when the fluorescent and luciferase brain organoids are cultured to the 60th-70th day, the neurotrophic factors BDNF and GDNF are added to activate the neurons in the organoids, and then different concentrations of KCl are added respectively; for luciferase reporter gene brain organoids, the culture medium of the dual luciferase organoids is collected every 48 hours to determine and count the luciferase content; for fluorescent protein reporter gene brain organoids, the fluorescence intensity graph of d2GFP is taken every 48 hours.

[0073] like Figure 6 As shown, it is a diagram of testing neurochemical compounds based on brain organoid model, where A is a schematic diagram of the testing method of neurochemical compounds in brain organoid model, Gau: Gaussia, Cyp: Cypridina, B is the change of luciferase secretion after treatment with different concentrations of KCl, C is the change of green fluorescence after treatment with different concentrations of KCl, where Two-Way ANOVA (two-way analysis of variance, a statistical method used to study the effect of two or more independent variables on one dependent variable), n = 8 (the actual total number of observations is 8). Figure 6 It can be seen that the platform can sensitively reflect the changes in the strength of neuronal activity.

[0074] In this example, since the neuronal membrane potential is very sensitive to changes in extracellular K+ concentration, increasing the extracellular K+ concentration causes the neurons to depolarize, and KCl can excite the neurons. Therefore, by increasing the extracellular K+ concentration, we observe whether the brain organoids can quickly and sensitively respond to changes in neuronal activity, and verify whether the brain organoid model can be used to test the effects of compounds on neuronal activity. The fluorescence intensity of the luciferase reporter gene brain organoid and the fluorescent protein reporter gene brain organoid is shown in Figure 1. Figure 6 As shown in A, in the range of 0-50mM, as the concentration increases, the fluorescence ratio of Gaussia and Cypridina, which indicates neuronal activity, changes as shown in Figure 6 As shown in C.

[0075] The steps of compound testing in the neurotoxic compound screening platform include: adding BDNF and GDNF to activate neurons after the brain organoid neurons have matured; changing the medium and collecting samples after adding 10 μM of the test compound respectively; changing the medium and collecting samples on the first, third and fifth days after adding the compound, and observing the changes in green fluorescence and luciferase.

[0076] like Figure 7 The figure shows the neurochemical compound test based on the brain organoid model, where A shows the changes in luciferase secretion after treatment with different types of compounds. n=8, and B shows the changes in green fluorescence after treatment with different types of compounds.

[0077] In order to further test whether the organoid model can be used to test the effects of compounds on neuronal activity, different compounds were tested using the constructed platform. Among them, the effective chemical components of a variety of FDA-approved neurological disease treatment drugs were selected, including anti-epileptic drugs Gabapentin, 4-A minute opyridine, Rufinamide, local anesthetics Bupivacaine, Parkinson's drugs Rotigotine, Alzheimer's drugs Memantine, etc. Figure 7 As shown in A and B, 4-aminopyridine and Gabapentin had no significant effect on neuronal activity, while Bupivacaine, Rufinamide, Rotigotine and Memantine Hydrochlotide caused a significant decrease in neuronal activity. In contrast, Aripiprazole Lauroxil increased neuronal activity within 72 hours, but decreased after 96 hours; by observing the green fluorescence graph, neurons showed obvious excitement after the addition of the compound, but the toxicity of the compound led to increased cell death, increased cell fragments and decreased fluorescence intensity after 72 hours. At the same time, due to individual differences between brain organoids, changes in luciferase can more accurately and high-throughput observe changes in neuronal activity.

[0078] The above results show that we can use this neuronal activity screening platform to test the effects of different compounds on neurons for a long time without destroying brain organoids, by real-time fluorescence observation or testing the amount of luciferase secreted into the culture medium. In this experiment, no significant changes in neuronal excitability were observed after treatment with 10μM 4-A-opyridine, so neurons may be insensitive to 10μM 4-A-opyridine and show neuroprotective effects. This is consistent with past reports: 50μM and 2500μM 4-A-opyridine have neuroprotective effects on cerebellar granule neurons (CGNs) without obvious toxicity. We observed that after treating brain organoids with 10μM Bupivacaine, the platform showed reduced fluorescence and reduced luciferase secretion. This is consistent with previous reports: In rat hippocampal CA1 neurons, patch clamp was used to observe that Bupivacaine has an inhibitory effect on glycine and glutamate-induced currents. Rufinamide was found in past studies to selectively bind to the intermediate inactived state (Ii) of Na+ channels and inhibit resurgent currents in a dose-dependent manner. Rotigotine and Memantine have a protective effect on excitotoxic necrosis caused by glutamate. They were observed to have an inhibitory effect on neuronal activity in brain organoids. Therefore, the neurotoxic compound screening platform can intuitively observe changes in neuronal activity, and is not limited to 2D cell models, better simulates the in vivo environment, and can be continuously observed under long-term drug administration conditions.

[0079] Furthermore, the first expression element is used to mark the activated neurons specifically: after the first expression element is expressed, it indicates that the neuron cells have matured and produced fluorescent substances.

[0080] In this example, the E-SARE, ArcMini, d2GFP and PURO gene sequences were introduced into the expression element so that the green fluorescent protein was stably expressed after neuron activation, and the first expression element (AAVS1-Puro-E-SARE-ArcMini-d2GFP) was obtained. The map of the first expression element is shown in FIG. Figure 3 shown.

[0081] Furthermore, the first expression element is specifically: an E-SARE enhancer element in series with an ArcMini promoter in series with a fluorescent substance element.

[0082] Furthermore, the fluorescent substance element is specifically a fluorescent protein element, and the fluorescent protein element produces a specific color after expression.

[0083] Furthermore, the second expression element is used to achieve measurable measurement of neuron activity as follows: the second expression element is expressed to produce substance one only after the neuron matures, and the second expression element produces substance two during the entire cell growth stage, and the activity of the neuron can be measured by detecting the amount of substance one and substance two.

[0084] Furthermore, the second expression element includes: a promoter associated with neuron activation, a sub-element one expressed after being activated by a promoter associated with neuron activation, a promoter that is continuously and stably expressed in cells, and a sub-element two expressed after being activated by a promoter that is continuously and stably expressed in cells; the sub-element one produces substance one after expression, and the sub-element two produces substance two after expression.

[0085] Furthermore, the sub-element 1 is specifically: E-SARE enhancer element in series with ArcMini promoter in series with Gaussia luciferase.

[0086] Furthermore, the second sub-element is specifically: CAG promoter tandemly connected to secretory luciferase Cypridina luciferase.

[0087] Furthermore, the substance one is specifically: Gaussia luciferase produced by expressing an E-SARE enhancer element in series with an ArcMini promoter in series with Gaussia luciferase; the substance two is specifically: Cypridina luciferase produced by expressing a CAG promoter in series with a secretory luciferase Cypridina luciferase.

[0088] Furthermore, the platform for screening neurotoxic compounds is tested, specifically: for the first cerebral organoid, no neurotrophic factors are added in the early stage of culture, and neurotrophic factors are added when the neurons are mature, and the neuronal activity is verified by comparing the fluorescence expression; for the second cerebral organoid, neurotrophic factors are added at different culture stages and after the neurons are mature, and the values ​​of chemical fluorescence generated by the corresponding substrate are collected on an ELISA instrument, and then the ratio of the two is analyzed to obtain the changing trend of the ratio of Gaussia protein and Cypridina protein expression, and the activity state of the neurons is judged by comparing the changing trend of the ratio of Gaussia protein and Cypridina protein expression, thereby verifying the reliability of the platform; the neurotrophic factors include: BDNF and GDNF.

[0089] In this embodiment, if Figure 2As shown in the figure, a neurotoxic compound screening platform based on brain organoids is constructed, where A is a schematic diagram of the construction of d2GFP cell line and dual luciferase reporter cell line, and B is a schematic diagram of brain organoid culture. Gaussia luciferase is a luciferase derived from marine organisms (such as large marine copepods in Hawaiian waters) and has a secretory signal peptide, so it can be secreted outside the cell through the endoplasmic reticulum. Gaussia luciferase has high luminescence intensity, which is thousands of times that of other luciferases, and does not require ATP for the reaction, has high stability, and is highly tolerant to temperature, pH, etc.; Cypridina luciferase, also known as Cypridina luciferase (cLuc), is a luciferase derived from marine organisms; CAG promoter is an artificially constructed combined promoter composed of cytomegalovirus (CMV) early enhancer elements and chicken beta-actin promoter. It is widely used to drive high-level expression of genes in mammalian cells and has a strong promoter ability. In the experiment, the CAG promoter was used to drive the efficient expression of Cypridina luciferase in various cell types and tissues, which can facilitate cell labeling and tracking. The ArcMini gene expression system driven by the E-SARE regulatory element promoter makes the activated neurons express Gaussia luciferase, while another secretory luciferase, Cypridina luciferase, is driven by the CAG promoter and is continuously and stably expressed in all cells. Figure 2 As shown in A.

[0090] like Figure 2 As shown in B, the neurotrophic factors GDNF (glial cell-derived neurotrophic factor) and BDNF (brain-derived neurotrophic factor), which play an important role in neuronal maturation and activity, were added at specific time points in the culture to activate neurons and verify the reliability of the organoid model.

[0091] In summary, the embodiments of the present application establish a screening platform for neurotoxic compounds based on brain organoids, which can quickly and easily perform drug screening, test the toxicity of various compounds to neurons, etc., can effectively screen candidate drugs at an earlier stage, reduce the time on failed projects and the overall cost of drug development, achieve real-time monitoring of neuronal activity, and observe the dynamic changes of neuronal activity in organoids and responses to drug treatment for a long time without damaging the organoids.

[0092] Those skilled in the art will appreciate that embodiments of the present invention may be provided as methods, systems, or computer program products. Therefore, the present invention may take the form of a complete hardware embodiment, a complete software embodiment, or an embodiment combining software and hardware. Moreover, the present invention may take the form of a computer program product implemented on one or more computer-usable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0093] The present invention is described with reference to flowcharts and / or block diagrams of methods, devices (systems), and computer program products according to embodiments of the present invention. It should be understood that each process and / or block in the flowchart and / or block diagram, as well as the combination of processes and / or blocks in the flowchart and / or block diagram, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, an embedded processor, or other programmable data processing device to produce a machine, so that the instructions executed by the processor of the computer or other programmable data processing device generate instructions for implementing the processes in the flowchart and / or block diagram. Figure 1 A process or multiple processes and / or boxes Figure 1 A device that provides the functions specified in a block or multiple blocks.

[0094] These computer program instructions may also be stored in a computer-readable memory capable of directing a computer or other programmable data processing device to operate in a specific manner, so that the instructions stored in the computer-readable memory produce an article of manufacture including an instruction device, which implements the process Figure 1 A process or multiple processes and / or boxes Figure 1 A function specified in one or more boxes.

[0095] These computer program instructions can also be loaded onto a computer or other programmable data processing device so that a series of operating steps are executed on the computer or other programmable device to produce a computer-implemented process, thereby providing instructions for implementing the process. Figure 1 A process or multiple processes and / or boxes Figure 1 The steps for the functions specified in one or more boxes.

[0096] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0097] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for screening neurotoxic compounds based on a brain organoid model, characterized in that: The following steps are involved: Stable human embryonic stem cell lines were constructed and divided into two groups, namely the first cell line and the second cell line; Using gene editing technology to insert a first expression element into the genome of cells of the first cell line, wherein the first expression element is used to mark activated neurons; Inserting a second expression element into the genome of a cell of a second cell line using gene editing technology, wherein the second expression element is used to achieve measurable neuronal activity; Differentiating and culturing the edited first cell line and the edited second cell line to obtain a first cerebral organoid and a second cerebral organoid, respectively; The first and second cerebral organoids were used as experimental carriers to construct a screening platform for neurotoxic compounds; Test the neurotoxic compound screening platform, obtain validation results and provide feedback.

2. The method for screening neurotoxic compounds based on the brain organoid model according to claim 1, characterized in that: The first expression element is used to mark the activated neurons specifically: after the first expression element is expressed, it indicates that the neuron cells have matured and produced fluorescent substances.

3. The method for screening neurotoxic compounds based on the brain organoid model according to claim 1, characterized in that: The first expression element is specifically: an E-SARE enhancer element in series with an ArcMini promoter in series with a fluorescent substance element.

4. The method for screening neurotoxic compounds based on the brain organoid model according to claim 1, characterized in that: The fluorescent substance element is specifically a fluorescent protein element, which produces a specific color after being expressed.

5. The method for screening neurotoxic compounds based on the brain organoid model according to claim 1, characterized in that: The second expression element is used to achieve measurable neuronal activity in the following way: the second expression element is expressed to produce substance one only after the neuron matures, and the second expression element produces substance two during the entire cell growth stage. The activity of the neuron can be quantified by detecting the amount of substance one and substance two.

6. The method for screening neurotoxic compounds based on the brain organoid model according to claim 5, characterized in that: The second expression element comprises: a promoter associated with neuron activation, a sub-element 1 expressed after being activated by the promoter associated with neuron activation, a promoter stably and continuously expressed in cells, and a sub-element 2 expressed after being activated by the promoter stably and continuously expressed in cells; The sub-element 1 produces substance 1 after being expressed, and the sub-element 2 produces substance 2 after being expressed.

7. The method for screening neurotoxic compounds based on the brain organoid model according to claim 6, characterized in that: The sub-element 1 is specifically: E-SARE enhancer element in series with ArcMini promoter in series with Gaussia luciferase.

8. The method for screening neurotoxic compounds based on the brain organoid model according to claim 6, characterized in that: The second sub-element is specifically: CAG promoter tandem secretory luciferase Cypridina luciferase.

9. The method for screening neurotoxic compounds based on the brain organoid model according to claim 6, characterized in that: The substance 1 is specifically: Gaussia luciferase produced by the expression of E-SARE enhancer element in series with ArcMini promoter in series with Gaussia luciferase; The second substance is specifically: Cypridina luciferase produced by expressing the secretory luciferase Cypridina luciferase in series with the CAG promoter.

10. The method for screening neurotoxic compounds based on the brain organoid model according to claim 1, characterized in that: The neurotoxic compound screening platform is tested as follows: For the first cerebral organoid, no neurotrophic factors were added in the early stage of culture. Neurotrophic factors were added when the neurons matured, and the neuronal activity was verified by comparing fluorescence expression; For the second cerebral organoids at different culture stages and after the neurons to be cultured mature, add neurotrophic factors, collect Gaussia luciferase and Cypridina luciferase, and collect the values ​​of chemical fluorescence produced by the corresponding substrate on a microplate reader. Then analyze the ratio of the two to obtain the changing trend of the ratio of Gaussia protein and Cypridina protein expression. By comparing the changing trend of the ratio of Gaussia protein and Cypridina protein expression, the activity state of neurons can be judged, thereby verifying the reliability of the platform; The neurotrophic factors include: BDNF and GDNF.

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