Method for detecting activity of neurons with asymptomatic anemia based on brain organoid

Abstract

The invention discloses an asymptomatic neuronal activity detection method based on brain organoid, and relates to the technical field of cell activity detection. The asymptomatic neuronal activity detection method based on the brain organoid comprises the following steps: constructing a recombinant transposon vector; performing differentiation culture; and adding a to-be-detected medicine. According to the invention, TDP43M337V and TDP43Q331KcDNA sequences are respectively connected in series with a CAG promoter, then a first plasmid and a second plasmid are respectively inserted into chromosomes in a cell line A and a cell line B to obtain a first brain organ and a second brain organ, and after the first brain organ and the second brain organ are mature, the first brain organ and the second brain organ are separated from each other. The to-be-detected medicine is added into the first brain organoid and the second brain organoid, and the TDP43 protein content and change are respectively detected to verify the effect of the to-be-detected medicine on the gradual freezing disease, so that the effect of more accurately detecting the change of the neuronal activity based on the gradual freezing disease is achieved; the problem that in the prior art, the accuracy rate of neuronal activity change detection based on the asymptomatic disease is low is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell activity detection, and in particular to a method for detecting the activity of ALS neurons based on brain organoids. Background Art

[0002] Neurological diseases are diseases that occur mainly in the central nervous system or peripheral nervous system. Their main symptoms include disorders of sensation, consciousness and movement, including epilepsy, schizophrenia, Alzheimer's disease and Huntington's disease. At present, many neurological diseases have been found to be related to abnormal neuronal activity. Abnormal neuronal activity is mainly divided into neuronal excitation / inhibition imbalance, abnormal neuronal excitation and neuronal inhibition / death, and different types of abnormal neuronal activity may lead to different types of neurological diseases.

[0003] The balance between excitation / inhibition (E / I) in neuronal synaptic inputs and neural circuits is important for normal brain development and function. E / I balance usually refers to the stable global activity level in a specific neural circuit. Individual neurons may experience temporary imbalances, and these neurons are dynamically changing. At the level of individual neurons, the number of excitatory and inhibitory synapses on each cortical pyramidal neuron affects the E / I balance. In large-scale neural networks, the excitation determined by excitatory neurons and the inhibition determined by inhibitory neurons jointly maintain the E / I balance. In simple terms, if the excitation level exceeds the inhibition level, overexcitation occurs until the excitation threshold is reached or the neural inhibitory signal in the neural network is enhanced until a new "balanced" state. Conversely, if inhibition exceeds excitation, electrical activity is inhibited. Changes in E / I balance are mainly manifested as changes in the relative activity of the two types of excitatory / inhibitory neurons in the cortical circuit, leading to the occurrence of a variety of neurological / psychiatric diseases. A variety of brain diseases are associated with neuronal E / I imbalance, including autism spectrum disorders (ASDs) and Rett syndrome.

[0004] Amyotrophic lateral sclerosis, also known as Lou Gehrig's disease, is a neurodegenerative disease characterized by the loss of motor neurons in the motor cortex, brainstem and spinal cord. Clinical manifestations include muscle weakness, spasticity, atrophy, and impaired speech, swallowing and breathing. The global incidence rate is 6-9 cases per 100,000 people, and the incidence rate has been increasing year by year. The disease is a complex disease. About 10% to 21% of patients are affected by single gene factors, but the onset of most patients is determined by the interaction of multiple genetic and environmental risk factors. More than 30 genes have been identified as being associated with the onset or increased risk of ALS, including the gene encoding TDP43 protein. In most patients, ALS manifests as TAR DNA-bindingprotein 43 (TDP43) proteinopathy, which is mainly characterized by abnormal aggregation of phosphorylated or ubiquitinated TDP43 protein in the cytoplasm of ALS neurons and loss of localization in the nucleus. TDP43 is a 43kDa protein encoded by the TAR DNA Binding Protein (TARDBP) gene. It is a DNA and RNA binding protein mainly expressed in the cell nucleus. Its expression is regulated by nuclear localization signals and transfer signals and is affected by the physiological state of the body. TDP43 is mainly located in the cell nucleus and participates in regulating RNA processing, including transcriptional regulation, splicing and microRNA processing. Under pathological conditions such as TARDBP mutation, TDP43 protein is cleared from the cell nucleus and aggregated in the cytoplasm, and is highly phosphorylated or ubiquitinated, resulting in a significant decrease in its solubility. TDP43 in the cytoplasm is truncated into insoluble C-terminal fragments, which are more likely to aggregate in the cytoplasm, thereby inducing cytotoxicity or binding to intracellular RNA, leading to neuronal degeneration. Among the known mutations, the TDP43 protein carrying the M337V mutation (TDP43M337V) has a longer half-life in cells, and ALS patients with this mutation develop the disease earlier. TDP43Q331K is also a common ALS mutation type. Mice that overexpress human TDP43Q331K show age-dependent pathological phenotypes. Overexpression of TDP43 can cause increased pathological symptoms and mortality in ALS, while pathogenic mutations can exacerbate these phenotypes.

[0005] There are currently a variety of methods used to study neuronal activity, commonly used ones include calcium imaging, electrophysiological recording, optogenetics, electroencephalography, and magnetoencephalography.

[0006] Electrophysiological techniques mainly use electrophysiology, patch clamp and other techniques to record and analyze the potential changes, conduction rate, and ion channel activity of neurons from whole animals or isolated tissues, organs, and cells, including intracranial electrophysiology, cell electrophysiology, and organoid electrophysiology. Electrophysiology is the "gold standard" for studying neuronal signal transduction. Combined with the rapid development of microscopy technology, neuronal signals can be measured and controlled. Electrophysiological tools have high sensitivity and time resolution, allowing us to study the characteristics of single ion channels and the activities of hundreds of cells in neuronal networks. However, our understanding of the mammalian brain is limited. Most neural circuits are composed of thousands of neurons, which are interconnected in a highly distributed manner. This makes the nervous system not operated independently by a single neuron. We need to study the activities of many or most neurons in a circuit. Specific movements are determined by the overall activity of the neuronal population in the motor cortex, so we hope to record the activity changes of the entire neuronal population in the experiment. The patch clamp technique is limited by its single channel and single cell characteristics. Currently, electrophysiology uses multi-electrode arrays (MEA) to increase the sampling density in experiments. This method has certain limitations. As a 3D structure, brain organoids are difficult to fix during MEA recording, and organoid recordings can only be performed briefly. The limited number of active electrodes used leads to certain restrictions on efficiency and certain inter-group differences, and has high requirements for equipment and personnel.

[0007] Calcium imaging technology uses calcium indicators (such as genetically encoded calcium indicators) to monitor changes in calcium ion concentrations in neurons, thereby indirectly reflecting changes in neuronal activity. This technology can provide activity information at the single-cell level and is suitable for in vivo and in vitro experiments, but it has low temporal resolution and cannot directly measure the electrical activity of neurons. It is also limited by the inability of calcium indicators to penetrate cells deep within organoid spheres, which has certain limitations for imaging deep neurons.

[0008] Optogenetics uses light-sensitive proteins (such as channelrhodopsin) combined with genetic technology to control the activity of specific neurons by stimulating them with light. This method can achieve precise activation or inhibition of specific neurons. It has high temporal and spatial resolution, but has high requirements for equipment and operation. At the same time, the light penetration depth is limited, which makes it difficult to operate deep neurons.

[0009] Electroencephalography (EEG) and magnetoencephalography (MEG) record the electrical activity of the cerebral cortex by placing electrodes on the scalp and measure the magnetic field generated by the brain to record neuronal activity. Both methods are non-invasive and can be used for human research, but the spatial resolution of EEG is low and the equipment for magnetoencephalography is expensive.

[0010] 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:

[0011] In the prior art, the accuracy of detecting changes in neuronal activity in ALS is low due to high equipment requirements, high costs, and the fact that it can only measure the activity of single or partial neurons. Summary of the invention

[0012] The embodiments of the present application solve the problem of low accuracy in detecting changes in neuronal activity in the prior art by providing a method for detecting neuronal activity in ALS based on brain organoids, thereby achieving more accurate detection of changes in neuronal activity in ALS.

[0013] The present application embodiment provides a method for detecting the activity of ALS neurons based on brain organoids, comprising the following steps: S1, TDP43 M337V 、TDP43 Q331K cDNA sequences were connected in series with CAG promoter to construct TDP43 M337V 、TDP43 Q331K The recombinant transposon vectors of cDNA are respectively recorded as the first plasmid and the second plasmid; S2, using transposase to insert the first plasmid and the second plasmid into the chromosomes of cell line A and cell line B, respectively, and differentiate and culture to obtain the first cerebral organoid and the second cerebral organoid, respectively; S3, after the first cerebral organoid and the second cerebral organoid are mature, add the drug to be tested to the first cerebral organoid and the second cerebral organoid, and detect the TDP43 protein content and changes respectively to verify the effect of the drug to be tested on ALS.

[0014] Furthermore, both cell line A and cell line B are cell lines that can verify the degree of toxicity of the drug to be tested on brain organoids.

[0015] Furthermore, the cell line A and / or cell line B are constructed by inserting a first expression element into the genome of the cell line cells using gene editing technology, and the first expression element is used to mark activated neurons; the first expression element is used to mark activated neurons specifically as follows: after expression, the first expression element indicates that the neuronal cells are mature and produce fluorescent substances.

[0016] Furthermore, the cell line A and / or cell line B are constructed by inserting a second expression element into the genome of the cell line cells using gene editing technology, and the second expression element is used to achieve quantifiable neuronal activity; the second expression element is used to achieve quantifiable neuronal activity specifically as follows: the second expression element is expressed to produce substance one only after the neurons mature, and the second expression element produces substance two throughout the entire cell growth stage, and the activity of the neurons can be quantified by detecting the amounts of substance one and substance two.

[0017] Furthermore, the cell line A and / or cell line B are constructed in the following manner: a stably transformed human embryonic stem cell line is constructed and divided into two groups, namely a first cell line and a 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; the first expression element is used to mark activated neurons in the following manner: after expression, the first expression element indicates that the neuronal cells have matured and produce fluorescent substances; 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 quantifiable neuronal activity; the second expression element is used to achieve quantifiable neuronal activity in the following manner: the second expression element is expressed to produce substance one only after the neurons mature, and the second expression element produces substance two throughout the entire cell growth stage, and the activity of the neurons can be quantified by detecting the amounts of substance one and substance two.

[0018] 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; the fluorescent substance element is specifically: a fluorescent protein element, and the fluorescent protein element produces a specific color after expression.

[0019] Furthermore, the second expression element includes: a promoter related to neuronal activation, a sub-element one expressed after being activated by a promoter related to neuronal 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; the sub-element one is specifically: an E-SARE enhancer element in series with an ArcMini promoter in series with Gaussia luciferase; the sub-element two is specifically: a CAG promoter in series with a secretory luciferase Cypridina luciferase; the substance one is specifically: Gaussia luciferase produced after the expression of the E-SARE enhancer element in series with an ArcMini promoter in series with Gaussia luciferase; the substance two is specifically: Cypridina luciferase produced after the expression of the CAG promoter in series with the secretory luciferase Cypridina luciferase.

[0020] Furthermore, the transposase is Sleeping Beauty.

[0021] Furthermore, the first cerebral organoid and the second cerebral organoid were subjected to immunofluorescence staining respectively, and the TDP43 protein content and changes were determined by the staining colors.

[0022] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0023] 1. By using TDP43 M337V 、TDP43 Q331K The cDNA sequence is concatenated with the CAG promoter, and then the first plasmid and the second plasmid are inserted into the chromosomes of cell line A and cell line B respectively using transposase, and the first cerebral organoid and the second cerebral organoid are differentiated and cultured to obtain the first cerebral organoid and the second cerebral organoid respectively. Then, after the first cerebral organoid and the second cerebral organoid are mature, the drug to be tested is added to the first cerebral organoid and the second cerebral organoid, and the TDP43 protein content and changes are detected respectively to verify the effect of the drug to be tested on ALS, thereby realizing the effect of the drug on ALS and the toxicity of the drug to neurons, and further realizing more accurate detection of changes in neuronal activity based on ALS, effectively solving the problem of low accuracy in detecting changes in neuronal activity in the prior art.

[0024] 2. By using gene editing technology to insert the first expression element into the genome of cell line cells, and then using gene editing technology to insert the second expression element into the genome of cell line cells, the reliability of platform detection of ALS-based neuronal maturation is improved, thereby achieving an improvement in the efficiency of platform detection of ALS-based neurons.

[0025] 3. By using gene editing technology to insert the first expression element into the genome of the first cell line cells, and then using gene editing technology to insert the second expression element into the genome of the second cell line cells, the accuracy of obtaining the genome is improved, thereby improving the reliability of genome construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A flowchart of a method for detecting neuronal activity of ALS based on brain organoids provided in an embodiment of the present application;

[0027] Figure 2 The AAVS1-Puro-E-SARE-Arcmini-d2GFP plasmid map provided in the examples of this application;

[0028] Figure 3The AAVS1-Puro-E-SARE-Arcmini-Gaussia-CAG-Cypridina plasmid map provided in the examples of this application;

[0029] Figure 4 The plasmid map provided for the examples of this application is shown in the figure;

[0030] Figure 5 A neurotoxic compound screening platform based on brain organoids provided in the embodiments of the present application;

[0031] Figure 6 A validation diagram of the brain organoid neurotoxic compound screening platform provided in the embodiments of the present application;

[0032] Figure 7 Diagram for testing neurochemical compounds based on brain organoid models;

[0033] Figure 8 The neurochemical compound testing and change diagram based on the brain organoid model provided in the embodiments of the present application;

[0034] Fig. 9 A diagram of constructing a disease model of ALS provided in an embodiment of the present application;

[0035] Fig.10 A graph showing the test results of the neuron activity platform of the overexpression ALS disease model provided in the embodiments of the present application;

[0036] Fig.11 This is a diagram of the immunostaining results of the apoptosis marker protein caspase3 of the TDP43 overexpressing organoid D100 provided in the examples of the present application;

[0037] Fig.12 This is a diagram of drug testing for neuronal activity in an ALS disease model overexpressing neuron activity provided in an embodiment of the present application. DETAILED DESCRIPTION

[0038] The present invention provides a method for detecting the activity of ALS neurons based on brain organoids to solve the problem of low accuracy in detecting changes in neuronal activity in the prior art. M337V 、TDP43 Q331K The cDNA sequence was concatenated with the CAG promoter, and then the first plasmid and the second plasmid were inserted into the chromosomes of cell line A and cell line B respectively using transposase, and differentiated and cultured to obtain the first cerebral organoid and the second cerebral organoid respectively. Then, after the first cerebral organoid and the second cerebral organoid matured, the drug to be tested was added to the first cerebral organoid and the second cerebral organoid, and the TDP43 protein content and changes were detected to verify the effect of the drug to be tested on ALS, thereby achieving more accurate detection of changes in neuronal activity based on ALS.

[0039] The technical solution in the embodiment of the present application is to solve the problem of low accuracy in detecting changes in neuronal activity, and the overall idea is as follows:

[0040] By integrating TDP43 M337V 、TDP43 Q331K The cDNA sequence was concatenated with the CAG promoter, and then the first plasmid and the second plasmid were inserted into the chromosomes of cell line A and cell line B, respectively, to obtain the first cerebral organoid and the second cerebral organoid. Then, after the first cerebral organoid and the second cerebral organoid matured, the drug to be tested was added to the first cerebral organoid and the second cerebral organoid, and the TDP43 protein content and changes were detected to verify the effect of the drug to be tested on ALS, thereby achieving a more accurate characterization of the changes in neuronal activity.

[0041] 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.

[0042] like Figure 1 As shown in the figure, it is a flow chart of a method for detecting neuronal activity of ALS based on brain organoids provided in an embodiment of the present application, the method comprising the following steps: S1, constructing a recombinant transposon vector: TDP43 M337V 、TDP43 Q331K cDNA sequences were connected in series with promoters to construct TDP43 M337V 、TDP43 Q331K The recombinant transposon vectors of cDNA are respectively recorded as the first plasmid and the second plasmid; S2, differentiation culture: use transposase to insert the first plasmid and the second plasmid into the chromosomes of cell line A and cell line B, respectively, and differentiate and culture to obtain the first cerebral organoid and the second cerebral organoid, respectively; S3, adding the drug to be tested: after the first cerebral organoid and the second cerebral organoid are mature, add the drug to be tested to the first cerebral organoid and the second cerebral organoid, and detect the TDP43 protein content and changes respectively to verify the effect of the drug to be tested on ALS.

[0043] In this example, vector construction

[0044] (1) Construction of AAVS1-Puro-E-SARE-Arcmini-d2GFP vector

[0045] The E-SARE, Arcmini, d2GFP and PURO gene sequences were introduced into the plasmid to stably express green fluorescent protein after neuronal activation, and the AAVS1-Puro-E-SARE-Arcmini-d2GFP plasmid was obtained, such as Figure 2Shown is a map of the AAVS1-Puro-E-SARE-Arcmini-d2GFP plasmid provided in the examples of this application.

[0046] (2) Construction of AAVS1-Puro-E-SARE-Arcmini-Gaussia-CAG-Cypridina vector 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, the quantifiable activity of neurons can be achieved. Figure 3 As shown, it is the AAVS1-Puro-E-SARE-Arcmini-Gaussia-CAG-Cypridina plasmid map provided in the examples of this application.

[0047] The constructed vector plasmid and gRNA vector plasmid were electroporated into the H9 human embryonic stem cell line, and Puro was used for drug screening. Single clones were selected for subculture, and genotype identification was performed when the cell fusion reached 80%; The Sleeping Beauty Transposase system consists of Sleeping Beauty (SB) transposase and transposon, which can simply and effectively achieve stable expression of exogenous genes;

[0048] Expression vector construction

[0049] (1) Construction of mut-TDP43 overexpression vector

[0050] The TDP43M337V and TDP43Q331K cDNA sequences are respectively connected in series with the CAG promoter, wherein the promoter can also be other promoters with similar functions, such as CMV, EF1a, Ubc, etc., to construct a recombinant transposon vector carrying TDP43M337V and TDP43Q331K cDNA, such as Figure 4 As shown, the plasmid map provided in the examples of this application is shown in the figure. (A. IR-CAG-TDP43Q331K-IR plasmid map. B. IR-CAG-TDP43M337V-IR plasmid map)

[0051] System electroporation:

[0052] After verifying the reliability of the SB system in N2a cells, the system was used to construct a target gene overexpression cell line in H9. The recombinant transposon vector constructed in (1) and (2) was mixed with Zeocin plasmid and SB100 plasmid in a ratio of 4:1:1, and a total of 6 μg was electroporated into 40wH9 cells in a 10 cm dish.

[0053] After screening with Zeocin, single clones were selected for culture and identification. Cells were collected to extract RNA and protein, and qPCR and WB were performed to identify the cell line.

[0054] Furthermore, both cell line A and cell line B are cell lines that can verify the degree of toxicity of the drug to be tested on brain organoids.

[0055] Cell line A and / or cell line B are constructed by inserting a first expression element into the genome of the cell line cells using gene editing technology, and the first expression element is used to mark activated neurons; the first expression element is used to mark activated neurons specifically: after expression, the first expression element indicates that the neuronal cells are mature and produce fluorescent substances.

[0056] Cell line A and / or cell line B are constructed as follows: a second expression element is inserted into the genome of the cell line cells using gene editing technology, and the second expression element is used to achieve quantifiable neuronal activity; the second expression element is used to achieve quantifiable neuronal activity specifically as follows: the second expression element is expressed to produce substance one only after the neurons mature, and the second expression element produces substance two throughout the entire cell growth stage, and the activity of the neurons can be quantified by detecting the amounts of substance one and substance two.

[0057] The construction method of cell line A and / or cell line B is as follows: a stably transformed human embryonic stem cell line is constructed and divided into two groups, namely a first cell line and a 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; the first expression element is used to mark activated neurons specifically as follows: after expression, the first expression element indicates that the neuronal cells are mature and produce fluorescent substances; 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 quantifiable neuronal activity; the second expression element is used to achieve quantifiable neuronal activity specifically as follows: the second expression element is expressed to produce substance one only after the neurons mature, and the second expression element produces substance two throughout the entire cell growth stage, and the activity of the neurons can be quantified by detecting the amounts of substance one and substance two.

[0058] It should be added 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; the fluorescent substance element is specifically: a fluorescent protein element, which produces a specific color after expression.

[0059] The second expression element includes: a promoter related to neuronal activation, a sub-element one expressed after being activated by a promoter related to neuronal 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; after the expression of sub-element one, substance one is produced, and after the expression of sub-element two, substance two is produced; sub-element one is specifically: an E-SARE enhancer element in series with an ArcMini promoter in series with Gaussia luciferase; sub-element two is specifically: a CAG promoter in series with a secretory luciferase Cypridina luciferase; substance one is specifically: Gaussia luciferase produced after the expression of E-SARE enhancer element in series with an ArcMini promoter in series with Gaussia luciferase; substance two is specifically: Cypridina luciferase produced after the expression of CAG promoter in series with a secretory luciferase Cypridina luciferase.

[0060] In this example, luciferase assay:

[0061] (1) Preparation of substrates: Prepare the Gaussia substrate coelenterazine and Cypridina substrate vargulin powders according to the instructions; add 98 μL of acidified ethanol solution (20 μL / mL 3N HCl) to dissolve every 500 μg of coelenterazine to prepare a 12 mM coelenterazine stock solution; add 200 μL of acidified n-butanol solution to every 500 μg of vargulin, and add 2-3 μL of 3N HCl solution until the yellow color disappears, then dilute 500-fold with PBS and place at room temperature away from light for 30 min for use.

[0062] (2) Diluting the culture medium: According to the preliminary experiment, the test culture medium was diluted with PBS by the gradient dilution method. The Gaussia sample was diluted 50 times and the Cypridina sample was diluted 2000 times. They were added to the ELISA plate, with 20 μL of the test sample in each well.

[0063] (3) ELISA detection: Add 50 μL of luciferase substrate working solution to each well, record the luminescence at 480 nm on an ELISA reader, and record the value.

[0064] Data processing: The ratios of Gaussia fluorescence readings and Cypridina fluorescence readings of the experimental group and the control group were calculated respectively, and then the fold changes of the experimental group and the control group were calculated; two-way analysis of variance (ANOVA) was used for significance analysis, and P < 0.05 was considered to be significantly different; after analyzing the data, expression histograms were prepared using GraphPad Prism 9 software; the standard deviation was to reflect the repeatability of the data.

[0065] Neuronal activity visualization and high-throughput screening platform construction:

[0066] By combining the short-lived green fluorescent protein d2GFP and the secreted luciferases Gaussia and Cypridina, a brain organoid platform for visualization of neuronal activity and high-throughput screening was constructed; through gene editing technology, the E-SARE-Arcmini-d2GFP and E-SARE-Arcmini-Gaussia-CAG-Cypridina expression elements were inserted into the "safe harbor" AAVS1 site on the genome of the H9 human embryonic stem cell line, and two cell lines were constructed: 1) Green fluorescent protein reporter cell line. d2GFP was connected in series after E-SARE, so that the activated neurons expressed green fluorescence. 2) Dual luciferase reporter cell line. The secreted luciferase encoding gene Gaussia luciferase was connected in series after E-SARE, so that the activated neurons expressed Gaussia luciferase, while the other secreted luciferase Cypridina luciferase was driven by the CAG promoter and continuously and stably expressed in all cells ( Figure 5 Using these two gene-edited cell lines, we will culture human brain organoids ( Figure 5 B), and at specific time points in culture, neurotrophic factors GDNF (glial cell-derived neurotrophic factor) and BDNF (brain-derived neurotrophic factor) that play an important role in neuronal maturation and activity were added to activate neurons and verify the reliability of the organoid model, such as Figure 5 As shown, a neurotoxic compound screening platform based on brain organoids is constructed as provided in an embodiment of the present application (A. Schematic diagram of the construction of d2GFP cell line and dual luciferase reporter cell line. B. Schematic diagram of brain organoid culture).

[0067] Validation of neuronal activity visualization and high-throughput screening platform:

[0068] Next, the constructed organoid platform was experimentally verified. The results showed that in the cultured d2GFP organoids, no green fluorescence was observed in the early stages of culture (day 20, 40, 60, D20, D40, D60) without the addition of neurotrophic factors. However, after the addition of BDNF and GDNF neurotrophic factors on D60, strong green fluorescence expression could be observed in the organoids within 8 hours ( Figure 6 Middle A). Immunofluorescence staining of d2GFP organoids at D70 revealed that GFP was significantly co-localized with the neuronal marker protein MAP2 and the synaptic marker protein PSD95 ( Figure 6 B). The dual-luciferase organoids were collected and cultured in D20, D40, and D60 culture media without adding neurotrophic factors, and in D70 culture media with BDNF and GDNF neurotrophic factors. The luciferase-catalyzed oxidation of their respective substrates produced bioluminescence values ​​using a microplate reader. The magnitude of the fluorescence value indicates the expression level of the two enzymes. The results showed that after adding BDNF and GDNF, the fluorescence value increased significantly, and the activity of neurons was significantly enhanced ( Figure 6 In addition, through WB analysis of organoids at different culture stages, it was observed that the expression trends of Gaussia protein and Cypridina protein were consistent with the results of the microplate reader test ( Figure 6 D). Figure 6 As shown, it is a verification diagram of the brain organoid neurotoxic compound screening platform provided in the examples of the present application (A. Representative images of the bright field (BF) and green fluorescence field (GFP) of d2GFP brain organoids on the 20th, 40th, 60th and 70th days of culture. Scale bar: 500 μm. B. Immunofluorescence staining of MAP2, GFP and PSD95 of d2GFP brain organoid D70, MAP2: neuronal marker, PSD95: synaptic marker. Scale bar: 50 μm. C. Changes in the fluorescence ratio of Gaussia / Cypridina of dual-luciferase brain organoids at different time points. n=8. D. Immunoblotting of Gaussia and Cypridina proteins of dual-luciferase brain organoids at different time points, GAPDH is the internal reference protein). In summary, the genetically engineered brain organoids can quickly and sensitively exhibit neuronal activity.

[0069] Brain organoid compound toxicity screening platform KCl gradient test:

[0070] Since neuronal membrane potential is very sensitive to changes in extracellular K+ concentration, increasing extracellular K+ concentration causes neurons to depolarize, and KCl can excite neurons

[88] . In order to verify whether our brain organoid model can be used to test the effects of compounds on neuronal activity, we increased the extracellular K+ concentration by adding different concentrations of KCl (0, 10, 25, 50 mM) to the culture medium and observed whether the brain organoids could respond quickly and sensitively to changes in neuronal activity. When the fluorescent and luciferase brain organoids were cultured to D60-70, the neurotrophic factors BDNF and GDNF were added to activate the neurons in the organoids, and then different concentrations of KCl were added. For luciferase reporter gene brain organoids, the culture medium of the dual-luciferase organoids was collected every 48 hours for luciferase content determination and statistics; for fluorescent protein reporter gene brain organoids, the fluorescence intensity graph of d2GFP was taken every 48 hours ( Figure 7 The results showed that after KCl treatment, the green fluorescence intensity increased significantly and increased with the increase of KCl concentration. At the same time, in the range of 0-50mM, with the increase of concentration, the fluorescence ratio of Gaussia and Cypridina, which indicates neuronal activity, also increased significantly ( Figure 7 Therefore, our platform can sensitively reflect the changes in the intensity of neuronal activity. Figure 7 Shown is a diagram of neural compound testing based on the brain organoid model (A. Schematic diagram of the neural compound testing method in the brain organoid model, Gau: Gaussia, Cyp: Cypridina. B. Changes in luciferase secretion after treatment with different concentrations of KCl. C. Changes in green fluorescence after treatment with different concentrations of KCl. n=8. Scale bar: 500μm. Two-Way ANOVA, *P<0.5, **P<0.01, ****P<0.0001).

[0071] Brain Organoid Compound Toxicity Screening Platform Compound Testing:

[0072] 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. A variety of effective chemical components of FDA-approved drugs for the treatment of neurological diseases were selected, including anti-epileptic drugs Gabapentin, 4-Aminopyridine, Rufinamide, local anesthetics Bupivacaine, Parkinson's drugs Rotigotine, Alzheimer's drugs Memantine, etc. After the brain organoid neurons matured, we added BDNF and GDNF to activate the neurons, and then added 10μM of the test compounds, and changed the medium and collected samples at D0, D1, D3, and D5 after the addition of the compounds. According to the changes in green fluorescence and luciferase, it was found that 4-Aminopyridine and Gabapentin had no significant effect on neuronal activity, while Bupivacaine, Rufinamide, Rotigotine, and MemantineHydrochlotide caused a significant decrease in neuronal activity. In contrast, AripiprazoleLauroxil increased neuronal activity within 72h, but decreased after 96h. By observing the green fluorescence image, we found that after the addition of the compound, the neurons showed obvious excitement, but the toxicity of the compound led to increased cell death, increased cell fragments and decreased fluorescence intensity after 72 hours ( Figure 8 At the same time, due to individual differences between brain organoids, changes in luciferase allow us to observe changes in neuronal activity more accurately and with high throughput; Figure 8 As shown, the neurological compound test and change diagram based on the brain organoid model provided in the embodiment of the present application (A. Changes in luciferase secretion after treatment with different types of compounds. n=8. B. Changes in green fluorescence after treatment with different types of compounds. Scale bar: 500μm. Two-Way ANOVA, *P<0.5, **P<0.01, ****P<0.0001).

[0073] Through this neuronal activity screening platform, the effects of different compounds on neurons can be tested for a long time by real-time fluorescence observation or testing the amount of luciferase secreted into the culture medium without destroying brain organoids. In this experiment, no significant changes in neuronal excitability were observed after treatment with 10μM 4-Aminopyridine, so neurons may be insensitive to 10μM 4-Aminopyridine and show neuroprotective effects. This is consistent with past reports: 50μM and 2500μM 4-Aminopyridine 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 secretion of luciferase. 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 previous studies to selectively bind to the intermediate inactivedstate (Ii) of Na+ channels and inhibit resurgent currents in a dose-dependent manner. Rotigotine and Memantine have protective effects on excitotoxic necrosis caused by glutamate. They have also been observed to have inhibitory effects on neuronal activity in brain organoids. Therefore, our system 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.

[0074] Furthermore, there are a variety of transposon systems to choose from, for example, systems are available: piggyBac, Sleeping Beauty and Tol2, etc. Different transposons correspond to different transposases, for example, the transposase is SleepingBeauty; by immunofluorescence staining of the first cerebral organoid and the second cerebral organoid, respectively, the TDP43 protein content and changes are determined by the color after staining.

[0075] In this example, a brain mut-TDP43 overexpression organoid ALS model was constructed and validated:

[0076] Overexpression of TDP43 mutants by the SB transposon system to construct an ALS disease model ( Fig. 9 WB results confirmed that TDP43 protein expression was significantly increased in cell lines and cultured brain organoids ( Fig. 9The results of immunofluorescence staining of brain organoids showed that the expression of TDP43 in the overexpression group was significantly increased compared with the control group ( Fig. 9 Part C); therefore, a cell line overexpressing mut-TDP43 was successfully constructed and induced to differentiate into human brain organoids; Fig. 9 The figure shows the construction of the ALS disease model provided in the examples of the present application. (A. Schematic diagram of the construction of the TDP43 overexpressing cell line; B. WB results of TDP43 overexpressing cells and organoids; C. TDP43 immunofluorescence staining on the 60th day of culture. Scale bar: 50 μm.)

[0077] Construction and validation of ALS neuronal activity screening platform:

[0078] Next, based on the constructed ALS model, a neuronal activity screening platform was built; the d2GFP and Luciferase expression vectors were electroporated into the TDP43-overexpressing cell line, thereby constructing an ALS neuronal activity screening platform; using this platform, disease phenotype observations and drug testing experiments were carried out. First, the cell line was cultured into adult brain organoids. As the neurons gradually matured, BDNF and GDNF were added at D60 to activate the neurons. On days 0, 1, 3, and 5, for the fluorescent protein reporter gene brain organoids, the fluorescence intensity graphs of d2GFP were taken; for the luciferase reporter gene brain organoids, the culture medium of the dual-luciferase organoids was collected, and the luciferase content was determined and counted. The results showed that brain organoid neurons overexpressing TDP43Q331K and TDP43M337V exhibited strong excitotoxicity, and their green fluorescence intensity was significantly increased ( Fig.10 The changes of luciferase were also significantly increased compared with the control group ( Fig.10 (Part B).

[0079] These brain organoids were further cultured to observe the effects of excitotoxicity on neurons caused by overexpression of mut-TDP43. When these brain organoids were cultured to D100, they were tested again. Based on the changes in green fluorescence intensity and luciferase, it was found that the green fluorescence intensity was significantly reduced ( Fig.10 The results of luciferase assay showed the changes of neuronal activity more intuitively and sensitively ( Fig.10 (section D).

[0080] like Fig.10As shown, the test result diagram of the neuron activity platform of the overexpression ALS disease model provided in the embodiment of the present application, wherein A. Changes in green fluorescence intensity of D60 after adding BDNF and GDNF on different days. B. Changes in luciferase ratio of D60 after adding BDNF and GDNF on different days. n=8. C. Changes in green fluorescence intensity of D100 after adding BDNF and GDNF on different days. D. Changes in luciferase ratio of D100 after adding BDNF and GDNF on different days. n=8. Scale bar: 50μm. Two-Way ANOVA, *P<0.5, **P<0.01, ***P<0.001, ****P<0.0001.

[0081] This suggests that the mutated TDP43 protein can cause neurons to become overexcited, leading to excitotoxicity, followed by inactivation or even death. This is consistent with previous studies that have reported that excitotoxicity mediated by excessive stimulation of postsynaptic glutamate receptors is the main pathophysiological mechanism of ALS. According to electrophysiological studies, glutamate signaling in ALS changes, and the release of glutamate from presynaptic terminals increases the excitotoxicity of ALS tissue.

[0082] Neuronal hyperexcitability was found in motor neurons cultured from ALS mice or iPSC cells with mutant SOD1, C9orf72, etc. Similarly, the strength-duration time constant of motor neurons was found to be abnormal in ALS patients. constant, SDTC); it has been confirmed that cortical hyperexcitability is an early and inherent feature of ALS patients; TDP43 maintains mitochondrial homeostasis by regulating the processing of mitochondrial transcripts, but this is disturbed in the case of mutations; TDP43 aggregates in the nucleus may sequester the nuclear genome of mitochondrial protein-specific microRNAs and proteins encoded by the nuclear genome, thereby leading to mitochondrial dysfunction, aggravating oxidative stress, and possibly increasing excitotoxic damage to motor neurons; in mouse and cell models, it has been confirmed that the misplaced expression of TDP43 leads to synaptic dysfunction and neuronal hyperexcitability in the motor cortex; in order to further verify the effect of mut-TDP43 overexpression on neurons, D100 brain organoids were immunofluorescently stained and co-stained with apoptosis marker protein Caspase-3 and neuronal marker protein MAP2. The results showed that apoptotic cells increased significantly in human brain organoids overexpressing mut-TDP43, such as Fig.11As shown, the immunostaining result of caspase3, apoptosis marker protein of D100 in TDP43 overexpressed organoids provided in the examples of the present application is consistent with the result of increased apoptosis found when TDP43Q331K was overexpressed in SH-5Y5Y cells. In summary, by long-term observation of the effect of mut-TDP43 overexpression on neuronal activity, it can sensitively and intuitively reflect the changes in neuronal activity, providing a new method for the study of neurological diseases.

[0083] Drug testing based on ALS disease screening platform:

[0084] Next, this model will be used for drug testing to test compounds with potential therapeutic effects on ALS. Digoxin and Lanatoside C (Lan C) are both cardiac glycoside drugs. The main mechanism of action of Digoxin is through blocking the sodium pump and Na+-K+-ATPsae, and Lan C can also act on Na+-K+-ATPsae. Studies have found that both Digoxin and Lan C can reduce the density of TDP43 aggregates in iPSCs of ALS patients. Therefore, these two compounds will be tried for drug testing on human brain organoids that overexpress mut-TDP43.

[0085] The organoids were divided into groups of 8 each, and D70 ALS human brain organoids (TDP43Q331K and TDP43M337V) and normal human brain organoids (control) were treated with 0.02 μM Digoxin and Lan C, respectively. Fresh culture medium was replaced and neurotrophic factors BDNF and GDNF were added every week for one month, and photos were taken and culture medium was collected every two weeks. Fig.12 As shown, the neuronal activity drug test diagram of the overexpression ALS disease model provided in the embodiment of the present application (A. Changes in green fluorescence after treating brain organoids (D70) with different compounds; scale: 500 μm. B. Changes in the CAG-TDP43Q331K luciferase ratio. n=8. B. Changes in the CAG-TDP43M337V luciferase ratio. n=8). By observing the changes in green fluorescence, at D98, the green fluorescence intensity of the drug-added group was higher than that of the TDP43Q331K and TDP43M337V blank groups ( Fig.12 The changes in luciferase more significantly reflected the reduction in neuronal inactivation after Digoxin and Lan C treatment ( Fig.12 This indicates that Digoxin and Lan C have a certain neuroprotective effect on neuronal overexcitation caused by abnormal TDP43 aggregation. In summary, Digoxin and Lan C may become potential ALS therapeutic drugs.

[0086] It should be added that the specific process of culturing human brain organoids is as follows:

[0087] (1)Embryoid bodies (EB)formation:

[0088] (1) Pretreatment of 96-well plate: Add 150 μL of 1% PVA polyvinyl alcohol to each well and leave at room temperature for about 30 min.

[0089] (2) Prepare the culture medium: Calculate the required volume of culture medium, 150 μL per well. Add RC at a ratio of 1:100 and place in a 15 mL centrifuge tube for later use.

[0090] (3) Cell treatment: First, remove the cell culture medium in the 6-well plate, add an appropriate amount of 0.5 μM EDTA, incubate at 37°C for 4 min, then remove the EDTA, add 500 μL Accutase, incubate at 37°C for 4 min, add an equal amount of culture medium for neutralization, gently blow down the cells, place them in a centrifuge tube, centrifuge at 200 g for 2 min, discard the supernatant, resuspend the cells in 1 mL of culture medium, and count the cells. Calculate the total number of cells required with 9,000 cells per well, pipette the required cells into the centrifuge tube prepared in (2), and mix by inversion.

[0091] (4) Sample loading and centrifugation: Adjust the range of the sample gun to 150 μL and pour the prepared cell suspension into the sample loading tank. Discard the Anti-adherence in the 96-well plate and add 150 μL of cell suspension to each well. Cover with sealing film and centrifuge at 100g for 5 minutes. Remove the sealing film and place in a cell culture incubator for culture.

[0092] (ii) Matrigel embedding:

[0093] (1) Thaw Matrigel in an ice box at 4°C overnight. Cut the sealing film into small pieces and expose to UV light for later use.

[0094] (2) Embedding: Cut a small hole in the 200μL pipette tip, suck out the organoids in the 96-well plate one by one, and place the organoids on the sealing film. Then use the pipette to suck away the culture medium around the organoids one by one. Use the pipette tip to take out the dissolved Matrigel, about 20μL per organoid, and be careful not to let the organoid dry out. Use the pipette tip to gently move the organoid so that it is in the middle of the Matrigel droplet.

[0095] The culture dish was placed at 37°C for 20 min to allow the Matrigel gel to completely solidify. The Matrigel gel clot was blown off the sealing film with 20 mL of Imp-A, and the sealing film was removed with tweezers. The dish was then placed in a cell culture incubator for culture.

[0096] (III) Organoid medium replacement:

[0097] (1) Change the medium to mTeSR medium on D3 after EB production.

[0098] (2) The Neural Induction medium was then replaced on D5, D7, and D11.

[0099] (3) On D13, cells were embedded with Matrigel and the Imp-A culture medium was replaced.

[0100] (4) Replace Imp-A medium at D16.

[0101] On D21, Imp+A medium was replaced and the culture medium was transferred to a shaker for culture. Imp+A medium was replaced once a week thereafter.

[0102] In summary, the present invention embodiment adopts TDP43 M337V 、TDP43 Q331K The cDNA sequence is concatenated with the CAG promoter, and then the first plasmid and the second plasmid are inserted into the chromosomes of cell line A and cell line B respectively using transposase, and the first cerebral organoid and the second cerebral organoid are differentiated and cultured to obtain the first cerebral organoid and the second cerebral organoid respectively. Then, after the first cerebral organoid and the second cerebral organoid are mature, the drug to be tested is added to the first cerebral organoid and the second cerebral organoid, and the TDP43 protein content and changes are detected respectively to verify the effect of the drug to be tested on ALS, thereby realizing the effect of the drug on ALS and the toxicity of the drug to neurons, and further realizing more accurate detection of changes in neuronal activity based on ALS, effectively solving the problem of low accuracy in detecting changes in neuronal activity in the prior art.

[0103] 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.

[0104] 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.

[0105] 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 comprising 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.

[0106] 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.

[0107] 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.

[0108] 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 detecting neuronal activity in ALS based on brain organoids, characterized in that: The following steps are involved: S1, TDP43 M337V 、TDP43 Q331K cDNA sequences were connected in series with promoters to construct TDP43 M337V 、TDP43 Q331K The recombinant transposon vectors of cDNA were denoted as the first plasmid and the second plasmid; S2, using transposase to insert the first plasmid and the second plasmid into the chromosomes of cell line A and cell line B, respectively, and differentiate and culture to obtain the first cerebral organoid and the second cerebral organoid, respectively; S3. After the first cerebral organoid and the second cerebral organoid have matured, adding the drug to be tested to the first cerebral organoid and the second cerebral organoid, and detecting the content and change of TDP43 protein respectively to verify the effect of the drug to be tested on ALS.

2. A method for detecting neuronal activity in ALS based on brain organoids as claimed in claim 1, characterized in that: The cell line A and the cell line B are both cell lines that can verify the degree of toxicity of the drug to be tested on brain organoids.

3. A method for detecting neuronal activity in ALS based on brain organoids as claimed in claim 2, characterized in that: The cell line A and / or cell line B are constructed as follows: Using gene editing technology to insert a first expression element into the genome of a cell line cell, wherein the first expression element is used to mark activated neurons; 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.

4. A method for detecting neuronal activity in ALS based on brain organoids as claimed in claim 2, characterized in that: The cell line A and / or cell line B are constructed as follows: Inserting a second expression element into the genome of a cell line cell using gene editing technology, wherein the second expression element is used to achieve measurable neuronal activity; 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.

5. A method for detecting neuronal activity in ALS based on brain organoids as claimed in claim 2, characterized in that: The cell line A and / or cell line B are constructed as follows: 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; 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; 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; 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. A method for detecting neuronal activity in ALS based on brain organoids as claimed in claim 3 or 5, 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; The fluorescent substance element is specifically a fluorescent protein element, which produces a specific color after being expressed.

7. A method for detecting neuronal activity in ALS based on brain organoids as claimed in claim 4 or 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 expression, and the sub-element 2 produces substance 2 after expression; The sub-element 1 is specifically: E-SARE enhancer element in series with ArcMini promoter in series with Gaussia luciferase; The second sub-element is specifically: CAG promoter tandem secretory luciferase Cypridina luciferase; 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 CAG promoter in series with the secretory luciferase Cypridina luciferase.

8. A method for detecting neuronal activity in ALS based on brain organoids as claimed in claim 1, characterized in that: The transposase is Sleeping Beauty.

9. A method for detecting neuronal activity in ALS based on brain organoids as claimed in claim 1, characterized in that: By performing immunofluorescence staining on the first and second cerebral organoids, respectively, the TDP43 protein content and changes were determined by the color after staining.

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