C. elegans with tdp-43 aggregation by optogenetic stimulation and method for preaparing the same

Abstract

The present invention relates to a Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation and a method for producing the same. The present invention provides a Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

Description

Technology Field

[0001] The present invention relates to a Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation and a method for producing the same. Background Technology

[0002] The incidence of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FD) is steadily increasing in today's aging society, yet there is currently no clear cure. Although the clinical symptoms of these diseases differ slightly, they share similar pathological characteristics. In these neurodegenerative diseases, the progressive loss of neurons and dysfunction occur due to the pathological degeneration of specific proteins in the central or peripheral nervous system.

[0003] ALS and FD are fatal neurodegenerative diseases that induce defects in the spinal cord and motor neurons, as well as in neurons within the frontal and temporal lobes, respectively. In the degenerated neurons of postmortem tissues from ALS and FTD patients, a deficiency of the nuclear Transactive Response Element DNA-binding Protein 43kDa (TDP-43) or TDP-43 aggregates are found in the cytoplasm. Furthermore, these TDP-43 aggregates have also been observed in neurons of patients with Limbic-Predominant Age-Related TDP-43 Encephalopathy (LATE), a new type of dementia associated with Alzheimer's disease. Neurodegenerative diseases involving such aggregated pathological TDP-43 protein in the cytoplasm, nucleus, and processes of neurons are collectively referred to as TDP-43 proteinopathy. In patients with hereditary ALS, TDP-43 aggregates resulting from more than 50 missense mutations in the TARDBP gene encoding TDP-43 have been identified, and additionally, characteristics of TDP-43 proteinopathy have been observed in over 90% of patients with sporadic ALS. Despite the high pathological correlation between these neurodegenerative diseases and TDP-43, the neurodegenerative mechanism mediated by TDP-43 in the onset of the disease has not been clearly elucidated. Therefore, this study aims to identify the molecular characteristics of TDP-43 aggregates and to elucidate the pathogenesis of TDP-43 proteinopathy and the resulting neurological damage mechanisms.

[0004] TDP-43, belonging to the heterogeneous nuclear ribonucleoprotein family, is a DNA / RNA binding protein essential for the survival of neurons. TDP-43 contains two RNA recognition motifs (RRMs), a nuclear localization signal (NLS), and a nuclear export signal (NES) (see Fig. 1). Although TDP-43 is primarily localized within the nucleus, it can freely move between the nucleus and the cytoplasm. TDP-43 undergoes homo-oligomerization via its N-terminal and binds to DNA / RNA, primarily regulating the stabilization and expression of the corresponding DNA / RNA. The C-terminal of TDP-43 possesses an intrinsically disordered region (IDR) containing a glycine-rich low complexity (LCD) / prion-like domain (PrLD). Through this region, TDP-43 interacts with proteins and RNA, enabling it to undergo liquid-liquid phase separation (LLPS), a protein phase separation process. LLPS is a phenomenon in which various proteins and RNA form a single liquid-like compartment depending on the intracellular environment.

[0005] The primary characteristic of TDP-43 proteopathies is the translocation of nuclear TDP-43 into the cytoplasm, leading to mislocalization and the formation of detergent-insoluble pathological aggregates containing hyperphosphorylated and ubiquitinated TDP-43. However, the molecular mechanisms underlying the formation of these pathological TDP-43 aggregates have not yet been clearly elucidated. Under normal physiological conditions, RRMs inhibit LLPS by binding to target RNA and increasing protein solubility. In contrast, mutations are relatively prevalent in the LCD-coding region of TARDBP in ALS / FTD; it is hypothesized that this stabilizes interactions between TDP-43 molecules and increases aggregation tendencies, thereby accelerating abnormal LLPS. RNA-binding proteins possessing IDRs that frequently undergo LLPS are primarily encapsulated within stress granules. Stress granules are transient, membrane-less organelles where cellular molecules that have undergone modification due to stress condense. Stress granules reversibly inhibit the expression of proteins and RNA by isolating damaged RNA-binding proteins within the cell, as well as their associated mRNA and ribosomal subunits. In TDP-43 proteinopathy, it is still unclear whether aggregate formation is a result of independent aggregation caused by abnormal LLPS or a result of TDP-43 being recruited into stress granules and exceeding its limits. Furthermore, previous studies have reported the observation of mutations in TARDBP's RRM along with LCD mutations in TARDBP. It is believed that TDP-43, lacking the ability to bind to DNA / RNA, fails to bind to RNA and is consequently recruited into stress granules, becoming encapsulated within the aggregates.

[0006] TDP-43 influences various RNA regulatory processes, such as RNA trafficking and stabilization, by preferentially binding to intron sequences or 3'UTR stem loop structures of RNA rich in uracil and guanine. Aggregations of abnormal TDP-43 in the cytoplasm induce the depletion of TDP-43 in the nucleus, leading to a loss of RNA processing function. Furthermore, recent studies indicate that TDP-43 significantly affects the mRNA splicing of STMN2, a microtubule regulatory protein. TDP-43 stabilizes microtubules by removing the cryptic exon of stathmin-2, thereby enabling the expression of normal STMN2. The loss of function in the nucleus of TDP-43 prevents the inhibition of the stathmin-2 cryptic exon, leading to the depletion of normal STMN2 and consequently the loss of STMN2 function. Consequently, the loss of function in the nucleus of TDP-43 can cause microtubule dysregulation and induce neuronal damage.

[0007] Many studies have analyzed the degree of motor neuron damage after overexpressing, knockout, or mutant TDP-43 to understand the pathological factors induced by TDP-43. The toxicity of TDP-43 is highly correlated with the expression levels of wild-type and mutant TDP-43 in various cell and animal models. However, these models could not accurately elicit TDP-43 aggregates, which are the primary characteristic of TDP-43 proteinopathy. Therefore, it is unclear whether the observed motor neuron phenotype is causally linked to the formation of TDP-43 aggregates. In this study, optogenetic technology was utilized to overcome these technical limitations and effectively induce aggregate formation while maintaining the function and properties of TDP-43 in vivo.

[0008] In recent research, an optogenetic technique has been developed that facilitates the study of the protein LLPS phenomenon. This technique, called OptoDroplet, is a method for inducing light-activated protein phase separation and utilizes a variant of cryptochrome 2 derived from Arabidopsis thaliana (Cry2olig; Taslimi et al., 2014). Previous studies have demonstrated that OptoDroplet technology works well not only in cultured cells but also in living biological models such as flies and zebrafish. Previous research showed that optoDroplet technology could easily induce TDP-43 aggregation, which was similar to the aggregates observed in motor neurons of ALS patients. Furthermore, these TDP-43 aggregates induced motor neuron degeneration, leading to phenotypes such as severe motor deficits and paralysis in biological models. Based on this, this study developed opto-TDP-43 by combining TDP-43 with Cry2olig, which can form TDP-43 aggregates upon light. It is hypothesized that Caenorhabditis elegans can more easily induce optoTDP-43 aggregation using external illumination because it is smaller than other in vivo models and transparent at all developmental stages. Therefore, this study aimed to analyze the neurodegenerative mechanism of TDP-43 pathological phase separation by constructing a neurodegenerative ALS in vivo model capable of screening pathological changes in motor neurons following optoTDP-43 aggregation. Prior art literature

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[0041] 33. Jo, M., et al., The role of TDP-43 propagation in neurodegenerative diseases: integrating insights from clinical and experimental studies. Experimental & Molecular Medicine, 2020. 52(10): p. 1652-1662. The problem to be solved

[0042] The object of the present invention is to provide a Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

[0043] In addition, the object of the present invention is to provide a method for producing Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

[0044] In addition, the objective of the present invention is to provide a method for screening candidate therapeutic agents for TDP-43 proteinopathy using Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

[0045] The purposes of the present disclosure are not limited to those mentioned above, and other purposes and advantages of the present disclosure not mentioned may be understood from the following description and will be more clearly understood from the embodiments of the present disclosure. Furthermore, it will be readily apparent that the purposes and advantages of the present disclosure can be realized by the means and combinations thereof set forth in the claims. means of solving the problem

[0046] The present invention provides a Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

[0047] The above optogenetic stimulation may involve transforming Caenorhabditis elegans with plasmid DNA containing nucleic acid encoding a fusion protein containing a CRY2 variant linked to the C-terminus of TDP-43, and photostimulating Caenorhabditis elegans.

[0048] The above fusion protein may include a detectable marker.

[0049] The above marker may be any one of a fluorescent protein, a reporter enzyme, a transcription factor, a radioisotope binding protein, and a bioluminescent protein.

[0050] The above fluorescent protein is a green fluorescent protein, a cyan fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein, a red fluorescent protein (e.g., mCherry), or any combination thereof.

[0051] The present invention also provides a method for producing a C. elegans in which TDP-43 aggregation is induced, comprising the step of optogenetically stimulating the C. elegans.

[0052] The optogenetic stimulation step may include 1) transforming a Caenorhabditis elegans with plasmid DNA containing nucleic acid encoding a fusion protein containing a CRY2 variant linked to the C-terminus of TDP-43; and 2) photostimulating the transformed Caenorhabditis elegans.

[0053] The above fusion protein may include a detectable marker.

[0054] The above marker may be any one of a fluorescent protein, a reporter enzyme, a transcription factor, a radioisotope binding protein, and a bioluminescent protein.

[0055] The above photo-stimulating step may be performed using a light source of 450 to 480 nm.

[0056] The present invention also provides a method for screening candidate therapeutic agents for TDP-43 proteinopathy using Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

[0057] The above screening method may include the step of administering a candidate therapeutic agent for TDP-43 proteinopathy to Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation; and the step of selecting a candidate therapeutic agent that exhibits an effect of improving TDP-43 proteinopathy by comparison with a group of Caenorhabditis elegans administered a control substance.

[0058] The above TDP-43 proteinopathy may be a disease mediated by the accumulation in the cytoplasm due to the aggregation of TDP-43 protein or mislocalization of TDP-43 protein.

[0059] The above TDP-43 proteinopathy disease may be selected from the group consisting of amyotrophic lateral sclerosis (ALS); frontotemporal lobar dementias, Lewy-body dementia, Parkinson's disease, Perry syndrome and ALS Parkinsonism-dementia complex of Guam, Huntington's disease, myopathies and sporadic inclusion body myositis. Effects of the invention

[0060] The present invention can provide a Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

[0061] In addition, the present invention can provide a Caenorhabditis elegans in which TDP-43 aggregation is induced without applying excessive stress.

[0062] In addition, the present invention can provide a method for producing Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

[0063] In addition, the present invention can provide a method for screening candidate therapeutic agents for TDP-43 proteinopathy using Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation. Brief explanation of the drawing

[0064] Figure 1 shows a schematic representation of the human TDP-43 protein domain, in which the TDP-43 protein includes two RNA-recognition motifs (RRM1, RRM2), a nuclear localization signal (NLS), a nuclear export signal (NES), and a low complexity domain (LCD) or pion-like domain (PrLD). Figure 2 shows a schematic diagram of OptoDroplet, an optogenetic clustering tool, where Cry2olig is an optogenetic protein, and when Cry2olig is exposed to blue light, LLPS (liquid-liquid phase separation) and reversible oligomerization proceed. The excitation wavelength of Cry2olig is 450 nm. Figure 3 is a schematic diagram illustrating the construction of an optogenetic ALS model in Caenorhabditis elegans, where A shows a diagram of the transplant gene (tg) containing opto-TDP-43, mCherry::Cry2olig, and mCherry::TDP-43, where the unc-25 promoter varies depending on the motor neuron. B shows the microinjection of the transplant gene into Caenorhabditis elegans. The green circle represents the co-injection marker (gcy-8p::tag-gfp), and the red circle represents the transformed DNA structure (Tg[mCherry::Cry2olig], Tg[mCherry::TDP-43], or Tg[Opto-TDP-43]). Figure 4 illustrates an optogenetic C. elegans ALS model inducing cytoplasmic aggregation of TDP-43, where A represents the blue light illumination paradigm. Transgenic C. elegans were exposed to blue light for 20 minutes and then to darkness for 5 minutes. These conditions were repeated four times. NGM plates were placed 10 cm above a 470 nm LED. B shows fluorescence microscopy images of GABAergic motor neurons in C. elegans without blue light (left panel) or with blue light exposure (right panel). GABAergic motor neurons consist of a nucleus, a cell body, and a neurotransmitter. The white frame contains a magnified image of the cell body. Opto-TDP-43 and Cry2olig aggregated in vesicles of the cell body (yellow arrow) and neurotransmitter (white arrow). The white scale bar represents 20 μm for all images. Figure 5 indicates that the cytoplasmic aggregation of TDP-43 is independent of Cry2olig clusters. A shows fluorescence microscopy images (top panel) and inversion images (bottom panel) of GABAergic motor neurons in Caenorhabditis elegans after light exposure. Opto-TDP-43 aggregated abundantly in the cytobody around the nucleus (yellow arrow). In contrast, Cry2olig formed several small inclusions in the cytoplasm (yellow arrow) and neurites (white arrow). The scale bar represents 20 μm. B shows a representative image of the GABAergic motor neuron cell body in Caenorhabditis elegans after light exposure. Opto-TDP-43 formed larger spots compared to Cry2olig. The white scale bar represents 5 μm. C shows a comparison of the size and number of spots generated from opto-TDP-43 or Cry2olig in the cell body. n=10 means 10 different nematodes per blue light. Figure 6 indicates that the inclusion of opto-TDP-43 shares similar functions with pathological TDP-43 aggregates, where A is a schematic diagram of the experimental procedure and B is a fluorescence microscopy image of GABAergic motor neurons over time. Opto-TDP-43 inclusion (left panel) was maintained in the cell bodies and neurites for up to 6 hours after light exposure. Cry2olig clusters (right panel) resolved 2 hours after light exposure. White arrow: inclusion, yellow arrow: inclusion in other neurons. The scale bar represents 20 μm for all images. Figure 7 illustrates that Opto-TDP-43 induces chronic motor deficits in C. elegans, where A is a time-series image of nematodes performing a single thrash ("lateral swim") in a liquid medium. Here, a thrash is defined as moving completely to one side and returning to the original position. B shows the number of thrashes per 30 seconds measured for adult N2 (WT) and transgenic animals. The total sample size was 80 obtained from three independent experiments. Data are expressed as mean ± SEM. Statistics: t-test; not significant (ns), ***p < 0.001. Figure 8 shows a comparison of pathological TDP-43 inclusion formation in TDP-43 proteinopathy and an optoDroplet-mediated TDP-43 model, which explains the behavior of the TDP-43 protein within the motor neurons of Caenorhabditis elegans. Under normal conditions, TDP-43 is primarily localized to the nucleus and traverses between the nucleus and the cytoplasm. In the cytoplasm, TDP-43 regulates RNA by forming oligomers through its N-terminal domain but does not form inclusions. In TDP-43 proteinopathy, TDP-43 undergoes excessive oligomerization due to LCD mutations. This abnormal assembly depletes TDP-43 in the nucleus and forms pathological aggregates in the cytoplasm. In a blue light-free optogenetic Caenorhabditis elegans ALS model, opto-TDP-43 acts similarly to the wild-type TDP-43 protein. However, upon exposure to blue light, opto-TDP-43 undergoes rapid Cry2olig-mediated oligomerization, inducing TDP-43 LCD-mediated oligomerization. Due to extended light exposure, opto-TDP-43 eventually forms large, persistent aggregates very similar to those seen in TDP-43 proteinopathy. Specific details for implementing the invention

[0065] The present invention will be explained in more detail below through examples and experimental examples. However, these examples and test examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples and test examples.

[0067] Materials and Methods

[0068] 1. C. elegans strain and maintenance

[0069] Caenorhabditis elegans is Escherichia coli(OP50) was cultured in Nematode Growth Media (NGM) and maintained at 20°C. Transgenic nematodes were maintained at 25°C to improve heritability to the next generation. The wild type used in this study was the N2 Bristol strain. Nematode CL6049 expressing Human TDP-43 (NM_007375.4) was provided by the Caenorhabditis Genetics Center (CGC, University of Minnesota, MN, USA). The nematodes used in this study are summarized in Table 1.

[0070]

[0072] 2. Polymerase Chain Reaction (PCR)

[0073] PCR was performed to induce genetic recombination with each DNA fragment. For nematodes, template DNA was extracted by inoculating 3–5 specimens into 2X lysis buffer and reacting at 60°C for 1 hour followed by 95°C for 15 minutes. Q5 High-Fidelity DNA Polymerase (New England Biolabs, NEB) was used to construct all DNA fragments into blunt ends to facilitate Gibson cloning. PCR was performed using a mixture of 5X Q5 reaction buffer, 10 mM dNTPs, 10 μM forward / reverse primers, Q5 High-Fidelity DNA Polymerase, 5x Q5 GC enhancer, and nuclease-free water. The PCR conditions were set as follows: initial denaturation at 98°C for 30 seconds, denaturation at 98°C for 10 seconds, annealing at 50–72°C for 30 seconds, and extension at 72°C for 30 seconds / kb. At this time, annealing was performed using the touch-down method, decreasing by 0.5℃ per cycle. The primers used in this study are summarized in Table 2.

[0074]

[0075] 3. TA Cloning & TOPO Cloning

[0076] The pCR8 / GW / TOPO TA Cloning Kit (Invitrogen) was used to construct an entry vector expressing TDP-43. First, to add adenine to the 3' end of the TDP-43 DNA, the DNA and Taq polymerase were mixed and incubated at 72°C for 10 minutes. Next, this reaction mixture was mixed with salt solution, water, and the TOPO vector, and incubated at room temperature for 1 hour. The cloning product was amplified through transformation.

[0078] 4. Gateway Cloning

[0079] Gateway LR Clonase II Enzyme mix (Invitrogen) was used to construct an expression vector capable of expressing TDP-43 at the unc-25 promoter. A destination vector containing the unc-25 promoter was provided by another laboratory. The entry vector, destination vector, 5X LR Clonase Reaction buffer, and TE buffer were mixed and reacted at 25°C for 1 hour. The cloning product was amplified through transformation.

[0081] 5. Gibson Cloning

[0082] To bind mCherry to the N-terminal of TDP-43 within the expression vector, primers were designed so that the N-terminal of TDP-43 overlapped with the C-terminal of mCherry, and the N-terminal of mCherry overlapped with the vector's unc-25 promoter. Similarly, to bind Cry2olig to the C-terminal of TDP-43, primers were designed so that the C-terminal of TDP-43 overlapped with the N-terminal of Cry2olig, and the C-terminal of Cry2olig overlapped with the vector's 3' untranslated region (3' UTR). To ensure high cloning efficiency, the overlap length of all primers was designed to be 30 bp. The DNA fragments obtained via PCR were ligated using NEBuilder HiFi DNA Assembly Master Mix (NEB). 10 μl of the DNA mix to be ligated was mixed with 10 μl of NEBuilder HiFi DNA Assembly Master Mix and incubated at 50°C for 1 hour. The cloning product was amplified through transformation.

[0084] 6. Site-directed mutagenesis

[0085] The Q5 Site-directed Mutagenesis Kit (NEB) was used to derive mCherry-TDP-43 and mCherry-Cry2olig from opto-TDP-43, a plasmid DNA produced via cloning. Primers were designed to delete TDP-43 or Cry2olig from opto-TDP-43 (mCherry::TDP-43::Cry2olig), and PCR was performed for each. Subsequently, the PCR products, 2X KLD Reaction Buffer, 10X KLD Enzyme Mix (Kinase, Ligase, DpnⅠ), and Nuclease-free Water were mixed and incubated at room temperature for 1 hour. The cloning products were amplified through transformation. The plasmid DNAs used in this study are summarized in Table 3.

[0088]

[0090] 7. Transformation

[0091] All cloned plasmid DNA contains a selective marker. Transformation was performed to select fully cloned plasmid DNA using the selective marker and to obtain it in large quantities. NEB stable cells (C3040H, NEB) were thawed on ice for 10 minutes, and then 2 μl of 100 pg-100 ng plasmid DNA was added and mixed. The mixture was incubated on ice for 30 minutes, followed by a heat shock at 42°C for 30 seconds. 350 μl of Stable Outgrowth Medium (SOC) was added, and the cells were incubated for 1 hour in a shaking incubator at 250 rpm at 37°C. Since the plasmid DNA used in this study contains an ampicillin resistance gene, the mixture was plated onto solid LB plates containing ampicillin and incubated overnight in the incubator.

[0093] 8. Plasmid DNA preparation

[0094] Prior to conducting this experiment, the transformed colonies from solid LB medium were transferred to liquid LB medium the previous day and cultured in a shaking incubator at 37°C rotating at 250 rpm. Plasmid DNA was extracted using the PureLink Quick Plasmid Miniprep Kit (K210010, Invitrogen). 3-4 mL of the LB culture cultured in liquid LB medium was harvested by centrifuging at 13,000 rpm. 250 μl of R3 buffer (Resuspension buffer) was added along with RNase A to break up the cell pellet, after which 250 μl of L7 buffer (Lysis buffer) was added and the mixture was gently shaken. 350 μl of N4 buffer (Precipitation buffer) was added to the mixture, and the mixture was centrifuged at 12,000 rpm for 10 minutes. The supernatant containing plasmid DNA was transferred to a spin column and centrifuged at 12,000 rpm for 1 minute, then washing buffer was added and centrifuged at 12,000 rpm for 1 minute. The DNA in the column was eluted with pure water and obtained by centrifuging at 12,000 rpm for 2 minutes.

[0096] 9. Restriction enzyme digestion and Sanger sequencing

[0097] The newly constructed plasmid DNA via cloning was verified through restriction enzyme treatment and DNA sequencing (Macrogen). For quantification of the plasmid DNA, the concentration was set to 1000 ng / μl, and 2 μl of Hind III (ELPIS BIOTECH) and 10X Reaction Buffer 2 (ELPIS BIOTECH) were added and incubated at 37°C for 4 hours. The cleaved size of the reaction product was confirmed by electrophoresis on a 1% agarose gel.

[0099] 10. Microinjection

[0100] Microinjection was performed to produce transgenic Caenorhabditis elegans. Before microinjection, glass capillaries (Narishige GD-1) were placed in Narishige PC-100 to construct microneedles. The temperature for constructing the microneedles was set to 58°C–60°C. 1 μl of plasmid DNA mix (co-injection marker and transgenic DNA) was injected into the completed microneedles.

[0101] A slide glass was placed on a microscope (ZEISS Axio Vert.A1) for microinjection, covered with a cover glass, and a drop of Halocarbon Oil 700 (SIGMA) was added. Then, the tip of the microneedle was gently tapped to break it open so that DNA could be released. The DNA injection pressure was set using a microinjector (Tritech Research).

[0102] To observe nematodes under a microscope, pads were prepared by boiling triple-distilled water with 2% agarose. Halocarbon oil was dropped onto the pad, and a wild-type nematode was placed on it to fix it in place. After focusing the nematode and the microneedle under the microscope, the nematode was moved by gently moving the gliding table toward the tip of the needle. The microneedle was inserted into the nematode's gonad to inject the plasmid DNA mix. The nematodes were rescued in M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 85.5 mM NaCl, 1 mM MgSO4) and cultured at 25°C. The transgenic nematodes produced in this study are summarized in Table 4.

[0103]

[0105] 11. Microscopy

[0106] Nematodes transformed by microinjection were detected using a fluorescence dissecting microscope (LEICA TL3000 Ergo). mCherry expression was observed using a fluorescence optical microscope (LEICA DM 2000) after placing 2 μl of M9 buffer on a 10% agarose pad, covering the nematodes with a cover glass, and observing the results. The fluorescence images presented in this study were captured using a LEICA DFC 7000gt.

[0108] 12. Blue light illumination

[0109] After transferring nematodes expressing Opto-TDP-43, mCherry::Cry2olig, and mCherry::hTDP-43 to each NGM, they were placed 10 cm below a blue light LED panel (Edmund Optics, #66-831) for photostimulation. The wavelength of the LED panel used was 470 nm.

[0111] 13. Thrashing assay

[0112] A thrashing assay was performed to evaluate the motility of Caenorhabditis elegans. 1-day-old adult nematodes were transferred to an NGM free of OP50, and 2 μl of M9 buffer was added. After acclimatizing the nematodes for 1 minute, the number of swims over 30 seconds was measured. Motility was evaluated for 15–20 nematodes per set. All analyses were performed at room temperature.

[0114] 14. Statistics analysis

[0115] Statistical significance was determined using Graphpad Prism software (Version 9.03), and p-values ​​of 0.05 or lower were considered significant. The Unpaired Student t-test was used to determine statistical significance in data comparing two variables.

[0116] First, when only the TDP-43 protein was expressed, TDP-43 was localized to the nucleus of motor neurons regardless of the presence or absence of blue light, and no specific inclusions of TDP-43 were found in the cell body or axon (Fig. 4B). Therefore, cytoplasmic aggregates, which are a pathological characteristic of TDP-43 proteinopathy, could not be observed in the motor neurons of nematodes expressing the TDP-43 protein. This is an expected result because the TDP-43 used in this study is a normal protein, not an ALS-inducing mutant, and previously published research results have also reported that TDP-43 exhibits a similar expression pattern observed within the nucleus.

[0117] Next, when only the optogenetic Cry2olig protein was expressed, contrary to expectations, it was observed that the TDP-43-free Cry2olig protein formed small inclusions throughout the cell even without exposure to blue light (Fig. 4B). This suggests that the Cry2olig protein underwent oligomerization for a short period of time as exposure to natural light, including blue light, was not completely blocked. Even when exposed to blue light, the Cry2olig protein was observed to form inclusions in both the cytoplasm and neurites (Fig. 4B).

[0118] Finally, under dark conditions, opto-TDP-43 was mostly distributed in the nucleus and partially aggregated in the cytoplasm. This cytoplasmic aggregation is considered to be a transient phenomenon resulting from the physical properties of Cry2olig upon exposure to natural light, similar to the Cry2olig protein. Interestingly, upon exposure to blue light, not only did the number and size of cytoplasmic inclusions of opto-TDP-43 increase significantly compared to before, but inclusions were also observed forming in neurites located close to the cell body (Fig. 4B). Based on these results, it is hypothesized that while TDP-43 was restricted to the nucleus under dark conditions due to its nucleus-localized nature, the optogenetic properties of Cry2olig exposed to blue light enhanced aggregation between TDP-43 LCDs, ultimately inducing TDP-43 mislocalization and contributing to inclusion formation. Therefore, in this study, we were able to induce the formation of abnormal inclusions through optogenetic regulation without significantly increasing the expression level of TDP-43.

[0120] 3. Opto-TDP-43 aggregates are independent of Cry2olig aggregates.

[0121] Following exposure to blue light, both Cry2olig and opto-TDP-43 proteins formed inclusions. These results suggest that these are reversible inclusions transiently formed by Cry2olig, rather than pathological aggregates of opto-TDP-43. In this study, the locations and morphologies of the two types of inclusions were compared to understand the differences between opto-TDP-43 and Cry2olig inclusions within nematode motor neurons. First, the locations of inclusions were investigated up to a point 20 μm away from the nucleus of motor neurons after blue light exposure. Cry2olig inclusions formed indiscriminately in the cytoplasm and neurites (Fig. 5A). Interestingly, opto-TDP-43 inclusions were mostly not found in neurites but were formed primarily in the cell body (Fig. 5A). This is presumed to be a result of the fact that Cry2olig proteins, which are widely distributed in neurons, have no positional restrictions on aggregation and could easily form inclusions in various locations, whereas opto-TDP-43 primarily exits from the nucleus and aggregates.

[0122] Importantly, the inclusions of Opto-TDP-43 found in the cytoplasm showed several significant differences compared to the inclusions of Cry2olig proteins (Fig. 5B). Although Opto-TDP-43 had fewer inclusions than Cry2olig proteins, it formed larger inclusions (Figs. 5B and C). The inclusions of Opto-TDP-43 were observed in a large, clumping form (Fig. 5B); this form is presumed to be due to irreversible aggregation following protein recruitment triggered by Cry2olig within Opto-TDP-43, resulting in increased inclusion size. Previous studies have reported that the initial recruitment of TDP-43 acts as a seed that further aggregates surrounding TDP-43 due to structural features within the TDP-43, such as the N-terminal domain or LCD. In this study, it is hypothesized that the initial aggregation of TDP-43 induced by Cry2olig was accelerated, leading to the formation of large aggregates. Therefore, these results indicate that the inclusions in opto-TDP-43 are likely irreversible aggregates, unlike the multiple inclusions in Cry2olig.

[0124] 4. Opto-TDP-43 aggregates have characteristics similar to pathological TDP-43 aggregates.

[0125] To determine whether aggregates formed upon exposure to blue light possess the characteristics of irreversible pathological aggregates observed in ALS patients, we examined the differences between opto-TDP-43 aggregates and Cry2olig protein aggregates following blue light exposure. After exposing nematodes expressing each protein to blue light, we observed the patterns of aggregate changes over time. At this time, to observe how opto-TDP-43 changes not only in the cell body but also in the neurites, we also observed caudal motor neurons, which have higher protein expression than other motor neurons. The locations of opto-TDP-43 aggregates and Cry2olig aggregates were observed at time intervals of 30 minutes, 2 hours, and 6 hours after blue light exposure (Fig. 6A). Both Cry2olig aggregates and opto-TDP-43 aggregates formed 30 minutes after blue light exposure were maintained in both the cell body and the neurites (Fig. 6B). Interestingly, 2 hours after blue light exposure, the aggregation of Cry2olig protein disappeared, but opto-TDP-43 aggregates persisted or increased in size (Fig. 6B), and opto-TDP-43 aggregates remained until 6 hours after blue light exposure (Fig. 6B). Furthermore, opto-TDP-43 aggregates were strongly observed in the cell bodies of neurons, and these remained strongly even after 6 hours following blue light exposure (Figs. 6A to C).

[0126] Consequently, once formed, opto-TDP-43 aggregates were not easily degraded, and even after a long time had passed since the blue light exposure stopped, the aggregates were maintained or even increased in size. These results are quite similar to previously reported research findings and indicate that the opto-TDP-43 aggregates in this study also undergo irreversible aggregation dependent on TDP-43. Therefore, the opto-TDP-43 aggregates produced in this study not only possessed molecular characteristics very different from Cry2olig aggregates but were also similar to the characteristics of pathological TDP-43 aggregates observed in TDP-43 proteinopathy.

[0128] 5. The formation of Opto-TDP-43 aggregates is correlated with motility disorders in Caenorhabditis elegans.

[0129] Since actual ALS patients lose motor function due to progressive muscle atrophy, the motor ability of nematodes was evaluated to determine whether the formation of opto-TDP-43 aggregates within motor neurons correlates with motor impairment. Blue light exposure was conducted in the same manner as in previous experiments, and subsequently, the motor ability of transgenic nematodes was evaluated using a thrashing assay. The thrashing assay is a method that measures the frequency of swimming movements by placing nematodes in a liquid, allowing for easy identification of nematodes with motor defects (Fig. 7A). In this study, adult nematodes were placed in M9 buffer, and the number of swims per 30 seconds was measured. One hour after blue light exposure, nematodes expressing Cry2olig and opto-TDP-43 exhibited a motor defect phenotype, excluding wild-type and TDP-43-expressing nematodes (Fig. 7B). Surprisingly, when the motility of the nematodes was evaluated 6 hours after light exposure, the motility of the nematodes expressing Cry2olig recovered, whereas the motility of the nematodes expressing opto-TDP-43 continued to decline (Fig. 7B). In particular, compared to Cry2olig, the nematodes expressing opto-TDP-43 exhibited a phenotype of slow swimming with their bodies bent and curled to one side.

[0130] In Figure 5, aggregates of Cry2olig and opto-TDP-43 initially formed inclusions, but opto-TDP-43 inclusions remained even after 6 hours of blue light exposure. In this context, motor defects were overcome over time in nematodes expressing Cry2olig after blue light exposure, whereas motor defects were continuously induced in nematodes expressing opto-TDP-43 regardless of time. Cry2olig inclusions were observed to cause acute neurotoxicity, whereas opto-TDP-43 aggregates are presumed to cause chronic neurotoxicity because they persisted compared to Cry2olig aggregates. Therefore, this correlation suggests that the formation and accumulation of opto-TDP-43 aggregates may contribute to the induction of motor defects.

[0132] argument

[0133] To understand TDP-43 proteopathies, this study constructed an optogenetic ALS in vivo model capable of forming cytoplasmic TDP-43 aggregates, a pathological feature observed in ALS / FTD. Previous research aimed to elucidate the mechanisms of neurological damage by regulating TDP-43 expression in various in vitro and in vivo settings or by inducing TDP-43 aggregation under excessive stress conditions. Consequently, there were limitations in identifying the clear causes of neurological damage because TDP-43 aggregation was induced under excessive stress conditions. In this study, to investigate the relationship between pathological TDP-43 aggregation and the mechanism of neurological damage, we successfully developed an optogenetic opto-TDP-43 system capable of forming inclusions using Cry2olig, whose aggregation is regulated by light (Fig. 3). In other words, we succeeded in inducing TDP-43 aggregation at any desired time using only a small amount of light, without applying excessive stress.

[0134] Interestingly, in motor neurons of nematodes, TDP-43 protein was localized to the nucleus; however, opto-TDP-43 exited the nucleus after light exposure and formed aggregates on the cell body and neurites, similar to the aggregates observed in neurons of actual ALS patients (Fig. 4). Previous studies have hypothesized that neuronal damage is caused by a deficiency in the nuclear function of TDP-43, the formation of cytoplasmic aggregates, or both processes. Since the opto-TDP-43 developed in this study also exhibits a deficiency of nuclear TDP-43 along with the simultaneous formation of cytoplasmic aggregates (Fig. 5), it is expected to serve as a highly useful animal model for TDP-43 proteinopathy.

[0135] These opto-TDP-43 aggregates exhibited characteristics similar to pathological aggregates; compared to Cry2olig, which forms small inclusions in clusters throughout the motor neuron, Opto-TDP-43 formed large, broad inclusions mainly in the cell body (Fig. 5). In actual ALS patients' neurons, relatively few large aggregates are mainly observed rather than numerous small aggregates. This indicates that opto-TDP-43 inclusions are more similar to pathological aggregates of TDP-43 proteinopathy than to transient aggregates like Cry2olig.

[0136] Another characteristic of pathological aggregates in TDP-43 proteinopathy is their sustained accumulation. The initial pathological aggregation of TDP-43 is irreversible and possesses a seeding ability that recruits RNA along with other surrounding RNA-binding proteins. Consequently, the sustained accumulation of TDP-43 in the cytoplasm gradually accelerates, leading to an expansion in aggregate size. In this study, we confirmed that the size of opto-TDP-43 aggregates was maintained or increased over time. These results suggest that although opto-TDP-43 aggregates are mediated by Cry2olig, they are subsequently maintained through TDP-43-dependent mechanisms.

[0137] Pathological aggregates observed in TDP-43 proteinopathy exhibit various biochemical characteristics. When TDP-43 forms pathological aggregates, phosphorylation often occurs at the C-terminal (S409 / 410), and the resulting aggregates become ubiquitinated. Furthermore, these aggregates are insoluble and do not dissolve easily. Although this study indirectly demonstrated that the opto-TDP-43 aggregates induced in this research are similar to pathological aggregates,

[0138] Patients with ALS develop muscle atrophy due to the progressive loss of motor neurons, which leads to paralysis and eventually death. In this study, to investigate the correlation between opto-TDP-43 aggregates and motor impairment in nematodes, the motor ability of nematodes was evaluated over time after the formation of aggregates. Motor defects resulting from TDP-43 aggregation in in vivo models were observed consistently in previous studies. Surprisingly, in this study as well, nematodes expressing opto-TDP-43 exhibited persistent motor dysfunction (Fig. 7). These results are significantly consistent with those of previous studies and suggest that opto-TDP-43 aggregates, which remain persistently in motor neurons after blue light exposure, have a high correlation with motor defects.

[0139] In this study, an optogeneic nematode ALS model was constructed using optoDroplet technology, and opto-TDP-43 aggregates were found to be significantly similar to the pathological aggregates observed in TDP-43 proteinopathy. Importantly, the study demonstrated that the mislocalization and irreversible aggregates of TDP-43 protein are consequently associated with the induction of motility disorders in nematodes. Therefore, the optogeneic nematode ALS model constructed in this study appears to serve as a useful tool for understanding TDP-43 proteinopathy.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 A method for producing a C. elegans with irreversible TDP-43 aggregation induced, comprising the step of optogenetically stimulating the C. elegans, wherein the optogenetically stimulating step comprises: preparing an expression vector by combining a CRY2 variant with the C-terminus of TDP-43, combining the N-terminus of TDP-43 with the C-terminus of mCherry, and combining the N-terminus of mCherry with the unc-25 promoter; preparing a plasmid DNA into which the expression vector is inserted; injecting the plasmid DNA into the C. elegans to transform it; and photostimulating the transformed C. elegans. Claim 6 A method for producing a Caenorhabditis elegans with irreversible TDP-43 aggregation induced, wherein, in claim 5, the transformation step involves injecting the plasmid DNA into the Caenorhabditis elegans via microinjection, and the plasmid DNA is injected at a concentration of 0.1 to 25 ng / μl. Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 A method for screening candidate therapeutic agents for TDP-43 proteinopathy, comprising the steps of: preparing a C. elegans in which irreversible TDP43 aggregation is induced according to claim 5; and using the C. elegans in which irreversible TDP-43 aggregation is induced prepared in the step. Claim 11 A screening method according to claim 10, wherein the step of using the above-mentioned Caenorhabditis elegans comprises: a step of administering a candidate therapeutic agent for TDP-43 proteinopathy to Caenorhabditis elegans in which irreversible TDP-43 aggregation is induced by optogenetic stimulation; and a step of selecting a candidate therapeutic agent that exhibits an effect of improving TDP-43 proteinopathy compared to a group of Caenorhabditis elegans administered a control substance. Claim 12 A screening method according to claim 11, characterized in that the above-mentioned TDP-43 proteinopathy is a disease mediated by the aggregation of TDP-43 protein or accumulation in the cytoplasm due to mislocalization of TDP-43 protein. Claim 13 A screening method according to claim 12, characterized in that the TDP-43 proteinopathy disease is amyotrophic lateral sclerosis (ALS) or frontotemporal lobar dementias.

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