KR20240111554A - C. elegans with tdp-43 aggregation by optogenetic stimulation and method for preaparing the same - Google Patents

Abstract

The purpose of the present invention is to provide a caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation. Additionally, the purpose of the present invention is to provide a manufacturing method of a caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation, and to provide a method for screening candidate therapeutics for TDP-43 proteinopathy diseases using a caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation. In the caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation, the optogenetic stimulation can involve transforming the caenorhabditis elegans with plasmid DNA containing a nucleic acid encoding fusion protein including a CRY2 variant linked to the C-terminus of TDP-43, and applying light stimulation to the caenorhabditis elegans. Additionally, the fusion protein can include a detectable marker, and the marker can be one selected from the group consisting of a fluorescent protein, a reporter enzyme, a transcription factor, a radiolabeled protein, and a bioluminescent protein.

Description

Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation and method for producing the same {C. ELEGANS WITH TDP-43 AGGREGATION BY OPTOGENETIC STIMULATION AND METHOD FOR PREAPARING THE SAME}

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

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, but there is currently no clear treatment. The clinical symptoms of these diseases are all slightly different, but they share similar pathological characteristics. In these neurodegenerative diseases, the gradual loss and dysfunction of nerve cells occurs due to pathological degeneration of specific proteins in the central nervous system or peripheral nervous system.

ALS and FD are fatal neurodegenerative diseases that induce defects in neurons in the spinal cord, motor neurons, and frontal and temporal lobes, respectively. In degenerated nerve cells in postmortem tissues of ALS and FTD patients, a lack of Transactive response element DNA-binding Protein 43kDa (TDP-43) protein in the nucleus or TDP-43 aggregates in the cytoplasm are found. Additionally, these TDP-43 aggregates were also observed in neurons of patients with Limbic-predominant Age-related TDP-43 Encephalopathy (LATE), a new type of dementia along with Alzheimer's disease. As such, neurodegenerative diseases involving pathological TDP-43 protein aggregated in the cytoplasm, nucleus, and neurites of nerve cells are collectively referred to as TDP-43 proteinopathy. In hereditary ALS patients, aggregates of TDP-43 due to more than 50 missense mutations were identified in the TARDBP gene encoding TDP-43. In addition, characteristics of TDP-43 proteinopathy were observed in more than 90% of sporadic ALS patients. Despite the high pathological correlation between these neurodegenerative diseases and TDP-43, the mechanism of neurodegeneration caused by TDP-43 in disease development has not been clearly identified. Therefore, in this study, we aimed to determine the molecular characteristics of TDP-43 aggregates and reveal the pathogenesis of TDP-43 proteinopathy and the resulting mechanism of nerve damage.

TDP-43, belonging to the heterogeneous nuclear ribonucleoprotein family, is a DNA/RNA binding protein essential for the survival of nerve cells. TDP-43 contains two RNA recognition motifs (RRM), a nuclear localization signal (NLS), and a nuclear export signal (NES) (see Figure 1). TDP-43 is primarily localized within the nucleus, but can freely shuttle between the nucleus and cytoplasm. TDP-43 forms homo-oligomerization through the N-terminal and binds to DNA/RNA, mainly controlling the stabilization and expression of the DNA/RNA. The C-terminal of TDP-43 has an intrinsically disordered region (IDR) containing a glycine-rich low complexity (LCD)/prion-like domain (PrLD). TDP-43 can interact with proteins and RNA through this region and undergo liquid-liquid phase separation (LLPS), a protein phase separation process. LLPS is a phenomenon in which several proteins and RNA form a liquid-like compartment depending on the intracellular environment.

The main characteristic of TDP-43 proteinopathy is that TDP-43 in the nucleus escapes into the cytoplasm and mislocalization occurs, resulting in the formation of insoluble (detergent-insoluble) pathological aggregates containing hyperphosphorylated and ubiquitinated TDP-43. However, the molecular mechanism that forms these pathological TDP-43 aggregates has not yet been clearly identified. Under normal physiological conditions, RRM inhibits LLPS by binding to target RNA and increasing protein solubility. However, in ALS/FTD, there are relatively many mutations in the LCD coding region of TARDBP, which is presumed to accelerate abnormal LLPS by making the interaction between TDP-43 more stable and increasing the aggregation tendency. RNA-binding proteins that have IDRs and commonly undergo LLPS are mainly contained in stress granules. Stress granules are temporary membraneless organelles in which cellular molecules that have been modified by exposure to stress condense. Stress granules reversibly inhibit protein and RNA expression by sequestering damaged RNA-binding proteins as well as their associated mRNAs and ribosomal subunits within the cell. In TDP-43 proteinopathy, it is still unclear whether the formation of aggregates is the result of independent aggregation by abnormal LLPS or the result of TDP-43 being recruited to stress granules and forming beyond their limits. In addition, in a previous study, it was reported that in addition to the LCD mutation of TARDBP, a mutation in the RRM of TARDBP was also observed. It is believed that TDP-43, which lacks the ability to bind DNA/RNA, is unable to bind RNA and is recruited together into stress granules, thereby being incorporated into aggregates.

TDP-43 affects various RNA regulatory processes such as RNA trafficking and RNA stabilization by preferentially binding to intron sequences or 3'UTR stem loop structures of RNA rich in uracil and guanine. Abnormal TDP-43 aggregates in the cytoplasm induce depletion of TDP-43 in the nucleus, resulting in loss of RNA processing function. Additionally, recent studies have shown that TDP-43 has a significant effect on mRNA splicing of STMN2, a microtubule regulatory protein. TDP-43 stabilizes microtubules by removing the cryptic exon of stathmin-2 and expressing normal STMN2. Loss of function of TDP-43 in the nucleus is unable to repress the cryptic exon of stathmin-2, leading to depletion of normal STMN2 and loss of function of STMN2. As a result, loss of function of TDP-43 in the nucleus can cause dysregulation of microtubules and induce neuronal damage.

In many studies, to determine the pathological factors caused by TDP-43, the degree of damage to motor neurons was analyzed after overexpressing TDP-43 and expressing knockout or mutant TDP-43. The toxicity of TDP-43 is highly correlated with wild-type and mutant TDP-43 expression levels in various cell and animal models. However, these models were unable to accurately derive TDP-43 aggregates, the main 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 used to overcome these technical limitations and effectively form aggregates while maintaining the function and properties of TDP-43 in vivo.

In recent research, optogenetic technology has been developed to facilitate the study of protein LLPS phenomenon. This technology, called OptoDroplet, uses a variation of cryptochrome 2 (Cry2olig; Taslimi et al., 2014) derived from Arabidopsis thaliana as a method to induce light-activated protein phase separation. Through previous research, OptoDroplet technology has been proven to work well not only in cultured cells, but also in living models such as live flies and zebrafish. In previous studies, TDP-43 could be easily aggregated using optoDroplet technology, which was similar to the aggregates seen in motor neurons of ALS patients. Additionally, these TDP-43 aggregates caused degeneration of motor neurons, leading to severe motor defects and paralysis-like phenotypes in the in vivo model. In this study, based on this, we developed opto-TDP-43, which can form TDP-43 aggregates by light by combining TDP-43 and Cry2olig. Because Caenorhabditis elegans is smaller than other biological models and is transparent at all stages of development, it is believed that aggregation of optoTDP-43 can be more easily induced by external lighting. Therefore, in this study, we attempted to analyze the neurodegenerative mechanism of pathological phase separation of TDP-43 by constructing a neurodegenerative ALS biological model that can screen for pathological changes in motor neurons due to aggregation of opto-TDP-43.

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The purpose of the present invention is to provide Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

Additionally, the purpose of the present invention is to provide a method for producing Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

Additionally, the purpose of the present invention is to provide a method for screening candidate treatments for TDP-43 proteinopathy disease using Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

The objects of the present disclosure are not limited to the purposes mentioned above, and other objects and advantages of the present disclosure that are not mentioned can be understood by the following description and will be more clearly understood by the examples of the present disclosure. Additionally, it will be readily apparent that the objects and advantages of the present disclosure can be realized by the means and combinations thereof indicated in the patent claims.

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

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

The fusion protein may contain a detectable marker.

The marker may be any one of fluorescent proteins, reporter enzymes, transcription factors, radioisotope binding proteins, and bioluminescent proteins.

The fluorescent protein is green fluorescent protein, cyan fluorescent protein, blue fluorescent protein, yellow fluorescent protein, red fluorescent protein (eg, mCherry), or any combination thereof.

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

The optogenetically stimulating step includes 1) transforming Caenorhabditis elegans with plasmid DNA containing a 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.

The fusion protein may contain a detectable marker.

The marker may be any one of fluorescent proteins, reporter enzymes, transcription factors, radioisotope binding proteins, and bioluminescent proteins.

The photostimulating step may be photostimulating using a light source of 450 to 480 nm.

The present invention also provides a method for screening candidate treatments for TDP-43 proteinopathy disease using Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

The screening method includes administering a candidate therapeutic agent for TDP-43 proteinopathy disease to Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation; And it may include the step of selecting a candidate therapeutic agent that shows an effect of improving TDP-43 proteinopathy disease compared to the Caenorhabditis elegans group administered the control substance.

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

The TDP-43 proteinopathy disease includes amyotrophic lateral sclerosis (ALS); frontotemporal lobar dementias, Lewy-body dementia, Parkinson disease, Perry syndrome and ALS Parkinsonism-dementia complex of Guam ), Huntington's disease, myopathies, and sporadic inclusion body myositis.

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

Additionally, the present invention can provide Caenorhabditis elegans in which TDP-43 aggregation is induced without applying excessive stress.

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

Additionally, the present invention can provide a method for screening candidate treatments for TDP-43 proteinopathy disease using Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation.

Figure 1 shows a schematic representation of the human TDP-43 protein domain, in which the TDP-43 protein has two RNA-recognition motifs (RRM1, RRM2), a nuclear localization signal (NLS), and a nuclear It contains a nuclear export signal (NES) and a low complexity domain (LCD) or prion-like domain (PrLD).
Figure 2 shows a schematic diagram of OptoDroplet, an optogenetic clustering tool. 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 showing the construction of an optogenetic ALS model in Caenorhabditis elegans, where A shows a diagram of the transgene (tg) including opto-TDP-43, mCherry::Cry2olig, and mCherry::TDP-43. . Here, the unc-25 promoter is motor neuron specific. B represents transgene microinjection into Caenorhabditis elegans. Green circles represent co-injection markers (gcy-8p::tag-gfp) and red circles represent transgenic DNA constructs (Tg[mCherry::Cry2olig], Tg[mCherry::TDP-43], or Tg[Opto-TDP- 43].
Figure 4 shows an optogenetic C. elegans ALS model that induces cytoplasmic aggregation of TDP-43, where A represents the paradigm of blue light illumination. Transgenic C. elegans were exposed to blue light for 20 minutes and dark for 5 minutes. These conditions were repeated four times. The NGM plate was placed 10 cm above the 470 nm LED. B shows fluorescence microscopy images of GABAergic motor neurons in Caenorhabditis elegans without blue light (left panel) or with blue light exposure (right panel). GABAergic motor neurons are composed of a nucleus, cell body, and neurites. The white frame contains a close-up photo of the cell body. Opto-TDP-43 and Cry2olig aggregated in vesicles of cell bodies (yellow arrows) and neurites (white arrows). White scale bar represents 20 μm for all images.
Figure 5 shows that cytoplasmic aggregation of TDP-43 is independent of Cry2olig clusters, and A shows a fluorescence microscopy image (top panel) and inverted image (bottom panel) of GABAergic motor neurons of Caenorhabditis elegans after light exposure. Opto-TDP-43 was abundantly aggregated in the cell body around the nucleus (yellow arrow). In contrast, Cry2olig formed several small inclusions in the cytoplasm (yellow arrows) and neurites (white arrows). Scale bar represents 20 μm. B shows a representative image of the cell body of a GABAergic motor neuron in Caenorhabditis elegans after light exposure. Opto-TDP-43 formed larger spots compared to Cry2olig. White scale bar represents 5 μm. C shows 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 shows that inclusion of optical TDP-43 shares similar functions with pathological TDP-43 aggregates, A is a schematic diagram of the experimental procedure, and B is a fluorescence microscopy image of GABAergic motoneurons over time. Opto-TDP-43 inclusions (left panel) were retained in cell bodies and neurites for up to 6 h after light exposure. Cry2olig clusters (right panel) were resolved after 2 hours of exposure to light. White arrows: embedded, yellow arrows: embedded in other neurons. Scale bar represents 20 μm for all images.
Figure 7 shows that Opto-TDP-43 causes chronic locomotor deficits in C. elegans, A is a time series image of nematodes performing a single thrash (“lateral swim”) in liquid media. Here, thrash moves completely to one side and returns to its original position. B represents the number of thrashes per 30 seconds measured for adult N2 (WT) and transgenic animals. The total sample size was 80 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 the optoDroplet-mediated TDP-43 model, which describes the behavior of the TDP-43 protein inside motor neurons of Caenorhabditis elegans. Under normal conditions, TDP-43 is mainly localized in the nucleus and shuttles between the nucleus and cytoplasm. TDP-43 regulates RNA by forming oligomers through its N-terminal domain in the cytoplasm, but does not form inclusions. In TDP-43 proteinopathy, TDP-43 undergoes excessive oligomerization due to LCD mutations. This abnormal assembly depletes TDP-43 from the nucleus and forms pathological aggregates in the cytoplasm. In the optogenetic C. elegans ALS model without blue light, opto-TDP-43 behaves similarly to the wild-type TDP-43 protein. However, upon exposure to blue light, opto-TDP-43 undergoes rapid Cry2olig-mediated oligomerization, leading to TDP-43 LCD-mediated oligomerization. With extended light exposure, opto-TDP-43 eventually forms large, persistent aggregates very similar to the aggregates seen in TDP-43 proteinopathy.

Hereinafter, the present application will be described in more detail through examples and experimental examples. However, these examples and test examples are for illustrative purposes only and the scope of the present application is not limited to these examples and test examples.

Materials and Methods

1. C. elegans strain and maintenance

C. elegans was maintained at 20°C in Nematode Growth Media (NGM) in which Escherichia coli (OP50) was cultured. Transgenic nematodes were maintained under conditions of 25 °C to improve heritability to the next generation. The wild type used in this study is 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.

2. Polymerase Chain Reaction (PCR)

PCR was performed to perform genetic recombination with each DNA fragment. In the case of nematodes, template DNA was extracted by aliquoting 3-5 animals into 2X lysis buffer and reacting at 60°C for 1 hour and 95°C for 15 minutes. Q5 High-Fidelity DNA Polymerase (New England Biolabs, NEB) was used to prepare all DNA fragments with blunt ends to facilitate Gibson cloning. PCR was performed by mixing 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. PCR conditions were set to 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 carried out using a touch down method in which the temperature was lowered by 0.5°C per cycle. Primers used in this study are summarized in Table 2.

3. TA Cloning & TOPO Cloning

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

4. Gateway Cloning

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

5. Gibson Cloning

To bind mCherry to the N-terminal of TDP-43 in the expression vector, primers must be used so that the N-terminal of TDP-43 overlaps with the C-terminal of mCherry, and the N-terminal of mCherry overlaps with the unc-25 promoter of the vector. was produced. In the same way, in order to bind Cry2olig to the C-terminal of TDP-43, the C-terminal of Cry2olig is bound to the 3' untranslated region (3' of the vector) so that the C-terminal of TDP-43 and the N-terminal of Cry2olig overlap. A primer was designed to overlap with UTR). For high cloning efficiency, the overlap length of all primers was designed to be 30bp. DNA fragments obtained through PCR were linked using NEBuilder HiFi DNA Assembly Master Mix (NEB). 10 μl of the DNA mix to be linked was mixed with 10 μl of NEBuilder HiFi DNA Assembly Master Mix and reacted at 50°C for 1 hour. Cloning products were amplified through transformation.

6. Site-directed mutagenesis

The Q5 Site-directed Mutagenesis kit (NEB) was used to derive mCherry-TDP-43 and mCherry-Cry2olig from plasmid DNA, opto-TDP-43, produced through cloning. Primers were designed to delete TDP-43 or Cry2olig from Opto-TDP-43 (mCherry::TDP-43::Cry2olig), and PCR was performed for each. Afterwards, the PCR product, 2X KLD Reaction buffer, 10X KLD Enzyme mix (Kinase, Ligase, DpnⅠ), and Nuclease-free water were mixed and reacted at room temperature for 1 hour. Cloning products were amplified through transformation. The plasmid DNA used in this study is summarized in Table 3.

7. Transformation

All cloned plasmid DNA contains a selective marker. Completely cloned plasmid DNA was selected using a selective marker and transformation was performed to obtain it in large quantities. NEB stable cells (C3040H, NEB) were thawed on ice for 10 minutes, then 2 μl of 100 pg-100 ng plasmid DNA was added, and the DNA was mixed. The mixture was reacted on ice for 30 minutes and then heatshocked at 42°C for 30 seconds. 350 μl of Stable Outgrowth Medium (SOC) was added and cultured in a 37°C shacking incubator rotating at 250 rpm for 1 hour. Since the plasmid DNA used in this study contains an ampicillin resistance gene, the mixture was spread on a solid LB plate containing ampicillin and incubated overnight in an incubator.

8. Plasmid DNA preparation

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

9. Restriction enzyme digestion and Sanger sequencing

The newly produced plasmid DNA through cloning was confirmed through restriction enzyme treatment and DNA sequencing (Macrogen). Quantification of plasmid DNA was set to 1000 ng/μl and treated with 2 μl of HindⅢ (ELPIS BIOTECH) and 10X Reaction buffer 2 (ELPIS BIOTECH) and reacted at 37°C for 4 hours. The cut size of the reactant was confirmed by electrophoresis on a 1% agarose gel.

10. Microinjection

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

A slide glass was placed on a microscope for microinjection (ZEISS Axio Vert.A1), covered with a cover glass, and a drop of Halocarbon oil 700 (SIGMA) was added. Afterwards, the tip was broken by gently tapping to allow the DNA to come out of the microneedle. The injection pressure of DNA was set using a microinjector (Tritech Research).

To view nematodes under a microscope, a pad was made by adding 2% agarose to tertiary distilled water and boiling it. Halocarbon oil was dropped on the pad and one wild-type nematode was placed there and fixed. After focusing the nematode and microneedle on the microscope, the nematode was moved by gently moving the gliding table toward the tip of the needle. A microneedle was inserted into the gonad of the nematode and the plasmid DNA mix was injected. The nematodes were rescued in M9 (22mM KH2PO4, 42mM Na2HPO4, 85.5mM NaCl, 1mM MgSO4) buffer and cultured at 25°C. The transgenic nematodes produced in this study are summarized in Table 4.

11. Microscopy

Nematodes transformed by microinjection were found using a fluorescence dissecting microscope (LEICA TL3000 Ergo). The expression of mCherry was observed using a fluorescence optical microscope (LEICA DM 2000) after dropping 2 μl of M9 buffer on a 10% agaraose pad and covering the nematodes with a cover glass. The fluorescence images presented in this study were taken using the LEICA DFC 7000gt.

12. Blue light illumination

Nematodes expressing Opto-TDP-43, mCherry::Cry2olig, and mCherry::hTDP-43 were transferred to each NGM and placed 10 cm below a blue light LED panel (Edmund Optics, #66-831) for light stimulation. The wavelength of the used LED panel is 470 nm.

13. Thrashing assay

A thrashing assay was performed to evaluate the locomotor ability of Caenorhabditis elegans. 1 day old adult nematodes were transferred to NGM without OP50 and then 2 μl M9 buffer was added. After acclimatizing the nematodes for 1 minute, the swimming number for 30 seconds was measured. The locomotor capacity of 15-20 nematodes per set was evaluated. All analyzes were performed at room temperature.

14.Statistical analysis

Statistical significance was performed using Graphpad Prism software (Version 9.03), and a p value of 0.05 or less was considered significant. Unpaired student t-test was used to determine statistical significance in data comparing two variables.

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

Next, when only the optogenetic Cry2olig protein was expressed, contrary to expectations, the Cry2olig protein without TDP-43 was observed to form small inclusions throughout the cell even without exposure to blue light (Figure 4B). It is assumed that oligomerization of Cry2olig protein occurred for a short period of time due to failure to completely prevent exposure to natural light, including blue light. Even when exposed to blue light, Cry2olig protein was observed to form inclusions in both the cytoplasm and neurites (Figure 4B).

Lastly, opto-TDP-43 was mostly distributed in the nucleus under dark conditions and partially aggregated in the cytoplasm. This cytoplasmic aggregation phenomenon is believed to be a temporary phenomenon that occurs due to the physical properties of Cry2olig when exposed to natural light, just like the Cry2olig protein. Interestingly, when opto-TDP-43 was exposed to blue light, not only did the number and size of cytoplasmic inclusions significantly increase compared to before, but it was also observed that inclusions were formed in neurites that were close to the cell body (Figure 4B). These results show that, although under dark conditions, TDP-43 was restricted in the nucleus by its nuclear localization properties, the optogenetic properties of Cry2olig exposed to blue light enhanced the aggregation of TDP-43 between LCDs, ultimately leading to mislocalization of TDP-43. It is assumed that it may have contributed to the formation of inclusions by inducing . Therefore, in this study, it was possible to form abnormal inclusions through optogenetic control without significantly increasing the expression level of TDP-43.

3. Aggregation of Opto-TDP-43 is independent of that of Cry2olig.

After exposure to blue light, both Cry2olig protein and opto-TDP-43 protein formed inclusions. These results are presumed to be reversible inclusions transiently formed by Cry2olig rather than pathological aggregates of opto-TDP-43. In this study, the positions and shapes of the two inclusions were compared to understand the differences between opto-TDP-43 inclusions and Cry2olig protein inclusions in nematode motor neurons. In this study, we first investigated the location of inclusions at a distance of 20 μm from the nucleus of motor neurons after exposure to blue light. Inclusions of Cry2olig protein were formed without distinction between cytoplasm and neurites (Figure 5A). Interestingly, most inclusions of opto-TDP-43 were not found in neurites and were mainly formed in the cell body (Figure 5A). This is presumed to be the result of the Cry2olig protein, which is widely spread in nerve cells, being able to easily form inclusions in various places due to the absence of locational restrictions for aggregation, but opto-TDP-43 mainly exits from the nucleus and aggregates.

Importantly, the inclusions of Opto-TDP-43 found in the cytoplasm had several significant differences compared to those of the Cry2olig protein (Figure 5B). Opto-TDP-43 had fewer inclusions than the Cry2olig protein, but formed larger inclusions (Figure 5B and C). Inclusions of Opto-TDP-43 were observed in a large, aggregate-like form (Figure 5B). This form occurs after protein recruitment is triggered by Cry2olig in Opto-TDP-43, and then aggregation becomes irreversible, forming inclusions. It is estimated that the size has increased. In a previous study, the initial recruitment of TDP-43 was reported to have the role of a seed to further aggregate surrounding TDP-43 due to structural features such as the N-terminal domain or LCD within TDP-43. In this study, it is assumed that the initial aggregation of TDP-43 by Cry2olig was accelerated to form large aggregates. Therefore, these results indicate that the inclusions of opto-TDP-43 are likely to be irreversible aggregates, unlike the multiple inclusions of Cry2olig.

4. Aggregates of Opto-TDP-43 have similar characteristics to pathological TDP-43 aggregates.

To determine whether the aggregates formed when Opto-TDP-43 was exposed to blue light had the characteristics of the irreversible pathological aggregates observed in ALS patients, what differences were there between the aggregates of opto-TDP-43 and the Cry2olig protein after exposure to blue light? I checked to see if it was visible. After exposing nematodes expressing each protein to blue light, changes in aggregates over time were observed. At this time, in order to observe how opto-TDP-43 changes not only in the cell body but also in neurites, we observed caudal motor neurons with higher protein expression than other motor neurons. The time intervals were divided into 30 minutes, 2 hours, and 6 hours after exposure to blue light, and the positions of opto-TDP-43 aggregates and Cry2olig aggregates were observed (Figure 6A). Both Cry2olig aggregates and opto-TDP-43 aggregates formed after 30 minutes of blue light exposure were maintained in the cell body and neurites (Figure 6B). Interestingly, after 2 hours of exposure to blue light, Cry2olig protein aggregation disappeared, but the aggregates of opto-TDP-43 persisted or increased in size (Figure 6B), and the aggregates of opto-TDP-43 persisted until 6 hours after exposure to blue light. remained (B in Figure 6). In addition, opto-TDP-43 aggregates were observed very strongly in the cell bodies of nerve cells, and they remained strong even 6 hours after exposure to blue light (Figure 6, A to C).

As a result, once formed, the aggregates of opto-TDP-43 were not easily decomposed, and the aggregates were maintained or even increased in size even a long time after blue light exposure stopped. These results are quite similar to previously reported research results, and indicate that the opto-TDP-43 aggregates in this study also undergo irreversible aggregation in a TDP-43-dependent manner. Therefore, the opto-TDP-43 aggregates produced in this study not only had very different molecular characteristics from Cry2olig aggregates, but were also similar to the characteristics of pathological TDP-43 aggregates seen in TDP-43 proteinopathy.

5. The formation of Opto-TDP-43 aggregates is correlated with movement disorders in C. elegans.

Since ALS patients actually 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 in motor neurons is correlated with nematode motor impairment. Blue light exposure was performed in the same manner as the previous experiment, and the locomotor ability of the transgenic nematodes was then evaluated using a thrashing assay. Thrashing assay is a method of measuring the frequency of swimming movements of nematodes in liquid, making it easy to identify nematodes with movement defects (A in Figure 7). In this study, adults were placed in M9 buffer and the number of swimming times per 30 seconds was measured. At 1 hour after exposure to blue light, nematodes expressing Cry2olig and opto-TDP-43, excluding the wild type and nematodes expressing TDP-43, showed a phenotype of locomotor defects (Figure 7B). Surprisingly, when the locomotor ability of nematodes was evaluated 6 hours after light exposure, the locomotor function of nematodes expressing Cry2olig was recovered, but the locomotor function of nematodes expressing opto-TDP-43 was observed to continue to decrease. (B in Figure 7). In particular, compared to Cry2olig, nematodes expressing opto-TDP-43 showed a slow swimming phenotype with their bodies bent to one side and curled.

In Figure 5, the aggregates of Cry2olig and opto-TDP-43 initially formed inclusions, but the inclusions of opto-TDP-43 remained even 6 hours after exposure to blue light. In this context, in nematodes expressing Cry2olig, locomotor defects were overcome over time after exposure to blue light, but in nematodes expressing opto-TDP-43, locomotor defects were continuously induced regardless of time. Cry2olig inclusions were observed to cause acute neurotoxicity, whereas opto-TDP-43 aggregates were maintained more persistently than Cry2olig aggregates, so they were presumed to cause chronic neurotoxicity. Therefore, this correlation suggests that the formation and accumulation of aggregates of opto-TDP-43 may contribute to causing motor deficits.

Argument

To understand TDP-43 proteinopathy, in this study, we created an optogenetic ALS biological model capable of forming cytoplasmic TDP-43 aggregates, a pathological feature observed in ALS/FTD. Previous studies have attempted to reveal the mechanisms of nerve damage by regulating the expression of TDP-43 in various in vitro/in vivo or by inducing aggregation of TDP-43 under excessive stress conditions. Therefore, because aggregation of TDP-43 was induced under excessive stress conditions, there was a limitation in specifying the clear cause of nerve damage. In this study, to investigate the relationship between the pathological aggregation of TDP-43 and the mechanism of nerve damage, we successfully developed an optogenetic opto-TDP-43 system that can form inclusions using Cry2olig, whose aggregation is controlled by light. (Figure 3). In other words, we succeeded in inducing the aggregation of TDP-43 at any desired time using a little light without applying excessive stress.

Interestingly, in nematode motor neurons, all TDP-43 proteins were localized to the nucleus, but opto-TDP-43 escaped from the nucleus and was located in the cell body and neurites after light exposure, similar to the aggregates seen in actual ALS patient neurons. Aggregates were formed (Figure 4). Previous studies have speculated that TDP-43's lack of nuclear function, formation of cytoplasmic aggregates, or both processes cause neuronal damage. The opto-TDP-43 developed in this study is also expected to be used as a very useful animal model for TDP-43 proteinopathy, as a deficiency of TDP-43 in the nucleus is observed simultaneously with the formation of cytoplasmic aggregates (Figure 5).

These aggregates of opto-TDP-43 showed characteristics similar to pathological aggregates. Compared to Cry2olig, which forms small inclusions in clusters throughout motor neurons, Opto-TDP-43 mainly formed large and wide inclusions in the cell body. (Figure 5). In fact, in the nerve cells of ALS patients, a relatively small number of large aggregates are mainly observed rather than many small aggregates. This indicates that the inclusions of opto-TDP-43 are more similar to the pathological aggregates of TDP-43 proteinopathy than to transient aggregates such as Cry2olig.

Another characteristic of pathological aggregates in TDP-43 proteinopathy is their persistent accumulation. The initial pathological aggregation of TDP-43 is irreversible, and it has the seeding ability to recruit other surrounding RNA binding proteins and RNA. Accordingly, the continuous accumulation of TDP-43 in the cytoplasm gradually accelerates and the size of the aggregate expands. In this study, it was confirmed that the size of opto-TDP-43 aggregates was actually maintained or increased over time. These results suggest that although aggregation of opto-TDP-43 is induced through Cry2olig, the aggregation is maintained through a TDP-43-dependent mechanism.

Pathological aggregates seen in TDP-43 proteinopathy have various biochemical characteristics. When TDP-43 forms pathological aggregates, in many cases, phosphorylation occurs at the C-terminal (S409/410), and the formed aggregates are ubiquitinated. Additionally, this aggregate has the characteristic of being insoluble and not easily soluble. Although it was indirectly demonstrated that the opto-TDP-43 aggregates induced in this study were similar to pathological aggregates.

ALS patients develop muscle atrophy due to the gradual loss of motor neurons, which further leads to paralysis and eventually death. In this study, to determine the correlation between opto-TDP-43 aggregates and nematode movement disorders, aggregates were formed and the nematode movement ability was evaluated over time. The motor defects caused by TDP-43 aggregation in the in vivo model were also found in previous studies. Surprisingly, in this study, nematodes expressing opto-TDP-43 continued to have impaired motor function (Figure 7). These results are quite consistent with those of previous studies and suggest that aggregates of opto-TDP-43 persistently remaining in motor neurons after blue light exposure are highly correlated with motor deficits.

In this study, an optogenetic nematode ALS model was created using optoDroplet technology, and the aggregates of opto-TDP-43 were quite similar to the pathological aggregates seen in TDP-43 proteinopathy. Importantly, we showed that mislocalization and irreversible aggregation of TDP-43 protein are ultimately associated with the induction of motility disorders in nematodes. Therefore, the optogenetic nematode ALS model created in this study is expected to be used as a useful tool for understanding TDP-43 proteinopathy.

Claims (13)

Caenorhabditis elegans in which TDP-43 aggregation was induced by optogenetic stimulation. According to paragraph 1,
The little elegans is
The optogenetic stimulation involves transforming Caenorhabditis elegans with plasmid DNA containing a nucleic acid encoding a fusion protein containing a CRY2 variant linked to the C terminus of TDP-43, and photostimulating the Caenorhabditis elegans. Little elegans. According to paragraph 2,
Caenorhabditis elegans, wherein the fusion protein contains a detectable marker. According to paragraph 3,
The marker is any one of a fluorescent protein, a reporter enzyme, a transcription factor, a radioisotope binding protein, and a bioluminescence protein, Caenorhabditis elegans. A method for producing Caenorhabditis elegans in which aggregation of TDP-43 is induced, comprising the step of optogenetically stimulating Caenorhabditis elegans. According to clause 5,
The optogenetic stimulation step is
1) Transforming Caenorhabditis elegans with plasmid DNA containing a nucleic acid encoding a fusion protein containing a CRY2 variant linked to the C terminus of TDP-43; and
2) A method for producing C. elegans in which aggregation of TDP-43 is induced, comprising the step of photostimulating the transformed C. elegans. According to clause 5,
A method for producing Caenorhabditis elegans, wherein the fusion protein includes a detectable marker. In clause 7,
The marker is any one of a fluorescent protein, a reporter enzyme, a transcription factor, a radioisotope binding protein, and a bioluminescent protein. A method of producing Caenorhabditis elegans. According to clause 6,
A method of producing C. elegans in which aggregation of TDP-43 is induced, wherein the photostimulating step is photostimulating using a light source of 450 to 480 nm. Method for screening candidate treatments for TDP-43 proteinopathy disease using Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation. According to clause 10,
Administering a candidate treatment for TDP-43 proteinopathy disease to Caenorhabditis elegans in which TDP-43 aggregation is induced by optogenetic stimulation; and
A screening method comprising the step of selecting a candidate therapeutic agent showing an effect of improving TDP-43 proteinopathy disease compared to a group of Caenorhabditis elegans administered a control substance. According to clause 11,
A screening method, wherein the TDP-43 proteinopathy disease is a disease mediated by aggregation of TDP-43 protein or accumulation in the cytoplasm due to mislocalization of TDP-43 protein. According to clause 12,
The TDP-43 proteinopathy disease includes amyotrophic lateral sclerosis (ALS); frontotemporal lobar dementias, Lewy-body dementia, Parkinson disease, Perry syndrome and ALS Parkinsonism-dementia complex of Guam ), Huntington's disease, myopathies, and sporadic inclusion body myositis.