阅读说明：本技术 用于als的突变型fus模型 (Mutant FUS model for ALS ) 是由 张雪 贾怡昌 于 2017-03-22 设计创作，主要内容包括：一种ALS模型,一种筛选用于治疗ALS的药物的方法以及一种构建ALS模型的方法,其中所述ALS模型表达突变型FUS,并且所述突变型FUS是FUS-R521C。(An ALS model, a method of screening for a drug for treating ALS, and a method of constructing an ALS model, wherein the ALS model expresses a mutant FUS, and the mutant FUS is FUS-R521C.)

1. An ALS model, wherein the ALS model expresses mutant FUS.

2. The model of ALS of claim 1, wherein the mutant FUS is FUS-R521C, wherein the mutation at FUS-R521 is the most frequent FUS mutation in human ALS.

3. The ALS model of claim 1, wherein the ALS model is an animal, a tissue, or a cell.

4. The ALS model of claim 3, wherein the tissue or cell is isolated from the animal.

5. The ALS model of claim 3, wherein cells are obtained as follows:

introducing a plasmid into a recipient cell, wherein the plasmid carries a nucleotide that expresses the FUS-R521 mutation, wherein the recipient cell is isolated from a human.

6. The ALS model of claim 3, wherein the cells are primary cells or embryonic stem cells.

7. The ALS model of claim 6, wherein the primary cell is a neuronal cell.

8. The ALS model according to claim 3 or 4, wherein the animal is a C57BL/6J mouse.

9. The ALS model of claim 8, wherein the animal is obtained as follows:

the FUS-R521C KI mutant mice were backcrossed with C57BL/6J wild-type mice for at least 5 generations.

10. The ALS model of claim 9, wherein the animal is obtained as follows:

the FUS-R521C KI mutant mice were backcrossed for 10 generations with C57BL/6J wild-type mice.

11. A method of screening for a drug for treating ALS, the method comprising:

stress treating the ALS model according to any one of claims 1 to 10;

contacting a candidate agent with the stress-treated ALS model;

determining the candidate agent as the drug for treating ALS based on a change in the model of ALS before and after the contact.

12. The method of claim 11, wherein the stress treatment comprises at least one of oxidative stress, endoplasmic reticulum stress, and mitochondrial stress.

13. The method of claim 12, wherein said model of ALS is a model of cells, contacted cells having at least one of said changes indicates that said candidate agent is said agent for treating ALS:

(1) increased cell survival rate;

(2) reduction of mutant FUS in the cytoplasm;

(3) reduction in the number and size of stress particles; and

(4) decreased ubiquitin-positive inclusion bodies.

14. The method of claim 12, wherein the ALS model is an animal model and an increase in locomotor activity, increased hind limb strength, increased axonal number or motor and learning capacity of peripheral motor neurons in the contacted animal indicates that the candidate agent is a drug for treating ALS.

15. A method of constructing an ALS model, the method comprising:

the FUS gene in the wild-type counterpart was mutated into a mutant FUS gene of ALS patients.

16. The method of claim 15, wherein the FUS gene mutation is FUS-R521C.

17. The method of claim 16, wherein FUS-R521C is obtained by means of directed mutagenesis.

18. The method of claim 16, wherein the directed mutagenesis is achieved by at least one of the following techniques:

homologous recombination of CRISPR-CAs9 and mouse zygotes or embryonic stem cells.

Technical Field

Embodiments of the present disclosure relate generally to biomedicine, and more particularly, to an ALS model, a method of screening for a drug for treating ALS, and a method of constructing an ALS model.

Background

Emerging evidence suggests that abnormal RNA metabolism, including gain of RBP function, loss of RNA helicase function, and mis-processing of pre-mRNA splicing, can lead to neurodegenerative diseases. Among them, mutations in genes encoding two structurally similar RBPs (TDP-43 and FUS) are associated with ALS and FTD, which have genetic and pathological overlap. More remarkably, although many of them do not carry these two RBP mutations, ubiquitin-positive and mislocalized TDP-43 and FUS were found in most ALS and FTD, which highlights the critical role of RBP dysfunction in pathogenesis. However, the disease mechanism of neurodegenerative diseases caused by dysfunction of these RBPs is still largely unknown.

Recently, among many other RBPs, both TDP-43 and FUS, which contain a Low Complexity Domain (LCD), also known as the Intrinsic Disorder Region (IDR), have been identified in the untranslated cytoplasmic mRNA complex, also identified by Stress Granules (SG), which are structures: it usually occurs under stress conditions to temporarily halt the initiation of translation of cytoplasmic mRNA. Like TDP-43 and FUS, mutations in other RBP genes have also been associated with neurodegenerative diseases including ALS and FTD. In ALS and FTD patient specimens, mis-localized TDP-43 co-localized with SG markers, whereas in cultured cells, over-expressed mutant TDP-43 and FUS were found in stress-induced SG. Mutations in VCP, a gene encoding a protein involved in SG clearance by autophagy, are also closely associated with ALS and FTD. Taken together, these data suggest that the wrong processing of SG may be the cause of the disease. However, it is not clear at present how endogenous levels of wild-type and mutant RBPs behave and function in SG formation and phase transition, particularly in disease target neurons facing stress attack, in part because of the urgent need for rational cellular and animal models.

In the past, great efforts have been made to create animal models of ALS by over-expressing recombinant DNA carrying mutations found in the ALS family. Although these transgenic animal models greatly deepen our understanding of the disease mechanism, one might argue that the potential artifact in disease models driven by ectopic overexpression of muteins, such as human SOD1-G93A, is 40-fold over-expressed in mice, which is one of the most popular ALS mouse models. Similar transgenic strategies have been applied to generate TDP-43 and FUS ALS models. However, overexpression of wild-type TDP-43 and FUS also produced a motor phenotype similar to that of the mutant transgenes in many animal species (including flies, mice and rats), suggesting a potential artifact of pathogenesis in these models. In addition, transgenic strategies have other considerations, including undefined genomic insertion sites, unstable copy numbers, potential disruption of genome integrity, ectopic expression patterns driven by exogenous promoters, and lack of endogenous splicing regulation.

Therefore, great efforts are required to create animal models for ALS.

Disclosure of Invention

Embodiments of the present disclosure seek to address, at least to some extent, at least one of the problems presented in the prior related art, or to provide the consumer with a useful commercial choice.

Embodiments of a first broad aspect of the disclosure provide an ALS model. According to some embodiments, wherein the model of ALS expresses mutant FUS. The ALS model described herein has defined genomic insertion sites, stable copy number, genomic integrity, endogenous splicing regulation, and no ectopic expression pattern driven by exogenous promoters. The ALS model can be widely used for ALS disease mechanism research and screening of drugs for treating ALS.

According to some embodiments, the ALS model may further comprise at least one of the following additional technical features.

According to some embodiments, the mutant FUS is FUS-R521C, wherein the mutation in FUS-R521 is the most common FUS mutation in human ALS. The ALS model can be widely used for researching human ALS disease mechanism and screening drugs for treating human ALS.

According to some embodiments, the model of ALS is an animal, tissue, or cell. According to a specific embodiment of the present invention, an animal, tissue or cell that is a model of ALS can be widely used for the study of ALS disease mechanism and screening of drugs for treating ALS.

According to some embodiments, the tissue or cell is isolated from an animal. According to a specific embodiment of the present invention, the animal expresses the mutant FUS, and tissues or cells isolated from the animal also express the mutant FUS. Animals, tissues or cells isolated from animals, which are models of ALS, can be widely used for the study of ALS disease mechanisms and the screening of drugs for treating ALS.

According to some embodiments, the cells are obtained as follows: introducing a plasmid into a recipient cell, wherein the plasmid carries a nucleotide that expresses the FUS-R521 mutation, wherein the recipient cell is isolated from a human. The cells obtained as described above can be widely used for the study of the mechanism of ALS disease and the screening of drugs for treating ALS.

According to some embodiments, the cells are primary cells or embryonic stem cells. According to a particular embodiment of the invention, both primary cells and embryonic stem cells have an ALS-like stress response under stress treatment. Both primary cells and embryonic stem cells can be widely used for disease mechanism research of ALS and screening of drugs for treating ALS.

According to some embodiments, the primary cell is a neuronal cell. Amyotrophic Lateral Sclerosis (ALS) is a specific disease that leads to neuronal death that controls autonomic muscles. Thus, ALS-like stress responses of neuronal cells are more typical.

According to some embodiments, the animals are C57BL/6J mice. The C57BL/6J mouse strain background has been widely used for generating mouse disease models, disease mechanism studies, drug safety assessment and drug efficacy assessment.

According to some embodiments, the animal is obtained as follows: the FUS-R521C KI mutant mice were backcrossed with C57BL/6J wild-type mice for at least 5 generations. Due to the off-target effect that the inventors' Crispr/Cas9 approach used to generate FUS-R521C KI mutant mouse ALS models may produce, the inventors backcrossed the KI mutant line to the C57BL/6J wild-type to avoid the potential off-target introduced by the Crispr/Cas9 approach to the maximum extent. Five generations of backcrossing will drive 97% of the genome of about KI mutant mice to a C57BL/6J wild type background.

According to some embodiments, the animal is obtained as follows: FUS-R521C KI mutant mice were backcrossed to C57BL/6J wild-type mice for 10 generations. Due to the off-target effect that the inventors' Crispr/Cas9 approach used to generate FUS-R521C KI mutant mouse ALS models may produce, the inventors backcrossed the KI mutant line to the C57BL/6J wild-type to avoid the potential off-target introduced by the Crispr/Cas9 approach to the maximum extent. Ten generations of backcrossing will drive 99.9% of the genome of about KI mutant mice to a C57BL/6J wild type background.

Embodiments of a second broad aspect of the disclosure provide a method of screening for a drug for treating ALS. According to some embodiments, the method comprises: stress-treating the ALS model; and contacting the candidate agent with the stress-treated ALS model; based on the change in the ALS model before and after the contact, the candidate agent is judged to be a drug for treating ALS. According to the method of the embodiment of the present invention, a drug for treating ALS can be effectively screened.

According to some embodiments, the method may further comprise at least one of the following additional technical features.

According to some embodiments, the stress treatment comprises at least one of oxidative stress, endoplasmic reticulum stress, and mitochondrial stress. More specifically, oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and the ability of biological systems to readily detoxify reactive intermediates or repair resulting damage. Disturbances in the normal redox state of a cell can cause toxic effects by producing peroxides and free radicals that damage all components of the cell, including proteins, lipids and DNA. Endoplasmic reticulum stress is activated in response to the accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum. Mitochondrial stress is a condition that leads to mitochondrial dysfunction (genetic, environmental factors, aging), including oxidative stress due to increased mitochondrial-derived ROS production, decreased cellular energy metabolism, and disruption of the apoptotic response, all leading to some of the major downstream cytopathic consequences observed for mitochondrial-based diseases.

According to some embodiments, the model of ALS is a model of cells, and the contacted cells have at least one of the following changes indicating that the candidate agent is a drug for treating ALS: (1) increased cell survival rate; (2) reduction of mutant FUS in cytoplasm; (3) reduction in the number and size of stress particles; and (4) reduction of ubiquitin-positive inclusion bodies. The inventors have surprisingly found that mutant FUS are prone to mis-localization in the cytosol under stress treatment. Mutant FUS that are mislocalized in the cytosol form stress particles and even ubiquitin-positive inclusion bodies. The above molecular mechanisms lead to cell death and a range of ALS diseases. Therefore, if a candidate agent can increase the cell survival rate or decrease the mutant FUS in the cytoplasm, for example, to remove the mutant FUS mislocalized in the cytosol or prevent the mutant FUS from escaping the nucleus, or prevent the formation of stress particles or even ubiquitin-positive inclusion bodies, it can be judged that the candidate agent can block the progression of ALS, and it can also be judged as a drug for treating ALS. In fact, the inventors of the present application have also confirmed this through experiments.

According to some embodiments, the ALS model is an animal model and an increase in locomotor activity, increased hindlimb strength, increased axonal number of peripheral motor neurons, or increased motor and learning abilities of the contacted animal indicates that the candidate agent is a drug for treating ALS. The inventors found that in stress treatment, the ALS animal model has reduced voluntary activity and hind limb muscle strength, and anxiety is increased. Thus, if a candidate agent can enhance voluntary activity and hind limb muscle strength in an animal, or reduce anxiety behavior, the candidate agent can be judged to be a drug to be used in the treatment of ALS. In fact, the inventors of the present application have also confirmed this through experiments.

Embodiments of the second broad aspect of the disclosure provide a method of constructing an ALS model. According to some embodiments, the method comprises: the FUS gene in the wild-type counterpart was mutated into a mutant FUS gene of ALS patients. The ALS model constructed using the method of the present invention has defined genomic insertion sites, stable copy number, genomic integrity, endogenous splicing regulation, and no ectopic expression pattern driven by exogenous promoters. The ALS model can be widely used for ALS disease mechanism research and screening of drugs for treating ALS.

According to some embodiments, the method may further comprise at least one of the following additional technical features.

According to some embodiments, the FUS gene is mutated to FUS-R521C. FUS-R521C is the most common mutation in human ALS. The ALS model expressing FUS-R521C is more typical.

According to some embodiments, FUS-R521C is obtained by means of directed mutagenesis. The directional mutation of FUS into FUS-R521C can avoid the non-directional mutation. The ALS model constructed by the method has a more definite genome insertion site and a more stable copy number. The ALS model can be widely used for ALS disease mechanism research and drug screening for treating ALS.

According to some embodiments, the directed mutation is achieved by homologous recombination of CRISPR-CAs9 and mouse zygotes or embryonic stem cells by at least one of the following techniques. CRISPR-CAs9 and homologous recombination can more successfully obtain FUS-R521C directed mutation.

According to some embodiments, when the inventors introduced the FUS-R513C mutation, they also introduced additional synonymous mutations to create a new PstI cleavage site that was constructed in the locus, which made future genotyping easier.

The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The figures and the detailed description that follow more particularly exemplify illustrative embodiments.

Additional aspects and advantages of embodiments of the present disclosure will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of embodiments of the present disclosure.

Drawings

These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows the generation of mFUS-R513C KI mutant mice of ALS using CRISPR/Cas9,

wherein the content of the first and second substances,

a shows the arrangement of the last 12 amino acids of FUS in different mammalian species. These amino acids are highly conserved from rodents to humans. NCBI accession number: sapiens (NP _004951.1), b.taurus (XP _005224884.1), r.norvegicus (NP _001012137), and m.musculus (NP _ 631888.1). The human FUS sequence was used as a reference for amino acid positioning. R521 is marked with an asterisk.

B shows the mouse genomic structure of the FUS gene. Mouse FUS R513 corresponds to human R521. Two nucleotide mutations, red-labeled, were introduced into the FUS locus 2bp upstream of the mouse PAM site (dark black 3 capital letters). The underlined genomic DNA sequence corresponds to the gRNA sequence.

C shows a DNA chromatogram;

d shows gel electrophoresis. The PCR product including the insert was treated with PstI. +/+ denotes wild type C57BL/6J mice, C/+ and C/C denote heterozygous and homozygous mFUS-R513C KI mice; and

e shows FUS expression in various tissues of mice. Tissues from 8-month old wild type (+/+) and mFUS-R513C KI mutant (C/C) mice were blotted with home-made FUS antibodies.

FIG. 2 shows that aged mFUS-R513C mutant KI mice exhibit hypokinesia and a reduced number of motor fibers,

wherein the content of the first and second substances,

a shows the distance traveled as measured by open field (cleversys. topscan behavioral analysis system, usa) and demonstrates a significant decrease in the distance traveled by senescent heterozygous (C/+) and homozygous (C/C) KI mutants (6.5 months), but not in young (4 months of age) mutant animals. 4 months old, n ═ 9 (+/+ and C/+), respectively, male. 6.5 months old, n 25(+/+), n 16(C/+), n 12(C/C), male. In the open field test, the standing time (seconds within 10 minute intervals) of hind limbs of aging (6.5 months of age) mice was calculated. The values are expressed as mean ± SEM. P <0.05, p <0.01 (single variance analysis or t-test, SPSS). NS, no statistical significance;

b shows a significant reduction in standing time for the heterozygous (C/+) and homozygous (C/C) KI mutant groups at 6.5 months of age compared to the wild type (+/+) group. The values are expressed as mean ± SEM. P <0.05, p <0.01 (single variance analysis or t-test, SPSS). NS, no statistical significance;

c and D show the performance of wild type (+/+), heterozygous (C/+) and/or homozygous (C/C) KI mutant mice at 4 months of age (C) and 6.5 months of age (D) in rotarod experiments (4 days interval, Med Associates Inc., usa). The residence time on the rotarod was significantly reduced in aged KI mutant (C/+ and C/C, 6.5 months of age) mice, but not in younger (4 months of age) mutant animals. 4 months old, n ═ 8 (+/+ and C/+), respectively, male. 6.5 months old, n 25(+/+), n 14(C/+), n 15(C/C), male. The values are expressed as mean ± SEM. P <0.05, p <0.01 (single variance analysis or t-test, SPSS). NS, no statistical significance;

e shows toluidine blue stained cross sections of femoral nerve motor branches of 8 month old wild type (+/+) and KI mutant (C/C) animals. Axonal degeneration (arrows) is shown in the high magnification image. On a scale bar, the low magnification image is 50 μm and the high magnification image is 100 μm. The diameter of the red dot in the low magnification image is 5 μm, which is used to measure the fiber size; and

f shows a significant reduction in the number of nerve fibers of the larger size (> 5 μm diameter) of the femoral motor branch, but not of the smaller size (<5 μm), in KI mutant (C/C, 8 months old) animals compared to wild type (+/+) littermate control animals. In (A, B, C and D), the values are expressed as mean. + -. SEM. P <0.05, p <0.01 (single variance analysis, SPSS). NS, no statistical significance. The values are expressed as mean ± SEM (n ═ 3). P <0.01 (t-test, SPSS), and

FIG. 3 shows that when cultured motoneurons are subjected to stress attack, the mutant R513C FUS (but not the wild type) migrates into SG, and causes the mutant SG to be defective in decomposition,

wherein the content of the first and second substances,

a shows representative immunostaining images of wild type (+/+) or KI mutant (C/C) cultured motor neurons (3DIV) that were either treated with AS (sodium arsenite, 1mM, 1 hour) or recovered from AS treatment (changed to no AS medium and cultured for an additional hour after AS treatment). After AS treatment, TIA1 positive SG was formed in both wild-type and KI mutant neurons. However, mutant SG alone was TIA1 and FUS positive after AS treatment or recovery. The scale bar is 20 μm. MOCK, vehicle control group;

b shows the percentage of cells containing TIA1 positive SG after AS treatment (AS) or recovery (AS +1 hour). After recovery, the percentage in the wild-type group decreased significantly, but there was no significant difference in the KI mutant group, indicating that there was a SG breakdown defect in SG containing mutant FUS. The values are expressed as mean. + -. SEM (n.gtoreq.3). P <0.05, p <0.01 (t-test, SPSS). NS, no statistical significance;

c shows the percentage of SG positive for both TIA1 and FUS after AS treatment or recovery. Each AS-induced mutant SG was double positive. In contrast, none of the AS-induced wild-type SG were double positive. The values are expressed as mean. + -. SEM (n.gtoreq.3). P <0.05, p <0.01 (t-test, SPSS). NS, no statistical significance;

d shows SG counts per cell. After recovery, the number of SG in wild-type neurons was significantly reduced, but there was no significant difference in the mutant group, further supporting mutant SG breakdown defects. The values are expressed as mean. + -. SEM (n.gtoreq.3). P <0.05, p <0.01 (t-test, SPSS). Ns, no statistical significance; and

e shows the AS-induced SG for different size classes. We classify the sizes of SGs into three groups: small size (<1 μm), medium size (. gtoreq.1 and <2 μm) and large size (. gtoreq.2 μm). After recovery, large size SG and most of small size SG were cleared in the wild type group. In contrast, the size of mutant SG was not effectively decomposed after recovery. The values are expressed as mean. + -. SEM (n.gtoreq.3). P <0.05, p <0.01 (t-test, SPSS). NS, no statistical significance.

Fig. 4 shows that paraquat (paraquot) treatment significantly increased the formation of mutant type SG, and caused the mutant FUS to relocate into SG,

wherein the content of the first and second substances,

a shows wild type (+/+) and KI mutant (C/C) cultured motor neurons (3DIV) were treated with paraquat (para., 1mM, 8 hours) and stained with TIA1 and FUS antibodies. Note that most TIA1 positive mutant SG were FUS positive. The scale bar is 20 μm.

B shows statistical results from the experiments shown in (a). Paraquat treatment significantly increased the formation of mutant SGs and relocated mutant FUS to SG in cultured motor neurons. The values are expressed as mean. + -. SEM (n.gtoreq.3). P <0.01 (t-test, SPSS).

Figure 5 shows that prolonged stress challenge converts SG into ubiquitin-positive inclusion bodies,

wherein the content of the first and second substances,

a shows representative immunostaining images of wild type (+/+) or KI mutant (C/C) cultured motor neurons (3DIV) treated with AS (sodium arsenite, 1mM) for 4 hours and 6 hours (4 and 6 hr). After long-term stress treatment, wild FUS still maintains its nuclear localization. In contrast, after AS treatment, ubiquitin-positive inclusions were shown in KI mutant neurons, and these inclusions were also FUS-positive. Asterisks indicate ubiquitin-positive inclusion bodies in DAPI-negative cell debris. The scale bar is 20 μm;

b shows the percentage of cells containing both FUS and ubiquitin positive inclusion bodies after 1 hour, 2 hours and 4 hours AS treatment. Double positive inclusion bodies were only present in KI mutant motor neurons, but not in wild-type motor neurons. The values are expressed as mean. + -. SEM (n.gtoreq.3);

inclusion body counts for each cell are shown in C. Prolonged stress attacks increase the number of ubiquitin-positive inclusion bodies in mutant (C/C) Motor Neurons (MNs). The values are expressed as mean. + -. SEM (n.gtoreq.3);

d shows the number of inclusions of different size classes. The inventors divided the size of inclusion bodies into three groups: small size (<1 μm), medium size (. gtoreq.1 and <2 μm) and large size (. gtoreq.2 μm). Prolonged stress challenge increased the size of the inclusion bodies of mutant MNs. The values are expressed as mean. + -. SEM (n.gtoreq.3); and

e shows cell debris containing FUS and ubiquitin-positive inclusion bodies. The inventors noted that in mutant MNs facing prolonged stress challenge (AS, 1mM, 2 hours or 4 hours), DAPI negative cell debris appeared positive for both FUS and ubiquitin. These points represent the percentage of ubiquitin-positive fragments normalized to the total number of cells in each image of at least three independent experiments.

FIG. 6 shows that stress treatment induced dramatic hypokinesia, FUS mislocalization, ubiquitin upregulation, and FUS positive stress particle formation in mutant spinal cords,

wherein the content of the first and second substances,

a shows a schedule for intragastric administration of Arsenite (AS) in mice;

b shows the time of stance of the hind limb in open field (seconds within a 10 minute interval) before and after exposure to AS. One month after intragastric administration of AS, the standing time of mFUS-R513C KI mice decreased significantly, demonstrating a dramatic decline in locomotion. n-5 (for +/+ and C/C, respectively), male. The values are expressed as mean ± SEM. P <0.05, p <0.01 (t-test, SPSS);

c shows FUS mislocalization and ubiquitin upregulation in ChAT positive motor neurons in KI mutant animals induced by stress treatment. Arrows mark typical neurons with FUS mislocalization and ubiquitin upregulation. Section of spinal cord at the 4 th to 6 th sections of lumbar vertebra. Scale bar, low magnification image 50 μm, high magnification image 20 μm;

d shows that stress treatment induced upregulation in both eIF3g, stress particle markers, and the formation of FUS and eIF3g double positive stress particles in the anterior horn of the KI mutant spinal cord. Section of spinal cord at the 4 th to 6 th sections of lumbar vertebra. The scale bar is 20 μm; and

FIG. 7 shows an established FUS reporter cell line for screening compounds capable of reversing FUS mispositioning.

Wherein the content of the first and second substances,

a shows the stable expression of GFP-tagged wild-type (hFUS) and mutant FUS (FUS-R521C) in Hela cells infected with lentiviral particles;

b shows that wild-type FUS faithfully localized in the nucleus without stress treatment, while mutant FUS showed cytoplasmic mislocalization. Both wild-type and mutant FUS showed cytoplasmic localization after arsenite treatment. The scale bar is 20 μm.

Detailed Description

Embodiments of the present disclosure will be described in detail. The embodiments described herein with reference to the drawings are illustrative, exemplary, and are provided for a general understanding of the present disclosure. The embodiments should not be construed as limiting the disclosure. Throughout the specification, the same or similar elements and elements having the same or similar functions are denoted by similar reference numerals.

The following examples are provided so that the invention may be more fully understood. It should be understood, however, that these embodiments merely provide a method of practicing the invention, and that the invention is not limited to these embodiments.

The related method is as follows

Materials and methods:

mice, behavioral testing of mice and intragastric administration

To generate the FUS-R513C KI mouse strain, the inventors PCR-amplified the target sequence in C57BL/6J (JAX, stock number 000664) mouse genomic DNA. The donor DNA fragment contained the R513C mutation (tcg to ctg) and left and right homology arms (about 1kb) on both sides, respectively. Donor DNA, gRNA (gcgagcacagacaggatcgcAGG, PAM site shown in uppercase) and Cas9 mRNA were injected into C57BL/6J embryos. The injected embryos were transferred to the ampulla of the oviduct of a pseudopregnant ICR (JAX, stock number 009122) female recipient. The correct genotype progeny were backcrossed with C57BL/6J for at least five generations to establish the line.

For open field behavioral testing, a single animal was placed in the center of the open field area (60cm x 60cm) and tracked by the TopScan behavioral analysis system (cleversys, usa) at 10 minute intervals with multiple parameters including total distance, average speed, and distance traveled in the central area. Anxiety levels are measured by the distance traveled by the center area divided by the total distance traveled. Vertical jumping activity was measured with the total standing time of the hind limb during the 10 minute interval of the open field test.

Performance in the rotarod experiment was measured by an automated system (Med Associates, Inc). Briefly, animals were placed on an accelerating spindle (4-40 rpm) for 5 minutes each, with 3 consecutive trials per day. A 20 minute rest period was set between each trial. The test was repeated for four days. When a mouse falls from the spindle within a 5 minute interval, the system automatically calculates the time to fall from the spindle. Residence time was calculated by subtracting the drop time from 5 minutes, and the average of residence times from 3 consecutive trials per day was used for statistical analysis.

For intragastric administration, the mice are first weighed and then gently held head back to form a line from the neck to the esophagus. The sodium arsenite solution was dosed at 2 μ g/g body weight from a round tip feeding tube 4 times per week. After each dose, mice were returned to their cages and monitored for at least 5 minutes. Animals were tested weekly for open field testing to monitor their movements.

Since 2014, animal facilities at the university of Qinghua have received full certification by the International Association for the protection and evaluation of animal laboratories (AAALAC). All animal protocols were approved by the university of Qinghua animal protection and use Committee (IACUC) under the guide for laboratory animal protection and use (eighth edition, NHR). Both C57BL/6J and ICR mice were purchased from the Charles river laboratory, Beijing, China.

Cell culture and stress induction

For motoneuron cultures, briefly, spinal cords were dissected from E13.5 mouse embryos and digested with papain (Sigma, 1:200) and EDTA (1mM) for 30 min at 37 ℃ in Neurobasal (Invitrogen) as described previously. During the digestion, DNAse I (Sigma, 10. mu.g/ml) was added over the last 10 minutes. After digestion, the tissue suspension was passed through a 40 μm filter. The resulting cell pellet was resuspended in 1ml HBSS (Invitrogen) containing EDTA (0.5mM) and a layer of 5ml Optiprep (Sigma, 10%) was superimposed on top of the suspension layer. After centrifugation at 400 × g for 25 minutes, motor neurons were enriched in the top 1ml volume. Motoneurons were cultured in Neurobasal containing B27(Invitrogen), horse serum (10% v/v; Sigma), glutamine-1 (1X; Invitrogen) and were characterized by immunostaining with anti-ChAT (rabbit, Millipore) and anti-Tuj-1 (mouse, Beyotime, China) antibodies. For the induction of stress particles, cultured motor neurons (3DIV) were treated with sodium arsenite (AS, 1mM) and paraquat (1mM) for a certain period of time depending on the purpose of the experiment.

Femoral nerve dissection and axon counting

Femoral nerve dissection was performed as previously described. Before dissection, the femoral nerve was exposed and fixed briefly in sacrificed mice (0.1M cocoate buffer containing 2% glutaraldehyde/2% paraformaldehyde). Isolated nerves were fixed overnight in the same fixative. Plastic embedding and transmission electron microscopy of dissected nerves were performed by standard procedures. Nerve sections were stained with toluidine blue and examined by light microscopy.