WO2018170794A1 - Mutant fus model for als - Google Patents

Mutant fus model for als Download PDF

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WO2018170794A1
WO2018170794A1 PCT/CN2017/077708 CN2017077708W WO2018170794A1 WO 2018170794 A1 WO2018170794 A1 WO 2018170794A1 CN 2017077708 W CN2017077708 W CN 2017077708W WO 2018170794 A1 WO2018170794 A1 WO 2018170794A1
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fus
als
mutant
model
cells
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PCT/CN2017/077708
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French (fr)
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Xue ZHANG
Yichang Jia
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Tsinghua University
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Priority to PCT/CN2017/077708 priority Critical patent/WO2018170794A1/en
Priority to CN201780091089.9A priority patent/CN110770336B/en
Priority to EP17902030.0A priority patent/EP3601541A4/en
Publication of WO2018170794A1 publication Critical patent/WO2018170794A1/en

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Definitions

  • Embodiments of the present disclosure generally relate to biological medicine, more particularly, to a ALS model, a method for screening a medicament for treating ALS and a method for constructing ALS model.
  • RNA metabolism abnormalities including gain-of-function of RBPs, loss-of-function of RNA helicases, and misprocessing of pre-mRNA splicing, lead to neurodegenerative diseases.
  • mutations in genes encoding two structurally similar RBPs, TDP-43 and FUS have been associated with ALS and FTD, the two neurodegenerative disorders sharing genetic and pathological overlaps.
  • the ubiquitin-positive and mislocalized TDP-43 and FUS were found in a large proportion of ALS and FTD, even though many of them do not carry these two RBP mutations, underscoring the critical roles of RBP dysfunction in the pathogenesis.
  • the disease mechanisms underlying neurodegeneration caused by dysfunction of these RBPs are still largely unknown.
  • TDP-43 and FUS among many other RBPs, which contain low complexity domain (LCD) also known as intrinsically disordered region (IDR)
  • LCD low complexity domain
  • IDR intrinsically disordered region
  • SG stress granule
  • mutations in those RBP genes have also been linked to neurodegenerative diseases, including ALS and FTD.
  • ALS and FTD patient specimen the mislocalized TDP-43 co-localized with SG markers, and in cultured cells, overexpressed mutant TDP-43 and FUS were found in the SG induced by stresses.
  • VCP a gene encodes protein involved in autophagic clearance of SGs
  • ALS a gene encodes protein involved in autophagic clearance of SGs
  • Embodiments of the present disclosure seek to solve at least one of the problems existing in the related art to at least some extent, or to provide a consumer with a useful commercial choice.
  • Embodiments of a first broad aspect of the present disclosure provide a ALS model. According the embodiments, wherein the ALS model express mutant FUS.
  • the ALS model described herein have certain genome insertion sites, stable copy numbers, genome integrity, endogenous splicing regulation and no ectopic expression pattern driven by exogenous promoter.
  • the ALS model can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
  • the ALS model may further include at least one of the following additional technical features.
  • the mutant FUS is FUS-R521C, wherein the mutations at the FUS-R521 are the most frequent FUS mutations in human ALS.
  • the ALS model can be more widely used in the human ALS disease mechanisms research and screening a medicament for treating ALS in human.
  • the ALS model is animals, tissue or cells.
  • the animal, tissue or the cell as the ALS model can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
  • the tissue or cells are separated from the animals.
  • the animals express FUS mutant, also the tissue or cells separated from the animals expressing mutant FUS.
  • the animal, tissue or the cell separated from the animals as the ALS model can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
  • cells are obtained as follows: introduction of plasmid into recipient cells, wherein the plasmid carrying nucleotide expressing FUS-R521 mutation, wherein the recipient cells are separated from human.
  • the cells obtained as above can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
  • the cells are primary cells or embryonic stem cells.
  • both primary cells and embryonic stem cells have ALS like stress response under stress treatment. Both primary cells and embryonic stem cells can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
  • the primary cells are neuronal cells.
  • Amyotrophic lateral sclerosis (ALS) is a specific disease that causes the death of neurons which control voluntary muscles. Therefore, neuronal cells’A LS like stress response is more typical.
  • the animals are C57BL/6J type mice.
  • C57BL/6J mouse strain background has been widely used for generating mouse disease models, disease mechanism studies, drug safety assessment, and drug efficacy evaluation.
  • the animals are obtained as following: backcrossing the FUS-R521C KI mutant mouse to C57BL/6J wild type mice at least 5 generations. Due to the potential target-off effect by Crispr/Cas9 means, which the inventors used for generating the FUS-R521C KI mutant mouse ALS model, the inventors backcross the KI mutant line to C57BL/6J wild type to maximally avoid the potential target-off introduced by Crispr/Cas9 means. Five generation backcross will drive about the KI mutant mouse 97%genome to C57BL/6J wild type background.
  • the animals are obtained as following: backcrossing the FUS-R521C KI mutant mouse to C57BL/6J wild type mice 10 generations. Due to the potential target-off effect by Crispr/Cas9 means, which the inventors used for generating the FUS-R521C KI mutant mouse ALS model, the inventors backcross the KI mutant line to C57BL/6J wild type to maximally avoid the potential target-off introduced by Crispr/Cas9 means. Ten generation backcross will drive about the KI mutant mouse 99.9%genome to C57BL/6J wild type background.
  • Embodiments of a second broad aspect of the present disclosure provide a method for screening a medicament for treating ALS.
  • the method comprising: stress treatment of the ALS model described above; contacting candidate reagent with the stress treated ALS modesl; based on the change of the ALS models before and after the contacting, the candidate reagent is judged to be the medicament for treating ALS.
  • the medicine for treating ALS can be screened out effectively.
  • the method may further include at least one of the following additional technical features.
  • the stress treatment comprising at least one of oxidative stress, endoplasmic reticulum stress and mitochondrial stress.
  • the oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA.
  • the endoplasmic reticulum stress is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum.
  • mitochondrial stresses are circumstances (genetics, environmental factors, aging) that result in mitochondrial dysfunction, including oxidative stress due to increased production of mitochondria-derived ROS, declines in cellular energy metabolism, and disruption of apoptotic responses are some of the major downstream cellular consequences leading to the observed pathology of mitochondrial-based diseases.
  • the ALS model is cell model, that the contacted cells having at least one of the change described below is an indication for the candidate reagent is a medicament for treating ALS, (1) increased cell survival; (2) reduction of mutant FUS in cytoplasm; (3) reduction of stress granule number and size; and (4) reduction of ubiquitin-positive inclusion.
  • the inventor accidentally discovered that mutant FUS is prone to be mislocalized in cytosol under stress treatment. Cytosol mislocalized mutant FUS form stress granules and even ubiquitin-positive inclusion. The molecular mechanism described above leads to cell death and a series of ALS disorders.
  • the candidate reagent could increase cell survival rate, or reduce the mutant FUS in cytoplasm, eg, either clear away cytosol mislocalized mutant FUS or block mutant FUS out of nucleus, or block forming stress granules even ubiquitin-positive inclusion, the candidate reagent can be judged to block ALS course and can be judged as the medicament for treating ALS. In fact, the inventor of this application also confirmed this point by experiment.
  • the ALS model is animal model, that the contacted animals having increased locomotor activity, increased hindlimb strength, number of axons of peripheral motor neurons, or increased sports and learning ability is an indication for the candidate reagent is a medicament for treating ALS.
  • the inventor discovered that the ALS animal model’s locomotor activity and hindlimb muscle strength were reduced, and the anxiety was increased under stress treatment. Therefore, if the candidate reagent could increase animal’s locomotor activity and hindlimb muscle strength, or reduce the anxiety behavior, the candidate reagent can be judged as the medicament for treating ALS. In fact, the inventor of this application also confirmed this point by experiment.
  • Embodiments of a second broad aspect of the present disclosure provide a method for constructing ALS model.
  • the method comprising: mutation of FUS gene in wild-type counterpart to mutant FUS gene of ALS patient.
  • the ALS model constructed using method of the present invention have certain genome insertion sites, stable copy numbers, genome integrity, endogenous splicing regulation and no ectopic expression pattern driven by exogenous promoter.
  • the ALS model can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
  • the method may further include at least one of the following additional technical features.
  • the FUS gene mutation is FUS-R521C.
  • FUS-R521C is the most frequent mutation in human ALS.
  • the ALS model expressing FUS-R521C is more typical.
  • FUS-R521C is obtained by the way of orthomutation. Orthomutation of FUS to FUS-R521C, non directional mutation can be avoided.
  • the ALS model constructed using the method have more certain genome insertion sites, more stable copy numbers.
  • the ALS model can be more widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
  • the orthomutation is realized through at least one of the following techniques, CRISPR-CAs9 and homologous recombination of mouse zygotes or embryonic stem cells.
  • CRISPR-CAs9 and homologous recombination can obtain FUS-R521C orthomutation more successfully.
  • Fig. 1 shows generation of mFUS-R513C KI mutant mouse for ALS by CRISPR/Cas9
  • A shows alignment of last 12 amino acids of FUS in different mammal species.
  • the amino acids are highly conserved from rodent to human.
  • the human FUS sequences are used as a reference for amino acid positioning.
  • the R521 is labeled with asterisk.
  • the mouse FUS R513 corresponds to human R521.
  • Two-nucleotide mutations labeled in red were introduced into mouse FUS locus 2bp upstream of PAM site (3 capital letters in dark black) .
  • the underlined genomic DNA sequences correspond to gRNA sequences.
  • D shows gel electrophoresis.
  • the PCR products including the insertion were treated with PstI. +/+, wild type C57BL/6J mouse, C/+ and C/C, hetero-and homozygous for mFUS-R513C KI mice; and
  • E shows the expression of FUS in mouse various tissues.
  • the tissues from wild type (+/+) and mFUS-R513C KI mutant (C/C) mice at 8 months of age were blotted with a homemade FUS antibody,
  • Fig. 2 shows the aged mFUS-R513C mutant KI mice showed motor decline and the reduced number of motor nerve fibers
  • Open field test the standing time (seconds in 10-minute interval) on the hind legs in aged (6.5 months) mice was calculated. The value are presented as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01 (one-way ANOVA or t-test, SPSS) . NS, no statistical significance;
  • E shows toluidine blue-stained cross sections of femoral nerve motor branches from wild type (+/+) and KI mutant (C/C) animals at 8 months of age.
  • Axon degeneration (arrow) was shown in high magnification image.
  • Scale bar 50 ⁇ m in low and 100 ⁇ m in high magnification images.
  • the diameter of a red dot in low magnification image is 5 ⁇ m, which is used for the fiber size measurement. ) ;
  • F shows the big size (diameter ⁇ 5 ⁇ m) , but not the small size ( ⁇ 5 ⁇ m) , nerve fiber numbers of femoral motor branches were significantly reduced in the KI mutant (C/C, 8 months) animals, compared to their wild type (+/+) littermate controls.
  • Fig. 3 shows the mutant R513C FUS, but not wild type, moves into SGs and results in the mutant SG disassembly defect when the cultured motor neurons facing the stress challenge
  • A shows representative immunostaining images from the wild type (+/+) or KI mutant (C/C) cultured motor neurons (3 DIV) treated with AS (sodium arsenite, 1mM, 1hr) or recovered from AS treatment (changed to AS-free medium and cultured for another hour after AS treatment) .
  • the TIA1-positive SGs were formed in both wild type and KI mutant neurons after AS treatment. However, only the mutant SGs were both TIA1-and FUS-positive after AS treatment or after recovery.
  • Scale bar 20 ⁇ m.
  • MOCK vehicle control groups
  • D shows the count number of SGs per cell.
  • the SG number was significantly reduced in the wild type neurons after recovery, but kept no significant difference in the mutant groups, further supporting the mutant SG disassembly defect.
  • the value are presented as mean ⁇ SEM (n ⁇ 3) . *p ⁇ 0.05, **p ⁇ 0.01 (t-test, SPSS) . NS, no statistical significance; and
  • E shows the AS-induced SGs in different size categories.
  • the size of SGs into three groups: small ( ⁇ 1 ⁇ m) , medium ( ⁇ 1, and ⁇ 2 ⁇ m) , and large ( ⁇ 2 ⁇ m) sizes.
  • small ( ⁇ 1 ⁇ m) After recovery, the big size and the most of small size of SGs were cleared up in wild type groups. In contrast, after the recovery, those sizes of mutant SGs were not effectively disassembled.
  • the value are presented as mean ⁇ SEM (n ⁇ 3) . *p ⁇ 0.05, **p ⁇ 0.01 (t-test, SPSS) . NS, no statistical significance.
  • Figure 4 shows paraquot treatment significantly increased the mutant SG formation and the relocation of the mutant FUS into SGs
  • A shows the wild type (+/+) and KI mutant (C/C) cultured motor neurons (3 DIV) were treated with paraquot (Para., 1mM, 8hrs) , and stained with TIA1 and FUS antibodies. Note the majority of TIA1-positive mutant SGs were FUS-positive. Scale bar, 20 ⁇ m.
  • B shows the statistic results from the experiments shown in (A) .
  • the paraquot treatment significantly increased the mutant SG formation, and the relocation of mutant FUS into the SGs in the cultured motor neurons.
  • the value are presented as mean ⁇ SEM (n ⁇ 3) . **p ⁇ 0.01 (t-test, SPSS) .
  • Figure 5 shows The prolonged stress challenge converts the SGs into the ubiquitin-positive inclusions
  • A shows representative immunostaining images from the wild type (+/+) or KI mutant (C/C) cultured motor neurons (3 DIV) treated with AS (sodium arsenite, 1mM) for 4 and 6 hours (4 and 6hrs) .
  • AS sodium arsenite, 1mM
  • the wild type FUS still kept its nuclear localization.
  • the ubiquitin-positive inclusions were shown in the KI mutant neurons after AS treatment, and these inclusions were FUS-positive as well.
  • the asterisk marked the ubiquitin-positive inclusion in the DAPI-negative cell debris.
  • Scale bar 20 ⁇ m;
  • B shows the percentage of cells contained both FUS-and ubiquitin-positive inclusions after the 1-, 2-, and 4-hour AS treatment.
  • the double-positive inclusions only appeared in the KI mutant motor neurons, but not the wild type ones.
  • the value are presented as mean ⁇ SEM (n ⁇ 3) ;
  • C shows the count number of inclusions per cell.
  • the prolonged stress challenge increased the number of ubiquitin-positive inclusions in the mutant (C/C) motor neurons (MNs) .
  • the value are presented as mean ⁇ SEM (n ⁇ 3) ;
  • D shows the inclusion number in different size categories.
  • the inventors categorized the size of inclusions into three groups: small ( ⁇ 1 ⁇ m) , medium ( ⁇ 1, and ⁇ 2 ⁇ m) , and large ( ⁇ 2 ⁇ m) sizes.
  • the prolonged stress challenge increased the size of inclusions in the mutant MNs.
  • the value are presented as mean ⁇ SEM (n ⁇ 3) ;
  • E shows the cell debris containing the FUS-and ubiquitin-positive inclusions.
  • the invenors noticed that the DAPI-negative cell debris both FUS-and ubiquitin-positive appeared in the mutant MNs facing the prolonged stress challenge (AS, 1mM, 2 or 4hrs) .
  • the dots represented the percentage of the ubiquitin-positive debris normalized to the total number of cells in the individual imagines from at least three independent experiments.
  • Figure 6 shows the stress treatment induced sharp motor decline, FUS mislocalization, ubiquitin upregulation, and FUS-positive stress granule formation in the mutant spinal cords
  • A shows the schedule for intragastric administration of arsenite (AS) in mice
  • C shows the stress treatment induced FUS mislocalization and ubiquitin upregulation in the ChAT-positive motor neurons in the KI mutant animals.
  • the typical neurons with FUS mislocalization and ubiquitin upregulation were labeled by arrows. Sections from lumbar 4-6 spinal cords. Scale bar, 50 ⁇ m in low and 20 ⁇ m in high magnification images;
  • D shows the stress treatment induced the upregulation of eIF3g, a stress granule marker, and the formation of FUS and eIF3g double positive stress granules in the KI mutant spinal cord ventral horns. Sections from lumbar 4-6 spinal cords. Scale bar, 20 ⁇ m; and
  • Figure 7 shows establish FUS reporter cell lines for screening for compounds that are able to reverse FUS mislocalization.
  • A shows the stable expression of GFP labelled wild type (hFUS) and mutant FUS (FUS-R521C) in Hela cells infected with the lenti-viral particles;
  • mice mouse behavior tests, and intragastric administration.
  • the inventors For the generation of the FUS-R513C KI mouse line, the inventors PCR amplified the target sequences from the C57BL/6J (JAX, Stock No. 000664) mouse genomic DNA.
  • the donor DNA fragment contains the R513C mutation (tcg to ctg) , and the flanking left and right homology arms ( ⁇ 1kb) , respectively.
  • the donor DNA, gRNA (gcgagcacagacaggatcgcAGG, PAM site capitalized) , and Cas9 mRNA were injected into C57BL/6J embryos.
  • the injected embryos were transferred into the oviduct ampulla of the pseudo-pregnant ICR (JAX, Stock No. 009122) female recipients.
  • the right genotype offsprings were backcrossed to C57BL/6J for at least five generations to establish the line.
  • Open Field behavior test the single animal was placed in the center of an open field area (60 ⁇ 60 cm) and tracked with multiple parameters, including total distance, average speed, and distance traveled in the center region, by TopScan behavioral analysis system (CleverSys., USA) in a 10-minute interval. Anxiety level was measured by distance traveled in the center region divided by total distance traveled . The vertical rearing activity was measured by total standing time on hind legs in the 10-minute interval of Open Field test. The rotarod performance was measured by an automated system (Med Associates, Inc) . In brief, the animal was placed on an accelerating spindle (4-40rpm) in 5 minutes per trail and 3 consecutive trials per day. A 20-minute break was set in between each trial. The test was repeated for four days.
  • the fall time from the spindle was auto-calculated by the system when the mouse fell off the spindle within the 5-minute interval.
  • the stay time was calculated by subtraction of the fall time from the 5 minutes, and the mean value of the stay time from 3 consecutive trials per day was used for statistical analysis.
  • mice For the intragastric administration, weight the mouse first and gently hold the mouse head back to create a straight line through the neck to esophagus.
  • the sodium arsenite solution was administrated by a rounded tip feeding tube at the dose of 2 ⁇ g/g body weight, 4 times/week. After each administration, the mice were returned back to the cages and monitored for at least 5 minutes. Animals went through Open Field test once every week to monitor their locomotion.
  • the spinal cords were dissected from E13.5 mouse embryos and digested with papain (Sigma, 1: 200) with EDTA (1mM) in Neurobasal (Invitrogen) at 37°C for 30 minutes. During the digestion, DNAse I (Sigma, 10 ⁇ g/ml) were added in the last 10 minutes. After the digestion, the tissue suspension was passed through the 40 ⁇ m strainer. The resulting cell pellet were re-suspended in 1ml HBSS (Invitrogen) with EDTA (0.5mM) , and the suspension was layered over a cushion of 5 ml Optiprep (Sigma, 10%) .
  • the motor neurons were enriched in the top 1ml volume.
  • the motor neurons were grown in Neurobasal with B27 (Invitrogen) , horse serum (10%v/v; Sigma) , Glutamax-1 (1 ⁇ ; Invitrogen) , and characterized by immunostaining with anti-ChAT (rabbit, Millipore) and anti-Tuj-1 (mouse, Beyotime, China) antibodies.
  • B27 Invitrogen
  • horse serum 10%v/v; Sigma
  • Glutamax-1 (1 ⁇ ; Invitrogen
  • anti-ChAT rabbit, Millipore
  • anti-Tuj-1 mimouse, Beyotime, China
  • Dissection of femoral nerves was performed as described previously.
  • the femoral nerve was exposed in the sacrificed mice and briefly fixed (2%glutaraldehyde/2%paraformaldehyde in 0.1 M cacodylate buffer) before dissection.
  • the isolated nerves were postfixed overnight in the same fixative.
  • Dissected nerves were processed for plastic embedding and transmission electron microscopy by standard procedures. Nerve cross sections were stained with toluidine blue and examined by light microscopy.
  • the inventors employed the CRISPR-Cas9-based knock-in (KI) approach. There are three reasons for them to choose hFUS-R521C to generate KI ALS mouse model.
  • the R521C mutation locates in FUS C-terminal, a non-classical PY nuclear localization signal (NLS) , in which clusters more than half of ALS-associated FUS mutations. Among these mutations, R521C is the most frequent one and accounts for 30%of the total.
  • the R521C mutation has been found in both familiar and sporadic ALS patients with high disease penetration.
  • the FUS R521C transgenic mouse and rat models present strong ALS-like pathological features.
  • the inventors To generate the KI mouse line, the inventors first analyzed the FUS protein conservation across species ( Figure 1A) .
  • the C-terminal NLS encoded by the last exon of mouse FUS is identical to that of human (515GEHRQDRRERPY526) .
  • the mouse corresponding position for human R521 is R513.
  • the inventors PCR amplified the target sequences from the C57BL/6J (JAX, Stock No. 000664) mouse genomic DNA.
  • the donor DNA fragment contains the R513C mutation (tcg to ctg) , and the flanking left and right homology arms ( ⁇ 1kb) , respectively.
  • the donor DNA, gRNA (gcgagcacagacaggatcgcAGG, PAM site capitalized) , and Cas9 mRNA were injected into C57BL/6J embryos.
  • the injected embryos were transferred into the oviduct ampulla of the pseudo-pregnant ICR (JAX, Stock No. 009122) female recipients.
  • the correct genome insertion and germ line transmission were confirmed by breeding the KI founders with C57BL/6J wild type mice ( Figure 1C and 1D) . Meanwhile, the inventors backcrossed the KI mutant mouse to C57BL/6J at least 5 generations, in order to reduce the potential off-target effect introduced by CRISPR/Cas9.
  • the right genotype offsprings were backcrossed to C57BL/6J for at least five generations to establish the line.
  • the inventors observed that the expression level of mutant FUS was slightly upregulated in spinal cord (Figure 1E) , suggesting that the human R521C mutation may increase the protein stability and/or decrease the turnover rate in the target tissue.
  • Aged mFUS-R513C mice showed motor decline and peripheral axon degeneration.
  • the degenerating motor axons of femoral nerve were observed in the homozygous KI mutant animals (Fig. 2E) .
  • the inventors found that the numbers of large-diameter ( ⁇ 5 ⁇ m) fibers, but not the small ones ( ⁇ 5 ⁇ m) , were significant reduced in the homozygous KI mutant mice (Fig. 2E and 2F) , suggesting that the fibers with fast conduction velocity are majorly affected by the expression of mutant FUS.
  • the cultured KI mutant motor neuron models the disease.
  • SGs formation of SGs is context-specific and induced by various stress conditions, including ER, oxidative, and mitochondria stresses.
  • TIA1 cytotoxic granule-associated RBP, a SG marker
  • AS sodium arsenite
  • ALS pathology The hallmark of ALS pathology is the formation of ubiquitin-positive inclusions containing TDP-43 and FUS.
  • pathology is related to SG misprocessing.
  • the inventors when we extended the AS treatment to 4 or even 6 hours, the inventors not only observed the mislocalization of mutant FUS into cytosolic SGs, but co-localization of the mutant FUS with ubiquitin, a key regulator for proteasome-mediated degradation (Figure 5A) .
  • the formation of ubiquitin-positive inclusion only appeared in the mutant, but not wild type, motor neurons, and the extent of inclusion formation was increased along with the treatment time (Figure 5B and 5C) .
  • the inventors performed the intragastric administration of arsenite in aged mFUS-R513C KI mice ( Figure 6A) .
  • the standing time on the hind legs was decreased in both wild type and mutant KI groups after 1 or 2-month exposure of arsenite, the hind leg weakness was much more severe in the mutant KI group after 1-month exposure ( Figure 6B) .
  • the stress exposure induced clear FUS mislocalization and ubiquitin upregulation in the mFUS-R513C KI spinal cord motor neurons, but not in that of wild type animals that were exposed the same dose of arsenite as the KI mutant mice ( Figure 6C) .
  • the inventors stained wild type and KI mutant spinal cord sections with antibody for eIF3g, a key subunit of the eukaryotic translation initiation factor 3 (eIF3) complex and also often used as a SG marker.
  • eIF3g a key subunit of the eukaryotic translation initiation factor 3
  • Figure 6D the signals largely colocalized with mislocalized mutant FUS
  • the inventors generated Hela cell lines that stably express wild type and mutant FUS-R521C with N-terminal fused with fluorescent protein, GFP.
  • the expression of GFP-tagged wild type FUS and FUS-R521C was detected by western blot with both GFP and FUS antibodies ( Figure 7A) .
  • the expression level of exogenous GFP-tagged FUS was comparable with the endogenous FUS.
  • the subcellular localization of stably expressed FUS was confirmed by GFP fluorescence. Without stress treatment, the subcellular distribution of wild type FUS was faithfully localized in nucleus measured by the GFP fluorescence ( Figure 7B) .
  • the FUS-R521C was majorly localized in the cytoplasm. After 0.25mM arsenite treatment for 1 hour, the observed the both wild type and mutant FUS moved into SGs.
  • the FUS-R513C knock-in ALS mouse model ( Figure 2) , and cell derived from the KI mouse line ( Figure 3-5) , and the FUS subcellular localization reporter cell lines ( Figure 7) the inventors established are rational disease models for testing the potential drug efficacy for ALS, for screening for compounds as potential drug for ALS, and for evaluating the environmental risk factors for ALS, and et al.
  • the travel distance in open field, the hind leg standing time, the stay time on rotarod, and the motor axon number count can be used for the purposes described above.
  • mutant FUS For cells derived from the KI mouse line, for example, the motor neurons, the presence of mutant FUS in the SGs, the stress-induced formation of TIA1 positive SGs, and recovery-induced SG disassembly can be used as the evaluation parameters for the purposes described above.
  • potential drugs for ALS inhibition of mutant FUS moving into SGs and recovery of the disassembly defect can be used for drug efficacy evaluation.
  • potential ALS risk factors the induction of mutant FUS-positive SGs, the SG formation and disassembly defects can be used for the risk estimation.
  • the subcellular location of wild type and mutant FUS can be used for the purposes described above.
  • mutant FUS mislocalization and reduce of SG formation can be used for drug efficacy evaluation.
  • potential ALS risk factors the induction of mutant FUS-positive SGs, the SG formation and disassembly defects can be used for the risk estimation.

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Abstract

An ALS model, a method for screening a medicament for treating ALS and a method for constructing ALS model, wherein the ALS model expresses mutant FUS and the mutant FUS is FUS-R521C.

Description

Mutant FUS model for ALS TECHNICAL FIELD
Embodiments of the present disclosure generally relate to biological medicine, more particularly, to a ALS model, a method for screening a medicament for treating ALS and a method for constructing ALS model.
BACKGROUND
Emerging evidence suggests that RNA metabolism abnormalities, including gain-of-function of RBPs, loss-of-function of RNA helicases, and misprocessing of pre-mRNA splicing, lead to neurodegenerative diseases. Among them, mutations in genes encoding two structurally similar RBPs, TDP-43 and FUS, have been associated with ALS and FTD, the two neurodegenerative disorders sharing genetic and pathological overlaps. More strikingly, the ubiquitin-positive and mislocalized TDP-43 and FUS were found in a large proportion of ALS and FTD, even though many of them do not carry these two RBP mutations, underscoring the critical roles of RBP dysfunction in the pathogenesis. However, the disease mechanisms underlying neurodegeneration caused by dysfunction of these RBPs are still largely unknown.
Recently, TDP-43 and FUS, among many other RBPs, which contain low complexity domain (LCD) also known as intrinsically disordered region (IDR) , have been identified in a non-translating cytoplasmic mRNA complex, also known as stress granule (SG) , a structure that often appears under stress conditions to temporarily cease the cytosolic mRNA translation initiation. Like TDP-43 and FUS, mutations in those RBP genes have also been linked to neurodegenerative diseases, including ALS and FTD. In ALS and FTD patient specimen, the mislocalized TDP-43 co-localized with SG markers, and in cultured cells, overexpressed mutant TDP-43 and FUS were found in the SG induced by stresses. The mutations in VCP, a gene encodes protein involved in autophagic clearance of SGs, have been also closely associated with ALS and FTD. Taken together, these data argued that the misprocessing of SG may contribute the disease etiology. However, how the endogenous level of wild type and mutant RBPs behave and function in the SG formation and phase change, especially in the disease target neurons facing the stress challenge, is unclear, partially due to the emergent needs for rational cell and animal models.
In the past, significant efforts have been made to generate animal models for ALS by overexpressing recombinant DNA carrying mutations found in ALS families. Although those transgenic animal models significantly gain our insights into the disease mechanisms, people may argue the potential artifacts driven by ectopic overexpression of mutant proteins in the disease  models, like 40-time overexpression of human SOD1-G93A in mouse, one of the most popular ALS mouse models. Similar transgenic strategy was applied for generation of TDP-43 and FUS ALS models. However, overexpression of wild type TDP-43 and FUS also generate similar motor phenotypes as that of the mutant transgenes in many animal species, including fly, mouse, and rat, suggesting the potential artifacts in pathogenesis in these models. Besides, the transgenic strategy has other caveats, including uncertain genome insertion sites, unstable copy numbers, potential disruption of genome integrity, ectopic expression pattern driven by exogenous promoter, and lack of endogenous splicing regulation.
Therefore, significant efforts need to be made to generate animal models for ALS.
SUMMARY
Embodiments of the present disclosure seek to solve at least one of the problems existing in the related art to at least some extent, or to provide a consumer with a useful commercial choice.
Embodiments of a first broad aspect of the present disclosure provide a ALS model. According the embodiments, wherein the ALS model express mutant FUS. The ALS model described herein have certain genome insertion sites, stable copy numbers, genome integrity, endogenous splicing regulation and no ectopic expression pattern driven by exogenous promoter. The ALS model can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
According the embodiments, the ALS model may further include at least one of the following additional technical features.
According the embodiments, the mutant FUS is FUS-R521C, wherein the mutations at the FUS-R521 are the most frequent FUS mutations in human ALS. The ALS model can be more widely used in the human ALS disease mechanisms research and screening a medicament for treating ALS in human.
According the embodiments, the ALS model is animals, tissue or cells. According to the specific embodiment of the present invention, the animal, tissue or the cell as the ALS model can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
According the embodiments, the tissue or cells are separated from the animals. According to the specific embodiment of the present invention, the animals express FUS mutant, also the tissue or cells separated from the animals expressing mutant FUS. The animal, tissue or the cell separated from the animals as the ALS model can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
According the embodiments, cells are obtained as follows: introduction of plasmid into  recipient cells, wherein the plasmid carrying nucleotide expressing FUS-R521 mutation, wherein the recipient cells are separated from human. The cells obtained as above can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
According the embodiments, the cells are primary cells or embryonic stem cells. According to the specific embodiment of the present invention, both primary cells and embryonic stem cells have ALS like stress response under stress treatment. Both primary cells and embryonic stem cells can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
According the embodiments, the primary cells are neuronal cells. Amyotrophic lateral sclerosis (ALS) is a specific disease that causes the death of neurons which control voluntary muscles. Therefore, neuronal cells’A LS like stress response is more typical.
According the embodiments, the animals are C57BL/6J type mice. C57BL/6J mouse strain background has been widely used for generating mouse disease models, disease mechanism studies, drug safety assessment, and drug efficacy evaluation.
According the embodiments, the animals are obtained as following: backcrossing the FUS-R521C KI mutant mouse to C57BL/6J wild type mice at least 5 generations. Due to the potential target-off effect by Crispr/Cas9 means, which the inventors used for generating the FUS-R521C KI mutant mouse ALS model, the inventors backcross the KI mutant line to C57BL/6J wild type to maximally avoid the potential target-off introduced by Crispr/Cas9 means. Five generation backcross will drive about the KI mutant mouse 97％genome to C57BL/6J wild type background.
According the embodiments, the animals are obtained as following: backcrossing the FUS-R521C KI mutant mouse to C57BL/6J wild type mice 10 generations. Due to the potential target-off effect by Crispr/Cas9 means, which the inventors used for generating the FUS-R521C KI mutant mouse ALS model, the inventors backcross the KI mutant line to C57BL/6J wild type to maximally avoid the potential target-off introduced by Crispr/Cas9 means. Ten generation backcross will drive about the KI mutant mouse 99.9％genome to C57BL/6J wild type background.
Embodiments of a second broad aspect of the present disclosure provide a method for screening a medicament for treating ALS. According the embodiments, the method comprising: stress treatment of the ALS model described above； contacting candidate reagent with the stress treated ALS modesl； based on the change of the ALS models before and after the contacting, the candidate reagent is judged to be the medicament for treating ALS. According to the method of the embodiment of the invention, the medicine for treating ALS can be screened out effectively.
According the embodiments, the method may further include at least one of the following  additional technical features.
According the embodiments, the stress treatment comprising at least one of oxidative stress, endoplasmic reticulum stress and mitochondrial stress. More specifically, the oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. The endoplasmic reticulum stress is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum. The mitochondrial stresses are circumstances (genetics, environmental factors, aging) that result in mitochondrial dysfunction, including oxidative stress due to increased production of mitochondria-derived ROS, declines in cellular energy metabolism, and disruption of apoptotic responses are some of the major downstream cellular consequences leading to the observed pathology of mitochondrial-based diseases.
According the embodiments, the ALS model is cell model, that the contacted cells having at least one of the change described below is an indication for the candidate reagent is a medicament for treating ALS， (1) increased cell survival； (2) reduction of mutant FUS in cytoplasm； (3) reduction of stress granule number and size； and (4) reduction of ubiquitin-positive inclusion. The inventor accidentally discovered that mutant FUS is prone to be mislocalized in cytosol under stress treatment. Cytosol mislocalized mutant FUS form stress granules and even ubiquitin-positive inclusion. The molecular mechanism described above leads to cell death and a series of ALS disorders. Therefore, if the candidate reagent could increase cell survival rate, or reduce the mutant FUS in cytoplasm, eg, either clear away cytosol mislocalized mutant FUS or block mutant FUS out of nucleus, or block forming stress granules even ubiquitin-positive inclusion, the candidate reagent can be judged to block ALS course and can be judged as the medicament for treating ALS. In fact, the inventor of this application also confirmed this point by experiment.
According the embodiments, the ALS model is animal model, that the contacted animals having increased locomotor activity, increased hindlimb strength, number of axons of peripheral motor neurons, or increased sports and learning ability is an indication for the candidate reagent is a medicament for treating ALS. The inventor discovered that the ALS animal model’s locomotor activity and hindlimb muscle strength were reduced, and the anxiety was increased under stress treatment. Therefore, if the candidate reagent could increase animal’s locomotor activity and hindlimb muscle strength, or reduce the anxiety behavior, the candidate reagent can be judged as the medicament for treating ALS. In fact, the inventor of this application also confirmed this point by experiment.
Embodiments of a second broad aspect of the present disclosure provide a method for constructing ALS model. According the embodiments, the method comprising: mutation of FUS gene in wild-type counterpart to mutant FUS gene of ALS patient. The ALS model constructed using method of the present invention have certain genome insertion sites, stable copy numbers, genome integrity, endogenous splicing regulation and no ectopic expression pattern driven by exogenous promoter. The ALS model can be widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
According the embodiments, the method may further include at least one of the following additional technical features.
According the embodiments, the FUS gene mutation is FUS-R521C. FUS-R521C is the most frequent mutation in human ALS. The ALS model expressing FUS-R521C is more typical.
According the embodiments, FUS-R521C is obtained by the way of orthomutation. Orthomutation of FUS to FUS-R521C, non directional mutation can be avoided. The ALS model constructed using the method have more certain genome insertion sites, more stable copy numbers. The ALS model can be more widely used in the ALS disease mechanisms research and screening a medicament for treating ALS.
According the embodiments, the orthomutation is realized through at least one of the following techniques, CRISPR-CAs9 and homologous recombination of mouse zygotes or embryonic stem cells. CRISPR-CAs9 and homologous recombination can obtain FUS-R521C orthomutation more successfully.
According the embodiments, when the inventors introduce FUS-R513C mutation, they also introduced an additional synonymous mutation to generate a novel PstI cutting site built into the locus, which make the 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 which follow more particularly exemplify illustrative embodiments.
Additional aspects and advantages of embodiments of present disclosure will be given in part in the following descriptions, become apparent in part from the following descriptions, or be learned from the practice of the embodiments of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following descriptions made with reference to the accompanying drawings, in which:
Fig. 1 shows generation of mFUS-R513C KI mutant mouse for ALS by CRISPR/Cas9,
wherein,
A shows alignment of last 12 amino acids of FUS in different mammal species. The amino acids are highly conserved from rodent to human. The NCBI accession number for H. sapiens (NP_004951.1) , B. Taurus (XP_005224884.1) , R. norvegicus (NP_001012137) , and M. musculus (NP_631888.1) . The human FUS sequences are used as a reference for amino acid positioning. The R521 is labeled with asterisk.
B shows the mouse genome structure of FUS gene. The mouse FUS R513 corresponds to human R521. Two-nucleotide mutations labeled in red were introduced into mouse FUS locus 2bp upstream of PAM site (3 capital letters in dark black) . The underlined genomic DNA sequences correspond to gRNA sequences.
C shows DNA chromatograms；
D shows gel electrophoresis. The PCR products including the insertion were treated with PstI. +/+, wild type C57BL/6J mouse, C/+ and C/C, hetero-and homozygous for mFUS-R513C KI mice； and
E shows the expression of FUS in mouse various tissues. The tissues from wild type (+/+) and mFUS-R513C KI mutant (C/C) mice at 8 months of age were blotted with a homemade FUS antibody,
Fig. 2 shows the aged mFUS-R513C mutant KI mice showed motor decline and the reduced number of motor nerve fibers,
wherein,
A shows the travel distance measured by Open Field (TopScan behavioral analysis system, CleverSys., USA) and the significant decline of travel distance was documented in aged hetero-(C/+) and homozygous (C/C) KI mutant (6.5 months) , but not in the younger (4 months) mutant animals. 4 months, n＝9 (+/+ and C/+, respectively) , male. 6.5 months, n＝25 (+/+) , n＝16 (C/+) , n＝12 (C/C) , male. In Open field test, the standing time (seconds in 10-minute interval) on the hind legs in aged (6.5 months) mice was calculated. The value are presented as mean ±SEM. *p <0.05, **p <0.01 (one-way ANOVA or t-test, SPSS) . NS, no statistical significance；
B shows the standing time was significantly reduced in the heterozygous (C/+) and homozygous (C/C) KI mutant groups at 6.5 month of age, compared to the wild type (+/+) group. The value are presented as mean ± SEM. *p <0.05, **p <0.01 (one-way ANOVA or t-test, SPSS) . NS, no statistical significance；
C and D shows Rotarod performance (4-day interval, Med Associates Inc., USA) was carried out in wild type (+/+) , the hetero- (C/+) and/or homozygous (C/C) KI mutant mice at 4 (C) and 6.5 (D) months of age. The stay time on the rotarod was significantly decreased in the aged KI mutant (C/+ and C/C, 6.5 months) mice, but not in the younger (4 months) mutant animals. 4 months, n＝8 (+/+ and C/+, respectively) , male. 6.5 months, n＝25 (+/+) , n＝14 (C/+) , n＝15  (C/C) , male. The value are presented as mean ± SEM. *p <0.05, **p <0.01 (one-way ANOVA or t-test, SPSS) . NS, no statistical significance；
E shows toluidine blue-stained cross sections of femoral nerve motor branches from wild type (+/+) and KI mutant (C/C) animals at 8 months of age. Axon degeneration (arrow) was shown in high magnification image. Scale bar, 50 μm in low and 100 μm in high magnification images. The diameter of a red dot in low magnification image is 5 μm, which is used for the fiber size measurement. ) ； and
F shows the big size (diameter ≥ 5 μm) , but not the small size (< 5 μm) , nerve fiber numbers of femoral motor branches were significantly reduced in the KI mutant (C/C, 8 months) animals, compared to their wild type (+/+) littermate controls. In (A, B, C and D) , the value are presented as mean ± SEM. *p <0.05, **p <0.01 (one-way ANOVA, SPSS) . NS, no statistical significance. The value are presented as mean ± SEM (n＝3) . **p <0.01 (t-test, SPSS) , and
Fig. 3 shows the mutant R513C FUS, but not wild type, moves into SGs and results in the mutant SG disassembly defect when the cultured motor neurons facing the stress challenge,
wherein,
A shows representative immunostaining images from the wild type (+/+) or KI mutant (C/C) cultured motor neurons (3 DIV) treated with AS (sodium arsenite, 1mM, 1hr) or recovered from AS treatment (changed to AS-free medium and cultured for another hour after AS treatment) . The TIA1-positive SGs were formed in both wild type and KI mutant neurons after AS treatment. However, only the mutant SGs were both TIA1-and FUS-positive after AS treatment or after recovery. Scale bar, 20 μm. MOCK, vehicle control groups；
B shows the percentage of cells contained TIA1-positive SGs after AS treatment (AS) or after the recovery (AS+1hr) . After the recovery, the percentage was significantly decreased in the wild type groups, but kept no significant difference in the KI mutant groups, suggesting the SG disassembly defect in the SGs containing mutant FUS. The value are presented as mean ± SEM (n≥3) . *p <0.05, **p <0.01 (t-test, SPSS) . NS, no statistical significance；
C shows the percentage of SGs were both TIA1-and FUS-positive after AS treatment or after recovery. Every AS-induced mutant SGs were double positive. In contrast, none of the AS-induced wild type SGs was double positive. The value are presented as mean ± SEM (n≥3) . *p <0.05, **p <0.01 (t-test, SPSS) . NS, no statistical significance；
D shows the count number of SGs per cell. The SG number was significantly reduced in the wild type neurons after recovery, but kept no significant difference in the mutant groups, further supporting the mutant SG disassembly defect. The value are presented as mean ± SEM (n≥3) . *p <0.05, **p <0.01 (t-test, SPSS) . NS, no statistical significance； and
E shows the AS-induced SGs in different size categories. We categorized the size of SGs into three  groups: small (<1μm) , medium (≥1, and <2μm) , and large (≥2μm) sizes. After recovery, the big size and the most of small size of SGs were cleared up in wild type groups. In contrast, after the recovery, those sizes of mutant SGs were not effectively disassembled. The value are presented as mean ± SEM (n≥3) . *p <0.05, **p <0.01 (t-test, SPSS) . NS, no statistical significance.
Figure 4 shows paraquot treatment significantly increased the mutant SG formation and the relocation of the mutant FUS into SGs,
wherein,
A shows the wild type (+/+) and KI mutant (C/C) cultured motor neurons (3 DIV) were treated with paraquot (Para.， 1mM, 8hrs) , and stained with TIA1 and FUS antibodies. Note the majority of TIA1-positive mutant SGs were FUS-positive. Scale bar, 20 μm.
B shows the statistic results from the experiments shown in (A) . The paraquot treatment significantly increased the mutant SG formation, and the relocation of mutant FUS into the SGs in the cultured motor neurons. The value are presented as mean ± SEM (n≥3) . **p <0.01 (t-test, SPSS) .
Figure 5 shows The prolonged stress challenge converts the SGs into the ubiquitin-positive inclusions,
wherein,
A shows representative immunostaining images from the wild type (+/+) or KI mutant (C/C) cultured motor neurons (3 DIV) treated with AS (sodium arsenite, 1mM) for 4 and 6 hours (4 and 6hrs) . After the prolonged stress treatment, the wild type FUS still kept its nuclear localization. In contrast, the ubiquitin-positive inclusions were shown in the KI mutant neurons after AS treatment, and these inclusions were FUS-positive as well. The asterisk marked the ubiquitin-positive inclusion in the DAPI-negative cell debris. Scale bar, 20 μm；
B shows the percentage of cells contained both FUS-and ubiquitin-positive inclusions after the 1-, 2-, and 4-hour AS treatment. The double-positive inclusions only appeared in the KI mutant motor neurons, but not the wild type ones. The value are presented as mean ± SEM (n≥3) ；
C shows the count number of inclusions per cell. The prolonged stress challenge increased the number of ubiquitin-positive inclusions in the mutant (C/C) motor neurons (MNs) . The value are presented as mean ± SEM (n≥3) ；
D shows the inclusion number in different size categories. The inventors categorized the size of inclusions into three groups: small (<1μm) , medium (≥1, and <2μm) , and large (≥2μm) sizes. The prolonged stress challenge increased the size of inclusions in the mutant MNs. The value are presented as mean ± SEM (n≥3) ； and
E shows the cell debris containing the FUS-and ubiquitin-positive inclusions. The invenors noticed that the DAPI-negative cell debris both FUS-and ubiquitin-positive appeared in the  mutant MNs facing the prolonged stress challenge (AS, 1mM, 2 or 4hrs) . The dots represented the percentage of the ubiquitin-positive debris normalized to the total number of cells in the individual imagines from at least three independent experiments.
Figure 6 shows the stress treatment induced sharp motor decline, FUS mislocalization, ubiquitin upregulation, and FUS-positive stress granule formation in the mutant spinal cords,
wherein,
A shows the schedule for intragastric administration of arsenite (AS) in mice；
B shows the standing time (seconds in 10-minute interval) on the hind legs in Open Field before and after AS exposure. The sharp motor decline was documented by the significant drop of the standing time in the mFUS-R513C KI mice after one month of AS intragastric administration. n＝5 (+/+ and C/C, respectively) , male. The value are presented as mean ± SEM. *p <0.05, **p <0.01. (t-test, SPSS) ；
C shows the stress treatment induced FUS mislocalization and ubiquitin upregulation in the ChAT-positive motor neurons in the KI mutant animals. The typical neurons with FUS mislocalization and ubiquitin upregulation were labeled by arrows. Sections from lumbar 4-6 spinal cords. Scale bar, 50 μm in low and 20 μm in high magnification images；
D shows the stress treatment induced the upregulation of eIF3g, a stress granule marker, and the formation of FUS and eIF3g double positive stress granules in the KI mutant spinal cord ventral horns. Sections from lumbar 4-6 spinal cords. Scale bar, 20 μm； and
Figure 7 shows establish FUS reporter cell lines for screening for compounds that are able to reverse FUS mislocalization.
wherein,
A shows the stable expression of GFP labelled wild type (hFUS) and mutant FUS (FUS-R521C) in Hela cells infected with the lenti-viral particles；
B shows without stress treatment, wild type FUS faithfully located in nucleus, but mutant FUS showed cytoplasmic mislocalization. After arsenite treatment, both the wild type and mutant FUS showed cytoplasmic localization. Scale bar, 20 μm.
DETAILED DESCRIPTION
Reference will be made in detail to embodiments of the present disclosure. The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present disclosure. The embodiments shall not be construed to limit the present disclosure. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions.
The following examples are provided so that the invention might be more fully understood.  However, it should be understood that these embodiments merely provide a method of practicing the present invention, and the present invention is not limited to these embodiments.
The related methods are described as follows:
Materials and Methods:
Mice, mouse behavior tests, and intragastric administration.
For the generation of the FUS-R513C KI mouse line, the inventors PCR amplified the target sequences from the C57BL/6J (JAX, Stock No. 000664) mouse genomic DNA. The donor DNA fragment contains the R513C mutation (tcg to ctg) , and the flanking left and right homology arms (～1kb) , respectively. The donor DNA, gRNA (gcgagcacagacaggatcgcAGG, PAM site capitalized) , and Cas9 mRNA were injected into C57BL/6J embryos. The injected embryos were transferred into the oviduct ampulla of the pseudo-pregnant ICR (JAX, Stock No. 009122) female recipients. The right genotype offsprings were backcrossed to C57BL/6J for at least five generations to establish the line.
For Open Field behavior test, the single animal was placed in the center of an open field area (60 × 60 cm) and tracked with multiple parameters, including total distance, average speed, and distance traveled in the center region, by TopScan behavioral analysis system (CleverSys., USA) in a 10-minute interval. Anxiety level was measured by distance traveled in the center region divided by total distance traveled . The vertical rearing activity was measured by total standing time on hind legs in the 10-minute interval of Open Field test. The rotarod performance was measured by an automated system (Med Associates, Inc) . In brief, the animal was placed on an accelerating spindle (4-40rpm) in 5 minutes per trail and 3 consecutive trials per day. A 20-minute break was set in between each trial. The test was repeated for four days. The fall time from the spindle was auto-calculated by the system when the mouse fell off the spindle within the 5-minute interval. The stay time was calculated by subtraction of the fall time from the 5 minutes, and the mean value of the stay time from 3 consecutive trials per day was used for statistical analysis.
For the intragastric administration, weight the mouse first and gently hold the mouse head back to create a straight line through the neck to esophagus. The sodium arsenite solution was administrated by a rounded tip feeding tube at the dose of 2μg/g body weight, 4 times/week. After each administration, the mice were returned back to the cages and monitored for at least 5 minutes. Animals went through Open Field test once every week to monitor their locomotion.
The animal facility at Tsinghua university has been fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) since 2014. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Tsinghua university based on Guide for the Care and Use of Laboratory Animals  (Eighth Edition, NHR) . The C57BL/6J and ICR mice were purchased from Charles River Laboratories, Beijing, China.
Cell cultures and stress induction.
For motor neuron culture, as previously described, in brief, the spinal cords were dissected from E13.5 mouse embryos and digested with papain (Sigma, 1: 200) with EDTA (1mM) in Neurobasal (Invitrogen) at 37℃ for 30 minutes. During the digestion, DNAse I (Sigma, 10μg/ml) were added in the last 10 minutes. After the digestion, the tissue suspension was passed through the 40 μm strainer. The resulting cell pellet were re-suspended in 1ml HBSS (Invitrogen) with EDTA (0.5mM) , and the suspension was layered over a cushion of 5 ml Optiprep (Sigma, 10％) . After centrifugation at 400 × g for 25 minutes, the motor neurons were enriched in the top 1ml volume. The motor neurons were grown in Neurobasal with B27 (Invitrogen) , horse serum (10％v/v； Sigma) , Glutamax-1 (1 ×； Invitrogen) , and characterized by immunostaining with anti-ChAT (rabbit, Millipore) and anti-Tuj-1 (mouse, Beyotime, China) antibodies. For stress granule induction, the cultured motor neurons (3 DIV) were treated with sodium arsenite (AS, 1mM) and paraquot (1mM) for certain hour dependent on the experiment purposes.
Femoral nerve dissection and axon count.
Dissection of femoral nerves was performed as described previously. The femoral nerve was exposed in the sacrificed mice and briefly fixed (2％glutaraldehyde/2％paraformaldehyde in 0.1 M cacodylate buffer) before dissection. The isolated nerves were postfixed overnight in the same fixative. Dissected nerves were processed for plastic embedding and transmission electron microscopy by standard procedures. Nerve cross sections were stained with toluidine blue and examined by light microscopy.
EXAMPLES
Generation of mFUS-R513C mouse model for ALS.
The inventors employed the CRISPR-Cas9-based knock-in (KI) approach. There are three reasons for them to choose hFUS-R521C to generate KI ALS mouse model. First, the R521C mutation locates in FUS C-terminal, a non-classical PY nuclear localization signal (NLS) , in which clusters more than half of ALS-associated FUS mutations. Among these mutations, R521C is the most frequent one and accounts for 30％of the total. Second, the R521C mutation has been found in both familiar and sporadic ALS patients with high disease penetration. Third, the FUS R521C transgenic mouse and rat models present strong ALS-like pathological features.
To generate the KI mouse line, the inventors first analyzed the FUS protein conservation across species (Figure 1A) . The C-terminal NLS encoded by the last exon of mouse FUS is identical to that of human (515GEHRQDRRERPY526) . The mouse corresponding position for human R521 is R513. Downstream of mouse R513 triplet codon, a PAM site for Cas9  recognition appears (Figure 1B) . The inventors PCR amplified the target sequences from the C57BL/6J (JAX, Stock No. 000664) mouse genomic DNA. The donor DNA fragment contains the R513C mutation (tcg to ctg) , and the flanking left and right homology arms (～1kb) , respectively. The donor DNA, gRNA (gcgagcacagacaggatcgcAGG, PAM site capitalized) , and Cas9 mRNA were injected into C57BL/6J embryos. The injected embryos were transferred into the oviduct ampulla of the pseudo-pregnant ICR (JAX, Stock No. 009122) female recipients. The correct genome insertion and germ line transmission were confirmed by breeding the KI founders with C57BL/6J wild type mice (Figure 1C and 1D) . Meanwhile, the inventors backcrossed the KI mutant mouse to C57BL/6J at least 5 generations, in order to reduce the potential off-target effect introduced by CRISPR/Cas9. The right genotype offsprings were backcrossed to C57BL/6J for at least five generations to establish the line. The inventors observed that the expression level of mutant FUS was slightly upregulated in spinal cord (Figure 1E) , suggesting that the human R521C mutation may increase the protein stability and/or decrease the turnover rate in the target tissue.
Aged mFUS-R513C mice showed motor decline and peripheral axon degeneration.
Unlike early-onset motor disability and death seen in previously reported wild type and mutant FUS transgenic mice, the inventors failed to observe progressive paralysis and shorter life span in our aged KI animals (data not shown) . However, a slight decline of spontaneous locomotor activity detected by open field assay was seen in FUS KI heterozygous group at 4 months of age, and significant motor decline was documented in the both hetero-and homozygous mutant mice at 6.5 months of age (Figure 2A) . The motor ability decline was also documented by the standing time on the hind legs, a more sensitive measurement for hind leg weakness. At 6.5 month of age, both FUS KI heterozygous and homozygous groups showed significant drop of the standing time, compared to the wild type group (Figure 2B) . The motor ability decline was further confirmed by the performance on rotarod in the mutant groups. Although the stay time on the rotarod was slightly declined in the 4-month old FUS KI heterozygous group (Figure 2C) , by 6.5 months of age, both hetero-and homozygous mutant mice had shown further drop of stay time on rotarod, significantly at  day  2 and 3 in comparison with wild type littermates (Figure 2D) .
The motor ability decline in the aged KI mice was further echoed by the motor axon reduction. At 8 months of age, the degenerating motor axons of femoral nerve were observed in the homozygous KI mutant animals (Fig. 2E) . By counting the axon number, the inventors found that the numbers of large-diameter (≥5μm) fibers, but not the small ones (<5μm) , were significant reduced in the homozygous KI mutant mice (Fig. 2E and 2F) , suggesting that the fibers with fast conduction velocity are majorly affected by the expression of mutant FUS.
The cultured KI mutant motor neuron models the disease.
Emerging evidence suggests that FUS, together with other RBPs, are protein components of SG, and mutations in these RBPs have been associated with ALS among other neurodegenerative diseases, suggesting that the misprocessing of SG is pathogenic. To test the hypothesis, the inventors cultured the isogenic motor neurons from the spinal cords of E13.5 homozygous KI and their wild type littermates.
Formation of SGs is context-specific and induced by various stress conditions, including ER, oxidative, and mitochondria stresses. In our motor neuron cultures, the inventors observed the formation of cytosolic stress granules, which were positive for TIA1 (cytotoxic granule-associated RBP, a SG marker) in both wild type and homozygous KI mutant groups, after an hour sodium arsenite (AS) treatment (Figure 3A) . As negative controls, the SG formation was never seen in either wild type or KI mutant motor neurons without AS treatment, neither were seen the FUS cytosol mislocalization (Figure 3A and 3B) . However, the percentage of cells containing SGs was increased in the KI mutant motor neurons, suggesting that the mutant motor neurons are prone to form SGs (Figure 3B) . Most strikingly, every single SG was both TIA1-and FUS-positive in the KI mutant cultures, but none of SG was double-positive in the wild type culture groups, indicating a stress-induced relocalization of mutant FUS into SGs (Figure 3C) .
The dynamics of AS-induced SGs was further evidenced by the disassembly of SGs after the recovery from AS treatment (the replacement of AS-containing medium with AS-free medium) . In the wild type cultures, the percentage of neurons containing SGs and the number of SGs per se were significantly reduced after the 1-hour recovery (Figure 3B and 3D) . However, in the KI mutant motor neuron cultures, these numbers showed no significant drop after the recovery (Figure 3B and 3D) , suggesting that the SGs containing the mutant FUS carry major SG disassembly defect. The mutant SG disassembly defect was further documented by counting the different size of SGs in wild type and mutant FUS neurons after the AS treatment and after the recovery. After the AS treatment, the inventors only observed a slight increase of the medium size (≥1, and <2μm) of SGs in the mutant neurons (Figure 3E) . However, after the 1-hour recovery, although the wild type neurons cleared up all the big size (≥2μm) and the most of small size (<1μm) SGs, the KI mutant neurons failed to disassemble the big size SGs and the most of small size ones (Figure 3E) . Similar to AS treatment, the inventors observed the increased mutant SG formation and the increased relocalization of the mutant FUS into SGs, when the motor neurons were treated with paraquat, a stress inducer and herbicide widely used worldwide (Figure 4A and 4B) .
The hallmark of ALS pathology is the formation of ubiquitin-positive inclusions containing TDP-43 and FUS. However, whether the pathology is related to SG misprocessing is unknown.  Interestingly, when we extended the AS treatment to 4 or even 6 hours, the inventors not only observed the mislocalization of mutant FUS into cytosolic SGs, but co-localization of the mutant FUS with ubiquitin, a key regulator for proteasome-mediated degradation (Figure 5A) . The formation of ubiquitin-positive inclusion only appeared in the mutant, but not wild type, motor neurons, and the extent of inclusion formation was increased along with the treatment time (Figure 5B and 5C) . In addition, the size of the inclusions was also increased with the treatment time, and large proportion of the inclusions size were over 1μm after the AS treatment (Figure 5D) . Strikingly, with the prolonged stress treatment, the inventors observed the inclusions appeared not only inside of the neurons but some cell debris-like structure (Figure 5A and 5D) , and the degree of these inclusions in debris increased with the treatment time (Figure 5E) , reminiscent of the stubborn inclusions in the late stage of ALS.
The stress treatment induced sharp motor decline, ALS-like pathologies, and formation of mutant FUS-positive SGs in the KI mice challenged with stress.
To test whether the mutant FUS disrupts SG processing in the pathogenesis in vivo, the inventors performed the intragastric administration of arsenite in aged mFUS-R513C KI mice (Figure 6A) . Although the standing time on the hind legs was decreased in both wild type and mutant KI groups after 1 or 2-month exposure of arsenite, the hind leg weakness was much more severe in the mutant KI group after 1-month exposure (Figure 6B) . Importantly, the stress exposure induced clear FUS mislocalization and ubiquitin upregulation in the mFUS-R513C KI spinal cord motor neurons, but not in that of wild type animals that were exposed the same dose of arsenite as the KI mutant mice (Figure 6C) . To examine whether the mislocalized mutant FUS moves into SGs to disrupt the SG processing, the inventors stained wild type and KI mutant spinal cord sections with antibody for eIF3g, a key subunit of the eukaryotic translation initiation factor 3 (eIF3) complex and also often used as a SG marker. Excitingly, the eIF3g was upregulated in the ventral horns of the aged KI mice exposed with arsenite, and the signals largely colocalized with mislocalized mutant FUS (Figure 6D) , suggesting the mutant FUS moves into SGs after stress exposure similar to our key observation in the cultured motor neurons. Combining no obvious eIF3g upregulation and ALS-like pathologies in the spinal cords in the aged
Figure PCTCN2017077708-appb-000001
mFUS-R513C mice without stress treatment, the inventors concluded that mFUS-R513C synergizes with stress to lead to pathologies in vivo by disrupting the SG processing.
Establish FUS subcellular localization reporter cell lines
In order to screen for compounds that are able to reverse the mislocalization of FUS, the inventors generated Hela cell lines that stably express wild type and mutant FUS-R521C with N-terminal fused with fluorescent protein, GFP. The expression of GFP-tagged wild type FUS  and FUS-R521C was detected by western blot with both GFP and FUS antibodies (Figure 7A) . The expression level of exogenous GFP-tagged FUS was comparable with the endogenous FUS. The subcellular localization of stably expressed FUS was confirmed by GFP fluorescence. Without stress treatment, the subcellular distribution of wild type FUS was faithfully localized in nucleus measured by the GFP fluorescence (Figure 7B) . However, the FUS-R521C was majorly localized in the cytoplasm. After 0.25mM arsenite treatment for 1 hour, the observed the both wild type and mutant FUS moved into SGs.
Model application examples
The FUS-R513C knock-in ALS mouse model (Figure 2) , and cell derived from the KI mouse line (Figure 3-5) , and the FUS subcellular localization reporter cell lines (Figure 7) the inventors established are rational disease models for testing the potential drug efficacy for ALS, for screening for compounds as potential drug for ALS, and for evaluating the environmental risk factors for ALS, and et al. For the KI ALS mouse model, the travel distance in open field, the hind leg standing time, the stay time on rotarod, and the motor axon number count can be used for the purposes described above. For cells derived from the KI mouse line, for example, the motor neurons, the presence of mutant FUS in the SGs, the stress-induced formation of TIA1 positive SGs, and recovery-induced SG disassembly can be used as the evaluation parameters for the purposes described above. For potential drugs for ALS, inhibition of mutant FUS moving into SGs and recovery of the disassembly defect can be used for drug efficacy evaluation. For potential ALS risk factors, the induction of mutant FUS-positive SGs, the SG formation and disassembly defects can be used for the risk estimation. For the established FUS subcellular localization reporter cell lines, the subcellular location of wild type and mutant FUS can be used for the purposes described above. For potential drugs for ALS, inhibition of mutant FUS mislocalization and reduce of SG formation can be used for drug efficacy evaluation. For potential ALS risk factors, the induction of mutant FUS-positive SGs, the SG formation and disassembly defects can be used for the risk estimation.
Reference throughout this specification to “an embodiment, ” “some embodiments, ” “one embodiment” , “another example, ” “an example, ” “aspecific example, ” or “some examples, ” means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments, ” “in one embodiment” , “in an embodiment” , “in another example, ” “in an example, ” “in a specific example, ” or “in some examples, ” in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore,  the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.
Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments can not be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims (18)

  1. A ALS model, wherein the ALS model express mutant FUS.
  2. The ALS model of claim 1, wherein the mutant FUS is FUS-R521C, wherein mutations at the FUS-R521 are the most frequent FUS mutations in human ALS.
  3. The ALS model of claim 1, wherein the ALS model is animals, tissue or cells.
  4. The ALS model of claim 3, wherein the tissue or cells are separated from the animals.
  5. The ALS model of claim 3, wherein cells are obtained as follows:
    Introduction of plasmid into recipient cells, wherein the plasmid carrying nucleotide expressing FUS-R521 mutation, wherein the recipient cells are separated from 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 cells are neuronal cells.
  8. The ALS model of claim 3 or 4， wherein the animals are C57BL/6J type mice.
  9. The ALS model of claim 8， wherein the animals are obtained as following:
    backcrossing the FUS-R521C KI mutant mouse to C57BL/6J wild type mice at least 5 generations.
  10. The ALS model of claim 9， wherein the animals are obtained as following:
    backcrossing the FUS-R521C KI mutant mouse to C57BL/6J wild type mice 10 generations.
  11. A method for screening a medicament for treating ALS, comprising:
    stress treatment of the ALS model described in any of the claims 1～10；
    contacting candidate reagent with the stress treated ALS models；
    based on the change of the ALS models before and after the contacting, the candidate reagent is judged to be the medicament for treating ALS.
  12. The method of claim 11, wherein the stress treatment comprising at least one of oxidative stress, endoplasmic reticulum stress and mitochondrial stress.
  13. The method of claim 12, wherein the ALS model is cell model， that the contacted cells having at least one of the change described below is an indication for the candidate reagent is a medicament for treating ALS,
    (1) increased cell survival；
    (2) reduction of mutant FUS in cytoplasm；
    (3) reduction of stress granule number and size； and
    (4) reduction of ubiquitin-positive inclusion.
  14. The method of claim 12, wherein the ALS model is animal model, that the contacted animals having increased locomotor activity, increased hindlimb strength, number of axons of peripheral motor neurons, or increased sports and learning ability is an indication for the  candidate reagent is a medicament for treating ALS.
  15. A method for constructing ALS model, comprising:
    mutation of FUS gene in wild-type counterpart to mutant FUS gene of ALS patient.
  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 the way of orthomutation.
  18. The method of claim 16, wherein the orthomutation is realized through at least one of the following techniques,
    CRISPR-CAs9 and homologous recombination of mouse zygotes or embryonic stem cells.
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