KR101591265B1 - A Method for Screening an Agent for Treating a Neuro-Degenerative Disease - Google Patents

Abstract

A method of screening for a therapeutic agent for a degenerative neurological disease that reduces the accumulation of stress granules in a fused in sarcoma mutant, comprising: (a) expressing a FUS mutant protein in a cell; (b) treating the cell with an oxidative stressor; (c) contacting the test substance with the cell; (d) selecting a substance that reduces FUS positive stress granule accumulation as a therapeutic agent for degenerative neurological diseases as compared to a control group in which the test substance is not treated.

Description

TECHNICAL FIELD The present invention relates to a method for screening an agent for treating neurodegenerative diseases,

The present invention relates to a method for screening a therapeutic agent for a neurodegenerative disease. More specifically, the present invention relates to a method for screening a therapeutic agent for treating a degenerative nervous system disease based on the pathological mechanism of stress granule accumulation and auto-digestive action associated with FUS (fused in sarcoma) mutations will be.

Amyotrophic lateral sclerosis (ALS) is a disorder characterized by progressive loss of upper and lower motor neurons in the brain cortex, brain stem, and spinal cord. (Bento-Abreu et al, (2010) Eur J Neurosci 31 (12) : 2247-2265), eventually leading to paralysis and death. Most ALS are idiopathic, but about 10% are inherited (Andersen et al, (2011) Nat Rev Neurol 7 (11) : 603-615).

Several mutations, including SOD1 , TARDBP , FUS / TLS, VAPB , OPTN , VCP , UBQLN2 and C9ORF72 , are known to be associated with ALS (Ticozzi et al., (2011) Arch Ital Biol 149 (1) : 65-82).

FUS ( FUS / TLS ) is a DNA / RNA binding protein involved in DNA repair, gene transcription, and RNA splicing (Dormann et al, (2013) Mol Cell Neurosci . ), The N-terminus of FUS is associated with transcriptional activity, while the C-terminus is known to be involved in protein and RNA binding. It has also been found that FUS has cognate sites for the transcription factors AP2, GCF, Sp1.

FUS has been reported to move back and forth in the cytoplasm for post-mitotic neurons, mRNA transfer to dendrites, and local protein synthesis at synapses responsive to synaptic activity (Aoki et al, (2012) Acta Neuropathol 124 (3) : 383-394; Fujii et al, (2005) Curr Biol 15 (6) : 587-593) and the identification of FUS RNA targets in the cerebral cortex has been shown to regulate the splicing of mRNA sequences important for neuron integrity and signaling (Lagier -Tourenne et al, 2012, Nat Neurosci 15 (11) : 1488-1497).

FUS mutations have been found in ALS and Frontotemporal Lobar Dementia (FTLD), a typical early dementia mutation of the RNA-binding gene.

In addition, several missense mutations and nonsense mutations in FUS have been reported to be associated with ALS, FTLD, and haploidy (Huey et al, 2012, Neurobiol Aging 33 (5) : 1016E1019-1017; Rajput et al., 2013, Eur J Neurol ). Most of these mutations are in the C-terminal domain, which shows the function of the Nuclear Localization Signal (NLS). Therefore, it has been reported that this FUS mutation prevents the FUS from migrating to the nucleus, causing it to be abnormally located in the cytoplasm and thus forming FUS-positive aggregates (Dormann et al., 2012, EMBO J 31 (22) : 4258 -4275).

In addition, the FUS-positive cell content in the cytoplasm was observed in the brain of a person with idiopathic ALS or FTLD suggesting that the FUS positive cell inclusion is likely to play a role in the development of this disease (Mori et < RTI ID = 0.0 > al., 2012, Neuropathol Appl Neurobiol 38 (4) : 322-328).

However, little is known about the mechanisms involved in the abnormal location and aggregation of these FUS mutant proteins found in patients with ALS or FTLD, and there is no such treatment.

Also, it is not known at all whether these ALS-induced mutant proteins are related to FUS-positive stress granules in degenerative brain diseases or the role of autolysis in the accumulation of FUS-positive stress granules .

Stress granule (SG) is a dense aggregate of proteins and RNA that appears in the cytoplasm under stress conditions, and may function to protect RNA from harmful conditions. In other words, it might function to protect the information encoded in the RNA sequence by accumulating the RNA sequence with a dense globule. In addition, stress granules composed of mRNA, RNA binding protein, and RNP mainly receive and protect mRNA from transcription initiation under stress conditions, but stress granules in which protein aggregates are abnormally retained are involved in some neuronal functions in cytoplasm or neurons Lt; RTI ID = 0.0 > mRNA < / RTI > and RNA binding proteins.

Stress granules have been reported to actively assemble and disintegrate (Li et al, 2013, J Cell Biol 201 (3) : 361-372), have been reported to have a dynamic structure that is formed under stress conditions and degrades after stress has ceased (Li et al, 2013, J Cell Biol 201 (3) : 361-372).

Recently, abnormal accumulation of the stress granule marker PABP (Poly (A) -binding protein) and FUS-positive cytoplasmic content in the brain region affected by ALS has been confirmed (Dormann et al, 2012, EMBO J 31 (22) : 4258-4275). In addition, several cell-level studies of mutations in FUS missense revealed that cytoplasmic FUS is located in the stress granules and accumulates in the cytoplasmic region (Li et al, 2013, J Cell Biol 201 (3) : 361- 372).

Other studies have shown that FUS / ALS assembly into stress granules is rapid and reversible, and FUS is involved in the pro-survival process in response to stress conditions (Sama et al, 2013, J Cell Physiol 228 (11) : 2222-2231). In addition, it has been shown that FUS C-truncation mutant proteins affect the assembly or degradation of stressed granules (Baron et al, 2013, Mol Neurodegener 8 (1) : 30).

However, how FUS mutations are associated with the accumulation of stressed granules and how such accumulation is regulated has not been clearly elucidated.

Autophagy is a pathway for large-scale degradation of cytoplasmic components such as RNA, proteins, or organelles in lysosomes (Lee, 2012, Exp Neurobiol 21 (1) : 1-8; Nixon, 2013, Nat Med 19 (8) : 983-997), and quality control of proteins and cell organelles.

The self-digestive pathway is known to be associated with degenerative neurological diseases involving several cytoplasmic inclusions, and in some degenerative neurological diseases, accumulation of aggregates and damaged cellular organelles, resulting in impaired cellular homeostasis and signaling in neurons It is known. In addition, oxidative stress is closely related to the onset of ALS and is known to be important for disease progression (Barber & Shaw, 2010, Free Radic Biol Med 48 (5) : 629-641).

On the other hand, eukaryotic stress granules were found to be treated by autogenous digestion in yeast (Buchan et al, 2013, Cell 153 (7) : 1461-1474).

Trehalose, known as a new mTOR (mechanistic target of rapamycin, a serine / threonine kinase) non-dependent self-extinguishing enhancer, has recently been shown to delay the progression of ALS in motor neurons, (Castillo et al., 2013, Autophagy 9 (9): 1308-1320), which may be an expected strategy for treating related ALS.

However, the role of auto-digestion in the accumulation of ALS-associated FUS-positive stress granules has not been known so far.

The inventors of the present application studied the mechanisms involved in degenerative nervous system diseases such as ALS or FTLD by FUS mutant proteins, and found that the FUS mutation inhibited accumulation in stress granules and inhibited decomposition from stress granules, The inventors confirmed that the therapeutic agent for ALS or FTLD can be screened and completed the present invention.

The present invention provides a method for screening for a therapeutic agent for a degenerative neurological disease which reduces accumulation of stress granules in a fused in sarcoma mutant,

(a) expressing a FUS mutant protein in a cell;

(b) treating the cell with an oxidative stressor;

(c) contacting the test substance with the cell;

(d) selecting a substance that reduces FUS positive stress granule accumulation as a therapeutic agent for degenerative neurological diseases as compared to a control group in which the test substance is not treated.

The candidate substance group that can be used in the treatment of the degenerative nervous system diseases that inhibits the accumulation of FUS mutations in stress granules or inhibits the decomposition disturbance from stress granules can be screened by the method for screening therapeutic agents for degenerative neurological diseases according to the present invention.

Figure 1a shows the results of western blot analysis (WT: wild type, R521C: R521C mutation) in which flag-FUS (WT) or flag-FUS (R521C) was expressed in HEK 293T cells.
Figure 1B shows the location of FUS and PABP as images of neurons expressing flag-FUS (WT) or flag-FUS (R521C) immunostained with anti-flag antibody or anti-PABP antibody, respectively. (SA: sodium arsenite treatment, Recovery: removal of sodium arsenate treatment, PABP: stress granule marker, DAPI: nuclear localization, arrow: FUS positive stress granule, scale bar:
Figure 1c shows the location of the FUS at each oxidative stress condition (non-presence / presence / after treatment) as an image of a neuron expressing flag-FUS (WT) or flag-FUS (R521C).
Figure 1d shows the percentage of neurons with FUS positive stress granules in each oxidative stress condition (non-presence / presence / treatment) in flag-FUS (WT) or flag-FUS (R521C) expressing neurons (Values are mean ± SEM, Turkey's multiple comparison test after one way AVOVA, *** p <0.001, ns: no significance, n = 3).
Figure 2a is an image of a neuron expressing flag-FUS (WT) or flag-FUS (R521C) expressed with RFP-LC3.
Figure 2B is a bar graph showing percent (%) of neurons with FUS positive stress granules in the RFP-LC3 autoclaved body (mean ± SEM, multiple comparison test in Turkey after one-way analysis, ** p <0.001, n = 3).
Figure 2c shows the effect of anti-FLAG or anti-LC3 from HEK 293T cell lysate expressing either FUS (WT) or FUS (R521C) in the presence or absence of NH ₄ Cl (10 mM) Western blot analysis.
FIG. 3A is a block diagram of a WT or an atg5 ^{- / -} flag-FUS (WT) or a flag-FUS (R521C) This is an image showing the intracellular distribution of FUS or PABP in cells transfected with MEFs.
Figure 3b is a flag-FUS (WT) or flag-FUS (R521C) WT or atg5 ^{- / -} (%) Of cells with FUS-positive stress granules in cells transfected with MEFs (values are mean ± SEM of three independent replicates, multiple comparison tests of Turkey after one-way ANOVA, ** p < 0.01, *** p < 0.01).
Figure 3c is a Western blot analysis showing the result of co-transfecting Flag-FUS (WT) or FUS (R521C) into cortical neurons cultured with atg7 siRNA or scrambled siRNA.
Figure 3d shows the effect of atg7 siRNA Or FUS (WT) or FUS (R521C) with scrambled siRNA, indicating FUS positive stress granules (Sc siRNA: scrambled siRNA).
Figure 3e shows that atg7 siRNA Or percentage of neurons with FUS positive stress granules in neurons expressing FUS (WT) or FUS (R521C) with scrambled siRNA (values are mean ± SEM of 3 independent replicates, Multiple comparison test of Turkey after variance analysis, *** p <0.001).
Figure 4a shows FUS positive stress granules as images of neurons expressing FUS (R521C) and atg7 siRNA (or scrambled siRNA) with (or without) rapamycin. (Rapa: Rapamycin treatment)
Figure 4b is a bar graph showing the percentage of neurons with FUS positive stress granules in neurons expressing FUS (R521C) and atg7 siRNA (or scrambled siRNA) with (or without) rapamycin Mean ± SEM of independent repetition, one-way ANOVA, multiple comparison test of Turkey, *** p <0.001, ns: no significance, arrow: FUS positive stress granule).
FIG. 4C shows a neurite outgrowth as a representative image of a neuron expressing FUS (R521C) or FUS (WT) together with GFP (arrow indicates neurite outgrowth, reference: 20 mu m).
Figure 4d is a bar graph showing percent (%) of neurons with neurite dysplasia (values are mean ± SEM of 4 independent replicates, one-way ANOVA, multiple comparison test of Turkey, *** p <0.001).

Hereinafter, the present invention will be described in detail in order to facilitate understanding of the present invention.

The inventors of the present application have found that FUS mutation can treat degenerative nervous system diseases by inhibiting excessive accumulation of stress granules and confirmed that this mechanism can be used for the screening of a therapeutic agent for degenerative nervous system diseases.

The present invention provides a method for screening for a therapeutic agent for a degenerative neurological disease which reduces accumulation of stress granules in a fused in sarcoma mutant,

(a) expressing a FUS mutant protein in a cell;

(b) treating the cell with an oxidative stressor;

(c) contacting the test substance with the cell;

(d) selecting a substance that reduces FUS positive stress granule accumulation as a treatment for a degenerative neurological disease disease as compared with a control group in which the test substance is not treated.

The FUS mutation may be a FUS (R521C) mutation.

The step (a) is a step of expressing a FUS mutant protein in a cell. FUS mutant proteins can be expressed, for example, by transfecting HEK293T cells or neuronal cells with vectors containing FUS mutant cDNA. The FUS mutation can be produced using the primers of SEQ ID NO: 1 or SEQ ID NO: 2 below.

SEQ ID NO: 1 : sense-5'-CGCCCAAGCTTATGGCCTCAAACGATTATAC-3 '

SEQ ID NO: 2 : Antisense-5'-CGCGGATCCTTAATACGGCCTCTCCCTGCAATCCT-3 '

The step (b) is a step of treating the oxidative stress-inducing substance to the cells. The oxidative stress can be induced, for example, by the addition of sodium arsenite drug during cell culture.

The step (c) is a step of bringing the test substance into contact with the cell. There is no particular limitation so long as the FUS mutation expressed in the cell can be checked whether or not the accumulation of stress granules can be confirmed by treating the test substance, and the test substance may be added to the cultured neuron at the specific growth time and incubated.

The step (d) is a step of selecting a substance that reduces the accumulation of FUS positive stress granules as a therapeutic agent for degenerative neurological diseases as compared with a control group in which the test substance is not treated. Reducing the FUS positive stress granule accumulation may be due to inhibiting the accumulation of FUS mutations in stress granules or inhibiting FUS mutations from interfering with the degradation of FUS positive stress granules.

Reducing the FUS positive stress granule accumulation may be due to activation of autophagy.

Reducing the FUS positive stress granule accumulation may be confirmed by a reduction in abnormal structural changes in neuronal cells. The abnormal structural change of the neuron may be a division of the neuron.

The measurement of reducing the FUS positive stress granule accumulation can be used without limitation, but it can be measured, for example, by confirming and quantifying the number of FUS positive stress granules by performing immunocytochemistry. Thereafter, the degree of decrease in FUS positive stress granule accumulation can be measured by comparing the quantification results with the control group without the test substance.

If the test substance decreases the accumulation of FUS-positive stress granules compared to the control group which does not treat the test substance after the result of the above measurement method, the screening method according to the present invention can be used as a therapeutic agent for the desired degenerative neurological disease .

The degenerative nervous system disease is characterized by being caused by nerve cell damage. Examples of degenerative nervous system diseases caused by nerve cell damage include amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis Frontotemporal Lobar Dementia (FTLD) or embryo can be included.

The material screened through the mechanism can be used for the preparation of a composition for the prevention or treatment of degenerative neurological diseases through activation of auto-digestion.

Example

The following examples further illustrate the present invention, but the present invention is not limited by these examples.

[Experimental Method and Condition]

One. cDNA  Plasmid production

FUS (WT) human cDNA was obtained from Addgene (Cambridge, MA, USA). For expression in animal cells, R521C cDNA was prepared using the primers of SEQ ID NO: 1 or SEQ ID NO: 2, which contain mutations.

SEQ ID NO: 1: sense-5'-CGCCCAAGCTTATGGCCTCAAACGATTATAC-3 '

SEQ ID NO: 2: Antisense-5'-CGCGGATCCTTAATACGGCCTCTCCCTGCAATCCT-3 '

Each cDNA of FUS (WT) or FUS (R521C) was then subcloned into the BamHI / HindIII site of the 3Xflag CMV7.1 vector (Sigma, St Louis, MO USA).

2. Cell culture, transfusion, and immunocytochemistry

Cerebral neuronal cells of ICR mouse (DAEIL, Korea) were transfected into the cortical neurons of Lee, JA, and FB Gao (Lee, J., A. Beigneux, ST Ahmad, SG Young, and FB Gao. 2007. Curr Biol 17: 1561-7. 2009. J Neurosci 29: 8506-11.).

300-500 ng of each cDNA plasmid was added to cortical neuron cells for 3 or 5 days (3 or 5 days in vitro (DIV)) after the cultivation, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA USA) .

Atg7 siRNA was transfected into cultured cortical neurons (DIV3) 3 days after cultivation to inhibit autogenous digestion (Ban et al, 2013, Mol Cell Biol 33 (19): 3907-3919). Sodium arsenate (Sigma, St. Louis, Mo., USA, 0.5 mM) or rapamycin (Sigma, St. Louis, MO, USA, 10 nM) was added to the culture medium 48 hours after the transfection.

For immunocytochemistry, neurons, atg5 knockout ( atg5 - / - ) MEFs (mouse embryonic fibroblasts), or wild type (WT) MEFs (Kuma et al, 2004) were mixed with 0.1% Triton X 100 ) For 5 minutes, and then blocked with 3% bovine serum albumin (BSA) at room temperature for 1 hour. Cells were treated with anti-FLAG antibody (Sigma, St. Louis, Mo., USA, 1: 100), anti-PABP antibody (abCAM, USA, 1: 100), anti-ATG7 antibody (Santa Cruz Biotech, Santa Cruz Incubated for 1 hour at room temperature with anti-rabbit secondary antibody (Jackson Labs, Bar Harbor, ME, USA).

3. Western Blasting

Whole cell lysates were prepared in lysis buffer as described in Ban et al, 2013, Mol Cell Biol 33 (19): 3907-3919. Cell lysis solution (50 ㎍ protein) was incubated with anti-LC3 antibody (Novus Biologicals, Littleton, CO, USA, 1: 5000), anti-PABP PABP (abCAM, Cambridge, MA, USA, (1: 10,000-20,000) and HRP-conjugated anti-rabbit or anti-mouse secondary antibody (1: 10,000-20,000).

[ Example  One] FUS ( R521C ) Mutation under oxidative stress Mutant of FUS  Evaluation of effects on accumulation in stress granules

(One) FUS  Identification of Protein Expression and Stability

To investigate the mechanism by which specific mutations of the fus gene cause ALS-related neurodegeneration, One of the most frequent point mutations among the fus gene mutations, the R521C missense mutation (Dormann & Haass, 2013, Mol Cell Neurosci ) was selected.

In order to confirm their expression and protein stability, flag-FUS (WT) or flag-FUS (R521C) were expressed in HEK 293T cells. Cells were transfected for 24 hours and then treated with cycloheximide (CHX) to inhibit new protein synthesis (Colombo et al, 1965, Biochem Biophys Res Commun 18: 389-395). Cell lysates were obtained at 0, 12, 24, and 48 hours after CHX treatment and Western blot analysis was performed using anti-flag antibody. As shown in FIG. 1A, FUS (R521C) protein was found to be more resistant to protein degradation than FUS (WT), while wild type (WT) protein was reduced while FUS (R521C) protein level remained at 48 hours.

(2) FUS  Protein Intracellular  Investigate location of expression

To investigate the intracellular location of FUS (R521C) in post-mitotic neurons, flag-FUS (WT) or flag-FUS (R521C) were cultured in cultured rat cortical neurons for 5 days DIV5, in vitro days). The results of the experiment are shown in Fig. 1B.

As shown in FIG. 1B, most of the FUS (WT) was located in the nucleus 24-48 hours after transfection. FUS (R521C) was also located mainly in the nucleus, but a part was also located in the cytoplasm (Fig. 1B). However, FUS (R521C) positive stress granules were rarely detected in cultured neurons.

This indicates that the formation of FUS positive stress granules may be initiated by environmental factors such as oxidative stress during aging (FIG. 1B and FIG. 1C).

(3) oxidation stress FUS ( R521C ) Of protein Intracellular  Location impact study

In addition, FUS (WT) or FUS (R521C) expressing neurons were also used to assess the effect of oxidative stress on the intracellular location of ALS-associated R521C in cultured neurons (Bosco et al, 2010, Hum Mol Sodium arsenite (0.5 mM, 1 hr) inducing oxidative stress and formation of stress granules (0.5 mM, 1 hr) was added to the culture medium 24 hours after transfection, as described in Genet 19 (21) : 4160-4175 . After incubation with sodium arsenate for 1 hour, neurons were incubated for 20 minutes in fresh medium without sodium arsenite for recovery from oxidative stress. The results of confirming protein expression in each neuron are shown in Fig. 1B and Fig. 1C.

As shown in Fig. 1B, FUS (WT) is a stress-granulating marker, PABP (Poly (A) -binding protein) in response to oxidative stress (Bentmann et al, 2012, J Biol Chem 287 (27): 23079-23094) Positive stress granules.

In addition, as shown in FIG. 1d, FUS binding to stress granules was elevated in FUS (R521C) -expressing neurons compared to FUS (WT) -expressing neurons, indicating that the R521C mutation was associated with the formation of FUS- Lt; / RTI &gt;

(4) R521C  Mutation FUS  Effects of positive stress granules on the assembly mechanics

We investigated whether the R521C mutation regulates the assembly mechanics of FUS positive stress granules. As shown in FIG. 1c and FIG. 1d, the FUS (WT) positive stress granules were significantly reduced at 20 minutes after sodium arsenate removal, indicating that the FUS was degraded from the stress granules (FIG. 1d). However, FUS (R521C) positive stress granules remained at 20 min after oxidative stress elimination, indicating that the R521C mutation interfered with the degradation of FUS from oxidative stress induced stress granules (Fig. 1B, Fig. 1C, And Fig. 1d). The FUS mutation not only increased the binding of FUS to the granules under stress conditions, but also prevented FUS from rapidly separating from the stress granules after stress. Therefore, the normal epidemiological impairment of stress granules in response to stress during aging and excessive accumulation of FUS positive stress granules may be the cause of ALS progression.

As a result, it was confirmed that the ALS-associated FUS (R521C) mutation enhanced the binding of FUS to stress granules during oxidative stress and interfered with their dynamics during recovery from oxidative stress.

[ Example  2] Self-digestion and FUS - Relationship between accumulation of positive stress granules

We performed the following experiments to determine whether auto-digestion is associated with the accumulation of FUS-positive stress granules in a reproduced cell model of ALS with FUS mutation (R521C).

First, flag-FUS (WT) or flag-FUS (R521C) was co-transfected with cultured cortical neurons with RFP-LC3, an autogranular somatic marker (Ban et al, 2013, Mol Cell Biol 33 ) : 3907-3919). However, no prominent RFP-LC3 progenitor cells were observed in FUS (WT) or FUS (R521C) expressing neurons under oxidative stress (data not shown).

Next, neurons expressing FUS (WT) or FUS (R521C) were exposed to oxidative stress (0.5 mM, sodium chloride) in the presence of ammonium chloride (NH ₄ Cl) (10 mM) 0.0 &gt; 1 &lt; / RTI &gt; hr), and RFP-LC3 positive autologous phagocytes were examined in the presence of ammonium chloride. As a result, the FUS-positive stress granules were located together with the RFP-LC3 positive autophagic bodies under the suppression of lysosomal degradation during oxidative stress (Figs. 2A and 2B), and as a result, the FUS positive stress granules inhibited lysosomal degradation And accumulate in the host organism.

The FUS (R521C) to perform a western blot analysis using the LC3 antibody under the presence or absence of NH ₄ Cl to say that the RFP-LC3-positive self-how accumulated in the phagocytic vesicles, flag-FUS (WT) or flag-FUS ( R521C) expressing cells.

As shown in FIG. 2C, LC3-II accumulates in the presence of NH ₄ Cl, indicating that the autopoietic pathway is active in FUS (WT and R521C) expressing cells.

As a result, some degenerative nervous system diseases are associated with dysfunction of self-digestive degradation, but FUS-positive stress granules do not appear to impair the self-digestive pathway.

[ Example  3] Self-digestion-deficiency FUS  Investigation on the accumulation of positive stress granules

To further investigate the role of self-extinguishing action in the excessive accumulation of FUS (R521C) positive stress granules, either flag-FUS (WT) or flag-FUS (R521C) were added to WT MEFs or atg5 ^{- / -} MEFs were transfected. FUS-positive stress granules were scarcely detected in WT MEFs, but accumulated in a significant portion of atg5 ^{- / -} MEFs (FIGS. 3A and 3B). In addition, more FUS (R521C) positive stress granules accumulate in atg5 ^{- / -} MEFs than FUS (WT) positive stress granules, suggesting that the accumulation of FUS (R521C) (Fig. 3A and Fig. 3B).

To further confirm that the assembly process of FUS-positive stress granules is regulated by the self-digestive pathway, atg7 (Komatsu et al, 2007, Proc Natl , 2007), an essential component of self- Acad Sci USA 104 (36): 14489-14494) was knocked down to examine the assembly of stress granules. Specifically, Flag-FUS (WT) or Flag-FUS (R521C) was co-transfected with cultured cortical neurons with either scrambled siRNA or atg7 siRNA. 48 hours after transfection, neurons were treated with sodium arsenate (0.5 mM, 1 hr). The results of expression of Flag-FUS (WT) or Flag-FUS (R521C) in neurons with atg7 siRNA or scrambled siRNA are shown in Fig.

As shown in Figures 3D and 3E, more FUS (WT) positive or FUS (R521C) positive stress granules accumulate in autolytic depleted neurons, as compared to control neurons (scrambled siRNA) Were involved in the regulation of FUS - positive stress granules.

[ Example  4] The activation of self-digestion FUS ( R521C ) Effect on expression neurons

(1) activation of self-digestion FUS  Effect of positive stress on the formation of granules

To investigate the direct role of self-digestion in the reduction of FUS (R521C) positive stress granules, rapamycin, an inhibitor of mTOR (mechanistic target of rapamycin, serine / threonine kinase) (Liang, 2010, Cell &lt; RTI ID = 0.0 &gt; Death Differ 17 (12) : 1807-1815).

Specifically, Flag-FUS (R521C) was transfected into cultured cortical neurons with either atg7 siRNA or scrambled siRNA (Sc siRNA). 48 hours after transfection, neurons were exposed to sodium arsenite (0.5 mM) in the presence or absence of rapamycin (10 nM). Neurons were fixed and immunostained with anti-flag or anti-PABP antibodies.

As shown in Figs. 4A and 4B, rapamycin treatment reduced the accumulation of FUS (R521C) positive stress granules.

Rapamycin can affect some intracellular pathways involved in protein synthesis or cell signaling, including autophagy. Therefore, in order to further confirm that the specific activation of rapamycin-induced auto-digestion affects the reduction of stress granules, atg7 siRNA was transfected into R521C-expressing neurons to inhibit auto-digestion, It was tested whether it could reduce the number of granules.

As also shown in Figures 4A and 4B, FUS (R521C) expressing neurons Treatment with rapamycin in the presence of atg7 siRNA did not reduce the number of FUS (R521C) positive stress granules during oxidative stress. These results suggest that the specific activation of autophagy by rapamycin leads to a decrease in the number of stress granules.

(2) Effect of activation of self-digestion on neurons

The inventors have found that FUS (R521C) induces neurodegeneration in response to oxidative stress in mitosis-prone neurons compared to FUS (WT) and decreases FUS (R521C) mutation by decreasing stress granulation by activation of autolysis And whether they could escape the induced neurodegeneration.

Specifically, Flag-FUS (R521C) or Flag-FUS (WT) was transfected with cultured cortical neurons together with GFP (green fluorescent protein). 48 hours after transfection, neurons were exposed to oxidative stress with sodium arsenate (0.5 mM) in the presence or absence of rapamycin (10 nM) for 1 hour and then washed with fresh medium. At 24 hours after washing, the morphology of the neurons was visualized as GFP.

As shown in FIG. 4c and FIG. 4d, neurite outgrowth was hardly observed in FUS (WT) -expressing neurons, while FUS (R521C) -expressing neurons showed remarkable neurite outgrowth.

In addition, it was confirmed that the neurite division induced by exposure to oxidative stress in FUS (R521C) -expressing neurons was significantly reduced when the autophagic action was activated by rapamycin treatment.

Taken together, these results suggest that autogenous digestion can reduce excessive accumulation of FUS-positive stress granules and reduce neurite regression associated with FUS pathology.

<110> Hannam University Institute for Industry-Academia Cooperation <120> A Method for Screening an Agent for Treating a Neuro-Degenerative          Disease <130> P140240 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 31 <212> DNA <213> Homo sapiens <400> 1 cgcccaagct tatggcctca aacgattata c 31 <210> 2 <211> 35 <212> DNA <213> Homo sapiens <400> 2 cgcggatcct taatacggcc tctccctgca atcct 35

Claims (7)

A method of screening for a therapeutic agent for a degenerative neurological disease that reduces the accumulation of stress granules of a mutant FUS (fused in sarcoma) (R521C)
(a) expressing a FUS mutant protein in a cell;
(b) treating the cell with an oxidative stressor;
(c) contacting the test substance with the cell;
(d) a substance exhibiting a decrease in FUS-positive stress granule accumulation and a decrease in the abnormal structural change of neuronal cells due to the activation of autophagy as compared with the control group in which the test substance is not treated is selected as a therapeutic agent for degenerative neurological diseases &Lt; / RTI &gt; delete 2. The method of claim 1, wherein said FUS (R521C) mutation is made using the primers of SEQ ID NO: 1 or SEQ ID NO: 2. The method according to claim 1, wherein reducing the FUS positive stress granule accumulation is by inhibiting the accumulation of FUS mutations in stress granules or inhibiting FUS mutations from interfering with the degradation of FUS positive stress granules. delete delete The method of claim 1, wherein the degenerative neurological disease is Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Dementia (FTLD), or embryo.

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