TR201903865A2 - USE OF INTERFERONES FOR THE TREATMENT OF AMYOTROPHIC LATERAL SCLEROSIS - Google Patents

Abstract

The invention relates to the use of interferon, preferably interferon-alpha, in the treatment of amyotrophic lateral sclerosis (ALS), particularly ALS caused by a mutation in the NEK1 gene. The invention further relates to a method developed for the treatment of a patient affected by ALS, comprising the steps of: (a) selecting a patient carrying a NEK1 variant, i.e., the NEK1 mutation, (b) providing the patient with a therapeutically effective amount of interferon, preferably interferon. -alfa giving.

Description

DESCRIPTION USE OF INTERFERON FOR THE TREATMENT OF AMYOTROPHIC LATERAL SCLEROSIS TECHNICAL FIELD The present invention relates to the treatment of amyotrophic lateral sclerosis (ALS), particularly NEC-associated ALS. BACKGROUND ALS, also known as Lou Gehrig's disease, is a neurodegenerative motor neuron disease that mostly affects adults between the ages of 40-65 and results from the deterioration and death of motor neurons in the brainstem and spinal cord. Early symptoms of ALS include muscle weakness, involuntary contraction, and cramps. In advanced stages of the disease, individuals lose their ability to control voluntary muscles. In the final stages, they usually die due to respiratory failure. ALS is a relatively rare disease, its prevalence is approximately every year. However, both the socioeconomic and personal burden of the disease is serious. The drugs used in ALS disease, which has very limited treatment options, relatively slow down the progression of the disease rather than eliminating or treating it. Most patients die within 3-4 years following the appearance of symptoms. Approximately 5-10% of ALS is familial, meaning it is passed from one generation to the next and shows Mendelian inheritance. The remaining 90-95% of patients are sporadic cases with no familial history (Hardiinan, O. et al., Nature Reviews Neurology (2011), it was discovered. Since this date, more than 30 genes have been associated with ALS (Robberecht, W. et al., Advances in Genoinics and Genetics (2015), 5, 327), unfortunately these sections were insufficient to cure or eliminate ALS because the genes identified were functionally and structurally very different. It is still not fully known how it causes the same disease phenotype. A common point of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and ALS is the protein seen in neurons. Proteins perform vital metabolic, structural or enzymatic functions not only in neurons but in all cells of the body. The three-dimensional conformations (structures) of proteins enable them to perform these functions; therefore, it is necessary for the proteins to fold correctly, that is, to acquire their three-dimensional structure. Under some conditions, due to genetic or environmental factors, proteins may misfold. Misfolded proteins lose their solubility in the cell cytoplasm, aggregate, and ultimately cause cell death (Taylor, J.P. et al., Nature (2016). Not only can they no longer fulfill their vital functions, but they can also cause cellular toxicity due to their tendency to form toxic aggregates. These mechanisms in every cell prevent proteins from misfolding and destroy misfolded proteins when they do. Depending on the type of protein, its amount, or the cause of its misfolding, these cells may not always be protected from toxicity or death by protein misfolding and its inappropriate consequences. The first mechanism involves molecular chaperons, which guide the correct folding of proteins synthesized in ribozoin. Molecular chaperons sometimes recognize intermediate protein forms that are already misfolded and help them refold correctly, using ATP. may be (Hartl, F.U. et al., Nature provides protection against proteins that can lead to toxic aggregates by directly eliminating misfolded proteins. This mechanism is a type of post-translational protein modification (PTM) that tags proteins for degradation by the Ubiquitin-Proteasome System (UPS), which carries out the ubiquitination of misfolded proteins and subsequent destruction within the proteazoin complex. When both mechanisms mentioned are insufficient to prevent protein misfolding, aggregate formation occurs. In recent years, researchers have revealed that there is a strong connection between ALS disease and SUMOlation, another ubiquitin-like modification (Foran, E. et al., Neuromolecular Medicine (2013), 15(4), SUMOlation, SUMO (Small Ubiquitin-like Modifier) It involves the covalent binding of a small peptide called ubiquitin, very similar to ubiquitin, to selected target proteins through a series of enzymatic reactions. SUMOlation is used by eukaryotic cells to change many important properties of proteins, including their stability, solubility, enzymatic activity and intracellular localization. (Hay, R.T., Molecular Cell (2005), they undergo SUMO modification on specific lysine amino acids in which ip refers to a hydrophobic amino acid, K to the target lysine, X to any amino acid, D/S to the negatively charged aspartic or glutamic acid . (Gareau, J.R. et al., Nature Reviews Molecular Cell Biology The vertebrate genome contains 3 functional SUMO paralogs: SUMO1, SUM02, and SUMO3. The sequences of SUM02 and SUMO3 are 95% identical, so together they are called SUM02/3 (Martin, S. et al., Nature Reviews) contains one of the consensus motifs, which itself is subject to SUMOlation. As a result, poly-ubiquitin, which is commonly seen in ubiquitination Similar to poly-SUMO chains, SUM01 acts as a chain terminator when added to the end of a growing poly-SUMO chain, since it cannot be SUMOized with SUM02/3 (Hay, R.T., Molecular Cell (2005), 861; Seeler, J-S. et al. ., Nature Reviews Molecular Cell Biology (2003), 4(9), Ubiquitin and SUMO peptides have 18% identity and differ greatly in their N-terminal regions. SUMOlation of a target protein is an enzymatic process consisting of 3 steps, very similar to ubiquitination. In the first step, the SUMO peptide is activated by an E1 enzyme (SAEl/SAEZ). The activated SUMO peptide is then transferred to an E2 conjugation enzyme called UBC9. Unlike the ubiquitination system, in which dozens of different E2 enzymes function, protein SUMOlation requires only one E2 enzyme: UBC9. UBC9 can directly add SUMO to target proteins, although an E3 ligase is not required for this, E3 ligase can facilitate this process in some cases. Depending on the number of lysines targeted in conjugation and the type of SUMO, three types of SUMO can occur: mono-, multi- or poly-SUMOlation (Hickey, CM. et al., Nature Reviews Molecular Cell Biology) can be cleaved by proteases, (J.R. et al., Nature Reviews Molecular Cell Protein SUMOlation is a highly dynamic modification and is involved in essential cellular modifications such as transcription, DNA repair and replication. Consistent with its role in these important processes, SUMOlation is also essential for life. Blocking SUMOlation by embryonic or conditional knockout of the UBC9 gene causes death in mice (Nacerddine, K. et al., Developmental Cell (2005), 9(6), 769). Intuitions that cause irregularities in the SUMO system have been associated with many diseases, such as cancer (Seeler, J-S. et al., Nature). Researchers have demonstrated that critical ALS-related proteins are targets for SUMO induction. For example, while SUMOlization of SOD-1 and TDP43 proteins increases the stability of both proteins, FUS has been shown to have a SUMOl E3 ligase activity on the tumor suppressor Ebpl protein (Fei, E., et al., Biochemical and Biophysical Research). FUS itself is also modified by SUMO, and this modification may control the E3 ligase activity of FUS (Oh, S.-M. et al., in addition to its effects under stress conditions, whose dysregulation has also been associated with ALS pathogenesis). It also plays an important role in response mechanisms (Dangoumau, A. et SUMOlation can affect the functions, enzymatic activities, intracellular localization, interaction with other proteins and solubility of proteins; in addition, it can control protein stability. RNF4 is a ubiquitin that only recognizes SUMOylated proteins. RNF4 is an E3 ligase and recognizes SUMOylated proteins and then marks them with ubiquitin, ensuring their degradation by the proteasome. In short, protein SUMOlation, especially the SUM02/3 modification, may be a trigger for protein degradation with the help of RNF4 (Xu, Y. et al., Nature Commun. (2014), 5, 4217; In the past, researchers found that protein-based organelles located in the nucleus of cells, called PML nuclear bodies or granules (PML NBs: BroMyelocytic Leukemia Nuclear Eodies), contain protein that facilitates SUMOlation of proteins and function as catalytic chambers that impose the hyper-SUMOlanin process of the proteins they contain. Some of these hyper-SUMOlated proteins in PML particles are then subjected to poly-ubiquitination by the RNF4 enzyme, which also enters PML particles, and are eventually destroyed (Sahin, U. et al., The Journal of Cell Biology (2014), 204(6) , 931) PML is actually a tumor suppressor protein and has been associated with acute promyelocytic leukemia (APL), a rare type of bone marrow cancer (de The, H. et al., The Journal of Cell Biology (2012), 198(l), 11). In APL disease (t15-17), chromosomal translocation produces a fusion oncoprotein called PML/RARA (fusion oncoprotein consists of PML and RARA: retinoic acid alpha receptor). PML/RARA disrupts the retinoic acid signaling pathway, which is essential for hematopoietic cell differentiation. It also prevents the formation of PML particles. The drug combination of arsenic trioxide and retinoic acid currently used in the treatment of APL triggers the destruction of the PML/RARA oncoprotein, thus it can cure most APL patients at a high rate (de Th'e, H. et al., Nature Reviews Cancer function apoptosis). The p53 signal transduction pathway regulates activities such as aging, redox balance and anti-viral (Sahin, U. et al., The Journal of Pathology (2014), 234(3)). PML protein comes together under oxidation conditions, that is, when it is oxidized, forms the outer shell of PML particles by cross-linking and gives these particles a hollow, spherical form. PML proteins in the outer shell undergo high SUMO modification. Partner proteins interact with the SUMOized PML in the outer shell through the SUMO-interacting motifs (SIM) they carry. As a result of this interaction, the global structure Sahin, U. et al., Nucleus (2014), 5(6), 499). Thanks to the funds collected as a result of the "ice bucket challenge", which has become very popular and attracted attention in recent years, and a worldwide scientific collaboration called the MinE project, a new gene associated with ALS was discovered: NEK] (NIMA-associated kinase 1). Recently Many mutations on NEK have been associated with ALS (Kenna, K.P. et al., Nature). It belongs to a family of protein kinases (NEK family) consisting of members (Monroe, G.R. et al., a member of which is involved in functions ranging from control of the cell cycle to mitosis). (Quarmby, L.M. et al., Journal of cell science) and its activity has been shown to increase significantly, especially as a result of DNA damage. Although NEKl is mostly located in the cytoplasm, it enters the cell nucleus following DNA breakage and damage and colocalizes with other DNA damage repair proteins. NEK1 activates the cell cycle checkpoint kinases CHK] and CHKZ, which are involved in DNA damage repair (Chen, Y. et al., Cell Cycle deficiency causes failures in DNA damage repair and therefore genomic instability). ) has been shown to cause (Chen, Y. et al., Molecular cancer (2011), 10(l), 5). In addition to all these functions, NEKI has also been genetically associated with polycystic kidney disease (Upadhya, P. et al., Proceedings of the National Academy of Sciences of the United States of America. Approximately 3% of all ALS cases are caused by mutations in the NEKI gene. A recent study showed that following induction of DNA damage in motor neuron cells obtained from a patient carrying the NEK1 loss-of-function mutation, there was damage accumulation well above the expected level (Higelin, J. et al., Stem Cell Research (2018), 30, 150). Despite all these developments, it is not known how NEK1 mutations cause ALS and it is not possible to provide treatment for a disease without understanding the underlying mechanism. As a result, there is a great need to reveal the biological mechanism of mutant NEK1 and develop a treatment option for ALS. The present invention is directed to the use of interferon, preferably interferon alfa, in the treatment of a neurodegenerative disease, especially ALS. In one embodiment of the present invention, the ALS discussed is an ALS caused by a mutation in the NEK1 gene (also called NEKI-associated or NEK1-related ALS). In another embodiment of the present invention, the mutation mentioned above includes a premature termination codon (i.e. p.Arg812Ter mutation) at amino acid position 812. In another embodiment of the present invention, the interferon used in the treatment of the above-mentioned disorders is selected from the group consisting of interferon alpha, interferon beta and interferon gamma, preferably interferon alfa. The present invention also relates to a method of treating patients affected by ALS and comprising the steps of: (a) selecting a patient identified as being affected by ALS, (b) administering to the patient a therapeutically effective amount of interferon, preferably interferon alfa. In another embodiment, a method of treating the patient affected by ALS is provided, the The method consists of the following steps: (a) selecting a NEK1 variant ALS patient (i.e., an ALS patient with a mutation in the NEK1 gene), (b) administering to the patient a therapeutically effective amount of interferon, preferably interferon alfa. Brief Description of the Drawings Figure 1. A clipped N. (a) Representative images of WT NEK1 and tNEK1. (b) Agarose gel showing NEK1 bands obtained after PCR amplification and DpnI digestion. Amplified DNA is marked with an arrow. (c) Sequence chromatogram of the mutant plasmid. Checked bases indicate that the point mutation was successfully introduced. (d) The mutation was confirmed at the protein level by anti-GST Western blot (a GST tag was fused to the coding sequence to facilitate detection of NEK1). Figure 2. Subcellular locations of WT and tNEK1 differ. HEK293 cells were transfected with WT or tNEK1 plasmids. Cells were stained using anti-GST antibody (green). Both WT and tNEK1 carry a GST tag. Cell nuclei were stained with DAPI (blue). Red arrows indicate TNEK1 aggregates. Samples were viewed under a confocal microscope. Figure 3. TNEK1 forms aggregate-like structures. HEK transfected with plasmid expressing GST-tNEK1 was stained with. Images were obtained using a confocal microscope. Figure 4. tNEK1 exhibits loss of solubility and accumulates in the insoluble protein fraction. (a) Schematic representation of two separate lysate fractions. (b) HEK293 cells were transfected with the indicated plasmids. Cells were first lysed (soluble fraction) using an NP-4O lysis buffer. Using ultracentrifugation, the supernatant was separated from the pellet, which was then extracted in buffer containing SDS (insoluble fraction). Samples were analyzed by Western blot using anti-GST antibodies to detect GST-tagged WT NEK1 or tNEK1. Figure 5. NEK1 can be SUMO1 by SUMO1, and SUMO1 mutants with disrupted SUMO1 consensus motif have reduced SUMO1. WT NEK] or NEKI mutants with disrupted SUMO1 consensus motifs were transfected into HEK293 cells together with the SUMO1 plasmid. Following immunoprecipitation using anti-GST antibody, the SUMO level on NEK1 was visualized by Western blotting using anti-SUMOI antibody. Figure 6. Excessive (hyper) SUMOlization was detected in the ALS-associated spliced NEK1 mutant (tNEK1). WT NEK1 or tNEK1 were cotransfected with SUMOI, followed by precipitation of urine from cells and immunoblotted with anti-SUMOI antibody. It can be seen that the tNEKl run in the last well is extremely (hyper) SUMOrated. Figure 7. SUMO deficiency does not alter the aggregation propensity of WT or tNEK9. HEK293 cells were transfected with the indicated plasmids. The protocol described in Figure 4 was applied, except that the pellet was extracted in a tainpon containing urea instead of SDS (insoluble fraction). Western blotting was performed with the indicated antibody. NEK1 forms were detected using anti-GST antibody due to their GST tags. Actin was used as a loading control. NEK1- 3KR: NEK1 variant in which all 3 SUMOlation consensus motifs are disrupted (lysines are converted to arginine). ZKR-tNEK: tNEK1 variant in which both SUMOlanina consensus motifs are disrupted. NEK1-3KR or 2KR-tNEK1 cannot undergo SUMO modification (Figure 5). Figure 8. PML NBs selectively call tNEK1, whereas WT do not call NEKPI. HEK293 cells were transfected with plasmids encoding GST-tagged WT or tNEK1. They were stained with anti-GST (green) and anti-PML (red) antibodies for observation under a confocal microscope. Cell nuclei were stained with DAPI (blue). To accelerate and stimulate the formation of PML particles and partner protein uptake, cells were treated with 2 μM arsenic trioxide for 2 h. White arrows indicate complete co-localization Figure 9. PML triggers SUMO modification of tNEK1. (a) HEK293 cells were transfected with the indicated plasinids. The arrows point to tNEKl forms with increased mass, most likely due to SUMOration. PML overexpression triggers SUMOlation of tNEK. (b) GST-tNEK1 and GFP-SUMO1 plasmids were transiently expressed in HEK293 cells. Cells were subjected to immunofluorescence analysis. Anti-GST (green) and anti-PML (red) antibodies were used, and cell nuclei were stained with DAPI (blue). (+) means that two or three different wavelength channels are combined and displayed. Figure 10. PML particles internalize tNEK1 in response to IFN treatment. HEK293 cells were transfected with GST-tagged tNEK1. For immunofluorescence analyses, tNEK1 and PML NBs were stained with anti-GST (green) and anti-PML (red) antibodies, respectively. DAPI (blue) was used for nuclei staining. Arrows indicate full co-localization and arrowheads indicate contact between PML NBs and tNEKl. Full co-localization was only evident when protein degradation was stopped with MG132, indicating that tNEK1 entering PML NBs under normal conditions is destroyed. Figure 11. tNEK1 protein level decreases as a result of IFN treatment. HEK293 cells expressing tNEK1 were treated with AS, IFN and AS/IFN combination for 48 hours (AS: ZiiM, IFN. The arrow on the left indicates that the tNEK1 protein level decreased as a result of IFN treatment. Cells were also treated with ZuM MG132 to stop protein degradation. MG132 It was found to blunt the activity of IFN and prevent the decrease in tNEK1 level, which indicates that IFN causes the degradation of tNEK1 protein in the proteasome. PML Western blot shows that PML production increases in response to IFN treatment (Stadler, M. et al., Oncogene (1995), 11). (l2), 2565) Figure 12. Interferon treatment does not affect NEK1 mRNA level. HEK293 cells expressing NEK1 were treated with IFN for 48 hours, RNA was isolated and qPCR analysis was performed. Figure 13. PML is required for IFN-driven tNEK1 degradation. HEK293 cells were transfected with the indicated siRNAs and pretreated with IFN. Cells were transfected with GST-tagged tNEK1 the next day. Numbers indicate the percentage of tNEK1 protein remaining in treated relative to untreated cells. Arrows (red) indicate the lFN°-induced decrease in tNEK1 protein level, which is blunted (green) in the absence of PML. Figure 14. tNEK1 selectively recruited to PML particles is more sensitive to IFN-driven degradation than WT NEK1. HEK293 cells were transfected with plasmids encoding GST-tagged WT or tNEK1. At 48 h after transfection (and IFN treatment), cells were lysed and Western blotted using the indicated antibodies. IFN: used at 2000 IU/ml. Double wells were run for each group. Figure 15. IFN treatment attenuates tNEK1 aggregation. HEK293 cells were transfected as indicated and treated with IFN. Lysate fractionation was performed using the same protocol as described in Figure 4. IFN treatment greatly reduces the amount of insoluble tNEK1 (arrow). Figure 16. tNEK1 aggregates are eliminated in a proteasome-dependent manner by IFN treatment. HEK293 cells expressing GST-tNEK1 were stained using tNEK1 anti-GST antibody (green) for analyzes only. White arrows indicate cells containing aggregates. Figure 17. SUMOlation is necessary for the degradation of IFN g'id'i'iml'u tNEK1. HEK293 cells were transfected with plasmids encoding either tNEK1 or ZKR-tNEK1 (both GST-tagged) and treated with IFN. ZKR-tNEKl is a non-SUMO variant of tNEKl. While IFN treatment induces tNEK1 degradation (arrow), ZKR-tNEKl degradation cannot be triggered. DETAILED DESCRIPTION OF THE INVENTION The main purpose of this invention is to reveal the underlying mechanism of ALS and to provide treatment of neurodegenerative diseases, especially ALS. For this purpose, a series of experiments were carried out by the owners of the present invention, a new hypothesis was created with the results of each experiment, and other experiments were carried out to test this hypothesis. As stated in the background of the invention, 'An important question in this field is how mutations on functionally and structurally different proteins lead to the same disease (in this case ALS) phenotype. To find an answer to this question, the present inventors aimed to focus on a recently found NEK mutation. The following steps outline the planned path to success in finding a new treatment for ALS. Details of the experiments are included in the experiments and results section of the description. The first aim was to determine whether NEK1 mutants form protein aggregates that can initiate and sustain ALS pathogenesis, respectively. For this purpose, the necessary experiments have been determined and carried out. As a result, while aggregate-like formations were not observed in cells expressing wild-type (WT) NEK1, these structures were observed to be present in cells expressing mutant NEK1. After observing aggregate-like structures in cells expressing truncated NEK1 (mutant NEKI), the second task was to investigate the propensity of tNEK1 to form aggregations. After carefully designing the necessary experiments, the inventors found that tNEK1 suffered a significant loss of solubility, forming distinct cellular aggregates in the process. Following the results of these experiments, as well as considering that defects in SUMOlamination were observed to trigger neurodegenerative diseases, the inventors hypothesized that defects in SUMOlamination of NEK1 (or tNEK1) might contribute to tNEK1 aggregation and loss of solubility. Therefore, the next aim was to test the effect of NEK1 SUMOlation on the solubility of the protein. The results of the experiments showed that; tNEK1, like many other ALS-related proteins, had a high tendency to aggregate, which could produce toxicity in the motor neurons of ALS patients. The inventors of the present invention then reasoned that tNEK1 could also enter PML particles (NBs) because it remains trapped in the cell nucleus. Ainino acid motifs called SUMO-interaction motif (SIM), which have previously been shown to encourage co-proteins to enter PML particles, are also present on NEKI. Recently, PML particles have also been shown to selectively recall proteins that are misfolded and tend to lose solubility. All this makes tNEK1 a strong candidate for the PML partner protein. Moreover, the inventors have previously shown that tNEK1 is hyperSUMOrated, just like the phenomenon observed in other PML partner proteins. After detailed analysis, PML particles selectively called the mutant (truncated) tNEK1 protein. With the addition of the finding that tNEK1 was recruited to PML particles, especially under oxidation conditions, the inventors decided to investigate whether tNEK1 hyper-SUMOlation was truly a PML-dependent process. After many experiments, the obtained data supported the hypothesis that PML particles recruit tNEK1 and initiate hyper-SUMOlation in situ. Finally, after uncovering the underlying pathogenesis mechanism of the disease, the inventors of the present invention focused on finding a molecule that could treat ALS. As a result, interferon has been found to be effective in the treatment of ALS, especially in ALS caused by the NEK1 mutation, and more specifically in ALS caused by the p.Arg812Ter mutation. Interferons are cytokines that show antiviral effects. All interferons function as ligands of specific cell surface receptors and have antiviral effects. It regulates the transcription of hundreds of interferon-stimulated genes that encode proteins with antitumor, antimicrobial and immunomodulatory effects. There are three main types of interferon: interferon alpha, interferon beta and interferon gamma. More specifically, it provides a molecule for the treatment of ALS caused by a mutation in the NEKI gene. The present invention further provides a method of treating an ALS patient exhibiting a) NEK1 mutation and b) tNEK1 protein expression; . This method involves administering to the patient a therapeutically effective amount of interferon, preferably interferon alfa. EXPERIMENTS AND RESULTS 1. MATERIALS 1.1. Equipment Equipment used in experiments for the purposes of this invention: Agarose Gel Electrophoresis System (EASY-CAST, Thermo Fisher, USA), Autoclaves, Carbon Dioxide Tank, Cell Culture Dishes, Cell Culture Incubator, Cell Scraper, Centrifuges (Ultracentrifuge JZMC, Beckman, USA VWR CTlSRE , Japan Allegra Cell, Bio-Rad, USA), Thermal Blocks (Block heater aiialog, VWR, USA), Ice Machine, Inverted Microscope (Zl Axio Observer, Zeiss, USA), Laboratory Flasks, Laminar Flow Cabinet, Magnetic Stirrer, Microfuge Tubes, Micropipettes , Micropipette Tips, Microscopes (Inverted Microscope, Nikon, Eclipse TSlOO, Netherlands, Fluorescence Microscope, Observer.Zl, Zeiss, Germany), Microwave Oven, Multi-Well Dishes, Nitrocellulose Membrane (Amersham, GE Life Sciences, England), Oven, PCR Tubes (, Pipettor, Pipette Tips (Bulk), Pipette Tips (filtered), Power Supply, Real-Time Quantitative PCR System (Bioneer Exicycler, Republic of Korea), Refrigerators, Rotor, Serological Pipettes, Shakers (Orbital shaker ANALOG, VWR, USA) , Softwares (Quantity One, Bio-Rad, ITALY Image] , Image Analysis Software, NIH, USA , Thermal Cycler, Vortex, Water Purification, Water Purification System, Watmann Filter Paper-Extra Thick. HEK293 cells. 1.3. Plasmids and Primers Table 1.1: Plasmids. Structure Origin Spine Flag-PML Addgene #59742 pRKS 1V/pRK5 pEBG NEKl tvl University of Dundee, DU41359 pEBG Hugues de The, College de France . _ GFP-SUMO] Provided by Unknown. His-SUMO] Provided by the Unknown. Table 1.2: Sequences of Primers and siRNAs. Oligo ID Sequence (5'-3') Application p.Arg8 12Ter- CTACAACTGAATGACATACAGTG SYM p.Arg812Ter- R CACTGTATGTCATTCAGTTGTAG SYM NEK] -qPCR- F AATGCTCAGAAAGGCGTTTTGT qPCR N EK] -qPC R R GGGGTCCCTATGCAAGTTCG qPCR GGAGCGAGATCCCTCCAAAAT qPCR GGCTGTTGTCATACTTCTCATGG qPCR si CTRL UUCUCCGAACGUGUCACGUTT silencing si PML AAGAGTCGGCCGACTTCTGGT silencing NEKl . 1 -F SYM NEKl . 1 -R SYM NEKl .2-F SYM NEKl .2-R CCATATATTTCTCTTCTCCATTTAGC TTC SYM NEKl .3-F CAGATGTCATTGAGACTGGAAGGAAAT SYM NEK] .3-R ATTTCCTTCCAGTCTCAATGACATCTG SYM 1.4. General Kits and Enzymes Table 1. 3: Kits and Enzymes. Name Vendor Complete Mini Protease Inhibitor Cocktail Roche, Switzerland DirectzolTM RNA MiniPrep Plus Zymo Research, USA DNA Ladder (1 kb) NEB, USA Dpnl endonuclease NEB, USA ECL-Fenlto Thermo, USA ECL-Pico Thermo, USA PageRuler Dyed Protein Ladder Thermo, USA Q5 High Quality DNA Polylinease NEB, USA SensiFAsrTM cDNA Synthesis Kit Bioline, UK SensiFASTTM SYBR® No-ROX Kit Bioline, UK ZymoPURETM MaxiPrep Kits Zymo Research, USA ZymoPURETM MidiPrep Kits Zymo Research, USA ZymoPURETM MiniPrep Kits Zymo Research, US 1.5 Chemicals Acetic Acid, Acrylamide, Agar, Agarose, Ammonium Persulfate (APS), Ampicillin, Arsenic, ß-Mercaptoethanol, Sigir Serum Albumin (SSA), Bromophenol Blue, Calcium chloride dehydrate, Cycloheximide, DAPI, Disodium hydrogen phosphate, DMSO, Dulbecco's Modified Eagles Culture Broth, EDTA, Ethanol, Ethidium Bromide, Fetal Sigir Serum (FSS), FluoroshieldTM, Glycerol, Glycine, HEPES buffered saline solution (HBS), Hydrochloric acid, HiPerFect Transfection agent, Interferon alpha, Kanamycin, LB fluid , Methanol, MG132, Paraformaldehyde, Penicillin/Streptomycin (100X), Potassium chloride, Potassium dihydrogen phosphate, Sodium Chloride (NaCl), Sodium Dodecyl Sulphate (SDS), Sodium Hydroxide, TEMED, Tris-BaZ , Triton X-100, Tween 20, Ure. 1.6 . Western Blotting Buffers and Antibodies NP-4O buffer was supplemented with protease inhibitor cocktail before each use. Urea tainpon was divided into small volumes and stored at -80 °C. As an exception, mouse PML antibody was prepared in 5% SSA instead of skim milk in TBS-T. Unless specified, all antibodies in the table below were used for western blotting and at the concentrations indicated. Table 1.4: Western Blot Solutions and Buffers. Solution/Buffer Content 137 mM NaCl 2 mM EDTA 50 mM Tris-HCl, pH 7.4 9 M Urea 2.5 mM EDTA Urea Buffer 2.5 inM EGTA 2001nM TrisHCl, pH 6.8 50 mM EDTA 375 mM TrisHCl pH 8 .8 0/0 8 Separation Gel 0.1% (w/v) SDS Acrylamide: Bisacrylamide (8% 21/11) NP-40 Buffer 4X Laemmli Buffer 0.125 mM TrisHCl pH 6.8 Acrylamide.' Bisacrylamide (4% 21/11) 10X SDS Running Buffer 10X Transfer Buffer 1)( Transfer Buffer Tween-20.1i IX Tris Buffered Saline (TBS-T) 50 mM Tris HCl, pH 7.4 150 mM NaCl Western B10t Blocking Solution Primary] Antibody Solution Secondary Antibody Solution Table 1.5: Antibodies. Antibody Vendor Source Dilution Cell Signalling Technologies, Aktin Tavsan 1: 1000 Cell Signaling Technologies, Flag Mouse 1: 1000 Cell Signaling Technologies, 1000: 1 GST Mouse 1 G, Cell Si. nallin Technolo ies, Mouse IgG, . Goat 1: Alexa Fluor 546 PML Santa Cruz Biotechnology, USA Mouse Rabbit IgG, . Thermo Fisher Scientific, USA Goat 1: Alexa Fluor 488 Tubulin Santa Cruz Biotechnology, USA Mouse 121000 2. METHODS To produce mutated plasmids, targeted mutagenesis was performed. Primers were purchased from Macrogen. Polymerase chain reaction (PCR) components and PCR conditions are shown in the tables below. 250-500 ng of template DNA was used for amplification. After PCR, the DNA template was digested using DpnI (6 al Cut Smart Tainpo + 1 al DpnI enzyme) for 3 hours at 37 oC. Then, 1 al of each sample was transformed into E. coli D1-I5alpha strain. For transformation, competent cells were incubated on ice for 30 min. This process was followed by a heat shock at 42 °C for 45 seconds. Afterwards, the samples were incubated on ice for 2 minutes. Then, 950 μl of LB was added to the matched cells, and these cells were incubated at 37 °C with shaking for 1 h. After incubation, competent cells were centrifuged and 900 µl of LB were discarded. The remaining 100 μl was resuspended and spread onto plates containing the appropriate antibiotic. These plates were incubated at 37 °C overnight. The next day, single colonies were selected and incubated with shaking at 37 °C overnight in LB containing appropriate antibiotics. Plasmid DNA was then isolated using the ZymoPURE Plasmid Miniprep Kit (Zymo Research, USA) according to the manufacturer's recommendations. Finally, the samples were sent to Macrogen for sequencing to confirm the mutations that had been successfully created. Table 2.1: PCR Components. Component Volume (µl) Q5 reaction buffer (NEB) 5 dNTP (10 mM) 1 Forward primer (10 µM) 1.25 Reverse primer (10 HM) 1.25 template DNA l Q5 high quality DNA polymerase (NEB) l dH2O 31 .5 Grand Total 50 Table 2.2: PCR Conditions. Temperature oC Time Cycle 98 30 sec 1 98 10 sec 55-65 30 sec 25 72 5 min 72 2 min 1 2 .2. Cell Culture was cultured in DMEM (Gibco, Thermo Fisher Scientific, USA) supplemented with HEK and Penicillin (100 U/mL)-Streptomycin (100 ug/mL) (Lonza, Switzerland). Cells were grown in 100 mm cell culture dishes at 37 0C in a humidified atmosphere containing 5% CO2. These cells were passaged every 2 or 3 days. 2. 3. Plasmid DNA and siRNA Transfections The day before plasmid DNA transfection, HEK293 cells were planted in an amount that would cover 50-70% of the culture vessel during transfection. 500 ng or 1 µg of plasmid DNA was grown in culture vessels with 12 or 6 wells, respectively. To transfect cells into 12-well plates, first add an appropriate amount of plasmid DNA. dissolved in it. Then, 30.5 m1 of 2M CaClz cooled in ice was added to the mixture. Following this, it was added to the mixture drop by drop. After 10 min of incubation, the transfection mixture was gently distributed into the cells. For siRNA transfection, HEK293 cells were planted at 105 cells per well. At the same time, 20 nM (1.2 pl) siRNA was dissolved in culture broth without FSS and antibiotics in a total volume of 100 μl. Then, 1.5 μl of HiPerFect transfection agent (Qiagen, Germany) was added to the solution, this mixture was mixed by vortex and left at room temperature for 10 min. After incubation, the transfection mixture was added dropwise to the cells. DNA transfections were performed the day after siRNA transfection. 2 .4. Separation of Proteins into Soluble and Insoluble Fractions To determine the sedimentation tendencies of wild and mutant NEK1 proteins, these plasmids were expressed in HEK293 cells. At 48 h after transfection, first, cells were washed with PBS and then detached from the container by trypsin digestion. After collecting the cells, these cells were centrifuged at 2000 rpm for 2 min to get rid of trypsin, then washed once with PBS. The supernatant was then collected after centrifugation on ice for 30 minutes. This collected part was mixed with 4x laemmli tainpon and boiled for 10 minutes at 95 °C. The remaining lower part was either lysed (extracted) by incubating at 45 °C for 40 minutes using 75 μl of 9M urea buffer or with 100 μl of 2x laemmli buffer. stirred and boiled at 95 DC for 20 minutes. Upon lysis, the urea solution was mixed with 25 μl of 4x laernin buffer and boiled at 95 °C for 10 minutes. Finally, the samples were analyzed using the western blot technique. 2.5. Treatments Interferon was ordered from Roche. Arsenic and MG132 were purchased from Sigma Aldrich and Calbiochem, respectively. Interferon was used at 2000 IU/ml. MG132 was used to treat cells overnight or at 10 μM. 2.6. Cell Lysis and Western Blot Cell lysis was performed 48 hours after transfection by using 2x Laemmli buffer, the amount of which varied between 200-400 μl, to lyse the cells grown in 12 mm culture dishes. After incubation with shaking, samples were collected and boiled at 95 0C for 1 minute. If the samples were not used on the same day, they were stored in a -20 0C cabinet. Samples were centrifuged at highest speed for 10 minutes before being loaded onto the gel. 20 of each sample were loaded onto a 1.11 SDS-PAGE gel. Proteins were heated first at 70 V and then at 150 V. After the separation phase, the proteins were transferred to a nitrocellulose membrane (Amersham, GE Life Sciences, England) by wet transfer at 100 V for 3 hours. Then, the membranes were transferred to 1 hour at room temperature. After blocking with 5% nonfat milk dissolved in TBS-T, the membranes were incubated on the rotor overnight at 4 °C with primary antibodies prepared in blocking solution for 5 minutes with TBS-T. They were washed 3 times. Then, they were incubated with secondary antibodies prepared in blocking solution at room temperature for 1 hour. Finally, the membranes were washed 3 times with TBS-T for 5 minutes to remove non-specific bindings in the membranes. Fisher, USA) and protein bands were visualized using a chemiluminescence visualization system (Gbox Chemi, Syngene, United Kingdom). To analyze protein data, coverslips were placed in 12 mm culture dishes for genetools and immunofluorescence experiments, and then HEK293 cells were seeded on these coverslips. Cells were fixed using cold 4% PFA dissolved in PBS for 20 min at room temperature. After 2 washing steps with cold PBS, cells were incubated with PBS containing 0.25% Triton X-100 for 15 min to permeabilize the cell membranes. Then the cells were washed 3 times with PBS for 5 min. Cells were incubated for 1 hour at room temperature with primary antibodies prepared in blocking solution in a chamber dissolved in PBS-T for 1 hour at room temperature. When antibody incubation was finished, cells were first washed 3 times with PBS-T for 5 minutes. Cells were then incubated with fluorescent dye-conjugated secondary antibodies in block solution for 1 hour at room temperature in the dark. After secondary antibody incubation, washing was performed 3 times with PBS-T for 5 min. Next, cells were incubated with DAPI (1 μg/ml) for 3 min, followed by washing with PBS. Then, the coverslips were sealed to the slides using fluoroshield liquid. Finally, the lamellae were sealed with nail polish. Images were acquired using a confocal microscope (Leica TCS SP8, USA) and analyzed using the Image J and LASX software packages. 2 .8. RNA Isolation, cDNA Synthesis and qPCR To isolate RNA, Direct-zolTM RNA MiniPrep Plus (Zymo Research, USA) kit was used. Briefly, cells were washed once with cold PBS and collected into 1 ml of cold PBS. They were then transferred to 1.5 ml tubes and centrifuged at 3000 g for 3 min. Afterwards, the supernatant was removed and the cells were resuspended in 300 µl Tri agent. After suspension, an equal amount of ethanol was added to the mixture. This mixture was transferred to a spin column and centrifuged at 12000 g for 30 seconds. The spin column was then washed with 400 111 RNA wash buffer and DNaseI treated to get rid of DNA residues. Then, 3 washing steps were performed with RNA wash buffers and RNA was obtained using DNase/RNase-free water. After elution, a total of 1 μg of RNA was converted to cDNA using the SensiFASTTM cDNA Synthesis Kit (Bioline, United Kingdom). The following tables (Tables 2.3 and 2.4) describe the quantities of the components and PCR conditions. After PCR, cDNA was diluted 1:5-fold. SensiFASTTM SYBR ® No-ROX Kit (Bioline, United Kingdom) was used for qPCR analysis. PCR conditions and components were performed using the qPCR system in the tables below. Data were analyzed using the ZM& method. Table 2.3: cDNA Synthesis Components. Component Volume (pl) total RNA (1 Mg) variable 5x TransAmp Buffer 4 Reverse transcriptase 1 DNase/RNase-free water up to 20 Grand Total 20 Table 2.4: cDNA Synthesis PCR Conditions. Temperature oC Time Cycle 10 min 1 42 15 min 1 48 15 min 1 85 5 min 1 Table 2.5: qPCR Components. Component Volume (nl) SensiFast SYBR Mastermix (Bioline) 5 Forward primer (10 µM) 0.25 Reverse primer (10 HM) 0.25 Diluted cDNA l Grand Total 10 Table 2.6: qPCR Conditions. Temperature 0C Time Cycle 95 2 min 1 95 5 sec 6] 10 sec 40 72 10 sec 2.9. Immunoprecipitation (IN) assay: Cells transfected with NEKI or tNEK1 together with SUMO1 or SUMO2 plasmids were washed with solution containing 10 mM N-ethylmaleimide (NEM) in PBS. Cells; After lysis with buffer containing 2% SDS, 20 mM NEM, protease inhibitor cocktail (PIK) and 50 mM Tris pH: 8, sonication was applied. Lysates were then diluted 10-fold with RIPA buffer containing 250 mM NaCl, 50 mM Tris pH: 8, 1% NP-40, and PIK. Lysates were first clarified by minute A/G bead incubation at room temperature. Purified lysates were incubated with GST antibody for 2 hours at 4 °C. After incubation of the lysate, antibody, and beads, the beads were washed 5 times with RIPA buffer and once with PBS and eluted with laeinmli buffer. Samples analyzed by Western blot 3. EXPERIMENTS AND RESULTS To elucidate how NEK1 mutations drive ALS pathogenesis, one of the most common NEK1 variants found in ALS patients was generated. This mutant contains a premature termination/stop codon at amino acid position 812 and produces a protein called truncated NEK1 (tNEK1) by its inventors (Brenner, D., K. Et. al. "NEK1 mutations in familia] amyotrophic lateral sclerosis", Brain, Vol. 139, No. 5, pp. e28-e28, 5 2016) (Figure 1a). In order to create this ALS-related NEK1 mutant, targeted inutagenesis was performed on wild type (WT: Wild type) NEK1 using appropriate primers (2.1. targeted mutagenesis), as described in detail under the methods heading above. After the polymerase chain reaction, cleavage was performed with DpnI endonuclease enzyme to destroy WT NEK1. PCR amplification and elimination of the WT plasmid was confirmed by agarose gel electrophoresis (Figure 1b), and insertion of the desired mutation was confirmed by sequencing (Figure 10). Finally, the mutation was also confirmed at the protein level. For this, HEK293 cells were transfected with plasmids encoding WT or tNEK1. Lysates were collected and Western blotted 48 h after transfection. As expected, WT NEK1 appeared to be around 190 kDa, whereas the tNEK1 protein signal was observed to be around 130 kDa due to truncation (Figure 1d). 3.2. Subcellular localization of WT and spliced NEK1: tNEK1 is confined to the nucleus. The first step to investigate the effect of this ALS-associated NEK1 mutation is to examine the subcellular localization of the mutant protein. For this, HEK293 cells were transfected with plasmids encoding WT or tNEK1. At 24 hours after transfection, cells were fixed and WT and tNEK1 proteins were visualized under immunofluorescence and confocal microscopy using anti-GST antibody (both WT and tNEK1 variant were tagged with the GST peptide at their N-terminal ends). The results showed a dramatic difference in the localization of the two proteins: WT NEK1 is predominantly in the cytoplasm, while tNEK1 is confined to the nucleus, which can be explained by the loss (due to truncation) of two nuclear export signals located at the C-terminal end of the protein (Figure 2). Interestingly, aggregate-like structures were observed in tNEK1-expressing cells, all of which were seen within the cell nucleus. Similar structures were not observed in cells expressing WT NEK1. 3.3. tNEK1 precipitates significantly and forms aggregates. In immunofluorescence experiments, aggregate-like structures were detected in the nuclei of cells expressing tNEK1, but these structures were not detected in cells expressing WT NEK1. For a more detailed examination, HEK293 cells expressing tNEK1 were stained with anti-GST antibody 24 hours after transfection, and 2D and 3D images were obtained using a confocal microscope (Figure 3). The results confirmed the presence of aggregate structures in cells expressing tNEK1. The number and size of the aggregates, whose boundaries are quite distinct, vary in different cells and are found only in the nucleus, which is consistent with the confinement of tNEK1 in the nucleus. tNEK1 aggregates were not seen in all cells, indicating that aggregate formation is a dynamic process. The potential of the aggregates 3.4. tNEK1 protein loses its solubility. In order to investigate the aggregation tendency of tNEK1 in more detail, HEK293 cells were transfected with WT or tNEK1 encoding plasinids. The cells were then blasted with NP-4O, a relatively weak, nonionic detergent. NP-40 extracts soluble proteins but is ineffective at dissolving protein aggregates. Then, insoluble proteins were precipitated by ultracentrifugation, followed by extraction in SDS, a strong and ionic detergent (= insoluble fraction). Fractions containing soluble (extracted in NP-40) or insoluble (extracted in SDS, insoluble with NP-40) proteins were then run on SDS-PAGE, and the presence of WT NEK1 or tNEK1 proteins in these fractions was then detected by Western blotting. was done. The results showed that more than half of the tNEK1 protein was found in the insoluble fraction, while only an insignificant and very small amount of WT NEK1 was found in this fraction (Figure 4). Almost all of the WT NEK1 was seen in the (soluble) fraction extracted with NP-40. In conclusion, both immunofluorescence analyzes and the biochemical extraction experiments mentioned above indicated that tNEK1 suffered a serious loss of solubility and formed intracellular aggregates. 3. 5. tNEKl is exposed to excessive (hyper) SUMOlation. It has previously been shown that SUMOlation colocalizes with aggregate-like structures formed by proteins associated with neurodegeneration. In addition, some important proteins associated with neurodegeneration, such as huntingtin, ataxin-1, tau and alpha-synuclein, are also known to be SUMOregulated. Therefore, the inventors examined the potential connection between "SUMOization" and "tNEK1-related ALS pathogenesis". The researchers first wanted to show that the NEK protein was indeed modified by SUMO. There is no previous data available on this subject. HEK293 cells were transfected with plasmids encoding GST-NEK1 either alone or together with SUMO1-GFP. NEK1 protein was precipitated from the cell lysate using GST antibody (immunoprecipitation/immunoprecipitation: IP), and its SUMO modification was detected by Western blotting using anti-SUMO1 or anti-SUMO2 antibodies (n=3). Figure 5 shows that GST-NEK1 is SUMO1ed by SUMO1. Since it is known that the SUMO peptide covalently binds to lysines located within a specific consensus motif on target proteins, researchers performed in silico analyzes on NEK1 and identified 3 lysines with the potential to be modified with SUMO: K385, K465, and K9l4. By targeted mutagenesis, all 3 lysines were converted to arginine and their SUMOization potential was eliminated. From this stage on, NEKI, NEK] carrying the K385R mutation. one; NEK1, NEK1.2, carrying the K465R mutation; NEK1 carrying the K914R mutation was called NEK1.3, and NEK1 carrying all 3 mutations together was called NEK1.3KR. In order to determine the SUMOlation levels of these mutants, it was determined that each of the motifs mentioned above contributed to the SUMOlation of NEKl, and it was shown that the SUMOlation of NEKI was abolished as a result of the disruption of all 3 motifs. Following the detection that the NEKl protein was SUMOrated, it was also tested whether ALS-related mutations changed the SUMOlation of NEKl. For this purpose, the above-mentioned IP and then SUMOI Western blot tests were performed. Interestingly, it was shown that the ALS-related tNEK1 mutant was exposed to a much more severe hyper-SUMOlanin (excessive SUMOization) than normal (Figure 6). 3.6. Investigation of the effect of SUMOlation on the solubility of NEK. Therefore, the solubility of target proteins is greatly increased as a result of modification with SUMO. In the laboratory, during recombinant protein expression in bacteria, SUMO can be fused (fused) to related proteins to increase their solubility. As noted above, SUMOlation defects have been observed in proteins that lead to many neurodegenerative diseases, including ALS. The inventors of the present invention hypothesized that a defect in the SUMOlation of NEK1 (or tNEK1) might contribute to the loss of solubility and aggregation of tNEK1. To test the effect of NEK1 SUMOlation on the solubility of this protein, non-SUMOylated forms of both WT and tNEK1 were generated by converting their consensus target lysines to arginines (Figure 5). The lysine-arginine conversion SUMOlation preserves the positive charge on the consensus motif, does not affect the correct folding and function of the protein, but prevents SUMO from covalently binding to this motif. Both SUMOlable and non-SUMOlable WT or tNEK1 proteins were expressed in HEK293 cells. As mentioned above, soluble or insoluble NEK1 and tNEK1 protein ratios were analyzed by NP-4O or SDS extraction. This time, urea was used instead of SDS in order to better extract the insoluble forms of tNEKl. As seen in previous experiments, while WT NEK1 is found in the completely soluble (NP-40 extracted) fraction, tNEKl is mostly extracted with SDS or urea, that is, there is no difference in solubility between the WT or tNEKl forms that cannot be SUMOrated. These results indicate that tNEKl aggregation is not due to a defect in SUMOlation. This indicates that it is probably due to misfolding of the protein due to the ciliary at the C-terminal end. As a result, it has been shown by the inventors that tNEK1 has a tendency to accumulate and aggregate that is strong enough to cause toxicity in the motor neurons of ALS patients. 3.7. tNEK1 enters PML granules The inventors of the present invention thought that tNEK1 could be a PML partner protein that enters PML granules, based on the following observations: l) trapping of tNEK1 within the cell nucleus, 2) presence of SUMO-interaction motifs (SIM) on tNEK1. (it has previously been shown that these motifs are elements that transport proteins into PML particles), 3) PML particles can selectively recognize misfolded proteins, which strengthens the possibility that tNEK1, which is likely to be misfolded due to clipping, is a PML partner, 4) tNEK1 is able to selectively recognize other proteins. To test whether tNEK1 enters PML granules by hyperSUMOlation (Figures 5 and 6), tNEK1 (or WT NEKI as a control) was transiently expressed in HEK293 cells and its intracellular localization. analyzed in detail (under a confocal microscope). As a result of these analyses, a partial co-localization was observed between tNEK1 and PML particles. Arsenic trioxide (arsenic, AS) treatment triggers the formation of PML particles and the recruitment of partner proteins. In cells given 2 µM AS for two hours, tNEKP was observed to be completely within the PML particles (full co-localization). WT NEKl with 11 SIM motifs was not detected in the particles under these conditions. These results indicate that PML particles selectively call for the spliced NEK1 mutant (tNEK1) (Figure 8), most likely because both tNEK1 is trapped in the cell nucleus and possibly because it is misfolded, triggering SUMOlation. Following the results from previous experiments, the present The inventors investigated whether tNEK1 hyper-SUMOlation is truly a PML-dependent process. For this purpose, the effect of PML overexpression on tNEK1 SUMOlation was examined, as SUMOylated tNEK1 forms can be easily detected covalently to the target protein on SDS-PAGE. Each bound SUMO peptide increases the mass of the protein by 18 kDa. tNEK1, SUMO1 and PML proteins were transiently expressed by transfection in HEK293 cells. 48 hours after transfection, the cells were separated on SDS-PAGE and then analyzed by Western blot. In conditions where in was overexpressed together with tNEK1, high molecular mass forms of tNEK1 emerged, possibly representing SUMO-modified tNEK1. The emergence of multiple bands (ladder pattern) also indicates that tNEK1 is probably polySUMO (Figure 9a). That is, PML overexpression leads to tNEK SUMO. In addition, to shed light on the intracellular localization of SUMOylated tNEK1, tNEK1 was overexpressed along with GFP-SUMOI in HEK293 cells and tNEK1/GFP-SUMOI co-localization was analyzed. Immunofluorescence method was used for this analysis. Remarkably, tNEK1 was seen co-localized with GF β-SUMOl only in PML particles (Figure 9b). This indicates that SUMOylated tNEK is present within PML particles. Consequently, these data indicate that PML particles recruit tNEK into their interior (in situ). ) indicates that this leads to excessive (hyper) SUMOlation of the protein. 3.9. Entry of tNEKP into PML particles as a result of IFN treatment. After it was shown that tNEKP enters PML particles, especially under oxidative stress conditions caused by arsenic treatment, researchers developed another PML particle-inducing drug/molecule. They tested interferon alpha[beta]1 (IFN) after HEK293 cells were transfected with tNEK1 and then treated with IFN for 24 hours. After the cells were fixed, tNEK1 and PML particles were detected by immunofluorescence. In control cells, tNEK was observed to touch the PML particles from the outside, complete co-localization was not detected. As a result of the IFN treatment, although the contact between tNEK1 and PML particles increased, tNEKl could be detected within the particles. Following this, the next hypothesis of the inventors was that tNEK entered the PML particles. It was found to be cleaved in situ (tNEK is cleaved as soon as it enters the particles, making detection of the protein within these particles impossible). This phenomenon has been demonstrated for many PML partner proteins, such as the HTLV-1 Tax precursor protein. Therefore, the inventors investigated the cells for both IFN and proteasomal degradation. Remarkably, as a result of the IFN/MG132 combination treatment, tNEK1 was observed in PML particles, and it was determined that tNEK accumulated in PML particles under these conditions and showed complete co-localization. According to these results, IFN indeed increases tNEK1 recruitment in PML particles and causes their final destruction (Figure 10). MG132 treatment alone also slightly increased tNEK1 signals in PML NB particles. This result indicates that there is a basal level of tNEK1 degradation in PML particles even in the absence of IFN treatment, which can be strongly induced by IFN treatment. 3.10. Induction of degradation of tNEK1 by IFN So far, the inventors of the present invention have shown that tNEK1, which has a tendency to aggregate, enters PML granules. TNEKl entering the particles is destroyed as a result of hyper-SUMOization, probably catalyzed by PML. Although some tNEK1 is degraded in this way at the basal level in quiescent cells under normal conditions, this process can be dramatically induced by IFN treatment. To investigate the degradation of tNEK in more detail, HEK293 cells expressing tNEK were treated with AS and IFN. This time, tNEK1 protein level was monitored by Western blot. Indeed, it was observed that the tNEK protein level decreased significantly as a result of IFN treatment (Figure 11). AS treatment did not trigger tNEK1 degradation. The increase in PML protein expression and PML granule formation occurs within approximately 24 hours following IFN treatment. qPCR analyzes showed that IFN treatment did not cause any decrease in NEK1 mRNA level and confirmed that lFN affected the stability of tNEK1 protein, but not tNEK1 mRNA and gene expression (WT and tNEK1 expressions are expressed on the same plasmid type and from the same promoter) (Figure 12) . In conclusion, these data demonstrate that IFN inuainelein triggers the proteasomal degradation of the ALS-associated tNEK protein. 3.11. IFN-driven tNEK1 degradation is a PML-dependent process. The next step was to test whether the IFN-inducible tNEK1 degradation was indeed facilitated by PML. For this purpose, PML expression was silenced with siRNA in HEK293 cells expressing tNEK1. It was observed that in the absence of PML, the effect of IFN was almost completely lost, that is, tNEK1 destruction did not occur as a result of IFN treatment (Figure 13). These data support that tNEK1 is indeed destroyed in situ by PML within PML particles and that this degradation is induced by IFN. At the same time, it was observed that IFN treatment destroyed tNEK1 much more efficiently than WT NEK1 (Figure 14). In other words, it was shown that WT NEK1, which does not normally enter PML particles, was not as sensitive to IFN treatment as tNEK1. 3.12 . IFN treatment/treatment attenuates tNEK1 aggregation. If it causes edema, it may also reduce aggregation caused by tNEK1. To test this exciting idea, the inventors analyzed NP-40-extracted (soluble) or urea-extracted (insoluble) protein fractions as indicated above from HEK293 cells treated (or not) with IFN and determined that tNEK1 Examined the solubility level. Remarkably, as a result of IFN treatment, tNEK1 protein was almost not detected in the insoluble (i.e., urea-extracted) fraction. These data indicate that IFN treatment triggers the degradation of the tNEK1 mutant and does not allow its aggregation (Figure 15). Effect of IFN treatment on tNEK1 aggregates So far, the inventors have shown that IFN treatment reduces the amount of insoluble tNEK1 by triggering the degradation of aggregation-prone tNEK1. The next step was to examine the fate of tNEK1 aggregates in cells treated with IFN. GST-tNEK1 was also expressed in HEK293 cells. After treatment, they were stained with anti-GST antibody for immunofluorescence analysis and the previously mentioned aggregate-like structures were examined (shown in Figure 3). Remarkably, simultaneous treatment with MG132, which blocks tNEK1 aggregates, blunted the effect of IFN[deg.] All these results support that the aggregate protein can prevent the aggregation of IFN guided proteazomal destruction (Figure 3.14. IFN guided Tnekl Difference It has been shown to be dependent on PML (and therefore on PML particles) and the proteasome. These results indicate that the "PML/SUMO/RNF4-mediated protein degradation mechanism" described in the introduction probably constitutes the mechanistic basis of the IFN-driven tNEK1 degradation. Finally, this is the mechanism of tNEK1 SUMOlation. To test its essential role in the process, the inventors expressed the non-SUMOlating form of tNEK1 (the 2KR-tNEK1 mutant mentioned in Figure 7 above) in HEK293 cells. Indeed, as shown in Figure 173, while IFN treatment reduced the tNEK1 protein level, it did not cause a significant change in the 2KR-tNEK1 protein level. In conclusion, these data suggest that SUMOlation of tNEK1 is a mechanism for the IFN-driven degradation of this protein within PML particles. TR

Claims (1)

1.CLAIMS. Interferon for use in the treatment of a neurodegenerative disorder. . The interferon for use according to claim 1, wherein the neurodegenerative disorder is amyotrophic lateral sclerosis (ALS). . Interferon for use according to claim Z, wherein ALS is caused by a mutation in NEK1. . Interferon for use according to claim 3, wherein the mutation involves a premature stop codon at amino acid position 812. . Interferon for use according to claims 1 to 4, wherein the interferon is selected from the group consisting of interferon-alpha, interferon-beta and interferon-gamma. . Interferon for use according to claim 5, wherein the interferon is interferon-alpha. . Interferon for use according to claims 2 to 6, wherein the ALS exhibits inhibited NEKI (tNEK1) protein expression. . A method of treating a patient with ALS exhibiting a NEK1 mutation, administering to the patient a therapeutically effective amount of 9. A method according to claim 8, wherein the mutation comprises a premature stop codon at amino acid position 812. 10. A method according to claim 8 or 9, wherein the interferon is administered in a therapeutically effective amount upon detection of a mutation in the NEK1 gene. TR TR TR