Targeting ALS-FUS aggregation with proteasome inhibitors

Abstract

Provided are methods of treating a subject having a disease characterized by accumulation of FUS -associated aggregates by administering to the subject a therapeutically effective amount of a proteasome inhibitor, an agent which upregulates SAT1, or an agent which upregulates spermine, thereby treating the subject. Also provided are proteasome inhibitors, an agent which upregulates SAT1, or an agent which upregulates spermine for use in treating a subject having a disease characterized by accumulation of FUS-associated aggregates, and methods of reducing accumulation of FUS-associated aggregates in cells of a subject, the method comprising contacting the cells of the subject with a proteasome inhibitor an agent which upregulates SAT1, or an agent which upregulates spermine, thereby reducing the accumulation of FUS-associated aggregates in the cells of the subject.

Description

[0001] TARGETING ALS-FUS AGGREGATION WITH PROTEASOME INHIBITORS

[0002] RELATED APPLICATION / S

[0003] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 545,225 filed on October 23, 2023, the contents of which are incorporated herein by reference in their entirety.

[0004] SEQUENCE LISTING STATEMENT

[0005] The ASCII file, entitled 101490.xml, created on October 22, 2024, comprising 44,561 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0006] FIELD AND BACKGROUND OF THE INVENTION

[0007] The present invention, in some embodiments thereof, relates to methods of reducing FUS- associated aggregates in the nervous system and, more particularly, but not exclusively, to a methods of treating ALS and FTD.

[0008] Amyotrophic lateral sclerosis, ALS, a devastating fatal disease affecting motor neurons, has no effective treatment or cure. The growing diversity of causative proteins in ALS makes the characterization of mechanisms and pathways involved highly challenging. To date, many genes have been implicated in ALS, the most well-known of which are SOD-1 (superoxide dismutase 1), C9orf72, TDP-43 (TAR DNA-binding protein) and FUS (FUS RNA binding protein).

[0009] A unifying hallmark of many of the ALS-causative proteins, as is true for many other neurodegenerative diseases (NDs), is their misfolding and aggregation in affected neurons. Accordingly, targeting the process of aggregation has been proposed as a means to slow down the course of ALS. Aggregates driven by distinct proteins can differ in their biophysical properties, or their aggregation process. Many ALS drivers, including FUS, naturally form condensates, which are termed “stress granules” in response to stress conditions. While FUS is naturally localized to the nucleus, where it binds RNA and regulates splicing and other transcriptional and post- transcriptional processes, stress granules are transiently formed in the cytosol in response to a variety of stress conditions. They contain a variety of nuclear and cytosolic RNA binding proteins, which bind and protect mRNAs from translation during stress. These condensates are transient, and dissolve in the course of recovery from stress. The stress granules are known to form in a liquid-liquid phase separation (LLPS) process (Mateju, D., 2017). However, the same LLPS has the capability to seed irreversible pathological condensation, in a process termed “liquid-to- solid” phase separation, causing the accumulation of irreversible aggregates of some of these proteins in the disease.

[0010] The protein quality control (PQC) network is comprised of hundreds of proteins which act to maintain protein homeostasis at the face of external and physiological challenges (Bukau, B., et al. 2006). At the heart of the PQC network, molecular chaperones are the primary regulators of protein folding and aggregation in the cell, and their function and regulation in various NDs are of major interest (Kampinga, H. et al., 2016). The proteotoxic stress response, aka, the heat shock response (HSR), is the pathway which activates the transcription factor HSF-1 in response to stress, leading to a vast induction of Hsp70 genes transcription, as well as additional specific chaperones, to protect cells from proteotoxicity (Akerfelt, M., et al., 2010). Nevertheless, Hsp70s themselves are problematic as therapeutic targets, due to their centrality, and the fact that their recognition of client proteins is promiscuous and highly widespread (Kampinga, H. H. et al. 2010). Several chaperone families have undergone a major expansion throughout evolution, with the notion of functional diversification and optimization towards specific targets (Kampinga, H. H. et al. 2010), and specific chaperones were shown to be modifiers of HTT aggregation in Huntington’s disease (Hageman, J., et al. 2010).

[0011] PQC regulation of ALS- associated aggregation has been previously demonstrated (Crippa, V. et al., 2010; Lackie, R. E., et al., 2017; Yu, H., et al., 2021; Lu, S., et al., 2022; Rozales, K., et al., 2022), and an emerging notion in the field posits that subtype- specific PQC regulation is required to battle aggregation of different ALS drivers.

[0012] Rozales K, et al. 2022 used a highly quantitative FACS-based assay (the PulSA assay), to examine the effects of different PQC proteins on the aggregation of mutant FUS, and found that induction of the classical HSR had no beneficial effect on aggregation of FUS harboring ALS- related mutations (Rozales K, et al. 2022). They further showed that augmentation of specific chaperones, i.e., the full-length (FL) DNAJB14 and DNAJB12, but not the naturally-occurring short isoforms, substantially protects from mutFUS aggregation, in an HSP70-dependent manner. In contrast, DNAJB12-short alleviates, while DNAJB12-FL aggravates, HTT-polyQ aggregation (Rozales K, et al. 2022).

[0013] In addition, although both TDP-43 and FUS are RNA-binding proteins, and both are known stress granule components, their aggregation is mutually exclusive. These molecular features, and other evidences, underscore the need to investigate specific therapies tailored for different ALS drivers.

[0014] A common paradigm states that ND-related aggregation-prone proteins are cleared via the UPS (Ubiquitin Proteasome System) and the Autophagy systems (Ciechanover, A. et al. 2015; Dantuma, N. P. et al. 2014), and therefore inhibition of these systems should enhance aggregation. Indeed, proteasome inhibition was shown to aggravate aggregation in the case of the Huntington’s disease-associated HTT-polyQ23(Li, X., et al. 2010), TDP-43 (Scotter, E. L., et al., 2014) and other ND-related proteins (Ciechanover, A. et al. 2015; Dantuma, N. P. et al. 2014).

[0015] In the past 15 years, RNA-binding proteins (RBPs) were found to be aggregated in ALS, the most well-known of which are TDP-43 and FUS (Kabashi, E., et al., 2008; Kwiatkowski, T. J., Jr., et al., 2009; Sreedharan, J., et al., 2008), and mutations in a myriad of RBPs were identified as causative of familial ALS (Taylor, J. P., et al., 2016).

[0016] Mutations in the FUS gene are known to cause familial ALS (type 6), and patients carrying point mutations in the C-terminus of FUS show neuronal cytoplasmic FUS-positive inclusions, whereas in healthy controls, FUS is predominantly nuclear (Caroline Vance, et al., Science, 2009 323(5918): 1208- 1211). In addition, patients with FUS mutations appear to have earlier symptom onset, a higher rate of bulbar onset, and shorter duration of symptoms than those with SOD1 mutations (Y n J, et al. “Frameshift and novel mutations in FUS in familial amyotrophic lateral sclerosis and ALS / dementi a”. Neurology. 2010; 75:807—14). Mutations in FUS are also found in ALS patients with additional atypical features such as mental retardation, parkinsonism, dementia, and essential tremor [reviewed in Hong-Fu Li et al., “Genotype-phenotype correlations of amyotrophic lateral sclerosis” Translational Neurodegeneration volume 5, Article number: 3 (2016)).

[0017] Several proteostasis drugs have been studied in the context of ALS in recent years. Nonetheless, while some have shown great promise in animal models and small-scale subtype specific studies (Benatar, M., et al., 2018; Kieran, D., et al., 2004), they have failed to get to the clinic when tested on ALS patients in general.

[0018] SUMMARY OF THE INVENTION

[0019] According to an aspect of some embodiments of the present invention there is provided a method of treating a subject having a disease characterized by accumulation of FUS -associated aggregates, the method comprising administering to the subject a therapeutically effective amount of a proteasome inhibitor, thereby treating the subject.

[0020] According to an aspect of some embodiments of the present invention there is provided a proteasome inhibitor for use in treating a subject having a disease characterized by accumulation of FUS -associated aggregates.

[0021] According to an aspect of some embodiments of the present invention there is provided a method of reducing accumulation of FUS-associated aggregates in cells of a subject, the method comprising contacting the cells of the subject with a proteasome inhibitor, thereby reducing the accumulation of FUS -associated aggregates in the cells of the subject.

[0022] According to an aspect of some embodiments of the present invention there is provided a method of treating a subject having a disease characterized by accumulation of FUS -associated aggregates, the method comprising administering to the subject a therapeutically effective amount of an agent which upregulates spermidine / spermine N1 -acetyltransferase 1 (SAT1) or spermine, thereby treating the subject.

[0023] According to an aspect of some embodiments of the present invention there is provided a composition-of-matter comprising an agent which upregulates spermidine / spermine Nl- acetyltransferase 1 (SAT1) or spermine, for use in treating a subject having a disease characterized by accumulation of FUS -associated aggregates.

[0024] According to an aspect of some embodiments of the present invention there is provided a method of reducing accumulation of FUS-associated aggregates in cells of a subject, the method comprising contacting the cells of the subject with an agent which upregulates spermidine / spermine N1 -acetyltransferase 1 (SAT1) or spermine, thereby reducing the accumulation of FUS-associated aggregates in the cells of the subject.

[0025] According to an aspect of some embodiments of the present invention there is provided a method of treating a subject having a disease characterized by accumulation of FUS-associated aggregates, the method comprising administering to the subject a therapeutically effective amount of Withaferin A (WA), thereby treating the subject.

[0026] According to an aspect of some embodiments of the present invention there is provided Withaferin A (WA) for use in treating a subject having a disease characterized by accumulation of FUS-associated aggregates.

[0027] According to an aspect of some embodiments of the present invention there is provided a method of reducing accumulation of FUS-associated aggregates in cells of a subject, the method comprising contacting the cells of the subject with Withaferin A (WA), thereby reducing the accumulation of FUS-associated aggregates in the cells of the subject.

[0028] According to an aspect of some embodiments of the present invention there is provided an in-vitro method of selecting a molecule for treatment of a disease characterized by accumulation of FUS-associated aggregates, the method comprising:

[0029] (a) contacting cells carrying a mutation in the FUS gene which causes mis-folding of the FUS with the molecule,

[0030] (b) determining presence and / or level of FUS-associated aggregates in the cells prior to and following the contacting, wherein a reduction in level of the FUS-associated aggregates following the contacting indicates that the molecule is suitable for the treatment of the disease characterized by accumulation of FUS-associated aggregates.

[0031] According to some embodiments of the invention, the method further comprising administering to the subject a therapeutically effective amount of an agent which upregulates spermidine / spermine N1 -acetyltransferase 1 (SAT1) or spermine.

[0032] According to some embodiments of the invention, the method further comprising administering to the subject a therapeutically effective amount of Withaferin A (WA).

[0033] According to some embodiments of the invention, the subject has a mutation in the FUS gene which causes mis-folding of the FUS.

[0034] According to some embodiments of the invention, the method further comprising identifying in a DNA of the subject a mutation in the FUS gene which causes mis-folding of the FUS prior to administering.

[0035] According to some embodiments of the invention, the proteasome inhibitor penetrates through the blood brain barrier (BBB).

[0036] According to some embodiments of the invention, the proteasome inhibitor is formulated in a formulation for enhancing penetration of the proteasome inhibitor through the blood brain barrier (BBB).

[0037] According to some embodiments of the invention, the agent is formulated in a formulation for enhancing penetration of the agent through the blood brain barrier (BBB).

[0038] According to some embodiments of the invention, the wherein the formulation comprises a nano-carrier.

[0039] According to some embodiments of the invention, the wherein the nano-carrier comprises a Poly(lactic-co-glycolic acid) (PLGA) nanoparticle, or an albumin nanoparticle.

[0040] According to some embodiments of the invention, the proteasome inhibitor is coadministered with a P-glycoprotein (P-gp) inhibitor.

[0041] According to some embodiments of the invention, the proteasome inhibitor is Carfilzomib, Ixazomib, Marizomib, Oprozomib, Delanzomib, ixazomib, delanzomib, KZR-616, NNU546, NNU219, MLN9708, ONX0912, or MG-132.

[0042] According to some embodiments of the invention, the proteasome inhibitor is is Carfilzomib, Ixazomib, Marizomib, Oprozomib, Delanzomib, ixazomib, delanzomib, KZR-616, NNU546, NNU219, MLN9708, ONX0912, MG-132 or Bortezomib.

[0043] According to some embodiments of the invention, the proteasome inhibitor is

[0044] Withaferin A. According to some embodiments of the invention, the proteasome inhibitor is Marizomib.

[0045] According to some embodiments of the invention, the proteasome inhibitor is Velcade (Bortezomib).

[0046] According to some embodiments of the invention, the FUS-associated aggregates comprise a mis-folded FUS protein.

[0047] According to some embodiments of the invention, the mis-folded FUS protein is caused by a mutation in the FUS gene.

[0048] According to some embodiments of the invention, the method further comprising identifying in a DNA of the subject a mutation in the FUS gene which causes mis-folding of the FUS.

[0049] According to some embodiments of the invention, the mutation in the FUS gene encodes a mutated FUS protein selected from the group consisting of S513P, R514G / S, G515C, E516V, H517D / P / Q, R518G / K, Q519X, Q519IfsX9, R521C / G / H / L / S, R522G, R524W / T / S, P525L, S96del, P431L, R502EfsX15, G504WfsX12, G507D, K510E / R, K510WfsX517, S462F, M464I, G466VfsX14, G472VfsX57, Y485AfsX514, R487C, R495X, G492EfsX527, R495EfsX527, R495QfsX527, G497AfsX527, R377W, G399V, S402_P411delinsGGGG, Q290X,

[0050] G175_G176insGG, G187S, G191S, G206S, R216C, S221del, G225V, G230C, G230del, R234C / L, R244C, M254V, P525L S96del, P106L, S115N, G144_Y149del, G156E, and S57del.

[0051] According to some embodiments of the invention, the FUS-associated aggregates are present in neurons of the subject.

[0052] According to some embodiments of the invention, the neurons are motor-neurons of the subject.

[0053] According to some embodiments of the invention, the neurons are cortical neurons of the subject.

[0054] According to some embodiments of the invention, the FUS-associated aggregates are present in at least one of the four anatomical regions selected from the group consisting of bulbar, cervical, thoracic, and lumbar regions.

[0055] According to some embodiments of the invention, the subject is diagnosed with Amyotrophic lateral sclerosis (ALS).

[0056] According to some embodiments of the invention, the subject is diagnosed with Frontotemporal dementia (FTD).

[0057] According to some embodiments of the invention, the cells are neuron cells.

[0058] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0059] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0060] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0061] In the drawings:

[0062] Figs. 1A-C, adopted from Rozales K, et al. 2022, depict FACS-based quantification and separation of FUS aggregate-containing cells. Figure 1A demonstrates HEK293T cells expressing either YFP (yellow fluorescence protein)-tagged FUS-WT (left image) or FUS-R521H (right image), which generates cytoplasmic aggregates (FUS-R521H). Figure IB - Illustration of the PulSA method (described in Ramdzan, Y. M., et al. Nat Methods 9, 467-470). Aggregatecontaining cells have distinct fluorescence peak features analyzed by FACS. Figure 1C - Raw data for PulSA show an additional cell population in FUS-R521H-YFP expressing cells compared to FUS-WT- YFP cells, corresponding to aggregate-containing cells.

[0063] Figs. 2A-B depict modulation of ALS-FUS aggregation. Figure 2A - Outline of the screening framework used in Rozales K, et al. 2022 and the FACS-based aggregation modulation score. Chaperones were introduced together with YFP-tagged mutFUS into HEK293T cells in multi-well plates, and were later subjected to FACS analysis to quantify the fraction of aggregatecontaining cells (AGG+). These numbers were used to calculate the Aggregation Modulation score: the log2 ratio of AGG+ cells in the presence of a modulator compared to the control. 95% confidence intervals (CI, dashed lines) used to assess significance. Figure 2B - Heat shock treatments with various regimens did not protect from mutFUS aggregation. Grey lines - 95% CI. Adopted from Rozales K, et al. 2022.

[0064] Fig. 3A depicts RNA-seq data for heat shock or MG- 132 treated HEK293T cells. The log2 fold changes of PQC genes were compared. Shown is a scatter plot of log2 fold change of MG- 132 18 hours vs control (x-axis), compared to mild heat shock (42°C) 8 hours vs control (y-axis). Dashed lines: + / -1 (fold change of 2). PQC families are color coded. Fig. 3B - Aggregation modulation scores for cells expressing mutFUS or HTT-polyQ, treated with different HSR inducers / proteasome inhibitors (or DMSO as control). On the left, the plot shows the FUS-R521H-YFP aggregation modulation scores for cells expressing mutFUS with different treatments, including MG-132 and Velcade (Bortezomib), which exhibit unexpected aggregation inhibition. On the right, the plot shows the aggregation modulation scores for cells expressing HTT-polyQ, demonstrating an exacerbating effect on aggregation. Dashed lines represent 95% Cis ***p < 0.003, *p < 0.05, empirical p-value (see Methods). Grey dashed lines - 95% CI. *** - p<0.003.

[0065] Figs. 4A-B depict aggregation modulation scores of cells in the presence of various proteasome inhibitors. Figure 4A - Cells co-expressing FUS-R521H-YFP treated with different doses of Withaferin A (WA) for 18 hours show aggregation alleviation assayed by PulSA analysis. Figure 4B - FUS-R521H-YFP Aggregation modulation scores in cells expressing DsRed that underwent HSF1 knockdown (SiHSFl) vs. siControl cells in addition to cells only transfected with FUS-R521H-YFP and DsRed with no further SiRNA interference. All cells were treated with MG- 132 10 pM for 18 hours and subjected to PulSA analysis. Dashed lines represent 95% Cis ***p < 0.003, *p < 0.05, empirical p-value (see Methods). “uM” = pM (micro-molar).

[0066] Figs. 5A-D - Heat shock showed no aggregation amelioration. Figure 5 A - Cell viability percentages of MG-132 and DMSO control, assayed by DAPI exclusion using FACS. Data are presented as mean values + / - SEM of n = 3 biologically independent experiments in FUS-R521H- YFP. Figure 5B - HEK293T cells co-transfected with FUS-R521H-YFP and DsRed, and then treated with different heat stress regimens; Heat shock of 44 °C for 1.5 hours with additional 18 hours recovery at 37 °C, 44 °C for 2 hours, or 42 °C for 8 hours, followed by PulSA analysis at 48 hours post transfection and right after the treatment. All three regimens showed no aggregation amelioration. Data are presented as mean values + / - SEM for n = 6 biologically independent samples for the recovery experiment, and n = 5 for 2 hours at 44 °C and 8 hours at 42 °C. Figures 5C-D - Cells co-expressing FUS-R521H-YFP were treated with varying doses of Bortezomib (as shown in Figure 5C) and Marizomib (as shown in Figure 5D) for 18 hours, resulting in a reduction of aggregation, as determined by PulSA analysis.

[0067] Fig. 6 is a histogram depicting fold changes in mRNA levels of the HSF1 gene normalized to the HPRT were assayed using qPCR for HSF1 knockdown samples (siHSFl) and control samples (siControl).

[0068] Figs. 7A-F depict proteasome-inhibitors enhancing PQC mRNAs. Figure 7A - Plotting the log-fold change (LFC) of gene expression for cells treated with MG-132 (10 pM for 18 hours) compared to control cells against the LFC gene expression of cells subjected to heat shock (42°C for 8 hours) compared to their respective controls. Chaperones families are taken from Brehme et al 2014. Figure 7B - The left side of the plot represents FUS-R521H-YFP aggregation modulation scores in cells expressing FUS-R521H-YFP that underwent BAG3 knockdown (SiBAG3) vs. siControl. On the right side of the plot FUS-R521H-YFP aggregation modulation scores are shown for cells co-expressing FUS-R521H-YFP with either DsRed or BAG3. Data are presented as mean values ± SEM of n = 3 / 3 and 5 / 5 biologically independent samples (for siControl / siBAG3 and DsRed / BAG3, respectively). Figure 7C - BAG3 mutant isoforms. The human BAG3 gene consists of 575 amino acids featuring a conserved BAG domain at its C-terminus (schematically represented as “BAG3-FL (full length) in Figure 7C). The conserved BAG domain functions as nucleotide exchange factors (NEFs) binding to the ATPase domain of heat shock protein 70 and facilitating the release of HSP70 clients in collaboration with DNAJ co-chaperones. The N-terminus domain contains two tryptophan-tryptophan (WW) domain. BAG3 also contains a PxxP (proline-rich region) repeat and a two conserved IPV (Isoleucine-Proline- Valine) domains. BAG3-AC: C- terminus lacking BAG3-isoform, BAG3-AN: N-terminus lacking BAG3-isoform, Q488A mutation in BAG domain. Figure 7D - FUS-R521H-YFP aggregation modulation scores for BAG3 isoforms. Data are presented as mean values ± SEM of n = 5 biologically independent experiments for the different isoforms presented. Figure 7E - mutFUS aggregation modulation scores in cells co-expressing DsRed and FUS-R521H-YFP that underwent BAG3 knockdown vs. siControl cells, once treated with MG- 132 and once with DMSO. Data are presented as mean values ± SEM of n = 3 biologically independent replicates. Source data are provided as a Source data file. Figure 7F -Autophagy inhibitor Bafilomycin A (200 nM for 18 hours) was introduced in cells co-transfected with FUS-R521H-YFP and DsRed, treated with either MG-132 (10 pM for 18 hours) or DMSO as a control (the score was divided by the values of the control). In addition, Bafilomycin A (200 nM for 18 hours) was introduced into samples co-transfected with FUS-R521H-YFP and BAG3, comparing them to control samples treated with DMSO as a control. Subsequently, PuLSA analysis was conducted, and data are presented as mean values ± SEM of n = 3 biologically independent replicates. The histogram presented in Figure 7F shows aggregation modulation score of cells treated with MG132 alone (“MG132”); Balfilomycin A alone (“Balfilomycin A”), combination of Balfilomycin A and MG132 (“Balfilomycin A+MG132”), BAG3 alone (“BAG3”), and the combination of BAG3 and Balfilomycin A (“BAG3+ Balfilomycin A”). Source data are provided as a Source data fileDashed lines represent Ci 95%. Dashed lines represent 95% Cis ***p < 0.003, *p < 0.05, empirical p-value (see General Materials and Experimental Methods).

[0069] Figs. 8A-D show that BAG3-mediated protection of FUS aggregation is HSP70 dependent. Figure 8A - FLAG-tagged BAG3 or DsRed co-expressed with FUS-R521H-YFP (green). Immunofluorescence imaging (IF) with anti-FLAG antibody (pink), DAPI marks nuclei (blue). Shown are representative images out of n = 3 biologically independent experiments. Figure 8B - Fold changes in mRNA levels of the BAG3 gene normalized to the HPRT were assayed using qPCR for BAG3 knockdown samples (siBAG3) and control samples (siControl). Figure 8C - CoIP for different BAG3 flag tagged mutant isoforms either co-transfected in cells expressing mutFUS (R521H) or WT FUS or without anything (-), WB for HSP70 antibody showing no interaction between BAG3-AC or DsRed and reduced interaction between HSP70 and BAG3-AN or Q488A. (See General Materials and Experimental Methods). Figure 8D - Signal intensity quantification of HSP70 bound to the BAG3 different isoforms, all normalized to FLAG signal. Data are presented as mean values + / - SEM of n = 3 biologically independent samples. Densitometry was performed using Fiji.

[0070] Figs. 9A-E show that proteasome inhibitors mediate a unique transcriptional program that is distinct from the one mediated by heat shock. Figure 9 A - Systematically plotting the log-fold change (LFC) of gene expression for cells treated with MG-132 (10 pM for 18 hours) compared to control cells against the LFC gene expression of cells subjected to heat shock (42°C for 8 hours) compared to their respective controls. In grey, all background genes after TPM (Transcript Per Million) thresholding. In blue, strict MG-132 unique program, that contain genes that were significantly changed after MG-132 treatment for both 8 hours and 18 hours (Deseq2- |LFC> 11, Deseq2-padj<0.01) and weren’t significant after HS treatment (Deseq2-|LFC<1|, Deseq2- padj>0.05). SAT1, red labeled, is among the strict program. Figure 9B - FUS-R521H-YFP Aggregation modulation scores for cells co-expressing FUS-R521H-YFP and DsRed compared to cells co-expressing FUS-R521H-YFP and SAT1. Data are presented as mean values ± SEM of n = 4 biologically independent samples. Source data are provided as a Source data file. Figure 9C - mutFUS Aggregation modulation scores in cells co-expressing DsRed and FUS-R521H-YFP that underwent SAT1 knockdown vs. siControl cells, once treated with MG-132 and once with DMSO. Data are presented as mean values ± SEM of n = 3 biologically independent replicates. Figure 9D - FUS-R521H-YFP Aggregation modulation scores for SAT1 mutant isoforms. Figure 9E - Shown is a quantitative polyamine assay to assess alterations in polyamine levels in cells treated with MG- 132 compared to those treated with DMSO as a control for n = 3 independent experiments, results were normalized to total protein measurements by BCA. Dashed lines represent 95% Cis. Data are presented as mean values ± SEM of n = 4 biologically independent samples. Source data are provided as a Source data file. Dashed lines represent Ci 95%. Dashed lines represent 95% Cis ***p < 0.003, *p < 0.05, empirical p-value (see Methods). Figs. 10A-C depict influencing SATl-mediated rescue mechanisms in (ALS) by modulating the polyamines pathway. Figure 10A - mRNA levels of the SAT1 gene normalized to the HPRT were assayed using qPCR for SAT1 knockdown samples (siSATl) and control samples (siControl). Figure 10B - Cells were co-transfected with DsRed and FUS-R521H-YFP and treated with varying concentrations (5 pM, 20 pM, 100 pM) of polyamines for 72 hours then subjected to PulSA analysis (Spermine (SP), Spermidine (SPD), Putrescine (PUT)). Figure 10C - Aggregation modulation scores within samples co-transfected with FUS-R521H-YFP and DsRed, treated with either MG-132 (10 pM, 18 hours) or Berenil (100 pM, 18 hours).

[0071] Figs. 11A-B depict that proteasome inhibitors show differential modulation of mutant FUS aggregation in primary neurons. Figure 11A - Confocal microscopy images of live neurons showing a complex FUS-R521H-YFP aggregation pattern. Figure 11B - A viability chart was generated using propidium iodide incorporation to depict the percentage of dead cells among live- imaged neurons over approximately 20 hours. Live cell analysis was performed using Incucyte (as described in General Materials and Experiments Methods below). Control = navy blue line; Marizomib = light blue line; MG 132 = grey line.

[0072] Figs. 12A-D show that proteasome inhibitors show differential modulation of mutant FUS aggregation in primary neurons. Figure 12A - Image analysis of thousands of live neurons showed significantly lower FUS-R521H-YFP aggregation in both Marizomib and MG-132 treated neurons compared to their respective controls. Data are presented as mean values ± SEM of the fraction of aggregate-positive cells in N = 3 biologically independent experiments for each treatment (see B). Experiment mean aggregation fraction was calculated including all fields of both treatment (either MG-132 or Marizomib, depending on the experiment) and respective control samples. Subsequently, all sample values were normalized to their respective experiment mean. The averaged normalized means seen in Figure 12A demonstrated a reduction in aggregation of 36% and 32% in MG-132 and Marizomib treatments, respectively. Figure 12B - Fraction of aggregatepositive neurons, data are presented as mean values + / - SEM of each of N=3 biologically independent experiments, containing n=831, 1185, 278 or 741, 957, 555 for MG-132 / control neurons respectively, or n=1077, 411, 165 or 801, 709, 148 for Marizomib / control neurons respectively. Mean fraction of aggregate-containing cells was consistently and significantly lower in all three biological replicates for each treatment vs. control. (p=0.0095, 1.56e-5, and 7.46e-5 for the three experiments of MG-132 respectively, or p=0.00041, 0.0405, and 0.00086 for the three experiments of Marizomib respectively, calculated using t-test, two-sided, non- adjusted). Figure 12C - Neuronal cultures infected with FUS-R521H-YFP and treated with MG-132 (right panel) or DMSO as a control (left panel). Shown are maximum-projection of confocal fluorescent images (using Fiji), of representative fields out of N = 3 biologically independent samples. Scale bar: 50 pm. Figure 12D - Polyamine metabolomics was performed to assess alterations in polyamine levels in cells treated with Marizomib compared to control samples, results were normalized to total protein measurements by BCA.

[0073] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0074] The present invention, in some embodiments thereof, relates to methods of treating a subject having a disease characterized by accumulation of FUS-associated aggregates such as ALS and FTD and, more particularly, but not exclusively, to proteasome inhibitors for use in treating ALS and FTD.

[0075] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and / or methods set forth in the following description and / or illustrated in the drawings and / or the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0076] A common paradigm states that ND-related aggregation-prone proteins are cleared via the UPS (Ubiquitin Proteasome System) and the Autophagy systems, and therefore inhibition of these systems should enhance aggregation. Indeed, proteasome inhibition was shown to aggravate aggregation in the case of the Huntington’s disease-associated HTT-polyQ (Ganassi et al., 2016), TDP-43 (Bracher et al., 2015) and other ND-related proteins (Santagata et al., 2012; Mathew et al., 2001), along with their role in inhibiting protein degradation leading to misfolded protein accumulation.

[0077] In sharp contrast to the common paradigm, the present inventors have uncovered that proteasome inhibitors can be used to inhibit aggregation of FUS harboring ALS -associated mutations.

[0078] Importantly, while proteasome inhibitors aggravated the aggregation of HTT-polyQ (Example 1, Figure 3B, right side), counterintuitively, proteasome inhibitors of various types significantly inhibited FUS aggregation in cell line models (Example 1, Figures 3B, 5C, 5D) and primary neurons (Example 6, Figures 12A-C). The present inventors demonstrate that treatment with the proteasome inhibitors did not affect cell viability (Example 1, Figure 5A). Notably, heat shock treatment did not have any beneficial effect on ALS-FUS aggregation (Example 1, Figures 2B and 5B).

[0079] The present inventors found that proteasome inhibitor effect is largely transcriptionally mediated through HSF1 (Example 2, Figure 4B). Cells which were co-transfected with FUS- R521H-YFP and siControl and were further treated with MG-132 exhibit alleviation of aggregation, similar to the aggregation alleviation of samples treated solely with MG-132 (Figure 4B). In contrast, cells which were co-transfected with FUS-R521H-YFP and siHSF exhibit minimal alleviation of aggregation (Figure 4B).

[0080] Example 4 of the Examples section which follows further shows that MG132-induced aggregation clearance is partially mediated by autophagy as the addition of the autophagy inhibitor Bafilomycin A resulted in a modest aggregation alleviation (Figure 7F).

[0081] Example 5 of the Examples section which follows shows the identification of genes which are upregulated in the presence of the MG 132 proteasome inhibitor but are un-changed under heat shock conditions (Figure 9A), such as SAT1 (spermidine / spermine N1 -acetyltransferase 1), which exhibited a substantial protective effect with approximately 80% aggregation alleviation (Figure 9B). In addition, siSATl in MG-132-treated samples led to a reduction in aggregation inhibition by approximately 35% (Figure 9C) compared to cells treated with MG-132 and siControl. The present inventors have further showed that mutations in SAT1, such as E152K, R19A and L156F negatively impacted SATl's ability to reduce mutFUS aggregation, thereby highlighting the significance of the polyamines pathway in SATl-mediated inhibition of FUS aggregation (Figure 9D). Similarly, Berenil, which reduces SAT1 activity, hindered FUS aggregation inhibition by MG-132 (Figure 10C).

[0082] Furthermore, treatment with spermine resulted in a significant alleviation of aggregation (Figure 10B).

[0083] Example 6 of the Examples section which follows, shows that proteasome inhibitors such as MG-132 and Marizomib inhibit FUS aggregation and affect polyamine metabolism in primary neurons (Figures 12A-C).

[0084] The present inventors have envisaged that blood-brain-barrier (BBB) permeable proteasome inhibitors, such as Marizomib, can be used to specifically target ALS-FUS or FTD- FUS as a therapeutic intervention. The present inventors have further the use of the non-BBB permeable proteasome inhibitors such as Bortezomib (Velcade, an FDA-approved drug for multiple myeloma), in combination with P-glycoprotein (P-gp) inhibitors (such as Tariquidar) to specifically penetrate the BBB and target ALS-FUS or FTD-FUS as a therapeutic intervention.

[0085] In addition, the findings that SAT1 and spermine significantly alleviate FUS aggregation (e.g., Figure 9B and 10B) suggest the use thereof as therapeutic interventions in ALS-FUS or FTD- FUS.

[0086] According to an aspect of some embodiments of the invention, there is provided a method of treating a subject having a disease characterized by accumulation of FUS-associated aggregates, the method comprising administering to the subject a therapeutically effective amount of a proteasome inhibitor, thereby treating the subject.

[0087] As used herein the phrase “a disease characterized by accumulation of FUS -associated aggregates” refers to a pathology which onset or progression is driven by an abnormal accumulation of the FUS -associated aggregates.

[0088] As used herein the phrase “FUS-associated aggregates” refers to protein aggregates formed by mutated FUS protein and aggregate in the cells.

[0089] The FUS (fused in sarcoma) gene encodes a multifunctional protein component of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex which is involved in pre-mRNA splicing and the export of fully processed mRNA to the cytoplasm. The FUS protein belongs to the FET family of RNA-binding proteins.

[0090] According to some embodiments of the invention, the disease, which is characterized by accumulation of FUS-associated aggregates, is Amyotrophic lateral sclerosis (ALS). According to some embodiments, the subject is diagnosed with Amyotrophic lateral sclerosis (ALS).

[0091] Amyotrophic lateral sclerosis is a progressive neurological disorder affecting motor neurons in the brain and spinal cord with initial manifestation of muscle weakness, twitching and atrophy, and leading eventually to losing the ability to control voluntary movements such as walking, talking, chewing and breathing. ALS can be diagnosed using various tools such as electromyography (EMG), nerve conduction study (NCS), a needle electrode which records electrical activity in muscle fibers and magnetic resonance imaging (MRI) of the brain and spinal cord.

[0092] The clinical presentation of ALS depends on the genetic etiology of the disease. Table 1 below summaries various ALS phenotypes resulting from different genes or gene loci and having different modes of inheritance.

[0093] Table 1

[0094] Table 1: “AR” - autosomal recessive; “AD” - autosomal dominant; “XLD” - X-linked dominant;

[0095] “MIM” - the unique 6 digit number ID for each “entry” (gene or disease / phenotype).

[0096] According to some embodiments of the invention, the subject who is diagnosed with ALS does not have a mutation in SOD1, and does not have SOD1 aggregates.

[0097] According to some embodiments of the invention, the disease, which is characterized by accumulation of FUS- associated aggregates, is Frontotemporal dementia (FTD). According to some embodiments, the subject is diagnosed with Frontotemporal dementia (FTD). Frontotemporal dementia relates to a group of brain diseases affecting the frontal and temporal lobes of the brain, associated with atrophy and shrinkage of these brain lobes. Symptoms include, among others, changes in personality, inappropriate social behavior, impulsive or emotionally indifferent. In more rare cases, symptoms can also include unusual movements such as tremor, rigidity, muscle spasms, weakness and twitches, falls or trouble walking, poor coordination, difficulties in swallowing, and inappropriate laughing or crying.

[0098] The term “treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and / or causing the reduction, remission, regression of a pathology or extending survival. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

[0099] According to some embodiments of the invention, treating refers to increasing survival of the subject having the disease and being treated with the therapeutic agent of some embodiment of the invention by at least six months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or more as compared to a control subject having the disease characterized by accumulation of FUS -associated aggregates who is not treated by the therapeutic agent of some embodiments of the invention.

[0100] According to some embodiments of the invention, the control subject having the disease characterized by accumulation of FUS -associated aggregates who is not treated by the therapeutic agent of some embodiments of the invention has the same mutation in the FUS gene as the subject having the same disease and being treated by the therapeutic agent of some embodiments of the invention.

[0101] According to some embodiments of the invention, the FUS-associated aggregates comprise a mis-folded FUS protein.

[0102] The wild-type FUS protein isoform has 526 amino acids (isoform 1) and its sequence is set forth by GenBank Accession No. NP_004951 (SEQ ID NO: 21), encoded by the mRNA sequence set forth by GenBank Accession No. NM_004960 (SEQ ID NO: 22). Other shorter FUS protein isoforms, which result from in-frame alternative splicing include for example, isoform 2 [sequence is set forth by GenBank Accession No. NP_001164105.1 (SEQ ID NO: 23), encoded by the mRNA sequence set forth by GenBank Accession No. NM_001170634.1 (SEQ ID NO:24)], and isoform 3 [sequence is set forth by GenBank Accession No. NP_001164408.1 (SEQ ID NO: 25), encoded by the mRNA sequence set forth by GenBank Accession No. NM_001170937.1 (SEQ ID NO:26)].

[0103] Protein mis-folding can be evaluated and quantified in-vitro. For example, the percentage of aggregate-containing cells can be quantified using the PuLSA method and can be compared these percentages of cells treated with DMSO as a control. Aggregation modulation scores for each treatment can be calculated as Log2(%AGG+(treatment) / %AGG+(control)), e.g., as exemplified herein in Figures 3B, 4A-B, 5C-D, 7B, 7E-F, 9B-D and 10B-C).

[0104] For the PulSA method about 48 hours following transfection with the exogenous polynucleotides of interest (e.g., the FUS-WT-YFP, FUS-R521H-YFP, FUS-R518K-YFP or DsRed constructs) the cells are trypsinized, resuspended in media with serum, placed on ice, and cell viability is determined (e.g., using a DAPI viability dye). DAPI is an impermeant DNA dye which is largely excluded from live cells. Cells are then subjected to Flow cytometry in order to differentiate between subpopulations with diffused cellular fluorescence and those with protein aggregates. To obtain Aggregation modulation scores, cells of appropriate size are considered during the FACS analysis, and non-viable cells (which are positive for DAPI) are excluded. The percentage of cells that show a fluorescent aggregate pattern (AGG+) is quantified, and compared these percentages to cells co-transfected with DsRed as a control. Aggregation modulation scores for each agent or molecule are calculated as Log2(%AGG+ (agent or treatment) / %AGG+ (control)). Confidence intervals (CI) (95%, appear as dashed lines) are calculated according to the variations between different DsRed co-transfected cells replicates, such that they represent twice the standard deviation of Aggregation modulation scores calculated between different replicates of DsRed performed on the same day. Agents or molecules are considered as significant modulators if their average Aggregation modulation scores + (or -) SEM is below (or above) the 95% CI. Specifically, an empirical p-value is indicated such that p < 0.05 if these values are below the 95% CI, and p < 0.003 if these values are below the 99.7% CI (z.e., 3 times STD).

[0105] Additional methods of qualifying protein folding or misfolding are described in Jad Walters et al., 2009, Methods Enzymol. 2009; 455: 1-39; and Katherine Do et al., 2024, “Adaptation of the protein misfolding cyclic amplification (PMCA) technique for the screening of anti-prion compounds”, FASEB J. 38(14):e23843; each of which is fully incorporated herein by reference. These include, but are not limited to X-ray crystallography; protein misfolding cyclic amplification (PMCA); intrinsic probes such as by fluorescence emission and circular dichroism (CD); extrinsic probes, vibrational circular dichroism (VCD), which extends the CD spectroscopy into the infrared and near infrared ranges, and detects differences in attenuation of left and right circularly polarized light passing through a sample. VCD which involves Fourier transform (FT) instruments, provide powerful means for determining protein conformations in solution even for very large protein molecules. It is noted that VCD studies of proteins can be combined with X-ray diffraction data for protein crystals, FT-IR (Fourier-transform infrared spectroscopy) data for protein solutions in heavy water (D2O), or quantum computations. According to some embodiments, the mutated FUS protein aggregates in the cytoplasm of the cells.

[0106] According to some embodiments, the mutated FUS protein aggregates in the nucleus.

[0107] In some embodiments of the invention, the FUS -associated aggregates accumulate in neuronal cells, e.g., motor neuron cells.

[0108] According to some embodiments of the invention, the mis-folded FUS protein is caused by a mutation in the FUS gene.

[0109] As used herein, the phrase “a mutation in the FUS gene” refers to any mutation in the DNA sequence of the FUS gene, which encodes a mutated FUS polypeptide resulting in a mis-folded FUS protein.

[0110] According to some embodiments of the invention, the mutation in the FUS gene causes an altered secondary and / or tertiary protein structure as compared to the secondary and / or tertiary protein structure of the wild-type FUS protein set forth by SEQ ID NO: 21 (GenBank Accession No. NP_004951.1).

[0111] According to some embodiments of the invention, the altered secondary and / or tertiary protein structure is a mis-folded FUS protein (when compared to the natural folding of the wildtype FUS polypeptide set forth by SEQ ID NO: 21) which causes the accumulation of FUS- associated aggregates characterizing the disease.

[0112] Non-limiting examples of such mutations include a missense mutation, z.e., a mutation which changes an amino acid residue in the protein with another amino acid residue; a nonsense mutation, z.e., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein; a frame-shift (fs) mutation, z.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non-functional protein; a promoter mutation, z.e., a mutation in a promoter sequence, usually 5' to the transcription start site of a gene, which results in down-regulation of a specific gene product; a regulatory mutation, z.e., a mutation in a region upstream or downstream, or within a gene, which affects the expression of the gene product; a deletion mutation (del), z.e., a mutation which deletes coding nucleic acids in a gene sequence and which may result in a frame-shift mutation or an in-frame mutation (within the coding sequence, deletion of one or more amino acid codons); an insertion mutation (ins), z.e., a mutation which inserts coding or non-coding nucleic acids into a gene sequence, and which may result in a frameshift mutation or an in-frame insertion of one or more amino acid codons; an inversion, z.e., a mutation which results in an inverted coding or non-coding sequence; a splice mutation z.e., a mutation which results in abnormal splicing or poor splicing; and a duplication mutation, z.e., a mutation which results in a duplicated coding or non-coding sequence, which can be in-frame or can cause a frame-shift.

[0113] The following list comprises non-limiting examples of FUS mutations which cause misfolding of FUS, and which are associated with ALS or FTD: S513P, R514G / S, G515C, E516V, H517D / P / Q, R5I8G / K, Q519X, Q519IfsX9, R521C / G / H / L / S, R522G, R524W / T / S, P525L, S96del, P431L, R502EfsX15, G504WfsX12, G507D, K510E / R, K510WfsX517, S462F, M464L G466VfsX14, G472VfsX57, Y485AfsX514, R487C. R495X. G492EfsX527, R495EfsX527, R495QfsX527, G497AfsX527, R377W, G399V, S402JP411delinsGGGG, Q290X,

[0114] G175_ G176insGG, G187S. G191S, G206S, R216C, S221del, G225V, G230C, G230del, R234C / L, R244C, M254V, P525L S96del, P106L, S115N, G144_Y149del, G156E, and / or S57del.

[0115] The above annotated mutations (based on the acceptable nomenclature) describe the effect of a genomic mutation in the FUS gene on the encoded FUS protein, hence, the mutations specify the mutated amino acids (using a single-letter symbol) and their positions in the FUS protein relative to the amino acid sequence of the wild type FUS protein (the longer isoform 1, SEQ ID NO: 21), with the resulting mutated amino acid(s). For example, the “R521C / G / H / L / S” mutation reflects a missense mutation in which the amino acid “R” at position 521 of the FUS protein is replaced by another amino acid which is either “C”, “G”, “H” “L” or “S”. Another example is the “S221de1” mutation which reflects a deletion (del) of amino acid “S” at position 221 of the FUS protein.

[0116] According to some embodiments of the invention, the disease which is characterized by accumulation of FUS -associated aggregates, such as ALS or FTD, can be diagnosed by DNA mutation analysis, for identifying presence of mutations which are associated with or causing misfolding of FUS and accumulation of FUS -associated aggregates.

[0117] Methods of identifying DNA mutations in a subject are well known in the art, and include for example, subjecting a genomic DNA, cell-free DNA (e.g., exosome analysis) or an mRNA of the subject to DNA sequencing, electrophoresis, an enzyme-based mismatch detection assay, an hybridization assay such as in-situ hybridization, Southern blot, Northern Blot or dot blot analysis, as well as PCR, RT-PCR, RNase protection, and primer extension. It is noted that genome sequencing involves assessing both the coding and noncoding regions of the genome, and exosome sequencing is limited to the protein coding regions of the genes (which comprises about 1-2% of the genome). Sequence alterations in the FUS gene can also be determined at the protein level using e.g. chromatography, electrophoretic methods, immunodetection assays such as ELISA and western blot analysis and immunohistochemistry.

[0118] According to some embodiments of the invention, the subject has a mutation in the FUS gene which causes mis-folding of the FUS protein.

[0119] According to some embodiments, the mutated FUS protein is caused by a mutation in the FUS gene.

[0120] According to some embodiments of the invention, the method further comprising identifying in a DNA of the subject a mutation in the FUS gene which causes mis-folding of the FUS prior to administering the therapeutic agents of some embodiments of the invention.

[0121] The method, according to an aspect of some embodiments of the invention, is effected by administering to the subject a therapeutically effective amount of a proteasome inhibitor.

[0122] According to an aspect to some embodiments of the invention, there is provided a proteasome inhibitor for use in treating a subject having a disease characterized by accumulation of FUS -associated aggregates.

[0123] As used herein the phrase “a proteasome inhibitor” refers to any molecule which can inhibit the activity of a 26S and / or 20S proteasome in degrading ubiquitinated proteins resulting in inhibition of FUS-associated aggregates formation in the cell.

[0124] According to some embodiments of the invention, the proteasome inhibitor is a direct inhibitor of proteasome activity.

[0125] According to some embodiments of the invention, the proteasome inhibitor is an indirect inhibitor of proteasome activity.

[0126] According to some embodiments, the proteasome inhibitor molecule inhibits the formation of FUS-associated aggregates in the cell.

[0127] According to some embodiments, the proteasome inhibitor is Carfilzomib, Ixa omib, Marizomib, Oprozomib, Delanzomib, ixazomib, delanzomib, KZR-616, NNU546, NNU219, MLN9708, ONX0912, Bortezomib, or MG-132.

[0128] According to some embodiments, the proteasome inhibitor is Velcade (Bortezomib).

[0129] According to some embodiments, the proteasome inhibitor is Marizomib.

[0130] According to some embodiments, the proteasome inhibitor is Withaferin A.

[0131] According to some embodiments, the proteasome inhibitor penetrates through the blood brain barrier (BBB). According to some embodiments of the invention, the method further comprising administering to the subject a therapeutically effective amount of an agent which upregulates spermidine / spermine N1 -acetyltransferase 1 (SAT1) or spermine.

[0132] According to an aspect to some embodiments of the invention, there is provided a method of treating a subject having a disease characterized by accumulation of FUS -associated aggregates, the method comprising administering to the subject a therapeutically effective amount of an agent which upregulates spermidine / spermine N1 -acetyltransferase 1 (SAT1) or spermine, thereby treating the subject.

[0133] According to an aspect to some embodiments of the invention, there is provided a composition-of-matter comprising an agent which upregulates spermidine / spermine Nl- acetyltransferase 1 (SAT1) or spermine, for use in treating a subject having a disease characterized by accumulation of FUS -associated aggregates.

[0134] As used herein the phrase “an agent which upregulates spermidine / spermine Nl- acetyltransferase 1 (SAT1)” refers to any molecule which increases the expression level of SAT1 and / or the activity of SAT1 in reducing FUS-associated aggregates in a cell.

[0135] The term “SAT1”, as used herein, refers to the spermidine / spermine N1 -acetyltransferase 1 (also known as “diamine N-acetyltransferase 1”), which belongs to the acetyltransferase family, and is a rate-limiting enzyme in the catabolic pathway of polyamine metabolism. It catalyzes the acetylation of spermidine and spermine, and is involved in the regulation of the intracellular concentration of polyamines and their transport out of cells. The SAT1 protein (the sequence is available GenBank Accession NO. NP_002961.1, SEQ ID NO: 27) is encoded by the SAT1 gene (Gene ID NO: 6303) and is transcribed from the SAT1 mRNA (GenBank Accession NO. NM_002970.4, SEQ ID NO: 28).

[0136] Upregulation of SAT1 can be effected at the genomic level (e.g., activation of transcription via promoters, enhancers, regulatory elements), at the transcript level (e.g., correct splicing, polyadenylation, activation of translation) or at the protein level (e.g., post-translational modifications, interaction with substrates and the like). Non-limiting examples of upregulation agents are described herein under.

[0137] According to some embodiments of the invention, the agent which upregulates SAT1 may be an exogenous polynucleotide sequence (e.g., a heterologous polynucleotide) designed and constructed to express SAT1 or at least a functional portion of the SATE

[0138] The phrase “functional portion” as used herein refers to part of the SAT1 protein which exhibits functional properties of the enzyme such as binding to a polyamine substrate and / or reducing FUS-associated aggregates in a cell. According to some embodiments of the invention, the functional portion of SAT1 comprises the amino acid(s) E152, R19, and / or L156 (numbering refers to the amino acid sequence of the wild type SAT1 (SEQ ID NO: 27).

[0139] According to some embodiments of the invention, the exogenous polynucleotide sequence may be a DNA or RNA sequence encoding a SAT1 molecule (e.g. SEQ ID NO: 28).

[0140] According to some embodiments of some embodiments of the invention the agent which upregulates SAT1 is spermine and / or spermidine.

[0141] The increased level of SAT1 can be determined at the mRNA level (e.g., by in-situ hybridization, RT-PCR, Northern blot analysis), or at the protein level (e.g., by FACS analysis, immune-fluorescence analysis, Western blot analysis, immune-precipitation analysis).

[0142] As used herein the phrase “an agent which upregulates spermine” refers to any molecule which increases the level of spermine in the cell.

[0143] The term “spermine”, as used herein, refers to a polyazaalkane that is tetradecane in which the carbons at positions 1, 5, 10 and 14 are replaced by nitrogens (by decarboxylation of amino acids), having a chemical formula of C10H26N4, and a CAS number 71-44-3, or a naturally occurring or synthetic analogue of same. Spermine is an important polyamine which is found in all living organisms, and has broad actions on cellular metabolism, has a role as an antioxidant, an immunosuppressive agent and a fundamental metabolite.

[0144] Methods of quantifying the levels of spermine include for example, liquid chromatographymass spectrometry (LC-MS / MS), or nuclear magnetic resonance (NMR) metabolomics to determine plasma metabolome (e.g., described in Stefan Senekowitsch et al., Nutrients. 2023; 15(8): 1852; which is fully incorporated herein by reference).

[0145] According to some embodiments of the invention, an agent which increases the level of spermine in the cell is spermidine.

[0146] According to some embodiments of the invention, an agent which increases the level of spermine in the cell is spermine.

[0147] According to some embodiments of the invention, an agent which increases the level of spermine in the cell is a synthetic spermine analogue.

[0148] Synthetic analogues of spermine include, but are not limited to, canavalmine, DENSpm (N(1),N(1 l)-diethylnorspermine), CPENSpm (N(l)-ethyl-N(l l)-[(cyclopropyl)-methy]-4,8- diazaundecane), CHENSpm (N(l)-ethyl-N(l l)-[(cycloheptyl)methy]-4,8-diazaundecane), and Ivospemin.

[0149] According to an aspect to some embodiments of the invention, there is provided a method of treating a subject having a disease characterized by accumulation of FUS-associated aggregates, the method comprising administering to the subject a therapeutically effective amount of Withaferin A, thereby treating the subject.

[0150] As used herein the term “Withaferin A” (also known as “WA”) refers to a compound having IUPAC (22R)-4p,27-Dihydroxy-5,6P:22,26-diepoxy-5P-ergosta-2,24-diene- 1,26-dione, optionally CAS 5119-48-2 or a synthetic analogs of same.

[0151] Non-limiting examples of synthetic or semi-synthetic analogs of WA include withalongolide A, a C-19 hydroxylated congener of withaferin A; withalongolide A 4,27- diacetate; and withalongolide A 4,19,27-triacetate. WA analogs can be qualified by their their cytotoxic activity against various cancer cell lines such as head and neck squamous cell carcinoma (HNSCC, JMAR), breast cancer cells (MDA-MB-231), melanoma (SKMEL-28), and colon cancer cells (DR081-1), as well as against normal fetal lung fibroblast (MRC-5) cells (e.g., as described in Hashim F Motiwala et al., 2013, “Synthesis and Cytotoxicity of Semisynthetic Withalongolide A Analogues” ACS Med Chem Lett. 4(11): 1069-1073, which is fully incorporated herein by reference).

[0152] It is noted that WA has the ability to penetrate the BBB when administered orally or intraperitoneally Dar NJ., et al., 2015, “Pharmacologic overview of Withania somnifera, the Indian Ginseng”. Cell Mol Life Sci.72: 4445-4460), and it was well-tolerated by patients with osteosarcoma when administered orally (Pires et al., 2020; “Safety and pharmacokinetics of Withaferin-A in advanced stage high grade osteosarcoma: a phase I trial”. J Ayurveda Integr Med. 11: 68-72).

[0153] According to some embodiments, the proteasome inhibitor is formulated in a formulation for enhancing penetration of the proteasome inhibitor through the blood brain barrier (BBB).

[0154] According to some embodiments, the agent which upregulates SAT1 or spermine is formulated in a formulation for enhancing penetration of the agent through the blood brain barrier (BBB).

[0155] According to some embodiments, the Withaferin A is formulated in a formulation for enhancing penetration of the agent through the blood brain barrier (BBB).

[0156] According to some embodiments, the formulation comprises a nano-carrier, which improves drug transport through the BBB.

[0157] Nano-carriers, such as nanoparticles are well known in the art and can assist the penetration of a drug through the BBB. These include, but are not limited to, polymeric NP, lipid-based nanoformulations such as solid lipid nanoparticles (SLN) and liposomes, polymeric micelles- based nanoformulations such as Pluronic micelles, dendrimers, metals, quantum dots, nanogels, magnetic NPs (MNPs) composed of an iron oxide core, extracellular vesicles (EVs), and liposome- like natural carriers. NP-based formulations are usually a colloidal system made of polymers, lipids, or other large macromolecules such as albumin, and they can cross the BBB, e.g., through membrane transcytosis or bypass efflux transporters. NPs that have been studied for brain delivery include polymeric NPs such as poly(D,L-lactide-co-glycolide) (PLGA) and poly(butyl- cyanoacrylate) (PBCA). With respect to polymeric NP it was found that rod-like polymeric nanoparticles showed superior BBB permeation compared to spherical counterparts of same volume (Reviewed in Yo Han Song et al., 2023. Advances in Colloid and Interface Science, Vol. 320, 103008; Andrew M Hersh et al., Int J Mol Sci. 2022, 23(8):4153, Kaining Zhi et al., 2021, Pharmaceutics. 13(4):5; each of which is fully incorporated herein by reference in its entirety). Albumin nanoparticles can penetrate the BBB via the mechanisms of SPARC- and gp60-mediated biomimetic transport (Tingting Lin et al., 2016, ACS Nano, Vol. 10, issue 11; Tincu lurciuc CE, et al., 2023. Polymers (Basel). 15:3969, each of which is fully incorporated herein by reference in its entirety).

[0158] According to some embodiments, the nano-carrier comprises a Poly(lactic-co-glycolic acid) (PLGA) nanoparticle.

[0159] According to some embodiments, the nano-carrier comprises an albumin nanoparticle.

[0160] According to some embodiments, the proteasome inhibitor or the composition-of-matter of some embodiments of the invention is co-administered with a P-glycoprotein (P-gp) inhibitor.

[0161] A non-limiting example of a suitable P-glycoprotein (P-gp) inhibitor is Tariquidar.

[0162] According to an aspect to some embodiments of the invention, there is provided a method of reducing accumulation of FUS -associated aggregates in cells of a subject, the method comprising contacting the cells of the subject with a proteasome inhibitor, thereby reducing the accumulation of FUS -associated aggregates in the cells of the subject.

[0163] According to an aspect to some embodiments of the invention, there is provided a method of reducing accumulation of FUS -associated aggregates in cells of a subject, the method comprising contacting the cells of the subject with an agent which upregulates spermidine / spermine N1 -acetyltransferase 1 (SAT1) or spermine, thereby reducing the accumulation of FUS -associated aggregates in the cells of the subject.

[0164] According to an aspect to some embodiments of the invention, there is provided a method of reducing accumulation of FUS -associated aggregates in cells of a subject, the method comprising contacting the cells of the subject with Withaferin A, thereby reducing the accumulation of FUS -associated aggregates in the cells of the subject.

[0165] Contacting cells with a molecule (e.g., a potential drug molecule, or an approved drug molecule) such as a proteasome inhibitor, an agent which upregulates spermidine / spermine Nl- acetyltransferase 1 (SAT1), an agent which upregulates spermine, or Withaferin A, can be performed by any in vitro conditions including for example, adding the molecule to cells of a subject (e.g., a primary cell culture, a cell line) or to a biological sample comprising same (e.g., a fluid, liquid which comprises the cells) such that the molecule is in direct contact with the cells. According to some embodiments of the invention, the cells of the subject are incubated with the molecule. The conditions used for incubating the cells are selected for a time period / concentration of cells / concentration of molecule / ratio between cells and the molecule and the like which enable the molecule to induce cellular changes, such as changes in reducing FUS aggregates from the cells.

[0166] According to an aspect to some embodiments of the invention, there is provided an in-vitro method of selecting a molecule for treatmet of a disease characterized by accumulation of FUS- associated aggregates, the method comprising:

[0167] (a) contacting cells carrying a mutation in the FUS gene which causes mis-folding of the FUS with the molecule,

[0168] (b) determining presence and / or level of FUS-associated aggregates in the cells prior to and following the contacting, wherein a reduction in level of the FUS-associated aggregates following the contacting indicates that the molecule is suitable for the treatment of the disease characterized by accumulation of FUS-associated aggregates.

[0169] According to some embodiments of the invention, the cells are neuron cells.

[0170] According to some embodiments of the invention, the cells are motor neuron cells.

[0171] According to some embodiments of the invention, the cells are from the frontal and temporal lobes of the brain.

[0172] Following is a list of agents capable of upregulating the expression level and / or activity of SAT1.

[0173] To express exogenous SAT1 in mammalian cells, a polynucleotide sequence encoding SAT1 (e.g., SEQ ID NO: 27) or a functional portion thereof is preferably ligated into a nucleic acid construct suitable for mammalian cell expression. Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.

[0174] It will be appreciated that the nucleic acid construct of some embodiments of the invention can encode SAT1 homologues which exhibit the desired activity (z.e., reduce FUS-associated aggregates). Such homologues can be, for example, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical to SEQ ID NO:27, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals -9.

[0175] Constitutive promoters suitable for use with some embodiments of the invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoters suitable for use with some embodiments of the invention include for example neuron- specific promoters such as the neurofilament promoter (e.g., described in Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), the synapsin 1 promoter (described in Vera Lukashchuk et al., 2016, Mol. Ther. Methods Clin. Dev. 3: 15055; which is fully incorporated herein by reference), and motor neuron specific promoters such as Hb9 which was shows to derive gene expression mainly within the motor-related areas of the brain stem (described in Vera Lukashchuk et al., 2016, Supra).

[0176] The nucleic acid construct (also referred to herein as an "expression vector") of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.

[0177] The nucleic acid construct of some embodiments of the invention typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention.

[0178] Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated.

[0179] Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations that are suitable for some embodiments of the invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.

[0180] In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

[0181] Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of SAT1 mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11- 30 nucleotides upstream. Termination and polyadenylation signals that are suitable for some embodiments of the invention include those derived from SV40.

[0182] In addition to the elements already described, the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

[0183] The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.

[0184] The expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

[0185] Examples for mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[0186] Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0187] As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. Thus, the type of vector used by some embodiments of the invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein. For example, bone marrow cells can be targeted using the human T cell leukemia virus type I (HTLV-I) and kidney cells may be targeted using the heterologous promoter present in the baculovirus Autographa californica nucleopolyhedro virus (AcMNPV) as described in Liang CY et al., 2004 (Arch Virol. 149: 51-60).

[0188] Recombinant viral vectors are useful for in vivo expression of SAT1 since they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0189] Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses. Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non- viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral construct such as a retroviral construct includes at least one transcriptional promoter / enhancer or locus -defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.

[0190] An agent capable of upregulating SAT1 may also be any compound which is capable of increasing the transcription and / or translation of an endogenous DNA or mRNA encoding the SAT1 and thus increasing endogenous SAT1 activity.

[0191] Thus, for example an agent capable of upregulating SAT1 may be the mRNA molecule itself which encodes the SAT1. mRNAs therapies are advantageous as they utilize a cell’s translational machinery to code production of the desired protein in vivo, saving time and expense on purification procedures.

[0192] Methods of stabilizing mRNA are known in the art and include modulation of the length of the polyadenine tail found at the 3” end of the mRNA transcript. Alternativley, or additionally, the mRNA cap found at the molecule’s 5’ end can be modified. The naturally occurring cap structure typical in mammalian cells has a tendency to be improperly incorporated into mRNAs synthesized in vitro, rendering them less effective. Synthetic “anti-reverse cap analogs” (e.g. those commercially available at Thermo Fisher Scientific) can prevent this misincorporation, which results in more stable mRNA with improved translational efficiency. In order to reduce immunogenicity, substitution of particular nucleotides can be exchanged with chemically modified alternatives such as 5-methylcytosine or pseudoruidine. Such substiutions can mute the immune response whilst also bolstering the stability of the mRNA and efficiency of translation. Other exemplary chemically modified nucleotides are described herein above. In certain cases, particular nucleotides can be incorporated into the mRNA to increase immunogenicity. This may be particularly relevant for vaccine therapy.

[0193] Alternatively, or additionally, the mRNA may be encapsulated in lipid-based particles to enhance fusion with the lipid cell membrane.

[0194] An agent capable of upregulating SAT1 may also be an exogenous polypeptide including at least a functional portion (as described hereinabove) of the SAT1.

[0195] An agent capable of upregulating SAT1 activity is spermine or a spermine synthetic analogue.

[0196] An agent capable of upregulating SAT1 activity is spermidine or a spermidine synthetic analogue.

[0197] Upregulation of SAT1 can be also achieved by introducing at least one SAT1 substrate. Non-limiting examples of such agents include DENSpm (N1,N 11 -diethylnorspermine, which is a poly amine analogue).

[0198] The proteasome inhibitors, the agents which upregulate SAT1 or spermine of some embodiments of the invention, or the Withaferin A of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

[0199] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0200] Herein the term "active ingredient" refers to the proteasome inhibitor(s), the agent(s) which upregulate(s) SAT1 or spermine of some embodiments of the invention, or the Withaferin A of some embodiments of the invention accountable for the biological effect.

[0201] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0202] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0203] Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0204] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0205] Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

[0206] Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

[0207] Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0208] Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0209] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0210] For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0211] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0212] Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0213] For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0214] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0215] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0216] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[0217] The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0218] Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g., the proteasome inhibitor(s), the agent(s) which upregulate(s) SAT1 or spermine of some embodiments of the invention, or the Withaferin A of some embodiments of the invention) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., ALS or FTD) or prolong the survival of the subject being treated.

[0219] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0220] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0221] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-l).

[0222] Dosage amount and interval may be adjusted individually to provide brain and / or spinal cord levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0223] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0224] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0225] Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

[0226] As used herein the term “about” refers to ± 10 %.

[0227] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0228] The term “consisting of’ means “including and limited to”.

[0229] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0230] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0231] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0232] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0233] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0234] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0235] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0236] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0237] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0238] EXAMPLES

[0239] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0240] GENERAL MATERIALS AND EXPERIMENTAL METHODS

[0241] HEK293T cells were cultured in standard DMEM, supplemented with 10% fetal bovine serum (FBS), 1% Penicillin-Streptomycin, at 37 °C, 5% CO2 incubator.

[0242] To assay aggregation, cells were seeded in 6-well plates (3xl05cells per well), and one day after seeding the cells were co-transfected with 1250 ng (nanogram) pcDNA3-DsRed and 1250 ng pTrex-FUS-R521H-YFP using PEI transfection reagent (Thermo Fisher Scientific), as described in Rozales K, et al. 2022.

[0243] For HTT-polyQ samples, cells were seeded at 25,000 cells per well in 96 well plates one day before transfection, and transfected with 200 ng of pTrex-HTT-134Q-GFP DNA and 200 ngr of pcDNA3-DsRed DNA.

[0244] All cells were assayed for aggregation levels using FACS 48 hours post transfection, as detailed in Rozales K, et al. 2022.

[0245] Treatments with MG-132 (10 pM), Velcade (10 pM), Withaferin A (6 pM) were added for 18 hours before FACS was performed. The percentage of aggregate-containing cells was quantified using the PuLSA method, compared these percentages of cells treated with DMSO as a control. Aggregation modulation scores for each treatment were calculated as Log2(%AGG+(treatment) / %AGG+(control)), as described in Rozales K, et al. 2022. Each experiment was performed in 3 independent biological replicates on different dates, with additional several biological replicates on the same day. Graphs represent mean and STE for all replicates. Confidence intervals (95%, appear as dashed lines).

[0246] Cloning and plasmid preparation

[0247] FUS-WT-YFP, FUS-R521H-YFP, and FUS-R518K-YFP constructs were performed as described in Rozales, K., et al., 2022.

[0248] FLAG-tagged BAG3 was generated as described in Rozales et al. 2022. The different mutant isoforms were generated in house, Q488A mutation was prepared by utilizing QuikChange 2 Site-Directed Mutagenesis Kit to introduce the point mutation, substituting glutamine with alanine. Additionally, the BAG3-AC isoform and the AN-BAG3 were both generated from the full- length isoform (WT isoform) using PCR and gateway cloning (see primers in Table 2 below).

[0249] SAT1 (WT) was cloned from HEK293T genomic DNA using PCR (see primers Table 2) into a pcDNA3.1-ccdb-FLAG plasmid. SAT1 point mutations (R19A, L156F, E152K) were also generated in house based on the WT SAT1 by utilization of the QuikChange 2 Site-Directed Mutagenesis Kit, primers are attached in Table 1 .

[0250] Cell culture and transfection

[0251] HEK293T cells were grown in standard DMEM supplemented with 10% FBS (fetal bovine serum), 1% Penicillin- Streptomycin in 37 °C, 5% CO2 incubator. For the FUS transfections, HEK293T cells (3X105cells per well) were seeded on 6-well plates and transfected using PEI transfection reagent (Thermo Fisher Scientific) one day after seeding, according to the manufacturer instruction, with 1250 ng (nano-gram) of FUS DNA and 1250 ng of chaperone DNA / dsRed as control. Cells were assayed 48 hours following the transfection. PulSA method

[0252] At 48 hour after transfection, cells were trypsinized, resuspended in media with serum, placed on ice, and DAPI regent was added (5 pM) in order to assay cell viability. Cells were then taken to Flow cytometry in order to differentiate between subpopulations with diffused cellular fluorescence and those with protein aggregates, as described in Rozales et al. 2022. To obtain Aggregation modulation scores, cells of appropriate size were considered during the FACS analysis, and non-viable cells (positive for DAPI) were excluded. The present inventors have then quantified the percentage of cells that showed a fluorescent aggregate pattern (AGG+), and compared these percentages to cells co-transfected with DsRed as a control. Aggregation modulation scores for each chaperone were calculated as Log2(%AGG+ (chaperone or treatment) / %AGG+ (control)). Confidence intervals (CI) (95%, appear as dashed lines) were calculated according to the variations between different DsRed co-transfected cells replicates, such that they represent twice the standard deviation of Aggregation modulation scores calculated between different replicates of DsRed performed on the same day, as described in Rozales et al. 2022. Chaperones or samples were called as significant modulators if their average Aggregation modulation scores + (or -) SEM was below (or above) the 95% CI. Specifically, an empirical p- value was indicated such that p < 0.05 if these values were below the 95% CI, and p < 0.003 if these values were below the 99.7% CI (z.e., 3 times STD).

[0253] RNA-seq and data analyses

[0254] RNA-seq was performed as described in Rozales et al. 2022. Scatter plot of log2 fold change (LFC) of MG-132 18 hours vs control (x-axis), compared to mild heat shock (42°C) 8 hours vs control (y- axis). Fold change was calculated using Deseq2 package (Love, M. I., et al., 2014, “Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2”, Genome Biol. 15, 550). In grey (Figure 9A), all background genes after TPM (Transcript Per Million) thresholding. In blue, strict MG-132 unique program, that contain genes that were significantly changed after MG-132 treatment for both 8 hours and 18 hours (Deseq2- |LFC>1|, Deseq2-padj<0.01) and weren’t significant after HS treatment (Deseq2-|LFC<1|, Deseq2-padj>0.05). SAT1, red labeled, is among the strict program. RNA-seq presented in (Figure 3A), shows a scatter plot of log2 fold change of MG-132 18 hours vs control (x-axis), compared to mild heat shock (42°C) 8 hours vs control (y- axis). Presented are Chaperones families taken form Brehem et al., 2014 (“A chaperome subnetwork safeguards proteostasis in aging and neurodegenerative disease”, Cell Rep. 9, 1135- 50). Immunofluorescence staining

[0255] Cells were cultured on cover-slips and transfected as above. Two days post transfection, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton-xlOO in IF buffer (5% FCS, 2% BSA in PBS), then immunostained by primary anti-FLAG antibody (Sigma- Aldrich, F1804) followed by secondary AlexaFluor 647 Donkey Anti-Mouse (Jackson) antibody to image FLAG-tagged chaperones. The far-red secondary antibody was used to eliminate fluorescence overlap between the YFP / GFP-tagged aggregates and the chaperones. The cells were additionally stained with DAPI to mark nuclei.

[0256] Co-immunoprecipitation

[0257] For co -immunoprecipitation (co-IP) of FLAG-tagged chaperones, lysis buffer was prepared as follows: 50 mM HEPES pH 7.9, 0.5% Triton X-100, 5% glycerol, 150 mM NaCl and 2 mM EDTA and Protease inhibitors (Sigma-Aldrich P5726). Wash buffer was prepared as follows: 50 mM HEPES pH 7.9, 1% Triton X-100, 5% glycerol, 150 mM NaCl and 2 mM EDTA. Cells were transfected as above. At 48 hours post transfection, cells were placed on ice, washed with ice- cold PBS, lysis buffer was added and cells were collected by scraping. Lysates were centrifuged (8200 g for 10 minutes at 4 °C), and supernatant was transferred to new tubes. Samples were IP-ed using 10 pl of EZ view Red anti-FLAG M2 Affinity Gel beads (Sigma- Aldrich, F2426), which were added to a sample volume containing equal number of 260 nm OD units. Additional co-IP buffer was added to reach a 600 pl volume for each sample and samples with beads were slowly rotated 3 h at 4 °C. Beads were then washed 7 times with 1 ml of wash buffer. Following the last wash, the beads were resuspended in equal volumes of wash buffer, protein sample buffer was added, and the samples were boiled at 100 °C for 5 minutes. Equal volumes were then run on an 8% or a 12.5% gel, and immunoblotted using anti-FLAG (Sigma- Aldrich, Fl 804), anti-HSC70 / HSP70 (Enzo Life Sciences, N27F34) .

[0258] Proteasome inhibitors and other drugs used

[0259] Cell were seeded in 6-well plates at a density of 350,000 cells per well. One day after seeding, cells were co-transfected with FUS-R521H-YFP and DsRed. At 30 hours post transfection, cells were treated with different materials as following:

[0260] (1) MG-312 (Sigma-Aldrich M7449-1ML Ready Made Solution) at a final concentration of 10 pM (or as otherwise indicated), DMSO was used as a control for MG- 132, same goes for HTT samples in case of MG- 132.

[0261] (2) Marizomib (Sigma- Aldrich SML1916-100UG) at 80 nM (or as otherwise indicated) for both HEK293T cells and primary neuronal cells.

[0262] (3) Velcade aka Bortezomib (selleckchem S 1013) at 10 pM. (4) Withaferin A (Sigma- Aldrich W4394) - cells were exposed to serial doses of WA (2 pM, 4 pM, 6 pM) for 12 hours and 18 hours, respectively. At 48 hours after transfection, all cells were subjected to PulSA aggregation modulation analysis.

[0263] The present inventors used the autophagy inhibitor Bafilomycin A (Sigma- Aldrich B1793- 2UG) - (200 nM for 18hours) in cells co-transfected with FUS-R521H-YFP and DsRed, treated with either MG-132 (10 pM for 18 hours) or DMSO as a control, or introduced Bafilomycin A (200 nM for 18 hours) into samples co-transfected with FUS-R521H-YFP and BAG3, comparing them to control samples treated with DMSO. Subsequently, the present inventors conducted PulSA analysis.

[0264] Berenil was introduced to samples co-transfected with FUS-R521H-YFP and DsRed, that were either treated with MG-132 (10 pM, 18 hours) or not (Benreil- Sigma-Aldrich D7770).

[0265] Natural polyamines (Sigma-Aldrich, Spermine S4264, Spermidine S0266, Putrescine P5780) were added to cells that were co-transfected with DsRed and FUS-R521H-YFP at varying concentrations (5 pM, 20 pM, 100 pM) for 72 hours then subjected to PulSA analysis.

[0266] Metabolomics analysis - Detection and quantification of cisplatin using liquid chromatography-mass spectrometry (LC-MS).

[0267] Sample preparation: For HEK293T samples, the present inventors used a 6-well plate, while for neurons, the present inventors used 24-well plates coated with poly-D-Lysine. The plates were placed on ice, and the medium was discarded. The cells were then washed once with PBS at room temperature and ice-cold (-20°C) metabolite extraction solution (MeOH:ACN: Water, 5:3:2, v / v / v) was added to each well. The plates were shaken in at 4 °C for 20 minutes to complete the metabolite extraction process. After extraction, every three replicates were combined (4 wells for primary neurons were combined) into one sample to enrich the population. The combined samples were centrifuged at 4 °C for 15 minutes at maximum speed. The supernatant was transferred into a clean Eppendorf tube, dried down, and re-suspended in extraction solution. The samples were then stored at -80 °C until liquid chromatography-mass spectrometry analysis was performed. The samples were run in SIM mode (of the mass spec for metabolomics) to increase sensitivity. Subsequently, the samples were normalized to protein quantification using a BCA kit.

[0268] Analysis method: LC-MS metabolomics analysis was performed using DionexTM ultra high-performance liquid chromatography (UPLC) system coupled to Q-ExactiveTM Orbitrap Mass Spectrometer (ThermoFisher Scientific) was used with a resolution of 70,000 at 200 mass / charge ratio (m / z), in electrospray ionization at positive mode (3500 v) was used at tSIM mode for spermine, spermidine, putrescine and their acetylated forms to increase sensitivity (e.g. the following m / z were followed). AGC target was set to 5E04 with max IT of 200 ms. RF level was set to 50, Sheath and Aux gas was set to 30 and 13 respectively (arbitraty units) with capillary tube temperature at 263 °C and Aux gas heater temperature at 425 °C. HPLC setup consisted of SeQuant® ZIC®-cHILIC TM (100 mm x 2.1 mm, 3 pm. 10 pL of biological extracts were injected and the compounds were separated with mobile phase consisting of water with 0.1% formic acid (mobile phase A) and of mobile phase B composed of Acetonitrile. Flow rate and column temperature were maintained at 0.4 mL / minute and 45°C, respectively, for a total run time of 16 minutes. Starting at 95% mobile phase B with a gradient lasting 10 minutes until reaching 90% mobile phase A. Polyamines and its acetylated forms were detected using a mass accuracy below 5 ppm. Xcalibur (ThermoFisher Scientific) was used for data acquisition. Data Analysis was performed using Skyline Daily246 where mass accuracy and chromatographic retention time were used for identification and peak integration using an in-house authentic standards library. Peak intensities were normalized to ug protein.

[0269] Small interfering RNA

[0270] Cells were seeded at 150,000 cells per 6-well plate one day before transfection. siRNA transfections were performed using RNAiMAX lipofactamine reagent (Thermo Fisher Scientific), according to the manufacturer’s protocol, with siRNA concentration of 15 nM. One day after transfection of the siRNA (Dharmacon Smartpool, L-012109-02-0005 for siHSFl, L-011957-00- 0005 for siBAG3 and L-010382-00-0005 for siSATl) or siControl (Dharmacon Smartpool, 001810-10-05 or 020585-01-0005), cells were co-transfected with 1250 ng of FUS-R521H and 1250 ng of DsRed using PEI. Cells were analyzed 48 hours later.

[0271] Neuronal cultures, infections, and image analysis

[0272] All viruses were packaged with the AAV-PHP.B capsid as described in Rozales et al. 2022. All viruses were then tittered using qPCR in the same assay and dosage consistency was kept between infections. Isolation and culturing of rat primary cortical neurons were done as previously described, and received an ethics approval from the Technion, Israel (Ethics certificate No. IL- 130- 09-17). Briefly, P0 neonates, from timed-pregnant white coat female Sprague Dawley female rats (Charles River, SAS SD, strain code 400), were euthanized and their cortex was dissected and dissociated. Then, 150,000 cells were plated in 24-well flat bottom plates on coverslips (12 mm, thickness -0.1 mm) coated with Poly-D-Lysine (Sigma- Aldrich P7886-50MGP) in Minimum essential medium (MEM), glutamine-free based Neuronal growth medium (5% fetal bovine serum, 2% B27, 1% Glutamax (100x, Invitrogen), 2% 1 mol / L d-glucose, 0.1% pL serum extender (BD Biosciences, Cat. No. 355006)). Cultures were incubated at 37 °C and 5% CO2. At 5 days after plating, media was changed and supplemented with cytosine arabinosine (Ara-C, Sigma, C6645) at a final concentration of 4 pM, to reduce the growth of glia in the cultures. Cultures were infected the same day with pAAV-FUS-R521H-YFP. After that media was changed every 2 days, and cells were imaged 6 days after infection. Image analysis was performed as described in Rozales et al. 2022. For image analysis of neurons treated with Marizomib and MG-132, the present inventors infected neuronal rat cells with pAAV-FUS-R521H-YFP using the same infection conditions and culturing media as mentioned previously. Subsequently, the cells were treated with 18 hours of marizomib (80 nM) or MG-132 (7.5 pM) along with their respective controls. Following treatment, the cells underwent confocal imaging using the same imaging parameters as described earlier. The present inventors utilized a modified version of the image analysis classifier to accurately detect the aggregation of these cell sets. In the MG-132 set, the present inventors identified 741, 957, and 555 different cells from each control set, and 831, 1185, and 278 from the MG-132-treated cells. In the marizomib set, a total of 1077, 411, and 156 neurons were treated with Marizomib and 801, 709 and 148 cells were their respective controls.

[0273] Incucyte for neuron

[0274] Isolation and culturing of rat primary hippocampal neurons were done as previously described, then 150,000 cells were plated in 24-well flat bottom plates coated with Poly-D-Lysine (Sigma-Aldrich P7886-50MGP) using the same growth media and conditions described in culturing section. At 5 days after plating, media was replaced as described above and cultures were infected with pAAV-FUS-R521H-YFP, after that media was changed every 2 days, until cells were imaged 6 days after infection. 18 hours before imaging cells were treated with 80 nM of marizomib or 7.5 pM MG-132 in addition to propidium iodide solution (Sigma-Aldrich - P4864-10ML, 1.0 mg / ml in water) that was further diluted 1:1000 in samples. Live imaging was done using Incucyte SX5 System, a high throughput continuous live cell imager. Nikon Plan Fluor objective was used in xlO / 0.30 magnification, and image analysis for each channel (Green and Orange) was done using the built-up Incucyte® SX5 Live-Cell Analysis System.

[0275] Table 2: List of primers

[0276] EXAMPLE 1

[0277] PROTEASOME INHIBITOR TREATMENT SIGNIFICANTLY REDUCES FUS AGGREGATION

[0278] Protein homeostasis, also known as proteostasis, is crucial for cellular function and is upheld by a tightly regulated protein quality control (PQC) system. This system, including the ubiquitin-proteasome system (UPS), molecular chaperones and co-chaperones, plays a critical role in ensuring the degradation of misfolded and detrimental protein species. Despite the significance of these components, the exact mechanisms orchestrating their interplay remain only partially elucidated. In this study, the present inventors investigated the influence of molecular chaperone functionality on the modulation of protein aggregation and its potential mediation by the proteasome.

[0279] Experimental results

[0280] Effect of proteasome inhibitor on protein aggregation

[0281] The present inventors employed MG- 132, a direct proteasome inhibitor, which causes the accumulation of misfolded proteins such as HTT-polyQ and TDP-43 due to impaired turnover mechanisms (Li, X., et al., 2010. “Inhibiting the ubiquitin-proteasome system leads to preferential accumulation of toxic N-terminal mutant huntingtin fragments”. Hum Mol Genet 19, 2445-2455).

[0282] HTT-polyQ aggregation was enhanced upon treatment with MG- 132 - Indeed, when HTT-134Q (poly Q) HEK293T cells were treated for 18 hours with 10 pM of MG-132, the anticipated exacerbating effect on aggregation was shown (Figure 3B, right side). Thus, the HTT- polyQ aggregation was in fact enhanced upon treatment with MG-132 (Figure 3B).

[0283] Proteasome inhibitor drugs significantly protect from mutant FUS aggregation - When mutFUS-HEK293T models were subjected to identical conditions of MG-132 treatment an unexpected reduction in mutFUS aggregation by approximately 30% was observed as compared to the DMSO control (Figure 3B, left side), despite no significant impact on cell viability (Figure 5A).

[0284] Heat chock treatment does not have any beneficial effect on ALS-FUS aggregation - The effect of the MG-132 proteasome inhibitor on FUS aggregation was particularly surprising, especially considering the failure of heat shock to elicit a similar effect on aggregation (Figures 2B and 5B). Thus, the present inventors found that, while heat shock treatment did not yield any beneficial effects on ALS-FUS aggregation (Figure 2B), strikingly, treatment with MG-132 had a significant protective effect on mutant FUS aggregation (Figure 3B, left side).

[0285] Additional proteasome inhibitors have a similar inihibitory effect on FUS aggregation - To ascertain the specificity of the unexpected aggregation inhibition by MG-132, the present inventors evaluated the impact of alternative proteasome inhibitors. Employing Velcade (Bortezomib), an FDA-approved therapeutic agent for refractory multiple myeloma, and Marizomib, an irreversible proteasome inhibitor, for 18 hours, significant reductions in mutFUS aggregation were shown (Figure 3B, and Figures 5C-D).

[0286] Withaferin A (WA), a natural compound which is a steroidal lactone sourced from the medicinal plant "Acnistus arborescens", was identified through high-throughput screening for its ability to induce the HSF1 activation (Santagata, S., et al. 2012. “Using the heat-shock response to discover anticancer compounds that target protein homeostasis”. ACS Chem Biol 7, 340-349), and was shown to inhibit proteasome function (Khan, S., et al., 2012, “Withaferin A induces proteasome inhibition, endoplasmic reticulum stress, the heat shock response and acquisition of thermotolerance”. PEoS One 7, e50547, Khedgikar, V., et al. 2013, “Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone”. Cell Death Dis 4, e778). As shown in Figures 3B (left side) and Figure 4A, Withaferin A also showed a significant prevention of mutant FUS aggregation. Thus, HEK293T cells were exposed to WA for 18 hours, and PulSA analysis revealed that WA was able to alleviate aggregation (Figure 4A). These results demonstrate that proteasome inhibitor treatment significantly reduces FUS aggregation, and suggest a potential involvement of the activation of the proteotoxic stress response as a downstream pathway.

[0287] These results show that while the proteasome inhibitor drugs significantly protect from mutant FUS aggregation, in the case of HTT-polyQ, MG-132 leads to enhancement of aggregation.

[0288] MG-132 treatment induces some PQC genes exclusively - Using RNA-seq, the present inventors compared the cellular response (in the form of gene expression profiles) in cells treated with heat shock (Figure 2B) to those treated with the proteasome inhibitor MG-132 (Figure 3B), which is also known to activate a proteotoxic stress response (Bush, K. T., et al., 1997, “Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermos tolerance”. J Biol Chem 272, 9086-9092). Figure 3A presents the results of the RNA-seq analysis. While many of the activated genes were shared, some were specifically induced in response to MG-132. The results presented in Figure 3A demonstrate that MG-132 treatment induces some PQC genes exclusively.

[0289] Thus, these results clearly demonstrate that different inducers of the proteotoxic stress response, including small molecule FDA-approved drugs, have strikingly different and unexpected protective effects in the case of ALS-FUS, in contrast to other neurodegeneration-related aggregating proteins examined. EXAMPLE 2

[0290] FUS AGGREGATION INHIBITION BY PROTEASOME INHIBITORS IS TRANSCRIPTIONALLY MEDIATED

[0291] Experimental results

[0292] To directly ask whether the mechanism underlying MG-132 alleviation of mutFUS aggregation is transcriptionally-mediated, the present inventors utilized small interfering RNA (siRNA) targeting HSF1. The depletion of HSF1 endogenous levels was aimed at uncovering its involvement in this process, supported by previous literature indicating that MG-132 proteasome inhibitor treatment elicits a transcriptional program which resembles the proteotoxic stress response, and is mediated by HSF1 [Mathew, A., et al., 2001. “Stress-specific activation and repression of heat shock factors 1 and 2”, Mol Cell Biol. 21, 7163-71]. siHSFl reduces levels of endogenous HSF1 - As shown in Figure 6, siHSFl was able to reduce the endogenous levels of HSF1 by 70%, while siControl maintains normal levels of HSF1.

[0293] Cells which were co-transfected with FUS-R521H-YFP and siControl and were further treated with MG-132 exhibit alleviation of aggregation - The results revealed that cells cotransfected with FUS-R521H-YFP alongside siControl, which were treated with MG-132 (as described in the General Materials and Experimental Methods), exhibited a similar aggregation alleviation as samples treated solely with MG-132 (Figure 4B).

[0294] Cells which were co-transfected with FUS-R521H-YFP and siHSF exhibit minimal alleviation of aggregation - Samples treated with MG-132 on the background of siHSFl demonstrated minimal (non-significant) alleviation of aggregation (Figure 4B).

[0295] Considering that classical heat shock was unable to provide any aggregation protection (Figure 5B), yet silencing of the major transcription factor for heat shock proteins impaired MG- 132 ability to alleviate aggregation (Figure 4B), without being bound by any theory the present inventors have hypothesized that proteasome inhibitors induce a unique transcriptional program, distinct from that mediated by heat shock, which confers protection against FUS aggregation.

[0296] EXAMPLE 3

[0297] THE CHAPERONE BAG3 PLAYS A ROLE IN PROTEASOME INHIBITOR-MEDIATED INHIBITION OF FUS AGGREGATION

[0298] Experimental Results

[0299] To substantiate the hypothesis that MG132-mediated rescue of aggregation is orchestrated via a unique transcriptional program, the present inventors have primarily focused on identifying mRNA transcripts implicated in this unique transcriptional program. First, the present inventors have examined particularly mRNAs associated with the protein quality control family. The present inventors have initiated an RNA-seq experiment, treating HEK293T cells with MG- 132, or with heat shock, for different durations. Then the log-fold change (LFC) of gene expression was plotted, focusing on chaperone mRNAs, in cells treated with MG-132 (10 pM for 18 hours) compared to control cells, against the LFC of gene expression in cells subjected to heat shock (42°C for 8 hours) compared to their respective controls (Figure 7A). The present inventors were particularly interested in genes within the protein quality control repertoire notably upregulated in response to the MG-132 proteasome inhibitor but not in heat shock (Figure 7A). While such genes were scarce, several genes were induced more in the presence of MG-132 compared to heat shock. Of particular interest was the induction of the BAG3 gene (Gene ID 9531, GeneBank accession Nos. NM_004281 (SEQ ID NO: 29) and NP_004272 (SEQ ID NO: 30) for the mRNA and polypeptide sequence, respectively).

[0300] BAG3 does not exhibit any colocalization with mutFUS aggregates - Since co-chaperones like BAG3 have been previously documented to be involved in regulating in FUS and TDP-43 phase separation, i.e. the disassembly of stress granules alongside HSPB8 [Ganassi, M., et al., 2016, “A Surveillance Function of the HSPB8-BAG3-HSP70 Chaperone Complex Ensures Stress Granule Integrity and Dynamism”, Mol. Cell. 63, 796-810], the present inventors investigated possible colocalization through immunofluorescent (IF) imaging. IF revealed that cells coexpressing FUS-R521H-YFP together with flag-tagged BAG3 did not exhibit any colocalization of BAG3 with mutFUS aggregates (Figure 8A).

[0301] Knockdown of BAG3 causes a significant exacerbation of aggregation - To further inspect the protective role of BAG3 against mutFUS aggregation, the present inventors employed siRNAs to silence BAG3 expression, which was able to reduce its endogenous levels by 97% (Figure 8B) and assessed its impact on aggregation using FACS-PuLSA. The results demonstrate a significant exacerbation of aggregation following BAG3 knockdown (Figure 7B), underscoring the critical role of B AG3 in aggregation protection.

[0302] BAG3-mediated protection of FUS aggregation is HSP70 dependent - BAG3, a nucleotide exchange factor (NEFs), features a conserved BAG domain at its C-terminus. The conserved BAG domain functions in binding to the ATPase domain of heat shock protein 70 [Bracher, A. et al., 2015 (“The nucleotide exchange factors of Hsp70 molecular chaperones”, Front Mol Biosci. 2, 10)] and facilitates the release of HSP70 clients in collaboration with DNAJ co-chaperones [Bracher, A. et al., 2015, Supra, Rosenzweig, R., et al., 2019 (“The Hsp70 chaperone network”, Nat Rev Mol Cell Biol. 20, 665-680)]. To further inspect the role of BAG3 in FUS aggregation regulation, the present inventors generated a BAG3-isoform lacking the C-terminus (BAG3-AC, Figure 7C). The interaction of BAG3-FL or BAG3-AC with HSP70 was tested via co-IP, and the results show that while BAG3-FL interacted with HSP70, the C-terminal truncation completely abrogated this interaction (Figures 8C-D). When employing the PuLSA method on cells cotransfected with FUS-R521H-YFP and BAG3-AC, the present inventors found that this mutant showed a reversed effect, enhancing FUS aggregation (Figure 7D).

[0303] The Q488A point mutation in the BAG domain reduces the binding of BAG3 to HSP70 - To gain a comprehensive understanding of the contribution of HSP70 interactions to the rescue phenotype and the significance of an intact C-terminal domain, the present inventors introduced a Q488A point mutation in the BAG domain (as described in Gentilella, A. et al., 2011 (“BAG3 expression in glioblastoma cells promotes accumulation of ubiquitinated clients in an Hsp70- dependent manner”, J Biol Chem. 286, 9205-15). This mutant exhibited reduced binding to HSP70 (Figures 8C-D) and this trend was consistent irrespective of the expression of either FUS-WT-YFP (indicated as “WT” in Figure 8C) or FUS-R521H-YFP (indicated as “R521H” in Figure 8C).

[0304] The Q488A mutation reduces ability of the BAG3 to protect cells from FUS-R521H-YFP aggregation - Then the present inventors tested the functional effects of the Q488A mutation on FUS-R521H-YFP aggregation. The data showed that this mutant lost its ability to protect cells from FUS-R521H-YFP aggregation (Figure 7D). Thus, it seems that HSP70 binding to BAG3 is crucial for its function in alleviating FUS-R521H-YFP aggregation.

[0305] Proteasome inhibitor-mediated FUS aggregation protection is abrogated when BAG3 is limiting - After observing a significant upregulation of BAG3 mRNA expression in the presence of MG-132 (Figure 7A) and recognizing its important contribution to aggregation inhibition, the present inventors explored its role further by silencing BAG3 using siRNAs. In this experiment, the present inventors treated samples transfected with either siBAG3 or siControl with MG-132 or DMSO as a control (Figure 7E). Interestingly, while samples treated with siBAG3 and DMSO aggravated FUS aggregation, and siControl and MG-132 samples demonstrated significant alleviation of FUS aggregation, other samples exhibited no significant changes. These results indicate that BAG3 plays a critical role in the activation of the MG132 stress response pathway finally leading to aggregation protection. EXAMPLE 4

[0306] MG132-INDUCED AGGREGATION CLEARANCE IS PARTIALLY MEDIATED BY AUTOPHAGY

[0307] Since the autophagy-lysosome system and ubiquitin-proteasome system (UPS) are the two main routes that cells use for degrading intracellular proteins, the present inventors aimed to explore the potential relationship between UPS inhibition and the induction of autophagy flux. To investigate this, the present inventors employed the autophagy inhibitor Bafilomycin A (200 nM for 18 hours) in cells co-transfected with FUS-R521H-YFP and DsRed, treated with either MG- 132 (10 pM for 18 hours) or DMSO as a control.

[0308] Experimental Results

[0309] Treatment with MG- 132 and Bafilomycin A resulted in a modest aggregation alleviation

[0310] - The results revealed that treatment Bafilomycin A individually led to aggregation aggravation by approximately 50% (Figure 7F). Surprisingly, when MG-132 was combined with Bafilomycin A, there was a modest, yet significant aggregation alleviation of around 20%. Without being bound by any theory, this suggests that potentially, autophagy may play a partial role in the observed inhibitory effect of MG-132 on FUS aggregation.

[0311] BAG3 devoid of the h" domain (BAG3-AN) loses the ability to inhibit FUS aggregation

[0312] - The present inventors aimed to further investigate the contribution of autophagy through BAG3 to FUS aggregation alleviation. The N-terminal domain of BAG3 contains two tryptophantryptophan (WW) domains known to be involved in BAG3-mediated autophagy (Merabova, N., et al., 2015. “WW domain of BAG3 is required for the induction of autophagy in glioma cells”, J Cell Physiol. 230, 831-41). The present inventors investigated the contribution of the WW domain to FUS aggregation by testing a BAG3 construct with a WW-domain truncation (AN, Figure 7C). Figure 7D shows that indeed, in the absence of the WW domain, BAG3 loses its ability to inhibit FUS aggregation.

[0313] BAG3 devoid of the IUV domain (BAG3-AN) exhibits reduced binding to HSP70 - It is noted that this BAG3-AN also showed hampered HSP70 binding compared to the FL (full-length) isoform (Figure 8D), and thus the present inventors cannot rule out that this impairment is responsible for the loss of BAG3 ability to confer FUS aggregation alleviation.

[0314] In the presence of autophagy inhibitors BAG3 loses the ability to inhibit FUS aggregation - To further investigate the connection of autophagy and BAG3, the present inventors introduced Bafilomycin A into samples co-transfected with FUS-R521H-YFP and BAG3, comparing them to control samples treated with DMSO. The results revealed that in the presence of autophagy inhibitors, BAG3 lost its ability to inhibit FUS aggregation (Figure 7F). EXAMPLE 5 IDENTIFYING ADDITIONAL MRN A TARGETS WITH DIFFERENTIAL RESPONSES TO MG-132 COMPARED TO HEAT SHOCK

[0315] Experimental Results

[0316] Identification of genes which are upregulated in the presence of the MG132 proteasome inhibitor by are un-changed under heat shock conditions - The present inventors used the RNA- seq data, and systematically looked into genes that were notably upregulated (with |LFC>2| and a significant DESEQ2 p-value) in the presence of the MG-132 proteasome inhibitor, which were completely unchanged under Heat shock conditions, aiming to detect additional mRNAs which are part of MG-132 transcriptional program which is distinct from the one mediated by heat shock. Examining all mRNAs, the present inventors found that -150 potential candidates answered these criteria (Figure 9A).

[0317] SAT1 has a substantial protective effect by aggregation alleviation - The present inventors investigated the potential effects of these top MG-132-induced genes on FUS-R521H-YFP aggregation, and found that SAT1, which was induced by MG-132 treatment by -25 fold, exhibited a substantial protective effect with approximately 80% aggregation alleviation (Figure 9B).

[0318] SAT1 belongs to the acetyltransferase family, functioning as a rate-limiting catalytic enzyme crucial for maintaining polyamine homeostasis [Pegg, A. E. (2008) “Spermidine / spermine- N(l)-acetyltransferase: a key metabolic regulator”, Am J Physiol Endocrinol Metab. 294, E995- 1010]. It facilitates the transfer of the acetyl group from acetyl coenzyme A to the N1 position of either spermidine or spermine, the acetylated derivatives can be substrates for either reverse oxidation by acetylpolyamine oxidase enzyme (APAO) to putrescine, or to be excreted out of cells by a diamine transporter.

[0319] SATl-mediated regulation of mutF US aggregation - The present inventors investigated the mechanism underlying the potential SAT1 involvement in MG-132-mediated FUS aggregation inhibition. Initially, the present inventors utilized siRNA against SAT1 in cells co-transfected with FUS-R521H-YFP, followed by treatment with MG-132 or DMSO as a control. Introduction of siSATl into cells led to reduction of 74% of its endogenous levels in control cells, and -80% in MG-132 treated cells (Figure 10A). siSATl in MG-132-treated samples led to a reduction in aggregation inhibition by approximately 35% (Figure 9C) compared to cells treated with MG-132 and siControl. This observation further underscores the potential contribution of SAT1 in the pathway mediated by proteasome inhibitors against FUS aggregation. Mutations in SAT1 which affect binding to polyamines reduced the ability of S ATI to reduce mutFUS aggregation - The present inventors further investigated the potential interplay between the polyamines pathway and SATl-mediated rescue mechanisms in FUS aggregation. To address this, the present inventors generated various isoforms of SAT1, including the mutants E152K and R19A, which have been previously reported to exhibit reduced binding to polyamines [Coleman, C. S. & Pegg, A. E. (1997) “Proteasomal degradation of spermidine / spermine Nl- acetyltransferase requires the carboxyl-terminal glutamic acid residues”, J Biol Chem. 272, 12164- 9]. Additionally, the mutation L156F was chosen for its known effects on enzyme activity and substrate binding efficiency [McCloskey, D. E. & Pegg, A. E. (2003) “Properties of the spermidine / spermine N1 -acetyltransferase mutant L156F that decreases cellular sensitivity to the polyamine analogue Nl, Nl l-bis(ethyl)norspermine”, J Biol Chem. 278, 13881-7]. The findings revealed that all mutants negatively impacted SATl's ability to reduce mutFUS aggregation, thereby highlighting the significance of the polyamines pathway in SATl-mediated inhibition of FUS aggregation (Figure 9D).

[0320] Then to manipulate SAT1 activity, cells were exposed to natural polyamines (spermine, spermidine, putrescine), given the association of polyamine level disturbances with aging and disease processes [doi: 10.1134 / S 1819712422030059] , and their reported activity in inducing SAT1 levels [Lewandowski, N. M.,et al., 2010. “Polyamine pathway contributes to the pathogenesis of Parkinson disease”, Proc Natl Acad Sci U S A. 107, 16970-5].

[0321] Treatment with spermine resulted in a significant alleviation of aggregation - Cells expressing FUS-R521H-YFP were treated with varying concentrations (5 pM, 20 pM, 100 pM) of polyamines for 72 hours (Figure 10B). Remarkably, only spermine treatment at a concentration of 100 pM demonstrated a significant alleviation of aggregation.

[0322] Berenil, which reduces SAT1 activity, hindered FUS aggregation inhibition by MG-132 - Previously, it was shown that Berenil (diminazene aceturate), a compound which reduces SAT1 activity, increased alpha- synuclein inclusion formation in Parkinson's disease [Lewandowski et al., 2010, Supra]. To explore whether this phenomenon extends to ALS-FUS, the present inventors investigated aggregation modulation within cells expression FUS-R521H-YFP, treated with either MG-132 (10 pM, 18 hours), Berenil (100 pM, 18 hours), or both. Interestingly, Berenil hindered FUS aggregation inhibition by MG-132, further implicating SATl's central role in this effect (Figure 10C).

[0323] MG132 significantly affects the polyamines pathway - Following the establishment of a correlation between polyamines and SATl-mediated rescue mechanisms, the present inventors explored potential connections between polyamines levels and MG132. Polyamine Metabolomics were applied, to assess alterations in polyamine levels in cells treated with MG-132 compared to those treated with DMSO as a control. The obtained results demonstrated a significant increase in the levels of SATl's acetylated products, namely N1 -Acetylspermidine (17.9 fold) and Nl- Acetylspermine (7.5 fold), upon MG-132 treatment (Figure 9E). The levels of Spermine were decreased by 1.5-2 fold (Figure 9E). This observation suggested that MG-132 significantly alters the polyamines pathway .

[0324] EXAMPLE 6

[0325] PROTEASOME INHIBITORS INHIBIT FUS AGGREGATION AND AFFECT POLY AMINE METABOLISM IN PRIMARY NEURONS

[0326] The present inventors sought to examine the functional effects of two different proteasome inhibitors in primary neurons, MG-132, and Marizomib, which has been reported to cross the blood brain barrier (BBB) [Di, K., et al., 2016. “Marizomib activity as a single agent in malignant gliomas: ability to cross the blood-brain barrier”, Neuro Oncol. 18, 840-8].

[0327] Experimental Results

[0328] Rat neuronal cultures were infected with an AAV2 viral construct expressing FUS-R521H- YFP. Due to the complex phenotype of FUS-R521H-YFP aggregates (Figure 11A), as well as neuronal vulnerability which precluded FACS analysis, the present inventors subjected confocal microscopy images of live neurons to image analysis and analyzed aggregation in cells using a classifier that was trained to distinguish between nuclear fluorescent pattern and cells displaying the complex FUS-R521H-YFP aggregate phenotype (described in the General Materials and Experimental Methods above). Then, the percentage of aggregate-containing neurons out of infected cortical neurons was quantified in FUS-R521H-YFP neuronal cultures treated with either MG-132 or Marizomib six days after viral infection.

[0329] Marizomib was found to be less toxic to the neurons compared to MG132 - The present inventors note that Marizomib was less toxic to the neurons (Figure 1 IB).

[0330] MG-132 and Marizomib treated neurons showed significantly less FUS-R521H-YFP aggregation compared to control neurons - In total, the present inventors analyzed 2294 and 2253 FUS-R521H-YFP expressing neurons treated with MG-132 or DMSO as a control, respectively, and 1653 and 1658 cells treated with Marizomib and control respectively. Strikingly, it was observed that both MG-132 and Marizomib showed significantly less FUS-R521H-YFP aggregation compared to control neurons (Figures 12A-C). FUS-R521H-YFP neuronal cultures showed on average 36% less aggregate-containing cells when treated with MG-132 compared to control (Figures 12A-C, p = 1.81E-10, N = 3, see Methods), and 32% less aggregate-containing cells when treated with Marizomib compared to control (Figures 12A-B, p = 2.66E-06, N = 3, see Methods).

[0331] Testing the effect of proteasome inhibitors on the metabolism of polyamines in primary neurons - The present inventors treated primary neurons with Marizomib for 18 hours, and performed polyamine metabolomics. Indeed, it was found that, in neurons too, the levels of Nl- Acetylspermidine and Nl-Acetylspermine were significantly induced, and so were the levels of Putrecine, while the levels of Spermine was significantly reduced (Figure 12D).

[0332] Analysis and Discussion

[0333] The present inventors explored the involvement of the UPS system, which is part of the protein quality control mechanisms, in the modulation of FUS aggregation in ALS, starting by exploring the impact of proteasome inhibitors on the aggregation of mutFUS. The findings presented herein reveal an unexpected significant reduction in FUS aggregation following treatment with various proteasome inhibitors, including MG- 132, Velcade (Bortezomib), WA and Marizomib. This unexpected result contradicted the conventional understanding that proteasome inhibition leads to the accumulation of protein aggregates due to impaired degradation.

[0334] To elucidate the underlying mechanism of this phenomenon, the present inventors explored the involvement of molecular chaperones and transcriptional regulation. The results demonstrated that the alleviation of FUS aggregation by proteasome inhibitors was transcriptionally mediated, as evidenced by the fact that silencing of HSF1, a master regulator of the heat shock response, impaired the aggregation-alleviating effect of proteasome inhibitors. Additionally the present inventors noticed that MG-132 treatment caused upregulation in specific genes associated with the protein quality control pathway, of particular interest was the induction of BAG3, a co-chaperone of HSP70.

[0335] Prior studies have suggested that the induction of BAG3 by MG-132 is regulated by HSF1226. This aligns well with the findings of the present inventors indicating HSFl's role as a mediator in this distinctive transcriptional program, with BAG3 identified as one of the targets for MG-132 mediated rescue.

[0336] Additionally, BAG3 has been reported to facilitate the autophagic clearance of protein aggregation associated with different neurovegetative diseases, such as mutant Huntingtin, tau, a- synuclein, mutant SOD1, and a truncated form of TDP-43. These findings underscore the critical role of BAG3 in maintaining proteostasis and without being bound by any theory, it may suggest that dysregulation of BAG3 may contribute to the development of age-related neurodegenerative diseases. Consequently, these previously reported findings provide additional support for the present results demonstrating the ability of BAG3 to mitigate mutFUS aggregation, further suggesting BAG3 as a legitimate target of the MG- 132 aggregation-protective transcriptional program.

[0337] The present inventors have also demonstrated that the BAG3-AN mutant, which lacks its tryptophan-tryptophan (WW) domain, known to be crucial for BAG3-mediated autophagy, loses its capability to protect against mutFUS aggregation (Figure 7D). This underscores the significance of further inspecting autophagy as a mechanism underlying the BAG3 rescue, similar to what has been reported for other neurodegenerative disease-related proteins.

[0338] Furthermore, the results presented here revealed the potential involvement of the polyamines pathway, specifically of SAT1, in the rescue of FUS aggregation induced by proteasome inhibitors. Manipulating SAT1 and polyamine levels demonstrated a significant impact on the extent of aggregation modulation, underscoring the potential regulatory role of the polyamines pathway in protein homeostasis.

[0339] Polyamines were reported to play diverse and critical roles in cells, including the maintenance of neurophysiological functions within the central nervous system (CNS). Consequently, disturbances in polyamine levels and homeostasis have been associated with aging and disease processes, including neurodegenerative disorders like Alzheimer's disease and Parkinson's disease. For instance, evidence supporting the connection between altered polyamine metabolism and Parkinson's disease pathogenesis includes reduced SAT1 activity in the PD brain, indicating that increasing SAT1 activity reduces aSyn toxicity, while inhibiting SAT1 has the opposite effect. Similarly, in Alzheimer's disease (AD), the autophagy activator spermidine in mice has been shown to reduce neurotoxic soluble Ap expression and reduce AD-associated neuroinflammation .

[0340] Moreover, the present inventors were able to show that the findings presented here extended beyond cell culture models to primary neurons, where both MG- 132 and Marizomib demonstrated significant reductions in FUS aggregation (Figure 12A-D). This observation highlights the potential of proteasome inhibitors as therapeutic agents for neurodegenerative diseases characterized by protein aggregation.

[0341] Without being bound by any theory, the findings presented herein may imply the existence of an evolutionarily conserved network that co-regulates polyamines and heat shock proteins to efficiently manage the proteotoxic stress response.

[0342] Establishing such a link within the context of mutFUS aggregation alleviation could enhance the understanding of the potential crosstalk between the proteotoxic stress response in general (and perhaps BAG3-HSP70 in particular) and SATl-mediated rescue mechanisms, offering new insights for therapeutic development and elucidating the molecular basis of the disease .

[0343] The present inventors have elucidated the transcriptional, chaperone-mediated, and polyamine-dependent pathways involved in proteasome inhibitor-mediated aggregation reduction offering promising avenues for therapeutic intervention in mutFUS-ALS neurodegenerative disease.

[0344] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0345] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

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Claims

WHAT IS CLAIMED IS:

1. A method of treating a subject having a disease characterized by accumulation of FUS -associated aggregates, the method comprising administering to the subject a therapeutically effective amount of a proteasome inhibitor, thereby treating the subject.

2. A method of treating a subject having a disease characterized by accumulation of FUS -associated aggregates, the method comprising administering to the subject a therapeutically effective amount of an agent which upregulates spermidine / spermine N1 -acetyltransferase 1 (SAT1) or spermine, thereby treating the subject.

3. The method of claim 1, further comprising administering to the subject a therapeutically effective amount of an agent which upregulates spermidine / spermine Nl- acetyltransferase 1 (SAT1) or spermine.

4. A proteasome inhibitor for use in treating a subject having a disease characterized by accumulation of FUS -associated aggregates.

5. A composition-of-matter comprising an agent which upregulates spermidine / spermine N1 -acetyltransferase 1 (SAT1) or spermine, for use in treating a subject having a disease characterized by accumulation of FUS-associated aggregates.

6. A method of reducing accumulation of FUS-associated aggregates in cells of a subject, the method comprising contacting the cells of the subject with a proteasome inhibitor, thereby reducing the accumulation of FUS-associated aggregates in the cells of the subject.

7. A method of reducing accumulation of FUS-associated aggregates in cells of a subject, the method comprising contacting the cells of the subject with an agent which upregulates spermidine / spermine N1 -acetyltransferase 1 (SAT1) or spermine, thereby reducing the accumulation of FUS-associated aggregates in the cells of the subject.

8. The method of any one of claims 1, 3 and 6, or the proteasome inhibitor for use of claim 4, wherein said proteasome inhibitor penetrates through the blood brain barrier (BBB).

9. The method of any one of claims 1, 3 and 6, or the proteasome inhibitor for use of claim 4, wherein said proteasome inhibitor is formulated in a formulation for enhancing penetration of said proteasome inhibitor through the blood brain barrier (BBB).

10. The method of any one of claims 2-3 and 7, the composition-of-matter for use of claim 5, wherein said agent is formulated in a formulation for enhancing penetration of said agent through the blood brain barrier (BBB).

11. The method of claim 9 or 10, the proteasome inhibitor for use of claim 9, or the composition-of-matter for use of claim 10, wherein said formulation comprises a nano-carrier.

12. The method of claim 11, the proteasome inhibitor for use of claim 11, or the composition-of-matter for use of claim 11, wherein said nano-carrier comprises a Poly(lactic-co- glycolic acid) (PLGA) nanoparticle, or an albumin nanoparticle.

13. The method of any one of claims 1-3 and 6-12, the proteasome inhibitor for use of any one of claims 4 and 8, 11-12, or the composition-of-matter for use of any one of claims 5 and 10-12, wherein said proteasome inhibitor or said composition-of-matter is co-administered with a P-glycoprotein (P-gp) inhibitor.

14. The method of any one of claims 1, 3, 6, 8-9, and 11-13, or the proteasome inhibitor for use of any one of claims 4, 8, 9, and 11-13, wherein said proteasome inhibitor is Carfilzomib, Ixazomib, Marizomib, Oprozomib, Delanzomib, ixazomib, delanzomib, KZR-616, NNU546, NNU219, MLN9708, ONX0912, MG-132 or Bortezomib.

15. The method of any one of claims 1, 3, 6, 8-9, and 11-13, or the proteasome inhibitor for use of any one of claims 4, 8, 9, and 11-13, wherein said proteasome inhibitor is Withaferin A.

16. The method of any one of claims 1, 3, 6, 8-9, and 11-13, or the proteasome inhibitor for use of any one of claims 4, 8, 9, and 11-13, wherein said proteasome inhibitor is Marizomib.

17. The method of any one of claims 1, 3, 6, 8-9, and 11-13, or the proteasome inhibitor for use of any one of claims 4, 8, 9, and 11-13, wherein said proteasome inhibitor is Velcade (Bortezomib).

18. The method of any one of claims 1-3, and 7-17, the proteasome inhibitor for use of any one of claims 4, 8-9, and 11-17, or the composition-of-matter for use of any one of claims 5, and 10-13, wherein said FUS -associated aggregates comprise a mis-folded FUS protein.

19. The method, the proteasome inhibitor for use, or the composition-of-matter for use of claim 18, wherein said mis-folded FUS protein is caused by a mutation in the FUS gene.

20. The method of any one of claims 1-3, and 6-19, further comprising identifying in a DNA of the subject a mutation in the FUS gene which causes mis-folding of said FUS prior to administering.

21. The method of any one of claims 1-3, and 6-19, the proteasome inhibitor for use of any one of claims 4, 8-9, 11-19, or the composition-of-matter for use of any one of claims 5, 10- 13 and 18-19, wherein the subject has a mutation in the FUS gene which causes mis-folding of said FUS.

22. The method of claim 20 or 21, the proteasome inhibitor for use of claim 21, or the composition-of-matter for use of claim 21, wherein said mutation in the FUS gene encodes a mutated FUS protein selected from the group consisting of S513P, R514G / S, G515C, E516V, H517D / P / Q, R518G / K, Q519X, Q519IfsX9, R521C / G / H / L / S, R522G, R524W / T / S, P525L, S96del, P431L, R502EfsX15, G504WfsX12, G507D, K510E / R, K510WfsX517, S462F, M464I, G466VfsX14, G472VfsX57, Y485AfsX514, R487C, R495X, G492EfsX527, R495EfsX527, R495QfsX527, G497AfsX527, R377W, G399V, S402_P411delinsGGGG, Q290X,G175_ G176insGG, G187S. G191S, G206S, R216C, S221del, G225V, G230C, G230del, R234C / L, R244C, M254V, P525L S96del, P106L, S115N, G144_Y149del, G156E, and S57del.

23. The method of any one of claims 1-3, and 6-22, the proteasome inhibitor for use of any one of claims 4, 8-9, and 11-19, or the composition-of-matter for use of any one of claims 5, 10-13 and 18-19, wherein the subject is diagnosed with Amyotrophic lateral sclerosis (ALS).

24. The method of any one of claims 1-3, and 6-22, the proteasome inhibitor for use of any one of claims 4, 8-9, and 11-19, or the composition-of-matter for use of any one of claims 5, 10-13 and 18-19, wherein the subject is diagnosed with Frontotemporal dementia (FTD).

25. An in-vitro method of selecting a molecule for treatment of a disease characterized by accumulation of FUS- associated aggregates, the method comprising:(a) contacting cells carrying a mutation in the FUS gene which causes mis-folding of said FUS with the molecule,(b) determining presence and / or level of FUS-associated aggregates in the cells prior to and following said contacting, wherein a reduction in level of said FUS-associated aggregates following said contacting indicates that the molecule is suitable for the treatment of the disease characterized by accumulation of FUS-associated aggregates.

26. The method of claim 25, wherein said cells are neuron cells.

27. The method of claim 25 or 26, wherein said mutation in the FUS gene encodes a mutated FUS protein selected from the group consisting of S513P, R514G / S, G515C, E516V, H517D / P / Q, R518G / K, Q519X, Q519IfsX9, R521C / G / H / L / S, R522G, R524W / T / S, P525L, S96del, P431L, R502EfsX15, G504WfsX12, G507D, K510E / R, K510WfsX517, S462F, M464I, G466VfsX14, G472VfsX57, Y485AfsX514, R487C, R495X, G492EfsX527, R495EfsX527, R495QfsX527, G497AfsX527, R377W, G399V, S402_P411delinsGGGG, Q290X,G175_G176insGG, G187S, G191S, G206S, R216C, S221del, G225V, G230C, G230del, R234C / L, R244C, M254V, P525L S96del, P106L, S115N, G144_Y149del, G156E, and S57del.

Images