WO2018067662A1 - Protein amyloidogenesis and related methods - Google Patents

Abstract

Provided herein are methods of testing a drug for efficacy for treating an amyloid disease. In exemplary embodiments, the methods comprises (i) contacting cells with the drug before or after the cells are contacted with a stimulus that causes amyloid aggregate formation in the cells and (ii) assaying amyloid aggregate formation or amyloid aggregate disaggregation in the cells, wherein the drug has efficacy for treating the amyloid disease, when amyloid aggregate formation decreases or is delayed or amyloid aggregate disaggregation increases, upon contacting the cells with the drug.

Description

PROTEIN AMYLOIDOGENESIS AND RELATED METHODS

GRANT FUNDING

[0001] This invention was made with government support under Grant No.

1 R01 CA200676-01 awarded by the National Cancer Institute. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62/404,001 , filed on October 4, 2016, the content of which is incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety is a computer-readable

nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 1 ,348,251 byte ACII (Text) file named "51075_SeqListing.txt," created on October 3, 2017.

BACKGROUND

[0004] Eukaryotic cells are frequently exposed to adverse environmental stimuli including extreme temperatures, low oxygen availability and acidosis. Each stressor presents a unique risk to cellular sustainability, with the severity and duration dictating whether cells enact pro-survival or apoptotic responses. Numerous pathways have been identified to mitigate the effects of unfavorable growth conditions. High

temperature causes protein denaturation (Pinto et al., 1991 ), activating the heat shock and unfolded protein responses. These pathways enhance the expression of chaperone proteins to alleviate the burden of misfolded proteins on the cell (Lindquist, 1986;

Richter et al., 2010). The Hypoxia Inducible Factor family of transcription factors (Semenza and Wang, 1992; Wiesener et al., 1998) activate an array of genes that augment oxygen delivery and enhance glucose metabolism during periods of low [O₂] (Semenza, 2012). Regardless of the stimulus, the goal of stress responsive pathways is to sustain viability during adverse environmental conditions, repair associated damage and restore cellular homeostasis. [0005] Recently, noncoding RNAs (ncRNA) have emerged as important biological molecules in cellular adaptation to common stressors. Human Alu elements (Liu et al., 1995) and Satellite III transcripts (Jolly et al., 2004) are strongly induced in response to elevated temperatures. These ncRNAs reduce the burden on the protein folding machinery by impairing global transcription (Mariner et al., 2008) and mRNA maturation (Denegri et al., 2001 ), respectively. In response to DNA damage lincRNA-p21 represses apoptosis (Huarte et al., 2010), while Pint and TUG1 transcripts facilitate the epigenetic silencing of cell cycle factors (Khalil et al., 2009; Marin-Bejar et al., 2013), inhibiting proliferation until genomic integrity has been restored. Another class of inducible ncRNA expressed from the ribosomal intergenic spacer (rIGSRNA) regulate cellular dynamics by capturing and immobilizing proteins in nuclear foci (Audas et al., 2012; Mekhail et al., 2005). This reversible process enables cells to regulate protein mobility (Lippincott- Schwartz and Patterson, 2003; Misteli, 2001 ) in response to stressors. Interestingly, fragments of the Huntington protein and the RNA-binding protein Rim4 display repressed mobility upon adopting an amyloid-like state in physiological settings

(Berchowitz et al., 2015; Kayatekin et al., 2014).

[0006] Amyloids are a highly organized form of protein aggregation typically associated with human neuropathies, including Alzheimer's, Parkinson's and

Huntington's diseases. Under pathological settings, amyloids are believed to act in a dominant negative manner, converting native-fold species into irreversible β-sheet rich protein aggregates (Knowles et al., 2014). Physiological amyloids are quite uncommon compared to the native-fold, especially in higher eukaryotes. In mammals, functional amyloidogenesis has been associated with hormone storage (Maji et al., 2009), melanin production (Fowler et al., 2006), regulation of kinase activity (Li et al., 2012) and protein synthesis (Berchowitz et al. 2015). Yet, most proteins have an inherent amyloidogenic propensity and possess the capacity to adopt the amyloid-fold (Goldschmidt et al., 2010). Researchers have proposed the existence of kinetic and thermodynamic barriers (Baldwin et al., 201 1 ; Knowles et al., 2014), as well as active suppressor programs to prevent the conversion of proteins to a toxic amyloid state (Dobson, 1999). This line of reasoning implies that the amyloidogenic propensity of proteins is essentially an undesirable byproduct of polypeptide assembly that cells must actively prevent. Still, it remains puzzling why cells have not evolved a comprehensive program that exploits the broad ability of proteins to assume the amyloid-fold.

[0007] Presented for the first time are data supporting the existence of amyloid- bodies (A-bodies), which are inducible and reversible subnuclear foci composed of an array of different proteins that adopt an amyloid-like immobile/insoluble state. The data herein shows that physiological amyloidogenesis of proteins is facilitated by the interaction between the amyloid-converting motif (ACM) and inducible rIGSRNA. It is shown herein that cells activate physiological amyloidogenesis to store large quantities of proteins and enter a state of dormancy in response to stress. These data challenge the concept that amyloids are an infrequent and mostly toxic protein-fold and introduce physiological amyloidogenesis as a cell-wide post-translational process.

SUMMARY

[0008] The amyloid state of protein organization is typically associated with debilitating human neuropathies and seldom observed in physiology. The data presented herein support the existence of a systemic program that leverages the amyloidogenic propensity of proteins to regulate cell adaptation to stressors. On stimulus, cells assemble the Amyloid-bodies (A-bodies), nuclear foci containing heterogeneous proteins with amyloid-like biophysical properties. A discrete peptidic sequence, termed the amyloid-converting motif (ACM), is capable of targeting proteins to the A-bodies by interacting with ribosomal intergenic noncoding RNA (rIGSRNA). The pathological β-amyloid peptide, involved in Alzheimer's disease, displays ACM-like activity and undergoes stimuli-mediated amyloidogenesis in vivo. Upon signal termination, elements of the heat shock chaperone pathway disaggregate the A-bodies. Physiological amyloidogenesis enables cells to store large quantities of proteins and enter a dormant state in response to stressors. These data suggest that cells have evolved a post-translational pathway that rapidly and reversibly converts native-fold proteins to an amyloid-like solid phase.

[0009] Without being bound to any particular theory, the data presented herein support a method of testing drugs for efficacy to treat an amyloid disease, including human neuropathies, such as Alzheimer's, Parkinson's and Huntington's diseases. Accordingly, the present disclosure provides methods of testing a drug for efficacy for treating an amyloid disease. In exemplary aspects, the method comprises (i) contacting cells with the drug before or after the cells are contacted with a stimulus that causes amyloid aggregate formation in the cells and (ii) assaying amyloid aggregate formation or amyloid aggregate disaggregation in the cells, wherein the drug has efficacy for treating the amyloid disease, when amyloid aggregate formation decreases or is delayed or amyloid aggregate disaggregation increases, upon contacting the cells with the drug. In exemplary aspects, amyloid aggregate is an amyloid body and the method comprises (i) contacting cells with the drug before or after the cells are contacted with a stimulus that causes amyloid body formation in the cells and (ii) assaying amyloid body formation or amyloid body disaggregation in the cells, wherein the drug has efficacy for treating the amyloid disease, when amyloid body formation decreases or is delayed or amyloid body disaggregation increases, upon contacting the cells with the drug.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 (A)-1 (I): Uncovering the distinct biophysical properties of the cellular A-bodies. (A) Physiological amyloidogenesis is rapid and reversible. MCF-7 cells exposed to extracellular acidosis and returned to standard growth conditions, for the indicated times, were stained with Congo red and Hoechst (blue inset). (B) Nuclear foci stain positively with amyloid-specific dyes. Untreated or acidotic MCF-7 cells were stained with Amylo-Glo (blue), Thioflavin S (green) and/or Congo red (red). Selected regions (white box) were expanded below with merged image included (far right panel). Dashed circles represent nuclei. (C) Established proteins are targeted to the A-bodies. MCF-7 cells expressing VHL-GFP or POLD1 -GFP were grown under standard, hypoxic/acidotic conditions or recovered for 24 hours post-acidosis treatment. Acidotic cells were stained with Congo red. Selected regions (white box) were expanded

(below). (D) A-body targets are reversibly immobilized. VHL-GFP, POLD1 -GFP or GFP- B23 transfected MCF-7 cells were treated as above and bleached repeatedly for fluorescent loss in photobleaching. Quantification is presented as the mean relative intensity of at least 5 data sets. (E) Stimuli-specific ^solubilization of A-body

components. Insoluble proteins (Insol) were extracted from whole cell lysates (WCL) of untreated, acidotic (2 hrs), heat shocked (2 hrs), sodium arsenite (1 hr), cycloheximide (1 hr), thapsigargin (8 hrs) or H₂0₂ (8 hrs) treated cells. A-body components; VHL-GFP and endogenous POLD1 or the GAPDH and Histone H3 control proteins were detected by western blot. (F) Stimuli-induced A-bodies are proteinase K resistant. Transmission electron microscopy (TEM) of untreated, heat shocked (1 hr) and acidotic (1 hr) MCF-7 cells were left undigested or exposed to proteinase K. Proteinase K-resistant nuclear bodies are indicated (yellow arrow). (G) Proteinase K-resistant fibrils possess amyloidlike properties. Heat shocked MCF-7 cells were left undigested or proteinase K-treated prior to TEM visualization. Proteinase K-resistant structures were stained with Congo red or the amyloid fibril conformation-specific antibody OC. (H) Protein ^solubilization correlates with A-body assembly and disaggregation. MCF-7 cells were treated as above (A) and insoluble proteins were extracted from WCL. A-body targets/controls were detected as in (E). (I) Inhibition of rIGSRNA transcripts impairs amyloidogenesis. MCF-7 cells stably-expressing control or shRNA against rlGS₂₈RNA or rlGS₂2RNA were grown in acidosis permissive media or exposed heat shock, respectively, prior to Congo red staining. Dashed circles represent nuclei. White scale bars represents 20μιη. Black and white TEM scale box represent 1 μιη and 0.1 μιτι.

[0011] Figure 2(A)-2(E): A-bodies are unique rIGSRNA-seeded amyloidogenic structures. (A) A-bodies are separate from nuclear stress granules. MCF-7 cells transfected with control or rlGS₂2RNA-specific siRNA were exposed to heat shock. HSF-1 was detected in Congo red stained cells. (B) Additional cellular bodies are not amyloidogenic. MCF-7 cells were treated as indicated. Foci-specific markers were detected (green) in Congo red (red) stained cells. (C) Aggresomes do not contain proteins in an amyloid-like conformation. Untransfected or HDAC6-GFP expressing PC3 cells were treated with stimuli that induce aggresome (5μΜ MG132) or aggresome and A-body (transcriptional stress: 8μΜ MG132 + 4μΜ Actinomycin D) formation. The aggresome marker vimentin was detected in Congo red or Amylo-Glo stained cells. (D) Other cellular domains possess mobile proteins. Fluorescence recovery after photobleaching was performed on MCF-7 cells expressing TIA1 -GFP (sodium arsenite and heat shock - 1 hr), SC35-GFP (no treatment), Coilin-GFP (no treatment), HSF1 - GFP (heat shock - 1 hr), NONO-GFP (5μΜ MG132 - 17hrs) and VHL-GFP (heat shock - 1 hr). Quantified kinetics are presented as the mean relative intensity of at least 5 data sets. (E) A-body formation is mediated by rIGSRNA. PC3 cells transfected with siRNA targeting the indicated transcripts were exposed to heat shock (45 min) or 5μΜ MG132 (17 hrs) prior to detection of A-bodies (Congo red), nuclear stress granules (HSF1 ) or paraspeckles (NONO). The proportion of body positive cells was counted and compared to the control siRNA. Results are means and SEM (n=3). Significance was measured by Student's t-test; ^*p < 0.01 . Dashed circles represent nuclei. Selected regions (white box) were expanded below. White scale bars represents 20μπι.

[0012] Figure 3(A)-3(H): Heterogenic protein composition of the A-body.

[0013] (A) Acidosis induces the formation of the A-bodies. MCF-7 cells grown under normoxic-neutral (NN) and hypoxic-neutral (HN) as baseline controls, and hypoxic- acidotic (HA) conditions were stained with Congo red dye or fluorescence in situ hybridized with an anti-sense probe targeting rlGS₂sRNA. Nucleolar B23 (green) is inset. (B) Identification of the A-body residents. SILAC-MS analysis comparing proteins extracted from MCF-7 cells treated as above. Plot compares the log enrichment of HA:HN versus HA:NN. A-body-enriched (red) proteins fall in the upper right quadrant. (C) A-body components are biochemically similar to the total protein population. Scatter plot of protein size versus hydrophobicity. (D) A heterogeneous family of proteins are targeted to the A-bodies. The size, isoelectric point and hydrophobicity of total and amyloid-specific protein fractions is summarized. Values represent data set averages +/- SEM. (E) Localization of SILAC-MS candidates to the A-bodies. MCF-7 cells transfected with HAT1 -GFP, UAP56-GFP, cdk1 -GFP, Ku70-GFP, HDAC2-GFP and Fib- GFP were left untreated or exposed to extracellular acidosis prior to staining with Congo red. (F) Targets of the A-bodies are immobilized. Quantification of recovery after photobleaching kinetics for the proteins listed above (E) as the mean relative intensity of at least 5 data sets. (G) SILAC-MS candidates reversibly insolubilize in response to stimuli. MCF-7 cells were exposed to extracellular acidosis and returned to standard growth conditions, for the indicated times prior to harvesting WCL and insoluble proteins. HAT1 , cdkl , HDAC2, UAP56, GAPDH and Histone H3 were detected by western blotting. (H) Stimuli-specific ^solubilization of SILAC-MS candidates. WCL and insoluble proteins were extracted from untreated, acidotic (2 hrs), heat shocked (2 hrs), sodium arsenite (1 hr), cycloheximide (1 hr), thapsigargin (8 hrs) or H2O2 (8 hrs) treated cells. A-body targets/controls were detected as in (E). Dashed circles represent nuclei. Selected regions (white box) were expanded below. White scale bars represents 20pm.

[0014] Figure 4(A)-4(I): Identification of the amyloid converting motif that targets proteins to A-bodies.

[0015] (A) VHL can obtain an amyloid-like conformation in bacteria. GFP or VHL- GFP expressing MCF-7 cells left untreated or exposed to acidosis or BL21 cells were stained with Congo red and Hoechst. X-ray diffraction was performed on BL21 bodies.

(B) VHL contains fibril-forming peptidic regions. Results of ZipperDB analysis of full length VHL for fibrillation propensity. The Rosetta energy threshold of -23 kcal/mol is an indicator of fibril-positive regions. Truncated VHL fragments, used below, are indicated.

(C) Amyloidogenic fragments of VHL insolubilize GFP and produce SDS-resistant multi- mers. Fragments of VHL (above) fused to GFP were expressed in BL21 , prior to lysis and insoluble protein fractionation. Fusion proteins were detected with a GFP-specific antibody at low and high exposures to detect monomeric (low) and multi-meric (high) proteins. (D) Insoluble VHL fragments form bacterial inclusion bodies with an amyloidlike x-ray diffraction profile. BL21 expressing the VHL fragments (above) were fixed and stained with Congo red and Hoechst. Inclusion bodies were purified, where present, for x-ray diffraction. (E) Table summarizing inclusion body formation and targeting to the A- bodies (Figure 4D, 1 1 B, F and H) for the indicated regions of VHL, cdkl or POLD1 fused to GFP. (F) Regions of VHL can associate with rlGS₂sRNA. MCF-7 cells transfected with VHL or VHL mutants were exposed to acidosis for 1 hour prior to lysis and RNA immunoprecipitation. 28S rRNA and rlGS₂₈RNA were detected by RT-PCR. Exogenous VHL fragments and GAPDH were detected by immunoblotting. (G)

Generation of artificial ACM sequences. R/H-rich (orange) and amyloidogenic (purple) sequences derived from VHL (aACM^VHL) and POLD1 (aACM^P0LD1) were fused to generate artificial ACM motifs (sequence inset). The fibrillation propensity was calculated by ZipperDB. (H) Artificial ACMs are sufficient to create insoluble multi-mers. BL21 expressing GFP fused to the artificial ACMs (above) were lysed into soluble (+) and insoluble (-) fractions. (I) Amyloidogenic regions of VHL form 10nm amyloid-like fibrils. Peptides encoding VHL (104-140), aACM^VHL and the classic pathological β-amyloid were synthesized and incubated for 1 week at 37°C. Fibrils were detected by TEM. White and yellow scale bars represent 20μηι and 5μηι, respectively. Dashed circles represent nuclei (MCF-7) or whole cell (BL21 ).TEM scale box represent 10nm.

[0016] Figure 5(A)-5(H): The pathological β-amyloid peptide is a target of physiological amyloidogenesis

[0017] (A) Amino acid sequence and Rosetta energy profiles for β-amyloid (1 -40) and VHL (104-140). Secretase (α-, β-, and γ-) cleavage sites are indicated. (B) Stress- specific targeting of β-amyloid to the A-bodies. β-amyloid-GFP expressing MCF-7 cells were left untreated or exposed to acidosis, heat shock, sodium arsenite, cycloheximide, thapsigargin or H₂0₂ and stained with Congo red. (C) Immobilization of β-amyloid by acidosis and pcrlGS₂₈RNA. Quantification of fluorescence recovery after photobleaching kinetics for β-amyloid-GFP in untreated, acidotic or co-transfected (pcDNA3.1 or pcrlGS₂₈RNA) MCF-7 cells. Mean relative intensity of at least 5 data sets. (D)

Amyloidogenic stimuli insolubilize β-amyloid, not the non-pathological P3 peptide.

Insoluble proteins were extracted from MCF-7 cells exposed to the stimuli (above), β- amyloid-GFP, P3-GFP, GAPDH and Histone H3 were detected by western blotting. (E) rlGS ₈RNA is essential for the subnuclear targeting of β-amyloid. MCF-7 cells stably- expressing control or rlGS₂₈RNA-specific shRNA were transfected with a plasmid encoding β-amyloid-GFP and grown under hypoxic conditions in acidosis-permissive media. (F) β-amyloid and P3 possess amyloidogenic propensity. β-amyloid-GFP, P3- GFP and β-amyloid (1 -17)-GFP were expressed in BL21 cells prior to staining with Congo red and Hoechst. (G) The amino-terminus of β-amyloid is essential for rIGSRNA binding. RNA immunoprecipitation of acidified MCF-7 cells transfected with GFP, β- amyloid-GFP or P3-GFP. GAPDH mRNA and rlGS₂₈RNA were detected by RT-PCR. Exogenous proteins and GAPDH were detected by immunoblotting. (H) P3 is not targeted to the A-bodies. P3-GFP-expressing MCF-7 cells were untreated or exposed to acidotic conditions, prior to Congo red staining. Dashed circles represent nuclei (MCF- 7) or whole cell (BL21 ). Selected regions (white box) were expanded below. White scale bars represents 20μιη.

[0018] Figure 6(A)-6(F): Heat shock chaperones regulate A-bodies disaggregation [0019] (A) Amyloidogenesis is rapid and reversible. MCF-7 and PC-3 cells exposed to acidosis or heat shock and allowed to recover (times indicated) were stained with Congo red. The proportion of cells containing Congo red-positive structures was assayed relative to Hoechst-positive nuclei. (B) Heat shock proteins mediate the solubilization of A-body components. Insoluble proteins were extracted from untreated, acidotic or recovering MCF-7 cells treated with the protein synthesis inhibitor cycloheximide (Chx) or PDI (16F16), GRP94 (EGCG), Hsp70 (VER155008) or Hsp90 (17-AAG) inhibitors. POLD1 , cdkl , GAPDH and Histone H3 were detected by western blot. (C) Heat shock proteins are associated with the A-bodies during recovery. Table summarizing data in Figure 13B. (D) Heat shock proteins disaggregate the A-bodies. MCF-7 cells were exposed to acidotic (left panel) or heat shock (right panel) conditions for 3 hours, then returned to normal growth conditions for 2 or 4 hours in the presence 16F16, EGCG, VER, AAG or Wortmannin. The proportion of Congo red-positive cells was determined as above. (E) Congo red stained MCF-7 cells allowed to recover for 2 or 4 hours from a 3 hour acidosis exposure in the presence or absence of VER155008. (F) Hsp70 activity enhances β-amyloid release during recovery. β-amyloid-GFP- expressing MCF-7 cells were allowed to recover for 4 hours from a 3 hour heat shock exposure in the presence or absence of VER or Chx. Western blots of insoluble fractionation for β-amyloid-GFP and Histone H3 are included (lower left). Results are means and SEM (n=4). Significance was measured by Student's t-test; ^*p < 0.01 .

Dashed circles represent nuclei. White scale bars represents 20μιη.

[0020] Figure 7(A)-7(K): rlGSRNA/A-bodies induce cellular dormancy

[0021] (A) Functional classification of A-bodies constituents. SILAC-MS results were analyzed and grouped by function, percentages per group are indicated. (B)

Proliferative factors are reversibly targeted to the A-bodies. Untreated, acidotic and recovered MCF-7 cells were stained for endogenous POLA1 and cdkl (red) and the nucleolar marker B23 (green). Dashed circles represent nuclei. (C) Acidosis induces a reversible state of dormancy. 750,000 (MCF-7 and PC-3 - left axis) or 75,000 (WI-38 - right axis) cells were grown for 2 days, prior to the application of acidosis-permissive media and exposure to hypoxic (1 % 0₂) conditions. Cells were returned to standard growth conditions two days post-acidification. Cells were counted daily with trypan blue staining to ensure viability. (D) DNA synthesis is reversibly inhibited by acidosis. MCF-7, PC-3 and WI-38 cells grown under the indicated conditions were incubated with BrdU prior to fixation. BrdU positive cells were counted relative to Hoechst stained nuclei. (E- F) Inhibition of rlGS2sR A restores proliferative capacity to acidotic cells. MCF-7 cells stably-expressing two independent shRNA against rlGS₂sRNA (sh28#1 and sh28#2) or a control sequence (shCtrl) were exposed to normoxic-neutral, hypoxic-neutral or hypoxic-acidosis and cell counts were performed each day (E) or incubated with BrdU for the incorporation assay described above (F). (G) Amyloidogenesis preserves cell viability during extracellular stress. MCF-7 cells described above were grown under hypoxic conditions in standard (pH7.4) or acidosis-permissive (pH6.0) low glucose media for the indicated times. Viability was calculated as propidium iodide-positive versus Hoechst-positive nuclei. (H) Human breast invasive duct carcinomas and prostatic acinar contain cellular amyloids. Paraffin-embedded prostate and breast tumors were stained with the Amylo-Glo, UAP56 or HAT1 and the nucleolar marker B23. (I-J) Inhibition of NGS28RNA relieves tumor dormancy. Nude mouse xenograft assays with MCF-7 and PC-3 cells described above. Representative mice and excised tumors (I) are presented, with tumor volumes calculated weekly (J) (n=5). (K) Inhibition of rlGS₂8RNA prevents amyloidogenesis in situ, causing tumor necrosis. Paraffin- embedded MCF-7 andPC-3 tumor sections were stained for the SILAC-MS candidate POLA1 (red) and B23 (green), Amylo-Glo (blue) or hematoxylin/eosin. Results are means and SEM (n>4) with significance measured by Student's t-test; ^*p < 0.01 .

Selected regions (white box) were expanded below. White scale bars represents 20μηπ.

[0022] Figure 8(A)-8(J): Generation of the cellular A-body

[0023] (A) Specific cellular stimuli induce amyloidogenesis. VHL-GFP expressing MCF-7 cells were exposed to acidosis, heat shock, sodium arsenite, H₂0₂, thapsigargin, transcriptional stress or cycloheximide for the indicated times prior to Amylo-Glo staining (blue inset). (B) Amyloidophilic dyes co-localize with VHP-GFP to the A-bodies. MCF-7 cells expressing VHL-GFP were stained with Congo red (red), NIAD4 (red), Methyoxy-X04 (blue), BSB (blue) and Amylo-Glo (blue). Selected regions (white box) were expanded below with merged image included (far right panel). (C-D) The cellular A-bodies stains with numerous amyloid-specific dyes. (C) MCF-7, PC3 and (D) WI-38 cells were left untreated, exposed to extracellular acidosis, heat shock, transcriptional stress (actinomycin D and MG132) or allowed to recover under standard growth conditions, post-acidosis, for 24 hours. Formaldehyde-fixed cells were stained with the amyloid dyes; Congo red, Amylo-Glo, Thioflavin S, NIAD4, Methyoxy-X04 and BSB. (E) A-bodies can be detected in live cells. Unfixed MCF-7 cells treated, as above, were stained with Thioflavin S. (F) Amyloidogenesis and rIGSRNA are present in situ. Cryo- sectioned human brain and pancreas were stained with Congo red and B23. Congo red- positive (white arrows) and -negative (yellow arrows) A-bodies/nucleoli are indicated (left panel). RNA from human tissues were analyzed by RT-PCR for expression of the rlGSi₆RNA, NGS22RNA and rlGS₂sRNA transcripts, β -actin was used as a loading control (right panel). (G) Proteinase K resistant A-body fibers are composed of small fibril-like structures. MCF-7 cells, with or without proteinase K digestion, were treated for 1 hour at 43°C or in acidotic media and were visualized by transmission electron microscopy. Identical 300,000x magnification images are presented (left two panels) with potential fibril-like structures indicated (yellow dashed lines). (H) Proteinase K unmasks the amyloid epitope within A-bodies. Heat shock treated MCF-7 cells, with or without proteinase K digestion, were stained with the OC antibody, an antibody that detects the amyloid conformation independent of the amino acid sequence. No primary antibody was used as a negative control, with images taken at the same exposure as OC antibody samples. Cytoplasmic signal is non-specific signal associated with the primary antibody. (I) Amyloidogenesis correlates with rIGSRNA expression. MCF-7 were exposed to acidotic media or heat shock for the indicated times. Environmental stressors were removed after three hours and cells were allowed to recover. RT-PCR quantification of the rlGS₂8RNA, NGS22RNA and rlGSi₆RNA expression levels are presented, β-actin was used as a loading control. (J) Knockdown efficiency of the stable shRNA cell lines. MCF-7 parental, control, rlGS₂2RNA and rlGS₂₈RNA (two different target sites)-specific shRNA cell lines were tested for the knockdown efficiency of the rlGS₂₂RNA and rlGS₂₈RNA transcripts by RT-PCR. Actin and total RNA are loading controls. Dashed circles represent nuclei. White scale bars represents 20μιη. White TEM scale box represent 0.1 μιη.

[0024] Figure 9(A)-9(D): A-bodies are distinct from other subcellular structures [0025] (A-C) Other cellular structures do not stain with the amyloidophilic dye Congo red. MCF-7 (A) and PC-3 (B-C) cells were grown under the indicated conditions prior to fixation and detection of subcellular bodies with the noted antibody (green). All cells were co-stained with Congo red and overlaid images are presented, with magnified regions (white box) expanded below and presented as the green (left) red (middle) and merge channels. Grossly over-exposed Congo red staining is included (C) to highlight the lack of amyloidogenic dye binding to the aggresome. (D) Knockdown efficiency of siRNA targeting established IncRNA. PC-3 cells transfected with control or siRNA targeting rlGS₂₂RNA, rlGS₂₈RNA, NEAT1 , MALAT1 , HOTAIR and GAPDH mRNA were exposed to heat shock or 5μΜ MG132 conditions. The indicated transcripts were detected by RT-PCR. Actin and total RNA are loading controls. (E) Inhibition of rlGS22 NA and rlGS₂sRNA does not impair the formation of other cellular bodies. MCF- 7 cells stably expressing shRNA against rlGS₂2RNA (left panels) and rlGS₂₈RNA (right panels) were treated as indicated and stained for subcellular body specific markers (green) and Congo red (red). Dashed circles represent nuclei. Selected regions (white box) were expanded below. White scale bars represents 20 μιη.

[0026] Figure 10(A)-10(F): The A-body is a heterogeneous population of proteins with similar characteristics to the total detected proteome

[0027] (A) A heterogeneous family of proteins are targeted to the cellular A-bodies. Distribution of protein size (kDa) (left panel), isoelectric point (pi) (middle panel) and grand average of hydrophobicity (GRAVY) scores (right panel) for the total (838 proteins) and A-body-specific (184 proteins) populations. Isoelectric point and GRAVY scores were calculated using the ProtParam program (SIB ExPASy Bioinformatics Resource Portal). The GRAVY score for each protein was calculated as the sum of hydropathy values of all the amino acids, divided by the number of residues in the protein. (B) Validation of SILAC-MS candidate proteins. Normoxic/neutral,

hypoxic/neutral and hypoxic/acidotic treated MCF-7 cells were harvested and whole cell lysates (WCL), cytoplasmic (Cyto), nuclear (Nuc) and nucleolus/A-body fractions were analyzed by western blot with antibodies against the catalytic subunit of DNA

polymerase delta (POLD1 ), spliceosome RNA helicase (UAP56), 14-3-3ζ, elongation factor 1 β (eEF1 B2), transcription initiation factor 1 β (TIF-Ι β), histone acetyltransferase-1 (HAT-1 ) and cyclin-dependent kinase 1 (cdkl ). Nucleolar transcription factor-1 (UBF1 ) and cyclin G were used as loading controls. (C-D) SILAC-MS candidates co-localize with the amyloid dye Amylo-Glo. PC-3 (C) and MCF-7 (D) cells were grown under hypoxic/acidotic conditions and analyzed by indirect immunofluorescence microscopy for endogenous HAT1 , TIF-1 β, cdkl and PCNA. Amylo-Glo images of the same field were captured (blue). (E) SILAC-MS candidates are targeted to nuclear foci. PC3 cells were grown in normoxia/neutral, hypoxia/neutral and hypoxia/acidosis conditions and analyzed by indirect immunofluorescence microscopy for endogenous UAP56, HAT1 , TIF-1 β, ATP-dependent helicase (ATRX), DNA polymerase alpha catalytic subunit (POLA1 ), POLD1 , cdkl , replication factor C subunit 1 (RFC1 ), UBF1 and PCNA. (F) Localization of SILAC-MS candidates to subnuclear foci is dependent on rIGSRNA. MCF-7 cells stably-expressing shRNA against rlGS₂sRNA (sh28#1 ) or a control sequence were treated and stained for endogenous proteins as in (E). B23 (green) inset. Dashed circles represent nuclei. White scale bars, 20pm.

[0028] Figure 11(A)-11(H): Targets of the A-body possess amyloidogenic potential

[0029] (A) UAP56, RNF8 and cdkl target to nuclear foci and can obtain an amyloidlike conformation upon bacterial expression. UAP56-GFP, RNF8-GFP, cdk1 -GFP, Ran- GFP, cdk4-GFP or the nucleolar resident protein B23-GFP were expressed in MCF-7 cells left untreated or exposed to acidosis or BL21 cells prior to staining with Congo red and Hoechst. (B) The central region targets VHL to the A-body. 36 amino acid fragments of VHL were fused to GFP and expressed in MCF-7 cells untreated or exposed to acidosis. Subcellular distribution of the VHL fragments was detected by fluorescence microscopy (C) An RH cluster and amyloidogenic region is necessary for stress-specific ^solubilization. The indicated fragments of VHL or the artificial ACM (aACM) of VHL and POLD1 were fused to GFP and expressed in MCF-7 cells exposed to acidosis for 2 hours. Whole cell lysates (WCL) and insoluble fractions were harvested. Exogenous proteins were detected by western blot (GFP). GAPDH and Histone H3 were used as loading controls. (D) Tethering ACM or aACM sequences to GFP immobilize the fusion protein in the A-bodies. Quantification of recovery after photobleaching kinetics for the constructs listed as the mean relative intensity of at least 5 data sets. (E) Cdk1 , HAT1 and HDAC2 contains fibril-forming peptidic regions.

Results of the Rosetta-design program ZipperDB analysis of full length cdkl and fragments of cdkl , HAT1 and HDAC2 containing an R/H cluster in close proximity to regions with fibrillation propensity. The Rosetta energy threshold of -23 kcal/mol was used as an indicator of fibril-positive regions. Fragments fused to GFP are indicated with amino acid sequence bracketed. (F) Mapping the ACM of VHL and cdkl . VHL (amino acids: 104-140 and 122-140) or cdkl (amino acids: 100-130 and 100-1 19)-GFP were expressed in BL21 cells (stained with Congo red and Hoechst) or MCF-7 cells (stained with Hoechst) left untreated or exposed to acidosis. (G) An R/H cluster is essential for mammalian amyloidogenesis. HAT1 and HDAC2 fragments described above (E) were expressed in MCF-7 cells exposed to extracellular acidosis. (H) aACMs are sufficient to induce amyloidogenesis. aACM described above were expressed in MCF-7 cells left untreated, exposed to acidosis prior to fluorescence microscopy.

Hoechst is inset. BL21 cells expressing aACM-GFP were stained with Congo red and Hoechst. X-ray diffraction was performed on BL21 inclusion bodies. Dashed circles represent nuclei (MCF-7) or whole cell (BL21 ). White scale bars, 20μιτι.

[0030] Figure 12(A)-12(C): APP and β-amyloid are targets of physiological amyloidogenesis

[0031] (A) Schematic diagram of amyloid precursor protein (APP). Signal peptide and transmembrane domain are highlighted in yellow, -, β-, and γ-secretase cleavage sites are indicated. The location of various truncations used are presented. (B) β-amyloid is targeted to subnuclear foci during specific environmental stress. The pathological β- amyloid (1 -40) and non-pathological P3 (18-40) peptides were fused to GFP and expressed in MCF-7 cells left untreated or exposed to acidosis, heat shock,

thapsigargin, sodium arsenite, transcriptional stress (actinomycin D and MG132) or H₂0₂ for the indicated times. (C) Random 42 amino acid regions of APP are not targeted to A-bodies. The indicated regions of APP or 42 amino acid fragments of this proteins were fused to GFP and expressed in untreated or acidotic MCF-7 cells.

Dashed circles represent nuclei. White scale bars, 20μιη. [0032] Figure 13(A)-13(E): Physiological amyloidogenesis and disaggregation require rIGSRNA and heat shock protein activity

[0033] (A) Amyloid disintegration is caused by protein disaggregation, not

degradation. VHL-GFP expressing MCF-7 cells were left untreated, exposed to acidosis or recovered for 4 hours with no drugs, the Hsp70 inhibitor VER155008 (VER) or the protein synthesis inhibitor cycloheximide. (B) Heat shock proteins are present in A- bodies as cells recover from extracellular acidosis and thermal stress. MCF-7 cells recovering from acidosis or heat shock (2 hours) were stained for Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, Hsp105, HSF1 , GRP78, GRP94, Calnexin, Calreticulin, and Protein Disulfide Isomerase. Congo red is inset (C) Schematic diagram indicating the stressing, recovery and drug treatment of MCF-7 cells exposed to chaperone inhibitors in figure 5 (C-E). (E) Chaperone inhibitors do not effect viability during recovery. MCF-7 cells exposed to heat shock for 3 hours were allowed to recover in media containing no drugs or the chaperone inhibitors 16F16 (5μΜ), EGCG (5μΜ), VER155008 (40μΜ), 17- AAG (5μΜ) or VER155008+17-AAG, the autophagy inhibitor wortmanin (5μΜ) or the protein synthesis inhibitor cycloheximide (25μ9/ηιΙ) for 4 hours. Viability was detected by fluorescein diacetate (FDA) fluorescence intensity. (E) Chaperone inhibitor treatment does not induce physiological amyloidogenesis. MCF-7 cells expressing VHL-GFP were left untreated or exposed to the chaperone inhibitors 16F16 (5μΜ), EGCG (5μΜ), VER155008 (40μΜ), 17-AAG (5μΜ) or VER155008+17-AAG for 4 hours and stained with Hoechst (inset left) and Congo red (inset right). White scale bars, 20μπι.

[0034] Figure 14(A)-14(I): Acidosis induces cellular dormancy in an rlGS₂8RNA- dependent manner

[0035] (A) Hypoxia has minimal effect on cell proliferation. MCF-7, PC-3 and WI-38 cells were incubated under standard growth conditions (21 % 0₂ + pH 7.4) for two days, prior to being transferred to a hypoxic environment (1 % 0₂ + pH 7.4). 750,000 (MCF-7 and PC-3 - left axis) or 75,000 (WI-38 - right axis) cells were initially cultured and counted daily with Trypan Blue staining to ensure viability. (B) MCF-7 with impaired rlGS₂8RNA expression proliferate during hypoxia. MCF-7 cells stably-expressing two independent shRNA against rlGS₂₈RNA (sh28#1 and sh28#2) or a control sequence (shCtrl) were grown under the indicated conditions and counted as above. (C) Knockdown efficiency of the stable PC-3 shRNA cell lines. PC-3 parental, control and rlGS₂8 NA (two clonal lines)-specific shRNA cell lines were tested for the knockdown efficiency of the rlGS₂₈RNA transcripts by RT-PCR. Actin and total RNA are loading controls. (D-E) Inhibition of rlGS₂₈RNA restores proliferative capacity to acidotic PC-3 cells. PC-3 lines described above (C) were exposed to hypoxia in acidosis-permissive media for the indicated times. (D) Live cells were counted as described above or (E) BrdU was added 1 hour prior to fixation and BrdU detection. Percent BrdU incorporation was calculated as BrdU-positive nuclei divided by Hoechst-positive nuclei. (F)

Intracellular ATP levels are maintained during extracellular acidosis by rlGS₂₈RNA. MCF-7 cells stably-expressing two independent shRNA against rlGS₂₈RNA or a control sequence (shCtrl) were exposed to normoxic-neutral, hypoxic-neutral and hypoxic- acidosis conditions for 24 hours prior to assessment of intracellular ATP levels using the CellTiter-Glo Luminescent Assay. (G) Acidosis induces a state of cellular dormancy. MCF-7 cells stably-expressing control (shCtrl) or rlGS₂₈RNA (sh28#1 )-specific shRNA were grown under the indicated conditions for 24 hours, prior to the addition of nocodazole (24 hours) to induce a G₂/M phase arrest in proliferating cells. Flow cytometry analysis of propidium iodide stained cells was used to assess cell cycle stage. (H) Inhibition of rlGS₂₈RNA enhances PC-3 tumor growth. Nude mouse xenograft assays with PC-3 cells expressing no shRNA, shRNA against a control sequence or shRNA targeting rlGS₂₈RNA (n=4). Tumor volumes were determined at two weeks post- injection (left). Endogenous HAT1 was detected by indirect immunofluorescence microscopy (B23- inset green) and hematoxylin/eosin staining was performed on paraffin-embedded sections. (I) Inhibition of rlGS₂₈RNA impairs amyloidogenesis in mouse xenografts. Paraffin-embedded MCF-7 shControl or shrlGS₂₈RNA xenograft sections were stained with Congo red and fluorescent in situ hybridization was performed to detect rlGS₂₈RNA. B23 (green) is inset. Results are presented as means and SEM (n=3). Significance values were calculated by a two-tailed Student's t-test; ^*p shown < 0.01 . DETAILED DESCRIPTION

[0036] The present disclosure provides a method of testing a drug for efficacy for treating an amyloid disease. In exemplary embodiments, the method of testing a drug for efficacy for treating an amyloid disease comprises (i) contacting cells with the drug before or after the cells are contacted with a stimulus that causes amyloid aggregate formation in the cells and (ii) assaying amyloid aggregate formation or amyloid aggregate disaggregation in the cells. The drug has efficacy for treating the amyloid disease, when amyloid aggregate formation decreases or is delayed or amyloid aggregate disaggregation increases, upon contacting the cells with the drug.

[0037] As used herein, the term "amyloid disease" refers to any disease, disorder, or medical condition associated with or caused by amyloid aggregation or the presence of amyloid aggregates. As used herein, the term "amyloid aggregate" refers to a structure comprising one or more proteins that adopt an amyloid-like immobile/insoluble state. In exemplary aspects, the amyloid aggregate is an amyloid body. As used herein, the term "amyloid bodies" also referred to herein as "A-bodies" means inducible and reversible sub-nuclear foci composed of an array of different proteins that adopt an amyloid-like immobile/insoluble state. In exemplary aspects, the amyloid aggregate or amyloid body comprises one or more of the proteins listed in Table 1 . In exemplary aspects, the amyloid aggregate or amyloid body comprises two or more of the proteins listed in Table 1 .In exemplary aspects, each protein of the amyloid aggregate or amyloid body is present as amyloid, or in an amyloid fold conformation. As used herein, the term "amyloid" means a highly organized form of protein aggregation. In exemplary aspects, the amyloid disease is a neurodegenerative disease. In exemplary aspects, the amyloid disease is a human neuropathy, including, but not limited to, Alzheimer's, Parkinson's and Huntington's diseases.

[0038] As used herein, the term "treat," as well as words related thereto, do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods of treating an amyloid disease can provide any amount or any level of treatment. Furthermore, the treatment provided by the drug tested by the method of the present disclosure may include treatment of one or more conditions or symptoms or signs of the amyloid disease being treated. Also, the treatment provided by the drug tested by the method of the present disclosure may encompass slowing amyloid disease progression or slowing the onset of amyloid disease. For example, the drug identified by the method can treat the amyloid disease by virtue of slowing amyloid aggregate formation or slowing the progression of amyloid aggregate formation, increasing the occurrence and/or frequency and/or rate of amyloid aggregate disaggregation, and the like.

[0039] In exemplary aspects, the drug tested by the method of the present disclosure may treat amyloid disease by virtue of preventing or inhibiting amyloid aggregate formation. As used herein, the term "prevent" and words stemming therefrom

encompasses delaying the onset of the medical condition being prevented. In exemplary aspects, the method delays the onset of the medical condition by 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, two months, 4 months, 6 months, 1 year, 2 years, 4 years, or more. As used herein, the term "prevent" and words stemming therefrom encompasses reducing the risk of the medical condition being prevented. In exemplary aspects, the method reduces the risk of amyloid disease 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more. As used herein, the term "inhibit" and words stemming therefrom may not be a 100% or complete inhibition or abrogation. Rather, there are varying degrees of inhibition of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the drug tested by the method of the present disclosure may inhibit amyloid aggregate formation to any amount or level. In exemplary embodiments, the inhibition provided by the drug tested by the methods of the present disclosure is at least or about a 10% inhibition (e.g., at least or about a 20% inhibition, at least or about a 30% inhibition, at least or about a 40% inhibition, at least or about a 50% inhibition, at least or about a 60% inhibition, at least or about a 70% inhibition, at least or about a 80% inhibition, at least or about a 90% inhibition, at least or about a 95% inhibition, at least or about a 98% inhibition).

[0040] In exemplary aspects, step (ii) of the method comprises contacting the cells with an amyloid aggregate binding agent or amyloid body binding agent. In exemplary aspects, the method comprises (a) contacting cells with (i) a stimulus to cause amyloid aggregate formation in the cells, (ii) the drug, and (iii) an amyloid aggregate binding agent or amyloid body binding agent; and (b) detecting the level of amyloid aggregate binding agent or amyloid body binding agent bound to amyloid. In exemplary aspects, the drug has efficacy for treating the amyloid disease, when the amyloid aggregate binding agent or amyloid body binding agent bound to amyloid is not detected or the level of the amyloid aggregate binding agent or amyloid body binding agent bound to amyloid is lower than a control level.

[0041] In exemplary aspects, the stimulus that causes amyloid aggregate (e.g., amyloid body) formation in the cells is acidosis or heat shock exposure. In exemplary aspects, the stimulus is a 3-hour acidosis or heat shock exposure. Suitable methods of contacting the cells with a stimulus that causes amyloid aggregate (e.g., amyloid body) formation in the cells, e.g., acidosis, heat shock exposure, are provided herein. See, e.g., Example 1 .

[0042] In exemplary aspects, the cells are contacted with the drug, or a plurality of drugs, before the cells are contacted with the stimulus. In exemplary aspects, the ability of the drug to prevent or slow amyloid aggregate (e.g., amyloid body) formation is tested by the method of the present disclosure.

[0043] In alternative aspects, the cells are contacted with the drug (or the plurality thereof) after the cells are contacted with the stimulus. In exemplary aspects, the ability of the drug to cause amyloid aggregate (e.g., amyloid body) disaggregation is tested by the method of the present disclosure.

[0044] In exemplary aspects, the amyloid body binding agent is a dye or molecule which emits a signal when an amyloid aggregate (e.g., amyloid body) forms.

[0045] In exemplary embodiments, the method comprises quantifying the amount of signal emitted by the molecule. In exemplary aspects, the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of signal emitted by the molecule about 1 hour or less after the cells are contacted with the stimulus. In exemplary aspects, the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of signal emitted by the molecule about 2 hours after the cells are contacted with the stimulus. In exemplary aspects, the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of signal emitted by the molecule about 3 hours after the cells are contacted with the stimulus. In exemplary aspects, the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of signal emitted by the molecule about 4 hours after the cells are contacted with the stimulus. In exemplary aspects, the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of signal emitted by the molecule more than 4 hours after the cells are contacted with the stimulus.

[0046] In exemplary aspects, the dye is a colorimetric dye or a fluorescent dye. In exemplary aspects, the fluorescent dye is selected from the group consisting of: Congo Red, Thioflavin S, NIAD4, and Methoxyl-X04 and BSB ((Trans, Trans)-1 -Bromo-2,5- Bis-(3-Hydroxycarbonyl-4-Hydroxy)Styrylbenzene).

[0047] In exemplary embodiments, the method comprises quantifying the amount of fluorescence emitted by the dye. In exemplary aspects, the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye about 1 hour or less after the cells are contacted with the stimulus. In exemplary aspects, the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye about 2 hours after the cells are contacted with the stimulus. In exemplary aspects, the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye about 3 hours after the cells are contacted with the stimulus. In exemplary aspects, the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye about 4 hours after the cells are contacted with the stimulus. In exemplary aspects, the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye more than 4 hours after the cells are contacted with the stimulus. For example, the method in some aspects comprises quantifying the amount of fluorescence emitted by the dye about 6 to about 36 hours (e.g., about 8 to about 30 hours, about 10 to about 24 hours, about 12 to about 20 hours, about 14 to about 18 hours) after the cells are contacted with the stimulus.

[0048] In exemplary aspects, the cells are contacted with the drug after the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye about 1 hour or less after the cells are contacted with the drug. In exemplary aspects, the cells are contacted with the drug after the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye about 2 hours after the cells are contacted with the drug. In exemplary aspects, the cells are contacted with the drug after the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye about 3 hours after the cells are contacted with the drug. In exemplary aspects, the cells are contacted with the drug after the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye about 4 hours after the cells are contacted with the drug. In exemplary aspects, the cells are contacted with the drug after the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye more than 4 hours after the cells are contacted with the drug. For example, the method in some aspects comprises quantifying the amount of fluorescence emitted by the dye about 6 to about 36 hours (e.g., about 8 to about 30 hours, about 10 to about 24 hours, about 12 to about 20 hours, about 14 to about 18 hours) after the cells are contacted with the drug.

[0049] Neurodegenerative Disease

[0050] In exemplary aspects, the drug is tested for efficacy for treatment of a neurodegenerative disease. In exemplary aspects, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, other demyelination related disorders, senile dementia, subcortical dementia, arteriosclerotic dementia, AIDS-associated dementia, or other dementias, a central nervous system cancer, traumatic brain injury, spinal cord injury, stroke or cerebral ischemia, cerebral vasculitis, epilepsy, Huntington's disease, Tourette's syndrome, Guillain Barre syndrome, Wilson disease, Pick's disease, neuroinflammatory disorders, encephalitis, encephalomyelitis or meningitis of viral, fungal or bacterial origin, or other central nervous system infections, prion diseases, cerebellar ataxias, cerebellar degeneration, spinocerebellar degeneration syndromes, Friedreichs ataxia, ataxia telangiectasia, spinal dysmyotrophy, progressive supranuclear palsy, dystonia, muscle spasticity, tremor, retinitis pigmentosa, striatonigral degeneration, mitochondrial encephalo-myopathies, neuronal ceroid lipofuscinosis, hepatic encephalopathies, renal encephalopathies, metabolic encephalopathies, toxin-induced encephalopathies, or radiation-induced brain damage. In exemplary aspects, the neurodegenerative disease is a neuropathy.

[0051] Cells

[0052] With regard to the method of the present disclosure, the cells which are contacted with the drug may be of any cell type and can originate from any type of tissue, and can be of any developmental stage. The cells may be any eukaryotic cell type, e.g., plant, animal, fungi, algae. In exemplary aspects, the cells are animal cells. In exemplary aspects, the cells used in the method of the present disclosure are mammalian cells. In exemplary aspects, the mammalian cells are human cells.

[0053] In exemplary aspects, the cells are cultured cells and, in other aspects, the cells are primary cells, e.g., cells directly isolated from an organism. In exemplary aspects, the cells are adherent, while in other aspects, the cells are in suspension. In exemplary aspects, the cells are CHO cells, VERO cells, COS cells, and the like. In exemplary aspects, the cells are selected from the group consisting of: MCF-7, PC-3, A549, HEK293, C2C12, U87mg, NIH3T3, HCT1 16, PC-12 and Neuro-2A cells.

[0054] In exemplary aspects, the cells are part of a population of cells. The cells in exemplary aspects are a heterogeneous population. In alternative aspects, the cells are substantially homogeneous or a clonal population of cells.

[0055] Test Drugs

[0056] With regard to the method of the present disclosure, the drug tested for efficacy for treating an amyloid disease may be any type of drug or molecule. In exemplary aspects, the drug is a small molecule, a biomolecule, a polymer, a carbohydrate, or a lipid. In exemplary aspects, the drug is a biomolecule, such as, for example, a protein, a peptide, an amino acid, an antibody or fragment thereof, a nucleic acid comprising one or more deoxynucleotides or synthetic analogs thereof,

ribonucleotides or synthetic analogs thereof, or a combination thereof. In exemplary aspects, the drug is from a chemical library. In exemplary aspects, the method tests a chemical library of small molecules. Suitable chemical libraries are commercially available through vendors, such as Sanford Burnham (La Jolla, CA), Sigma-Aldrich (St. Louis, MO), and Selleck Chem (Houston, TX). In exemplary aspects, the drug binds to one or more of the proteins listed in Table 1 . In exemplary aspects, the drug binds to one or more of the proteins listed in Table 1 only when the protein is in an amyloid aggregate (e.g., amyloid body) or when the protein is in an amyloid fold conformation.

[0057] The method of the present disclosure is advantageous for the ability to test multiple drugs for efficacy for treating an amyloid disease in a relatively short period of time. In exemplary aspects, at least or about 20 drugs are simultaneously tested. In exemplary aspects, at least or about 75 drugs are simultaneously tested. In exemplary aspects, the method is capable of simultaneously testing a plurality of drugs within about 12 hours for efficacy for treating the disease. In exemplary aspects, the method is capable of simultaneously testing a plurality of drugs within about 6 hours for efficacy for treating the disease.

[0058] Controls

[0059] In exemplary aspects of the methods of the present disclosure, the drug is deemed as having efficacy for treating the amyloid disease, when amyloid aggregate (e.g., amyloid body) formation decreases or is delayed or amyloid aggregate (e.g., amyloid body) disaggregation increases, upon contacting the cells with the drug. In exemplary aspects, delayed amyloid formation, decreases in amyloid aggregate (e.g., amyloid body) formation or increases in amyloid aggregate (e.g., amyloid body) disaggregation is relative to a rate or level of amyloid formation or to a level of amyloid disaggregation of a control. In exemplary aspects, the control is the rate or level of amyloid formation or to a level of amyloid disaggregation achieved by stimulated cells in the absence of a drug. In exemplary aspects, such cells not exposed to any drug are contacted with a vehicle control, e.g., DMSO, PBS, and the like.

[0060] In exemplary aspects, when amyloid aggregate (e.g., amyloid body) formation or disaggregation is measured vis-a-vis measurement of an amyloid aggregate (e.g., amyloid body) binding agent bound to amyloid aggregates (e.g., amyloid bodies), the level at which the amyloid aggregate (e.g., amyloid body) binding agent is bound to amyloid is compared to a control level. In exemplary aspects, the control level is the level at which the amyloid aggregate (e.g., amyloid body) binding agent is bound to amyloid by stimulated cells in the absence of a drug. In exemplary aspects, such cells not exposed to any drug are contacted with a vehicle control, e.g., DMSO, PBS, and the like.

[0061] The following examples are given merely to illustrate the present invention and not in any way to limit its scope.

EXAMPLES

[0062] Proteins convert from a native-fold to an amyloid-like state in sub-nuclear amyloid-bodies (A-bodies) for adaptation to stressors. Cellular amyloids undergo efficient disaggregation by the hsp chaperone family upon stimuli termination. This physiological pathway causes reversible amyloidogenesis of the pathological β-amyloid peptide, which is associated with Alzheimer's disease.

[0063] The following examples highlight that (1 ) systemic protein amyloidogenesis forms the amyloid-bodies (A-bodies) on stimuli; (2) the amyloid-converting motif and ribosomal intergenic IncRNA mediate amyloidogenesis; (3) the heat shock chaperone pathway disaggregates the A-bodies; and (4) cells activate physiological

amyloidogenesis to enter a dormant state.

[0064] At least some of the data of the following examples are presented in Audas et al., Adaptation to Stressors by Systemic Protein Amyloidogenesis, Dev Ce// 39(2):155- 168 (2016). EXAMPLE 1

[0065] The following experimental procedures were carried out for the studies described in Examples 2-8,

[0066] Cell Lines, Treatments and Stains: MCF-7, PC-3 and WI-38 cell lines were purchased from the ATCC and propagated in the suggested media. Stable MCF-7 and PC-3 cell lines were previously described (Audas et al., 2012). Hypoxia was induced by incubating cells in an H35 Hypoxystation at 37°C in a 1 % 0₂, 5% C0₂ and N₂-balanced environment (HypOxygen). Acidosis was performed under hypoxic conditions by transferring cells to pH 6.0 media or growing cells in acidosis-permissive media as previously described (Audas et al., 2012). Heat shock was at 43°C for the indicated times. Sodium arsenite (125mM), H₂0₂ (300nM), thapsigargin (300nM), transcriptional stress (4μΜ Actinomycin D + 8μΜ MG132), rapamycin (5μΜ) and cycloheximide ^g/ml) were treated for the indicated times. The Hsp70 (VER-155008: 40μΜ), Hsp90 (17-AAG: 5μΜ), PDI (16F16: 5μΜ) and GRP94 (Epigallocatechin gallate: 5μΜ) chaperone inhibitors and autophagosome inhibitor (Wortmannin: 5μΜ) were added 30 minutes before the 2 or 4 hour recoveries or for 3 hours in untreated cells. All recovered cells were grown in standard growth media at 21 % 0₂ following stress treatment. Congo red (0.05%), Thioflavin S (0.002%), Amylo-Glo (1 X), NIAD4 (10μΜ) and Methoxyl-X04 (20μΜ) were used to stain formaldehyde-fixed cells. Proteinase K (O.^g/ml) treatment of methanol-fixed MCF-7 cells occurred at 25°C (1 hr).

[0067] Insoluble Fractionation: Mammalian or bacterial cells were re-suspended in NP40 buffer (50mM Tris-HCI + 150mM NaCI + 1 % NP40) and incubated at 25^QC for 5min. Lysates were sonicated 2x for 10sec at 25% power (QSonica) and aliquots were taken as whole cell lysate fractions. Lysates were pelleted at 8000rpm for 10min. The pellet was washed 2x, prior to addition of NP40 buffer and one sonication for 10sec at 25% power to fully re-suspend the insoluble fraction.

[0068] Bacterial Preparation: BL21 cultures were grown at 32°C (20hrs) prior to insoluble fractionation, inclusion body purification or staining. Microscopy was performed on fixed (4% formaldehyde 1 hr) and permeabilized (0.5% Triton 10 min) cells stained with Congo red and Hoechst. [0069] Fibrillation Propensity: Rosetta free energy scores for proteins and peptides was calculated using online ZipperDB software (UCLA) derived from published work (Goldschmidt et al., 2010).

[0070] Statistical analysis: Bar and line graphs represent the mean value from at least (n values indicated in figure legends) three independent replicates. Statistical analyses were performed with the error bars representing the standard error of the mean, p values were based on two-tailed Student's t-test with the significance level indicated in the figure legend.

[0071] Antibodies and dyes: Antibodies purchased were validated for western blotting and immunohistochemistry by the supplier. Monoclonal antibodies were used to detect 14-3-3ξ (QED Biosciences Inc.), B23 (Santa Cruz Biotechnology, sc-47725), eEF1 B2 (Abnova, H00001933-M10), cdkl (Boehringer Mannheim), Hsp27 (Cell

Signaling Technology, 2402S), Hsp40 (Cell Signaling Technology, 4871 ), Hsp60 (Cell Signaling Technology 1216S), Hsp90 (Cell Signaling Technology, 4877), VHL

(Oncogene Science, OP102), BiP/GRP78 (Cell Signaling Technology, 3177), Calnexin (Cell Signaling Technology, 2679), PDI (Cell Signaling Technology, 3501 ), PCNA (Santa Cruz Biotechnology, sc-25280), UBF1 (Santa Cruz Biotechnology, sc-13125), Vimentin (Santa Cruz Biotechnology, sc-6260), SC35 (Abeam, ab1 1826) and

p54nrb/NonO (EMD Millipore, 05-950), and polyclonal antibodies detected POLD1 (Santa Cruz Biotechnology, sc-8797), POLA1 (Santa Cruz Biotechnology, sc-48818), ATRX (Santa Cruz Biotechnology, sc-15408), RFC1 (Santa Cruz Biotechnology, sc- 20993), Hsp70 (Cell Signaling Technology, 4872), Hsp105 (Santa Cruz Biotechnology, sc-6241 ), HSF1 (Cell Signaling Technology, 4356), GRP94 (Santa Cruz Biotechnology, sc1794), Calreticulin (EMD Millipore, 208910), UAP56 (Abeam, ab47955), TIF-1 β (Santa Cruz Biotechnology, sc-33186), Fibrillarin (Santa Cruz Biotechnology, sc-25397), GFP (Abeam, ab290), HAT1 (Santa Cruz Biotechnology, sc-8752),cyclin G (Santa Cruz Biotechnology, sc-320), Histone H3 (sc-10809), Coilin (Santa Cruz Biotechnology, sc- 32860), TIA-1 (Santa Cruz Biotechnology, sc-1751 ), LC3 (MBL International), Dcpl a (Abeam, ab4781 1 ), HDAC2 (Santa Cruz Biotechnology, sc-7899), G3BP1 (Santa Cruz Biotechnology, sc-98561 ), PML (Abeam, ab53773), anti-amyloid fibrils OC (EMD

Millipore, AB2286), GAPDH (Santa Cruz Biotechnology, sc-25778). Secondary antibodies were HRP- or Alexa-conjugated (Life Technologies). Hoechst (Life Technologies), ethidium bromide (Sigma Aldrich), Congo red (Amresco), Thioflavine S (Sigma Aldrich), Amylo-Glo (Biosensis), NIAD4 (Biovision, 10μΜ), Methoxy-X04 (Biovision, 20μΜ), 1 -bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy) styrylbenzene (Anaspec, 200μΜ) and hematoxylin/eosin (Sigma) dyes were used.

[0072] Stable cell lines, siRNA and transfections: MCF-7 and PC-3 cells stably expressing control and rlGS2sRNA/rlGS22RNA specific shRNA were previously described¹. Transfections were performed using Effectene (Qiagen), according to manufacturer's protocol. The following siRNAs were purchased from Ambion: NEAT1 (n509900), MALAT1 (n51 1399), HOTAIR (n272230), Negative control #1 (4390844), GAPDH positive control (4390849) and custom sequences for NGS22RNA

(AGGCACTGTATTGCTACTGGGCT) and rlGS₂₈RNA

(AATCTAGACAGGCGGGCCTTGCT). PC3 cells were reverse-transfected using RNAiMAX (Life Technologies) and treated/harvested 48 hours post-transfection.

[0073] Plasmids: pFLAG-VHL-GFP, pFLAG-POLD1 -GFP, pYFP-Tial , pEGFP-B23, pEGFP-Fibrillarin, pFLAG-RNF8-GFP, pcDNA3.1 , pcGFP were previously generated¹. Full length cDNA was subcloned into pEGFP-C2 from the Addgene plasmids pcDNA3.1 -SC35-cMyc (K. Scotto: Addgene plasmid 44721 ) and pSCT-GAL93-NONO (S. Brown: Addgene plasmid 46325), while amino acids 1 -770 and 18-713 of APP were subcloned into the same plasmid from pCAX-FLAG-APP (D. Selkoe: Addgene plasmid 30154). The following plasmids were purchased from Addgene and used without modification: pEGFP.N1 -HDAC6 (T. Yao: Addgene plasmid 36188), pEGFP-coilin (G. Matera: Addgene plasmid 36906) and HSF1 -GFPN3 (S. Calderwood: Addgene plasmid 32538). The cDNA encoding UAP56, HDAC2, Ku70, cdkl , Ran, cdk4, HAT1 , β-amyloid (amino acids: 1 -42), β-amyloid (amino acids: 1 -17), P3 (amino acids: 18-42) and fragments of VHL (amino acids: 1 -213, 1 -36, 34-70, 69-105, 104-140, 122-140, 139-175 and 174-213), cdkl (amino acids: 100-1 19, 100-130), HDAC2 (amino acids: 1 -33 and 18-60), HAT1 (amino acids: 228-260 and 366-400) and APP (amino acids: 167-209, 209-251 , 543-585, 586-628, 629-671 and 723-770) was reverse transcribed from MCF- 7 RNA and inserted into the pET30-LIC (EMD Millipore) and pcTOPO (Life

Technologies) cloning vectors with carboxy-terminal GFP tags. Artifical Amyloid Converting Motifs (aACM) derived from POLD1 and VHL were created from linker sequences and sub-cloned into the pET30 and pcTOPO GFP plasmids.

[0074] Photobleaching: Fluorescence loss in photobleaching (FLIP) and

fluorescence recovery after photobleaching (FRAP) was performed as previously described² on a Zeiss LSM5 Pascal confocal microscope.

[0075] Bacterial inclusion body purification and x-ray diffraction: Bacterial inclusion body purification was modified from⁴ Briefly, 100ml cultures were grown for 20 hours at 32°C, then pelleted and re-suspended in 4ml bacterial lysis buffer (50mM Tris- HCI + 10OmM NaCI + 1 mM EDTA + 20mM PMSF + 0.1 % Triton X100) and incubated for 1 hour at 37°C with gentle agitation. Samples were sonicated on ice for 20 minutes (1 second on, 1 second off) at 40% power (QSonica Sonicators). NP40 (0.8%) was added to lysates and incubated at 4°C with gentle agitation for 1 hour, prior to a 1 hour DNase I treatment at 37°C. Samples were pelleted at 15,000rpm for 15 minutes, washed twice in lysis buffer and re-suspended in ddH₂0. Samples were checked by x- ray diffraction for amyloid content by pelleting down insoluble fractions and then pipetting 2-4 μΙ of the resulting slurry on mounted nylon loops (1 mm Cryoloops on Crystal Caps from Hampton Research). The resulting drops were allowed to dry in air for 2-3 days and then exposed on X-rays at room temperature. 10 minute exposures were taken on a Rigaku MicroMax 007HF generator using a MAR345 detector at the Scripps Florida X-ray Crystallography Core Facility. Strong reflections were seen at 4.7 A and 10 A, which are characteristic of the cross-beta sheet structure in amyloid fibrils⁵.

[0076] Transmission electron microscopy: 2% glutaraldehyde fixed cells were washed and post-fixed in 1 % osmium tetroxide. Samples were dehydrated in a series of ascending alcohols and embedded in Epon/Araldite and stained with uranyl acetate and lead citrate. Imaging occurred on a JEOL 1400 transmission electron microscope (Jeol, MA).

[0077] Immunofluorescence: Cells were seeded in 3.5 cm plates with 20-mm glass coverslips. Post-treatment, cells were fixed in 4% formaldehyde for 20 minutes and permeablized in 0.5% TritonX-100 for 5 minutes. Cells were incubated for 1 hour with the primary antibody (1 :100), washed and incubated for another hour with the secondary antibody (1 :300). Hoechst or ethidium bromide was added in PBS for 5 minutes. Congo red and Thioflavine S staining followed established protocols^6"8. All amyloidophilic dyes were stained used with formaldehyde-fixed cells to minimize background fluorescence. Cells were immersed in 0.05% Congo red or 0.002%

Thioflavine S solution for 15 minutes and then washed 3 times in ddH₂0. Amylo-Glo staining was modified from Schmued et al. 2012⁹. Fixed cells were rinsed in ddH₂0, immersed in 1 X Amylo-Glo staining solution for 10 minutes, washed in 0.9% saline for 5 minutes and briefly rinsed in ddH₂0. Slips were mounted and analyzed using an Axio Observer D1 inverted microscope. Images were captured in black and white using an AxioCam MRm and ZEN Pro 2012 software (Carl Zeiss). Artificial color was added using Photoshop CS6 (Adobe). Merged images were generated using ImageJ software. Artificially colored images were combined by Zprojection, with a max intensity projection type.

[0078] Amyloid-Body Purification: Five 15cm plates per condition were washed in PBS and cells were scraped off of the surface. Osmotic buffer (10mM HEPES-pH 7.9, 10mM KCI, 1 .2mM MgCfe, and 0.5mM DTT) was added and cells were incubated on ice for 5 minutes. Cells were dounced 10 times and pelleted. A sample of the supernatant was saved as the cytoplasmic fraction. Pellets were re-suspended in osmotic buffer, incubated on ice for 5 minutes, dounced 10 times and pelleted. Supernatant was discarded and a portion of the pellet was saved as the nuclear fraction. 1 % TritonX-100 was used to re-suspend the pellet and slurry was sonicated twice for 10 seconds at 25% power (QSonica Sonicators). Solution was pelleted, the supernatant was discarded and the remaining material was re-suspended in 400μΙ 65% Percoll + 35% 1 xNEH buffer (20mM HEPES-pH 7.4, 150mM NaCI, 0.2mM EDTA and 0.6% TritonX-100). Sample was layered with 400μΙ 55% Percoll + 45% 1 xNEH buffer, 400μΙ 45% Percoll + 55% 1 xNEH buffer and 400μΙ 35% Percoll + 65% 1 xNEH buffer. Percoll gradients were spun at 1000 times gravity for 90 minutes and harvested into 3 400μΙ fractions.

Fractions were analyzed by western blot for the nucleolar marker UBF1 and the established Amyloid-Body protein POLD1 . These molecules were present

predominantly at the 55-45% Percoll transition. [0079] Stable isotope labeling by amino acids in cell culture-mass

spectrometry: SILAC-labeling media was prepared using custom DMEM minus arginine and lysine (Athena Environmenal Sciences) supplemented with 5% dialyzed fetal calf serum (FCS) and 1 % penicillin/streptomycin. MCF-7 cells were grown for 14 days in SILAC media containing 84 μς/ιηΙ L-arginine and 146 μς/ιτιΙ L-lysine as follows: L-arginine and L-lysine (hypoxia-acidosis), L-arginine ¹³C₆ and L-lysine 4,4,5,5-D₄ (hypoxia-neutral) or L-arginine ¹³C₆ ¹⁵N₄ and L-lysine ¹³C₆ ¹⁵N₂ (normoxia-neutral) (Cambridge Isotope Labs). Normoxic-neutral cells were maintained at 21 % 0₂ in neutral media (pH7.4), hypoxic-neutral cells were transferred to 1 % 0₂ for 3 hours in pH7.4 media and hypoxic-acidotic cells were grown in pH 6.0 media at 1 % 0₂ for 3 hours. Following treatment, nucleolar detention centers were isolated, described below, and total protein was extracted using 2% SDS and combined at a 1 :1 :1 ratio between the three treatments. Combined extracts were run on a precast TGX 4-12% gradient gel (BioRad), stained with Simply Blue Safestain (Invitrogen) and the entire lane was excised into 12 slices. Peptides were extracted from an in-gel tryptic digestion of each gel splice and analyzed by mass spectrometry as previously described¹⁰. Protein identification and quantitation were performed using the program Peaks Studio 7.0 (Thermo Scientific). Identification was set to a false discovery rate of 1 % and proteins were considered to be enriched in the nucleoli of cells incubated under hypoxia-acidotic conditions if they possessed a minimum 2-fold hypoxia-acidosis:hypoxia-neutral and hypoxia-acidosis:normoxia-neutral ratio and significance (A) values. Protein sizes, grand average of hydrophobicity (GRAVY) and isoelectric point were calculated using the ProtParam program (SIB ExPASy Bioinformatics Resource Portal).

[0080] Western blotting: Western blots were performed using standard techniques. Transferred blots were probed with primary and secondary antibodies described above. Bands were detected by enhanced chemi-luminescence (Luminata Forte, Millipore) on x-ray film.

[0081] RNA extractions, analysis and immunoprecipitation: Purified human RNA from multiple tissue types was purchased from Clontech. RNA immunoprecipitation was carried out on transfected MCF-7 cells harvested in lysis buffer (100 imM NaCL, 0.5% Igepal, 20 mM Tris [pH7.6], 5 mM MgCI2, and 1 mM Na3V04) and sonicated prior to incubation with GFP antibody (Abeam, ab290) and ChIP grade Protein A/G magnetic beads (Pierce). Following washes, RNA was extracted from samples and 10% inputs for RT-PCR.RNA extractions, cDNA synthesis and semi-quantitative PCR were previously described¹.

[0082] Peptide synthesis and fibrillation assay: Peptides were synthesized by GenScript with the sequences: VHL(104-140) - GTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPS, β-amyloid - DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA and ACM^VHL- RRIHSYRLLVNQTELFV. Fibrillation was performed over a 1 week period at 37°C by incubating 1 m peptide in 10mM HCI. Fibrils were detected by transmission electron microscopy.

[0083] Cell proliferation, BrdU incorporation and Congo red assays: 750,000 (10 cm plates) or 400,000 (3.5 cm plates) MCF-7 or PC-3 were plated for the cell proliferation and BrdU incorporation assay, respectively. 75,000 (6 cm plates) and 40,000 (3.5cm plates) WI-38 cells were also plated for the cell proliferation and BrdU assay, respectively. Media were changed at the indicated times to standard (pH 7.4) or acidosis-permissive (pH6.0) media, described previously¹'¹², and grown at normoxic or hypoxic oxygen tensions for the indicated times. Post-acidification recovery was achieved by replacing acidosis-permissive media with standard media and a returning to normoxic conditions. Cell proliferation rates were determined by trypsinizing parallel plates and counting total cells for each time point for three independent repeats. Trypan blue was added to ensure variation was not due to excessive cell death. BrdU was incorporated for the last 20 minutes (MCF-7 and PC-3) or 60 minutes (WI-38) of each time point. Cells were fixed for 20 minutes at -20°C with an ethanol fixative (70% ethanol; 50mM glycine; pH 2.0), prior to BrdU staining according to manufacturer's protocol (Roche). The ratio of BrdU stained nuclei versus Hoechst stained nuclei was assessed by fluorescence microscopy. Congo red assays to detect the proportion of A- body-positive cells was performed by staining untreated/treated cells with Congo red and Hoechst. Minimal exposure times were selected to eliminate background auto- fluorescence. The percentage of Congo red-positive cells was determined by counting the number of cells with Congo red-positive nucleoli and dividing by the total number of Hoecht-positive cells. The proportion of nuclear stress granule- and paraspeckle- positive cells was performed in the same manner on immunostained cells. At least three random fields were counted for three independent replicates.

[0084] Flow cytometry: Flow cytometry was conducted on normoxic-neutral, hypoxic-neutral and hypoxic-acidotic cells. Acidosis-permissive media was prepared that reached a final pH of 6.5 and 6.0 as indicated. MCF-7 cells stably-expressing scramble or rlGS2sFlNA-specific shRNA were grown under the described conditions for 24 hours. Nocodazole (250nM) was added to one plate of each cell line under each condition and treatments continued for 24 hours. Flow cytometry was performed on 70% ethanol fixed cells, washed in phosphate-citrate buffer and treated with 50μΙ RNase A (Ι ΟΟμς/ιηΙ) and 450μΙ propidium iodide (δθμς/ιηΐ) for 15 minutes prior to analysis (Beckman Coulter Cyan ADP 9 Analyzer).

[0085] ATP and viability assay: Cellular ATP concentrations were measured using the CellTiter-Glo Luminescent Assay (Promega) on a LUMIstar Galaxy luminometer (BMG Labtechnologies) according to manufacturer's protocol. Viability was calculated by growing the stable MCF-7 cell lines in standard or acidosis-permissive media for the indicated times. Each day parallel plates were stained for 5 minutes with 2mM propidium iodide and Hoechst 33342. Cells were immediately imaged by fluorescence microscopy. The cell death ratio was determined by counting the number of propidium iodide positive cells per field versus Hoechst stained nuclei. Experiments were performed in triplicate.

[0086] Tumor Xenografts: Animal experiments were performed in accordance with the University of Ottawa Animal Care Committee (CMM-181 ) policy. 107 exponentially growing cells suspended in 200 μΙ_ PBS were injected subcutaneously into the flanks of 6-8 week old CD-1 nude female mice (Charles River). Each mouse was injected with a control (left flank) and experimental (right flank) cell line. Tumor growth was recorded blind to the cell lines injected with calibers and animals were killed when the end point was reached or 5 weeks post-injection. Post-mortem, tumors were harvested, measured, fixed in 10% formalin, paraffin-embedded, sliced and stained. [0087] Staining tissue sections and fluorescence in situ hybridization: Cryo- sections of normal human brain and pancreatic tissue were purchased (Amsbio).

Human breast and prostate tumor sections were purchased (Biochain) or obtained from excised masses, respectively. Experiments using human tumors were approved by the University of Miami - Miller School of Medicine institutional review board, with informed consent obtained prior to specimen acquisition. Human tumors, excised mouse xenograft masses were fixed in 10% formalin. All tissue was embedded in paraffin and sliced to a thickness of 3 μπι. Human prostate tumors were entirely submitted for histologic evaluation. Primary antibodies were used at a concentration of 1 :50 and secondary antibodies at 1 :150. Congo red and Amylo-Glo staining was performed as above. Hematoxylin/eosin staining was performed on each section. Fluorescence in situ hybridization was performed as described, and with probes previously described^5,9.

[0088] Statistical analysis: Bars represent the mean value from at least (n values indicated in figure legends) three independent replicates. Statistical analyses were performed with the error bars representing the standard error of the mean. Variances were noted to ensure that they were similar between the compared groups, p values were based on two-tailed Student's t-test with the significance level indicated in the figure legend.

EXAMPLE 2

[0089] This example demonstrates the identification and characterization of A-bodies as Nuclear protein foci with amyloid-like biophysical properties.

[0090] Highly mobile molecules establish functional networks by randomly diffusing in the cellular milieu in search of high affinity interactions (Lippincott-Schwartz and

Patterson, 2003; Misteli, 2001 ). On stimulus, inducible rIGSRNA immobilize proteins in nuclear foci to regulate cellular dynamics (Audas et al., 2012). As protein immobilization is a property associated with cellular amyloids (Berchowitz et al., 2015; Kayatekin et al., 2014), we hypothesized that rIGSRNA participate in the conversion of mobile/soluble native-fold proteins to their immobile/insoluble amyloid-like counterparts. To test this hypothesis, we first stained cells with Congo red, the quintessential amyloidophilic dye. As expected, untreated cells lacked Congo red-positive foci, owing to the absence of detectable amyloid-like protein structures (Figure 1A). Cells exposed to stimuli that induce protein immobilization (Audas et al., 2012) (Figure 8A) displayed subnuclear foci with strong Congo red signatures, revealing the existence of widespread protein organization with amyloid features (Figure 1A and Figure 8B-8D). Staining with additional dyes that recognize various biochemical features of amyloids, further highlighted the amyloidogenic properties of the nuclear foci in primary cultures and tumorigenic cells on stimuli (Figure 1 B and Figure 8B-8E) and in human tissues

(Figure 8F). These amyloidogenic cellular bodies co-localized with VHL and POLD1 , proteins that undergo stimulus-specific immobilization (Figure 1 C, D and Figure 8A, B) (Audas et al., 2012) and insolubilization (Figure 1 E). Stressors that do not target VHL to nuclear foci (sodium arsenite, cycloheximide, thapsigargin and H2O2) failed to generate insoluble structures that stained with amyloidophilic dyes (Figure 1 E and Figure 8A). The Congo red-positive foci were resistant to proteinase K (Figure 1 F, G), another amyloidogenic feature (McKinley et al., 1983), and the digestion revealed the presence of ~10nm fibers (Figure 1 G and Figure 8G), structures that have been predicted for cellular amyloids/solid phase transition (Weber and Brangwynne, 2012). These fibers were recognized by the OC antibody (Figure 1 G and Figure 8H), which targets the amyloid fibril conformation (Kayed et al., 2007). Stimuli termination caused the loss of nuclear amyloidogenic foci (Figure 1 A and Figure 8C, D) and correlated with the release (Figure 1 C) and re-solubilization (Figure 1 H) of targeted proteins, suggesting that this form of amyloidogenesis is rapid and reversible. The appearance of Congo red- positive foci correlated with the expression of stimuli-specific rIGSRNA (e.g. rlGS₂₂RNA induction by heat shock and NGS28RNA induction by acidosis) (Figure 8I). Silencing of rIGSRNA impaired or delayed the formation of amyloidogenic foci (Figure 11 and Figure 8J), suggesting a role for these IncRNA in nuclear amyloidogenesis. Put together, these data identify the Amyloid-bodies (A-bodies): rIGSRNA-dependent nuclear foci of immobilized proteins with amyloid-like properties.

[0091] The unique biophysical properties of A-bodies was apparent when compared to known subcellular foci (nuclear and cytoplasmic stress granule, Cajal bodies and nuclear speckles, paraspeckles, PML bodies, autophagosomes, Processing-bodies (P- bodies) and aggresomes). A-bodies are spatially distinct from other domains (Figure 2A-C and Figure 9A-C), possess affinity for amyloidophilic dyes (Figure 1 B and Figure 8B-8E) and harbor proteins in an immobile/solid-phase organization (Figure 1 D and 2D). This is in stark contrast to other subcellular domains, which failed to stain with Congo red/Amylo-Glo (Figure 2A-C and Figure 9A-C), display mobile/liquid-phase properties (Figure 2D) and do not form fiber-like structures under physiological settings (Weber and Brangwynne, 2012). Like A-bodies, nuclear stress granules and

paraspeckles are RNA-seeded domains. To confirm the specificity of the rIGSRNAs in the amyloidogenic process, we used siRNA to target several established IncRNAs (rlGS₂₂RNA, rlGS₂₈RNA, NEAT1 , MALAT1 and HOTAIR) and assayed for foci formation (Figure 9D). Inhibition of NGS22RNA delays heat shock-induced A-bodies, but has no effect on the genesis of other bodies under standard growth conditions (Figure 2A, E and Figure 9E). Impairing various IncRNA failed to disrupt the A-bodies, though NEAT1 depletion did reduce paraspeckle formation (Figure 2E), as previously observed

(Sasaki et al., 2009). These results highlight the cellular and biophysical properties of A- bodies as rIGSRNA-induced endogenous protein foci with amyloidogenic

characteristics.

[0092] Next, we analyzed the proteomic composition of acidotic A-bodies (Figure 3A) using SILAC-MS analysis. This revealed a large influx (Figure 3B) of heterogeneous proteins (Figure 3C, D and Figure 10A) into Congo red-positive nuclear foci, with >180 proteins over the 2 fold stringency threshold. We validated several of these molecules by western blotting (Figure 10B) and observed a re-localization of proteins from their original nuclear/cytoplasmic distribution to the A-bodies (Figure 3E and Figure 10C-F). This was accompanied by a loss of mobility (Figure 3F) and a reversible shift to the insoluble protein fraction (Figure 3G). Stressors that do not induce nuclear

amyloidogenesis (Figure 8A) did not insolubilize SILAC-MS candidates (Figure 3H). Silencing of rIGSRNA prevented the shift of tested SILAC-MS candidate proteins to the A-bodies (Figure 10F). This indicates that the amyloidogenic properties of the A-bodies were dependent on the accumulation of SILAC-MS proteins, with a proteomic composition indistinguishable from the typical cellular distribution of proteins in term of size, hydrophobicity and isoelectric point (Figure 3D and Figure 10A). These data demonstrate that A-bodies contain a heterogeneous family of proteins with amyloid-like properties.

EXAMPLE 3

[0093] This example demonstrates that the amyloid-converting motif immobilizes proteins in the A-bodies.

[0094] In principle, proteins that are immobilized in the A-bodies should possess amyloidogenic properties. We tested several immobile A-bodies constituents, including VHL, RNF8 (Audas et al., 2012) and the SILAC-MS identified cdkl and UAP56 for their amyloid propensity using an established bacterial in vitro assay (Garcia-Fruitos et al., 201 1 ; Wang et al., 2008). In this assay, proteins with amyloidogenic propensity are able to self-associate in amyloid-like structures likely due to their high concentration.

Bacterially expressed A-body proteins formed Congo red-positive aggregates (Figure 4A and Figure 11 A) containing crossed β-sheeted proteins, which generate the classic amyloid 4.7 and 10 A ringed x-ray diffraction profile (Figure 4A). Conversely, mobile B23 and SILAC-MS-negatives cdk4 and Ran failed to form Congo red-positive structures under identical conditions (Figure 11 A). Bioinformatics analysis of VHL

(Figure 4B) identified several regions with amyloidogenic propensity based on a Rosetta energy score lower than the established -23 kcal/mol threshold (Goldschmidt et al., 2010; Thompson et al., 2006). There was excellent correlation between the predicted fibrillation propensity of the VHL fragments (Figure 4B) and their ability to insolubilize GFP in bacteria (Figure 4C; bottom panel), form SDS-boiling resistant multi-mers - another hallmark of amyloids - (Figure 4C; top panel), and Congo red- positive inclusion bodies (Figure 4D). This correlation was also maintained in

mammalian cells, as fragments of VHL with low fibrillation propensity (Figure 4B) and poor amyloidogenic properties in bacteria (Figure 4C, D) failed to accumulate in insoluble A-bodies on stimulus (Figure 4E and Figure 11 B, C). In contrast, the three VHL fragments with strong amyloidogenic properties in bacteria did accumulate in A- bodies on stimulus, albeit to different degrees (Figure 4E and Figure 11 B). VHL fragment (104-140) was particularly efficient at targeting/immobilizing GFP within the A- bodies (Figure 4E and Figure 11 B-D). RNA immunoprecipitation analysis revealed that this fragment assembles efficiently with endogenous rlGS₂sRNA during acidosis compared to other amyloidogenic or non-amyloidogenic regions (Figure 4F). A closer examination of VHL (104-140) revealed the presence of an arginine/histidine (R/H) cluster in close proximity to a highly amyloidogenic stretch of amino acids (Figure 11 E, middle right panel). Bioinformatics analysis of the SILAC-MS candidate and A-body constituents cdkl , HAT1 and HDAC2 identified similar motifs (Figure 11 E). As expected, within bacterial settings all tested fragments containing regions that cross the Rosetta energy threshold formed amyloid-like inclusion bodies (Figure 4F and Figure 11 F). However, the presence of the R/H motif was essential for capture in the

endogenous A-bodies in response to environmental stimuli (Figure 4F and Figure 11C, D, F, G). We named these bipartite domains amyloidogenic converting motifs (ACM). Based on these data, we generated artificial ACM sequences composed of peptidic fragments from VHL and POLDI to create molecules that harbor an R/H cluster flanking an amyloidogenic domain from the same proteins, termed aACM^VHL and aACM^P0LD1, respectively (Figure 4G). These artificial molecules displayed amyloidogenic properties in bacteria and were efficiently captured by the A-bodies (Figure 4E, H and Figure 11 C, D, H). Synthetic peptides of VHL (104-140), or its artificial fragment, efficiently formed classic (Greenwald and Riek, 2010; Wang et al., 2008) -10 nm fibrils in vitro, similar to D-amyloid, with the artificial molecule possessing considerably more steric flexibility than the other peptides (Figure 41). Hence, the A-body targeted proteins examined in this study encode a bipartite domain capable of facilitating the conversion of proteins to an amyloid-like state.

EXAMPLE 4

[0095] This example demonstrates that the pathological β-amyloid displays ACM-like properties.

[0096] The possible implication of the amyloidgenic program in disease-associated amyloid formation was examined. Surprisingly, the prototypical pathological β-amyloid involved in Alzheimer's disease harbors an R/H-rich cluster flanking a highly

amyloidogenic domain analogous to VHL (100-140), and other ACM regions (Figure 5A and Figure 11 E, 12A). Under standard growth conditions, the β-amyloid-GFP fusion protein has a diffuse and mobile cellular distribution, indistinguishable from GFP alone (Figure 5B-D). Environmental stimuli that activate physiological amyloidogenesis

(Figure 8A) triggered the efficient capture, immobilization and ^solubilization of β- amyloid within the A-bodies (Figure 5B-D and Figure 12B) in a rIGSRNA-dependent manner (Figure 5E). The amyloid precursor protein (APP) can be cleaved by β- secretase to produce the pathological β-amyloid or β-secretase to generate the non- pathological P3 fragment (Figure 5A and Figure 12A), lacking the R/H cluster necessary for mammalian cell amyloidogenesis seen in other ACMs (Figure 11 F). P3 can form amyloid-like structures in bacteria (Figure 5F), however, it is unable to associate with rIGSRNA (Figure 5G) and failed to accumulate in the A-bodies in mammalian cells (Figure 5D, H and Figure 12B), similar to other regions of APP

(Figure 12C) and ACM that lack the R/H cluster (Figure 11 F, G). Hence, these data suggest that β-amyloid displays ACM-like properties and undergoes physiological amyloidogenesis in A-bodies in response to environmental stimuli.

EXAMPLE 5

[0097] This example demonstrates that cellular amyloidogenesis is a reversible process.

[0098] As seen in Figure 1 A, the restoration of standard growth conditions results in a rapid dissipation of the A-bodies (Figure 1 A, 6A), correlating with the release of proteins from the insoluble fraction (Figure 1 H, 3G) and a downregulation of rIGSRNA levels (Figure 8I). We investigated refolding, rather than degradation, as a mechanism of A-bodies disassembly since the proteins return to their original localization and retain their steady state levels in the presence of cycloheximide, a protein synthesis inhibitor (Figure 6B and Figure 13A). Subcellular analysis of prominent chaperones revealed that members of the heat shock protein family (Hsp27, Hsp 70 and Hsp90) are associated with A-bodies during and after stimuli termination (Figure 6C and Figure 13B), suggesting a role for these molecules in amyloid disaggregation. To assay the effects of these chaperones, we used readily available drug inhibitors (Figure 13C). We preferred this approach to RNA interference or CRISPR technology, as depletion of the hsp members results in a loss of cellular viability during stress treatment and is therefore uninformative. Inhibition of Hsp70 and Hsp90, with VER155008 (VER) and 17- allylamino-17-demethoxygeldanamycin (AAG), respectively, after A-body formation significantly impaired the disaggregation of Congo red-positive foci upon signal termination (Figure 6B, 6D and 6E and Figure 13D). Inhibition of other chaperones, GRP78 (Epigallocatechin gallate [EGCG]), Protein Disulfide Isomerase (16F16) and the autophagosome (Wortmannin), failed to prevent this process (Figure 6B, 6D and Figure 13D). A combinatory treatment of VER and 17-AAG delayed recovery in an additive manner (Figure 6D), suggesting that both Hsp70 and Hsp90 are involved in disassembly. The pathological β-amyloid peptide was also released from the A-bodies in an Hsp70 activity-dependent manner (Figure 6F). This suggests that fibrillation of this neurotoxic molecule can be reversed by elements of the hsp pathway, returning to its pre-stress localization, even in the presence of cycloheximide (Figure 6F). While the hsp response is necessary for disassembly of the A-bodies, its inhibition alone is insufficient to induce the accumulation of cellular amyloid foci (Figure 13E). These data demonstrate the disaggregation of the A-bodies is regulated by elements of the heat shock machinery, which reversibly switch proteins from the amyloid- to the native-fold.

EXAMPLE 6

[0099] This example demonstrates that rIGSRNA/A-bodies induce a state of cellular dormancy.

[00100] SILAC-MS analysis shown in Figure 3 revealed a large influx of proteins into the A-bodies with several constituents involved in cell cycle progression and DNA synthesis (Figure 7A, 7B and Figure 10E, F). As such, formation of the acidosis arrested proliferation (Figure 7C and Figure 14A) and DNA synthesis (Figure 7D) of cells in an A-body/rlGS2sRNA-dependent manner (Figure 7E, 7F and Figure 14B-G). This enables cells to remain viable during prolonged periods of extracellular acidosis (Figure 7G), highlighting the non-toxic/protective nature of physiological

amyloidogenesis. Restoration of neutral pH reverted these cells back to the untreated phenotype (Figure 7C, 7D), correlating with disaggregation of the A-bodies (Figure 1A). These hypoxic/acidotic conditions are prevalent within the tumor microenvironment and are believed to cause cancer cell dormancy (Giancotti, 2013; Sosa et al., 2014; Tannock and Rotin, 1989). This model system was used to assess for the presence and function of the A-bodies in an in vivo setting. Formalin fixed paraffin-embedded human tissues from human prostatic acinar and breast invasive duct carcinomas stained positive for the Amylo-Glo dye and the validated SILAC-MS proteins UAP56 and HAT1 (Figure 7H). To assess the effects of this amyloidogenic event we performed nude mouse xenograft assays with MCF-7 and PC-3 cells expressing control or rlGS₂₈RNA- specific shRNA (Figure 1J and Figure 14C). MCF-7 cells only form minimal masses when injected into nude mice, without the addition of external growth factors (Benz et al., 1992). Consistent with our model, shRNA-mediated silencing of rlGS₂8RNA enabled MCF-7 cells to form large necrotic tumors four weeks post-injection (Figure 71-K). PC-3 cells that generally form moderate sized masses also formed larger masses when rlGS₂₈RNA was inhibited (Figure 71 and Figure 14H). Silencing of rlGS₂₈RNA prevented capture of the validated SILAC-MS candidates POLA1 , HAT1 and the formation of Congo red/Amylo-Glo positive nuclear foci in the core of these tumors

(Figure 7K and Figure 14H, I). These data support the hypothesis that rlGS₂₈RNA- mediated A-body formation force cells to enter a state of dormancy, maintaining viability during periods of extracellular stress.

DISCUSSION

[00101] The following is a discussion of the results obtained in Examples 2-6.

[00102] This disclosure introduces A-bodies: rIGSRNA-seeded nuclear foci containing proteins possessing biophysical properties associated with an amyloid-like state. Formation of the A-bodies is rapid, reversible and plays a role in the ability of cells to enter a dormant state as an adaptive response to severe environmental insults.

Physiological amyloidogenesis represents a clever post-translational regulatory program, allowing for the prompt removal of a large family of heterogeneous proteins, without relying on complex covalent modifications or extensive protein degradation. In fact, A-body formation could be a physiological example of the liquid to solid phase transition of proteins, a process observed under in vitro settings (Kato et al., 2012) and attributed predominantly to pathological aggregates (Weber and Brangwynne, 2012). A- bodies are found in cells exposed to various stressors, the cores of tumors and normal human tissues, highlighting their ubiquitous nature. This challenges the widely held concept that amyloids are mostly aberrant/toxic aggregates and seldom observed in physiology compared to native-folded proteins. Based on these data, we suggest that the amyloid-fold should be considered, alongside the native-fold and unfolded, as a common protein organization in cell biology.

[00103] A-bodies join a group of established RNA-seeded cellular protein foci, including paraspeckles and nuclear stress granules (Chujo et al., 2016). Yet, they are currently unique in their biochemical properties as they include immobile proteins with amyloid-like properties. Other tested protein foci, such as stress granules and aggresomes, do not display amyloidogenic properties in a cellular context. rlGSRNA- impaired cells fail to produce A-bodies on stimuli but retain their ability to form other protein foci, highlighting the role of these IncRNA in amyloidogenic body formation. While the exact mechanisms remain unclear, we favor a model whereby rIGSRNA facilitate in vivo amyloidogenesis by operating as micro-concentrators of ACM- containing proteins. The ACM of proteins that we have studied so far harbor two distinct domains: an R/H rich sequence that flanks a highly amyloidogenic stretch of amino acids (Figure 12F). The amyloidogenic domains are independently capable of forming amyloids when overexpressed in bacteria, likely as a consequence of reaching a concentration threshold that initiates self-amyloidogenesis. In contrast, the

amyloidogenic domains require the flanking R/H-rich sequences for RNA-mediated amyloidogenic conversion in mammalian cells. This implies that endogenous rIGSRNA enable the amyloidogenic domains to reach a sufficient concentration by interacting with the R/H residues, triggering the initial fibrillation event followed by polymerization of proteins, consistent with several models of amyloid formation. Precisely how rIGSRNA facilitates A-bodies formation and whether a combination of other endogenous or exogenous factors can also activate cellular amyloidogenesis remains to be studied.

[00104] A notable observation from this study is that β-amyloid shares striking similarities with the ACM of several A-body targets and undergoes physiological amyloidogenesis on stimuli. Unlike the non-pathological P3 peptide, which lacks the R/H rich motif (because of its naturally occurring cleavage by β-secretase), β-amyloid is efficiently captured in A-bodies. On signal termination, β-amyloid and other physiological amyloids can be reverted back to their soluble form by Hsp70/Hsp90, without undergoing degradation. This implies that the amyloid state is not a

terminal/irreversible form of protein aggregation, even for pathological amyloid peptides. This cellular data is in good agreement with in vitro results showing a role for Hsp70 in disaggregation of β-amyloid and a-synuclein (Evans et al., 2006; Gao et al., 2015). While unproven, it is nonetheless tempting to speculate that pathological

amyloidogenesis may be explained by dysregulation of the rIGSRNA-Hsp system. The data also suggest that β-amyloid/ACM activity is not unique to APP but present in many proteins that undergo amyloidogenesis in A-bodies.

[00105] Conceptually, these observations fundamentally change our understanding of amyloids from mysterious, toxic and irreversible protein aggregates to a common and reversible polypeptide-fold. We envision the existence of multiple different adaptive A- bodies, with proteins encoding variants of the ACM specific to rIGSRNA, and possibly other IncRNA and environmental cues. This work opens several avenues of

investigation in protein folding, liquid to solid phase transition and cellular adaptation to stressors, while providing new conceptual insights in our efforts to resolve debilitating conditions associated with pathological amyloidogenesis, including Alzheimer's disease and diabetes.

EXAMPLE 7

[00106] This example demonstrates an exemplary method of the present disclosure.

[00107] Decreasing doses of a small molecule compound from the small molecule compound library obtained from Sanford Burnham (La Jolla, CA) are added to different wells containing human cells in a multi-well plate from left to right. In the right-most well is a vehicle control containing 0 μg of the small molecule compound. Each row of the plate represents a different small molecule compound of the library. Several plates are used to simultaneously test multiple small molecule compounds of the library.

[00108] The cells in each plate are stimulated to form amyloid bodies via a 3-hour acidosis or heat shock exposure as described in Example 1 . The cells are stained with Congo Red and the fluorescence from the Congo Red-stained cells is measured at different time points from 0-4 hours. Fluorescence at each time point is expressed as a %age of the fluorescence of the vehicle control.

[00109] Those rows having an increased %age of the fluorescence from left to right indicates the small molecule compound as one that has efficacy for inhibiting amyloid formation in cells. As amyloid formation is associated with and related to amyloid diseases, the small molecule compound is deemed as having efficacy for

prophylactically treating an amyloid disease.

EXAMPLE 8

[00110] This example demonstrates an exemplary method of the present disclosure.

[00111] This method is essentially the same as that described in Example 7, except that the small molecule compound is added after the cells are stimulated for amyloid formation. Briefly, cells in multiwall plates are stimulated to form amyloid bodies via a 3- hour acidosis or heat shock exposure as described in Example 1 . Amyloid bodies are formed within 1 -2 hours post-stimulus treatment.

[00112] Decreasing doses of a small molecule compound from the small molecule compound library obtained from Sanford Burnham (La Jolla, CA) are added to different wells containing human cells in a multi-well plate from left to right. In the right-most well is a vehicle control containing 0 μg of the small molecule compound. Each row of the plate represents a different small molecule compound of the library. Several plates are used to simultaneously test multiple small molecule compounds of the library.

[00113] The stimulated and treated cells are stained with Congo Red and the fluorescence from the cells is measured at different time points from 0-4 hours.

Fluorescence at each time point is expressed as a %age of the fluorescence of the vehicle control.

[00114] Those rows having an increased %age of the fluorescence from left to right indicates the small molecule compound as one that has efficacy for increasing amyloid disaggregation in cells. As amyloid disaggregation (breakdown of amyloid) is

associated with and related to treatment of amyloid diseases, the small molecule compound is deemed as having efficacy for treating an amyloid disease. TABLE 1

Description Accession

Nuclease-sensitive element-binding protein 1 OS=Homo sapiens GN=YBX1 PE=1 SV=3 P67809|YBOX1_HUMAN

T-complex protein 1 subunit delta OS=Homo sapiens GN=CCT4 PE=1 SV=4 P50991 |TCPD_HUMAN

Symplekin OS=Homo sapiens GN=SYMPK PE=1 SV=2 Q92797|SYMPK_HUMAN

TATA-binding protein-associated factor 2N OS=Homo sapiens GN=TAF15 PE=1 SV=1 Q92804|RBP56_HUMAN

Flavin reductase (NADPH) OS=Homo sapiens GN=BLVRB PE=1 SV=3 P30043|BLVRB_HUMAN

Heterogeneous nuclear ribonudeoprotein H OS=Homo sapiens GN=HNRNPH1 PE=1 SV=4 P31943|HNRH1_HUMAN

Heterogeneous nuclear ribonucleoproteins C1/C2 OS=Homo sapiens GN=HNRNPC PE=1 SV=4 P07910|HNRPC_HUMAN

WD repeat-containing protein 46 OS=Homo sapiens GN=WDR46 PE=1 SV=3 015213|WDR46_HUMAN

Nucleolin OS=Homo sapiens GN=NCL PE=1 SV=3 P19338|NUCL_HUMAN

Round spermatid basic protein 1 OS=Homo sapiens GN=RSBN1 PE=1 SV=2 Q5VWQ0|RSBN1_HUMAN

14-3-3 protein zeta/delta OS=Homo sapiens GN=YWHAZ PE=1 SV=1 P63104|1433Z_HUMAN

Hepatocyte nuclear factor 3-alpha OS=Homo sapiens GN=FOXA1 PE=1 SV=2 P55317|FOXA1_HUMAN

Beta-actin-like protein 2 OS=Homo sapiens GN=ACTBL2 PE=1 SV=2 Q562R1 IACTBL HUMAN

Heterogeneous nuclear ribonudeoprotein DO OS=Homo sapiens GN=HNRNPD PE=1 SV=1 Q14103|HNRPD_HUMAN

Heterogeneous nuclear ribonudeoprotein A3 OS=Homo sapiens GN=HNRNPA3 PE=1 SV=2 P51991 |ROA3_HUMAN

60S ribosomal protein L29 OS=Homo sapiens GN=RPL29 PE=1 SV=2 P47914|RL29_HUMAN

Zinc finger CCCH domain-containing protein 18 OS=Homo sapiens GN=ZC3H18 PE=1 SV=2 Q86VM9|ZCH18_HUMAN

Pre-mRNA-splicing factor ISY1 homolog OS=Homo sapiens GN=ISY1 PE=1 SV=3 Q9ULR0|ISY1_HUMAN

Heterogeneous nuclear ribonudeoprotein K OS=Homo sapiens GN=HNRNPK PE=1 SV=1 P61978|HNRPK_HUMAN

Vesicle-trafficking protein SEC22b OS=Homo sapiens GN=SEC22B PE=1 SV=4 075396 |SC22B_HU MAN

Heterogeneous nuclear ribonucleoproteins A2/B1 OS=Homo sapiens GN=HNRNPA2B1 PE=1 SV=2 P22626|ROA2_HUMAN

Tubulin-tyrosine ligase-like protein 12 OS=Homo sapiens GN=TTLL12 PE=1 SV=2 Q14166|TTL12_HUMAN

Poly(rC)-binding protein 1 OS=Homo sapiens GN=PCBP1 PE=1 SV=2 Q15365|PCBP1_HUMAN

Heterogeneous nuclear ribonudeoprotein AO OS=Homo sapiens GN=HNRNPAO PE=1 SV=1 Q13151 |ROA0_HUMAN

Putative RNA-binding protein 3 OS=Homo sapiens GN=RBM3 PE=1 SV=1 P98179|RBM3_HUMAN

Heterogeneous nuclear ribonudeoprotein C-like 1 OS=Homo sapiens GN=HNRNPCL1 PE=1 SV=1 O60812|HNRCL_HUMAN

RNA-binding protein 39 OS=Homo sapiens GN=RBM39 PE=1 SV=2 Q14498|RBM39_HUMAN

Transcription initiation factor TFIID subunit 6 OS=Homo sapiens GN=TAF6 PE=1 SV=1 P49848|TAF6_HUMAN

Putative RNA-binding protein Luc7-like 2 OS=Homo sapiens GN=LUC7L2 PE=1 SV=2 Q9Y383|LC7L2_HUMAN

Dystrophin OS=Homo sapiens GN--DMD PE=1 SV=3 P1 1532|DMD_HUMAN

Transcriptional adapter 2-beta OS=Homo sapiens GN=TADA2B PE=1 SV=2 Q86TJ2 |TAD2B_H UMAN

Vacuolar protein sorting-associated protein 13C OS=Homo sapiens GN=VPS13C PE=1 SV=1 Q709C8|VP13C_HUMAN

ELAV-like protein 1 OS=Homo sapiens GN=ELAVL1 PE=1 SV=2 Q15717|ELAV1_HUMAN

Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1 P60709|ACTB_HUMAN

Triosephosphate isomerase OS=Homo sapiens GN=TPI1 PE=1 SV=3 P60174|TPIS_HUMAN

ATP-dependent RNA helicase DDX39A OS=Homo sapiens GN=DDX39A PE=1 SV=2 O00148|DX39A_HUMAN

Melanoma-associated antigen D2 OS=Homo sapiens GN=MAGED2 PE=1 SV=2 Q9UNF1 MAGD2 HUMAN

60S ribosomal protein L19 OS=Homo sapiens GN=RPL19 PE=1 SV=1 P84098|RL19_HUMAN

60S acidic ribosomal protein P2 OS=Homo sapiens GN=RPLP2 PE=1 SV=1 P05387|RLA2_HUMAN

Serine/arginine-rich splicing factor 2 OS=Homo sapiens GN=SRSF2 PE=1 SV=4 Q01 130|SRSF2_HUMAN Description Accession

UPF0488 protein C8orf33 OS=Homo sapiens GN=C8orf33 PE=1 SV=1 Q9H7E9|CH033_HUMAN

Heterogeneous nuclear ribonucleoprotein M OS=Homo sapiens GN=HNRNPM PE=1 SV=3 P52272|HNRPM_HUMAN

Small nuclear ribonucleoprotein Sm D1 OS=Homo sapiens GN=SNRPD1 PE=1 SV=1 P62314|SMD1_HUMAN

Probable ATP-dependent RNA helicase DDX6 OS=Homo sapiens GN=DDX6 PE=1 SV=2 P26196|DDX6_HUMAN

Actin, alpha cardiac muscle 1 OS=Homo sapiens GN=ACTC1 PE=1 SV=1 P68032|ACTC_HU AN

Nucleosome assembly protein 1 -like 1 OS=Homo sapiens GN=NAP1 L1 PE=1 SV=1 P55209|NP1 L1_HUMAN

Poly(rC)-binding protein 2 OS=Homo sapiens GN=PCBP2 PE=1 SV=1 Q15366|PCBP2_HUMAN

40S ribosomal protein S7 OS=Homo sapiens GN=RPS7 PE=1 SV=1 P62081 |RS7_HU AN

Probable ATP-dependent RNA helicase DDX5 OS=Homo sapiens GN=DDX5 PE=1 SV=1 P17844|DDX5_HUMAN

Methylated-DNA-protein-cysteine methyltransferase OS=Homo sapiens GN=MGMT PE=1 SV=1 P16455|MGMT_HUMAN

General transcription factor IIF subunit 1 OS=Homo sapiens GN=GTF2F1 PE=1 SV=2 P35269|T2FA_HUMAN

Eukaryotic translation initiation factor 4H OS=Homo sapiens GN=EIF4H PE=1 SV=5 Q15056|IF4H_HUMAN

SAP domain-containing ribonucleoprotein OS=Homo sapiens GN=SARNP PE=1 SV=3 P82979|SARNP_HUMAN

Splicing factor 3A subunit 3 OS=Homo sapiens GN=SF3A3 PE=1 SV=1 Q12874|SF3A3_HUMAN

SURP and G-patch domain-containing protein 1 OS=Homo sapiens GN=SUGP1 PE=1 SV=2 Q8IWZ8|SUGP1_HUMAN

Eukaryotic translation initiation factor 3 subunit K OS=Homo sapiens GN=EIF3K PE=1 SV=1 Q9UBQ5|EIF3K_HUMAN

Transcription factor Sp1 OS=Homo sapiens GN=SP1 PE=1 SV=3 P08047|SP1_HUMAN

Transcription elongation factor B polypeptide 3 OS=Homo sapiens GN=TCEB3 PE=1 SV=2 Q14241 |ELOA1_HUMAN

Condensin-2 complex subunit D3 OS=Homo sapiens GN=NCAPD3 PE=1 SV=2 P42695|CNDD3_HUMAN

Hemicentin-2 OS=Homo sapiens GN=HMCN2 PE=2 SV=2 Q8NDA2 HMCN2 HUMAN

Nuclear factor related to kappa-B-binding protein OS=Homo sapiens GN=NFRKB PE=1 SV=2 Q6P4R8|NFRKB_HUMAN

Protein strawberry notch homolog 1 OS=Homo sapiens GN=SBN01 PE=1 SV=1 A3KN83|SBN01_HUMAN

Ral-GDS-related protein OS=Homo sapiens GN=RGL4 PE=2 SV=1 Q8IZJ4|RGDSR_HUMAN

Trinucleotide repeat-containing gene 18 protein OS=Homo sapiens GN=TNRC18 PE=1 SV=3 015417|TNC18_HUMAN

Hepatoma-derived growth factor-related protein 2 OS=Homo sapiens GN=HDGFRP2 PE=1 SV=1 Q7Z4V5|HDGR2_HUMAN

COMM domain-containing protein 3 OS=Homo sapiens GN=COMMD3 PE=1 SV=1 Q9UBI1 ICOMD3JHUMAN

Titin OS=Homo sapiens GN=TTN PE=1 SV=4 Q8WZ42|TITIN_HUMAN

U4/U6 small nuclear ribonucleoprotein Prp31 OS=Homo sapiens GN=PRPF31 PE=1 SV=2 Q8WWY3|PRP31_HUMAN

Eukaryotic translation initiation factor 5 OS=Homo sapiens GN=EIF5 PE=1 SV=2 P55010|IF5_HUMAN

Nucleosome assembly protein 1 -like 4 OS=Homo sapiens GN=NAP1 L4 PE=1 SV=1 Q99733|NP1 L4_HU AN

Coatomer subunit epsilon OS=Homo sapiens GN=COPE PE=1 SV=3 014579|COPE_HUMAN

Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 P81605|DCD_HUMAN

NAD(P)H dehydrogenase [quinone] 1 OS=Homo sapiens GN=NQ01 PE=1 SV=1 P15559|NQ01_HUMAN

Methyl-CpG-binding domain protein 3 OS=Homo sapiens GN=MBD3 PE=1 SV=1 095983|MBD3_HUMAN

ATP-dependent RNA helicase A OS=Homo sapiens GN=DHX9 PE=1 SV=4 Q08211 |DHX9_HUMAN

CD2 antigen cytoplasmic tail-binding protein 2 OS=Homo sapiens GN=CD2BP2 PE=1 SV=1 O95400|CD2B2_HUMAN

Hemoglobin subunit alpha OS=Homo sapiens GN=HBA1 PE=1 SV=2 P69905|HBA_HUMAN

Small nuclear ribonucleoprotein Sm D3 OS=Homo sapiens GN=SNRPD3 PE=1 SV=1 P62318|SMD3_HUMAN

Zinc finger CCCH-type with G patch domain-containing protein OS=Homo sapiens GN=ZGPAT PE=1 SV=3 Q8N5A5 |ZG PAT H U MAN

Transcriptional repressor p66-beta OS=Homo sapiens GN=GATAD2B PE=1 SV=1 Q8WXI9|P66B_HUMAN Description Accession

Ubiquitin-conjugating enzyme E2 E1 OS=Homo sapiens GN=UBE2E1 PE=1 SV=1 P51965|UB2E1_HU AN

Nucleolar protein 9 OS=Homo sapiens GN=NOP9 PE=1 SV=1 Q86U38|NOP9_HUMAN

Chromatin target of PRMT1 protein OS=Homo sapiens GN=CHTOP PE=1 SV=2 Q9Y3Y2|CHTOP_HUMAN

Survival of motor neuron-related-splicing factor 30 OS=Homo sapiens GN=SMNDC1 PE=1 SV=1 O75940|SPF30_HU AN

Putative small nuclear ribonucleoprotein G-like protein 15 OS=Homo sapiens GN=SNRPGP15 PE=5 SV=2 A8MWD9|RUXGL_HUMAN

Small nuclear ribonucleoprotein G OS=Homo sapiens GN=SNRPG PE=1 SV=1 P62308|RUXG_HUMAN

RNA-binding protein 8A OS=Homo sapiens GN=RBM8A PE=1 SV=1 Q9Y5S9|RBM8A_HUMAN

Glutathione S-transferase Mu 3 OS=Homo sapiens GN=GSTM3 PE=1 SV=3 P21266|GSTM3_HUMAN

40S ribosomal protein S17 OS=Homo sapiens GN=RPS17 PE=1 SV=2 P08708|RS17_HUMAN

40S ribosomal protein S17-like OS=Homo sapiens GN=RPS17L PE=1 SV=1 P0CW22|RS17L_HUMAN

Heterogeneous nuclear ribonucleoprotein U-like protein 1 OS=Homo sapiens GN=HNRNPUL1 PE=1 SV=2 Q9BUJ2|HNRL1_HUMAN

40S ribosomal protein S25 OS=Homo sapiens GN=RPS25 PE=1 SV=1 P62851 |RS25_HUMAN

Complement component 1 Q subcomponent-binding protein, mitochondrial OS=Homo sapiens GN=C1 QBP PE=1 SV=1 Q07021 |C1 QBP_HUMAN

RNA-binding protein EWS OS=Homo sapiens GN=EWSR1 PE=1 SV=1 Q01844|EWS_HUMAN

Small nuclear ribonucleoprotein Sm D2 OS=Homo sapiens GN=SNRPD2 PE=1 SV=1 P62316|SMD2_HUMAN

Ig lambda-1 chain C regions OS=Homo sapiens GN=IGLC1 PE=1 SV=1 P0CG04|LAC1_HUMAN

Ig lambda-2 chain C regions OS=Homo sapiens GN=IGLC2 PE=1 SV=1 P0CG05|LAC2_HUMAN

Ig lambda-3 chain C regions OS=Homo sapiens GN=IGLC3 PE=1 SV=1 P0CG06|LAC3_HUMAN

Ig lambda-6 chain C region OS=Homo sapiens GN=IGLC6 PE=4 SV=1 P0CF74|LAC6_HU AN

Ig lambda-7 chain C region OS=Homo sapiens GN=IGLC7 PE=1 SV=2 A0M8Q6 LAC7 HUMAN

Immunoglobulin lambda-like polypeptide 5 OS=Homo sapiens GN=IGLL5 PE=2 SV=2 B9A064|IGLL5_HUMAN

Histone-binding protein RBBP4 OS=Homo sapiens GN=RBBP4 PE=1 SV=3 Q09028|RBBP4_HUMAN

Ig kappa chain C region OS=Homo sapiens GN=IGKC PE=1 SV=1 P01834|IGKC_HUMAN

Zinc finger RNA-binding protein OS=Homo sapiens GN=ZFR PE=1 SV=2 Q96KR1 IZFR HUMAN

Serine/arginine-rich splicing factor 1 OS=Homo sapiens GN=SRSF1 PE=1 SV=2 Q07955|SRSF1_HUMAN

60S acidic ribosomal protein P1 OS=Homo sapiens GN=RPLP1 PE=1 SV=1 P05386|RLA1_HUMAN

Dual specificity protein phosphatase 3 OS=Homo sapiens GN=DUSP3 PE=1 SV=1 P51452|DUS3_HUMAN

Elongation factor 1 -delta OS=Homo sapiens GN=EEF1 D PE=1 SV=5 P29692|EF1 D_HUMAN

60S ribosomal protein L31 OS=Homo sapiens GN=RPL31 PE=1 SV=1 P62899|RL31_HUMAN

Homeobox protein HMX3 OS=Homo sapiens GN=HMX3 PE=2 SV=1 A6NHT5|HMX3_HU AN

60S ribosomal protein L1 1 OS=Homo sapiens GN=RPL11 PE=1 SV=2 P62913|RL11_HUMAN

Splicing factor 3A subunit 1 OS=Homo sapiens GN=SF3A1 PE=1 SV=1 Q15459|SF3A1_HUMAN

U5 small nuclear ribonucleoprotein 40 kDa protein OS=Homo sapiens GN=SNRNP40 PE=1 SV=1 Q96DI7|SNR40_HUMAN

Epithelial splicing regulatory protein 2 OS=Homo sapiens GN=ESRP2 PE=1 SV=1 Q9H6T0|ESRP2_HUMAN

40S ribosomal protein S3 OS=Homo sapiens GN=RPS3 PE=1 SV=2 P23396|RS3_HUMAN

E3 ubiquitin-protein ligase TRI 33 OS=Homo sapiens GN=TRIM33 PE=1 SV=3 Q9UPN9|TRI33_HUMAN

Pre-mRNA 3'-end-processing factor FIP1 OS=Homo sapiens GN=FIP1 L1 PE=1 SV=1 Q6UN15|FIP1_HUMAN

Signal recognition particle receptor subunit beta OS=Homo sapiens GN=SRPRB PE=1 SV=3 Q9Y5M8 SRPRB_HUMAN

RNA polymerase II subunit A C-terminal domain phosphatase OS=Homo sapiens GN=CTDP1 PE=1 SV=3 Q9Y5B0|CTDP1_HUMAN

THO complex subunit 6 homolog OS=Homo sapiens GN=THOC6 PE=1 SV=1 Q86W42|THOC6_HUMAN Description Accession

Protein DEK OS=Homo sapiens GN=DEK PE=1 SV=1 P35659|DEK_HUMAN

Collagen alpha-1 (V) chain OS=Homo sapiens GN=COL5A1 PE=1 SV=3 P20908|CO5A1_HUMAN

Protein phosphatase 1 G OS=Homo sapiens GN=PPM1 G PE=1 SV=1 015355|PP 1 G_HUMAN

Serine/arginine-rich splicing factor 5 OS=Homo sapiens GN=SRSF5 PE=1 SV=1 Q13243|SRSF5_HUMAN

14-3-3 protein beta/alpha OS=Homo sapiens GN=YWHAB PE=1 SV=3 P31946|1433B_HUMAN

Calnexin OS=Homo sapiens GN=CANX PE=1 SV=2 P27824|CALX_HUMAN

Y-box-binding protein 3 OS=Homo sapiens GN=YBX3 PE=1 SV=4 P16989|YBOX3_HUMAN

Ubiquitin thioesterase OTUBI OS=Homo sapiens GN=OTUB1 PE=1 SV=2 Q96FW1 |OTUB1_HUMAN

Elongation factor 1 -alpha 1 OS=Homo sapiens GN=EEF1A1 PE=1 SV--1 P68104|EF1A1_HUMAN

Putative elongation factor 1 -alpha-like 3 OS=Homo sapiens GN=EEF1A1 P5 PE=5 SV=1 Q5VTE0|EF1A3_HUMAN

Heterogeneous nuclear ribonucleoprotein L OS=Homo sapiens GN=HNRNPL PE=1 SV=2 P14866|HNRPL_HUMAN

Calreticulin OS=Homo sapiens GN=CALR PE=1 SV=1 P27797|CALR_HUMAN

REST corepressor 1 OS=Homo sapiens GN=RCOR1 PE=1 SV=1 Q9UKL0|RCOR1_HUMAN

60S ribosomal protein L4 OS=Homo sapiens GN=RPL4 PE=1 SV=5 P36578|RL4_HUMAN

MKI67 FHA domain-interacting nucleolar phosphoprotein OS=Homo sapiens GN=NIFK PE=1 SV=1 Q9BYG3 MK67I HUMAN

Malate dehydrogenase, mitochondrial OS=Homo sapiens GN=MDH2 PE=1 SV=3 P40926|MDH _HUMAN

60S ribosomal protein L23a OS=Homo sapiens GN=RPL23A PE=1 SV=1 P62750|RL23A_HUMAN

SURP and G-patch domain-containing protein 2 OS=Homo sapiens GN=SUGP2 PE=1 SV=2 Q8IX01 |SUGP2_HUMAN

DNA polymerase delta subunit 3 OS=Homo sapiens GN=POLD3 PE=1 SV=2 Q15054|DPOD3_HUMAN

Cleavage stimulation factor subunit 2 OS=Homo sapiens GN=CSTF2 PE=1 SV=1 P33240|CSTF2_HU AN

INO80 complex subunit E OS=Homo sapiens GN=INO80E PE=1 SV=1 Q8NBZ0|IN80E_HUMAN

RRP12-like protein OS=Homo sapiens GN=RRP12 PE=1 SV=2 Q5JTH9|RRP12_HUMAN

Ras GTPase-activating protein-binding protein 1 OS=Homo sapiens GN=G3BP1 PE=1 SV=1 Q13283|G3BP1_HUMAN

AT-rich interactive domain-containing protein 1A OS=Homo sapiens GN=ARID1A PE=1 SV=3 014497|ARI1A_HUMAN

Importin subunit alpha-3 OS=Homo sapiens GN=KPNA4 PE=1 SV=1 0006291 IMA3JHU MAN

Heat shock 70 kDa protein 12B OS=Homo sapiens GN=HSPA12B PE=2 SV=2 Q96MM6|HS12B_HUMAN

60S ribosomal protein L12 OS=Homo sapiens GN=RPL12 PE=1 SV=1 P30050|RL12_HUMAN

Heterogeneous nuclear ribonucleoprotein R OS=Homo sapiens GN=HNRNPR PE=1 SV=1 O43390|HNRPR_HUMAN

60S ribosomal protein L18a OS=Homo sapiens GN=RPL18A PE=1 SV=2 Q02543|RL18A_HUMAN

Electron transfer flavoprotein subunit alpha, mitochondrial OS=Homo sapiens GN=ETFA PE=1 SV=1 P13804|ETFA_HUMAN

LEM domain-containing protein 2 OS=Homo sapiens GN=LEMD2 PE=1 SV=1 Q8NC56|LEMD2_HUMAN

Tubulin beta-3 chain OS=Homo sapiens GN=TUBB3 PE=1 SV=2 Q13509|TBB3_HUMAN

A-kinase anchor protein 8 OS=Homo sapiens GN=AKAP8 PE=1 SV=1 043823|AKAP8_HUMAN

Extracellular glycoprotein lacritin OS=Homo sapiens GN=LACRT PE=1 SV=1 Q9G ZZ81 LAC RT_H U M AN

60S ribosomal protein L10a OS=Homo sapiens GN=RPL10A PE=1 SV=2 P62906|RL10A_HUMAN

Fibrinogen beta chain OS=Homo sapiens GN=FGB PE=1 SV=2 P02675|FIBB_HUMAN

U4/U6 small nuclear ribonucleoprotein Prp4 OS=Homo sapiens GN=PRPF4 PE=1 SV=2 043172|PRP4_HUMAN

Polymerase delta-interacting protein 3 OS=Homo sapiens GN=POLDIP3 PE=1 SV=2 Q9BY77|PDIP3_HUMAN

Catechol O-methyltransferase OS=Homo sapiens GN=COMT PE=1 SV=2 P21964|COMT_HUMAN

Negative elongation factor C/D OS=Homo sapiens GN=NELFCD PE=1 SV=2 Q8IXH7|NELFD_HUMAN REFERENCES ng references are cited throughout the present disclosure. olecular cell 45, 147-157 (2012).

Am Chem Soc 133, 14160-14163 (201 1 ).

ast Cancer Res Treat 24, 85-95 (1992).

l., Cell 163, 406-418 (2015).

chim Biophys Acta 1859, 139-146 (2016)

ol Biol Cell 12, 3502-3514 (2001 ).

rends Biochem Sci 24, 329-332 (1999)

iol Chem 281, 33182-33191 (2006).

LoS Biol 4, e6 (2006)

Cell 59, 781 -793 (2015).

t al., FEBS J 278, 2419-2427 (201 1 ).

Cell 155, 750-764 (2013).

al., Proc Natl Acad Sci U S A 107, 3487-3492 (2010). Riek, Structure 18, 1244-1260 (2010).

, 409-419 (2010).

ll Biol 164, 25-33 (2004).

149, 753-767 (2012).

, Proc Natl Acad Sci U S A 111, \ 2085-12090 (2014) l Neurodegener 2, 18 (2007).

c Natl Acad Sci U S A 106, 1 1667-1 1672 (2009).

Nat Rev Mol Cell Biol 15, 384-396 (2014).

0, 339-350 (2012).

nnu Rev Biochem 55, 1 151 -1 191 (1986).

artz and Patterson, Science 300, 87-91 (2003).

ic Acids Res 23, 1758-1765 (1995).

ce 325, 328-332 (2009).

l., Genome Biol 14, R104 (2013).

ol Cell 29, 499-509 (2008).

Cell 35, 57-62 (1983). 30. Mekhail et al., The Journal of cell biology 170, 733-744 (2005).

31 . Misteli, T. (2001 ). Science 291, 843-847.

32. Pinto et al., J Biol Chem 266, 13941 -13946 (1991 ).

33. Richter et al., ol Cell 40, 253-266 (2010).

34. Sasaki et al., Proc Natl Acad Sci U S A 106, 2525-2530 (2009).

35. Semenza, G.L. Cell 148, 399-408 (2012).

36. Semenza and Wang, Mol Cell Biol 12, 5447-5454 (1992).

37. Sosa et al., Nat Rev Cancer 14, 61 1 -622 (2014).

38. Tannock and Rotin, Cancer Res 49, 4373-4384 (1989).

39. Thompson et al., Proc Natl Acad Sci U S A 103, 4074-4078 (2006).

40. Wang et al., PLoS Biol 6, e195 (2008).

41 . Weber and Brangwynne, Cell 749, 1 188-1 191 (2012).

42. Wiesener et al., Blood 92, 2260-2268 (1998).

[00116] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[00117] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

[00118] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[00119] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[00120] Preferred embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted b

Claims

WHAT IS CLAIMED:

1 . A method of testing a drug for efficacy for treating an amyloid disease, comprising (i) contacting cells with the drug before or after the cells are contacted with a stimulus that causes amyloid aggregate formation in the cells and (ii) assaying amyloid aggregate formation or amyloid aggregate disaggregation in the cells, wherein the drug has efficacy for treating the amyloid disease, when amyloid aggregate formation decreases or is delayed or amyloid aggregate disaggregation increases, upon contacting the cells with the drug.

2. The method of claim 1 , wherein the amyloid aggregate comprises one or more of the proteins in Table 1 , each of which are in an amyloid fold conformation.

3. The method of claim 1 , wherein the amyloid aggregate comprises two or more of the proteins in Table 1 , each of which are in an amyloid fold conformation.

4. The method of any one of claims 1 to 3, wherein step (ii) comprises contacting the cells with an amyloid aggregate binding agent.

5. The method of claim 4, comprising:

a. contacting cells with (i) a stimulus to cause amyloid aggregate formation in the cells, (ii) the drug, and (iii) an amyloid aggregate binding agent; and b. detecting the level of amyloid aggregate binding agent bound to amyloid; wherein, when the amyloid aggregate binding agent bound to amyloid is not detected or the level of the amyloid binding agent bound to amyloid is lower than a control level, the drug has efficacy for treating the disease.

6. The method of any one of the previous claims, wherein the cells are contacted with the drug (or the plurality thereof) before the cells are contacted with the stimulus.

7. The method of any one of the previous claims, wherein the cells are contacted with the drug (or the plurality thereof) after the cells are contacted with the stimulus.

8. The method of any one of claims 4 to 7, wherein the amyloid aggregate binding agent is a dye which emits a signal when an amyloid aggregate forms.

9. The method of claim 8, wherein the dye is a colorimetric dye or fluorescent dye.

10. The method of claim 9, wherein the fluorescent dye is selected from the group consisting of: Congo Red, Thioflavin S, NIAD4, and Methoxyl-X04 and BSB ((Trans, Trans)-1 -Bromo-2,5-Bis-(3-Hydroxycarbonyl-4-Hydroxy)Styrylbenzene).

1 1 . The method of claim 9 or 10, comprising quantifying the amount of fluorescence emitted by the dye.

12. The method of any one of the previous claims, wherein the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye about 2 hours after the cells are contacted with the stimulus.

13. The method of any one of the previous claims, wherein the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye about 3 hours after the cells are contacted with the stimulus.

14. The method of any one of the previous claims, wherein the cells are contacted with the drug before the cells are contacted with the stimulus, and the method comprises quantifying the amount of fluorescence emitted by the dye about 4 hours after the cells are contacted with the stimulus.

15. The method of any one of the previous claims, wherein the stimulus is acidosis or heat shock exposure.

16. The method of claim 15, wherein the stimulus is a 3-hour acidosis or heat shock exposure.

17. The method of any one of the previous claims, wherein the drug is tested for efficacy as a therapeutic agent for treatment of a neurodegenerative disease.

18. The method of claim 17, wherein the neurodegenerative disease is Alzheimer's disease.

19. The method of any one of the previous claims, wherein the cells are mammalian cells.

20. The method of claim 19, wherein the mammalian cells are human cells.

21 . The method of claim 19 or 20, wherein the cells are selected from the group consisting of: MCF-7, PC-3, A549, HEK293, C2C12, U87mg, NIH3T3, HCT1 16, PC-12 and Neuro-2A cells.

22. The method of any one of the previous claims, wherein the method is capable of simultaneously testing a plurality of drugs for efficacy for treating the disease within about 12 hours.

23. The method of claim 22, wherein the method is capable of simultaneously testing a plurality of drugs for efficacy for treating the disease within about 6 hours.

24. The method of claim 22 or 23, wherein at least or about 20 drugs are simultaneously tested.

25. The method of claim 24, wherein at least or about 75 drugs are

simultaneously tested.

26. The method of any one of the previous claims, wherein the amyloid aggregate is an amyloid body.

27. The method of claim 26, wherein the amyloid body comprises one or more of the proteins in Table 1 , each of which are in an amyloid fold conformation.

28. The method of claim 26, wherein the amyloid body comprises two or more of the proteins in Table 1 , each of which are in an amyloid fold conformation.

29. The method of any one of the previous claims, wherein the drug binds to one or more of the proteins listed in Table 1 .

30. The method of claim 29, wherein the drug binds to one or more of the proteins listed in Table 1 only when the protein is in an amyloid aggregate or when the protein is in an amyloid fold conformation.

31 . The method of any one of the previous claims, wherein the level at which the amyloid aggregate binding agent is bound to amyloid is compared to a control level.

32. The method of claim 31 , wherein the control level is the level of the amyloid binding agent bound to amyloid when the cells are contacted with the stimulus in the absence of the drug(s).

33. The method of claim 32, wherein the control level is the level of the amyloid binding agent bound to amyloid when the cells are contacted with the stimulus in the absence of the drug(s) and with a vehicle control.