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Buck Institute Technology Summary: Chronic Disease Therapy with Anti-‐Aggregation Compounds
Background During aging there is a disruption to normal protein processing, leading to the deposit of insoluble misfolded proteins, or aggregates. In particular, protein aggregates are a hallmark of common neurodegenerative diseases, for example α-‐synuclein aggregation in Parkinson’s Disease and β-‐amyloid aggregation in Alzheimer’s Disease. For decades, compounds have been used to image these aggregates in postmortem brain tissue and more recently in patients, using modern imaging systems. Scientists at the Buck Institute hypothesized that these compounds may prevent aggregation in vivo and therefore have a therapeutic effect beyond their current diagnostic use.
The Technology Dr. Silvestre Alavez in the Lithgow Lab created a selective screen to analyze molecules traditionally employed in histopathology to stain amyloids in tissues, in the nematode worm C. elegans. This screen revealed that indeed some of these compounds not only bind protein fibrils but slow aggregation and increase lifespan in C. Elegans. Promising compounds included the turmeric component curcumin, the antibiotic rifampicin and other compositions. One compound, Thioflavin T (“ThT”), was found to extend the lifespan of worms by up to 60%, as well as decreasing the physiological aging process causing morbdity, in particular improving frailty by decreasing motor dysfunction (see graphs). These results are reproducible, occur in a dose-‐dependent manner and were published in the journal Nature in 2011.
ThT has been used for over 50 years as an imaging agent to understand the aggregation of soluble amyloid proteins into beta-‐sheet fibrils and has been explored as an in vivo diagnostic for imaging β-‐amyloid plaques. This is the first time that ThT and related compounds have been identified as potential therapeutics to reduce aggregation and improve healthy aging. The Lithgow lab has shown that this novel mechanism of action likely occurs through ramping up of the endogenous stress-‐response and protein degradation pathways.
While this finding was made in nematode worms, preliminary results in collaboration with researchers at the Barshop Institute suggest that HBX, a ThT structurally related compound that crosses the blood brain barrier, improves the prognosis in a mouse model of neurodegenerative disease. In addition, the Andersen lab at the Buck Institute has shown that HBX effectively suppresses neuronal inflammation associated with neurological
disease. These findings indicate a key role for the maintenance of protein homeostasis during aging.
Opportunity Research conducted in the Lithgow Lab at the Buck Institute represents the first example of amyloid-‐binding dyes for use in extending lifespan and reducing age-‐related frailty. Furthermore, these studies suggest a novel and critical role for protein aggregation in controlling physiological aging. ThT and related compounds have potential as therapeutic agents, improving healthspan by controlling protein aggregation in chronic human disease.
Work continues at the Buck regarding this new approach. The Buck seeks developmental partners who can collaborate and further develop these agents to treat the myriad diseases caused or exacerbated by aggregation. The Buck Institute has filed patents on these compounds and derivatives thereof for treatment of multiple age-‐related processes, such as frailty, and chronic diseases, such as arthritis, macular degeneration and cardiovascular disease.
The Buck Institute is the only free standing institute dedicated to aging and age-‐related research in the United States. We actively partner with industry to develop therapeutics, diagnostics or tools that make a difference. The Buck Institute welcomes interested parties to inquire regarding licensure or collaboration of this technology. For more information on this or another technology or opportunity, please contact:
Carlotta Duncan, Ph.D .
Business Development & Licensing Officer Technology Transfer, Buck Institute for Research on Aging. Phone -‐ 415-‐209-‐2000; cduncan@buckinstitute.org
LETTERdoi:10.1038/nature09873
Amyloid-binding compounds maintain proteinhomeostasis during ageing and extend lifespanSilvestre Alavez1, Maithili C. Vantipalli1, David J. S. Zucker1,2, Ida M. Klang1,3 & Gordon J. Lithgow1
Genetic studies indicate that protein homeostasis is a major contri-butor to metazoan longevity1. Collapse of protein homeostasisresults in protein misfolding cascades and the accumulation of inso-luble protein fibrils and aggregates, such as amyloids2. A group ofsmall molecules, traditionally used in histopathology to stain amy-loid in tissues, bind protein fibrils and slow aggregation in vitro andin cell culture3,4. We proposed that treating animals with such com-pounds would promote protein homeostasis in vivo and increaselongevity. Here we show that exposure of adult Caenorhabditiselegans to the amyloid-binding dye Thioflavin T (ThT) resulted ina profoundly extended lifespan and slowed ageing. ThT also sup-pressed pathological features of mutant metastable proteins andhuman b-amyloid-associated toxicity. These beneficial effects ofThT depend on the protein homeostasis network regulator heatshock factor 1 (HSF-1), the stress resistance and longevity transcrip-tion factor SKN-1, molecular chaperones, autophagy and proteoso-mal functions. Our results demonstrate that pharmacologicalmaintenance of the protein homeostatic network has a profoundimpact on ageing rates, prompting the development of novel thera-peutic interventions against ageing and age-related diseases.
The longevity of the nematode Caenorhabditis elegans is influencedby hundreds of genes including an insulin-like signalling pathway (ILS)that regulates the activities of the transcription factors FOXO-likeDAF-16 (ref. 5) and Nrf2-like SKN-1 (ref. 6). Together with the stressresponse transcription factor HSF-1, DAF-16 also regulates proteinhomeostasis and influences lifespan7–9, indicating that chemical modu-lation of protein homeostasis might slow ageing. We reasoned thatcompounds that have protein-fibril- and protein-aggregate-bindingproperties may affect age-related changes to protein homeostasis andtested a series of amyloid-binding proteins for lifespan effects. Wefound that exposing sterilized wild-type (N2) nematodes to the fibril-binding flavonoid ThT (4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,N-dimethylaniline chloride)10 at either 50 or 100mM throughoutadult life leads to an increase in median (60%) and maximal lifespan(43–78%; Fig. 1a, b, Supplementary Fig. 2 and Supplementary Table 1).The compound reduced age-specific mortality at all ages (P , 0.001,Fig. 1c) and slowed age-related decline in spontaneous movement(Fig. 1d), indicating improved health throughout adulthood. At higherdoses (500mM) ThT is toxic and shortens lifespan (Fig. 1a, b). Othercompounds with protein-aggregate-binding properties, including cur-cumin and rifampicin, increased lifespan to a lesser extent (up to 45%)(Supplementary Figs 3, 4). When ThT and curcumin treatments werecombined, we did not observe additive effects on lifespan (Supplemen-tary Fig. 5).
We then tested several compounds with similar structural features toThT, but with different pharmacological properties: 2-(2-hydroxyphe-nyl)-benzoxazole (HBX), 2-(2-hydroxyphenyl) benzothiazole (HBT) and2-(2-aminophenyl)-1H-benzimidazole (BM)11 (Supplementary Fig. 6).These compounds also extended the lifespan of adult nematodes (up to40%) but at concentrations significantly lower than ThT (Fig. 1e–g),
1Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, California 94945, USA. 2Department of Natural Sciences and Mathematics, Dominican University of California, San Rafael, California94901, USA. 3Karolinska Institute, Center for Biosciences at NOVUM, Department of Biosciences and Nutrition, Halsovagen 7, S-141 83 Huddinge, Sweden.
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Figure 1 | Amyloid-binding compounds extend C. elegans lifespan.a, Dose–response Kaplan–Meier survival curves of synchronously ageinghermaphrodite wild-type (N2) populations exposed to 0 mM (control) to500mM ThT at 20 uC. b, Per cent change in median lifespan of N2 populationscultured on 0–500mM ThT and curcumin. c, ln-linear plot of age-specificmortality rate with age for control and 50mM ThT-treated C. elegans. d, Effectof 50mM ThT and 100mM curcumin on motility of N2 worms evaluated as themean number of body bends in a 20-s period in 15 individual wormsthroughout life (upper panel) and after 12 days of treatment (lower panel) withThT and curcumin. Data are presented as bends min21 and represent theaverage of three independent experiments. *P , 0.0001. e–g, Dose–responseKaplan–Meier survival curves of synchronously ageing hermaphrodite N2populations exposed to 0mM (control) to 1mM of BM (e), HBT (f) and HBX(g) at 20 uC. Plots are representative of three independent experiments.
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indicating that the bioavailability and/or pharmacological properties ofThT-like compounds influence lifespan.
To test the effects of ThT on protein homeostasis we exploited twoC. elegans models of human proteotoxic disease: the strain CL4176(dvIs27[myo-3::Ab3–42 let 39UTR(pAF29); pRF4 (rol-6(su1006))])12,which expresses an aggregating amyloid-b(3–42) peptide (Ab(3–42))in muscle tissue13 and AM140 (rmIs132[P(unc-54) Q35::YFP]), whichexpresses a polyglutamine (polyQ) protein. Amyloid-b aggregates areassociated with lesions in Alzheimer’s disease, and polyQ aggregation is afeature in several neurological conditions14. When raised at 25uC, nema-todes expressing these proteins in muscle accumulate protein aggregatesand become paralysed. We found that 50mM ThT and 100mM curcu-min decreased the proportion of paralysed worms (Fig. 2a, b). Byimmunohistochemistry we found that ThT reduced Ab(3–42) aggrega-tion in vivo and preserved muscle integrity in CL4176 (Fig. 2e). We alsofound that ThT rescued Ab(3–42) aggregation-induced paralysis evenwhen nematodes were treated 18 h after the induction of aggregateformation, indicating that ThT can ameliorate detrimental effects dur-ing the development of the aggregate-related pathology (Supplemen-tary Fig. 7).
If amyloid-binding compounds extended lifespan throughimproved protein homeostasis, then we expected that they would influ-ence not only heterologous disease-related models but also nematodeproteins. We tested ThT and curcumin on mutant worms that expressmetastable proteins previously exploited as indicators of the proteinhomeostatic network capacity15. Strains carrying mutations in thegenes unc-52 (HE250 [unc-52(e669su250)II]) and unc-54 (CB1157[unc-54(e1157)I]) produce temperature-sensitive muscle proteinsUNC-52 (perlecan) and UNC-54 (myosin class II heavy chain),respectively, that exhibit altered structure and cause paralysis at25 uC15,16,17. We found that ThT suppressed paralysis of these mutants(Fig. 2c), prevented the disruption of the muscle sarcomeres (Sup-plementary Fig. 8) and restored perlecan organization (Fig. 2d). Weextended these observations to other temperature-sensitive missenseprotein-folding mutations expressed in the neuromuscular junctionand in the nervous system18. We found that ThT suppressed ethanolsensitivity in a strain carrying the gas-1(fc21) mutation in a subunit ofmitochondrial complex I and levamisole resistance in a strain carryingunc-63(x26), an a-subunit of the nicotinic acetylcholine receptor(Supplementary Fig. 9), indicating that ThT could act in a variety oftissues including the nervous system.
Because certain forms of dietary restriction suppress proteinaggregation and increase lifespan, we asked whether ThT acts as adietary restriction mimetic. We observed that ThT produces a smalldecrease in pharyngeal pumping rate (,15%) after 3 days of treat-ment, which could slightly decrease food intake. No difference wasdetected after 6 days of ThT treatment (Supplementary Fig. 16a). It isvery unlikely that this small difference could promote the major ThT-mediated lifespan extension we observe. ThT also increased the life-span of a strain carrying the eat-2(ad1116) mutation (Fig. 4c) thatcauses a major defect in pharyngeal pumping, thereby inducing adietary restriction lifespan extension19. Dilution of the bacterial foodsource also leads to lifespan extension by dietary restriction20. ThT at50mM was detrimental to lifespan in this dietary restriction model but1 and 10mM ThT increased lifespan by 24% (Supplementary Fig. 16b).As ThT increases lifespan in both genetic and nutrient-based modelsof dietary restriction, ThT-induced lifespan extension is at least in partindependent from dietary restriction.
We then considered whether ThT was interacting more directlywith homeostatic mechanisms. We exploited ThT fluorescence tovisualize the compound in vivo and observed a variable co-distributionof ThT with Ab(3–42) aggregates detected by immunolocalization,indicating a direct interaction between ThT protein misfolding cas-cades (Supplementary Fig. 10). Consequently, we tested for the pres-ence of amino acid sequence-independent oligomers of protein orpeptides prone to aggregation. We found that protein detected by an
antibody specific for such oligomers (A11) accumulated during normalageing and after heat shock, but was significantly decreased in bothCL4176 (Fig. 2e) and N2 strains (Fig. 2f) after ThT treatment, consistentwith ThT affecting protein misfolding cascades.
We reasoned that ThT may also require components of the proteinhomeostatic network activated by DAF-16 and HSF-1 to influenceprotein aggregation and lifespan8,21. We undertook a targeted pharma-cogenetic RNA interference (RNAi) screen of genes encoding several
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Figure 2 | ThT and curcumin rescue a paralysis phenotype and slow proteinaggregation in vivo. a, b, The paralysis phenotype associated with proteinaggregation is suppressed by 25mM ThT, 50mM ThT, 100mM curcumin inCL4176 (*P , 0.001, **P , 0.0001) expressing Ab(3–42) (a) and AM140(*P , 0.05, **P , 0.01) expressing polyQ (b) after 1 and 8 days at 25 uC,respectively. Error bars represent the mean 6 s.e.m. of four independentexperiments. c, Temperature-sensitive strain HE250 [unc-52(e669su250)II]after 36 h at 25 uC showing the typical paralysis phenotype (left upper panel)and the rescue elicited by 50mM ThT (right upper panel). Arrows indicate thehalos of clearance in the bacterial lawn characteristic of paralysed worms.Lower panel shows protection (6 s.e.m.) of the HE250 paralysis phenotype by50mM ThT, 100mM curcumin (Cur) and 100mM rifampicin (Rif).*P , 0.0001, **P , 0.01. n 5 4 independent experiments. d, Perlecanimmunolocalization showing disruption/aggregation pattern after 24 h at25 uC, as compared with worms raised at the permissive temperature (upperpanel), and the suppression of disruption by 50mM ThT treatment. Sixteen oftwenty worms showed similar perlecan distribution in three independentexperiments. Arrows indicate perlecan aggregates. Scale bar, 30mm.e, Immunolocalization of aggregation-prone soluble oligomeric protein (A11antibody, red) and Ab(3–42) (green) in the presence or absence of 50mM ThTin CL4176. Scale bar, 10mm. Error bars represent the mean 6 s.e.m., 11 wormsper group in 3 independent experiments. *P , 0.0001. f, Immunolocalizationof aggregation-prone soluble oligomeric protein (A11 antibody) in the presenceor absence of 50mM ThT and under heat shock (HS) in 11 days old wild-typeN2 worms. Scale bar, 20mm. Error bars represent the mean 6 s.e.m., 11 wormsper group, of 3 independent experiments. *P , 0.0001. Scale bar, 10mm.
LETTER RESEARCH
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A. Dose-‐dependent survival increase for worms with ThT treatment. B. Decreased paralysis of worms with ThT and curcumin treatment. Paralysis is detected by a halo-‐effect of bacteria around the control stressed worms unable to feed (asterisks) compared to the treated stressed worms with decreased frailty.
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