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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