WO2006106544A2

WO2006106544A2 - Use of nothobranchius furzeri as a model system for the characterization of drugs that control ageing

Info

Publication number: WO2006106544A2
Application number: PCT/IT2005/000753
Authority: WO
Inventors: Alessandro Cellerino
Original assignee: Lay Line Genomics S.P.A.
Priority date: 2005-04-07
Filing date: 2005-12-22
Publication date: 2006-10-12
Also published as: WO2006106558A3; EP1871427A2; ITRM20050169A1; WO2006106544A3; WO2006106558A2

Abstract

The present invention relates to the use of fish belonging to the family of Cyprinodontids (common name: killifish) as an animal model to study ageing-related diseases. The present invention further relates to a method for the characterization and identification of active substances which can modulate the onset of pathologies related to ageing.

Description

Use of Nothobranchius furzeri as a model system for the characterization of drugs that control ageing

TECHNICAL FIELD OF THE INVENTION The present invention relates to the use of fish belonging to the family of Cyprinodontids (common name: killifish) as an animal model to study ageing- related diseases. The present invention further relates to a method for the characterization and identification of active substances which can modulate the onset of pathologies related to ageing. PRIOR ART

Nothobranchius furzeri, member of the Cyprinodontid family (common name: killifish) belongs to a group of annual fish whose life expectancy in Nature is limited to a few months. The longest survival time of N. furzeri was described and characterised by Valdesalici and Cellerino (2003), and it is less than 12 weeks if bred in standard laboratory conditions at 25 ⁰C. This is the shortest captive lifespan ever recorded for a vertebrate. This lifespan is comparable with that of Drosophila melanogaster maintained at the same temperature (Kang et al., 2002). Other fish such as Zebrafish {Danio rerio), Nothobranchius guentheri, Nothobranchius rachovii, Cynolebias bellotti e Cynolebias nigripinnis have been proposed as model systems for the study of the biological mechanisms that control ageing (Liu and Walford, 1970; Markofsky and Perlmutter, 1972; Patnaik et al., 1994; Herrera and Jagadeeswaran, 2004). All these fish have a much longer life expectancy: Zebrafish can live up to 5 years (Gerhard et al., 2002), Nothobranchius guentheri over 16 months (Markofsky and Perlmutter, 1972). Nothobranchius rachovii lives 8.5 months in stagnant water, while in systems with rapid water recirculation it develops a pathology known as "gas bubble disease" which causes its death in less than 2 months (Herrera and Jagadeeswaran, 2004). Cynolebias bellotti lives over 2 years if maintained at 15°C (Liu and Walford, 1975) and Cynolebias nigripinnis lives 10 months if kept at 28°C (Herrera and Jagadeeswaran, 2004), which, however, is a temperature at least 15 ⁰C higher than the temperature measured in the natural habitats of these animals, which are cold-water fishes (Costa, 1996). Fish larvae, in particular larvae of Zebrafish or Medaka have been described (WO200404044241 ) as a "screening" model to investigate metabolism diseases, such as obesity and associated diseases (eating disorders, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, bilious disorders, cancer and sleep apnea). Nothobranchius furzeri, due to its exceptionally short life expectancy (12 weeks in laboratory conditions), exhibits significant advantages with respect to other vertebrate models, such as Zebrafish, Mus muscυlus and Cynolebias, which have been already used. In the present invention, it is demonstrated that Nothobranchius furzeri in its 12 weeks of life expresses a series of markers of ageing, which are similar and comparable to those described in humans. These markers include accumulation of lipofuscin, expression of senescence-associated /?-galactosidase, reduction in spontaneous locomotor activity and cognitive decay. Lipofuscin is a autofluorescent pigment which accumulates in the form of granular inclusions inside the neurons during ageing in the human brain (Glees and Hasan, 1976; Anderton, 1997) and in many other animals, including fish (Patnaik et al., 1994; Strauss, 1999). Moreover, during ageing, human fibroblasts lose their ability to replicate and this replicative blockade is coincident with the expression of an endogenous /?-galactosidase activity (Dimri et al., 1995). This activity is easily detectable in skin sections of elderly persons (Dimri et al., 1995) and it is considered a marker of cells that have lost the ability to replicate in vivo. The expression of senescence-associated /?-galactosidase was revealed during ageing of Zebrafish (Danio rerio) (Kishi et al., 2003). The decay of muscle strength and motor performance and a reduction in muscle mass (sarcopenia) is invariably a symptom associated with ageing in humans (Volpi et al., 2004) which is also observed in fish (Reznick et al., 2004). Lastly, memory decay is another sensitive marker of cerebral ageing (Anderton, 1997). Many genes that control ageing are expressed both in invertebrates, such as C. elegans and Drosophila, and in vertebrates (Barbieri et al., 2003; Cohen et al., 2004). However, some genes that control ageing, such as "microsomal transfer protein" (MTP) (Geesaman et al., 2003) e "p66shc" (Napoli et al., 2003), have been identified only in mammals. The present invention reports that these genes are also present in Nothobranchius furzeri. These genes can be targets for identifying drugs that modulate their activity.

Nothobranchius furzeri therefore represents a model that allows to characterise the function of known genes or products thereof in the modulation of longevity and the insurgence of pathologies related to ageing.

Lastly, Nothobranchius furzeri is a screening model for the identification and/or the characterisation of pharmacologically active substances that are able to prevent the insurgence of pathologies related to ageing. The authors have used as an example resveratrol. Resveratrol is a polyphenol of natural origin present in grape peels and in particularly high concentrations in red wine. Resveratrol has various biological actions, it acts as an anti-oxidant but it is also an activator of histone- deacetylase of the sirtuin family whose homologous in humans is the SIRT1 gene (Frye, 1999). Resveratrol is particularly interesting as a possible compound capable of preventing pathologies related to ageing because the administration of resveratrol extends the lifespan of S. cerevisae, C. elegans and D. melanogaster (Wood et al., 2004). However, the bodily organisation of these two invertebrates is totally different from that of humans and of other vertebrates. Moreover, Drosophila and C. elegans, unlike vertebrates, do not show cell renewal in adults and cannot represent models for the loss of replicative ability typical of vertebrate aging. For this reason, results harvested in these animal models are not applicable to the composition of drugs for the prevention and therapy of human pathology with slow and progressive course related to ageing.

In vertebrates, the sole treatment able to extend life expectancy described heretofore is a low calorie diet (Masoro, 2003). Hypocaloric diet activates the expression of SIRT1 (Cohen et al., 2004) and therefore the effects of resveratrol could be mediated by the same molecular pathways activated by hypocaloric diet. In S. cerevisae, C. elegans and D. melanogaster, the effects of resveratrol and those of caloric restriction require the presence of sirtuins (Wood et al., 2004). In patent application PCT WO0238141 , natural products like resveratrol, capsaicin, anandamide, etc. were suggested for the treatment of mild cognitiveimpaimnent, based on their effect on a) cellular beta-amyloid precursor proteins (APP) secreted in cultures of rat astrocytes and b) the concentration of total beta-amyloid (Aβ) in culture media of CHO cells transfected with an human APP construct. The patent does not include in vivo data (cognitive tests in both animal models and human patients) and it does not specifically demonstrates the effects of resveratrol on all ageing-related diseases. Additionally, the document does not provide any functional link between the effect of resveratrol on APP or on the concentration of Aβ and the potential improvement of mild cognitive impairment. Lastly, the hypothetical (and not demonstrated) effect of resveratrol on memory or on ageing-related deficits is mediated by an Aβ dependent action mechanism. In the patent EP 1.117.394, the authors describe a mixture of L- camitine or derivatives thereof with a tri-hydroxy or a trihydroxystilbene (for example resveratrol) to prepare a dietary supplement or a pharmaceutical product for the prevention and treatment of pathological forms due to neuron or cerebral alterations consequent to ageing, use of neurotoxic drugs, brain stroke, memory loss, pre-senile dementia and Alzheimer's disease (AD). The patent does not demonstrate a therapeutic effect of the combination of carnitine and resveratrol in ageing-related diseases. It does not include results on memory tests, or on other in vivo models of progressive degenerative diseases related to ageing. The patent application PCT WO03/103583 concerns the use of a broad class of compounds (which includes resveratrol) for therapeutic applications for neurological diseases in general, including motor neuron diseases. In this application, resveratrol is not a object of experimental studies, but it is inserted in the general description and in the claims.

The patent application PCT WO99/59561 describes that resveratrol is an inhibitor of neutrophil myeloperoxidase, and therefore it could protect tissues in pathologies involving chronic inflammations, including AD. Lastly, numerous publications demonstrate neuroprotective effects of resveratrol after acute damages such as ischemia or kainic acid intoxication (Virgili and Contestabile, 2000; Huang et al., 2001 ; Gupta et al., 2002; Sinha et al., 2002; Wang et al., 2002; lkeda et al., 2003; Morin et al., 2003; Seidman et al., 2003; Zhuang et al., 2003; Kiziltepe et al., 2004; Wang et al., 2004). However, the identity of physiopathological mechanisms linked to acute traumas and those that generate slow and progressive decay of cognitive abilities related to ageing is yet to be demonstrated. Up to the present day, there is no demonstration that a chronic and prolonged treatment with resveratrol prevents the insurgence of mild cognitiveimpairment. The method for the study of memory in Nothobrachius furzeri that is part of the present invention, provides a system for testing the effectiveness of resveratrol in preventing mild cognitive impairmen.

An additional illness related to ageing is sarcopenia: a progressive reduction in muscle mass that is observed during ageing (lannuzzi-Sucich et al., 2002; Greenlund and Nair, 2003; Volpi et al., 2004). This loss in muscle mass has two main consequences: the first one is a reduced protection of the bones and joints which raises the probability of fractures, the second one is a progressive loss of independence. Lastly, a reduced locomotor activity measured as step speed and ability to rise from a chair is predictive of more severe disabilities and hospitalisation (lannuzzi-Sucich et al., 2002; Greenlund and Nair, 2003; Volpi et al., 2004). For these reasons, sarcopenia is considered a pathology related to ageing that impacts the quality of life of elderly person and whose prevention would lead to considerable savings in health care expenditure. For example, the estimated annual cost of sarcopenia in the United States in 2000 was 18.5 billion Dollars (Janssen et al., 2004).

The therapies that have been proposed so far are physical exercise (Winett and Carpinelli, 2001 ; Greenlund and Nair, 2003), use of hormones (Bhasin, 2003) and of the trophic factor IGF-I (Renganathan et al., 1998; Greenlund and Nair, 2003) since all these factors have a trophic activity on muscles. Carnitine supplements in fooddid not prevent sarcopenia, in animal models(Derave et al., 2005). The method for measuring spontaneous locomotor activity that is a part of the present invention provides a system for testing the effectiveness of resveratrol in preventing sarcopenia. Therefore, presently the prior art lacks vertebrate models that allow to identify and/or characterise compounds or genes able to modulate longevity and/or the insurgence of functional deficits related to ageing that have slow and progressive course, comparable to what occurs during human ageing. Nothobranchius furzeri is an advantageous model because it is a vertebrate whose life expectancy is limited to 12 weeks. Therefore, it can be used rapidly to assay the effect of chronic treatments with natural and/or synthetic compounds on ageing and on longevity in a short time, i.e. about one month. DISCLOSURE OF THE INVENTION The present invention relates to models, genes and agents that are therapeutic and/or preventive of functional ageing. Functional ageing is a slow and progressive decrease in physiological efficiency of all systems of the body: cardiovascular, skeletal-muscular, respiratory, excretory, thermoregulatory and nervous (Harman, 2001 ). The result of this phenomenon, which takes place in every human being as age advances, is a progressive decay of all biological functions that lead to a reduction in quality of life and in independence, an increase in susceptibility to all traumas and stresses, an increased need for chronic treatments and hospitalization and an increase in the incidence of a large range of pathologies properly defined as pathologies related to ageing which may affect all organs and systems (Hazzard et al., 2003). It is important to emphasise that their slow, progressive nature is the distinctive characteristic of these pathologies. Therefore they can only be studied in animal models where the course of the symptoms is gradual and progressive (obviously, rapidity in the onset of the symptoms must be related to the rapidity of the life cycle of the analyzed species) and observed only during the late stages of the life of the organism. Two examples of pathologies related to ageing are sarcopenia and mild cognitive impairment (Hazzard et al., 2003). Another object of the present invention is a method for identifying and/or characterising a substance able to modulate the longevity and/or ageing of a vertebrate, compromising the steps of: a) exposing individuals belonging to the family of Cyprinodontids (common name: killifish) to said substance in effective manners and quantities; b) measuring in said individuals the longevity and/or a marker of ageing and comparing said measurements with negative control values; c) selecting the substance capable of positively modulating longevity and/or at least one marker of ageing.

For a positive modulation it is intended an increase in longevity and/or a reduction of the phenotype linked to ageing. Preferably, exposure to said substance is obtained by ingestion of semi-solid food that contains it and is able to release it.

Preferably, the markers of ageing belong to the following group: accumulation of lipofuscin in the brain and/or the liver, the expression of B-galactosidase associated to senescence, reduction in spontaneous locomotor activity, cognitive decay and neurofibrillary degeneration.

Preferably the individuals belong to the species Nothobranchiυs furzeri. Preferably, the substance is resveratrol. A further object of the invention is the use of resveratrol for the preparation of a pharmaceutical composition for the prevention and/or therapy of pathologies connected with senescence, loss of spontaneous locomotor activity, cognitive decay and/or neurofibrillary degeneration. Yet another object of the invention is the use of resveratrol for the preparation of a pharmaceutical composition, or as a food additive, for extending the longevity of a subject.

The present invention shall now be described in non limiting examples thereof, with particular reference to the following figures: Figure 1 : Lipofuscin accumulation in the brain and liver of Nothobranchius furzeri (GRZ) during ageing. In column A, the top photograph corresponds to a section of the optic tectumoptic tectum of a 4 week old Nothobranchius furzeri observed with confocal microscope at a wavelength of 488 nm. No accumulation of autofluorescent granules in the neurons is noted. The centre top photograph corresponds to a section of the optic tectum of a 9 week old Nothobranchius furzeri. The accumulation of autofluorescent granules autofluorescentin the cytoplasm of some neurons is readily apparent. The bottom photograph is a cortex of a 20 month old mouse shown as positive control. Column B, at the top, shows a section of liver of a 4 week old Nothobranchius furzeri, while the image at the bottom shows a section of a 9 week old Nothobranchius furzeri. Themicroscope magnification, the thickness of the optical sections laser- and photomultiplier-intesity are identical in all images.

Figure 2: Endogenous β-galactosidase activity in the skin of Nothobranchius furzeri (GRZ) during ageing. The photographs show coronal sections of the caudal peduncle. The specific reaction product is in blue. The black labelling identifies melanocytes and is due to the presence of endogenous melanin. In column A, sections of 4 week old animals are shown, whilst in column B sections of 9 week old animals are shown. The calibration bars correspond to 50 μm. Figure 3: Endogenous β-galactosidase activity in the eyes of Nothobranchius furzeri (GRZ) during ageing. The photos show cross sections of eyes with surronding connective tissue. The photos correspond to a 4 week old (A) and a 9 week old (B) Nothobranchius furzeri (GRZ). The specific labelling is indicated by the two arrows.

Figure 4: Spontaneous locomotor activity of Nothobranchius furzeri (GRZ) during ageing. Chart A shows the percentage of time spent in motion, chart B the average speed (cm/s) and chart C the maximum speed (cm/s). The x-axis shows age in weeks. 10 animals were analyzed for each age. The error bars are standard errors of means. The statistical test used is Kruskall-Wallis' ANOVA, calculated with Statistica ® (Statsoft, ^** = P<0.01 ).

Figure 5: Measurement of cognitive activity by means of active avoidance behavioral test of Nothobranchius furzeri (GRZ) during ageing. The chart A schematically shows the test apparatus. The tank for the test has a rectangular shape and is divided in two by a narrow passage. On both sides are positioned coloured lights. In this example the fish is on the left and the left light is turned on. If the fish moves into the right compartment within 15 seconds from the time the light is turned on, the test was counted as a success, otherwise as a failure. The chart B shows the learning curves expressed in performance index versus the number of trials. The performance index is calculated as the frequency of successes in the 10 previous trials. Hence, on the x-axis the curve starts from 11 and ends at 51. The error bars are standard errors. The line indicated as "chance" denotes the probability that a fish moves from one compartment to the other in response to light in the absence of training. The statistical significance of the difference between the two groups for each animal was calculated in two ways:

1) analyzing the learning curves with the Kolmogoroff-Smirnoff test (Statistica ®, Statsoft) which yielded a level of significance p<0.0001 ,

2) analyzing the percentage of successes in the last 10 tests and comparing the two groups by means of the Mann-Whitney test U of Statistica ®, Statsoft. The difference between the two test groups is significant at the level p<0.01.

Figure 6: Sequence homologies of the MTP gene between N. furzeri and other animals including humans. The figure shows the alignment of the amino acid sequences of p66shc and MTP in various species of vertebrates. They are identified by the initials of their genus and species. Nf, Nothobranchius furzeri; Tr, Takifugu rubripes (Fugu); Tn, Tetraodon nigroviridis (pufferfish); Dr, Danio rerio (zebrafish); Hs, Homo sapiens (man); Pp, Pongo pygmaeus (orang-utan); Pt, Pan troglodytes (chimpanzee); Mm, Mus musculus (mouse); Rn, Rattus norvegicus (rat); Cf, Canis familiaris (dog); Gg, Gallus gallus (chicken); Xl₁ Xenopus laevis. The numbers that follow the initials of each species indicate the position, in the complete sequence of the protein, of the first and last amino acidic residual of the region aligned to the cloned sequence in Nothobranchius furzeri. The areas of greater homology are indicated by the following symbols: " * " (identical amino acids), " : " (strongly similar amino acids) and " . " (weakly similar amino acids). The regions containing amino acidic residues indicated by the three above symbols can be considered "consensus sequences". The diagram under the sequence quantifies the level of homology for every position in the sequence. The alignments of the amino acidic sequences were obtained with the Clustal program, and they were edited with the Jalview program to even their ends.

Figure 7: Sequence homologies of the p66shc gene between N. furzeri and other animals including humans. The figure shows the alignment of the amino acidic sequences of p66shc in various species of vertebrates. They are identified by the initials of their genus and species. Nf, Nothobranchius furzeri; Tr, Takifugu rubripes (Fugu); Tn, Tetraodon nigroviridis (pufferfish); Dr, Danio rerio (zebrafish); Hs, Homo sapiens (man); Pp, Pongo pygmaeus (orang-utan); Pt, Pan troglodytes (chimpanzee); Mm, Mus musculus (mouse); Rn, Rattus norvegicus (rat); Cf, Canis familiaris (dog); Gg, Gallus gallus (chicken); Xl, Xenopus laevis. The numbers that follow the initials of each species indicate the position, in the complete sequence of the protein, of the first and last amino acidic residual of the region aligned to the cloned sequence in Nothobranchius furzeri. The areas of greater homology are indicated by the following symbols: " * " (identical amino acids), " : " (strongly similar amino acids) and " . " (weakly similar amino acids). The regions containing amino acidic residues indicated by the three above symbols can be considered "consensus sequences". The diagram under the sequence quantifies the level of homology for every position in the sequence. The alignments of the amino acid sequences were obtained with the Clustal program, and they were edited with the Jalview program to even their ends. Figure 8: Age-dependent survivorship comparison of fishes hatched the same day and raised at a constant temperature of 25 ⁰C (N=30) or moved to 22 ⁰C at age 4 weeks (N=30). Percent of animals survived is reported on the abscissa and age (in weeks) is reported on the ordinate. Figure 9: Age-dependent Loco-motor decay in fishes kept at constant 25 ⁰C (n=9) as compared with fishes moved to 22 ⁰C at age 4 weeks (n=8). (A) Average velocity and (B) percent of time spent moving Bars represent standard errors. Mann-Whithney U-test ^**=p<0.01 , ^***=p<0.001. The stars on the lines refer to comparisons of animal of different ages within the same experimental group. The stars on the points refer to comparisons of age-matched animals across groups.

Figure 10: Age-dependent cognitive decay measured in the active avoidance test in fishes kept at constant 25 ⁰C (n=9) as compared with fishes moved to 22 ⁰C at age 4 weeks (n=8). Columns represent the mean of individual learning scores. Bars represent standard errors. Mann-Whithney U-test ^**=p<0.01.

Figure 11 : (A) Age-dependent survivorship comparison of control untreated fishes, vehicle-fed fishes and resveratrol-fed fishes. The reference survival is the mean of 5 independent experiments for a total of 145 animals. The solid line reports the survival of 45 control-fed fishes (2 replicates). The other three graphs reports the survival in response to increasing doses of resveratrol. (B)

Age-dependent survivorship divided by gender. Only Control-fed and fishes fed with 120 μg/gr (0.5mM) resveratrol are reported. The abscissa reports the fraction of animals survived (survivorship), the ordinate the age (in weeks) .

Comparisons versus control performed with Log-Rank test: ns=not significant, ***=p<0.001.

Figure 12: Effect of resveratrol on the locomotor activity of Nothobranchius furzeri (GRZ). Chart A shows the percentage of time spent in motion, chart B the average speed (cm/s) and chart C the maximum speed (cm/s). The control groups comprise 10 animals, those treated with resveratrol 7 animals. The error bars are standard deviations. The significance levels are indicated as follows: ^*= P<0.05, *^*= P<0.01. The statistical test used is the ANOVA of Kruskall-Wallis that was calculated with Statistica ® (Statsoft). Figure 13: Effect of resveratrol on cognitive activity in the active avoidance test of Nothobranchius furzeri. The animals were subjected to training as described in the method section. The charts show the mean of the performance index reached at the end of fifty trials in the three test groups. The control group includes 10 animals, those treated with resveratrol 7 animals. The error bars are standard deviations. The statistical test used is Kruskall-Wallis' ANOVA, which was calculated with Statistica ® (Statsoft, ^** = P<0.01 ).

Figure 14: Fluoro-JadeB histochemistry representing neurofibrillary degeneration in an horizontal sections of stratum griseum superficiale of the optic tectum. The section is oriented so that the lateral surface is facing to the right and the medial suface is facing to the left. The specific reaction product is fluorescent green. (A) 5-weeks old fish. Note absence of specific signal (B) 9-weeks old control fish. Note the bright labeling in neuronal processes orthogonal to the tectal surface. (C) 9- weeks old resveratrol 0.5 mM-treated fish. (D) Quantification of signal intensity in 9-weeks old control fish (n=4) and 9-weeks old resveratrol 0.5 mM-treated fish (n=4). Values correspond to average grey levels expressed as arbitrary units. Bars represent standard errors. Student's t-test, ^**=p<0.01.

MATERIALS AND METHODS 1) Supply and breeding of Nothobranchius furzeri.

Nothobranchius furzeri of the Gona Re Zhou strain (GRZ) descend from fish collected in 1968 as described (Jubb, 1971 ). They originate from the National Park of Gona Re Zhou (Zimbabwe), alluvial plane of the Gulene river, co-ordinates: 21 40.2 S ; 31 2.4 E . The fish were collected using large fords and sieving water without sight, given the considerable turbidity of these pools. The fish are common and easy to capture in their typical location during the months of January-March if that year the rains were sufficient to fill the pools. Moreover, Nothobranchius furzeri of the Gona Re Zhou strain can be bought from Kenjiro Tanaka (http://www.ne.Jp/asahi/medaka-ken/k.t/framepage3-10.html).

The opening of the eggs, larvae growth and reproduction are described in Valdesalici and Cellerino (2003). The fish were maintained in a facility comprising sets of 60 litre tanks with centralised filtering, UV germicide lamp and micro- filtration (Aqua, Lucca), to make water quality homogeneous. 2) Biomarkers of ageing

The characterisation of biomarkers of ageing was conducted in Nothobranchius fυrzeri between 4 and 9 weeks for control animals and at 9 and 13 weeks for the animals treated with resveratrol. a) Survival

The method is identical to the one described by Valdesalici and Cellerino (2003). b) Lipofuscin accumulation Lipofuscin accumulation in the brain of Nothobranchius furzeri (GRZ) was measured with a confocal microscope (Leica, Germany) using a 488 nm laser according to the method described in (Belichenko et al., 1996). c) Expression of /?-qalactosidase associated to senescence

Sections of the caudal peduncle and of the eye of 9 and 4 week old Nothobranchius furzeri (GRZ) were taken. The tissues were treated according to the protocol described for zebrafish (Kishi et al., 2003). Beta-galactosidase accumulation is particularly evident in histological sections of the eye. d) Spontaneous locomotor activity

Nothobranchius furzeri (GRZ), were placed individually in tanks with a base of 35x15 cm and a water depth of 5 cm and were filmed for a period of 10 minutes. The films were analysed with the software Ethovision ® (Noldus, Wageningen, Netherlands) and the following parameters are quantified automatically by the software: percentage of time spent in motion, average speed (cm/s) and maximum speed (cm/s). e) Cognitive decay

To study the memory of Nothobranchius furzeri (GRZ) a protocol devised for zebrafish (Danio rerio) and based on a shuttle box was modified (Laudien et al., 1986; Pradel et al., 1999). The fish were placed, one at a time, in a 35x15x15 tank divided in to by a narrow passage. The fish were trained by turning on a red light in one of the two compartments and after 15 seconds they received a punishment (they were repeatedly touched with a plastic stick). The punishment ended after 20 seconds or as soon as the fish moved into the other compartment (Fig. 5a) the test started immediately with no pre-training. If the animal, following the lighting of the red light, moved to the other compartment before receiving the punishment, the test was counted as a success, whilst in case of failure to move and consequent punishment the test was counted as a failure. The test consisted of 50 consecutive trials for total duration of 50 minutes. To depict learning graphically, a parameter called performance index was calculated in the following way: at the n^th trial, performance index was calculated as the fraction of successes in the 10 previous trials or (s+ή/IO where s is the number of successes and f is the number of failures. Hence, the value at trial 11 corresponds to the average of the trials 1-10, the value at trial 21 to the average of the trials 11-20, etc. To test the differences between the groups, for each animal the performance index was calculated in the trial 41-50 that corresponds to the value number 51 of the chart. e) Neurofibrillary degeneration: Fluoro-JadeB histochemistry Neurofibrillary degeneration is a hallmark of human brain aging (Anderton, 1997) which is observed in fishes as well (Maldonado et al., 2002). Neurofibrillary degeneration can be visualized using the fluorescent dye Fluoro- jadeB (Schmued et al., 1997). The optic tectum was analysed as it represents a region particularly affected by aging in fish (Woodhead and Pond, 1984; Maldonado et al., 2002). Fluoro-JadeB histochemistry was performed following purchaser's instructions (Chemicon, Temencula, CA, USA). Preparations were examined with a Leica TCS-NT confocal microscope equipped with a krypton-argon laser, to quantify the presence of FluoroJade-B signal 3 fields for each slide were acquired by standardizing magnification, pinhole size, gain and offset of the confocal microscope; thickness of extended-focus images. Optical section of 10μm of thickness and a 488 nm laser were used. Pictures will be collected as 1024x1024 TIFF files. Signal intensity was quantified by Metamorph ® software as raw signal minus background.

3) Cloning the genes MTP and p66shc

To verify the presence and the expression of the genes MTP and p66shc in Nothobranchius furzeri (GRZ), degenerated primers were constructed, complementary to the regions of maximum conservation based on the sequences of Zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and fugu (Takifugu rubipes) that are available (Genomic BLAST, http://www.ncbi.nlm.nih.gov/sutils/genom tree.cgi) for each of these genes. The oligonucleotide sequences used are the following: oligo p66shc_3' : AAV GCC TGN CCD ATK GTR CTG AT (23-mer with 5 degenerated positions) SEQ ID No 1 oligo p66shc_5' : AAR CCY WCV MGD GGM TGG YTG CA (23-mer with 8 degenerated positions) SEQ ID No 2 oligo MTP 5': AHG ARA TGA AYA ART ACA TGC T (22-mer with 4 degenerated positions) SEQ ID No 3 oligo MTP 3': AHG ARA TGA AYA ART ACA TGC T (22-mer with 5 degenerated positions) SEQ ID No 4 conditions PCR p66shc:

5 min 94 ⁰C 5 cycles 94 ⁰C 45 s, 48 ⁰C 30 s, 72 ⁰C 30 s

25 cycles 94 ⁰C 45 s, 51 ⁰C 30 s, 72 ⁰C 30 s

15 min 72°C conditions PCR MTP:

5 min 94 ⁰C 5 cycles 94 ⁰C 45 s, 40 ⁰C 30 s, 72 ⁰C 30 s

25 cycles 94 ⁰C 45 s, 43 ⁰C 30 s, 72 ⁰C 30 s

15 min 72 ⁰C

4) Effect of water temperature

In poikilothermic animals, lowering ambient temperature prolongs lifespan (Partridge and Brand, 2005). This effect is well-documented in invertebrate aging models, and was also demonstrated in the American annual fish Austrolebias bellotii (Liu and Walford, 1975). A demonstration that lowering ambient temperature prolongs lifespan in N. furzeri would then demonstrate that short lifespan in this species is related to physiological aging. A group of 60 N. furzeri was hatched and raised at the standard temperature of

25°C. At age 4 weeks, when the fishes are sexually mature, they were divided into two groups. One group of 30 fishes remained at 25°C, while a second group of 30 fishes was moved to 22⁰C. Open-field exploration was also measured in N. furzeri raised at 22°C at 5 weeks and 9 weeks of age. In addition, age dependent decrease in cognitive function was measured in N. furzeri raised at 22°C using the active avoidance protocol described earlier.

5) Pharmacological treatment

Nothobranchius furzeri (GRZ) were bred as described in Valdesalici e Cellerino (2003). To test the effects of resveratrol on longevity in N. furzeri, the authors studied a total of 45 vehicle-fed control fish (2 replicates) and 110 experimental fish (5 replicates, 3 doses). Fish were hatched and raised according to Valdesalici & Cellerino (2003). At age 4 weeks, they were divided into two groups. The control group of fish were fed with bloodworms included in 5% gelatin mixed with 5% EtoH (the vehicle of resveratrol). The three control groups were fed with bloodworms included in 5% gelatin with the following concentrations of resveratrol: 24 μg/gr food (0.1 mM, n=30), 120 μg/gr food (0.5mM, n=60); 600 μg/gr food (2.5mM, n=20) The effect of treatment with resveratrol was determined on survival, spontaneous locomotor activity and cognitive decay as described in the previous paragraphs.

RESULTS 1) Biomarkers of ageing a) Lipofuscin accumulation

Figure 1A shows that in 9 week old Nothobranchius furzeri, but not in 4 week old Nothobranchius furzeri, autofluorescentautofluorescent granules of lipofuscin can be detected in the neurons. Similar results were obtained in liver sections within the hepatocytes shown in Fig. 1 B. b) Expression of β-qalactosidase associated to senescence

Histological analysis revealed the presence of senescence-associated β- galactosidase in cells both of the derma and of the eye in 9 week old animals but not in 4 week old animals (Fig. 2 and 3). The induction of senescence-associated yff-galactosidase is particularly evident in the connective tissue surrounding the eye (Fig. 3). c) Spontaneous locomotor activity Analysis of spontaneous locomotor activity demonstrates a progressive reduction in the percentage of time in motion, in average swimming speed and of maximum swimming speed (Fig. 4 A₁B₁C). In all three cases, statistical analysis (ANOVA di Kruskall-Wallis, Statistica ®, Statsoft) detected a significant effect of age. "Pairwise comparisons" noted a significantly lower time in motion in 9 week old fish than both in 6 week old and in 8 week old fish (P < 0.01 ). The average speed of 9 week old fish was significantly reduced relative to that of 6 week old fish (P < 0.01 ). Lastly, the maximum speed of 9 week old N. furzeri is significantly less than 7 week old fish (P < 0.01 ). d) Cognitive decay

The learning curves of 10 Nothobranchius furzeri (GRZ) at 5 weeks of age and 10 Nothobranchius furzeri (GRZ) at 9 weeks of age are shown in Fig. 5B. Old Nothobranchius furzeri learn the task more slowly and the difference between the two learning curves is statistically significant (Kolmogoroff-Smimofft est , p<0,0001 , Statistica ®, Statsoft). Moreover 5 week old fish reach a higher average success index after 50 tests (Mann-Withney U-test, p<0.01 ; Statistica ®, Statsoft)

2) Expression of the genes MTP and p66shc

In all cases, a highly homologous sequence to the human, zebrafish, fugu and pufferfish genes was univocally identified. The sequences of the amplified fragments are shown in Fig. 6 and 7. This result demonstrates that the genes that control the life expectancy of mammals and humans are also present in

Nothobranchius furzeri.

In particular, the comparison between the aminoacidic sequences of p66shc and

MTP of Nothobranchius furzeri and humans shows the following percentages of homology, considering respectively mutually identical and similar aminoacids: p66shc: 82% identity; 92% similarity

MTP: 62% identity; 81% similarity

3) Effect of a water temperature a) Effect on survival In poikilothermic animals, lowering ambient temperature prolongs lifespan (Partridge and Brand, 2005). This effect is well-documented in invertebrate aging models, and was also demonstrated in the American annual fish Austrolebias bellotii (Liu and Walford, 1975). A demonstration that lowering ambient temperature prolongs lifespan in N. furzeri would then demonstrate that short lifespan in this species is related to physiological aging.

A group of 60 N. furzeri was hatched and raised at the standard temperature of 25°C. At age 4 weeks, when the fishes are sexually mature, they were divided into two groups. One group of 30 fishes remained at 25°C, while a second group of 30 fishes was moved to 22⁰C.

The age-dependent survival of the two groups is reported in Fig. 8. Lowering ambient temperature increased longevity (Log Rank, p<0.01 ) by extending median lifespan from 9 to 12 weeks and maximum lifespan from 11 to 14 weeks. b) Effect on locomotor activity

Open-field exploration was also measured in N. furzeri raised at 22°C at 5 weeks and 9 weeks of age. The analysis revealed the increased longevity was tied with a significant preservation of motor function Fig. 9 A and B. c) Effect on cognitive functions

Age dependent decrease in cognitive function was measured in N. furzeri raised at 22⁰C using the active avoidance protocol described earlier. 9 weeks old N. furzeri raised at 22⁰C have the same performance of control N. furzeri at 5 weeks Fig.10.

4) Effect of pharmacological treatment a) Effect of resveratrol on survival

The lowest dose (24 μg/g, 0.1 mM) did not cause life extension, the medium dose caused a significant life-extension effect by increasing both the median and maximum lifespan (FIG. 11 A, Log-Rank, p<0.001 ). The highest dose (600 μg/g, 2.5 mM) was more effective than the medium dose (Log-Rank, p=0.012). The effects were identical in both genders (FIG. 11 B). These data demonstrate that addition of resvertarol to the food during fish adult life causes life-extension in a dose-dependent manner. b) Effect of resveratrol on spontaneous locomotor activity

The results are shown in Fig. 12. Performance in terms of percentage of time spent in motion, average speed and maximum speed of the 9 week old animals treated with resveratrol were much higher than those of the 9 week old control animals and comparable to those of the control animals at 5 weeks (ANOVA di Kruskall-Wallis, p<0.01 , Statistica ®, Statsoft). Treated animals were analysed again a 13 weeks. Their performance in terms of percentage of average speed, maximum speed and time spent in motion are comparable to those of the untreated control animals at 9 weeks. c) Effect of resveratrol on cognitive decay

The average performance index in the last 10 trials of the animals treated with resveratrol was compared to that of untreated control animals at 9 weeks and at 5 weeks of age. The results are shown in Fig. 13. The difference in the average performance index between control animals at 9 weeks and animals treated with resveratrol at 9 weeks is statistically significant (ANOVA of Kruskall-Wallis, p<0.01 , Statistica ®, Statsoft). The animals treated with resveratrol at 9 weeks have a similar performance to control animals at 5 weeks. d) Effect of resveratrol on neurofibrillary degeneration The optic tecum of 5 weeks old N. furzeri did not show any sign of neurofibrillary degeneration Fig. 14A. The optic tectum of control-fed 9 weeks old N. furzeri did show signs of neurofibrillary degeneration Fig. 14B and this effect is completely prevented by addition of 120 μg/gr (0.5 mM) of resevratrol to the food starting from the 4^th week of life Fig. 14C. Prevention of neurofibrillary degenration was confirmed by fluorescence analysis at the confocal microscope Fig. 14D.

CONCLUSIONS

1 ) Nothobranchius furzeri exhibits a series of biomarkers of ageing which are homologous to well described phenomena in humans. 2) The effects of temperature reduction and resveratrol administration in

Nothobranchius furzeri are identical to those of the model system C. elegans and

D. melanogaster, therefore Nothobranchius furzeri is an attractive vertebrate model to study the effects of natural or synthetic substances on ageing.

3) In this model, resveratrol slows the onset of neuro-muscular and cognitive pathologies caused by ageing and therefore resveratrol can be used to prevent the onset of sarcopenia and cognitive deficits during human ageing.

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Claims

1. A method for identifying and/or characterizing a substance able to modulate the longevity and/or ageing of a vertebrate, comprising the steps of: a) exposing individuals belonging to the family of Cyprinodontids (common name: killifish) to said substance in effective manners and quantities; b) measuring in said individuals the longevity and/or a marker of ageing and comparing said measurements with negative control values; c) selecting the substance capable of positively modulating longevity and/or at least one marker of ageing.

2. The method according to claim 1 , wherein exposure to said substance is obtained by ingestion of semi-solid food that contains it and is able to release it.

3. The method according to claim 1 , wherein the markers of ageing belong to the following group: accumulation of lipofuscin in the brain and/or the liver, expression of B-galactosidase associated to senescence, reduction in spontaneous locomotor activity, cognitive decay and neurofibrillary degeneration.

4. The method according to claims 1 through 3 wherein the individuals belong to the species Nothobranchius furzeri.

5. The method according to claim 4, wherein said substance is resveratrol.

6. Use of resveratrol for the preparation of a pharmaceutical composition for the prevention and/or therapy of pathologies connected with senescence, loss of spontaneous locomotor activity, cognitive decay and/or neurofibrillary degeneration.

7. Use of resveratrol for the preparation of a pharmaceutical composition for extending the longevity of a subject.