Simona De Blasi

Alberto Montesanto

Cinzia Martino

Serena Dato

Francesco De Rango

Amalia C Bruni

Vincenzo Mari

Emidio Feraco

Giuseppe Passarino
email: g.passarino@unical.it

Prof Giuseppe Passarino

APOE polymorphism affects episodic memory among non demented elderly subjects

Keywords: APOE, cognitive abilities, episodic memory, MMSE, cognitive decline, aging

The APOE ε4 allele has been associated with a number of neurodegenerative disorders e.g. Alzheimer's disease. Inconsistent results have been obtained for cognitive decline in 'normal' aging. We investigated whether specific aspects of cognitive decline were associated with APOE ε4 among 620 'healthy' elderly subjects living in Calabria, southern Italy. MMSE scores ranged from 11 to 30. A lower MMSE score was unrelated to APOE polymorphism, i.e. a global measure of cognition. However, poorer episodic memory was associated with APOE ε4, both registration (p=0.01) and recall (p=0.01). Temporal and spatial orientation, attention and calculation, language, and constructive function were not affected. We conclude that episodic memory, specifically, is adversely affected by APOE ε4 and urge evaluation of precise phenotypes in genetic association studies of cognitive decline in order to avoid inconsistent results due to phenotypic heterogeneity.

Introduction

The polymorphism of Apolipoprotein E leading to E2, E3, E4 isoforms [START_REF] Utermann | Polymorphism of apolipoprotein E. II. Genetics of hyperlipoproteinemia type III[END_REF][START_REF] Weisgraber | Human E apoprotein heterogeneity. Cysteine-arginine interchanges in the amino acid sequence of the apo-E isoforms[END_REF][START_REF] Rall | Human apolipoprotein E. The complete amino acid sequence[END_REF] has attracted the interest of many scientists due to its involvement in Alzheimer's disease (AD) and in a number of complex diseases [START_REF] Saunders | Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer's disease[END_REF][START_REF] Corder | Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families[END_REF][START_REF] Srinivasan | The apolipoprotein E epsilon4 allele selectively increases the risk of frontotemporal lobar degeneration in males[END_REF][START_REF] Bernardi | The effects of APOE and tau gene variability on risk of frontotemporal dementia[END_REF]). In addition, many studies have focused on the possible correlation between APOE polymorphism and cognitive performances in non demented subjects.

Among the most important results it is worth mentioning that [START_REF] Deary | Cognitive change and the APOE epsilon 4 allele[END_REF] found that APOE variability is not correlated with differences in mental ability in youth, but the allele ε4 is significantly associated with decreased mental ability at old age. In agreement, [START_REF] Wilson | The apolipoprotein E epsilon 4 allele and decline in different cognitive systems during a 6-year period[END_REF] found that the ε4 allele is associated with a more rapid decline in cognitive functions in the elderly, particularly episodic memory which allows the recollection of events and episodes of the recent past. A prominent effect of APOE variability on episodic memory has recently been confirmed by a study on a Swedish population (the Betula Study, [START_REF] Nilsson | The influence of APOE status on episodic and semantic memory: data from a populationbased study[END_REF]. However, the studies on the correlation between APOE and cognitive performances in the elderly have also shown some contrasting results (for a review see [START_REF] Savitz | Apolipoprotein E variants and cognition in healthy individuals: a critical opinion[END_REF]. Many reasons may explain the incoherence of the data so far obtained on the effects of APOE polymorphism in non demented old subjects. Among the others is the fact that the studies have been carried out in different populations, with different genetic backgrounds and different environments. In addition, in some cases different cognitive functions have not been tested separately but within inclusive tests. Indeed, recent analyses have highlighted the importance of disentangling the different items included in inclusive tests, as the crude computation of overall scores may be not fully representative of important phenotypic differences [START_REF] Antonelli-Incalzi | Construct validity of activities of daily living scale: a clue to distinguish the disabling effects of COPD and congestive heart failure[END_REF], 2008) In the last few years coordinated international surveys are showing that the quality of aging is quite different across Europe. Indeed, specific markers of the quality of aging showed that the age related physical decline is faster in southern than in northern Europeans [START_REF] Jeune | Handgrip strength among nonagenarians and centenarians in three European regions[END_REF]. This phenomenon may be due to different historic and demographic reasons (differences in childhood growth, lifestyle and public health care for old people). However biological reasons cannot be excluded. This clearly poses interesting questions to the researchers who are dealing with the effect of polymorphisms on age related phenotypes. In fact, genetic variants and environmental factors may interact in a population specific way in modulating some specific agerelated phenotypes [START_REF] De Benedictis | The unusual genetics of human longevity[END_REF].

In order to further explore the effect of APOE variability on the age related cognitive decline we analysed APOE genotypes and cognitive performances in non demented subjects aged 65-85 years, that is in the age range where there is a dramatic increase of cognitive impairment [START_REF] De Ronchi | Occurrence of cognitive impairment and dementia after the age of 60: a population-based study from Northern Italy[END_REF]). The study was carried out on a population sample from Calabria, a southern Italian region where previous surveys have highlighted that the aging decline, including the cognitive decline, is faster than in other European regions [START_REF] Jeune | Handgrip strength among nonagenarians and centenarians in three European regions[END_REF].

Materials and Methods

Samples

The sample we analyzed included 65-85 years old healthy subjects (N=620; 287 males and construction functions [START_REF] Folstein | Mini-mental state". A practical method for grading the cognitive state of patients for the clinician[END_REF]. Since the test is affected by age and educational status, the MMSE scores were normalized for these variables according to a standardised procedure [START_REF] Grigoletto | Norms for the Mini-Mental State Examination in a healthy population[END_REF]. A score of 23 was considered as a cut- words to the subject. Then, the subject was invited to repeat all the words he/she cought (Recognition and Registration test). After a few minutes where the subject was distracted with other questions, the interviewer asked the subject to repeat all the words she/he could remember of the 12-word list (Delayed Recall test). As the scores varied according to age and education (p<0.05 in both cases) we adjusted the scores with respect to these variables by using the standardized residuals of a linear regression model where the scores were used as dependent variable and age and education as independent variables. It is worth mentioning that these two tests have been introduced

in the questionnaire not from the beginning but after some time, in order to replicate preliminary observations on episodic memory. For this reason data for these tests were available for 271 subjects out of 620.

DNA Analysis

DNA was prepared from blood buffy-coats according to standard procedures and stored at -20°C until use. APOE genotyping (alleles 2, 3, 4) was carried out according to the protocol described in [START_REF] Carrieri | Mitochondrial DNA haplogroups and APOE4 allele are non-independent variables in sporadic Alzheimer's disease[END_REF].

Statistical analysis

Testing for independence in 2 by 2 table (Table 2) has been carried out by using Fisher's exact test. For larger than 2 by 2 tables (Tables 3 and4), because of the sparseness of the observed data, testing for independence has been performed by a

Monte Carlo approximation to Fisher's exact test [START_REF] Agresti | Exact conditional tests for cross-classifications[END_REF]. Statistical analyses were performed by using SPSS 15.0 (SPSS Inc., Chicago, IL). A significance level of = 0.05 was chosen in all the tests.

Results

In order to explore the influence of the APOE polymorphism on the preservation of cognitive functioning, we analyzed the association between APOE genotypes and MMSE scores.

The frequency of APOE genotypes and alleles are reported in Table 1. The frequencies observed in the present study are in agreement with those previously reported for the Calabrian population [START_REF] Carrieri | Mitochondrial DNA haplogroups and APOE4 allele are non-independent variables in sporadic Alzheimer's disease[END_REF].

MMSE scores, adjusted for age and education, > 23 or <= 23, was essentially the same for APOE ε4 carriers and APOE ε4 non carriers (Table 2; p=0.619).

We then determined whether any of the component cognitive domains were associated with APOE ε4 (Table 3). Both tests of episodic memory, registration (p=0.01) and recall (p=0.01) indicated poorer function among APOE ε4 carriers. None of the other cognitive domains (orientation, attention, language, and construction functions) was associated with APOE status. We did not correct for multiple comparisons because of previous studies indicating worse episodic memory for APOE ε4 subjects and because we then went on to replicate the finding.

In fact, in order to have an independent evidence of our findings we analyzed data for two tests specifically testing episodic memory: Recognition-and-registration test and Delayed recall test. In order to verify if allele ε4 affects episodic memory, the sample was partitioned into four groups according to the quartiles of the scores obtained for each test (Quartile 1 represented the group with the lowest score). This allowed us to replicate the finding of poorer episodic memory among APOE ε4 subjects for both registration (p=0.05) and recall (p=0.0046) (Table 4).

Discussion

APOE polymorphisms are certainly among the most studied human genetic variations, due to their role in individual risk for age related diseases, primarily for AD. A significant share of literature has also been dealing with the effects of APOE polymorphisms on the age related cognitive decline. Aim of the present study was to explore the relationship between APOE variation and age related cognitive decline in a southern European population characterized by a poor quality of aging [START_REF] Jeune | Handgrip strength among nonagenarians and centenarians in three European regions[END_REF].

We found that APOE variation is not correlated to the overall cognitive performance as evaluated by the MMSE test (Table 2). However, we found that the performances at different tests which specifically evaluate episodic memory were able to show a significant higher frequency of ε4 carriers among the subjects with the lowest performances (Tables 3 and4). These observations are in line with the results obtained by [START_REF] Nilsson | The influence of APOE status on episodic and semantic memory: data from a populationbased study[END_REF] in a population from Sweden and highlight the specific effect of APOE ε4 on episodic memory. This is particularly interesting if we consider the different environmental, social and biological backgrounds of the two populations examined. On the other hand, if we consider the importance of memory in the cognitive decline and, consequently, in the quality of aging, it is not surprising that ε4 may, as a side effect, lead to a proneness to frailty, as reported in other papers [START_REF] Passarino | A cluster analysis to define human aging phenotypes[END_REF][START_REF] Packard | Prospective Study of Pravastatin in the Elderly at Risk Group. Association between apolipoprotein E4 and cognitive decline in elderly adults[END_REF]).

The observations reported, might be possibly connected to the age-related reduction of hyppocampal volume which has been found to be more pronounced in ε4 carriers [START_REF] Cohen | Effect of apolipoprotein E genotype on hippocampal volume loss in aging healthy women[END_REF][START_REF] Lind | Reduced functional brain activity response in cognitively intact apolipoprotein E epsilon4 carriers[END_REF]). Indeed, a reduction of hyppocampus is associated to cognitive failures without objective cognitive impairment and to Mild Cognitive Impairment [START_REF] Van Norden | Subjective cognitive failures and hippocampal volume in elderly with white matter lesions[END_REF][START_REF] Martin | Evidence that volume of anterior medial temporal lobe is reduced in seniors destined for mild cognitive impairment[END_REF]. On turn, this phenomenon may be possibly derived from the decreased antioxidant activity of E4 (Myiata and Smith 1996) or from the altered use of the nutrients observed in ε4 carriers [START_REF] Reiman | Functional brain abnormalities in young adults at genetic risk for late-onset Alzheimer's dementia[END_REF]). In any case, our data have confirmed that APOE ε4 allele has specific deleterious effects on episodic memory and that, unlike other polymorphisms, its effects on the quality of aging is mostly independent from population specificity. In addition our findings urge for a clear definition of the studied phenotype to untangle the genetic component of complex traits.

  and 333 females; median ages 72 years in both cases). The sample has been recruited in the frame of different recruitment campaigns focused on the monitoring of the quality of aging in Calabria, which have been carried out by our research group between 2002 and 2007. All the subjects were considered healthy after a thorough clinical assessment with detailed clinical history, including MMSE, ADL, IADL, Physical tests, GDS and a set of the most common clinical haematological tests. Subjects with dementia and/or neurologic disorders were not included. The blood for DNA extraction was obtained at the same time of the medical visit, when also blood for clinical exams was extracted.All the subjects were born in Calabria and their ancestry in the region had been ascertained up to the grandparents generation. The vital status of subjects recruited is ascertained by our research group every 18 months. The questionnaire used for /biologia.unical.it/echa/documents/minute/First_Steering/QUEST_G2.pdf . Before the interview each subject consented to her/his phenotypic and genetic data to be used anonymously for genetic studies on aging (informed consent).Cognitive functionsCognitive functions were assessed by Mini Mental State Examination (MMSE) test a 30-item questionnaire that assesses orientation, episodic memory, attention, language,

  off for recognizing impaired or conserved cognitive functioning[START_REF] Rait | Prevalence of cognitive impairment: results from the MRC trial of assessment and management of older people in the community[END_REF][START_REF] Anderson | Effects of sociodemographic and health variables on mini-mental state exam scores in older Australians[END_REF]. Episodic memory was evaluated by two tests of immediate and delayed recall of a 12word list (McGue and Christensen 2001; Nilsson et al. 2006). The interviewer read 12

Table 1 .

 1 Genotypic and allelic frequencies of APOE polymorphism in the sample analyzed (N= 620).

	Genotype	Absolute frequency (Relative Frequency)
	2-2	3 (0.5)
	2-3	82 (13.2)
	2-4	10 (1.6)
	3-3	443 (71.5)
	3-4	81 (13.1)
	4-4	1 (0.2 )
	Allele	Absolute frequency (Relative Frequency)
	2	98 (7.9)
	3	1049 (84.6)
	4	93 (7.5)
	HWE 2 = 3.01 gdl = 3 p=0.390

Table 2 .

 2 Distribution of APOE 4 carriers and non carriers across the two groups defined on the basis of the MMSE scores.

		4 carriers	4 non carriers
	MMSE <23	26 (31.7%)	188 (34.9%)
	MMSE>23	56 (68.3%)	350 (65.1%)
	p-value		0.619
	p-value refers to a Fisher's exact test.

Table 3 .

 3 Distribution of APOE ε4 cariers and APOE ε4 non carriers across the groups defined by the scores obtained for each item included in the MMSE test. refer to 2-sided Montecarlo approximation to Fisher's exact test.

	MMSE items	Scores	4 carriers	4 non carriers
	Temporal Orientation	0 correct answers	0 (0.0%)	3 (0.6%)
		1 correct answers	0 (0.0%)	5 (0.9%)
		2 correct answers	6 (7.3%)	13 (2.4%)
		3 correct answers	0 (0.0%)	37 (6.9%)
		4 correct answers	14 (17.1%)	110 (20.4%)
		5 correct answers	62 (75.6%)	370 (68.8%)
	p-value			0.376
	Spatial Orientation	0 correct answers	0 (0.0%)	0 (0.0%)
		1 correct answers	1 (1.2%)	1 (0.2%)
		2 correct answers	3 (3.7%)	14 (2.6%)
		3 correct answers	9 (11.0%)	36 (6.7%)
		4 correct answers	18 (22.0%)	135 (25.1%)
		5 correct answers	51 (62.2%)	352 (65.4%)
	p-value			0.224
	Registration	0 correct answers	0 (0.0%)	1 (0.2%)
		1 correct answers	2 (2.4%)	2 (0.4%)
		2 correct answers	4 (4.9%)	7 (1.3%)
		3 correct answers	76 (92.7%)	528 (98.1%)
	p-value			0.009
	Attention and Calculation 0 correct answers	12 (14.6%)	87 (16.2%)
		1 correct answers	7 (8.5%)	74 (13.8%)
		2 correct answers	8 (9.8%)	55 (10.2%)
		3 correct answers	11 (13.4%)	66 (12.3%)
		4 correct answers	11 (13.4%)	60 (11.2%)
		5 correct answers	33 (40.2%)	196 (36.4%)
	p-value			0.295
	Recall	0 correct answers	18 (22.0%)	63 (11.7%)
		1 correct answers	9 (11.0%)	47 (8.7%)
		2 correct answers	23 (28.0%)	189 (35.1%)
		3 correct answers	32 (39.0%)	239 (44.4%)
	p-value			0.006
	Language	2 correct answers	0 (0.0%)	1 (0.2%)
		3 correct answers	0 (0.0%)	4 (0.7%)
		4 correct answers	3 (3.7%)	19 (3.5%)
		5 correct answers	16 (19.5%)	130 (24.2%)
		6 correct answers	63 (76.8%)	384 (71.4%)
	p-value			0.311
	Constructive function	0 correct answers	17 (20.7%)	102 (19.0%)
		1 correct answers	14 (17.1%)	84 (15.6%)
		2 correct answers	22 (26.8%)	144 (26.8%)
		3 correct answers	29 (35.4%)	208 (38.7%)
	p-value			0.526
	p-values

Table 4 .

 4 Distribution of APOE 4 carriers and non carriers across the four quartiles defined on the basis of the scores obtained to the Recognition and Registration test and to the Delayed Recall test.

	Memory test	Quartiles of scores	4 carriers	4 non carriers
	Recognition and Registration test	1 st Quartile	10 (33.3%)	58 (24.1%)
		2 nd Quartile	9 (30.0%)	59 (24.5%)
		3 rd Quartile	7 (23.3%)	61 (25.3%)
		4 th Quartile	4 (13.3%)	63 (26.1%)
	p-value			0.0501
	Delayed Recall test	1 st Quartile	14 (46.7%)	54 (22.4%)
		2 nd Quartile	6 (20.0%)	62 (25.7%)
		3 rd Quartile	5 (16.7%)	63 (26.1%)
		4 th Quartile	5 (16.7%)	62 (25.7%)
	p-value			0.0046

p-values refer to 2-sided Montecarlo approximation to Fisher's exact test. .

Acknowledgements

The work was supported by Fondi di Ateneo of the University of Calabria to GP.


Stijn Mouton
email: stijn.mouton@ugent.be

Maxime Willems

Bart P Braeckman

Bernhard Egger

Peter Ladurner

Lukas Schärer

Gaetan Borgonie

Keywords: Flatworm, Macrostomum lignano, Ageing, Stem cells, Rejuvenation

To study the several elements and causes of ageing, diverse model organisms and methodologies are required. The most frequently used models are Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster and rodents. All have their advantages and disadvantages and allow studying particular aspects of the ageing process. During the last few years, several ageing studies focussed on stem cells and their role in tissue homeostasis. Here we present a new model organism which can study this relation where other model systems fail. The flatworm Macrostomum lignano possess a dynamic population of likely totipotent somatic stem cells known as neoblasts. Several characteristics qualify M. lignano as a suitable model system for ageing studies in general and more specifically for gaining more insight in the causal relation between stem cells, ageing and rejuvenation.

Introduction

Ageing is a complex process that affects a wide variety of functions [START_REF] Arking | Biology of aging[END_REF]. This complexity is shown by the countless ageing theories that have been published, most of which are monistic in nature and focuse on one particular element (Semsei, 2000). Studying the different elements and causes of ageing requires diverse methodologies and model organisms, such as Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster and rodents. All of these models have their characteristic strengths and weaknesses (Figure 1).

During the last few years, several ageing studies have focussed on stem cells and their role in tissue homeostasis [START_REF] Rando | Stem cells, ageing and the question for immortality[END_REF][START_REF] Rossi | Stem cells and the pathways to aging and cancer[END_REF]Sharpless and DePinho, 2007).

These studies have shown that ageing is invariably accompanied by a diminished capacity to adequately maintain tissue homeostasis or to repair tissues after injury. Furthermore, the ageing of tissue-specific stem cells and progenitor cell compartments is believed to play a pivotal role in the decline of tissue and organ integrity and function in the elderly [START_REF] Rossi | Stem cells and the pathways to aging and cancer[END_REF]. However, studying the reciprocal influence between stem cells and the ageing process, especially in vivo, is difficult in the current model organisms. This is partly due to the following problems: the adult soma of C. elegans and D. melanogaster are either completely or largely post-mitotic, and the complexity and relative inaccessibility of the vertebrate stem cell population restricts in vivo analysis of stem cell functionality [START_REF] Sánchez Alvarado | Proposal for sequencing of a new target genome: white paper for a planarian genome project[END_REF]Sharpless and DePinho, 2007). These limitations are not present in flatworms, which possess a dynamic population of likely totipotent somatic stem cells known as neoblasts [START_REF] Dubois | Sur une nouvelle méthode permettant de mettre en évidence la migration des cellules de régénération chez les planaires[END_REF]Stephan-Dubois and Gilgenkrantz, 1960;[START_REF] Lange | Studies on the cellular basis of radiation lethality. III. The measurement of stem cell repopulation probability[END_REF]Lange, 1968a;[START_REF] Baguñà | Planarian neoblasts[END_REF]. In the past, several researchers already used flatworms to gain more insight into the causal relation between neoblasts, ageing and rejuvenation (see references in [START_REF] Haranghy | Ageing and rejuvenation in planarians[END_REF][START_REF] Haranghy | Ageing and rejuvenation in planarians[END_REF]Lange 1968a). While most of these studies used triclad species, we introduce Macrostomum lignano (Macrostomorpha) as a new model. We focus on this species because it has several advantages in comparison to triclads, making it possible to expand and improve the flatworm ageing research. These advantages are discussed below in the appropriate sections.

In the past, M. lignano has already been put forward as a model organism for stem cell biology, development, regeneration, and the study of sexual selection (Ladurner et al., 2005a;[START_REF] Ladurner | The stem cell system of the basal flatworm Macrostomum lignano[END_REF]. In this mini-review, we will briefly describe the species and its life history, and discuss the role of its stem cells in ageing and rejuvenation. We will also give an overview of the available experimental tools that allow a multidisciplinary approach for studying ageing in M. lignano.

Macrostomum lignano: description

M. lignano (Macrostomorpha, Rhabditophora) is a marine, free-living flatworm that can be found in the high-tide interstitial sand fauna on beaches of, for example, the Northern Adriatic Sea [START_REF] Ladurner | Spatial distribution and differentiation potential of stem cells in hatchlings and adults in the marine platyhelminth Macrostomum sp.: A Bromodeoxyuridine analysis[END_REF]Ladurner et al., 2005a). In the laboratory, it can be easily cultured and allows stocking in high densities due to its small size. M. lignano is cultured individually, in pairs or in groups in Petri dishes or multiwell plates in f/2, a nutrient-enriched artificial seawater medium at a salinity of 32‰ [START_REF] Guillard | Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran[END_REF]. The dishes are incubated at 20°C, with a 60% relative humidity and a 14h:10h or 13:11h light:dark cycle. Animals are fed with the diatom Nitzschia curvilineata that can be obtained from culture collections (Ladurner et al., 2008). The diatoms are grown under the same conditions as the worms and, if necessary for an experiment with controlled feeding, they can be easily quantified (Vizoso and Schärer, 2007).

M. lignano is a small animal of about 1-1.5 mm length consisting of roughly 25 000 cells [START_REF] Ladurner | Spatial distribution and differentiation potential of stem cells in hatchlings and adults in the marine platyhelminth Macrostomum sp.: A Bromodeoxyuridine analysis[END_REF]. Although individuals may appear brownish because of the gut content, they are highly transparent in squeeze preparations, allowing easy observation of all major tissues and organ systems (Figure 2A) [START_REF] Ladurner | The stem cell system of the basal flatworm Macrostomum lignano[END_REF]. The several organ systems are described in detail in Ladurner et al. (2005a), and here we focus only on the stem cell system.

The neoblasts are located in two bands along the lateral sides of the animal, merging in the tail plate (Figure 2B). Somatic neoblasts are also present along the post-pharyngeal commissure and a few, most likely gastrodermal neoblasts, can be found scattered along the midline of the body. Neoblasts are entirely absent in the rostrum which is the region anterior to the eyes. The neoblasts are located within the parenchyma and are the only dividing cells in the adult (see references in [START_REF] Ladurner | The stem cell system of the basal flatworm Macrostomum lignano[END_REF]. They can differentiate into all cell types, including germ cells, and maintain their own population [START_REF] Ladurner | Spatial distribution and differentiation potential of stem cells in hatchlings and adults in the marine platyhelminth Macrostomum sp.: A Bromodeoxyuridine analysis[END_REF]. Therefore, neoblasts play a key role during reproduction, development, tissue turnover and regeneration.

In an adult animal there are about 1 600 neoblasts, which is 6.5 % of the total cell number [START_REF] Bode | Immunogold labeled S-phase neoblasts, total neoblast number, their distribution and evidence for arrested neoblasts in Macrostomum lignano (Platyhelminthes, Rhabditophora)[END_REF]. The average number of somatic S-phase neoblasts is 435 ± 79 [START_REF] Ladurner | Spatial distribution and differentiation potential of stem cells in hatchlings and adults in the marine platyhelminth Macrostomum sp.: A Bromodeoxyuridine analysis[END_REF], which is 27% of the total somatic neoblast population [START_REF] Bode | Immunogold labeled S-phase neoblasts, total neoblast number, their distribution and evidence for arrested neoblasts in Macrostomum lignano (Platyhelminthes, Rhabditophora)[END_REF], and only a small fraction of about 20 -40 neoblasts are in mitosis [START_REF] Ladurner | The stem cell system of the basal flatworm Macrostomum lignano[END_REF]. The several techniques to study the neoblasts are discussed in the section "experimental toolbox".

During the last years, M. lignano has clearly entered the era of functional genomics. More than 15 000 expressed sequence tags (ESTs) have been generated [START_REF] Morris | The Macrostomum lignano EST database as a molecular resource for studying platyhelminth development and phylogeny[END_REF]Ladurner unpublished;Pfister pers. com.), in which a number of candidate genes known to be involved in ageing processes in other organisms were found (Table 1). This opens possibilities to study these ageing genes in M. lignano. There are ongoing efforts to increase the number of ESTs, to perform large scale whole mount in situ hybridization studies [START_REF] Pfister | The exceptional stem cell system of Macrostomum lignano: Screening for gene expression and studying cell proliferation by hydroxyurea treatment and irradiation[END_REF] and systematic mutagenesis screens.

Life history

of about 18 days (see references in [START_REF] Ladurner | The stem cell system of the basal flatworm Macrostomum lignano[END_REF]. Mature worms generally lay one egg per day, allowing fine-graded culturing which is essential for survival studies and experiments.

The fecundity and fertility pattern during adult lifespan are important parameters for ageing studies (Terzibasi et al., 2007). Preliminary data in M. lignano suggest that fertility declines with advancing age, although a post-reproductive period has not yet been observed: 40-weekold individuals are still able to produce eggs, but the number of offspring is lower in comparison to young adults (Mouton, unpublished observations).

M. lignano has a median lifespan of 205 ± 13 days (29.6 weeks) and a 90 th percentile (90% mortality) of 362 ± 41 days (51.7 weeks), determined from three replicate cultures each of 100 individuals. A detailed survival analysis, in which the lifespan, survival curve and mortality rate are discussed, is published in this issue (Mouton et al., submitted). M. lignano is the first flatworm species in which this set of demographic data is determined. In the existing flatworm ageing literature, only the maximum lifespan of the studied species, often triclads with a maximum lifespan of several years, is reported [START_REF] Haranghy | Ageing and rejuvenation in planarians[END_REF]Lange 1968a;Mouton et al., submitted). The availability of a basic set of demographic data is, however, a prerequisite for ageing research and this is now obtained for M. lignano.

Furthermore, the culture conditions of M. lignano are very standardised, making it possible to distinguish between physiological and pathological ageing, which was in the past often difficult in the used triclad species [START_REF] Haranghy | Ageing and rejuvenation in planarians[END_REF]Mouton et al., submitted).

Chronological and replicative ageing

It is known that post-mitotic cells experience chronological ageing, whereas proliferative cells experience both chronological and replicative ageing [START_REF] Rando | Stem cells, ageing and the question for immortality[END_REF]. Like all flatworms, M. lignano consists of both differentiated post-mitotic cells and totipotent proliferating neoblasts.

Replicative ageing and the limit of the replicative potential of neoblasts, however, have not yet been studied in M. lignano and other flatworms.

During tissue homeostasis, the neoblasts produce daughter cells that differentiate and replace aged and injured post-mitotic cells. Because different tissues have a different cellular turnover, the extent of cell renewal of the different tissues will vary. However, the turnover rates of the different tissues affect the proliferation rate of the neoblasts only as a total demand for new cells from the neoblast pool because the neoblasts pool is likely totipotent.

Already in 1930, Abeloos hypothesized that the main cause of ageing in flatworms is the insufficiency of tissue renewal to assure the permanent equilibrium between young and old cells in the somatic post-mitotic tissues. Our morphological ageing data support this hypothesis. With advancing age, the transparent body changes to an opaque grey, but inner organs can still be observed under a microscope. Body deformities appear such as small bulges in the epidermis, the presence of grooves in the head region, and liquid-filled cysts that tend to be present in all body regions (Figure 3). Furthermore, it is several times observed that individuals shrink during their last months of life, i.e. they decrease in size and lose their gonads (Figure 3B). These observations suggest that there is indeed an overall decline in tissue maintenance potential with advancing age. Tissue homeostasis can be studied by using continuous 5-bromo-2'-deoxyuridine (BrdU) exposure. By using this technique, [START_REF] Mouton | Gradual senescence in Macrostomum lignano: demographic data of a flatworm species[END_REF] demonstrated that about one-third of all epidermal cells are replaced every 2 weeks in adults of M. lignano. Further studies in which epidermal replacement is used as a parameter for measuring tissue homeostasis over the complete lifespan are underway (Mouton, unpublished data).

Experimental toolbox

Stem cell function is regulated by both intrinsic (cell autonomous) and extrinsic factors (for example surrounding tissue, stem cell niche and environment) during ageing. Moreover, these factors also interact with each other [START_REF] Rando | Stem cells, ageing and the question for immortality[END_REF]. Therefore, a large-scale multidisciplinary approach at all the levels of organization, from molecules and genes to tissues, organs and ultimately the whole organism is necessary. The essential experimental toolbox for this kind of research is available in M. lignano and will be discussed in this section.

M. lignano has a well characterized and easily stainable stem cell system [START_REF] Bode | Immunogold labeled S-phase neoblasts, total neoblast number, their distribution and evidence for arrested neoblasts in Macrostomum lignano (Platyhelminthes, Rhabditophora)[END_REF][START_REF] Egger | The regeneration capacity of the flatworm Macrostomum lignano -on repeated regeneration, rejuvenation and the minimal size needed for regeneration[END_REF][START_REF] Ladurner | Spatial distribution and differentiation potential of stem cells in hatchlings and adults in the marine platyhelminth Macrostomum sp.: A Bromodeoxyuridine analysis[END_REF][START_REF] Nimeth | Stem cell dynamics during growth, feeding, and starvation in the basal flatworm Macrostomum sp. (Platyhelminthes)[END_REF]. The morphology of the neoblasts has been described in detail by means of light and electron microscopy (see references in [START_REF] Ladurner | The stem cell system of the basal flatworm Macrostomum lignano[END_REF]. S-phase neoblasts can be single or double stained with the thymidine analogs BrdU, IdU and CldU. Dividing cells can be labelled with the antiphospho-histone H3 mitosis marker and double labelling with both this marker and a thymidine analog can be performed [START_REF] Ladurner | The stem cell system of the basal flatworm Macrostomum lignano[END_REF]. These markers make studying the distribution, migration and differentiation of the neoblasts possible by performing pulse, pulse-chase and continuous labelling experiments. Moreover, the number of neoblasts can be easily quantified by using the 3D counting method presented by Schärer et al. (2004). The fact that this can be done at any given time point is a major advantage to assess the relation between age and neoblasts. Such studies have already been undertaken and preliminary results suggest that the number of neoblasts does not decline with age in M. lignano (Mouton, unpublished results). However, it remains to be tested whether the dynamics of neoblast proliferation change with age. For this, animals can be incubated in colchicine to determine mitotic rate and hydroxyurea can be used to arrest neoblasts in early S-phase [START_REF] Nimeth | Stem cell dynamics during growth, feeding, and starvation in the basal flatworm Macrostomum sp. (Platyhelminthes)[END_REF].

Molecular germ line/stem cell markers (vasa, piwi) are also available [START_REF] Ladurner | The stem cell system of the basal flatworm Macrostomum lignano[END_REF][START_REF] Pfister | The exceptional stem cell system of Macrostomum lignano: Screening for gene expression and studying cell proliferation by hydroxyurea treatment and irradiation[END_REF], Pfister et al., 2008) and can be used for studying the mRNA expression by in situ hybridization and protein localization by specific antibody labelling [START_REF] Pfister | Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano[END_REF].

In most mammalian tissues, the relative inaccessibility of the stem cell population in vivo has made it difficult to determine whether a decline in stem cell function is correlated to the decline of regenerative capacities with ageing (Sharpless and DePinho, 2007). In flatworms, however, the neoblast population is accessible, and purification of the neoblast cell fraction is well established [START_REF] Hayashi | Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting[END_REF]. In fact, the rescue of lethally X-irradiated hosts after transplanting neoblasts from healthy donors is one of the most convincing experiments that suggests totipotency of triclad stem cells [START_REF] Dubois | Sur une nouvelle méthode permettant de mettre en évidence la migration des cellules de régénération chez les planaires[END_REF]Stephan-Dubois and Gilgenkrantz, 1960;Lange, 1968b;Lange 1968c;[START_REF] Lange | Studies on the cellular basis of radiation lethality. III. The measurement of stem cell repopulation probability[END_REF]Lange, 1969a;Lange 1969b;Báguñà et al. 1989). In M. lignano it would be possible to perform parabiotic pairings of injected "old" or "young" neoblasts into young or old X-ray irradiated hosts respectively. This would allow testing whether the putative diminished replicative potential of the neoblasts is primarily due to intrinsic ageing or to the effect of the ageing somatic environment. Similar experiments have recently been performed in mice and have shown the importance of a youthful systemic milieu to activate aged muscle stem cells [START_REF] Rando | Stem cells, ageing and the question for immortality[END_REF]. In flatworms, the importance of the somatic environment was shown by Lange (1968a) who studied ageing in the triclad Dugesia lugubris. The experimental toolbox available for M. lignano allows studying the causal relationship between the age of the somatic environment and the declining stem cell functionality in greater detail.

Other advantages of M. lignano are its small size, its simple well-described anatomy, and its transparency. This allows a relatively fast assessment of 1) the number of cells, 2) the general physical condition of the animal, and 3) the in vivo study of cells and organ systems during ageing. Moreover, the recent production and characterization of cell-and tissue-specific monoclonal antibodies (mAB) for this species (Ladurner et al., 2005b) will certainly favour future studies on ageing, especially in studies concerning cell differentiation potential and cell and tissue quantification during lifespan. For example, the number and the condition of gut cells, nerve fibres and clusters, epidermal cells, gland cells, muscle fibres, and spermatids can now be easily quantified and visualized over time. Because protocols for electron microscopy are well established for M. lignano, it is further possible to analyze tissues and cells ultrastructurally [START_REF] Bode | Immunogold labeled S-phase neoblasts, total neoblast number, their distribution and evidence for arrested neoblasts in Macrostomum lignano (Platyhelminthes, Rhabditophora)[END_REF]. Three life stages (i.e. 1, 6, and 9 months old) of M. lignano are currently being analyzed by means of electron microscopy (Mouton, unpublished) and future analyses will aim at following the ultrastructural morphology of cells, tissues and body deformities during its entire lifespan. Recently, high pressure freezing was used as a primary fixation technique for ultrastructural preparations in M. lignano (Egger et al. in review). This new tool will improve future ultrastructural immunocytochemistry studies aimed at characterizing ageing specific markers such as the p53 protein.

Lifespan extension and rejuvenation

The main goal of ageing research is to understand and eventually manipulate processes which lead to senescence and death. Essentially, increased lifespan can be achieved either by slowing down or even by reversing the ageing process. The latter phenomenon is called rejuvenation, known as the "fountain of youth" concept in myth and legend. In several flatworm species, a lifespan extension induced by starvation or repeated regeneration is observed. Several authors even suggest that starvation and repeated regeneration induce rejuvenation and bring individuals to a "younger" condition than at the start of the experiment (see references in [START_REF] Haranghy | Ageing and rejuvenation in planarians[END_REF]. The possibilities of M. lignano for studying lifespan extension and especially for studying rejuvenation will be discussed in this section. By using M. lignano, the relationship between reproduction and longevity can be readily addressed. This hermaphrodite flatworm allocates a substantial amount of energy into reproduction [START_REF] Schärer | Phenotypically plastic adjustment of sex allocation in a simultaneous hermaphrodite[END_REF]Vizoso and Schärer, 2007). Analogous to recent C. elegans work, efforts are being undertaken to use RNA interference for genes involved in gonad formation, which may make it possible to generate animals with reduced or absent spermatogenesis or oogenesis, or to generate animals completely devoid of gonads (K. Sekii, P. Ladurner and L. Schärer, unpublished). These manipulations are expected to open avenues to contribute to our understanding of how reproduction might regulate lifespan in this species.

Known lifespan-extending pathways (e.g. insulin/insulin-like growth factor 1 signalling pathway) could also be studied by using the same molecular technique.

One of the most interesting advantages of M. lignano as a stem cell model is the possibility of experimentally inducing additional stem cell proliferation through amputation and regeneration. In addition, somatic damage can be removed and post-mitotic tissues and the gonads can be renewed by inducing regeneration. M. lignano is capable of regenerating lost tissue anterior to the brain and posterior to the pharynx [START_REF] Egger | The regeneration capacity of the flatworm Macrostomum lignano -on repeated regeneration, rejuvenation and the minimal size needed for regeneration[END_REF]. Repeated amputation and subsequent regeneration of the same individual is not only possible, but apparently extends the lifespan as well [START_REF] Egger | The regeneration capacity of the flatworm Macrostomum lignano -on repeated regeneration, rejuvenation and the minimal size needed for regeneration[END_REF]. Over a period of more than two years (26.5 months), up to 59 consecutive amputations of the posterior part of single individuals were successfully performed and animals were consistently able to regenerate lost tissues between amputations (Egger, unpublished observations;[START_REF] Egger | The regeneration capacity of the flatworm Macrostomum lignano -on repeated regeneration, rejuvenation and the minimal size needed for regeneration[END_REF], Ladurner et al., 2008). Only the head, including the rostrum, brain and eyes, and the pharynx region were not amputated in the repeatedly regenerating animals. Interestingly, after about seven months, some animals showed signs of deterioration in the head region: grooves appeared in the rostrum and the pigmented eyes were lost one after the other. After about ten months, all test animals had lost both pigmented eyes and these were never found to be repaired or replaced during the remaining months of the experiment (Egger, unpublished observations). The additional stem cell proliferation possibly leads to proliferation-induced damage, and the loss of eyes and deterioration in rostrum could be an indication of distorted maintenance of the head, possibly causing the eventual death of the animal. However, it is important to note that at the end of the experiment the individuals were still able to regenerate. The renewal of all amputated tissues, including neoblasts, suggests an actual rejuvenation of the regenerated tissues. The next step is to assess whether the number of amputations, the amputation level and the age of amputated individuals influence the lifespan. Different hypotheses can be formulated as for the reasons for the extended lifespan due to repeated regeneration in M. lignano:

1) During regeneration, neoblasts lost by amputation are being renewed (Figure 4A). This is in contrast to normal tissue maintenance, where differentiated cells, but not necessarily neoblasts, are renewed. The possibility of stem cell renewal taking place in adult organisms can also be observed in obligatorily asexually reproducing animals. For example, the annelid Dorvillea bermudensis [START_REF] Åkesson | Two new Dorvillea species (Polychaeta, Dorvilleidae) with obligate asexuaI reproduction[END_REF] has been known to reproduce in laboratory cultures exclusively by transverse fission for more than 30 years.

2) During regeneration, all amputated tissues are completely renewed. Morphologically, the regeneration of small fragments of M. lignano presumably passes through several stages of postembryonic development again: after wound healing and blastema formation, the remaining fragment undergoes morphallactic changes, shrinking the remaining tissues (rostrum, pharynx) to proportions similar to a freshly hatched juvenile [START_REF] Egger | The regeneration capacity of the flatworm Macrostomum lignano -on repeated regeneration, rejuvenation and the minimal size needed for regeneration[END_REF][START_REF] Egger | Free-living flatworms under the knife: past and present[END_REF]. Subsequently, the juvenile and the regenerating animal face identical challenges: both must increase their size considerably by forming new tissue, and need to develop gonads and copulatory organs (Figure 4B). It appears likely, therefore, that during regeneration some of the same genetic pathways are used as in postembryonic and possibly even as in embryonic development (see [START_REF] Egger | Free-living flatworms under the knife: past and present[END_REF]. Monitoring gene expression during postembryonic development and regeneration will clarify this question.

3) Animals are experiencing so-called "mild stress" during amputation and regeneration (Figure 4C). Exposure to mild stress including, for example, irradiation, starvation, heat stress, hypergravity and free radicals, has been shown to increase the lifespan of cells and organisms [START_REF] Rattan | Hormesis in aging[END_REF]. Amputation can be considered a form of mild stress, at least in animals capable of regeneration.

All of these hypotheses are non-exclusive and could very well act together to increase the individual lifespan.

In some flatworms, starvation was also found to increase longevity. It is suggested that the smaller the fragment left to regenerate, or the longer the period of starvation is, the more pronounced the rejuvenation effect is (see references in [START_REF] Haranghy | Ageing and rejuvenation in planarians[END_REF]. Like most flatworms, M. lignano can cope with extended periods of starvation (months) by shrinkage (termed 'degrowth' in [START_REF] Baguñà | Mitosis in the intact and regenerating planarian Dugesia mediterranea n. sp. I. Mitotic studies during growth, feeding and starvation[END_REF][START_REF] Nimeth | Stem cell dynamics during growth, feeding, and starvation in the basal flatworm Macrostomum sp. (Platyhelminthes)[END_REF][START_REF] Pfister | Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano[END_REF].

During starvation, the animals shrink, absorb reproductive organs and other tissues, and assume a shape that strongly resembles a hatchling. The expression of the vasa-like gene macvasa in gonads and stem cells is greatly reduced but animals recover to normal morphology and gene expression upon refeeding [START_REF] Pfister | Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano[END_REF]). However, for M. lignano, it still needs to be tested whether starvation has a similar influence on longevity as regeneration.

In addition to starvation, the effects of dietary restriction can also be studied in M. lignano because it is possible to manipulate calorie uptake by limiting the food source without inducing starvation (=malnutrition) (Vizoso and Schärer 2007). Dietary restriction (defined as caloric restriction without malnutrition) slows ageing in yeast, flies, nematodes, fish and rodents. Sir2 is conserved from yeast to flies, worms and mammals [START_REF] Fabrizio | Sir2 blocks extreme life-span extension[END_REF] and may be involved in dietary restriction mediated determination of lifespan. We have studied the expression of a sir2-like gene in M. lignano. Surprisingly, Sir2 mRNA was found in a 13 certain cell type -called goblet cells -in the gastrodermis (Fig. 5). These cells can also be identified with the monoclonal antibody MDr-2 (Fig. 5) that was raised for M. lignano (Ladurner et al., 2005b).

Conclusion

M. lignano is a flatworm that can be easily cultured. It has a relatively short lifespan and an accessible stem cell population. Because an experimental toolbox is available for all levels of organisation, this species is an ideal model organism for obtaining more insight into the reciprocal influence between stem cells and the ageing process. Furthermore, this species is a suitable model system for studying lifespan extension and rejuvenation after starvation and repeated regeneration. evidence that testis size reflects testicular cell proliferation activity in the marine invertebrate, the free-living flatworm Macrostomum sp. Behav. Ecol. Sociobiol. 56, 420-425. Sharpless, N.E., DePinho, R.A., 2007. How stem cells age and why this makes us grow old. Nat. Rev. Mol. Cell. Biol. 8, 703-713. Semsei, I., 2000. On the nature of aging. Mech. Ageing Dev. 117, 93-108.

Stephan- Dubois, F., Gilgenkrantz, F. 1961. Régénération après transplantation chez la planaire Dendrocoelum lacteum. C.R. Soc. Biol. 155, 115-118. Terzibasi, E., Valenzano, D.R., Cellerino, A., 2007. The short-lived fish Nothobranchius furzeri as a new model system for aging studies. Exp. Gerontol. 42, 81-89. Vizoso, D.B., Schärer, L., 2007. Resource-dependent sex allocation in a simultaneous hermaphrodite. J. Evol. Biol. 20, 1046-1055. Rejuvenation is a fascinating and emerging field of interest. It can only be studied in a limited number of species, such as cnidarians, annelids and flatworms, which have the ability to regenerate and renew their body. As discussed in the text, it can be easily studied in M. lignano. M. lignano has a shorter maximum lifespan than the vertebrate models, except for Nothobranchius furzeri. This annual fish is a short-lived vertebrate which may be of biomedical relevance but does not have such an accessible stem cell system as M. lignano. The presence of a groove (black arrowheads) in the head region is a common deformity in old individuals. Grooves are external structures at the epidermis. It should be noted that in the literature both the terms 'urns' (Ladurner et al. 2005) and 'grooves' [START_REF] Egger | The regeneration capacity of the flatworm Macrostomum lignano -on repeated regeneration, rejuvenation and the minimal size needed for regeneration[END_REF] are used for these structures. (B) Another frequently observed deformity is the presence of a cyst (*). This is an internal structure which can be located anywhere in the body. Besides the presence of a cyst, this individual is also shrinking. This can be observed because the animal is small and the gonads are degenerating. (A) Adult animal with aged stem cells (red spots). Amputating a posterior body part causes the animal to regenerate lost tissues, including stem cells (green spots). (B) Genetic pathways (green) expressed during postembryonic development are possibly reactivated during regeneration and subsequent growth; some of these pathways are probably not expressed in adults (red). (C) Amputation itself can be considered a form of "mild stress" to animals with pronounced regeneration capacity and thus increase the lifespan.

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Fig. 1 :

 1 Fig. 1: Suitability of model organisms for studying the relation between stem cells and age. Essential characteristics for studying this conceptual theme are plotted against the maximum

Fig. 2 :

 2 Fig. 2: (A) Interference contrast photomicrograph and schematic drawing of M. lignano. The length of the animal is about 1 mm. Scalebar: 100µm. (B) BrdU picture of M. lignano. The

Fig. 3 :

 3 Fig. 3: Body deformities in 238 days old individuals of M. lignano. Scalebars: 100µm. (A)

Fig. 4 :

 4 Fig. 4: Possible explanations for increased longevity (rejuvenation) by means of repeated regeneration in M. lignano. All three presented hypotheses can play a role at the same time.

Fig. 5 :

 5 Fig. 5: In situ hybridization of a sir2-like gene of M. lignano. (A) The signal (blue) is located in the gut. (C) Higher levels of sir2-like mRNA are located in a special cell type called goblet

  Figure1

Table and Figure legends:Table 1 :

 and1 Selected Macrostomum lignano ESTs with homology to genes known to play a role in ageing.

Acknowledgements

We would like to thank Prof. Beatrix Grubeck-Loebenstein for inviting us to write this review. This research was funded by a PhD grant to S. M. and M. W. by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

During the initiation of this research, L.S. was supported by a Lise-Meitner fellowship (FWF, Austria), a start-up fellowship of the Tyrolean Science Foundation (TWF, Austria), and an advanced researcher fellowship (SNF, Switzerland). B. E. was supported by FWF project P16618 and P. L. by FWF project P18099.


Anne-Marie Miller

Alessia Piazza

Darren S D Martin

Michelle Walsh

Arkady Mandel

Anthony E Bolton

Marina A Lynch

la diffusion de documents scientifiques

Keywords: Long-term potentiation (LTP), hippocampus, caspase 3, amyloid-, phospholipid particles

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The deficit in long-term potentiation induced by chronic administration of amyloid-β-is attenuated by treatment of rats with a novel phospholipid-based drug formulation, VP025

INTRODUCTION

Deterioration in neuronal function has been consistently described in rats treated with A and one manifestation of this is a decrease in the ability of these animals to sustain LTP in the hippocampus. Data from this laboratory have established that the A -induced decrease in LTP is associated with microglial activation and increased concentration of inflammatory cytokines in hippocampus (Minogue et al., 2007). Data from a number of studies have shown that if microglial activation is attenuated, then the depression in LTP is also attenuated suggesting that the inflammatory conditions which prevail following A administration are detrimental to neuronal function [START_REF] Lynch | Eicosapentaenoic acid confers neuroprotection in the amyloid-beta challenged aged hippocampus[END_REF][START_REF] Lyons | IL-4 attenuates the neuroinflammation induced by amyloid-betain vivo and in vitro[END_REF]Minogue et al., 2007). A similar inverse correlation between maintenance of LTP and inflammatory changes has been identified in rats following treatment with the immune modulator, lipopolysaccharide (LPS) but these changes were prevented by treatment of rats with the polyunsaturated fatty acid, eicosapentaenoic acid [START_REF] Lonergan | Neuroprotective actions of eicosapentaenoic acid on lipopolysaccharide-induced dysfunction in rat hippocampus[END_REF] or by treatment of rats with a preparation of phospholipid nanoparticles incorporating phosphatidylserine [START_REF] Nolan | Evidence of a protective effect of phosphatidylserine-containing liposomes on lipopolysaccharide-induced impairment of long-term potentiation in the rat hippocampus[END_REF].

Phosphatidylglycerol shares some properties with phosphatidylserine and we have recently found that particles incorporating phosphatidylglycerol (VP025) attenuate the age-related and LPSinduced depression of LTP (Martin et al., submitted) and ameliorates the 6hydroxydopamine (6OHDA)-induced cell loss in the striatum [START_REF] Crotty | Neuroprotective effects of novel phosphatidylglycerol-based phospholipids in the 6-hydroxydopamine model of Parkinson' s disease[END_REF].

In addition to the inverse relationship between A -induced microglial activation and decreased LTP, we have found that decreased LTP is associated with activation of caspase 3 [START_REF] Minogue | Activation of the c-Jun N-terminal kinase signaling cascade mediates the effect of amyloid-beta on long term potentiation and cell death in hippocampus: a role for interleukin-1beta?[END_REF]. It is known from in vitro analysis that A induces neuronal stress and increases caspase 3 activation as a consequence of increased permeability of the mitochondrial membrane with the subsequent release of cytochrome c [START_REF] Green | The pathophysiology of mitochondrial cell death[END_REF][START_REF] Minogue | Activation of the c-Jun N-terminal kinase signaling cascade mediates the effect of amyloid-beta on long term potentiation and cell death in hippocampus: a role for interleukin-1beta?[END_REF]. Among the factors which contribute to loss of mitochondrial membrane patency and ultimately, activation of caspase 3, is ceramide, which is generated when sphingomyelinase hydrolyses the sphingophospholipid, sphingomyelin. It has been suggested that caspase 8 can act on membrane-associated sphingomyelinase in endosomes or lysosomes, and in this way lead to the generation of ceramide [START_REF] Smyth | prICE: a downstream target for ceramide-induced apoptosis and for the inhibitory action of Bcl-2[END_REF] which can increase mitochondrial membrane permeability and trigger cytochrome c release [START_REF] Roberts | Lysosomal cysteine proteases: structure, function and inhibition of cathepsins[END_REF][START_REF] Siskind | Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations[END_REF].

Here, we investigated the effects of chronic administration of A 1-40 and A 1-42 on LTP in perforant path-granule cell synapses and asked whether treatment of rats with VP025, which has been shown to have neuroprotective effects, might modulate A -induced changes. The data indicate that A treatment decreased LTP and that this, as well as the associated increase in caspase 3 activity, was attenuated in VP025-treated animals. The evidence is consistent with the proposal that the Ainduced increase in caspase 3 is dependent on activation of sphingomyelinase.

MATERIALS AND METHODS

Treatment of animals

Male Wistar rats aged 3-4 months (Bioresources Unit, Trinity College, Dublin, Ireland) were used in these studies. Animals were housed in groups of 4-6 at 22-23°C, and were maintained on a 12-hour light schedule, under veterinary supervision in the BioResources Unit. The experiments were performed under a license issued by the Department of Health and Children (Ireland), with the approval of the local ethics committee and in accordance with local guidelines.

Rats were randomly divided into 8 groups, half of which were treated with a cocktail of Aβ 1-40 + Aβ 1-42 , while the other half received the reverse peptide, Aβ 40-1 . Animals were anaesthetized with ketamine (75mg/kg) and xylazine (10mg/kg) and implanted with osmotic mini-pumps (model 2004, Alzet, USA). The pump was implanted subcutaneously in the mid-scapular region and was attached via polyvinylchloride tubing (Alzet, 0.69mm diameter) to a chronic indwelling cannula (Alzet, Infusion Kit II), which was positioned stereotaxically in the ventricle (0.9mm posterior to bregma, 1.3 lateral to the midline and 3.5mm ventral to the dura). The cannula was affixed to the skull using cryanoacetate gel and was secured in place by a smooth covering of dental cement (Stoelten, USA). Post-operative care included a subcutaneous injection of the analgesic Rimadil (5mg/kg). The pumps delivered a cocktail of Aβ 1-40 (26.9µM) and Aβ 1-42 (36.9µM; aggregated for 24 h at 25°C and 37°C for 48 h; Biosource, Belgium) or control peptide Aβ 40-1 (63.8 M) intracerebroventricularly at the rate of 0.25µl/h (± 0.05µl) for 28 days. Analysis of the A preparation by the thioflavin T fluorescent assay and gel electrophoresis revealed the presence of low oligomeric species; the predominant form (36% of the total) was the 13.5kDa species.

Rats which received reverse peptide (hereafter referred to as control-treated rats) and those which received Aβ 1-40 + Aβ 1-42 (hereafter described as Aβ-treated rats) were further subdivided. In one experiment, rats were injected intramuscularly with saline or VP025 (150 l; 1.2x10 7 particles/ml) 14, 13 and 1 day prior to implantation of the minipumps and delivery of A for 28 days (pretreated group); in this experiment there were 4 subgroups of rats, control-treated, A -treated, VP025-treated and A +VP025-treated. In the second experiment, saline or VP025 was injected 15, 16 and 27 days following surgery, i.e. 2 weeks after A treatment began (intervention group); this was to assess whether intervention with VP025 would ameliorate the effect of A .

Analysis of LTP

At the end of the period of A delivery, rats were anaesthetized with urethane (1.5mg/kg) and assessed for their ability to sustain LTP in perforant path-granule cell synapses as described previously [START_REF] Nolan | Role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus[END_REF]. A bipolar stimulating electrode was positioned in the perforant path (4.4mm lateral to lambda) and a unipolar recording electrode was positioned in the dorsal cell body region of the dentate gyrus (2.5mm lateral and 3.9mm posterior to bregma. Test shocks were delivered at 30 sec intervals, and after a period of recording which allowed stabilization, responses were recorded for 10 min before and 40 min after, tetanic stimulation (3 trains of stimuli; 250 Hz for 200 msec; 30 sec intertrain interval). At the end of the period of electrophysiological recording, rats were killed by decapitation, the brains were rapidly removed and the hippocampus and cortex were dissected free. Tissue was cross-chopped (350 m x 350 m) and stored at -80°C in Krebs buffer (composition in mM; NaCl 136, KCl 2.54, KH 2 PO 4 1.18, MgSO 4 .7H 2 O 1.18, NaHCO 3 16, glucose 10) with added CaCl 2 (1.13 mM) and 10% dimethyl sulphoxide (DMSO) until required for analysis.

Preparation of neuronal cultures

Primary cortical neurons were isolated and prepared from 1-day old Wistar rats as described previously [START_REF] Lyons | IL-4 attenuates the neuroinflammation induced by amyloid-betain vivo and in vitro[END_REF]. Briefly, cells were maintained in neurobasal medium (NBM; Gibco BRL) at 37°C in a humidified atmosphere containing 5% CO 2 :95% air. Media was replaced every 3 days and cells were grown in culture for up to 7 days prior to treatment. Cells were treated with Aβ 1-42 (10 M; aggregated according to manufacturer's instructions; Biosource, USA) for 24 h. In some cases, cells were coincubated for 24h with A in the presence or absence of the neutral sphingomyelinase inhibitor, GW4869 (20 M). GW4869 was dissolved in DMSO (0.001%). In other experiments, cells were incubated in the presence of ceramide (100 M; Sigma, UK) for 24h; ceramide was dissolved in DMSO (0.005%) and diluted with NBM (1:200 dilution) for cell treatment. In the experiments in which DMSO was used as diluent, the appropriate concentration of DMSO was used in the vehicle controls. At the end of the treatments, supernatants were removed and cells were harvested for analysis of activities of sphingomyelinase and caspase and for assessment of cell viability.

Assessment of caspase activity

Activities of caspases 3 and 8 were assessed in samples of cortical or hippocampal tissue and in cultured neuronal cells according to the manufacturer's instructions (Biomol, UK). Briefly, samples were equalized for protein concentration in assay buffer (50mM HEPES, 100mM NaCl, 0.1% CHAPS, 1mM EDTA, 10% glycerol; pH 7.4) to give a final concentration of 0.8mg/ml. Aliquots (25 l) of supplied enzyme solution were added to 96-well microplates, sample or assay buffer (25µl) was added and incubation continued at room temperature 21°C for 10 min. Substrate (Ac-DEVD-pNA (caspase-3) or Ac-IETD-pNA (caspase-8); 50µl; final concentration 200µM) was added to start the reaction and fluorescence was measured continuously for 6 min at 405nm. Data are presented as means ± SEM and are expressed as pNA produced (pmol/min/mg).

Assessment of sphingomyelinase activity

Sphingomyelinase activity was assessed in aliquots of cortical tissue. Samples were equalized for protein and sphingomyelinase activity was assessed using the Amplex Red assay kit (Molecular Probes, UK) according to the manufacturer's instructions. Briefly, a working solution (100 l) containing Amplex Red reagent (100µM), horseradish peroxidase (2U/ml), choline oxidase (0.2U/ml), alkaline phosphatase (8U/ml) and sphingomyelin (0.5mM) was added to samples or controls (100 l) and incubated for 30 min at 37°C. Fluorescence was measured (544nm excitation and 590 emission) at several time points (0, 5, 15, 25 and 60 min), values were corrected for background and mean fluorescent values were calculated.

Statistical analysis

Data were analysed using either the Student's t-test for independent means, or analysis of variance (ANOVA) or multifactorial ANOVA followed by Student Newman-Keuls or Bonferoni post hoc tests to determine which conditions were significantly different from each other. All data are expressed as means ± standard error of the means.

RESULTS

We report that delivery of a high frequency train of stimuli to the perforant path induced an immediate and sustained increase in population excitatory post synaptic potential (epsp) slope in control-treated rats, but treatment of rats with A led to a marked time-related decline in epsp slope; statistical analysis revealed a significant difference between control-treated and A -treated rats (***p < 0.001; ANOVA; Figures 1a and1b). Pretreatment of rats with VP025 significantly attenuated the Ainduced change ( +++ p < 0.001; ANOVA; Figure 1a), while VP025 treatment did not affect the response to tetanic stimulation. Similarly the A -induced depression in LTP was attenuated in animals which received VP025 2 weeks after A infusion began ( +++ p < 0.001; ANOVA; Figure 1b).

Hippocampal tissue prepared from these animals was assessed for caspase 3 activity and the data show that there was a significant A -induced effect in both experiments (***p < 0.001; ANOVA; Figures 1c and1d). Post hoc analysis indicated that the significant A -induced change was significantly attenuated in tissue prepared from A -treated rats which received VP025 as a pretreatment or as an intervention treatment ( +++ p< 0.001; ANOVA; Figures 1c and1d). Analysis of caspase 3 in cortical tissue prepared from the same rats revealed similar findings; thus there was a significant A effect (***p < 0.001; ANOVA; Figure 2c) and this Ainduced change was significantly attenuated in tissue prepared from rats which were pretreated with VP025 ( +++ p< 0.001; ANOVA; Figure 2c). The changes in activities of caspase 8 (***p < 0.001; ANOVA; Figure 2a) and sphingomyelinase (*p < 0.05; mixed factorial ANOVA; Figure 2b) mirrored the changes in caspase 3 with a significant A effect in each case that was attenuated in VP025-treated rats, although this attenuation was statistically significant only in the case of caspase 8 ( +++ p< 0.001; ANOVA; Figure 2a).

In an effort to understand the mechanism by which A induces activation of caspase 3, we undertook a series of in cultured neurons prepared from cortical tissue obtained from neonatal rats. We first showed that A significantly increased caspase 3 activity in these cells (***p < 0.001; ANOVA; Figure 2d) and that this effect was inhibited in cells which were incubated in A and the sphingomyelinase inhibitor, GW4869 ( ++ p< 0.01; ANOVA). This suggests that ceramide, which is the product of sphingomyelinase, mediates the effect of A and, consistent with this, we show that ceramide, like A , significantly increased caspase 3 activity (**p < 0.01; student t-test for independent means; Figure 2e). We also show that incubation of cells in the presence of A decreased cell viability (*p < 0.05; ANOVA; Figure 2f) and that this was inhibited by including GW4869 in the incubation medium ( + p < 0.05; ANOVA; Figure 2f; compared with A alone). Consistent with the view that the effects of A are mediated by sphingomyelinase is the finding that ceramide also significantly decreased cell viability (***p < 0.001; student t-test for independent means; Figure 2g).

DISCUSSION

Our objective was to assess whether a chronic infusion of A for 28 days would exert an inhibitory effect on LTP and to establish whether any A -induced effect might be modulated by pre-treatment or intervention treatment with VP025. We demonstrate that A significantly decreased LTP and increased caspase 3 activity and that VP025 inhibited these effects of A . The evidence suggests that the Ainduced activation of sphingomyelinase mediates the effect of A .

An important finding of the present study is that chronic infusion of A 1-40 and A 1- 42 inhibited LTP and while several groups have reported that acute exposure to different species of A inhibits LTP in vivo [START_REF] Freir | Blockade of long-term potentiation by beta-amyloid peptides in the CA1 region of the rat hippocampus in vivo[END_REF][START_REF] Klyubin | Soluble Arctic amyloid beta protein inhibits hippocampal long-term potentiation in vivo[END_REF][START_REF] Minogue | Activation of the c-Jun N-terminal kinase signaling cascade mediates the effect of amyloid-beta on long term potentiation and cell death in hippocampus: a role for interleukin-1beta?[END_REF][START_REF] Walsh | Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo[END_REF] and in vitro [START_REF] Freir | Blockade of long-term potentiation by beta-amyloid peptides in the CA1 region of the rat hippocampus in vivo[END_REF][START_REF] Puolivali | Hippocampal A beta 42 levels correlate with spatial memory deficit in APP and PS1 double transgenic mice[END_REF][START_REF] Saleshando | SB203580, the p38 mitogen-activated protein kinase inhibitor blocks the inhibitory effect of beta-amyloid on long-term potentiation in the rat hippocampus[END_REF][START_REF] Wang | Block of longterm potentiation by naturally secreted and synthetic amyloid beta-peptide in hippocampal slices is mediated via activation of the kinases c-Jun N-terminal kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well as metabotropic glutamate receptor type 5[END_REF], few studies have examined the effect of chronic infusion of A on hippocampal function. However one study reported that LTP in CA1 was decreased in hippocampal slices obtained from rats which received A 1-40 for 10-11 days [START_REF] Itoh | Impairments of long-term potentiation in hippocampal slices of betaamyloid-infused rats[END_REF] while others reported that hippocampal-dependent learning is depressed in rats treated chronically with A 1-40 or A 1-40 +A 1-42 [START_REF] Frautschy | Phenolic anti-inflammatory antioxidant reversal of Abeta-induced cognitive deficits and neuropathology[END_REF][START_REF] Nitta | Continuous infusion of betaamyloid protein into the rat cerebral ventricle induces learning impairment and neuronal and morphological degeneration[END_REF] and the findings of the present study are consistent with this. Here we coupled the A -induced inhibition of LTP with increased activity of caspase 3 in hippocampus; this is indicative of cell stress, although not necessarily cell death [START_REF] Garnier | Hypoxia induces caspase-9 and caspase-3 activation without neuronal death in gerbil brains[END_REF], and consistent with several reports which show that A increases cell stress in vivo and in vitro [START_REF] Boland | Abeta-mediated activation of the apoptotic cascade in cultured cortical neurones: a role for cathepsin-L[END_REF][START_REF] Clarke | The HMG-CoA reductase inhibitor, atorvastatin, attenuates the effects of acute administration of amyloidbeta1-42 in the rat hippocampus in vivo[END_REF][START_REF] Lyons | IL-4 attenuates the neuroinflammation induced by amyloid-betain vivo and in vitro[END_REF]. Importantly we show that treatment of rats with VP025 restores their ability to sustain LTP, even if treatment is not started until 2 weeks after A infusion begins.

Consistent with the relationship between LTP and caspase 3 activity, the data show that VP025 also reversed the A -induced increase in enzyme activity. These findings reveal a neuroprotective effect of VP025 and therefore provide support for our recent findings which demonstrated that VP025 overcame the inhibitory effects of age and LPS on LTP (Martin et al., submitted) and an earlier report which indicated that treatment of rats with VP025 attenuated the loss of dopaminergic neurons following 6OHDA injection [START_REF] Crotty | Neuroprotective effects of novel phosphatidylglycerol-based phospholipids in the 6-hydroxydopamine model of Parkinson' s disease[END_REF]. Interestingly VP015 exerted similar neuroprotective effects and attenuated LPS-and age-associated deficits in LTP [START_REF] Nolan | Evidence of a protective effect of phosphatidylserine-containing liposomes on lipopolysaccharide-induced impairment of long-term potentiation in the rat hippocampus[END_REF][START_REF] Nolan | Role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus[END_REF]; these effects were coupled with the antiinflammatory actions of VP015 which are shared by VP025 (Martin et al., submitted).

The A -induced increase in activity of caspase 3 in hippocampus was mirrored by a similar change in cortical tissue, which was accompanied by increased activity in caspase 8 and sphingomyelinase. The coupled increases in activation of these enzymes is consistent with the fact that the executioner caspase 3 is a major substrate for the initiator caspase 8 [START_REF] Stennicke | Pro-caspase-3 is a major physiologic target of caspase-8[END_REF], with the evidence which indicated that caspase 8 can activate sphingomyelinase [START_REF] Heinrich | Cathepsin D links TNF-induced acid sphingomyelinase to Bid-mediated caspase-9 and -3 activation[END_REF] and with the demonstration that TNF -induced apoptosis in oligodendrocytes is dependent on sequential activation of caspase 8, neutral sphingomyelinase and caspase 3 [START_REF] Testai | Regulation of sphingomyelinases in cells of the oligodendrocyte lineage[END_REF]. Numerous studies have reported that A increases caspase 3 activity [START_REF] Awasthi | Amyloid-beta causes apoptosis of neuronal cells via caspase cascade, which can be prevented by amyloid-betaderived short peptides[END_REF][START_REF] Boland | Abeta-mediated activation of the apoptotic cascade in cultured cortical neurones: a role for cathepsin-L[END_REF] and caspase 8 activity [START_REF] Awasthi | Amyloid-beta causes apoptosis of neuronal cells via caspase cascade, which can be prevented by amyloid-betaderived short peptides[END_REF] in cultured neurons while increases in both enzymes have also been reported in the AD brain [START_REF] Rohn | Caspase-9 activation and caspase cleavage of tau in the Alzheimer' s disease brain[END_REF].

In an effort to obtain additional evidence to support the proposal that sphingomyelinase plays a role in activating caspase 3, we undertook a series of experiments in vitro and demonstrate that, in cortical neurons, the A -induced activation of caspase 3 was blocked by the sphingomyelinase inhibitor GW4869, suggesting that ceramide may trigger the increase in caspase 3 activity. This was confirmed by the finding that the stimulatory effect of A on caspase 3 activity was mimicked by exposure of neurons to ceramide. We found that the A decreased cell viability and that this, like its effect on caspase 3 was attenuated by GW4869 and mimicked by ceramide. The findings are consistent with others which have shown that A increased ceramide in human primary neurons [START_REF] Jana | Fibrillar amyloid-beta peptides kill human primary neurons via NADPH oxidase-mediated activation of neutral sphingomyelinase. Implications for Alzheimer' s disease[END_REF]) and that ceramide increases activity of caspases 9 and 3 in cortical neurons [START_REF] Movsesyan | Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway[END_REF].

The present findings identify a causal relationship between activation of sphingomyelinase and caspase 3 and reveal that the A -associated decrease in LTP is coupled with upregulation of enzyme activity. Importantly, we identify a neuroprotective effect of VP025 and show that it attenuates the A -induced increase in caspase 3 and deficit in LTP. The data are consistent with the proposal that activation of sphingomyelinase activity, which we have also observed in the aged brain, is a key factor in modulating activation of caspase 3 and controlling neuronal function.

Figure 1 :

 1 Figure 1: VP025 treatment attenuates A induced changes in LTP and caspase 3 activity. (a) and (b) LTP in dentate gyrus was significantly decreased in rats which received A 1-40 and A 1-42 intracerebroventricularly for 28 days (***p < 0.001; ANOVA n = 4-6). Pretreatment of rats with VP025 14, 13 and 1 day prior to the start of A infusion inhibited the A -induced deficit in LTP (a) and similarly, the Ainduced decrease was inhibited when VP025 was given 15, 16 and 27 days after A infusion started (b; +++ p < 0.001; ANOVA; n = 4-6). (c) and (d) Caspase 3 activity was significantly increased in hippocampal tissue prepared from A -treated rats (***p < 0.001; ANOVA; n = 4-6) and both VP025 treatment schedules significantly inhibited this change ( +++ p < 0.001; ANOVA; n = 4-6).

Figure 2 :

 2 Figure 2: The A -induced increase in caspase 3 activity is accompanied by increased sphingomyelinase activity. (a)-(c) A significantly increased activities of caspases 8 (a) sphingomyelinase (b) and caspase 3 (c) in cortical tissue (*p < 0.05; multifactorial ANOVA; ***p < 0.001; ANOVA; n = 5-6); pretreatment of rats with VP025 14, 13 and 1 day prior to the start of A infusion inhibited these A -induced changes and the effects were significant in the case of caspases 8 and 3 ( +++ p < 0.001; ANOVA; n = 5). (d) A significantly increased caspase 3 in cortical neurons (***p < 0.001; ANOVA; n = 6) and this was inhibited by GW4869 (20 M; ++ p < 0.01; ANOVA; n = 5), GW4869 alone also decreased activity of caspase 3 ( p < 0.001; ANOVA; n = 5). (e) and (g) Incubation of cortical neurons in the presence of ceramide (100 M) significantly increased caspase 3 activity (e; **p < 0.01; student's t-test for independent means; n = 6) and decreased cell viability (g; ***p < 0.001; student's t-test for independent means; n = 6). (f) Incubation of cortical neurons in the presence of A (10 M) significantly decreased cell viability (*p < 0.05; ANOVA; n = 6) and this was significant attenuated by inclusion of GW4869 (20 M) in the incubation medium ( + p < 0.05; ANOVA; n = 6).

  Figure 1