Ángela González-Martínez

Belén Rosado

Pedro Pesini
email: pedropesini@araclon.com

María-Luisa Suárez

Germán Santamarina

Sylvia García-Belenguer

Ainara Villegas

Inmaculada Monleón

Manuel Sarasa

Franz Schubert

Plasma β-amyloid peptides in canine aging and cognitive dysfunction as a model of Alzheimer's disease

Keywords: Amyloid-beta protein, plasma, Alzheimer's disease, animal behavior, canine, ELISA, neurodegeneration, brain aging

Aging dogs naturally demonstrate cognitive impairment and neuropathology that models early Alzheimer's disease (AD). In particular, there is evidence that canine cognitive dysfunction syndrome (CDS) in aged dogs is accompanied by cortical deposition of Aβ peptides and neurodegeneration. Plasma Aβ levels have been examined in humans as putative biomarkers for AD, but to date, no similar studies have been conducted for canine dementia. The aim of the present study was to assess plasma Aβ1-42 and Aβ1-40 levels in a blind study using pet dogs that were either successfully aging or exhibiting CDS. The severity of cognitive impairment was assessed using an owner-based questionnaire. On average, young dogs presented significantly higher plasma levels of Aβ1-42 and Aβ1-40 than aged, cognitively unimpaired dogs. Notably, among aged dogs, the levels of Aβ1-42 and the Aβ42/40 ratio were significantly higher in those showing mild cognitive impairment than in either cognitively unimpaired or severely affected dogs. These results suggest that increased plasma Aβ1-42 levels and Aβ42/40 ratio could be a biomarker for canine cognitive dysfunction, which is considered an excellent natural model of early AD.

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Introduction

Dogs may naturally suffer age-related cognitive deficits that parallel several key aspects of early Alzheimer's disease (AD). There is evidence of β-amyloid (Aβ) deposits and amyloid angiopathy in the brain of aged dogs [START_REF] Borras | Age-related changes in the brain of the dog[END_REF][START_REF] Uchida | Immunohistochemical analysis of constituents of senile plaques and cerebro-vascular amyloid in aged dogs[END_REF]. Furthermore, the extent of Aβ deposition in the cerebral cortex has been shown to correlate with declines in select measures of cognitive function in aged beagle dogs and in aged pet dogs [START_REF] Colle | Vascular and parenchymal Abeta deposition in the aging dog: correlation with behavior[END_REF][START_REF] Rofina | Cognitive disturbances in old dogs suffering from the canine counterpart of Alzheimer's disease[END_REF]. Other neuropathological features of AD, such as neurodegeneration and oxidative damage, have also been reported in aged dogs [START_REF] Rofina | Cognitive disturbances in old dogs suffering from the canine counterpart of Alzheimer's disease[END_REF][START_REF] Bernedo | Cortical beta-amyloid pathology in the dog is accompanied with loss of serotonergic neurons[END_REF][START_REF] Head | Insights into A[beta] and Presenilin from a Canine Model of Human Brain Aging[END_REF][START_REF] Head | Oxidative damage increases with age in a canine model of human brain aging[END_REF][START_REF] Insua | Dogs with canine counterpart of Alzheimer's disease lose noradrenergic neurons[END_REF][START_REF] Pugliese | Canine cognitive deficit correlates with diffuse plaque maturation and S100beta (-) astrocytosis but not with insulin cerebrospinal fluid level[END_REF][START_REF] Siwak-Tapp | Region specific neuron loss in the aged canine hippocampus is reduced by enrichment[END_REF][START_REF] Skoumalova | The role of free radicals in canine counterpart of senile dementia of the Alzheimer type[END_REF][START_REF] Su | Magnetic resonance imaging of anatomic and vascular characteristics in a canine model of human aging[END_REF][START_REF] Tapp | Frontal Lobe Volume, Function, and β-Amyloid Pathology in a Canine Model of Aging[END_REF]. Furthermore, from the molecular perspective, it has been found extensive homology for the canine Aβ (100% similarity), its precursor protein (APP, 98%), and the enzymes for APP processing (92-100%) with their human counterparts [START_REF] Johnstone | Conservation of the sequence of the Alzheimer's disease amyloid peptide in dog, polar bear and five other mammals by cross-species polymerase chain reaction analysis[END_REF][START_REF] Sarasa | Cloning, sequencing and expression in the dog of the main APP isoforms and some of the enzymes related with their processing[END_REF]. Altogether, these data suggest the dog as an appropriate natural model for studying the biology of AD [START_REF] Sarasa | Natural non-trasgenic animal models for research in Alzheimer's disease[END_REF].

Research on the canine model of AD has been extensively conducted using laboratory beagle dogs rather than pet/companion dogs. However, the latter population may offer additional contributions to the investigation of AD because these dogs share a common environment with humans and, therefore, encounter similar environmental stressors during the aging process [START_REF] Cotman | The canine (dog) model of human aging and disease: dietary, environmental and immunotherapy approaches[END_REF]. Cognitive dysfunction syndrome (CDS) in elderly pet dogs is characterized by behavioral and cognitive deficits that fall into four main categories: sleep-wake cycle, social interaction, housetraining, and orientation [START_REF] Heath | Behaviour problems in geriatric pet[END_REF][START_REF] Ruehl | Canine cognitive dysfunction[END_REF].

Owner-based questionnaires show that CDS may affect more than 22% of geriatric dogs, and prevalence and severity increase strikingly with age [START_REF] Azkona | Prevalence and risk factors of behavioural changes associated with age-related cognitive impairment in geriatric dogs[END_REF][START_REF] Neilson | Prevalence of behavioral changes associated with age-related cognitive impairment in dogs[END_REF]. These data highlight the relevance of this canine syndrome, not only for research purposes as a model for AD but also plainly from a veterinary clinical point of view.

Increasingly, it appears that early diagnosis of AD, even at pre-symptomatic stages, is essential for effective therapeutic intervention. Therefore, there is an urgent need for

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4 reliable biomarkers that can detect the onset of brain amyloid pathology before irreversible neurodegeneration has taken place. Plasma Aβ1-42 and Aβ1-40 peptides have been proposed as non-invasive peripheral biomarkers to distinguish between cognitively healthy people and patients with mild cognitive impairment (MCI) with a high degree of sensitivity and specificity [START_REF] Assini | Plasma levels of amyloid beta-protein 42 are increased in women with mild cognitive impairment[END_REF][START_REF] Blasko | Conversion from cognitive health to mild cognitive impairment and Alzheimer's disease: prediction by plasma amyloid beta 42, medial temporal lobe atrophy and homocysteine[END_REF][START_REF] Cosentino | Plasma β-Amyloid and Cognitive Decline[END_REF][START_REF] Lopez | Plasma amyloid levels and the risk of AD in normal subjects in the Cardiovascular Health Study[END_REF][START_REF] Mayeux | Plasma Aβ40 and Aβ42 and Alzheimer's disease: relation to age, mortality, and risk[END_REF][START_REF] Mehta | Plasma and cerebrospinal fluid levels of amyloid beta proteins 1-40 and 1-42 in Alzheimer disease[END_REF][START_REF] Schupf | Peripheral Aβ subspecies as risk biomarkers of Alzheimer's disease[END_REF][START_REF] Sobow | Plasma levels of alpha beta peptides are altered in amnestic mild cognitive impairment but not in sporadic Alzheimer's disease[END_REF][START_REF] Van Oijen | Plasma Abeta(1-40) and Abeta(1-42) and the risk of dementia: a prospective case-cohort study[END_REF][START_REF] Xia | A specific enzyme-linked immunosorbent assay for measuring beta-amyloid protein oligomers in human plasma and brain tissue of patients with Alzheimer disease[END_REF]. However, considerable controversy remains in the field, and it is still too early to clearly establish the role of Aβ blood tests as a diagnostic tool for MCI and AD [START_REF] Blennow | Cerebrospinal fluid and plasma biomarkers in Alzheimer disease[END_REF][START_REF] Freeman | Plasma Abeta levels do not reflect brain Abeta levels[END_REF][START_REF] Graff-Radford | Association of low plasma Abeta42/Abeta40 ratios with increased imminent risk for mild cognitive impairment and Alzheimer disease[END_REF][START_REF] Hansson | Evaluation of plasma Abeta(40) and Abeta(42) as predictors of conversion to Alzheimer's disease in patients with mild cognitive impairment[END_REF][START_REF] Lambert | Association of plasma amyloid beta with risk of dementia: the prospective Three-City Study[END_REF][START_REF] Lewczuk | Amyloid beta peptides in plasma in early diagnosis of Alzheimer's disease: A multicenter study with multiplexing[END_REF][START_REF] Oh | Maximizing the potential of plasma amyloid-beta as a diagnostic biomarker for Alzheimer's disease[END_REF][START_REF] Sundelof | Plasma beta amyloid and the risk of Alzheimer disease and dementia in elderly men: a prospective, population-based cohort study[END_REF]. To date, no similar studies have been performed in pet dogs.

The aim of the present study was to assess the plasma levels of Aβ1-40 and Aβ1-42 in pet dogs with and without CDS. To this end, four groups of privately owned dogs were considered: young, middle-aged, cognitively unimpaired aged, and cognitively impaired aged animals. Taking into account our preliminary results in human patients, we hypothesized that plasma Aβ-peptides levels would differ among the different groups of animals and would relate to the presentation of cognitive dysfunction in the aged groups [START_REF] Pesini | Differences in beta amyloid pool in blood discriminate between amnesic mild cognitive impairment patients and healthy controls[END_REF].

Materials and methods

Study population

Two veterinary teaching hospitals (Universidad de Santiago de Compostela and Universidad de Zaragoza, Spain) contributed to the collection of cases. Dogs were recruited from animals in these hospitals' populations that were not referred for behavioral consultations at the time of admission; they were all small to medium-sized dogs usually living with their owners (i.e., pets). There were no selection criteria regarding neuter status, but the groups were sex-balanced (

Medical assessment

Prior to inclusion in the study for Aβ peptides analysis, all dogs were screened by routine physical and neurological examination, complete blood count, serum biochemistry and thyroid hormone measurement, and urinalysis when needed. Animals with primary organ system failure (other than brain degeneration), hypothyroidism, untreated Cushing's syndrome and seriously affected mobility were excluded from the study. Animals with severe loss of visual capacity were also excluded.

Cognitive assessment

Classification of cognitive status was carried out using an owner-based observational questionnaire (see Table 2). The design of the questionnaire was based on previously published models [START_REF] Colle | Vascular and parenchymal Abeta deposition in the aging dog: correlation with behavior[END_REF][START_REF] Heath | Behaviour problems in geriatric pet[END_REF][START_REF] Ruehl | Canine cognitive dysfunction[END_REF][START_REF] Azkona | Prevalence and risk factors of behavioural changes associated with age-related cognitive impairment in geriatric dogs[END_REF] and included a number of behavioral and cognitive items grouped into four categories: (a) sleep-wake cycle, (b) socio-environmental interaction, (c) housetraining and commands, and (d) disorientation. The questionnaires were administered to the owners by two doctors in veterinary medicine (DVM) who specialize in behavior (AG-M and BR). The owners of the aged dogs were asked to compare the dog's present behavior to its behavior prior to 9 years of age, when the dog was a younger adult. After matching the affected items (Yes or No answer), the owner

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6 was asked to grade the severity of the impairment for each category using a five-point scale (0 = non-impaired; 4 = severely impaired).

A dog was considered to be impaired in a category if at least one item was affected and that category scored ≥1 points. A dog was considered cognitively impaired when two or more categories were impaired and the total dysfunction score (TDS, the sum of scores attributed by the owner to each of the four categories) was ≥2 points. A score of 2 to 5 points was described as mild cognitive impairment (mCI) and ≥6 points as severe cognitive impairment (sCI). Simultaneously, using the information obtained during the screening, the researchers filled out the canine dementia questionnaire published by Rofina et al. [START_REF] Rofina | Cognitive disturbances in old dogs suffering from the canine counterpart of Alzheimer's disease[END_REF] for every animal. The dementia score obtained from that questionnaire is referred to hereafter as the Rofina dysfunction score (RDS).

Blood sampling and Aβ peptide analysis

Blood samples (5 ml) were collected from the jugular vein into polypropylene vials containing EDTA and a protease inhibitor cocktail (CompleteMini, Roche); they were centrifuged (2500 g, 4ºC, 15 min), aliquoted and coded without any reference to the age or cognitive state of the animals. The aliquots of plasma were immediately frozen at -80ºC and sent to the Araclon laboratory. Aβ1-42 and Aβ1-40 were measured in plasma using two specific ELISA sandwich kits (ABtest 40 and ABtest 42; Araclon Biotech Ltd., Zaragoza, Spain). Each sandwich kit is composed of an N-terminal Aβ-binding capture antibody and a C-terminal Aβ1-40 or Aβ1-42 binding detection antibodies, respectively. The samples and peptide standards were always assayed in triplicate. To test the reproducibility of the analysis, the ELISA was repeated on two different days.

Average coefficients of variation (CV) between the two assays were 7.84 % for Aβ1-42 and 13.07 % for Aβ1-40. Furthermore, comparing the series of measurements using the Wilcoxon test indicated that the measurements of Aβ1-42 levels, Aβ1-40 levels, and the

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Aβ42/40 ratio did not differ significantly between the two assays (p = 0.472, p = 0.162, and p = 0.277, respectively). Therefore, the averaged data from the two assays was used for further statistical analysis.

An additional 3-ml blood sample from each animal was collected for cell count and serum clinical biochemistry.

Statistical analysis

The

Results

Demographic data

Demographic information for each group is shown in Table 1. Non-significant differences were found for sex, reproductive status, and weight or body condition among groups. Moreover, the CU and CI groups did not differ significantly in age. The CU group showed significantly higher serum albumin concentrations than the CI group (4.12 g/dl ± 0.52 vs 3.74 g/dl ± 0.56, respectively; p = 0.01). This difference was

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ACCEPTED MANUSCRIPT 8 considered clinically irrelevant, and there were no other biochemical or hematological differences between the aged groups (data not shown).

Clinical diagnosis of canine CDS

Table 2 shows the frequency distribution of impaired behavioral categories and items in each group. The proportion of dogs with affected items in the CI group was significantly higher than in the CU group for all items except for "vocalizing at night" and "decrease in recognizing familiar people". Obviously, the CI group showed significantly higher TDS than the CU group (6.08 ± 3.26 vs 0.35 ± 0.48, respectively; p < 0.001) and the same occurred for the RDS (9.79 ± 6.70 vs 1.23 ± 1.91, respectively; p < 0.001). Furthermore, TDS was strongly correlated with the number of affected items (r = 0.91, p < 0.001) and RDS (r = 0.89, p < 0.001) in the aged groups (CU and CI).

Plasma Aβ1-40 and Aβ1-42

The mean Aβ peptide concentrations and Aβ42/40 ratio values for each group are shown in Table 3. The levels of Aβ1-42 and Aβ1-40 were correlated with each other across the study groups (r = 0.68; p < 0.001), but they did not correlate with any hematological or serum biochemical parameters (data not shown). The plasma concentration of Aβ peptides, particularly Aβ1-42, varied considerably between dogs, even within a given group, as denoted by the high standard deviations (SD).

The highest Aβ1-42, Aβ1-40 peptide concentrations and Aβ42/40 ratio were found in the youngest group (YG). Thus, the YG and CU groups differed significantly for both Aβ-peptides and Aβ42/40 ratio. The concentrations of Aβ1-42 and the Aβ42/40 ratio in the MA group were between those in the YG and CU, but the differences did not reach statistical significance (Fig 1A ).

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Within the aged animals, it was found that Aβ plasma levels differed with cognitive status. Thus, the CI group showed significantly higher Aβ1-42 levels and Aβ42/40 ratio, but not Aβ1-40 levels, than the CU group (Table 3). Nevertheless, because of the wide dispersion of the measurements, individual values for these two parameters showed considerable overlap between the two groups. This overlap compromised the capability of the tests to discriminate between CU and CI animals, with an area under the ROC curve (AUC) < 0.67 for the three parameters, and only statistically significant for Aβ1-42 (p = 0.02). Furthermore, measurements of Aβ1-42 levels, Aβ1-40 levels or Aβ42/40 did not correlate with the any dementia score in the aged groups (data not shown).

However, when we split the CI group, the severely impaired animals (sCI, n = 17)

showed a significantly lower Aβ1-42 level and Aβ42/40 ratios than the mildly impaired animals (mCI, n = 21, Table 4). Moreover, the average Aβ1-42 levels and Aβ42/40 ratio p ≤ 0.002) and RDS (r = 0.37 and 0.41, respectively; p ≤ 0.007).

Discussion

Plasma Aβ1-40 and Aβ1-42 in relation to age and cognitive dysfunction.

In the present study, classification of cognitive status was carried out using an ownerbased questionnaire after ruling out other medical causes of dementia-like changes. This

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ACCEPTED MANUSCRIPT 10 questionnaire included four behavioral categories that were graded in severity by the owner according to a five-point scale to obtain a total cognitive dysfunction score (TDS). Owner-based questionnaires, including psychometric scales, have already been used for measuring and phenotyping behavior in dogs [START_REF] Colle | Vascular and parenchymal Abeta deposition in the aging dog: correlation with behavior[END_REF][START_REF] Heath | Behaviour problems in geriatric pet[END_REF][START_REF] Ruehl | Canine cognitive dysfunction[END_REF][START_REF] Azkona | Prevalence and risk factors of behavioural changes associated with age-related cognitive impairment in geriatric dogs[END_REF]. The most affected categories in our geriatric animals were "sleep/wake cycle" and "socio-environmental interactions," but cognitively impaired dogs were significantly more frequently affected in all categories and in the vast majority of items than the healthy aged group. The number of affected items was strongly related to the above-mentioned TDS, suggesting that the severity assessment carried out by the owner is greatly dependent on the number of contexts in which the dog shows impairment.

The two groups of aged animals were highly homogeneous regarding age, sex and reproductive status distributions, weight or body condition. Furthermore, there were not any significant hematological or biochemical differences between these two aged groups except for serum albumin that appeared slightly increased in the CU with respect to the CI group. It has been reported that mild decreases of serum albumin is related to an increased risk of mortality, disability, sarcopenia and frailty in people over 65 years [START_REF] Bourdel-Marchasson | Interpreting routine biochemistry in those aged over 65 years: a time for change[END_REF]. However, this type of information is scarce in veterinary practice. Recently it has been reported an increasing mortality risk in dogs as albumin decreased from 40 to 15 g/L (i.e., a very substantial decrease) [START_REF] Hayes | The acute patient physiologic and laboratory evaluation (APPLE) score: a severity of illness stratification system for hospitalized dogs[END_REF]. However, it is important to note that this study was carried out in hospitalized dogs, not in the normal population. The subtle serum albumin difference between our two aged groups (4.12 g/dl ± 0.52 in CU vs 3.74 g/dl ± 0.56 in the CI) was considered clinically irrelevant.

The primary objective of the present study was to analyze plasma levels of Aβ1-42 and Aβ1-40 peptides in relation to age and cognitive dysfunction in pet dogs. The concentrations of these two Aβ isoforms were correlated, and Aβ1-40 levels were

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ACCEPTED MANUSCRIPT 11 higher than Aβ1-42 levels in all the study groups. Both these features agreed with the results reported in the majority of similar studies conducted in humans [START_REF] Lopez | Plasma amyloid levels and the risk of AD in normal subjects in the Cardiovascular Health Study[END_REF][START_REF] Mayeux | Plasma Aβ40 and Aβ42 and Alzheimer's disease: relation to age, mortality, and risk[END_REF][START_REF] Schupf | Peripheral Aβ subspecies as risk biomarkers of Alzheimer's disease[END_REF][START_REF] Van Oijen | Plasma Abeta(1-40) and Abeta(1-42) and the risk of dementia: a prospective case-cohort study[END_REF][START_REF] Selkoe | Alzheimer's disease: genes, proteins, and therapy[END_REF].

We found that plasma Aβ peptides levels tended to decrease with age in cognitively intact dogs (i.e., in young, middle-aged and cognitively unimpaired aged groups). In particular, dogs less than 4 years old showed significantly higher Aβ1-42 and Aβ1-40 levels, as well as higher Aβ42/40 ratio values, than those more than 9 years old. A recent study carried out in healthy beagles (4-16 years old) showed that Aβ1-42, but not Aβ1-40, levels decrease slightly in cerebrospinal fluid (CSF) as brain amyloid deposition increases with age [START_REF] Head | Amyloid-beta peptide and oligomers in the brain and cerebrospinal fluid of aged canines[END_REF]. Our results suggest that the observed reduction of plasma Aβ1-42 and Aβ1-40 concentrations in cognitively unimpaired aged dogs might also be a consequence of increasing brain amyloid deposition, although a decreased production of mentioned peptides could not be excluded. In contrast, higher Aβ1-42 plasma levels and Aβ42/40 ratio were detected in dogs suffering from CDS when compared with cognitively unimpaired dogs. Thus, it appeared that the onset of the clinical condition was accompanied by an increase in Aβ1-42 production that might reverse the decrease in Aβ peptides levels observed in successful aging. These results are consistent with numerous studies on AD that have reported a relationship between elevated plasma Aβ peptide levels and the disease in humans [START_REF] Assini | Plasma levels of amyloid beta-protein 42 are increased in women with mild cognitive impairment[END_REF][START_REF] Blasko | Conversion from cognitive health to mild cognitive impairment and Alzheimer's disease: prediction by plasma amyloid beta 42, medial temporal lobe atrophy and homocysteine[END_REF][START_REF] Cosentino | Plasma β-Amyloid and Cognitive Decline[END_REF][START_REF] Lopez | Plasma amyloid levels and the risk of AD in normal subjects in the Cardiovascular Health Study[END_REF][START_REF] Mayeux | Plasma Aβ40 and Aβ42 and Alzheimer's disease: relation to age, mortality, and risk[END_REF][START_REF] Mehta | Plasma and cerebrospinal fluid levels of amyloid beta proteins 1-40 and 1-42 in Alzheimer disease[END_REF][START_REF] Schupf | Peripheral Aβ subspecies as risk biomarkers of Alzheimer's disease[END_REF][START_REF] Sobow | Plasma levels of alpha beta peptides are altered in amnestic mild cognitive impairment but not in sporadic Alzheimer's disease[END_REF][START_REF] Van Oijen | Plasma Abeta(1-40) and Abeta(1-42) and the risk of dementia: a prospective case-cohort study[END_REF][START_REF] Xia | A specific enzyme-linked immunosorbent assay for measuring beta-amyloid protein oligomers in human plasma and brain tissue of patients with Alzheimer disease[END_REF]. From those studies, it is becoming increasingly clear that the elevation in Aβ blood levels is an early event that could precede the onset of cognitive symptoms and increase the risk of developing AD [START_REF] Demeyer | Diagnosis-independent Alzheimer disease biomarker signature in cognitively normal elderly people[END_REF][START_REF] Jack | Serial PIB and MRI in normal, mild cognitive impairment and Alzheimer's disease: implications for sequence of pathological events in Alzheimer's disease[END_REF][START_REF] Fagan | Cerebrospinal fluid tau/beta-amyloid(42) ratio as a prediction of cognitive decline in nondemented older adults[END_REF][START_REF] Frisoni | The clinical use of structural MRI in Alzheimer disease[END_REF][START_REF] Jack | Hypothetical model of dynamic biomarkers of the Alzheimer's pathological cascade[END_REF]. Notably, when we separated dogs with a CDS diagnosis into two subgroups according to the severity of the syndrome (i.e., mCI and sCI), new differences emerged. Thus, the mCI animals showed significantly higher Aβ1-42 levels and Aβ42/40 ratio values than the sCI dogs and the CU dogs; no significant differences

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12 appeared between these latter groups. In contrast, plasma Aβ1-40 levels did not change according to diagnosis or severity of CDS. Again, these results were similar to studies reporting that a faster cognitive decline and/or progression of AD were accompanied by decreases in plasma Aβ1-42 [START_REF] Assini | Plasma levels of amyloid beta-protein 42 are increased in women with mild cognitive impairment[END_REF][START_REF] Cosentino | Plasma β-Amyloid and Cognitive Decline[END_REF][START_REF] Mayeux | Plasma Aβ40 and Aβ42 and Alzheimer's disease: relation to age, mortality, and risk[END_REF][START_REF] Schupf | Peripheral Aβ subspecies as risk biomarkers of Alzheimer's disease[END_REF][START_REF] Sobow | Plasma levels of alpha beta peptides are altered in amnestic mild cognitive impairment but not in sporadic Alzheimer's disease[END_REF][START_REF] Xia | A specific enzyme-linked immunosorbent assay for measuring beta-amyloid protein oligomers in human plasma and brain tissue of patients with Alzheimer disease[END_REF]. In a pilot study including 40 participants, we recently found that human plasma Aβ1-42 and Aβ1-40 concentrations were significantly higher in MCI patients than in non-demented controls. Furthermore, in that study, we observed that, although the different markers of the Aβ pool in blood did not vary significantly between MCI and AD groups, they tended to decrease in the AD patients with the lower MMSE [START_REF] Pesini | Differences in beta amyloid pool in blood discriminate between amnesic mild cognitive impairment patients and healthy controls[END_REF]. The amyloid hypothesis states that AD pathology starts when Aβ peptides, particularly Aβ1-42, begin to aggregate and precipitate in the interstitial spaces of the brain. In this situation, diffusion of brain Aβ toward the ventricular system will be severely hampered, leading to a reduction in Aβ1-42 levels in the CSF that has been shown to herald cognitive decline [START_REF] Blennow | Cerebrospinal fluid and plasma biomarkers in Alzheimer disease[END_REF][START_REF] Demeyer | Diagnosis-independent Alzheimer disease biomarker signature in cognitively normal elderly people[END_REF][START_REF] Fagand | Cerebrospinal fluid biomarkers of Alzheimer's disease[END_REF][START_REF] Shaw | Cerebrospinal fluid biomarker signature in Alzheimer's disease neuroimaging initiative subjects[END_REF]. At the initial stage of the disease, probably without extensive capillary damage, the increased Aβ blood levels observed in humans and, in this study, in mCI dogs might reflect the increasing peptide levels in brain tissue. The subsequent drop of plasma Aβ1-42 observed in both canines and humans as the disease progresses could be related to the deposition of Aβ around capillaries, which would seal off the blood-brain barrier.

Thus, the results of the present work revealed a similar pattern of plasma Aβ changes in dogs and humans during the progression of cognitive impairment. This parallelism reinforces the similarities between canine CDS and AD and suggests that those dogs suffering mild cognitive impairment could model human MCI, whereas those displaying severe impairment could model early or mild sporadic AD. A longitudinal study (already in progress), rather than a cross-sectional approach, will help better explore these disease-related changes in plasma Aβ peptides in dogs.

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13 Some limitations still remain regarding classification of the cognitive status in aged dogs. Notably, the available tools and criteria for diagnosis of age-associated cognitive impairment, mild cognitive impairment and dementia in canines (or other animals) are not as developed as in humans. In addition, it is possible that a CDS diagnosis in dogs could include dementias other than AD-like dementia (e.g., vascular dementia). We found that our owner-based TDS was strongly correlated with the RDS obtained with the test of Rofina et al. [START_REF] Rofina | Cognitive disturbances in old dogs suffering from the canine counterpart of Alzheimer's disease[END_REF]. In RDS, the severity of cognitive impairment was not assessed by the owner, as it is in our questionnaire, but a pre-established score was arbitrarily assigned to each item. These authors showed a correlation between their dementia score and several brain lesions, including amyloid. In contrast, we did not find a correlation between plasma Aβ peptide levels and any cognitive score in our aged dogs (CU and CI groups). However, if we considered only the CU and mCI animals, plasma Aβ1-42 concentrations and the Aβ42/40 ratio significantly correlated with TDS, RDS and the total number of affected items. These results are congruent with the idea that changes in Aβ blood levels could be an early marker for the onset of brain amyloid pathology and cognitive impairment. At more advanced stages, probably after development of extensive cerebral amyloid angiopathy, plasma peptide levels would not correlate with ongoing brain pathology and cognitive dysfunction [START_REF] Jack | Serial PIB and MRI in normal, mild cognitive impairment and Alzheimer's disease: implications for sequence of pathological events in Alzheimer's disease[END_REF][START_REF] Frisoni | The clinical use of structural MRI in Alzheimer disease[END_REF][START_REF] Jack | Hypothetical model of dynamic biomarkers of the Alzheimer's pathological cascade[END_REF].

Furthermore, in spite of the uncertainties of the veterinary clinical criterion standard, the AUC obtained for plasma Aβ1-42 levels and the Aβ42/40 ratio would allow for discrimination between mCI and CU dogs with a sensitivity and specificity close to, although still lower than, that considered suitable for most AD biomarkers. In our opinion, this is of the greatest interest because, from any practical point of view, it is at these early stages of AD when the diagnoses should be improved, and the canine model would be particularly useful in this context. 1º) plasma Aβ was analyzed for the first time in relation to age in a pet dog population. 2º) aged dogs with cognitive dysfunction have higher Aβ42 plasma levels than controls. 3º) Aβ42 levels were higher in mildly cognitive impaired than in severely affected dogs. 4º) plasma Aβ42 could be an early biomarker for cognitive dysfunction in aged dogs. 5º) canine cognitive dysfunction syndrome appears as an excellent natural model for early AD.

A C C E P T E D

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  plasma concentration of Aβ1-42, Aβ1-40 and the Aβ-42/40 ratio were defined as dependent variables whose group mean values were compared using Mann-Whitney U tests. Correlations were analyzed using Spearman's rank correlation tests. Chi-square tests were used to compare the distribution of categorical demographic or cognitive variables among groups. The differences in serum biochemical parameters were assessed either by ANOVA or Kruskal-Wallis test, if the parameter distribution was parametric or non-parametric, respectively. Receiver operating characteristic (ROC) curves were used to assess the accuracy of the dependent variables in classifying the presence versus absence of cognitive impairment. Calculations were carried out using the statistical program SPSS 15.0 for Windows (SPSS, Inc., Chicago, IL, USA), and p ≤ 0.05 denoted statistical significance.

were 2 .

 2 5 times and 1.8 times greater, respectively, in the mCI than in the aged unimpaired CU group (p < 0.01) (Table4, Fig1B). In concordance with these results, the ability of the Aβ1-42 level and the Aβ42/40 ratio, but not the Aβ1-40 level, to discriminate between CU and mCI animals (instead of between CU and CI groups) substantially improved (AUC = 0.78; p < 0.001) (Fig 2A-C). Notably, when CU and mCI animals were plotted together (without the sCI), both the Aβ1-42 level and the Aβ42/40 ratio showed a weak but significant correlation with TDS (r = 0.44 and 0.47, respectively; p ≤ 0.001), total number of positive items (r = 0.42 and 0.53, respectively;

Fig 1

 1 Fig 1 Bar graphs for plasma Aβ1-42 and Aβ1-40 concentrations (in pg/ml) and for the

Table

  ).

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	In total, 88 animals were enrolled in the study. The subjects were sorted into four
	groups: i) young (YG, 1-4 years of age, n=9), ii) middle aged (MA, 5-8 years of age,
	n=10), iii) cognitively unimpaired aged (CU, ≥9 years of age, n=31), and iv) cognitively
	impaired aged (CI, ≥9 years of age, n=38). The animals were treated according to the European and Spanish legislations on animal handling (86/609/EU, Real Decreto 1201/2005) and the experiments and procedures were approved by the Ethical M A N U S C R I P T Committees of the University of Santiago and Zaragoza.
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	5

Table 1 .

 1 Demographic data in the studied canine population.

	Group		Males Females Neutered	Age (months)	Weight (Kg)	Body condition (1-5) †
			(%)		(%)		Mean ± SD	Mean ± SD	Mean ± SD
	YG	(n=9)	55.6	44.4	33.3		31.1 ± 17.7	11.8 ± 5.7	3.2 ± 0.4
	MA	(n=10)	30.0	70.0	40.0		83.2 ± 16.7	12.8 ± 10.6	3.2 ± 0.4
	CU	(n=31)	48.4	51.6	41.9		146.4 ± 35.2	12.2 ± 7.7	3.5 ± 0.8
	CI	(n=38)	60.5	39.5	42.1		152.6 ± 25.1	14.8 ± 9.2	3.5 ± 0.6
			p= 0.35 a	p= 0.97	a	p= 0.15 b	p= 0.59	c	p= 0.42	a
	YG: young; MA: middle aged; CU: cognitively unimpaired aged; CI: cognitively impaired aged.
	† Body condition scale: 1(too thin) -3(thin) -3(ideal) -4(heavy) -5(too heavy).

a Chi-square test, b Mann-Whitney U tes tbetween CU and CI groups, c Kruskal-Wallis test.

.

Table 2 .

 2 Frequency distribution (percentage) of impaired categories and items in each group. Middle aged; CU: cognitively unimpaired aged; CI: cognitively impaired aged. † p values corresponding to statistical differences between groups 3 and 4. *, p<0.05; **, p<0.01; ***, p <0.001; NS, Nonsignificant difference.

	Category	Items	YG	MA	CU	CI	p †
			(n=9)	(n=10)	(n=31)	(n=38)
	Sleep-wake cycle		0	0	25.8	81.6	***
		Walking/Pacing at night	0	0	0	42.1	***
		Vocalizing (barking/whining) at night	0	0	3.2	15.8	NS
		Sleeping less at night	0	0	0	31.6	**
		Sleeping noticeably more during the day	0	0	48.4	81.6	**
		Switching between insomnia and hypersomnia	0	0	0	23.7	**
	Socio-environmental interactions	0	0	9.7	97.4	***
		Decrease in greeting owners	0	0	0	50	***
		Decrease in soliciting attention from the owners	0	0	6.5	34.2	**
		Increase in following the owners around the house	0	10	19.4	47.4	*
		Decrease in playing with the owners	0	0	25.8	73.7	***
		Decrease in playing with other dogs	0	10	12.9	60.5	***
		Changes in personality (irritability, new fears,	0	0	9.7	68.4	***
		lack of interest on stimuli)
	House-training and commands	0	0	0	63.2	***
		Starting urinate/defecate in the house	0	0	0	42.1	***
		Decrease in signalling to go out for eliminating	0	0	0	23.7	**
		Decrease in urine marking (non-castrated males)	0	0	0	28.9	**
		Decrease in responding to prior learned	0	0	3.2	36.8	**
		commands
	Disorientation		0	0	6.5	68.4	***
		Staring into space (star gazing) or getting stuck	0	0	9.7	57.9	***
		Getting lost in the house or during routine walks	0	0	0	42.1	***
		Wandering (aimless walking) in the house	0	0	0	39.5	***
		Trying to pass through narrow places	0	0	0	23.7	**
		Standing at the wrong side of the door to go out	0	0	0	23.7	**
		Difficulty in navigating around or over obstacles	0	0	0	31.6	**
		Decrease in recognizing familiar people	0	0	0	7.9	NS
	YG: Young; MA:

Table 3 .

 3 Amyloid β-peptides concentrations (pg/µl) and Aβ42/40 ratios in each group of study. : young; MA: middle aged; CU: cognitively unimpaired aged; CI: cognitively impaired aged. Different letters indicate significant differences between groups (capital letters: p<0.001; lower case letters: p<0.05).

	Group	Aβ40	Aβ42	Aβ42/40
			Mean ± SD
	YG	88.7 ± 35.3 ma,cu	59.2 ± 58.6 cu	0.59 ± 0.38 CU
	MA	61.5 ± 19.4	31.4 ± 32.5	0.44 ± 0.29
	CU	64.7 ± 24.1	23.3 ± 12.0	0.35 ± 0.20
	CI	79.1 ± 33.0	46.1 ± 53.7 cu	0.52 ± 0.35 cu

YG

Table 4 .

 4 Amyloid β-peptides concentrations (pg/µl) and Aβ42/40 ratios in the aged groups. : cognitively unimpaired; mCI: mild cognitive impairment; sCI: severe cognitive impairment. Letters m/M indicate significant differences with regard to the mCI group (capital letters: p<0.01; lower case letters: p<0.05).

	Cognitive	Aβ40	Aβ42	Aβ42/40
	status
			Mean ± SD
	CU	64.7 ± 24.1	23.3 ± 12.0 M	0.35 ± 0.20 M
	mCI	82.1 ± 38.8	58.3 ± 65.5	0.65 ± 0.37
	sCI	75.3 ± 24.7	31.1 ± 29.5 m	0.37 ± 0.26 m

CU


Mario Ruiz
email: ruizgm@ibgm.uva.es

Diego Sanchez

Inmaculada Canal
email: inmaculada.canal@uam.es

Angel Acebes
email: a.acebes@cajal.csic.es

Maria D Ganfornina

D Glaz

Nlaz Mario Ruiz

Sex-dependent modulation of longevity by two Drosophila homologues of human Apolipoprotein D, GLaz and NLaz

Keywords: ApoD, Lazarillo, lifespan, aging, reproduction, stress-response 30 text pages, 7 figures, 2 tables, 3 supplementary figures, 2 supplementary tables

Apolipoprotein D (ApoD), a member of the Lipocalin family, is the gene most upregulated with age in the mammalian brain. Its expression strongly correlates with agingassociated neurodegenerative and metabolic diseases. Two homologues of ApoD expressed in the Drosophila brain, Glial Lazarillo (GLaz) and Neural Lazarillo (NLaz), are known to alter longevity in male flies. However, sex differences in the aging process have not been explored so far for these genes. Here we demonstrate that NLaz alters lifespan in both sexes, but unexpectedly the lack of GLaz influences longevity in a sex-specific way, reducing longevity in males but not in females. While NLaz has metabolic functions similar to ApoD, the regulation of GLaz expression upon aging is the closest to ApoD in the aging brain. A multivariate analysis of physiological parameters relevant to lifespan modulation uncovers both common and specialized functions for the two Lipocalins, and reveals that changes in protein homeostasis account for the observed sex-specific patterns of longevity.

The response to oxidative stress and accumulation of lipid peroxides are among their common functions, while the transcriptional and behavioral response to starvation, the pattern of daily locomotor activity, storage of fat along aging, fertility, and courtship behavior differentiate NLaz from GLaz mutants. We also demonstrate that food composition is an important environmental parameter influencing stress resistance and reproductive phenotypes of both Lipocalin mutants. Since ApoD shares many properties with the common ancestor of invertebrate Lipocalins, we must benefit from this global comparison with both GLaz and NLaz to understand the complex functions of ApoD in mammalian aging and neurodegeneration.

Research highlights

GLaz and NLaz alter longevity in a sex-specific manner.

Like ApoD, GLaz increases its expression in the aged brain, but only in females.

A multivariate analysis reveals specialized and redundant Lipocalin functions.

Response to oxidative stress and control of lipid peroxidation are common functions.

NLaz and GLaz alter metabolism and reproduction in different and complementary ways.

A C C E P T E D

M A N U S C R I P T

Introduction

Aging and the regulation of an organism lifespan are complex traits that are the result of multiple factors, many of which are clearly inheritable. This fact has guided the quest for genes that regulate longevity. Many genetic interventions that alter the length of life have been documented in model organisms ranging from nematodes to rodents (for recent reviews see Fontana and others 2010;[START_REF] Kenyon | The genetics of ageing[END_REF]. They reveal the involvement of a basic set of conserved pathways, being the nutrient sensing pathways (the insulin and insulin-like growth factor signalling (IIS) and the target of rapamycin (TOR) pathway) the best known examples (Biteau and others 2010;[START_REF] Broughton | Insulin/IGF-like signalling, the central nervous system and aging[END_REF][START_REF] Karpac | Insulin and JNK: optimizing metabolic homeostasis and lifespan[END_REF][START_REF] Wang | Autophagy in cellular growth control[END_REF]. These studies are leading the search for genes in humans that perform similar functions and can become candidates for pharmacological interventions to achieve a longer and healthier life. Either genetic association studies or transcriptome analysis are the main approaches to aging research in humans (Fontana and others 2010;[START_REF] Kenyon | The genetics of ageing[END_REF]Passtoors and others 2008). It is striking that in a meta-genome study where the changes in brain transcriptome have been compared between mice, monkeys and humans, Apolipoprotein D (ApoD) was revealed as the most robust agedependent up-regulated gene in the brain conserved across species (Loerch and others 2008). The same is true for a meta-array analysis using mouse, rat and human expression data in different tissues, where ApoD appears as the gene most consistently over-expressed with age (de Magalhaes and others 2009). Not surprisingly, the expression of ApoD is highly boosted by a collection of traumatic, pathological and degenerative nervous system conditions in humans as well as by cancer and age-related metabolic diseases (reviewed by Van Dijk and others 2006).

It is widely accepted that many of the genes that regulate longevity participate in defence or repair mechanisms that counteract the accumulation of damage to cell components with age. Thus, many of these genes are also thought to be important for the onset of a wide array of diseases where age is a major risk-factor. Neurodegenerative diseases are no exception. The study of ApoD and its homologous genes in model organisms should therefore prove relevant to the understanding of aging in general, and of the aging nervous system in particular.

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ApoD is a member of the Lipocalin family, small secreted lipid-binding proteins. At least three other Lipocalin genes are also expressed in the nervous system of mammals:

retinol binding protein, RBP (MacDonald and others 1990), L type prostaglandin D synthase, L-PGDS (Kanekiyo and others 2007), and Lipocalin 2 (also called NGAL) (Lee and others 2009). In Drosophila, two genes are the closest homologues to ApoD: Glial Lazarillo (GLaz) and Neural Lazarillo (NLaz). They are expressed in the nervous system in a cell-type specific manner (Sanchez and others 2000). A third Drosophila Lipocalin, Karl, is not expressed in the nervous system (Hull-Thompson and others 2009). Both GLaz and NLaz are known to regulate stress resistance and lifespan in male flies (Hull-Thompson and others 2009;Sanchez and others 2006b;Walker and others 2006): loss-of-function mutants have a reduced longevity while gain-of-function manipulations result in extended lifespan.

GLaz also influences fat storage (Sanchez and others 2006b) while NLaz regulates the metabolic response to stress by systemic inhibition of the IIS pathway (Hull-Thompson and others 2009). Protection against oxidative stress in the nervous system has also been demonstrated for mammalian ApoD (Ganfornina and others 2008), revealing a sufficient degree of functional conservation between ApoD and the Lazarillo genes expressed in the nervous system of Drosophila.

Single gene analyses of aging mutants often overlook pleiotropy and effects of other gene family members expressed in the same tissues. Also, in a few cases, the original interpretations derived from studies of classical longevity mutants (Lin and others 1998;Rogina and others 2000) have been challenged when other physiological, environmental (Baldal and others 2006a), or genetic (Toivonen and others 2007) factors have been taken

into account. This fact highlights the need for a comprehensive understanding of the many effects of single gene mutations, as well as of the gene-environment interactions that are relevant to aging and lifespan determination.

Particularly, sex differences in the aging process are a very relevant issue that has not been explored so far for these genes. Almost all the work published on this group of Lipocalins (ApoD/Lazarillo) has been performed in males only. This work was triggered by the consistent observation that one of the Drosophila Lipocalins (GLaz) alters longevity in a sex-dependent manner in any genetic background tested: only null mutant males reduce

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their longevity, while null mutant females live as long as wild type flies. The lack of NLaz, on the other hand, shortens lifespan in both sexes. This work is at the same time an attempt to understand the origin of such a sex-specific phenotype, and an exhaustive search for the physiological processes in which these Lipocalins intervene, with the final goal of uncovering the interesting labor division structure of the two Drosophila Lipocalins homologous to ApoD.

Material and Methods

Fly strains and husbandry.

Flies were grown in a temperature-controlled environmental incubator at 25ºC, 60%

relative humidity, under a 12h light-dark cycle.

Food recipes: (A) "Valladolid-standard" food: wet yeast 84 g/l, NaCl 3.3 g/l, agar 10 g/l, wheat flour 42 g/l, apple juice 167 ml/l, and propionic acid 5ml/l. (B) "Carolina"

Instant Food (Carolina Biological Supplies). (C) "Caltech" food: dry yeast 15 g/l, agar 4.5 g/l, dextrose 50 g/l, sucrose 25g/l, corn meal 83 g/l, phosphoric acid 0.6 ml/l, and propionic acid 4ml/l..

GLaz and NLaz null mutants were generated in a w 1118 background as previously described (Rong and others 2002;Sanchez and others 2006b). Mutations were outcrossed into a Canton-S wild type strain for five generations to obtain a homogeneous background with less than 5% of the original genomic background. Isogenic sister lines containing the wild type allele of GLaz and NLaz (line G10: GLaz +/+ , NLaz +/+ ), or the mutant allele (lines G2: GLaz -/-, NLaz +/+ and N5: GLaz +/+ , NLaz -/-) were selected by PCR screening for GLaz, and PCR followed by SceI restriction digest for NLaz. In a last round of outcrosses, a set of fly strains with white eyes were generated in the same way outcrossing with w 1118 -CS 10. The line w 1118 -CS 10 , a gift from Seymour Benzer (Caltech), is a 10 generation outcross of w 1118 into the Canton-S background. A set of lines with white eyes were thus generated: lines CGW and CNW14 as wild type isogenic controls (GLaz +/+ , NLaz +/+ ), GW line (GLaz -/-, NLaz +/+ ) and NW5 line (GLaz +/+ , NLaz -/-). Most of the experiments were carried out with G10 as wild type control and G2 and N5 as experimental lines unless otherwise noted. Salamanca).

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Wolbachia test.

The absence of infection by

Lifespan analysis.

At least 100 flies of each genotype were collected within 24h of eclosion, separated by sex under brief CO 2 anaesthesia, housed in groups of 25, and maintained at 25ºC. Dead flies were counted and surviving flies were transferred to new vials with fresh food twice a week.

Oxidative stress, starvation, and desiccation resistance.

Flies collected as described for the longevity analysis were separated by sex in groups of 25 when they were 3 days old. Application of different stressors was performed as follows.

Paraquat treatment: After a period of dry starvation (3 hours) flies were transferred to vials with filter papers soaked with 1 ml of 10% sucrose-20mM paraquat (Sigma).

Incubation proceeded in the absence of light and deaths were scored every 4-8 h.

Wet starvation treatment: Starting at 3 days of age, flies were transferred to vials with 1% agar in water. Dead flies were scored every 4-8 h.

Dry starvation treatment (desiccation):

Starting at 3 days of age, flies were transferred to empty vials. Dead flies were scored every 4-8 h.

Body weight, fat content, and protein content.

Total wet weight, dry weight after evaporation of water, and fat-free dry weight after extraction of neutral lipids with diethyl ether, was performed as previously described (Sanchez and others 2006b). The protein content per fly was determined with the

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microBCA kit (Pierce). Three independent experiments with measurements in triplicate were performed at 3 and 30 days of age.

Lipid peroxidation levels.

A spectrophotometric assay was used to determine the concentration of free malondialdehyde (MDA-586, Bioxytech) as previously described (Sanchez and others 2006b). Three independent experiments with measurements in triplicate were performed at 3 and 30 days of age.

Population reproductive output.

Three populations of 10 males x 10 virgin females per genotype were housed in 50 ml food bottles (250ml plastic bottle). Eggs laid were counted every day after transferring parents into a new bottle, and the number of adults emerging form each bottle was counted till the culture was exhausted. Egg and adult progeny production was scored during 7 days (age of parents: 5-11 days). An estimate of fecundity and fertility per female in the population per day was calculated.

Courtship behaviour and reproduction.

Courtship and mating tests were performed during 60 min using male-female pairs of 4-5 day-old flies under a watch glass used as an observation chamber, as described previously (Ferveur and others 1995). The courtship index (C.I.) represents the proportion of time a male spends actively courting the female for 10 min. Copulation features as mating time, mating latency and mating performance (%) were scored for 60 min. Offspring numbers and sex ratio (male/female) from each mated female that yielded progeny were scored during two weeks.

2.9. Hunger driven short-term food intake behaviour.

Two groups of 20 flies per sex, previously food-deprived for 19h on 1% agar in water, were fed for 5 minutes on 4ml of 10% sucrose, 1% agar, supplemented with 0.5% food dye FD&C Blue No.1 (E-133 European Union, provided by Proquimac). After feeding, the flies were anesthetized with CO 2 and homogenized in PBS. After a short spin to pellet tissue

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debris, absorbance of the homogenate was measured at 625nm. Background absorbance was corrected with fly samples of the same genotype subjected to the same procedure without the dye. The experiment was repeated twice and performed with the white-eyed fly lines (see above) to avoid disturbances of the absorbance spectrum due to the eye pigment.

Circadian spontaneous locomotor activity.

Individual flies at 3 days of age were transferred to monitor tubes containing fresh food.

Their locomotor activity was monitored using the Drosophila Activity Monitoring System (Trikinetics, Waltham, MA) with a cumulative sampling rate of 15 min, in a temperaturecontrolled environmental incubator at 25ºC, 60% relative humidity, under a 12h light-dark cycle. Five monitors were used, each housing 5 flies/sex/genotype and a sixth monitor recorded light, temperature and humidity. Monitoring was performed for 6 days, but analysis included data starting on day 3 (6 days of age) to avoid variations due to habituation to the housing conditions.

Climbing ability.

Flies were collected and separated by sex in groups of 10 as described for the longevity analysis. Tests were performed as previously described (Sanchez and others 2006b), after a minimum of 24 hours after anaesthesia, at 3 and 30 days of age.

qRT-PCR: GLaz and NLaz gene expression.

Flies of each genotype were collected within 24 hours of eclosion, separated by sex under brief CO 2 anaesthesia, and housed in groups of 25. They were transferred to fresh food vials twice a week until the day of processing (3, 30 and 60 days). When preceded by wet starvation, gene expression measurements were performed at 15 hours of treatment (see above). We separated heads and bodies. RNA extracted from heads should represent a good approximation to the expression in the brain, which makes most of the head tissue. RNA extracted from the body, which includes the relatively large thoracic ganglia, represents an approximation to the global expression level in whole flies.

Heads from 50 flies were cut and total RNA was extracted using TRIzol (Invitrogen).

RNA concentration was measured with a Nanodrop spectrophotometer. Reverse

A C C E P T E D M A N U S C R I P T

transcription was done with the PrimeScript TM RT reagent Kit (Takara) according to the manufacturer instructions by using Oligo-dT primers and random hexamers. Primers of equal amplification efficiency were designed for GLaz (5'-GCGAACAATCGAAGTTTTCC-3' and 5'-ACAAGATGGCGAAGTTCTCG-3'), NLaz

(5'-CGAGTACGCAGCCTATCCAT-3' and 5'-CCAGGTAGTTGGCCTTCGT-3') and the ribosomal protein L18 (RPL18, endogenous control) (5'-AGAACCGACCCCAAATCC-3' and 5'-CGACCACGATGGTAGACTCC-3'). Quintuplicate PCR reactions were performed for each RNA sample using the SYBR ® Premix Ex Taq TM (Takara) according to the manufacturer instructions in a Rotor-Gene RG-3000 thermal cycler (Corbett Research).

Cycling conditions were 30 sec 95º, (5 sec 95º, 15 sec 55º, 15 sec 72º) x 35.

Melting curves were established for all conditions to check for the absence of unspecific amplifications.

RNA transcription levels were determined by the method of direct comparison of C T values and relative quantities calculated by the ∆∆C T method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF].

Transcripts were normalized to RPL18 for each condition. We transform the data as Log 2 - ∆∆Ct and then represent them as fold changes. In this way, up-and down-regulations are symmetrically scaled around one. Statistically significant differences of pairwise gene transcriptional changes were evaluated with a Mann-Whitney U-test, using ∆C T of each replica (calculated by subtracting the average CT of the reference gene). The level of significance was set at p<0.05.

Statistics and principal component analysis.

Statistical analyses were performed with Statgraphics plus (v 5.0) software. Student's t-Test was used to assess two-sample comparisons (with the exception of qRT-PCR data, see above) with p<0.05 as threshold for significant changes.

To find patterns of covariation among all the physiological parameters measured, we performed a principal component analysis (PCA). The average of each parameter was arranged in two matrices (Tables S1-S2), one with 25 variables measured at 3 days of age, and another with 7 variables measured at 3 and 30 days of age. Neither longevity data nor Lipocalin expression data were included, in order to render the analysis "blind" to the genotype (the dependent variable) and lifespan (the variable we want to explain). The

A C C E P T E D M A N U S C R I P T

analysis was also blind to sex and age, since it is performed for 6 independent entries (2 sexes per genotype) or 12 independent entries (6 entries per age).

Results and Discussion

3.1. The nervous system Lipocalins GLaz and NLaz influence longevity in a sexspecific manner.

The loss of either GLaz (Sanchez and others 2006b) or NLaz (Hull-Thompson and others 2009) is known to reduce the lifespan of male flies. However, here we report that females reduce their lifespan when NLaz is absent, but their lifespan is similar to control female flies in the absence of GLaz (Fig. 1A,B). The same results are obtained before and after changing the genetic background for both mutations (from w 1118 to CantonS, not shown; median survival effects stated in Fig. 1 legend). The absence of Wolbachia infection, as a potential lifespan-reducing cause, was confirmed by PCR tests (see Fig. S1 and section 2.2.

in Methods).

These observations led us to search for causal links that might explain how GLaz regulates longevity differently in males and females, and to find the fine structure of functional relationships between the two nervous system Drosophila Lipocalins.

Male and female flies differ in their temporal and spatial regulation of Lipocalin genes expression.

Since both Lipocalins are expressed within and outside the nervous system (Hull-Thompson and others 2009; Sanchez and others 2000; Sanchez and others 2006b), we investigated how much of their expression is contributed by the brain in young flies. We focused our study on the expression changes in the brain, either through aging or upon stress. These data can then guide the comparisons between the fly Lipocalins and the mammalian homologue, ApoD, known to be the most up-regulated gene in the aged brain.

Brains of male flies have stable expression levels of both Lipocalins throughout life;

NLaz expression being higher than that of GLaz at all ages explored (Fig. 1C, triangles). In contrast, the brains of females show interesting and opposite expression changes with age:

GLaz increases and NLaz decreases with aging (Fig. 1C, circles). Curiously, it is the sum of

A C C E P T E D M A N U S C R I P T
the two Lipocalins what is kept roughly constant throughout aging in females. The increase of GLaz expression with age in female brains echoes the pattern followed by ApoD in mice, macaques, and humans (Loerch and others 2008).

The fact that NLaz expression in females shows an opposite pattern, suggests that different regulatory elements are responsible for the temporal control of each Lipocalin, and that GLaz gene regulation must have commonalities with the age-dependent networks controlling ApoD expression in the mammalian CNS. Also, these gene-regulation networks differ with sex for both Drosophila Lipocalin genes.

Interestingly, the level of Lipocalins in the brain of young flies correlate with their longevity reduction when each Lipocalin is missing. Note that GLaz expression is the lowest in young wild type females, and its absence does not alter longevity. These data support the idea that early adulthood represents a critical period for Lipocalin function, affecting parameters that will result in lifespan alterations later on.

The relative contribution of head and body in Lipocalin expression also varies with sex (Fig. 1D). In young females, GLaz has the highest ratio body/head. In males, the expression of both Lipocalins is higher in the brain than in the body, with NLaz again showing higher expression levels than GLaz.

Starvation triggers a differential expression of Lipocalins in the brain.

The males of both mutants had been already described as starvation sensitive (Hull- Given that longevity is the result of an intricate puzzle of interactions between genetic components and physiological and environmental variables, we need to uncover the full complexity of effects of a single mutation in order to learn how each gene alters the network of causal links underlying lifespan regulation.

With that in mind, we have undertaken an analysis of multiple parameters and their dependence on NLaz and GLaz expression. We have screened variables including metabolic (levels of various metabolites, stress-nutrition relationships), behavioral (reproduction, food intake, locomotor activity), as well as stress resistance, and analyzed them by principal component analysis (PCA). PCA has been successfully used in a multitrait study by Baldal and others (2006b) pertaining starvation resistance and aging evolution, and by Andersen and others (2005), in a study uncovering how heat stress and age induced maternal effects influence physiological parameters in the offspring. This type of analysis extracts features of the parameters variation as a set of new variables (the principal components, PCs) that are uncorrelated with one another and successively account for maximal amounts of variation. Our analysis was performed with 25 variables at 3d of age (Fig. 3A-B; Fig. S2A-B), and a subset of 7 variables mainly related to metabolic parameters at 30d of age (Fig. 3C, Fig. S2C-D). Values obtained for all variables are listed in supplementary tables (Tables S1-S2). The PCA has uncovered sex, genotype, and agespecific patterns.

Some life history parameters clearly separate genotypes. The two principal components explaining 69% of the variation in the sample studied at 3d of age separate wild type flies from either mutant, and in turn segregate each mutant into opposite corners of the multivariate space (Fig. 3A). This pattern reflects the existence of specialized functions

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separating Lipocalin mutants from each other (component 1), as well as common functions separating both mutants from the wild type (component 2). For example, lipid peroxidation and relative fat content contribute to the separation of wild type from either mutant (black labels in Fig. 3B). Variables in the domain of reproduction, locomotor activity, food intake and metabolism (relative water content, starvation resistance) separate GLaz from NLaz mutants (red and green labels in Fig. 3B).

Other life history parameters contribute to sex or age differences regardless of genotype (Fig. S2). Metabolic parameters (wet weight, protein content or lipid peroxidation), response to stress (starvation, desiccation or paraquat resistance) and diurnal vs. nocturnal locomotor activity are important variables in the separation of sexes in all genotypes (Fig. S2A-B). In the analysis of variables at two ages (Subset 2) it is interesting that only females are segregated by age in the component 1 by 2 plot (explaining 63% of de variance).

Metabolic parameters (relative water content in opposition to relative fat) and climbing ability have the most weight for the separation of young female flies from old female flies in all genotypes (Fig. S2C-D).

Sex-specific modulation of longevity by GLaz is dependent on metabolic parameters related to protein homeostasis.

Studying the multivariate space, we searched for components that would cause GLaz mutant females to cluster with WT females, and would therefore explain the observed longevity curves. Interestingly, the stronger association of GLaz -/-females with wild type flies arises in the PCA analysis performed for the subset of 7 variables measured at both 3 and 30d of age (Fig. 3C). The association only occurs for young GLaz mutant females, further supporting the idea that the function of Lipocalins in early adulthood is crucial for longevity determination. Young wild type females, with their low levels of GLaz expression, show a combination of parameters similar to that of GLaz -/-young females. Old GLaz mutant females, however, are displaced to the opposite corner of the component 2 by 3 plane of the multivariate space (arrows in Fig. 3C). The variables with more weight in the "mutant GLaz females -wild type" association are the protein content and relative fat free content (with a negative weight, red labels) and the wet weight and relative water content

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(with a positive weight, green labels). Variables related to lipids (relative fat content or lipid peroxidation levels) do not contribute to this association.

Our analysis indicates that the patterns of metabolism, especially during early adulthood, contribute importantly to the final outcome in longevity, resulting in a shorter lifespan in NLaz mutants and in GLaz mutant males, but a similar lifespan in GLaz mutant females and wild type flies. Some of the parameters included in our PCA analysis are further described and discussed hereafter.

Energy intake and homeostasis are altered through aging in the absence of

Lipocalins.

The loss-of-function mutants of both Lipocalins show pleiotropic effects at different levels of energy management (intake-balance-expenditure, Fig. 4A) that contribute to their segregation in the multivariate space (Fig. 3A-B). Particularly, the PCA analysis shows that energy management differences between genotypes are part of the functional specialization between both Lipocalins, and also contribute to the unaltered lifespan in females lacking GLaz (Fig. 3C).

In addition, the behavioral response to starvation is increased in young NLaz -/-flies, but not in GLaz -/-flies (Fig. 4B). We have previously described that overall weight in males is increased in the absence of NLaz (Hull-Thompson and others 2009), and decreased in the absence of GLaz (Sanchez and others 2006b). Here we show that NLaz -/-flies start life with a different body size and increase their fat content with age, while GLaz -/-mutants maintain a low fat content throughout life in both sexes (Fig. 4C). These results are in line with the food intake increase observed in NLaz mutant flies (Fig. 4B).

Energy storage (in the form of fat storage) is clearly altered in opposite directions by the two Lipocalins. However, only male and not female GLaz mutants shorten their lifespan.

On the other hand, NLaz mutants show both increased food intake (this work) and increased IIS activity (Hull-Thompson and others 2009). In order to understand the relevance of food storage and food intake in lifespan determination we need to know how Lipocalins influence energy expenditure. We have analyzed locomotor activity patterns and reproduction as the major forms of energy output.

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Lipocalins influence the patterns of daily energy expenditure in locomotor

activity.

The patterns of daily spontaneous locomotor activity also differ with genotype and sex (Fig. 5), showing complementary patterns. Overall activity is higher in GLaz -/-males (Fig. 5A,C), which are more active than wild type flies especially at night (Fig. 5D). NLaz -/- females are overall less active (Fig. 5B,C), but the difference is mostly due to diurnal activity (see Table 1).

Shortened resting periods at night have been reported to correlate with aging (reviewed by Grotewiel and others 2005). Our data show that the highest sex difference within a given genotype is the large increase in nocturnal activity of GLaz -/-males (Fig. 5D) compared to mutant females of the same genotype. Interestingly, GLaz -/-female activity is more similar to the wild type pattern (Fig. 5 and Table 1). One could argue that this effect will contribute to an accelerated senescence of GLaz -/-males and therefore a shortening of lifespan occurring only in males of this genotype. However, the lower nocturnal activity observed in males NLaz -/-is not accompanied by an increase in lifespan. Our data support the idea that the amount of activity per se does not correlate with longevity, in agreement with Koh and others (2006), who report sleep fragmentation rather than total sleep time as the relevant parameter influencing longevity.

Reproductive output and courtship behavior is differentially altered in each

Lipocalin mutant.

Reproductive parameters separate GLaz from NLaz mutants in the multivariate space analyzed at 3d of age (Fig. 3B). A lower fecundity is observed in both mutants (Fig. 6A), but fertility of NLaz -/-mutants does not differ from wild type (Fig. 6B). An estimation of developmental success, or "egg quality" (% of eggs reaching adulthood) is higher in NLaz -/- mutants and lower in GLaz -/-mutants compared to wild type flies (Fig. 6C). We can therefore discard the existence of major fertilization or developmental problems at least in NLaz mutants. However, given that GLaz mRNA is detected very early in embryogenesis (0-2 hours embryos, Sanchez and others 2000), the GLaz -/-eggs that do not reach adulthood

A C C E P T E D M A N U S C R I P T

might have developmental problems. Alternatively, they can be unfertilized eggs. We therefore tested whether mating is hampered in the absence of Lipocalins.

Mating behavior is indeed altered in both GLaz -/-and NLaz -/-flies. When paired with wild type females, males of both mutant genotypes are less involved in courting (Fig. 6D), and a lower percent of pairs actually mate (Table 2). Wild type males paired with mutant females (Fig. 6E) also display a deficient courtship, and therefore the alteration of behavior is dependent on both sexes. When NLaz -/-males and females pair, the courtship index is even lower (Fig. 6E), and mating success is strikingly low (Table 2), which would account for the low number of eggs per female observed in population experiments (Fig. 6A). On the contrary, pairs composed of two GLaz mutant flies are more effective in triggering the courtship behavior than pairs of GLaz -/-with wild type flies. This result would be compatible with pheromone signaling being off-set in GLaz mutants, both at the emission and reception levels. Interestingly, the GLaz or NLaz mutants who successfully mate show longer mating latencies (Table 2), but this effect is cancelled when mutant males are paired with wild type females.

The altered mating behaviors could modify the final reproductive output of the population. However, the fertility of successfully mated mutants does not differ from wild type values (Fig. S3A). Thus, we can conclude that in experiments performed in fly populations (Fig. 6B) the GLaz -/-eggs that do not reach adulthood are mostly unfertilized eggs, while the fertilized ones reach adulthood at a normal rate, discarding major developmental problems also for GLaz -/-mutants.

In addition, we analyzed the lifespan of virgin females, which results in a pattern identical to the one observed for mated females: similar in wild type and GLaz -/-and shortened in NLaz -/-mutants (Fig. S3B). These virgin females do lay eggs, although all of them are unfertilized. The putative differential constraint to lifespan is not therefore dependent on the energy spent by the female in producing eggs proper.

Environment-genotype interactions: Food composition influence on Lipocalins phenotypes.

Food composition is an important environmental parameter that influences stress resistance and reproductive output. We have performed a nutritional analysis of three common fly food recipes including the standard one used in our laboratory, and the one used by collaborators that have also worked on NLaz or GLaz function (Fig. 7A, see Methods).

Each recipe differs in the amount of yeast and the source of sugar. However, a clear pattern emerges. For wild type flies, the higher the proportion Net Fat / Total Carbohydrates (Fig. 7B), the higher the fertility (Fig. 7C) and the lower the oxidative stress resistance (Fig. 7D-F).

Characterizing this interaction in the fly strains used in our analysis has been important, since the GLaz mutation effects on reproduction are significantly higher in diets with high or medium Fat/Carbohydrate ratio ("Valladolid" and "Carolina" foods, Fig. 7C), while sensitivity to stress observed in both mutants is only observed in diets with medium to low Fat/Carbohydrate ratio ("Carolina" and "Caltech" foods, Fig. 7D-F shows the results for NLaz mutants).

Previous studies analyzing diet effects on lifespan (Grandison and others 2009b;

Skorupa and others 2008) have been performed with different wild type strains. Our set of experiments emphasizes the importance of controlling diet when characterizing the effects of single genes influencing lifespan (see also Baldal and others 2006a). The effects of Lipocalin genotype in both reproductive output and stress resistance are best observed when flies are fed on our standard "Valladolid food", which contains a medium Fat/Carbohydrate proportion.

It is interesting to note that the largest differences in the stress response upon changes in diet are observed in the wild type flies. Without NLaz, the differences between feeding regimes are reduced. These results should not be surprising giving the role of NLaz in the control of metabolism in response to environmental stress (Hull-Thompson and others 2009). In the absence of NLaz, the IIS pathway is activated and flies become less sensitive to diet modifications. On the contrary, when IIS activity is genetically reduced (Grandison and others 2009a), lifespan extension becomes insensitive to nutrient supplemented diets.

¿How do brain Drosophila Lipocalins help us to understand the aging process?

Several general ideas relevant to aging and lifespan determination are supported by our study.

Early adulthood is a crucial period for lifespan determination.

Experiments where the IIS pathway was manipulated at different time points in life, both in C. elegans (Dillin and others 2002) and Drosophila (Giannakou and others 2007), have

shown that in order for this pathway to influence lifespan, it needs to operate exclusively during adulthood, with such an influence decreasing at older ages. Two lines of evidence suggest that a brain Lipocalin critical period also exists early in adulthood for their influence on lifespan: (1) their temporal expression pattern in wild type flies, and (2) the clustering of GLaz mutant females with wild type flies only at young ages in the multivariate space of metabolic parameters.

This time period, critical for both IIS and Lipocalin function, is coincident with the reproductive period of the organism (both in worms and flies), and could be interpreted in the light of the disposable soma theory of aging [START_REF] Kirkwood | Evolution of ageing[END_REF], where natural selection forces are predicted to decline sharply once the organism is no longer engaged in passing its genes to the next generation [START_REF] Kirkwood | Understanding ageing from an evolutionary perspective[END_REF]. Lipocalin function would be under selection in early adulthood only, and subsequent consequences of Lipocalin expression on the aging brain or body would be exaptations of this early adulthood selected functions [START_REF] Gould | Exaptation -a missing term in the science of form[END_REF].

A In accordance to the disposable soma theory, our first working hypothesis was that GLaz mutant females maintain a lifespan similar to the wild type because they would be able to allocate more resources to maintenance and less to reproduction. We then confirmed that GLaz mutant females reproduce less and survive more. However, since NLaz mutant females reduce both fecundity and lifespan, we must conclude that survival and reproduction are in this particular case uncoupled.

Curiously, the PCA analysis shows that it is the alteration of metabolic parameters related to the protein content of the fly what mainly accounts for the observed sex differences in longevity. The final protein content and the fat free dry weight of the organism depends of protein synthesis, which has a strong evolutionary conservation among the genetic modifiers of aging (Smith and others 2008).

NLaz is also related to the control of metabolism. When NLaz is expressed in the fat body, it is able to repress IIS activity in systemic target tissues. Also, the loss of NLaz increases signaling through IIS (Hull-Thompson and others 2009). The experiments in this work predict that, in addition, NLaz must be a negative regulator of food intake. NLaz is thus expected to fulfill a role similar to Leptin in the nervous system, inhibiting food intake and promoting energy expenditure. Loss of NLaz mimics therefore the Leptin deficiency state in mouse models of obesity (Ingalls and others 1950;Maffei and others 1995).

Curiously, mice where the neural/brain specific insulin receptor is knocked out, exhibit increased food intake and mild adiposity (Bruning and others 2000), just as the NLaz mutant flies. A direct interaction of ApoD with the cytoplasmic portion of the Leptin receptor has been suggested (Liu and others 2001), although it is still questionable how a secreted Lipocalin can access the cytoplasmic side of the plasma membrane for such an interaction to occur.

In contrast with NLaz, GLaz is not regulated by JNK in the peripheral tissues (Hull-Thompson and others 2009). Here we demonstrate that it is instead induced by food deprivation in the brain, where no NLaz transcriptional response is detected. This pattern of differential transcriptional response in the brain further supports the existence of specialized functional domains for these two Lipocalins.

3.11. Two brain Lipocalins in Drosophila: a labor division strategy in longevity regulation.

Our work highlights that the two Drosophila Lipocalins expressed in the nervous system have both functional redundancies and specializations. The response to oxidative stress and accumulation of lipid peroxides are among their common functions, while the transcriptional and behavioral response to starvation, the pattern of daily locomotor activity, storage of fat along aging, fertility, and courtship behavior differentiate NLaz from GLaz mutants. This framework is guiding our current research, as more details need to be elucidated. However, we can already start questioning: how many of these shared or unique functions are conserved in the mammalian homologues also expressed in the brain?

The basal position of ApoD in the phylogenetic tree of vertebrate Lipocalins (Ganfornina and others 2000;Sanchez and others 2006a) suggests that ApoD shares many properties with the common ancestor of invertebrate Lipocalins. However, we have to take into account that neither GLaz nor NLaz is a true orthologue of ApoD. Our molecular phylogenetic analyses strongly suggest that the Drosophila Lipocalins originated from an independent duplication event, taking place within the invertebrate lineage. Subsequently, the resulting genes have diverged both in their protein coding sequence and their regulatory sequences.

Since ApoD in the adult mammalian brain is expressed in glial cells, one might be tempted to directly conclude that GLaz is the closest Drosophila homologue. Furthermore, the expression data we report in this work strongly suggest that GLaz regulation through aging is most similar to the robust increase of mammalian ApoD in the aged brain (de Magalhaes and others 2009; Loerch and others 2008). Interestingly, ApoD is most similar to GLaz in protein sequence, but to NLaz in the intron-exon structure of the gene (Sanchez and others 2003). We have also data showing that ApoD is up-regulated by oxidative stress in astrocytes, and that this induction is mediated through the JNK pathway (Bajo-Grañeras, Ganfornina and Sanchez, unpublished observations), which is comparable to the NLaz JNK-mediated induction by stress in Drosophila (Hull-Thompson and others 2009). Thus, if we want to extrapolate the Drosophila data to learn about the functions of ApoD in mammalian aging and neurodegeneration we must benefit from a global comparison with both GLaz and NLaz, as the one reported here.

To understand the multigenic control of aging, we need to take into account that a layer of complexity is added due to the fact that each gene has pleiotropic effects, and each one has differing degrees of specialization or redundancy with members of the same gene family. This fact represents a daunting complication for the task of predicting the actions of putative anti-aging or anti-neurodegeneration drugs. However, complexity should not keep us from investigating till we get a comprehensive understanding of the aging process.

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  Wolbachia pipiens in all fly strains was tested by PCR of genomic DNA extracts. The following primers against the rRNA-16S gene of Wolbachia were used: 5'-GAAGATAATGACGGTACTCAC-3', 5'-GTCAGATTTGAACCAGATAGA-3' and 5'-GTCACTGATCCCACTTTA-3'. Program: 2 min 94º; (30 sec 94º, 30 sec 60º, 45 sec 72º) x15; (30 sec 94. 30 sec 52, 75 sec 72º) x15; 7 min 72º. DNA from Wolbachia-infected Dilofilaria immitis worms was used as positive control. Primers and a positive control DNA were kindly provided by Dr. F. Simon (Univ.

  Thompson and others 2009; Sanchez and others 2006b), and NLaz expression is known to be induced by starvation(Hull-Thompson and others 2009). However, our previous works used different regimes of starvation (wet or dry starvation) and were performed only in males. Here we analyze in detail how brain expression of the two Lipocalins is altered upon food deprivation (without water deprivation, to avoid a more complex stress).Expression of NLaz and GLaz in the fly head is differentially induced upon food deprivation in wild type flies (Fig.2A,B). Interestingly, no induction of NLaz is observed in the head tissue where its expression is constitutively high. These data are compatible with NLaz working in the control of systemic metabolism and with the fat body tissue being a source of NLaz under stress conditions (as we had previously shown using a dryA C C E P T E D M A N U S C R I P Tstarvation paradigm) (Hull-Thompson and others 2009). In contrast, GLaz is highly induced in the head upon starvation, suggesting that GLaz might be involved in a nervous system regulatory loop triggered by nutrient deprivation. These results show the existence of differential domains of action of the two Lipocalins.3.4.A multivariate analysis of the physiological parameters that contribute to lifespan modulation by Lipocalins.

  Even though reproduction has been shown to correlate negatively with lifespan in many cases(Baldal and others 2006a; Tatar and others 2003;[START_REF] Toivonen | Endocrine regulation of aging and reproduction in Drosophila[END_REF], instances of genetic interventions that alter longevity and reproductive output independently have also being documented (Dillin and others 2002; Giannakou and others A C C E P T E D M A N U S C R I P T 2007) (Grandison and others 2009a; Partridge and others 2005), challenging the existence of a direct causal relationship between reproduction and lifespan. Lipocalin mutants support the latest view, since the sex differences observed in longevity can not be fully explained by a net reduction in the energy spent in the production of eggs. The control of courtship behaviors are part of the pleiotropic functions of the two nervous system Lipocalins clearly deserving future investigation.

  .2. The sex-specific lifespan modulation by Lipocalins is better explained by metabolic than by reproduction phenotypes.

Figure 1 .

 1 Figure 1. Lipocalin loss-of-function mutants show sex-specific patterns in longevity that can be related to their different spatiotemporal patterns of expression. (A) Male lifespan determination of GLaz -/-(G), NLaz -/-(N) compared to isogenic wild type flies (G10 line). Median survival time is reduced by 15.5% in the GLaz mutant (p<10 -5 ), and by 22.5% in the NLaz mutant (p<10 -5 ). (B) Lifespan is also reduced in NLaz females (30.8% reduction, p<10 -5 ), but not in GLaz mutant females (p=0.034). Reductions in median survival in the w1118 genetic background (not shown) were 27.8 and 34.3% for GLaz and NLaz mutant males, and 37.2% for NLaz mutant females. Log-rank test was used for statistical analysis. N=114-213/genotype. (C) Age effect in NLaz and GLaz transcription in wild type fly heads assayed by qRT-PCR. (D) Transcription levels of NLaz and GLaz in wild type body RNA extracts. The RPL18 gene is used as endogenous control.The expression level of GLaz in 3 day-old male heads is used as calibrator (Cal.) for both graphs. Symmetrical Fold Change was chosen for data representation for equal display of down-regulations and up-regulations. Statistical differences assayed by Mann-Whitney Utest. * p<0.05 ** p<0.01.

Figure 2 .

 2 Figure 2. Differential expression of Lipocalins in the brain upon starvation.(A) Changes in expression of GLaz and NLaz in the head of wild type male flies at 3 days of age in control conditions and after 15 hours of wet starvation treatment. (B) Changes in expression of both genes in the same conditions as in A, but in females. qRT-PCR using RPL18 as endogenous control and the value of GLaz in 3d wild type male heads as calibrator (Cal.) for both graphs. Symmetrical Fold Change was chosen for data representation for equal display of down-regulations and up-regulations. Statistical differences assayed by Mann-Whitney U-test. ** p<0.01.

Figure 3 .

 3 Figure 3. Principal Component Analysis of variables excluding Lipocalin gene expression and lifespan parameters.(A-B) Scatterplot and biplot of the two principal components explaining 69% of the variation in the sample of 25 variables measured in 3 day-old flies (Subset 1). The twodimensional space defined by these components separate wild type flies from either mutant,

Figure 4 .

 4 Figure 4. Energy intake and homeostasis are altered in Lipocalin mutants. (A) Variables exploring the flow of energy in Lipocalin mutants. (B) Behavioral response to starvation is increased in NLaz -/-but not in GLaz -/-mutants. (C) Age and genotype effect in relative fat content. GLaz -/-mutants have a constitutive reduction in their neutral fat stores while fat increases with age in NLaz -/-mutants. Values normalized with respect to wild type young males. Statistical differences assayed by Student's T-test. * p<0.05; ** p<0.01.

Figure

  Figure 5. Lipocalin mutants show sex-specific alterations in their circadian locomotor activity. (A-B) Average of three 24h periods of locomotor activity in wild type, GLaz -/-and NLaz -/- males (A) and females (B). (C) Average total locomotor activity is higher in GLaz -/-males and lower in NLaz -/-females . (D) Locomotor activity during the dark phase. GLaz -/-males are much more active at night than the wild type, while NLaz -/-males reduce their activity. N=25/sex/genotype. Statistical differences assayed by Student's T-test. * p<0.05; ** p<0.01.

  Fig. 3

Fig. 4 A

 4 Fig. 4

  Fig. 5

  Fig. 6

Fig. 7 A

 7 Fig. 7

Acknowledgments and financial support

We thank J.R. Acebes and E. Martin for technical assistance. We also thank our colleagues in the lab R. Bajo-Grañeras, A. Pérez-Castellanos, N. García-Mateo, and M. del Caño for their stimulating discussions, as well as A. Ferrús for his interesting questions and for laboratory support. This work was supported by start-up grants to D.S. and M.D.G. from the "Ramón y Cajal Program" (Ministerio de Ciencia y Tecnología -Spain, and Fondo Europeo de Desarrollo Regional -Fondo Social Europeo); the SGFRH Foundation; Junta de Castilla y Leon grant VA049A0, Ministerio de Educacion y Ciencia grant BFU2005-00522, and Ministerio de Ciencia e Innovacion grant BFU2008-01170 to M.D.G. and D.S.; Ministerio de Ciencia e Innovacion grant BFU2008-04683-C02-02 to I.C.; Ministerio de Ciencia e Innovacion grant BFU2009-12410/BMC and Fundación Cien Reina Sofía to A.A. Authors declare no conflict of interest exists in relation to the content of this manuscript.

Neither the author's institutions nor the funding agencies had a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors' contributions and approval of manuscript

MR performed the food intake and courtship behaviour experiments, contributed to carry out the longevity curves and the molecular biology analyses and participated in the design of the study, the statistical analysis, and in writing the manuscript. Average of three 24h periods of locomotor activity in wild type, GLaz -/-and NLaz -/-flies.

Sunrise and sunset are defined as the period of the light switch ±1h. N=25/sex/genotype. S1 andS2. (A) Fertility of successfully mated mutant pairs does not differ from wild type when scoring the offspring of mated couples from courtship behavior analysis (Fig. 6D-E). N= 34-52 pairs. Statistical differences assayed by Student's T-test (p<0.05): a Different from WT-M x WT-F pairs. (B) Lifespan of virgin females is reduced in NLaz -/-mutants (p<10 -5 ), but is similar to wild type in GLaz -/-mutants. Log-rank test was used for statistical analysis.

N=97-120/genotype.