Healthy Aging Biomarkers: The INSPIRE's Contribution.

The find solutions for optimizing healthy aging and increase health span is one of the main challenges for our society. A novel healthcare model based on integration and a shift on research and care towards the maintenance of optimal functional levels are now seen as priorities by the WHO. To address this issue, an integrative global strategy mixing longitudinal and experimental cohorts with an innovative transverse understanding of physiological functioning is missing. While the current approach to the biology of aging is mainly focused on parenchymal cells, we propose that age-related loss of function is largely determined by three elements which constitute the general ground supporting the different specific parenchyma: i.e. the stroma, the immune system and metabolism. Such strategy that is implemented in INSPIRE projects can strongly help to find a composite biomarker capable of predicting changes in capacity across the life course with thresholds signalling frailty and care dependence.

The INSPIRE Research Initiative: A Program for GeroScience and Healthy Aging Research Going from Animal Models to Humans and the Healthcare System.

Aging is the most important risk factor for the onset of several chronic diseases and functional decline. Understanding the interplays between biological aging and the biology of diseases and functional loss as well as integrating a function-centered approach to the care pathway of older adults are crucial steps towards the elaboration of preventive strategies (both pharmacological and non-pharmacological) against the onset and severity of burdensome chronic conditions during aging. In order to tackle these two crucial challenges, ie, how both the manipulation of biological aging and the implementation of a function-centered care pathway (the Integrated Care for Older People (ICOPE) model of the World Health Organization) may contribute to the trajectories of healthy aging, a new initiative on Gerosciences was built: the INSPIRE research program. The present article describes the scientific background on which the foundations of the INSPIRE program have been constructed and provides the general lines of this initiative that involves researchers from basic and translational science, clinical gerontology, geriatrics and primary care, and public health.

Towards a Large-Scale Assessment of the Relationship between Biological and Chronological Aging: The INSPIRE Mouse Cohort.

Aging is the major risk factor for the development of chronic diseases. After decades of research focused on extending lifespan, current efforts seek primarily to promote healthy aging. Recent advances suggest that biological processes linked to aging are more reliable than chronological age to account for an individual's functional status, i.e. frail or robust. It is becoming increasingly apparent that biological aging may be detectable as a progressive loss of resilience much earlier than the appearance of clinical signs of frailty. In this context, the INSPIRE program was built to identify the mechanisms of accelerated aging and the early biological signs predicting frailty and pathological aging. To address this issue, we designed a cohort of outbred Swiss mice (1576 male and female mice) in which we will continuously monitor spontaneous and voluntary physical activity from 6 to 24 months of age under either normal or high fat/high sucrose diet. At different age points (6, 12, 18, 24 months), multiorgan functional phenotyping will be carried out to identify early signs of organ dysfunction and generate a large biological fluids/feces/organs biobank (100,000 samples). A comprehensive correlation between functional and biological phenotypes will be assessed to determine: 1) the early signs of biological aging and their relationship with chronological age; 2) the role of dietary and exercise interventions on accelerating or decelerating the rate of biological aging; and 3) novel targets for the promotion of healthy aging. All the functional and omics data, as well as the biobank generated in the framework of the INSPIRE cohort will be available to the aging scientific community. The present article describes the scientific background and the strategies employed for the design of the INSPIRE Mouse cohort.

The INSPIRE Bio-Resource Research Platform for Healthy Aging and Geroscience: Focus on the Human Translational Research Cohort (The INSPIRE-T Cohort).

BACKGROUND: The Geroscience field focuses on the core biological mechanisms of aging, which are involved in the onset of age-related diseases, as well as declines in intrinsic capacity (IC) (body functions) leading to dependency. A better understanding on how to measure the true age of an individual or biological aging is an essential step that may lead to the definition of putative markers capable of predicting healthy aging. OBJECTIVES: The main objective of the INStitute for Prevention healthy agIng and medicine Rejuvenative (INSPIRE) Platform initiative is to build a program for Geroscience and healthy aging research going from animal models to humans and the health care system. The specific aim of the INSPIRE human translational cohort (INSPIRE-T cohort) is to gather clinical, digital and imaging data, and perform relevant and extensive biobanking to allow basic and translational research on humans. METHODS: The INSPIRE-T cohort consists in a population study comprising 1000 individuals in Toulouse and surrounding areas (France) of different ages (20 years or over - no upper limit for age) and functional capacity levels (from robustness to frailty, and even dependency) with follow-up over 10 years. Diversified data are collected annually in research facilities or at home according to standardized procedures. Between two annual visits, IC domains are monitored every 4-month by using the ICOPE Monitor app developed in collaboration with WHO. Once IC decline is confirmed, participants will have a clinical assessment and blood sampling to investigate markers of aging at the time IC declines are detected. Biospecimens include blood, urine, saliva, and dental plaque that are collected from all subjects at baseline and then, annually. Nasopharyngeal swabs and cutaneous surface samples are collected in a large subgroup of subjects every two years. Feces, hair bulb and skin biopsy are collected optionally at the baseline visit and will be performed again during the longitudinal follow up. EXPECTED RESULTS: Recruitment started on October 2019 and is expected to last for two years. Bio-resources collected and explored in the INSPIRE-T cohort will be available for academic and industry partners aiming to identify robust (set of) markers of aging, age-related diseases and IC evolution that could be pharmacologically or non-pharmacologically targetable. The INSPIRE-T will also aim to develop an integrative approach to explore the use of innovative technologies and a new, function and person-centered health care pathway that will promote a healthy aging.

[Analysis of cell-free DNA in maternal blood for detection of fetal trisomy 21 in high-risk population: Couples acceptance and grounds for refusal]. / Étude de l'ADN fœtal dans le sang maternel pour la détection de la trisomie 21 en population à risque accru : adhésion des couples et motifs de refus.

OBJECTIVES: To assess the determinants associated with the use of analysis of cell-free DNA in maternal blood for detection of trisomy 21 in high-risk women. MATERIALS AND METHODS: Prospective study conducted in a single center between July 15, 2014 and December 15, 2014 on 99 consecutive women with increased risk of trisomy 21 above 1/250. RESULTS: Analysis of cell-free DNA in maternal blood for detection of fetal trisomy 21 was proposed to 95 women out of 99, among them, 43 women (45.3%) required the test. Among these 43 women, 17 (38.6%) had a higher socio-economic status versus 10 (19.2%) among the women who did not request the test (P=0.03). The most common reason given by the 52 women who did not request the analysis of cell-free DNA was the cost, for 30 of them (57.7%), then because the test was not providing certainty for the diagnostic of trisomy 21 for 23 women (44.2%). CONCLUSION: Analysis of cell-free DNA on maternal blood for detection of trisomy 21 does not seem accepted by the majority of women. The cost is probably the main reason for not using this test, but it seems that the lack of diagnostic certainty is also an obstacle for some women.

[Comparison of two bedside tests performed on cervicovaginal fluid to diagnose premature rupture of membranes]. / Analyse comparative de deux tests diagnostiques de rupture prématurée des membranes dans les sécrétions cervico-vaginales.

OBJECTIVE: To compare the performance of two rapid tests for the diagnosis of premature rupture of membranes (PROM) based on the detection of the insulin-like growth factor-binding protein-1 (IGFBP-1) and placental α-microglobulin-1 (PAMG-1) in cervicovaginal secretions. METHODS: A case-control prospective study. Pregnant women between 24 and 41(6/7) weeks' of gestation, consulting for profuse amniotic fluid loss (group 1) or for other reasons without any rupture of membrane (group 2) were included in the study. Successively, AmniSure(®) test (PAMG-1) without speculum, and then Actim™Prom test (IGFBP-1) during speculum examination were performed during the same visit. RESULTS: Eighty subjects (40 in each group) were included between 25(1/7) to 41(1/7) weeks of gestation. AmniSure(®) diagnostic test demonstrated a sensitivity and specificity of 95 % (82.4-99.4) and 94.8 % (79.3-98) respectively and a positive and negative predictive value of 95 % (84.7-100) and 94.8 % (87.9-100) respectively. Actim™Prom diagnostic test demonstrated a sensitivity and specificity of 97.5 % (85.7-100) and 97.4 % (82.4-99.4) respectively and a positive and negative predictive value of 97.5 % (88.5-100) and 97.4 % (92.5-100) respectively. CONCLUSION: Both tests have similar performance to diagnose premature rupture of membranes.

Impaired neurogenesis, neuronal loss, and brain functional deficits in the APPxPS1-Ki mouse model of Alzheimer's disease.

Amyloid-ß peptide species accumulating in the brain of patients with Alzheimer's disease are assumed to have a neurotoxic action and hence to be key actors in the physiopathology of this neurodegenerative disease. We have studied a new mouse mutant (APPxPS1-Ki) line developing both early-onset brain amyloid-ß deposition and, in contrast to most of transgenic models, subsequent neuronal loss. In 6-month-old mice, we observed cell layer atrophies in the hippocampus, together with a dramatic decrease in neurogenesis and a reduced brain blood perfusion as measured in vivo by magnetic resonance imaging. In these mice, neurological impairments and spatial hippocampal dependent memory deficits were also substantiated and worsened with aging. We described here a phenotype of APPxPS1-Ki mice that summarizes several neuroanatomical alterations and functional deficits evocative of the human pathology. Such a transgenic model that displays strong face validity might be highly beneficial to future research on AD physiopathogeny and therapeutics.

Young hippocampal neurons are critical for recent and remote spatial memory in adult mice.

New granule cells are continuously generated throughout adulthood in the mammalian hippocampus. These newly generated neurons become functionally integrated into existing hippocampal neuronal networks, such as those that support retrieval of remote spatial memory. Here, we sought to examine whether the contribution of newly born neurons depends on the type of learning and memory task in mice. To do so, we reduced neurogenesis with a cytostatic agent and examined whether depletion of young hippocampal neurons affects learning and/or memory in two hippocampal-dependent tasks (spatial navigation in the Morris water maze and object location test) and two hippocampal-independent tasks (cued navigation in the Morris water maze and novel object recognition). Double immunohistofluorescent labeling of the birth dating marker 5-bromo-2'deoxyuridine (BrdU) together with NeuN, a neuron specific marker, was employed to quantify reduction of hippocampal neurogenesis. We found that depletion of young adult-generated neurons alters recent and remote memory in spatial tasks but spares non-spatial tasks. Our findings provide additional evidence that generation of new cells in the adult brain is crucial for hippocampal-dependent cognitive functions.

Brainstem glycinergic neurons and their activation during active (rapid eye movement) sleep in the cat.

It is well established that, during rapid eye movement (REM) sleep, somatic motoneurons are subjected to a barrage of inhibitory synaptic potentials that are mediated by glycine. However, the source of this inhibition, which is crucial for the maintenance and preservation of REM sleep, has not been identified. Consequently, the present study was undertaken to determine in cats the location of the glycinergic neurons, that are activated during active sleep, and are responsible for the postsynaptic inhibition of motoneurons that occurs during this state. For this purpose, a pharmacologically-induced state of active sleep (AS-carbachol) was employed. Antibodies against glycine-conjugated proteins were used to identify glycinergic neurons and immunocytochemical techniques to label the Fos protein were employed to identify activated neurons. Two distinct populations of glycinergic neurons that expressed c-fos were distinguished. One population was situated within the nucleus reticularis gigantocellularis (NRGc) and nucleus magnocellularis (Mc) in the rostro-ventral medulla; this group of neurons extended caudally to the ventral portion of the nucleus paramedianus reticularis (nPR). Forty percent of the glycinergic neurons in the NRGc and Mc and 25% in the nPR expressed c-fos during AS-carbachol. A second population was located in the caudal medulla adjacent to the nucleus ambiguus (nAmb), wherein 40% of the glycinergic cells expressed c-fos during AS-carbachol. Neither population of glycinergic cells expressed c-fos during quiet wakefulness or quiet (non-rapid eye movement) sleep. We suggest that the population of glycinergic neurons in the NRGc, Mc, and nPR participates in the inhibition of somatic brainstem motoneurons during active sleep. These neurons may also be responsible for the inhibition of sensory and other processes during this state. It is likely that the group of glycinergic neurons adjacent to the nucleus ambiguus (nAmb) is responsible for the active sleep-selective inhibition of motoneurons that innervate the muscles of the larynx and pharynx.

Tracing the glycogen cells with protocadherin 12 during mouse placenta development.

Among the different trophoblast subtypes of the mouse placenta, the glycogen cells (GC) are one of the trophoblast subtypes that invade the decidua. We previously established that GC specifically expressed protocadherin 12 (PCDH12). In this paper, we investigated the origin of the PCDH12-positive cells and we characterized their fate in the maternal tissues. Our data indicate that they directly originate from the central part of the ectoplacental cone at embryonic day (E) 7.5. PCDH12-positive cells start to accumulate glycogen from E10.5 and the first migrating cells could be observed from this age. Unlike other placental and decidual cells, GC do not express P-cadherin, which may explain their migration properties in this organ. In the decidua, GC settle in the vicinity of the maternal vascular sinuses but do not incorporate in the endothelium. By the end of gestation (E17.5), most GC islets of the decidua enter into a lytic phase and form large lacunae. These lacunae, filled with glycogen, may provide a substantial source of energy at the end of gestation or during delivery. Our data suggest that spongiotrophoblasts and GC are two independent lineages and we bring insights into GC migration and fate.

Gene control of synaptic plasticity and memory formation: implications for diseases and therapeutic strategies.

There has been nearly a century of interest in the idea that information is stored in the brain as changes in the efficacy of synaptic connections between neurons that are activated during learning. The discovery and detailed report of the phenomenon generally known as long-term potentiation opened a new chapter in the study of synaptic plasticity in the vertebrate brain, and this form of synaptic plasticity has now become the dominant model in the search for the cellular and molecular bases of learning and memory. Accumulating evidence suggests that the rapid activation of the genetic machinery is a key mechanism underlying the enduring modification of neural networks required for the laying down of memory. Here we briefly review these mechanisms and illustrate with a few examples of animal models of neurological disorders how new knowledge about these mechanisms can provide valuable insights into identifying the mechanisms that go awry when memory is deficient, and how, in turn, characterisation of the dysfunctional mechanisms offers prospects to design and evaluate molecular and biobehavioural strategies for therapeutic prevention and rescue.

Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces.

To examine the in vivo function of presenilin-1 (PS1), we selectively deleted the PS1 gene in excitatory neurons of the adult mouse forebrain. These conditional knockout mice were viable and grew normally, but they exhibited a pronounced deficiency in enrichment-induced neurogenesis in the dentate gyrus. This reduction in neurogenesis did not result in appreciable learning deficits, indicating that addition of new neurons is not required for memory formation. However, our postlearning enrichment experiments lead us to postulate that adult dentate neurogenesis may play a role in the periodic clearance of outdated hippocampal memory traces after cortical memory consolidation, thereby ensuring that the hippocampus is continuously available to process new memories. A chronic, abnormal clearance process in the hippocampus may conceivably lead to memory disorders in the mammalian brain.

Genetic analysis of learning behavior-induced structural plasticity.

It is well-documented that enriched environment and behavioral training can lead to improved learning and memory, as well as structural and morphological changes in the brain. It has been hypothesized that such experience-dependent behavioral improvement results from structural modifications that may represent some forms of possible memory substrates for these behavioral experiences. It was generally assumed until now that, like the activity-dependent structural plasticity observed in the developing brain, behavioral experience-induced structural plasticity would require the activation of the NMDA receptor, a molecular switch for learning and memory. Recent genetic and anatomical analyses reveal that behavioral experience-induced increases in spine and synapse density in the hippocampal CA1 region occur despite the deletion of the NMDA receptor in conditional knockout mice. Recent studies indicate that the molecular mechanism of behavioral experience-induced structural plasticity in the adult brain differs from that of the developing brain, and can be disassociated from the NMDA-mediated long-term potentiation (LTP) phenomenon. Deepening the understanding of the molecular mechanism of experience-induced structural plasticity should facilitate the study of the relationship between structural changes and memory formation. Using an integrated approach with genomic, genetic, and modern histological techniques should move us closer in this direction.

NMDA receptor-dependent synaptic reinforcement as a crucial process for memory consolidation.

The hippocampal CA1 region is crucial for converting new memories into long-term memories, a process believed to continue for week(s) after initial learning. By developing an inducible, reversible, and CA1-specific knockout technique, we could switch N-methyl-D-aspartate (NMDA) receptor function off or on in CA1 during the consolidation period. Our data indicate that memory consolidation depends on the reactivation of the NMDA receptor, possibly to reinforce site-specific synaptic modifications to consolidate memory traces. Such a synaptic reinforcement process may also serve as a cellular means by which the new memory is transferred from the hippocampus to the cortex for permanent storage.

Effects of environmental enrichment on gene expression in the brain.

An enriched environment is known to promote structural changes in the brain and to enhance learning and memory performance in rodents [Hebb, D. O. (1947) Am. Psychol. 2, 306-307]. To better understand the molecular mechanisms underlying these experience-dependent cognitive changes, we have used high-density oligonucleotide microarrays to analyze gene expression in the brain. Expression of a large number of genes changes in response to enrichment training, many of which can be linked to neuronal structure, synaptic plasticity, and transmission. A number of these genes may play important roles in modulating learning and memory capacity.

Role and origin of the GABAergic innervation of dorsal raphe serotonergic neurons.

Extracellular electrophysiological recordings in freely moving cats have shown that serotonergic neurons from the dorsal raphe nucleus (DRN) fire tonically during wakefulness, decrease their activity during slow wave sleep (SWS), and are nearly quiescent during paradoxical sleep (PS). The mechanisms at the origin of the modulation of activity of these neurons are still unknown. Here, we show in the unanesthetized rat that the iontophoretic application of the GABA(A) antagonist bicuculline on dorsal raphe serotonergic neurons induces a tonic discharge during SWS and PS and an increase of discharge rate during quiet waking. These data strongly suggest that an increase of a GABAergic inhibitory tone present during wakefulness is responsible for the decrease of activity of the dorsal raphe serotonergic cells during slow wave and paradoxical sleep. In addition, by combining retrograde tracing with cholera toxin B subunit and glutamic acid decarboxylase immunohistochemistry, we demonstrate that the GABAergic innervation of the dorsal raphe nucleus arises from multiple distant sources and not only from interneurons as classically accepted. Among these afferents, GABAergic neurons located in the lateral preoptic area and the pontine ventral periaqueductal gray including the DRN itself could be responsible for the reduction of activity of the serotonergic neurons of the dorsal raphe nucleus during slow wave and paradoxical sleep, respectively.

Enrichment induces structural changes and recovery from nonspatial memory deficits in CA1 NMDAR1-knockout mice.

We produced CA1-specific NMDA receptor 1 subunit-knockout (CA1-KO) mice to determine the NMDA receptor dependence of nonspatial memory formation and of experience-induced structural plasticity in the CA1 region. CA1-KO mice were profoundly impaired in object recognition, olfactory discrimination and contextual fear memories. Surprisingly, these deficits could be rescued by enriching experience. Using stereological electron microscopy, we found that enrichment induced an increase of the synapse density in the CA1 region in knockouts as well as control littermates. Therefore, our data indicate that CA1 NMDA receptor activity is critical in hippocampus-dependent nonspatial memory, but is not essential for experience-induced synaptic structural changes.

Genetic enhancement of learning and memory in mice.

Hebb's rule (1949) states that learning and memory are based on modifications of synaptic strength among neurons that are simultaneously active. This implies that enhanced synaptic coincidence detection would lead to better learning and memory. If the NMDA (N-methyl-D-aspartate) receptor, a synaptic coincidence detector, acts as a graded switch for memory formation, enhanced signal detection by NMDA receptors should enhance learning and memory. Here we show that overexpression of NMDA receptor 2B (NR2B) in the forebrains of transgenic mice leads to enhanced activation of NMDA receptors, facilitating synaptic potentiation in response to stimulation at 10-100 Hz. These mice exhibit superior ability in learning and memory in various behavioural tasks, showing that NR2B is critical in gating the age-dependent threshold for plasticity and memory formation. NMDA-receptor-dependent modifications of synaptic efficacy, therefore, represent a unifying mechanism for associative learning and memory. Our results suggest that genetic enhancement of mental and cognitive attributes such as intelligence and memory in mammals is feasible.

Origins of the glycinergic inputs to the rat locus coeruleus and dorsal raphe nuclei: a study combining retrograde tracing with glycine immunohistochemistry.

The amino acid glycine is a major inhibitory neurotransmitter in the brainstem and is likely involved in the tonic inhibition of the monoaminergic neurons during all sleep-waking stages. In order to determine the neurons at the origin of the glycinergic innervation of the two principal monoaminergic nuclei, the locus coeruleus and the dorsal raphe of the rat, we applied a double-labelling technique, combining retrograde transport of cholera-toxin B subunit with glycine immunohistochemistry. Using this technique, we found that the locus coeruleus and dorsal raphe nuclei receive a common glycinergic innervation from the ventral and ventrolateral periaqueductal grey, including the adjacent deep mesencephalic reticular nucleus. Small additional glycinergic inputs to these nuclei originated from the lateral paragigantocellular nucleus and the rostral ventromedial medullary reticular formation. The potential role of these glycinergic inputs in the control of the excitability of the monoaminergic neurons of the locus coeruleus and dorsal raphe nuclei is discussed.

Forebrain afferents to the rat dorsal raphe nucleus demonstrated by retrograde and anterograde tracing methods.

The dorsal raphe nucleus through its extensive efferents has been implicated in a great variety of physiological and behavioural functions. However, little is know about its afferents. Therefore, to identify the systems likely to influence the activity of serotonergic neurons of the dorsal raphe nucleus, we re-examined the forebrain afferents to the dorsal raphe nucleus using cholera toxin b subunit and Phaseolus vulgaris-leucoagglutinin as retrograde or anterograde tracers. With small cholera toxin b subunit injection sites, we further determined the specific afferents to the ventral and dorsal parts of the central dorsal raphe nucleus, the rostral dorsal raphe nucleus and the lateral wings. In agreement with previous studies, we observed a large number of retrogradely-labelled cells in the lateral habenula following injections in all subdivisions of the dorsal raphe nucleus. In addition, depending on the subdivision of the dorsal raphe nucleus injected, we observed a small to large number of retrogradely-labelled cells in the orbital, cingulate, infralimbic, dorsal peduncular, and insular cortice, a moderate or substantial number in the ventral pallidum and a small to substantial number in the claustrum. In addition, we observed a substantial to large number of cells in the medial and lateral preoptic areas and the medial preoptic nucleus after cholera toxin b subunit injections in the dorsal raphe nucleus excepting for those located in the ventral part of the central dorsal raphe nucleus, after which we found a moderate number of retrogradely-labelled cells. Following cholera toxin b subunit injections in the dorsal part of the central dorsal raphe nucleus, a large number of retrogradely-labelled cells was seen in the lateral, ventral and medial parts of the bed nucleus of the stria terminalis whereas only a small to moderate number was visualized after injections in the other dorsal raphe nucleus subdivisions. In addition, respectively, a substantial and a moderate number of retrogradely-labelled cells was distributed in the zona incerta and the subincertal nucleus following all tracer injections in the dorsal raphe nucleus. A large number of retrogradely-labelled cells was also visualized in the lateral, dorsal and posterior hypothalamic areas and the perifornical nucleus after cholera toxin b subunit injections in the dorsal part of the central raphe nucleus and to a lesser extent following injections in the other subdivisions. We further observed a substantial to large number of retrogradely-labelled cells in the tuber cinereum and the medial tuberal nucleus following cholera toxin b subunit injections in the dorsal part of the central dorsal raphe nucleus or the lateral wings and a small to moderate number after injections in the two other dorsal raphe nucleus subdivisions. A moderate or substantial number of labelled cells was also seen in the ventromedial hypothalamic area and the arcuate nucleus following cholera toxin injections in the dorsal part of the central dorsal raphe nucleus and the lateral wings and an occasional or small number with injection sites located in the other subdivisions. Finally, we observed, respectively, a moderate and a substantial number of retrogradely-labelled cells in the central nucleus of the amygdala following tracer injections in the ventral or dorsal parts of the central dorsal raphe nucleus and a small number after injections in the other subnuclei. In agreement with these retrograde data, we visualized anterogradely-labelled fibres heterogeneously distributed in the dorsal raphe nucleus following Phaseolus vulgaris-leucoagglutinin injections in the lateral orbital or infralimbic cortice, the lateral preoptic area, the perifornical nucleus, the lateral or posterior hypothalamic areas, the zona incerta, the subincertal nucleus or the medial tuberal nucleus. (ABSTRACT TRUNCATED)