Microbiome changes through the ontogeny of the marine sponge Crambe crambe.

BACKGROUND: Poriferans (sponges) are highly adaptable organisms that can thrive in diverse marine and freshwater environments due, in part, to their close associations with internal microbial communities. This sponge microbiome can be acquired from the surrounding environment (horizontal acquisition) or obtained from the parents during the reproductive process through a variety of mechanisms (vertical transfer), typically resulting in the presence of symbiotic microbes throughout all stages of sponge development. How and to what extent the different components of the microbiome are transferred to the developmental stages remain poorly understood. Here, we investigated the microbiome composition of a common, low-microbial-abundance, Atlantic-Mediterranean sponge, Crambe crambe, throughout its ontogeny, including adult individuals, brooded larvae, lecithotrophic free-swimming larvae, newly settled juveniles still lacking osculum, and juveniles with a functional osculum for filter feeding. RESULTS: Using 16S rRNA gene analysis, we detected distinct microbiome compositions in each ontogenetic stage, with variations in composition, relative abundance, and diversity of microbial species. However, a particular dominant symbiont, Candidatus Beroebacter blanensis, previously described as the main symbiont of C. crambe, consistently occurred throughout all stages, an omnipresence that suggests vertical transmission from parents to offspring. This symbiont fluctuated in relative abundance across developmental stages, with pronounced prevalence in lecithotrophic stages. A major shift in microbial composition occurred as new settlers completed osculum formation and acquired filter-feeding capacity. Candidatus Beroebacter blanensis decreased significatively at this point. Microbial diversity peaked in filter-feeding stages, contrasting with the lower diversity of lecithotrophic stages. Furthermore, individual specific transmission patterns were detected, with greater microbial similarity between larvae and their respective parents compared to non-parental conspecifics. CONCLUSIONS: These findings suggest a putative vertical transmission of the dominant symbiont, which could provide some metabolic advantage to non-filtering developmental stages of C. crambe. The increase in microbiome diversity with the onset of filter-feeding stages likely reflects enhanced interaction with environmental microbes, facilitating horizontal transmission. Conversely, lower microbiome diversity in lecithotrophic stages, prior to filter feeding, suggests incomplete symbiont transfer or potential symbiont digestion. This research provides novel information on the dynamics of the microbiome through sponge ontogeny, on the strategies for symbiont acquisition at each ontogenetic stage, and on the potential importance of symbionts during larval development.

High compositional and functional similarity in the microbiome of deep-sea sponges.

Sponges largely depend on their symbiotic microbes for their nutrition, health, and survival. This is especially true in high microbial abundance (HMA) sponges, where filtration is usually deprecated in favor of a larger association with prokaryotic symbionts. Sponge-microbiome association is substantially less understood for deep-sea sponges than for shallow water species. This is most unfortunate, since HMA sponges can form massive sponge grounds in the deep sea, where they dominate the ecosystems, driving their biogeochemical cycles. Here, we assess the microbial transcriptional profile of three different deep-sea HMA sponges in four locations of the Cantabrian Sea and compared them to shallow water HMA and LMA (low microbial abundance) sponge species. Our results reveal that the sponge microbiome has converged in a fundamental metabolic role for deep-sea sponges, independent of taxonomic relationships or geographic location, which is shared in broad terms with shallow HMA species. We also observed a large number of redundant microbial members performing the same functions, likely providing stability to the sponge inner ecosystem. A comparison between the community composition of our deep-sea sponges and another 39 species of HMA sponges from deep-sea and shallow habitats, belonging to the same taxonomic orders, suggested strong homogeneity in microbial composition (i.e. weak species-specificity) in deep sea species, which contrasts with that observed in shallow water counterparts. This convergence in microbiome composition and functionality underscores the adaptation to an extremely restrictive environment with the aim of exploiting the available resources.

Communality in microbial stress response and differential metabolic interactions revealed by time-series analysis of sponge symbionts.

The diversity and function of sponge-associated symbionts is now increasingly understood; however, we lack an understanding of how they dynamically behave to ensure holobiont stability in the face of environmental variation. Here, we performed a metatransciptomic analysis on three microbial symbionts of the sponge Cymbastela concentrica in situ over 14 months and through differential gene expression and correlation analysis to environmental variables uncovered differences that speak to their metabolic activities and level of symbiotic and environmental interactions. The nitrite-oxidizing Ca. Porinitrospira cymbastela maintained a seemingly stable metabolism, with the few differentially expressed genes related only to stress responses. The heterotrophic Ca. Porivivens multivorans displayed differential use of holobiont-derived compounds and respiration modes, while the ammonium-oxidizing archaeon Ca. Nitrosopumilus cymbastelus differentially expressed genes related to phosphate metabolism and symbiosis effectors. One striking similarity between the symbionts was their similar variation in expression of stress-related genes. Our time-series study showed that the microbial community of C. concentrica undertakes dynamic gene expression adjustments in response to the surroundings, tuned to deal with general stress and metabolic interactions between holobiont members. The success of these dynamic adjustments likely underpins the stability of the sponge holobiont and may provide resilience against environmental change.

Stress response of the marine sponge Scopalina sp.. Can microbial community composition predict sponge disease?

Disease has become an increasingly recognised problem in the marine environment, but our understanding of the factors that drive disease or our ability to predict its occurrence is limited. Marine sponges are known for their close associations with microorganisms, which are generally accepted to underpin sponge health and function. The aim of this study is to explore whether the microbial community composition of sponges can act as a predictor of disease occurrence under stressful environmental conditions. The development of a naturally occurring disease in the temperate sponge species Scopalina sp. was reproducibly recreated in a flow-through aquarium environment using increasing temperature stress. Throughout the experiments, four morphological health states were observed and described. Fingerprinting based on terminal restriction fragment length polymorphism of the bacterial community uncovered a statistically significant signature in healthy sponges prior to stress or apparent symptoms that correlated with the time it took for the disease to occur. This shows that the bacterial community composition of individual sponges can act as predictors of necrotic disease development. To the best of our knowledge, this is the first time a microbial signature of this nature has been reported in marine sponges and this finding can contribute to unravelling cause-effect pathways for stress-related dysbiosis and disease.

On the way to specificity - Microbiome reflects sponge genetic cluster primarily in highly structured populations.

Most animals, including sponges (Porifera), have species-specific microbiomes. Which genetic or environmental factors play major roles structuring the microbial community at the intraspecific level in sponges is, however, largely unknown. In this study, we tested whether geographic location or genetic structure of conspecific sponges influences their microbial assembly. For that, we used three sponge species with different rates of gene flow, and collected samples along their entire distribution range (two from the Mediterranean and one from the Southern Ocean) yielding a total of 393 samples. These three sponge species have been previously analysed by microsatellites or single nucleotide polymorphisms, and here we investigate their microbiomes by amplicon sequencing of the microbial 16S rRNA gene. The sponge Petrosia ficiformis, with highly isolated populations (low gene flow), showed a stronger influence of the host genetic distance on the microbial composition than the spatial distance. Host-specificity was therefore detected at the genotypic level, with individuals belonging to the same host genetic cluster harbouring more similar microbiomes than distant ones. On the contrary, the microbiome of Ircinia fasciculata and Dendrilla antarctica - both with weak population structure (high gene flow) - seemed influenced by location rather than by host genetic distance. Our results suggest that in sponge species with high population structure, the host genetic cluster influence the microbial community more than the geographic location.

Evidence of Vent-Adaptation in Sponges Living at the Periphery of Hydrothermal Vent Environments: Ecological and Evolutionary Implications.

The peripheral areas of deep-sea hydrothermal vents are often inhabited by an assemblage of animals distinct to those living close to vent chimneys. For many such taxa, it is considered that peak abundances in the vent periphery relate to the availability of hard substrate as well as the increased concentrations of organic matter generated at vents, compared to background areas. However, the peripheries of vents are less well-studied than the assemblages of vent-endemic taxa, and the mechanisms through which peripheral fauna may benefit from vent environments are generally unknown. Understanding this is crucial for evaluating the sphere of influence of hydrothermal vents and managing the impacts of future human activity within these environments, as well as offering insights into the processes of metazoan adaptation to vents. In this study, we explored the evolutionary histories, microbiomes and nutritional sources of two distantly-related sponge types living at the periphery of active hydrothermal vents in two different geological settings (Cladorhiza from the E2 vent site on the East Scotia Ridge, Southern Ocean, and Spinularia from the Endeavour vent site on the Juan de Fuca Ridge, North-East Pacific) to examine their relationship to nearby venting. Our results uncovered a close sister relationship between the majority of our E2 Cladorhiza specimens and the species Cladorhiza methanophila, known to harbor and obtain nutrition from methanotrophic symbionts at cold seeps. Our microbiome analyses demonstrated that both E2 Cladorhiza and Endeavour Spinularia sp. are associated with putative chemosynthetic Gammaproteobacteria, including Thioglobaceae (present in both sponge types) and Methylomonaceae (present in Spinularia sp.). These bacteria are closely related to chemoautotrophic symbionts of bathymodiolin mussels. Both vent-peripheral sponges demonstrate carbon and nitrogen isotopic signatures consistent with contributions to nutrition from chemosynthesis. This study expands the number of known associations between metazoans and potentially chemosynthetic Gammaproteobacteria, indicating that they can be incredibly widespread and also occur away from the immediate vicinity of chemosynthetic environments in the vent-periphery, where these sponges may be adapted to benefit from dispersed vent fluids.

Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater sponge Ephydatia muelleri.

The genomes of non-bilaterian metazoans are key to understanding the molecular basis of early animal evolution. However, a full comprehension of how animal-specific traits, such as nervous systems, arose is hindered by the scarcity and fragmented nature of genomes from key taxa, such as Porifera. Ephydatia muelleri is a freshwater sponge found across the northern hemisphere. Here, we present its 326 Mb genome, assembled to high contiguity (N50: 9.88 Mb) with 23 chromosomes on 24 scaffolds. Our analyses reveal a metazoan-typical genome architecture, with highly shared synteny across Metazoa, and suggest that adaptation to the extreme temperatures and conditions found in freshwater often involves gene duplication. The pancontinental distribution and ready laboratory culture of E. muelleri make this a highly practical model system which, with RNAseq, DNA methylation and bacterial amplicon data spanning its development and range, allows exploration of genomic changes both within sponges and in early animal evolution.

Vertical transmission of sponge microbiota is inconsistent and unfaithful.

Co-evolutionary theory predicts that if beneficial microbial symbionts improve host fitness, they should be faithfully transmitted to offspring. More recently, the hologenome theory of evolution predicts resemblance between parent and offspring microbiomes and high partner fidelity between host species and their vertically transmitted microbes. Here, we test these ideas in multiple coexisting host species with highly diverse microbiota, leveraging known parent-offspring pairs sampled from eight species of wild marine sponges (Porifera). We found that the processes governing vertical transmission were both neutral and selective. A neutral model was a better fit to larval (R2 = 0.66) than to the adult microbiota (R2 = 0.27), suggesting that the importance of non-neutral processes increases as the sponge host matures. Microbes that are enriched above neutral expectations in adults were disproportionately transferred to offspring. Patterns of vertical transmission were, however, incomplete: larval sponges shared, on average, 44.8% of microbes with their parents, which was not higher than the fraction they shared with nearby non-parental adults. Vertical transmission was also inconsistent across siblings, as larval sponges from the same parent shared only 17% of microbes. Finally, we found no evidence that vertically transmitted microbes are faithful to a single sponge host species. Surprisingly, larvae were as likely to share vertically transmitted microbes with larvae from other sponge species as they were with their own species. Our study demonstrates that common predictions of vertical transmission that stem from species-poor systems are not necessarily true when scaling up to diverse and complex microbiomes.

Delineation of ecologically distinct units of marine Bacteroidetes in the Northwestern Mediterranean Sea.

Bacteroidetes is one of the dominant phyla of ocean bacterioplankton, yet its diversity and population structure is poorly understood. To advance in the delineation of ecologically meaningful units within this group, we constructed near full-length 16S rRNA gene clone libraries from contrasting marine environments in the NW Mediterranean. Based on phylogeny and the associated ecological variables (depth and season), 24 different Bacteroidetes clades were delineated. By considering their relative abundance (from iTag amplicon sequencing studies), we described the distribution patterns of each of these clades, delimiting them as Ecologically Significant Taxonomic Units (ESTUs). Spatially, there was almost no overlap among ESTUs at different depths. In deep waters there was predominance of Owenweeksia, Leeuwenhoekiella, Muricauda-related genera, and some depth-associated ESTUs within the NS5 and NS2b marine clades. Seasonally, multi-annual dynamics of recurring ESTUs were present with dominance of some ESTUs within the NS4, NS5 and NS2b marine clades along most of the year, but with variable relative frequencies between months. A drastic change towards the predominance of Formosa-related ESTUs and one ESTU from the NS5 marine clade was typically present after the spring bloom. Even though there are no isolates available for these ESTUs to determine their physiology, correlation analyses identified the environmental preference of some of them. Overall, our results suggest that there is a high degree of niche specialisation within these closely related clades. This work constitutes a step forward in disentangling the ecology of marine Bacteroidetes, which are essential players in organic matter processing in the oceans.

Detecting signatures of a sponge-associated lifestyle in bacterial genomes.

Sponges interact with diverse and rich communities of bacteria that are phylogenetically often distinct from their free-living counterparts. Recent genomics and metagenomic studies have indicated that bacterial sponge symbionts also have distinct functional features from free-living bacteria; however, it is unclear, if such genome-derived functional signatures are common and present in different symbiont taxa. We therefore compared here a large set of genomes from cultured (Pseudovibrio, Ruegeria and Aquimarina) and yet-uncultivated (Synechococcus) bacteria found in either sponge-associated or free-living sources. Our analysis revealed only very few genera-specific functions that could be correlated with a sponge-associated lifestyle. Using different sets of sponge-associated and free-living bacteria for each genus, we could however show that the functions identified as 'sponge-associated' are dependent on the reference comparison being made. Using simulation approaches, we show how this influences the robustness of identifying functional signatures and how evolutionary divergence and genomic adaptation can be distinguished. Our results highlight the future need for robust comparative analyses to define genomic signatures of symbiotic lifestyles, whether it is for symbionts of sponges or other host organisms.

Unveiling the role and life strategies of viruses from the surface to the dark ocean.

Viruses are a key component of marine ecosystems, but the assessment of their global role in regulating microbial communities and the flux of carbon is precluded by a paucity of data, particularly in the deep ocean. We assessed patterns in viral abundance and production and the role of viral lysis as a driver of prokaryote mortality, from surface to bathypelagic layers, across the tropical and subtropical oceans. Viral abundance showed significant differences between oceans in the epipelagic and mesopelagic, but not in the bathypelagic, and decreased with depth, with an average power-law scaling exponent of -1.03 km-1 from an average of 7.76 × 106 viruses ml-1 in the epipelagic to 0.62 × 106 viruses ml-1 in the bathypelagic layer with an average integrated (0 to 4000 m) viral stock of about 0.004 to 0.044 g C m-2, half of which is found below 775 m. Lysogenic viral production was higher than lytic viral production in surface waters, whereas the opposite was found in the bathypelagic, where prokaryotic mortality due to viruses was estimated to be 60 times higher than grazing. Free viruses had turnover times of 0.1 days in the bathypelagic, revealing that viruses in the bathypelagic are highly dynamic. On the basis of the rates of lysed prokaryotic cells, we estimated that viruses release 145 Gt C year-1 in the global tropical and subtropical oceans. The active viral processes reported here demonstrate the importance of viruses in the production of dissolved organic carbon in the dark ocean, a major pathway in carbon cycling.

Temporal Stability and Composition of the Ocular Surface Microbiome.

To determine if there is a core ocular surface microbiome and whether there are microbial community changes over time, the conjunctiva of 45 healthy subjects were sampled at three time points over three months and processed using culture-dependent and -independent methods. Contaminant taxa were removed using a linear regression model using taxa abundances in negative controls as predictor of taxa abundances in subject samples. Both cultured cell counts and sequencing indicated low microbial biomass on the ocular surface. No cultured species was found in all subjects at all times or in all subjects at any one time. After removal of contaminant taxa identified in negative controls using a statistical model, the most commonly detected taxon was Corynebacterium (11.1%). No taxa were found in all subjects at all times or in all subjects in any one time, but there were 26 taxa present in at least one or more subjects at all times including Corynebacterium and Streptococcus. The ocular surface contains a low diversity of microorganisms. Using culture dependent and independent methods, the ocular surface does not appear to support a substantial core microbiome. However, consistently present taxa could be observed within individuals suggesting the possibility of individual-specific core microbiomes.

Integrated metabolism in sponge-microbe symbiosis revealed by genome-centered metatranscriptomics.

Despite an increased understanding of functions in sponge microbiomes, the interactions among the symbionts and between symbionts and host are not well characterized. Here we reconstructed the metabolic interactions within the sponge Cymbastela concentrica microbiome in the context of functional features of symbiotic diatoms and the host. Three genome bins (CcPhy, CcNi and CcThau) were recovered from metagenomic data of C. concentrica, belonging to the proteobacterial family Phyllobacteriaceae, the Nitrospira genus and the thaumarchaeal order Nitrosopumilales. Gene expression was estimated by mapping C. concentrica metatranscriptomic reads. Our analyses indicated that CcPhy is heterotrophic, while CcNi and CcThau are chemolithoautotrophs. CcPhy expressed many transporters for the acquisition of dissolved organic compounds, likely available through the sponge's filtration activity and symbiotic carbon fixation. Coupled nitrification by CcThau and CcNi was reconstructed, supported by the observed close proximity of the cells in fluorescence in situ hybridization. CcPhy facultative anaerobic respiration and assimilation by diatoms may consume the resulting nitrate. Transcriptional analysis of diatom and sponge functions indicated that these organisms are likely sources of organic compounds, for example, creatine/creatinine and dissolved organic carbon, for other members of the symbiosis. Our results suggest that organic nitrogen compounds, for example, creatine, creatinine, urea and cyanate, fuel the nitrogen cycle within the sponge. This study provides an unprecedented view of the metabolic interactions within sponge-microbe symbiosis, bridging the gap between cell- and community-level knowledge.

Particle-association lifestyle is a phylogenetically conserved trait in bathypelagic prokaryotes.

The free-living (FL) and particle-attached (PA) marine microbial communities have repeatedly been proved to differ in their diversity and composition in the photic ocean and also recently in the bathypelagic ocean at a global scale. However, although high taxonomic ranks exhibit preferences for a PA or FL mode of life, it remains poorly understood whether two clear lifestyles do exist and how these are distributed across the prokaryotic phylogeny. We studied the FL (<0.8 µm) and PA (0.8-20 µm) prokaryotes at 30 stations distributed worldwide within the bathypelagic oceanic realm (2150-4000 m depth) using high-throughput sequencing of the small subunit ribosomal RNA gene (16S rRNA). A high proportion of the bathypelagic prokaryotes were mostly found either attached to particles or freely in the surrounding water but rarely in both types of environments. In particular, this trait was deeply conserved through their phylogeny, suggesting that the deep-ocean particles and the surrounding water constitute two highly distinct niches and that transitions from one to the other have been rare at an evolutionary timescale. As a consequence, PA and FL communities had clear alpha- and beta-diversity differences that exceeded the global-scale geographical variation. Our study organizes the bathypelagic prokaryotic diversity into a reasonable number of ecologically coherent taxa regarding their association with particles, a first step for understanding which are the microbes responsible for the processing of the dissolved and particulate pools of organic matter that have a very different biogeochemical role in the deep ocean.

Validation of a new catalysed reporter deposition-fluorescence in situ hybridization probe for the accurate quantification of marine Bacteroidetes populations.

Catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH) is a powerful approach to quantify bacterial taxa. In this study, we compare the performance of the widely used Bacteroidetes CF319a probe with the new CF968 probe. In silico analyses and tests with isolates demonstrate that CF319a hybridizes with non-Bacteroidetes sequences from the Rhodobacteraceae and Alteromonadaceae families. We test the probes' accuracy in 37 globally distributed marine samples and over two consecutive years at the Blanes Bay Microbial Observatory (NW Mediterranean). We also compared the CARD-FISH data with the Bacteroidetes 16S rRNA gene sequences retrieved from 27 marine metagenomes from the TARA Oceans expedition. We find no significant differences in abundances between both approaches, although CF319a targeted some unspecific sequences and both probes displayed different abundances of specific Bacteroidetes phylotypes. Our results demonstrate that quantitative estimations by using both probes are significantly different in certain oceanographic regions (Mediterranean Sea, Red Sea and Arabian Sea) and that CF968 shows seasonality within marine Bacteroidetes, notably large differences between summer and winter that is overlooked by CF319a. We propose CF968 as an alternative to CF319a for targeting the whole Bacteroidetes phylum since it has better coverage, greater specificity and overall better quantifies marine Bacteroidetes.

Spatial and temporal variability among marine Bacteroidetes populations in the NW Mediterranean Sea.

The abundance and structure of Bacteroidetes populations at diverse temporal and spatial scales were investigated in the Northwestern Mediterranean Sea. At a temporal scale, their relative abundance exhibited a marked seasonality, since it was higher in spring and decreased in winter. Similarly, Bacteroidetes community structure encompassed three main groups (winter, spring and summer-fall), which mimicked global bacterioplankton seasonality. At the spatial scale, relative abundances were similar in all surface samples along an inshore-offshore transect, but they decreased with depth. Analysis of the community structure identified four markedly different groups mostly related to different depths. Interestingly, seasonal changes in abundance and community structure were not synchronized. Furthermore, richness was higher when Bacteroidetes were less abundant. The variability of Bacteroidetes contributions to community structure in the temporal and spatial scales was correlated with different environmental factors: day length was the most important factor at the temporal scale, and salinity at the spatial scale. The community composition in terms of phylotypes changed significantly over time and along the depth gradients, but season or depth-specific phylogenetic clusters were not identified. Delineation of coherent Bacteroidetes sub-clusters should help to uncover higher resolution patterns within Bacteroidetes, and explore associations with environmental and biological variables.

Evaluation of marine bacteroidetes-specific primers for microbial diversity and dynamics studies.

Assumptions on the matching specificity of group-specific bacterial primers may bias the interpretation of environmental microbial studies. As available sequence data continue growing, the performance of primers and probes needs to be reevaluated. Here, we present an evaluation of several commonly used and one newly designed Bacteroidetes-specific primer (CF418). First, we revised the in silico primer coverage and specificity with the current SILVA and RDP databases. We found minor differences with previous studies, which could be explained by the chosen databases, taxonomies, and matching criteria. We selected eight commonly used Bacteroidetes primers and tested them with a collection of assorted marine bacterial isolates. We also used the denaturing gradient gel electrophoresis (DGGE) approach in environmental samples to evaluate their ability to yield clear and diverse band patterns corresponding to Bacteroidetes phylotypes. Among the primers tested, CF968R did not provide satisfactory results in DGGE, although it exhibited the highest in silico coverage for Flavobacteria. Primers CFB560 and CFB555 presented undesirable features, such as requiring nested protocols or presence of degeneracies. Finally, the new primer CF418 and primer CF319a were used to explore the Bacteroidetes dynamics throughout a 1-year cycle in Mediterranean coastal waters (Blanes Bay Microbial Observatory). Both primers provided clear and diverse banding patterns, but the low specificity of CF319a was evidenced by 83.3 % of the bands sequenced corresponding to nontarget taxa. The satisfactory DGGE banding patterns and the wide diversity of sequences retrieved from DGGE bands with primer CF418 prove it to be a valuable alternative for the study of Bacteroidetes communities, recovering a wide range of phylotypes within the group.

Proteomic study of neuron and astrocyte cultures from senescence-accelerated mouse SAMP8 reveals degenerative changes.

Senescence-accelerated prone (SAMP) strain 8 mice suffer an earlier development of cognitive age-related pathologies and a shorter life span than conventional mice. Protein alterations in astrocytes, in addition to those in neurons, may contribute to neurodegenerative damage. We applied proteomics techniques to study cell-specific early markers of brain aging-related degeneration in SAMP8. The two-dimensional protein expression patterns of the SAMP8 neuron and astrocyte cultures were compared with those obtained from senescence-accelerated resistant mouse strain 1 cultures. Differentially expressed spots were identified by matrix-assisted laser desorption/ionization-time of flight peptide map fingerprinting and database search. Proteins belonged to cell pathways of energy metabolism, biosynthesis, cell transduction and signaling, stress response, and the maintenance of cytoskeletal functions. Most of the changes were cell type specific. However, there was a general increase in cell transduction, signaling, and stress-related proteins and a decrease in cytoskeletal proteins. In addition, neurons showed an increased expression of proteins involved in biosynthetic pathways. A number of the protein alterations have been previously reported in the brain tissue proteome of SAMP8, aged brain or Alzheimer's disease brain. Alterations in neuron and astrocyte proteoma indicated that both cell types are involved in the brain degenerative changes of SAMP8 mice. However, network analysis suggests that neuronal changes are more complex and have a greater influence.

Dysfunction of astrocytes in senescence-accelerated mice SAMP8 reduces their neuroprotective capacity.

Early onset increases in oxidative stress and tau pathology are present in the brain of senescence-accelerated mice prone (SAMP8). Astrocytes play an essential role, both in determining the brain's susceptibility to oxidative damage and in protecting neurons. In this study, we examine changes in tau phosphorylation, oxidative stress and glutamate uptake in primary cultures of cortical astrocytes from neonatal SAMP8 mice and senescence-accelerated-resistant mice (SAMR1). We demonstrated an enhancement of abnormally phosphorylated tau in Ser(199) and Ser(396) in SAMP8 astrocytes compared with that of SAMR1 control mice. Gsk3beta and Cdk5 kinase activity, which regulate tau phosphorylation, was also increased in SAMP8 astrocytes. Inhibition of Gsk3beta by lithium or Cdk5 by roscovitine reduced tau phosphorylation at Ser(396). Moreover, we detected an increase in radical superoxide generation, which may be responsible for the corresponding increase in lipoperoxidation and protein oxidation. We also observed a reduced mitochondrial membrane potential in SAMP8 mouse astrocytes. Glutamate uptake in astrocytes is a critical neuroprotective mechanism. SAMP8 astrocytes showed a decreased glutamate uptake compared with those of SAMR1 controls. Interestingly, survival of SAMP8 or SAMR1 neurons cocultured with SAMP8 astrocytes was significantly reduced. Our results indicate that alterations in astrocyte cultures from SAMP8 mice are similar to those detected in whole brains of SAMP8 mice at 1-5 months. Moreover, our findings suggest that this in vitro preparation is suitable for studying the molecular and cellular processes underlying early aging in this murine model. In addition, our study supports the contention that astrocytes play a key role in neurodegeneration during the aging process.