Mario Baumgart

Adult neurogenesis in the short‐lived teleost Nothobranchius furzeri: localization of neurogenic niches, molecular characterization and effects of aging

We studied adult neurogenesis in the short‐lived annual fish Nothobranchius furzeri and quantified the effects of aging on the mitotic activity of the neuronal progenitors and the expression of glial fibrillary acid protein (GFAP) in the radial glia. The distribution of neurogenic niches is substantially similar to that of zebrafish and adult stem cells generate neurons, which persist in the adult brain. As opposed to zebrafish, however, the N. furzeri genome contains a doublecortin (DCX) gene. Doublecortin is transiently expressed by newly generated neurons in the telencephalon and optic tectum (OT). We also analyzed the expression of the microRNA miR‐9 and miR‐124 and found that they have complementary expression domains: miR‐9 is expressed in the neurogenic niches of the telencephalon and the radial glia of the OT, while miR‐124 is expressed in differentiated neurons. The main finding of this paper is the demonstration of an age‐dependent decay in adult neurogenesis. Using unbiased stereological estimates of cell numbers, we detected an almost fivefold decrease in the number of mitotically active cells in the OT between young and old age. This reduced mitotic activity is paralleled by a reduction in DCX labeling. Finally, we detected a dramatic up‐regulation of GFAP in the radial glia of the aged brain. This up‐regulation is not paralleled by a similar up‐regulation of S100B and Musashi‐1, two other markers of the radial glia. In summary, the brain of N. furzeri replicates two typical hallmarks of mammalian aging: gliosis and reduced adult neurogenesis.

RNA-seq of the aging brain in the short-lived fish N. furzeri – conserved pathways and novel genes associated with neurogenesis

The brains of teleost fish show extensive adult neurogenesis and neuronal regeneration. The patterns of gene regulation during fish brain aging are unknown. The short‐lived teleost fish Nothobranchius furzeri shows markers of brain aging including reduced learning performances, gliosis, and reduced adult neurogenesis. We used RNA‐seq to quantify genome‐wide transcript regulation and sampled five different time points to characterize whole‐genome transcript regulation during brain aging of N. furzeri. Comparison with human datasets revealed conserved up‐regulation of ribosome, lysosome, and complement activation and conserved down‐regulation of synapse, mitochondrion, proteasome, and spliceosome. Down‐regulated genes differ in their temporal profiles: neurogenesis and extracellular matrix genes showed rapid decay, synaptic and axonal genes a progressive decay. A substantial proportion of differentially expressed genes (~40%) showed inversion of their temporal profiles in the last time point: spliceosome and proteasome showed initial down‐regulation and stress‐response genes initial up‐regulation. Extensive regulation was detected for chromatin remodelers of the DNMT and CBX families as well as members of the polycomb complex and was mirrored by an up‐regulation of the H3K27me3 epigenetic mark. Network analysis showed extensive coregulation of cell cycle/DNA synthesis genes with the uncharacterized zinc‐finger protein ZNF367 as central hub. In situ hybridization showed that ZNF367 is expressed in neuronal stem cell niches of both embryonic zebrafish and adult N. furzeri. Other genes down‐regulated with age, not previously associated with adult neurogenesis and with similar patterns of expression are AGR2, DNMT3A, KRCP, MEX3A, SCML4, and CBX1. CBX7, on the other hand, was up‐regulated with age.

Age-dependent regulation of tumor-related microRNAs in the brain of the annual fish Nothobranchius furzeri

MicroRNAs are regulators of gene expression. We used miRNA-seq by the Illumina platform to quantify and compare the temporal miRNA expression profiles in the brain of a short-lived (GRZ) and a longer-lived strain (MZM) of the annual fish Nothobranchius furzeri. We used fuzzy-c-means clustering to group miRNAs with similar profiles. In MZM, we found tumor suppressors with known negative interactions with MYC and/or positive interactions with TP53 among up-regulated miRNAs (e.g. miR-23a, miR-26a/b, miR-29a/b and miR-101a) in aged animals. Conversely, we found oncogenes which are MYC targets among down-regulated miRNAs (miR-7a, members of miR cluster 17∼92). These latter were previously shown to be regulated in human replicative aging. In addition, three regulated miRNAs (miR-181c, miR-29a and miR-338) are known to be age-regulated and to globally contribute to regulation of their targets in the human brain. Therefore, there appears to be a degree of evolutionarily conservation in age-dependent miRNA expression between humans and N. furzeri. GRZ showed specific regulation of some miRNAs, notably a marked up-regulation of miR-124, a miRNA important for neuronal differentiation. The two strains differ in their miRNA expression profiles already at sexual maturity. Short lifespan in GRZ could therefore be--at least partially--due to dysregulated miRNA expression.

Brain-derived neurotrophic factor: mRNA expression and protein distribution in the brain of the teleost Nothobranchius furzeri

BDNF (brain‐derived neurotrophic factor) is a member of the neurotrophin family and it is implicated in regulating brain development and function. The BDNF gene organization and coding sequence are conserved in all vertebrates. The present survey was conducted in a teleost fish, Nothobranchius furzeri, because it is an emerging model of aging studies due to its short lifespan and shows the high rate of adult neurogenesis typical of anamniotes. The present survey reports: 1) the identification and characterization of the cDNA fragment encoding BDNF protein, and 2) the localization of BDNF in the whole brain. BDNF mRNA expression was assessed by in situ hybridization, by employing an antisense RNA probe; BDNF protein was detected by employing a sensitive immunohistochemical technique, along with highly specific affinity‐purified antibodies to BDNF. Both BDNF mRNA and protein were detected in neurons and glial cells of all regions of the brain of N. furzeri. Interestingly, BDNF was localized also in brain areas involved in adult neurogenic activities, suggesting a specific role for this neurotrophic factor in controlling cell proliferation. These results provide baseline information for future studies concerning BDNF involvement in the aging processes of the teleost brain. J. Comp. Neurol. 522:1004–1030, 2014.

Conserved Senescence Associated Genes and Pathways in Primary Human Fibroblasts Detected by RNA-Seq.

Cellular senescence correlates with changes in the transcriptome. To obtain a complete view on senescence-associated transcription networks and pathways, we assessed by deep RNA sequencing the transcriptomes of five of the most commonly used laboratory strains of human fibroblasts during their transition into senescence. In a number of cases, we verified the RNA-seq data by real-time PCR. By determining cellular protein levels we observed that the age-related expression of most but not all genes is regulated at the transcriptional level. We found that 78% of the age-affected differentially expressed genes were commonly regulated in the same direction (either up- or down-regulated) in all five fibroblast strains, indicating a strong conservation of age-associated changes in the transcriptome. KEGG pathway analyses confirmed up-regulation of the senescence-associated secretory phenotype and down-regulation of DNA synthesis/repair and most cell cycle pathways common in all five cell strains. Newly identified senescence-induced pathways include up-regulation of endocytotic/phagocytic pathways and down-regulation of the mRNA metabolism and the mRNA splicing pathways. Our results provide an unprecedented comprehensive and deep view into the individual and common transcriptome and pathway changes during the transition into of senescence of five human fibroblast cell strains.

Similarities in Gene Expression Profiles during In Vitro Aging of Primary Human Embryonic Lung and Foreskin Fibroblasts.

Replicative senescence is of fundamental importance for the process of cellular aging, since it is a property of most of our somatic cells. Here, we elucidated this process by comparing gene expression changes, measured by RNA-seq, in fibroblasts originating from two different tissues, embryonic lung (MRC-5) and foreskin (HFF), at five different time points during their transition into senescence. Although the expression patterns of both fibroblast cell lines can be clearly distinguished, the similar differential expression of an ensemble of genes was found to correlate well with their transition into senescence, with only a minority of genes being cell line specific. Clustering-based approaches further revealed common signatures between the cell lines. Investigation of the mRNA expression levels at various time points during the lifespan of either of the fibroblasts resulted in a number of monotonically up- and downregulated genes which clearly showed a novel strong link to aging and senescence related processes which might be functional. In terms of expression profiles of differentially expressed genes with age, common genes identified here have the potential to rule the transition into senescence of embryonic lung and foreskin fibroblasts irrespective of their different cellular origin.

Regulation of microRNA expression in the neuronal stem cell niches during aging of the short-lived annual fish Nothobranchius furzeri

In the last decade, our group has intensively studied the annual fish Nothobranchius furzeri as a new experimental model in Biology specifically applied to aging research. We previously studied adult neuronal stem cells of N. furzeri in vivo and we demonstrated an age-dependent decay in adult neurogenesis. More recently we identified and quantified the expression of miRNAs in the brain of N. furzeri and we detected 165 conserved miRNAs and found that brain aging in this fish is associated with coherent up-regulation of well-known tumor suppressor miRNAs, as well as down-regulation of well-known onco miRNAs~– In the present work we characterized the expression of miR-15a, miR-20a, and microRNA cluster 17–92 in the principal neurogenic niches of the brain of young and old subjects of N. furzeri, by using in situ hybridization techniques, together with proliferating-cell nuclear antigen immuno-staining for a simultaneous visualization of the neuronal progenitors. We found that: (1) the expression of miR-15a is higher in the brain of old subjects and concentrates mainly in the principal neurogenic niches of telencephalon and optic tectum, (2) the expression of miR-20a is higher in the brain of young subjects, but more widespread to the areas surrounding the neurogenic niches, (3) finally, the expression of the microRNA cluster 17–92 is higher in the brain of young subjects, concentrated mainly in the principal neurogenic niches of telencephalon and cerebellum, and with reduced intensity in the optic tectum. Taken together, our data show that these microRNAs, originally identified in whole-brain analysis, are specifically regulated in the stem cell niche during aging.

Comparison of captive lifespan, age-associated liver neoplasias and age-dependent gene expression between two annual fish species: Nothobranchius furzeri and Nothobranchius korthause

Nothobranchius is a genus of annual fish broadly distributed in South-Eastern Africa and found into temporary ponds generated during the rain seasons and their lifespan is limited by the duration of their habitats. Here we compared two Nothobranchius species from radically different environments: N. furzeri and N. korthausae. We found a large difference in life expectancy (29- against 71-weeks of median life span, 40- against 80-weeks of maximum lifespan, respectively), which correlates with a diverse timing in the onset of several age dependent processes: our data show that N. korthause longer lifespan is associated to retarded onset of age-dependent liver-neoplasia and slower down-regulation of collagen 1 alpha 2 (COL1A2) expression in the skin. On the other hand, the expression of cyclin B1 (CCNB1) in the brain was strongly age-regulated, but with similar profiles in N. furzeri and N. korthausae. In conclusion, our data suggest that the different ageing rate of two species of the same genus could be used as novel tool to investigate and better understand the genetic bases of some general mechanism leading to the complex ageing process, providing a strategy to unravel some of the genetic mechanisms regulating longevity and age-associate pathologies including neoplasias.

MicroRNA 19a replacement partially rescues fin and cardiac defects in zebrafish model of Holt Oram syndrome

Holt-Oram Syndrome (HOS) is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene, a transcription factor capable of regulating hundreds of cardiac-specific genes through complex transcriptional networks. Here we show that, in zebrafish, modulation of a single miRNA is sufficient to rescue the morphogenetic defects generated by HOS. The analysis of miRNA-seq profiling revealed a decreased expression of miR-19a in Tbx5-depleted zebrafish embryos compared to the wild type. We revealed that the transcription of the miR-17-92 cluster, which harbors miR-19a, is induced by Tbx5 and that a defined dosage of miR-19a is essential for the correct development of the heart. Importantly, we highlighted that miR-19a replacement is able to rescue cardiac and pectoral fin defects and to increase the viability of HOS zebrafish embryos. We further observed that miR-19a replacement shifts the global gene expression profile of HOS-like zebrafish embryos towards the wild type condition, confirming the ability of miR-19a to rescue the Tbx5 phenotype. In conclusion our data demonstrate the importance of Tbx5/miR-19a regulatory circuit in heart development and provide a proof of principle that morphogenetic defects associated with HOS can be rescued by transient miRNA modulation.

Discovering mirna regulatory networks in holt-Oram syndrome Using a Zebrafish Model

MicroRNAs (miRNAs) are small non-coding RNAs that play an important role in the post-transcriptional regulation of gene expression. miRNAs are involved in the regulation of many biological processes such as differentiation, apoptosis, and cell proliferation. miRNAs are expressed in embryonic, postnatal, and adult hearts, and they have a key role in the regulation of gene expression during cardiovascular development and disease. Aberrant expression of miRNAs is associated with abnormal cardiac cell differentiation and dysfunction. Tbx5 is a member of the T-box gene family, which acts as transcription factor involved in the vertebrate heart development. Alteration of Tbx5 level affects the expression of hundreds of genes. Haploinsufficiency and gene duplication of Tbx5 are at the basis of the cardiac abnormalities associated with Holt–Oram syndrome (HOS). Recent data indicate that miRNAs might be an important part of the regulatory circuit through which Tbx5 controls heart development. Using high-throughput technologies, we characterized genome-widely the miRNA and mRNA expression profiles in WT- and Tbx5-depleted zebrafish embryos at two crucial developmental time points, 24 and 48 h post fertilization (hpf). We found that several miRNAs, which are potential effectors of Tbx5, are differentially expressed; some of them are already known to be involved in cardiac development and functions, such as miR-30, miR-34, miR-190, and miR-21. We performed an integrated analysis of miRNA expression data with gene expression profiles to refine computational target prediction approaches by means of the inversely correlation of miRNA–mRNA expressions, and we highlighted targets, which have roles in cardiac contractility, cardiomyocyte proliferation/apoptosis, and morphogenesis, crucial functions regulated by Tbx5. This approach allowed to discover complex regulatory circuits involving novel miRNAs and protein coding genes not considered before in the HOS such as miR-34a and miR-30 and their targets.