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.

Neurotrophin-4 in the brain of adult Nothobranchius furzeri.

Neurotrophin-4 (NT-4) is a member of the well-known family of neurotrophins that regulate the development of neuronal networks by participating in neuronal survival and differentiation, the growth of neuronal processes, synaptic development and plasticity, as well as myelination. NT-4 interacts with two distinct receptors: TrkB, high affinity receptor and p75 low-affinity neurotrophin receptor (p75(NTR)). In the present survey, we identified the gene encoding NT-4 in the teleost Nothobranchius furzeri, a model species for aging research. The identified gene shows a similarity of about 72% with medaka, the closest related species. The neuroanatomical localization of NT-4 mRNA is obtained by using an LNA probe. NT-4 mRNA expression is observed in neurons and glial cells of the forebrain and hindbrain, with very low signal found in the midbrain. This survey confirms that NT-4 is expressed in the brain of N. furzeri during adulthood, suggesting that it could also be implicated in the maintenance and regulation of neuronal functions.

Nerve growth factor in the adult brain of a teleostean model for aging research: Nothobranchius furzeri.

Nerve growth factor (NGF) acts on central nervous system neurons, regulating naturally occurring cell death, synaptic connectivity, fiber guidance and dendritic morphology. The dynamically regulated production of NGF beginning in development, extends throughout adult life and aging, exerting numerous roles through a surprising variety of neurons and glial cells. This study analyzes the localization of NGF in the brain of the teleost fish Nothobranchius furzeri, an emerging model for aging research due to its short lifespan. Immunochemical and immunohistochemical experiments were performed by employing an antibody mapping at the N-terminus of the mature chain human origin NGF. Western blot analysis revealed an intense and well defined band of 20 kDa, which corresponds to proNGF of N. furzeri. Immunohistochemistry revealed NGF immunoreactivity (IR) diffused throughout all regions of telencephalon, diencephalon, mesencephalon and rhomboencephalon. It was detected in neurons and in glial cells, the latter mostly lining the mesencephalic and rhomboencephalic ventricles. Particularly in neurons, NGF IR was localized in perikarya and, to a less extent, in fibers. The widespread distribution of proNGF suggests that it might modulate numerous physiological functions in the adult brain of N. furzeri. The present survey constitutes a baseline study to enhance the understanding of the mechanisms underlying the role of NGF during aging processes.

Genetic and morphological studies of Nothobranchius (Cyprinodontiformes) from Malawi with description of Nothobranchius wattersi sp. nov.

Molecular and morphological data were used to explore evolutionary differentiation among populations of Nothobranchius in the Lake Malawi-upper Shire River and the Lakes Chilwa-Chiuta drainage systems in Malawi. The aim of the study was to test the hypothesis that Nothobranchius of the Malawi-Shire system constitute a separate evolutionary group from Nothobranchius kirki. Mitochondrial and nuclear sequence data show a strongly supported phylogenetic split into two monophyletic groups separating the Lake Malawi basin fish from N. kirki. Unlike N. kirki, Lake Malawi-Shire fish do not deviate from neutrality and express an excess of rare haplotypes and mutations in terminal branches, characteristic of recently expanded populations. Further, the two groups significantly differ in morphology. Two body characters (dorsal-fin base length and pre-pelvic-pre-anal distance) are significantly different between the two species in both sexes. Several other characters are significantly different in either male or female comparisons with respect to both standard and head lengths, and robust morphological differentiation is detected by multivariate analysis. The two groups are readily distinguished on the basis of male colouration, especially in scale centres and the caudal fin. On the basis of this differentiation at the molecular and morphological levels, in addition to colouration, the Lake Malawi-Shire fish are hereby formally recognized as constituting a new species, Nothobranchius wattersi. This distinction is in agreement with the geomorphologic and recent climatic history in the region.

Phylogeny, genetic variability and colour polymorphism of an emerging animal model: the short-lived annual Nothobranchius fishes from southern Mozambique.

Nothobranchius are a group of small, extremely short-lived killifishes living in temporary savannah pools in Eastern Africa and that survive annual desiccation of their habitat as dormant eggs encased in dry mud. One mitochondrial (COI) and three nuclear (CX32.2, GHITM, PNP) loci were used to investigate the phylogenetic relationship of Nothobranchius species from southern and central Mozambique. This group shows marked variation in captive lifespan at both the inter- and intraspecific levels; lifespan varies from a few months to over a year. As their distribution encompasses a steep gradient between semi-arid and humid habitats, resulting in contrasting selection pressures on evolution of lifespan and associated life history traits, Mozambican Nothobranchius spp. have recently become a model group in studies of ageing, age-related disorders and life history evolution. Consequently, intraspecific genetic variation and male colour morph distribution was also examined in the recovered clades. Using Bayesian species tree reconstruction and single loci analyses, three large clades were apparent and their phylogenetic substructure was revealed at the inter- and intra-specific levels within those clades. The Nothobranchius furzeri and Nothobranchius orthonotus clades were strongly geographically structured. Further, it was demonstrated that male colour has no phylogenetic signal in N. furzeri, where colour morphs are sympatric, but is associated with two reciprocally monophyletic groups in Nothobranchius rachovii clade, where colour morphs are parapatric. Finally, our analysis showed that a polymorphism in the Melanocortin1 receptor gene (which controls pigmentation in many vertebrates and was a candidate gene of male colouration in N. furzeri) is unrelated to colour phenotypes of the study species. Our results raise significant implications for future comparative studies of the species and populations analysed in the present work.

Age-dependent remodelling of retinal circuitry.

We have investigated morphological changes in second-order neurons of the mouse retina during aging by using immunohistochemistry and electron microscopy. We observed sprouting of rod bipolar cells dendrites and horizontal cells arborizations: neuronal processes of both neuronal types showed irregular extensions beyond the outer plexiform layer, toward the outer limiting membrane, as well as into the outer nuclear layer (ONL). These processes were first observed in animals of 12 months of age and increased in numbers steadily until 24 months, which represent the last age examined. The ectopic processes are decorated by puncta immunoreactive for pre-synaptic markers typical of photoreceptor terminals juxtaposed to post-synaptic neurotransmitter receptors, demonstrating the presence of the entire molecular machinery of functional synapses. Electron microscopy confirmed that ectopic processes receive synapses from photoreceptor terminals. We conclude that during the second year of life retinal rod bipolar and horizontal cells undergo sprouting and form ectopic synapses in the ONL.

Why humans need type 5 phosphodiesterase inhibitors.

Erectile dysfunction (ED) is a widespread medical condition affecting millions of males at any age and requiring medical treatment. ED may simply reflect a limit of human physiology, yet ED equates to genetic death and the high prevalence of ED is a clear evolutionary paradox. Why is a condition which totally blocks reproduction so widespread? Epidemiology shows that impotence is a common symptom of almost all major diseases and male reproductive physiology is sensitive to general health and to environmental, psychological, and physical stressors. Moreover, erectile dysfunction is a predictor of myocardial infarction and stroke. Briefly, efficient erection is a marker of good health and good health prognosis. In the animal kingdom, mate choice is often based on extravagant and cumbersome physical traits (such as the peacock's tail). These traits, that are necessary for gene propagation, are at the same time efficient handicaps which reduce the survival of the carrier. What animal studies show, is that these handicaps can function as honest indicators of the individual's fitness. In other words, their expression is condition-dependent and only individuals with high phenotypic quality present the trait. By mating with males which express the trait, females indirectly select for individuals of superior fitness which will be inherited by the offspring. Erection is very clearly a condition-dependent trait in the human species. We suggest that the fragility of male sexual physiology is a sexually selected handicap which hampers the reproduction of individuals with lower phenotypic quality.

Psychobiology of facial attractiveness.

The last decade has witnessed an upsurge of interest in the research on facial attractiveness. The development of computer graphics has allowed to objectively investigate the conserved features of attractive faces. Averageness, symmetry and sex-specific traits have been associated with attractiveness. The effect of averageness is exemplified by blending a set of real faces into a chimeric face. This composite is more attractive than most of the faces used to create it. Beautiful faces are not simply average faces, however. If the female-specific features of a female composite face are enhanced, the resulting face is perceived as more attractive than the composite. In particular, smaller than average chin, smaller than average nose and higher than average forehead, all are traits associated with female's attractiveness. These traits have been interpreted as signs of high estrogen/testosterone ratio and therefore cues of high fertility. However, these same traits are also a species-specific characteristic of Homo sapiens that differentiates it from other hominid species. Preference for caricature of human features could represent a relic of species recognition mechanisms. Female preferences for male faces proved to be more variable than male preferences for female faces. Different facial traits are preferred in the choice of short-term and long-term partners. Preference for short term depend on the hormonal status and changes across the menstrual cycle and is influenced by contraceptive hormonal treatment. Psychological factors are also important sources of variance: female preferences correlate with self-perceived attractiveness, status in a relationship and degree of gender-conformity.

The dynamics of neuronal death: a time-lapse study in the retina.

Using time-lapse video microscopy, we have followed the process of neuronal death in an intact region of the mammalian nervous system. We show here the fast dynamics of nuclear fragmentation, which is over in <1 hr for neurons undergoing apoptosis in the living rat retina. Nuclear fragmentation is accompanied by a progressive raise of intracellular calcium and followed by erratic movement of the apoptotic cells, documenting their loss of adhesion.

Apoptosis in the developing visual system.

Programmed cellular death is a widespread phenomenon during development of the nervous system. Two classes of molecules are particularly important in the context of apoptosis control in the nervous system: intracellular effectors homologous to the Caenorhabditis elegans Ced-3, -4, and -9 proteins, which in mammals correspond to the proteases of the caspase family, Apaf-1, and the members of the Bcl-2 protein family, and neurotrophic factors. Retinal ganglion cells lend a convenient model system with which to investigate apoptosis in central neurons during development as well as after injury. In this review, we discuss the role of these molecules in the control of programmed cellular death in the retinotectal system. Transgenic animal models and expression studies have shown that caspases, Bcl-2, Bax, and possibly Bcl-X are necessary players for the control of programmed cellular death in retinal ganglion cells. Bax and caspase 3 expression in retinal ganglion cells is upregulated after injury, and inhibition of Bax or caspase 3 increases the survival of injured retinal ganglion cells. Neurotrophins can support the survival of injured retinal ganglion cells, but this effect is transient. The physiological role of neurotrophins in the development of the retinocollicular system seems more related to the topographic refinement of retinocollicular projections, a process that is mediated, at least partially, by selective elimination of retinal ganglion cells making inappropriate topographic projections.

Retinal ganglion cells with NADPH-diaphorase activity in the chick form a regular mosaic with a strong dorsoventral asymmetry that can be modelled by a minimal spacing rule.

We have identified a class of retinal ganglion cells in the chick retina that can be labelled by NADPH-diaphorase histochemistry. These cells have a remarkable topographic distribution, being restricted to the dorsal hemiretina, and form a highly regular mosaic, as revealed by the analysis of nearest neighbour distribution and Delaunay triangulation. Autocorrelation analysis of the mosaic of NADPH-diaphorase-positive retinal ganglion cells shows that the mosaic spatial organization could be generated with the single constraint that two elements cannot be closer than a given minimal distance (d(min)), which we confirmed by computer simulations. In contrast with what has been observed in other mosaics, here d(min) varies with cell density. However, the observed variation of the exclusion area is consistent with an original assembly of the mosaic with a constant d(min) (as is the case in other mosaics), followed by differential expansion of the retina during development.

Excess target-derived brain-derived neurotrophic factor preserves the transient uncrossed retinal projection to the superior colliculus.

During early postnatal development, a widespread ipsilateral projection to the superior colliculus is secondarily restricted to a small topographically defined region by elimination of ipsilaterally projecting retinal ganglion cells. Brain-derived neurotrophic factor (BDNF) has been proposed as the target-derived neurotrophic factor for retinal ganglion cells in several studies. Here we investigated the long-term effects of excess BDNF in the retinal ganglion cell target on naturally occurring retinal ganglion cell (RGC) elimination and on the restriction of the ipsilateral projection. To this end, sustained overexpression of BDNF was achieved in the postnatal superior colliculus using an adenoviral vector. While the total number of retinal ganglion cells in the adenovirus-BDNF treated animals was unchanged, a much higher proportion of RGCs retained a projection to the ipsilateral superior colliculus. We conclude that an excess of target-derived BDNF does not reduce the net amount of naturally occurring cell death in the retino-collicular system, but prevents the negative selection of retinal ganglion cells making inappropriate topographic connections.

Effects of brain-derived neurotrophic factor on the development of NADPH-diaphorase/nitric oxide synthase-positive amacrine cells in the rodent retina.

Amacrine neurons expressing nitric oxide synthase (NOS) contain brain-derived neurotrophic factor (BDNF) receptors and respond to exogenous BDNF [Klöcker, N., Cellerino, A. & Bähr, M. (1998) J. Neurosci., 18, 1038-1046]. We analysed the effects of BDNF on the development of neurons which express NOS in the mouse and rat retina. Rat pups received a total of three intraocular injections of BDNF at intervals of 48 h, starting at postnatal day 16 (P16), and were killed at P22. The retinas were stained for NADPH-diaphorase, a histological marker of NOS. NOS-expressing neurons were found in both the inner nuclear layer (INL) and the ganglion cell layer (GCL). Two classes of NOS-expressing neurons, type I and type II, had already been distinguished in the INL [Koistinaho, J. & Sagar, S.M. (1995) In Osborne, N.N. & Chader, G.J. (eds), Progress in Retinal and Eye Research, Vol. 15. Oxford University Press, pp. 69-87] and a third one in the GCL. Up-regulation of NADPH-diaphorase activity was observed after BDNF treatment. The number of type I neurons remained stable, whereas the number of type II neurons and NOS-positive neurons in the GCL increased significantly (P < 0.001). Type I and type II neurons were significantly larger in BDNF-treated retinas. Double-labelling experiments revealed that BDNF induces NADPH-diaphorase in dopaminergic neurons and amacrine cells displaced to the GCL, but not in retinal ganglion cells. In mice homozygous for a null mutation of the bdnf gene, the intensity of NADPH-diaphorase labelling in both somata and processes was reduced, but the number of labelled neurons was not dramatically reduced. These findings indicate that BDNF regulates the neurotransmitter phenotype of NOS-expressing amacrine neurons under physiological conditions, but is not required for their survival.

Retinal ganglion cell loss after the period of naturally occurring cell death in bcl-2-/- mice.

Over-expression of Bcl-2 is known to reduce the extent of retinal ganglion cell death during development as well as after axotomy. Here we investigated whether retinal ganglion cell (RGC) numbers are reduced in mice with a targeted inactivation of the bcl-2 gene. Compared with wild-type mice, adult bcl-2 null mutants have lost 29% of the retinal ganglion cell axons in the optic nerve. This reduction was almost fully established at P15, but not present at P10, which marks the end of the period of naturally occurring cell death. These observations, together with the previously reported late loss of primary motoneurons and peripheral neurons, point to a general physiological requirement for Bcl-2 soon after the period of naturally occurring cell death.

Brain-derived neurotrophic factor modulates the development of the dopaminergic network in the rodent retina.

Dopaminergic cells in the retina express the receptor for brain-derived neurotrophic factor (BDNF) (). To investigate whether BDNF can influence the development of the retinal dopaminergic pathway, we performed intraocular injections of BDNF during the second or third postnatal week and visualized the dopaminergic system with tyrosine hydroxylase (TH) immunohistochemistry. Both regimens of BDNF treatment caused an increase in TH immunoreactivity in stratum 1 and stratum 3 of the inner plexiform layer (IPL). D2 dopamine receptor immunoreactivity, a presynaptic marker of dopaminergic cells (), was also increased in stratum 1 and stratum 3 of the inner plexiform layer. These data suggest that BDNF causes sprouting of dopaminergic fibers in the inner plexiform layer. Other neurochemical systems, for example, the cholinergic amacrine cells, remained unaffected. Similar effects were observed after injections of neurotrophin-3 and neurotrophin-4, but not nerve growth factor. Analysis of whole-mounted TH-immunolabeled retinae revealed hypertrophy of dopaminergic cells (+41% in soma areas; p < 0.01) and an increase of labeled dopaminergic varicosities in stratum 1 of the IPL (+51%; p < 0.01) after BDNF treatment. The opposite was observed in mice homozygous for a null mutation of the bdnf gene: dopaminergic cells were atrophic (-22.5% in soma areas; p < 0.05), and the density of TH-positive varicosities in stratum 1 was reduced (57%; p < 0.01). We conclude that BDNF controls the development of the retinal dopaminergic network and may be particularly important in determining the density of dopaminergic innervation in the retina.

Free radical scavenging and inhibition of nitric oxide synthase potentiates the neurotrophic effects of brain-derived neurotrophic factor on axotomized retinal ganglion cells In vivo.

Brain-derived neurotrophic factor (BDNF) partially promotes the survival of axotomized retinal ganglion cells (RGCs). In analogy with in vitro experiments (; ), we tested whether neuroprotection by BDNF is limited by adverse effects as a consequence of excessive free radical formation. First, we investigated whether BDNF and the free radical scavenger N-tert-butyl-(2-sulfophenyl)-nitrone (S-PBN) cooperate in protecting RGCs from axotomy-induced death. Although systemic S-PBN treatment alone did not influence RGC survival after axotomy, it potentiated the neuroprotective effects of BDNF significantly. Single BDNF treatment rescued 27% of the RGCs, which otherwise would have died 14 d after optic nerve transection, whereas a combined treatment of BDNF and S-PBN improved this rescue rate up to 68%. We then investigated whether the adverse effects of BDNF could be ascribed to activation of nitric oxide synthase (NOS). We found colocalization of NOS and the BDNF receptor TrkB in the retina. NADPH-diaphorase reactivity, a reliable marker for NOS in the rat retina, increased after chronic BDNF treatment in vivo. Systemic application of the NOS-inhibitor N-omega-nitro-L-arginine-methylester (L-NAME) potentiated the neuroprotective action of BDNF (55% rescue rate). We conclude that activation of NOS is a pathological consequence of BDNF application, which reduces its neuroprotective potential. The observation that this adverse effect can be antagonized by systemic application of free radical scavengers could be of relevance for clinical applications of neurotrophins in human neurodegenerative diseases.

Brain-derived neurotrophic factor/neurotrophin-4 receptor TrkB is localized on ganglion cells and dopaminergic amacrine cells in the vertebrate retina.

The tyrosine kinase TrkB is a receptor for the neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). Retinal ganglion cells are responsive to BDNF, and TrkB has been localized in ganglion cells as well as in a subpopulation of amacrine cells in the retina of the chicken and the rat. In the present paper, we analyzed the distribution of TrkB immunoreactivity in the retina of marmoset monkeys, ferrets, rabbits, rats, mice, chickens, pigeons, barn owls, Pseudemys turtles, Xenopus frogs, goldfishes, and carps. TrkB antibodies gave a positive reaction in all of these vertebrates. TrkB immunoreactivity was detected in the majority of retinal ganglion cells. Some amacrine cells also contained TrkB immunoreactivity; they were located mainly at the vitreal border of the inner nuclear layer, and their relative abundance varied in the different species. Until now, no information has been available concerning the neurochemical identity of the amacrine neurons containing TrkB. In some species (marmoset monkeys, rats, pigeons), we observed that the morphology and location of TrkB-immunoreactive amacrine cells was reminiscent of that of the well-described dopaminergic cells. To determine whether dopaminergic amacrine cells contained TrkB immunoreactivity, we therefore performed double-labelling immunohistochemistry by using tyrosine hydroxylase (TH) antibodies in combination with TrkB antibodies in marmoset monkeys, rats, pigeons, Pseudemys turtles, and goldfishes. The most novel finding of the present paper is that, in all of these species, the majority of dopaminergic neurons were found to contain TrkB immunoreactivity. Dopaminergic neurons, on the other hand, represented only a fraction of the TrkB+ amacrine cells. Our data suggest that BDNF and/or NT-4 might modulate expression of TH in the retina and may therefore influence the retinal dopaminergic system. Whatever the action of TrkB ligands on the retinal dopaminergic system, it was conserved during vertebrate evolution.

Reduced size of retinal ganglion cell axons and hypomyelination in mice lacking brain-derived neurotrophic factor.

While brain-derived neurotrophic factor (BDNF) delays the death of axotomized retinal ganglion cells in rodents, it is unclear if it affects any aspect of the normal development of these cells. Here we examined the optic nerve of bdnf-/- mice. Axonal numbers were normal, but their diameter, as well as the proportion of myelinated axons, was reduced at postnatal day 20 (P20). In contrast, the facial nerve was not hypomyelinated. Expression levels of mRNAs coding for the myelin proteins PLP and MBP were substantially reduced in the hippocampus and cortex at P20, but not in the sciatic nerve. Intraventricular injections of BDNF into the ventricles of wild-type mice at P10 and P12 up-regulated expression of PLP in the hippocampus at P14. These results indicate a role of BDNF, discussed as indirect, in the control of myelination in the central nervous system.

The distribution of brain-derived neurotrophic factor and its receptor trkB in parvalbumin-containing neurons of the rat visual cortex.

We analysed the distribution of brain-derived neurotrophic factor (BNDNF) and its receptor trkB in the adult rat visual cortex, paying particular attention to a GABAergic neuronal subpopulation - the parvalbumin-positive cells. We found expression of trkB in the cell body and apical dendrite of pyramidal neurons and in the cell body of non-pyramidal neurons. Double labelling experiments revealed extensive colocalization of parvalbumin and trkB immunoreactivity in non-pyramidal neurons. Interestingly, the trkB-positive pyramidal neurons appeared surrounded by parvalbumin-labelled boutons. The use of double immunohistochemistry and in situ hybridization histochemistry showed that parvalbumin-positive neurons express trkB mRNA. BDNF mRNA was found in several cells. Coexpression of BDNF mRNA and parvalbumin immunoreactivity was extremely rare. These data strongly suggest that BDNF synthesized by cortical neurons acts as a postsynaptically derived factor for parvalbumin-positive neurons in the adult rat visual cortex.

The action of neurotrophins in the development and plasticity of the visual cortex.

Nerve growth factor (NGF) and the other members of the NGF gene family have been extensively characterized as neurotrophic factors. Recently a modulatory action of these neurotrophic factors on synapse efficacy has emerged. The developing visual system has provided a convenient model to test the role of neurotrophins on neural plasticity in vivo.