Impaired biogenesis of basic proteins impacts multiple hallmarks of the aging brain.

Aging and neurodegeneration entail diverse cellular and molecular hallmarks. Here, we studied the effects of aging on the transcriptome, translatome, and multiple layers of the proteome in the brain of a short-lived killifish. We reveal that aging causes widespread reduction of proteins enriched in basic amino acids that is independent of mRNA regulation, and it is not due to impaired proteasome activity. Instead, we identify a cascade of events where aberrant translation pausing leads to reduced ribosome availability resulting in proteome remodeling independently of transcriptional regulation. Our research uncovers a vulnerable point in the aging brain's biology - the biogenesis of basic DNA/RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity and the biosynthesis of macromolecules.

Distribution of Brain-Derived Neurotrophic Factor in the Brain of the Small-Spotted Catshark Scyliorhinus canicula, and Evolution of Neurotrophins in Basal Vertebrates.

Neurotrophins (NTFs) are structurally related neurotrophic factors essential for differentiation, survival, neurite outgrowth, and the plasticity of neurons. Abnormalities associated with neurotrophin-signaling (NTF-signaling) were associated with neuropathies, neurodegenerative disorders, and age-associated cognitive decline. Among the neurotrophins, brain-derived neurotrophic factor (BDNF) has the highest expression and is expressed in mammals by specific cells throughout the brain, with particularly high expression in the hippocampus and cerebral cortex. Whole genome sequencing efforts showed that NTF signaling evolved before the evolution of Vertebrates; thus, the shared ancestor of Protostomes, Cyclostomes, and Deuterostomes must have possessed a single ortholog of neurotrophins. After the first round of whole genome duplication that occurred in the last common ancestor of Vertebrates, the presence of two neurotrophins in Agnatha was hypothesized, while the monophyletic group of cartilaginous fishes, or Chondrichthyans, was situated immediately after the second whole genome duplication round that occurred in the last common ancestor of Gnathostomes. Chondrichthyans represent the outgroup of all other living jawed vertebrates (Gnathostomes) and the sister group of Osteichthyans (comprehensive of Actinopterygians and Sarcopterygians). We were able to first identify the second neurotrophin in Agnatha. Secondly, we expanded our analysis to include the Chondrichthyans, with their strategic phylogenetic position as the most basal extant Gnathostome taxon. Results from the phylogenetic analysis confirmed the presence of four neurotrophins in the Chondrichthyans, namely the orthologs of the four mammalian neurotrophins BDNF, NGF, NT-3, and NT-4. We then proceeded to study the expression of BDNF in the adult brain of the Chondrichthyan Scyliorhinus canicula. Our results showed that BDNF is highly expressed in the S. canicula brain and that its expression is highest in the Telencephalon, while the Mesencephalic and Diencephalic areas showed expression of BDNF in isolated and well-defined cell groups. NGF was expressed at much lower levels that could be detected by PCR but not by in situ hybridization. Our results warrant further investigations in Chondrichthyans to characterize the putative ancestral function of neurotrophins in Vertebrates.

Immunofluorescence and Aggresome Staining of Nothobranchius furzeri Cryosections.

Protein aggregation is a hallmark of many age-related pathologies and, in particular, of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. The teleost Nothobranchius furzeri shows the shortest median life span among all vertebrate animal models and has recently gained popularity as a convenient model for experimental approaches to aging. Immunofluorescence staining is the primary technique used to visualize the distribution of proteins in fixed cells and tissues and it has proven to be a powerful tool to study aggregates and proteins involved in neurodegenerative diseases. Specifically, immunofluorescence staining allows for precise localization of aggregates in specific cell types and can be used to identify the proteins constituting such aggregates. To facilitate the study of aggregate-related pathologies in the context of aging using the new model N. furzeri, we report a protocol to visualize general protein aggregates and specific proteins optimized for N. furzeri brain cryosections.

Whole-Brain Clearing and Immunofluorescence in Nothobranchius furzeri.

Tissue clearing techniques for three-dimensional reconstruction and imaging of entire organs and thick samples have become a popular and broadly used methodology, leading to the development of numerous protocols. Due to the complex cellular architecture of the brain and the wide spatial range of the connections that neurons may display, having the possibility to stain, image, and reconstruct neurons and/or neuronal nuclei in their entire extent can be crucial. However, this is hard to accomplish due to the natural opacity of the brain and the general thickness of the sample, posing a barrier to both imaging and antibody penetration. Nothobranchius furzeri has recently become a widely used model to study brain aging thanks to its short life span (3-7 mo), providing new opportunities to study the effects of aging on the brain and the role of aging in the development of neurodegenerative diseases. Here, we present a methodology to clarify and stain whole N. furzeri brains. This protocol is based on the ScaleA2 and ScaleS protocols developed and presented by Hama and colleagues, together with an in-house developed staining procedure for thick slices of tissues. ScaleS is a convenient and easy clearing technique based on sorbitol and urea that does not require particularly complex equipment, but due to the high urea concentration in some of the solutions, not all antigens are preserved. To overcome this issue, we developed a method that allows optimal staining of Nothobranchius furzeri brains before clarification.

Localization and Characterization of Major Neurogenic Niches in the Brain of the Lesser-Spotted Dogfish Scyliorhinus canicula.

Adult neurogenesis is defined as the ability of specialized cells in the postnatal brain to produce new functional neurons and to integrate them into the already-established neuronal network. This phenomenon is common in all vertebrates and has been found to be extremely relevant for numerous processes, such as long-term memory, learning, and anxiety responses, and it has been also found to be involved in neurodegenerative and psychiatric disorders. Adult neurogenesis has been studied extensively in many vertebrate models, from fish to human, and observed also in the more basal cartilaginous fish, such as the lesser-spotted dogfish, Scyliorhinus canicula, but a detailed description of neurogenic niches in this animal is, to date, limited to the telencephalic areas. With this article, we aim to extend the characterization of the neurogenic niches of S. canicula in other main areas of the brain: we analyzed via double immunofluorescence sections of telencephalon, optic tectum, and cerebellum with markers of proliferation (PCNA) and mitosis (pH3) in conjunction with glial cell (S100ß) and stem cell (Msi1) markers, to identify the actively proliferating cells inside the neurogenic niches. We also labeled adult postmitotic neurons (NeuN) to exclude double labeling with actively proliferating cells (PCNA). Lastly, we observed the presence of the autofluorescent aging marker, lipofuscin, contained inside lysosomes in neurogenic areas.

EdU and Immunofluorescence Staining of Nothobranchius furzeri Organotypic Cultures.

Immunofluorescence is a widely used technique to visualize the localization of proteins of interest. Nucleoside analogs, such as 5-ethynyl-2'-deoxyuridine (EdU), are incorporated into newly synthesized DNA and enable permanent labeling of newly divided cells. Both these techniques can be applied to long-term organotypic culture of Nothobranchius furzeri in a fashion similar to that already described for tissue sections. We report here our optimized method for immunofluorescence and EdU staining of N. furzeri organotypic slices.

Long-Term Brain Organotypic Cultures of the Turquoise Killifish Nothobranchius furzeri.

Organotypic culture is a well-established method for culturing ex vivo tissue samples. The advantages of culturing tissue slices for prolonged time periods ex vivo are numerous and consist primarily of the maintenance of the overall in vivo architecture of the isolated sample, the lack of the ematoencephalic barrier, and the ease of pharmacological treatments and interventions that can be conducted under controlled conditions as in in vitro systems such as cell cultures. Given the extremely short life span of Nothobranchius furzeri and the emergence of aging signs only after a few months of life, it is of particular interest to establish this protocol for N. furzeri as a potential method to study brain aging ex vivo.

Quantification of noradrenergic-, dopaminergic-, and tectal-neurons during aging in the short-lived killifish Nothobranchius furzeri.

Parkinson's disease (PD) is characterized by phosphorylation and aggregation of the protein α-Synuclein and ensuing neuronal death progressing from the noradrenergic locus coeruleus to midbrain dopaminergic neurons. In 2019, Matsui and colleagues reported a spontaneous age-dependent degeneration of dopaminergic neurons and an even greater neurodegeneration of the noradrenergic neurons in the short-lived killifish Nothobranchius furzeri. Given the great possible relevance of a spontaneous model for PD, we assessed neurodegeneration of noradrenergic and dopaminergic neurons in two further laboratory strains of N. furzeri. We implemented, for the first time in N. furzeri, a whole-brain clarification technique and proceeded to entire 3D nuclei reconstruction to quantify total cell numbers in two different stains of N. furzeri. In both strains, we observed that age-dependent neurodegeneration is limited to the locus coeruleus and does not involve the posterior tuberculum. We also applied 3D counting to the optic tectum, an area of active adult neurogenesis, and detected an increase of neurons with age. Our results confirm age-dependent neurodegeneration of noradrenergic neurons, a condition reminiscent of the presymptomatic stage of PD indicating that N. furzeri could be used in the future to identify modifying factors for age-dependent neurodegeneration and open the intriguing possibility that natural genetic variation may influence the susceptibility of dopaminergic neurons.

New lessons on TDP-43 from old N. furzeri killifish.

Frontotemporal dementia and amyotrophic lateral sclerosis are fatal and incurable neurodegenerative diseases linked to the pathological aggregation of the TDP-43 protein. This is an essential DNA/RNA-binding protein involved in transcription regulation, pre-RNA processing, and RNA transport. Having suitable animal models to study the mechanisms of TDP-43 aggregation is crucial to develop treatments against disease. We have previously demonstrated that the killifish Nothobranchius furzeri offers the advantage of being the shortest-lived vertebrate with a clear aging phenotype. Here, we show that the two N. furzeri paralogs of TDP-43 share high sequence homology with the human protein and recapitulate its cellular and biophysical behavior. During aging, N. furzeri TDP-43 spontaneously forms insoluble intracellular aggregates with amyloid characteristics and colocalizes with stress granules. Our results propose this organism as a valuable new model of TDP-43-related pathologies making it a powerful tool for the study of disease mechanism.

Antioxidants Prevent the Effects of Physical Exercise on Visual Cortical Plasticity.

BACKGROUND: Physical activity has been recently shown to enhance adult visual cortical plasticity, both in human subjects and animal models. While physical activity activates mitochondrial oxidative metabolism leading to a transient production of reactive oxygen species, it remains unknown whether this process is involved in the plasticizing effects elicited at the visual cortical level. RESULTS: Here, we investigated whether counteracting oxidative stress through a dietary intervention with antioxidants (vitamins E and C) interferes with the impact of physical exercise on visual cortex plasticity in adult rats. Antioxidant supplementation beyond the closure of the critical period blocked ocular dominance plasticity in response to eye deprivation induced by physical activity in adult rats. CONCLUSIONS: Antioxidants exerted their action through a mithormetic effect that involved dampening of oxidative stress and insulin-like growth factor 1 (IGF-1) signaling in the brain.

Age-Dependent Regulation of Notch Family Members in the Neuronal Stem Cell Niches of the Short-Lived Killifish Nothobranchius furzeri.

Background: The annual killifish Nothobranchius furzeri is a new experimental model organism in biology, since it represents the vertebrate species with the shortest captive life span and also shows the fastest maturation and senescence recorded in the laboratory. Here, we use this model to investigate the age-dependent decay of neurogenesis in the telencephalon (brain region sharing the same embryonic origin with the mammalian adult niches), focusing on the expression of the Notch pathway genes. Results: We observed that the major ligands/receptors of the pathway showed a negative correlation with age, indicating age-dependent downregulation of the Notch pathway. Moreover, expression of notch1a was clearly limited to active neurogenic niches and declined during aging, without changing its regional patterning. Expression of notch3 is not visibly influenced by aging. Conclusion: Both expression pattern and regulation differ between notch1a and notch3, with the former being limited to mitotically active regions and reduced by aging and the latter being present in all cells with a neurogenic potential, regardless of the level of their actual mitotic activity, and so is less influenced by age. This finally suggests a possible differential role of the two receptors in the regulation of the niche proliferative potential throughout the entire fish life.

Alternative Animal Models of Aging Research.

Most research on mechanisms of aging is being conducted in a very limited number of classical model species, i.e., laboratory mouse (Mus musculus), rat (Rattus norvegicus domestica), the common fruit fly (Drosophila melanogaster) and roundworm (Caenorhabditis elegans). The obvious advantages of using these models are access to resources such as strains with known genetic properties, high-quality genomic and transcriptomic sequencing data, versatile experimental manipulation capabilities including well-established genome editing tools, as well as extensive experience in husbandry. However, this approach may introduce interpretation biases due to the specific characteristics of the investigated species, which may lead to inappropriate, or even false, generalization. For example, it is still unclear to what extent knowledge of aging mechanisms gained in short-lived model organisms is transferable to long-lived species such as humans. In addition, other specific adaptations favoring a long and healthy life from the immense evolutionary toolbox may be entirely missed. In this review, we summarize the specific characteristics of emerging animal models that have attracted the attention of gerontologists, we provide an overview of the available data and resources related to these models, and we summarize important insights gained from them in recent years. The models presented include short-lived ones such as killifish (Nothobranchius furzeri), long-lived ones such as primates (Callithrix jacchus, Cebus imitator, Macaca mulatta), bathyergid mole-rats (Heterocephalus glaber, Fukomys spp.), bats (Myotis spp.), birds, olms (Proteus anguinus), turtles, greenland sharks, bivalves (Arctica islandica), and potentially non-aging ones such as Hydra and Planaria.

Nothobranchius annual killifishes.

Annual fishes of the genus Nothobranchius inhabit ephemeral habitats in Eastern and Southeastern Africa. Their life cycle is characterized by very rapid maturation, a posthatch lifespan of a few weeks to months and embryonic diapause to survive the dry season. The species N. furzeri holds the record of the fastest-maturing vertebrate and of the vertebrate with the shortest captive lifespan and is emerging as model organism in biomedical research, evolutionary biology, and developmental biology. Extensive characterization of age-related phenotypes in the laboratory and of ecology, distribution, and demography in the wild are available. Species/populations from habitats differing in precipitation intensity show parallel evolution of lifespan and age-related traits that conform to the classical theories on aging. Genome sequencing and the establishment of CRISPR/Cas9 techniques made this species particularly attractive to investigate the effects genetic and non-genetic intervention on lifespan and aging-related phenotypes. At the same time, annual fishes are a very interesting subject for comparative approaches, including genomics, transcriptomics, and proteomics. The N. furzeri community is highly diverse and rapidly expanding and organizes a biannual meeting.

MiR-29 coordinates age-dependent plasticity brakes in the adult visual cortex.

Visual cortical circuits show profound plasticity during early life and are later stabilized by molecular "brakes" limiting excessive rewiring beyond a critical period. The mechanisms coordinating the expression of these factors during the transition from development to adulthood remain unknown. We found that miR-29a expression in the visual cortex dramatically increases with age, but it is not experience-dependent. Precocious high levels of miR-29a blocked ocular dominance plasticity and caused an early appearance of perineuronal nets. Conversely, inhibition of miR-29a in adult mice using LNA antagomirs activated ocular dominance plasticity, reduced perineuronal nets, and restored their juvenile chemical composition. Activated adult plasticity had the typical functional and proteomic signature of critical period plasticity. Transcriptomic and proteomic studies indicated that miR-29a manipulation regulates the expression of plasticity brakes in specific cortical circuits. These data indicate that miR-29a is a regulator of the plasticity brakes promoting age-dependent stabilization of visual cortical connections.

Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation.

A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging in the short-lived vertebrate Nothobranchius furzeri combining transcriptomics and proteomics. We detected a progressive reduction in the correlation between protein and mRNA, mainly due to post-transcriptional mechanisms that account for over 40% of the age-regulated proteins. These changes cause a progressive loss of stoichiometry in several protein complexes, including ribosomes, which show impaired assembly/disassembly and are enriched in protein aggregates in old brains. Mechanistically, we show that reduction of proteasome activity is an early event during brain aging and is sufficient to induce proteomic signatures of aging and loss of stoichiometry in vivo. Using longitudinal transcriptomic data, we show that the magnitude of early life decline in proteasome levels is a major risk factor for mortality. Our work defines causative events in the aging process that can be targeted to prevent loss of protein homeostasis and delay the onset of age-related neurodegeneration.

Identification and Expression of Neurotrophin-6 in the Brain of Nothobranchius furzeri: One More Piece in Neurotrophin Research.

Neurotrophins contribute to the complexity of vertebrate nervous system, being involved in cognition and memory. Abnormalities associated with neurotrophin synthesis may lead to neuropathies, neurodegenerative disorders and age-associated cognitive decline. The genome of teleost fishes contains homologs of some mammalian neurotrophins as well as a gene coding for an additional neurotrophin (NT-6). In this study, we characterized this specific neurotrophin in the short-lived fish Nothobranchius furzeri, a relatively new model for aging studies. Thus, we report herein for the first time the age-related expression of a neurotrophin in a non-mammalian vertebrate. Interestingly, we found comparable expression levels of NT-6 in the brain of both young and old animals. More in detail, we used a locked nucleic acid probe and a riboprobe to investigate the neuroanatomical distribution of NT-6 mRNA revealing a significant expression of the neurotrophin in neurons of the forebrain (olfactory bulbs, dorsal and ventral telencephalon, and several diencephalic nuclei), midbrain (optic tectum, longitudinal tori, and semicircular tori), and hindbrain (valvula and body of cerebellum, reticular formation and octavolateral area of medulla oblongata). By combining in situ hybridization and immunohistochemistry, we showed that NT-6 mRNA is synthesized in mature neurons. These results contribute to better understanding the evolutionary history of neurotrophins in vertebrates, and their role in the adult brain.

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

[This corrects the article DOI: 10.3389/fncel.2014.00051.].

The clock is ticking. Ageing of the circadian system: From physiology to cell cycle.

The circadian system is the responsible to organise the internal temporal order in relation to the environment of every process of the organisms producing the circadian rhythms. These rhythms have a fixed phase relationship among them and with the environment in order to optimise the available energy and resources. From a cellular level, circadian rhythms are controlled by genetic positive and negative auto-regulated transcriptional and translational feedback loops, which generate 24h rhythms in mRNA and protein levels of the clock components. It has been described about 10% of the genome is controlled by clock genes, with special relevance, due to its implications, to the cell cycle. Ageing is a deleterious process which affects all the organisms' structures including circadian system. The circadian system's ageing may produce a disorganisation among the circadian rhythms, arrhythmicity and, even, disconnection from the environment, resulting in a detrimental situation to the organism. In addition, some environmental conditions can produce circadian disruption, also called chronodisruption, which may produce many pathologies including accelerated ageing. Finally, some strategies to prevent, palliate or counteract chronodisruption effects have been proposed to enhance the circadian system, also called chronoenhancement. This review tries to gather recent advances in the chronobiology of the ageing process, including cell cycle, neurogenesis process and physiology.

MicroRNA miR-29 controls a compensatory response to limit neuronal iron accumulation during adult life and aging.

BACKGROUND: A widespread modulation of gene expression occurs in the aging brain, but little is known as to the upstream drivers of these changes. MicroRNAs emerged as fine regulators of gene expression in many biological contexts and they are modulated by age. MicroRNAs may therefore be part of the upstream drivers of the global gene expression modulation correlated with aging and aging-related phenotypes. RESULTS: Here, we show that microRNA-29 (miR-29) is induced during aging in short-lived turquoise killifish brain and genetic antagonism of its function induces a gene-expression signature typical of aging. Mechanicistically, we identified Ireb2 (a master gene for intracellular iron delivery that encodes for IRP2 protein), as a novel miR-29 target. MiR-29 is induced by iron loading and, in turn, it reduces IRP2 expression in vivo, therefore limiting intracellular iron delivery in neurons. Genetically modified fish with neuro-specific miR-29 deficiency exhibit increased levels of IRP2 and transferrin receptor, increased iron content, and oxidative stress. CONCLUSIONS: Our results demonstrate that age-dependent miR-29 upregulation is an adaptive mechanism that counteracts the expression of some aging-related phenotypes and its anti-aging activity is primarily exerted by regulating intracellular iron homeostasis limiting excessive iron-exposure in neurons.