The Genetics of Aging: A Vertebrate Perspective.

Aging negatively impacts vitality and health. Many genetic pathways that regulate aging were discovered in invertebrates. However, the genetics of aging is more complex in vertebrates because of their specialized systems. This Review discusses advances in the genetic regulation of aging in vertebrates from work in mice, humans, and organisms with exceptional lifespans. We highlight challenges for the future, including sex-dependent differences in lifespan and the interplay between genes and environment. We also discuss how the identification of reliable biomarkers of age and development of new vertebrate models can be leveraged for personalized interventions to counter aging and age-related diseases.

Antiepileptic activity of preferential inhibitors of persistent sodium current.

OBJECTIVE: Evidence from basic neurophysiology and molecular genetics has implicated persistent sodium current conducted by voltage-gated sodium (NaV ) channels as a contributor to the pathogenesis of epilepsy. Many antiepileptic drugs target NaV channels and modulate neuronal excitability, mainly by a use-dependent block of transient sodium current, although suppression of persistent current may also contribute to the efficacy of these drugs. We hypothesized that a drug or compound capable of preferential inhibition of persistent sodium current would have antiepileptic activity. METHODS: We examined the antiepileptic activity of two selective persistent sodium current blockers ranolazine, a U.S. Food and Drug Administration (FDA)-approved drug for treatment of angina pectoris, and GS967, a novel compound with more potent effects on persistent current, in the epileptic Scn2a(Q54) mouse model. We also examined the effect of GS967 in the maximal electroshock model and evaluated effects of the compound on neuronal excitability, propensity for hilar neuron loss, development of mossy fiber sprouting, and survival of Scn2a(Q54) mice. RESULTS: We found that ranolazine was capable of reducing seizure frequency by approximately 50% in Scn2a(Q54) mice. The more potent persistent current blocker GS967 reduced seizure frequency by >90% in Scn2a(Q54) mice and protected against induced seizures in the maximal electroshock model. GS967 greatly attenuated abnormal spontaneous action potential firing in pyramidal neurons acutely isolated from Scn2a(Q54) mice. In addition to seizure suppression in vivo, GS967 treatment greatly improved the survival of Scn2a(Q54) mice, prevented hilar neuron loss, and suppressed the development of hippocampal mossy fiber sprouting. SIGNIFICANCE: Our findings indicate that the selective persistent sodium current blocker GS967 has potent antiepileptic activity and that this compound could inform development of new agents.

Life Span Assessment in the African Turquoise Killifish Nothobranchius furzeri.

The African turquoise killifish (Nothobranchius furzeri) is the shortest-lived vertebrate bred in captivity, with a median life span of 4-6 mo. Within its short life span, the killifish recapitulates critical aspects of human aging, including neurodegeneration and increased frailty. Developing standardized protocols for life span assessment in killifish is critical for identifying environmental and genetic factors that impact vertebrate life span. A standardized life span protocol should have low variability and high reproducibility, and it should enable comparison of life spans between laboratories. Here, we report our standardized protocol for measuring life span in the African turquoise killifish.

Husbandry of the African Turquoise Killifish Nothobranchius furzeri.

The African turquoise killifish (Nothobranchius furzeri) is an extremely short-lived vertebrate that has emerged as a powerful model organism for several research areas, including aging and embryonic diapause, which is the temporary suspension of embryonic development. The killifish research community is expanding and developing new solutions to improve the tractability of the killifish as a model system. Starting a killifish colony from scratch can present numerous challenges. In this protocol, we aim to highlight critical elements in building and maintaining a killifish colony. This protocol should help laboratories start a killifish colony and standardize aspects of killifish husbandry.

Rapid and precise genome engineering in a naturally short-lived vertebrate.

The African turquoise killifish is a powerful vertebrate system to study complex phenotypes at scale, including aging and age-related disease. Here, we develop a rapid and precise CRISPR/Cas9-mediated knock-in approach in the killifish. We show its efficient application to precisely insert fluorescent reporters of different sizes at various genomic loci in order to drive cell-type- and tissue-specific expression. This knock-in method should allow the establishment of humanized disease models and the development of cell-type-specific molecular probes for studying complex vertebrate biology.

The Jellyfish Cassiopea Exhibits a Sleep-like State.

Do all animals sleep? Sleep has been observed in many vertebrates, and there is a growing body of evidence for sleep-like states in arthropods and nematodes [1-5]. Here we show that sleep is also present in Cnidaria [6-8], an earlier-branching metazoan lineage. Cnidaria and Ctenophora are the first metazoan phyla to evolve tissue-level organization and differentiated cell types, such as neurons and muscle [9-15]. In Cnidaria, neurons are organized into a non-centralized radially symmetric nerve net [11, 13, 15-17] that nevertheless shares fundamental properties with the vertebrate nervous system: action potentials, synaptic transmission, neuropeptides, and neurotransmitters [15-20]. It was reported that cnidarian soft corals [21] and box jellyfish [22, 23] exhibit periods of quiescence, a pre-requisite for sleep-like states, prompting us to ask whether sleep is present in Cnidaria. Within Cnidaria, the upside-down jellyfish Cassiopea spp. displays a quantifiable pulsing behavior, allowing us to perform long-term behavioral tracking. Monitoring of Cassiopea pulsing activity for consecutive days and nights revealed behavioral quiescence at night that is rapidly reversible, as well as a delayed response to stimulation in the quiescent state. When deprived of nighttime quiescence, Cassiopea exhibited decreased activity and reduced responsiveness to a sensory stimulus during the subsequent day, consistent with homeostatic regulation of the quiescent state. Together, these results indicate that Cassiopea has a sleep-like state, supporting the hypothesis that sleep arose early in the metazoan lineage, prior to the emergence of a centralized nervous system.

C. elegans Stress-Induced Sleep Emerges from the Collective Action of Multiple Neuropeptides.

The genetic basis of sleep regulation remains poorly understood. In C. elegans, cellular stress induces sleep through epidermal growth factor (EGF)-dependent activation of the EGF receptor in the ALA neuron. The downstream mechanism by which this neuron promotes sleep is unknown. Single-cell RNA sequencing of ALA reveals that the most highly expressed, ALA-enriched genes encode neuropeptides. Here we have systematically investigated the four most highly enriched neuropeptides: flp-7, nlp-8, flp-24, and flp-13. When individually removed by null mutation, these peptides had little or no effect on stress-induced sleep. However, stress-induced sleep was abolished in nlp-8; flp-24; flp-13 triple-mutant animals, indicating that these neuropeptides work collectively in controlling stress-induced sleep. We tested the effect of overexpression of these neuropeptide genes on five behaviors modulated during sleep-pharyngeal pumping, defecation, locomotion, head movement, and avoidance response to an aversive stimulus-and we found that, if individually overexpressed, each of three neuropeptides (nlp-8, flp-24, or flp-13) induced a different suite of sleep-associated behaviors. These overexpression results raise the possibility that individual components of sleep might be specified by individual neuropeptides or combinations of neuropeptides.

Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.

Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering.