MicroRNAs as modulators of longevity and the aging process.

MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally repress translation or induce mRNA degradation of target transcripts through sequence-specific binding. miRNAs target hundreds of transcripts to regulate diverse biological pathways and processes, including aging. Many microRNAs are differentially expressed during aging, generating interest in their use as aging biomarkers and roles as regulators of the aging process. In the invertebrates Caenorhabditis elegans and Drosophila, a number of miRNAs have been found to both positive and negatively modulate longevity through canonical aging pathways. Recent studies have also shown that miRNAs regulate age-associated processes and pathologies in a diverse array of mammalian tissues, including brain, heart, bone, and muscle. The review will present an overview of these studies, highlighting the role of individual miRNAs as biomarkers of aging and regulators of longevity and tissue-specific aging processes.

High-throughput screening in the C. elegans nervous system.

The nematode Caenorhabditis elegans is widely used as a model organism in the field of neurobiology. The wiring of the C. elegans nervous system has been entirely mapped, and the animal's optical transparency allows for in vivo observation of neuronal activity. The nematode is also small in size, self-fertilizing, and inexpensive to cultivate and maintain, greatly lending to its utility as a whole-animal model for high-throughput screening (HTS) in the nervous system. However, the use of this organism in large-scale screens presents unique technical challenges, including reversible immobilization of the animal, parallel single-animal culture and containment, automation of laser surgery, and high-throughput image acquisition and phenotyping. These obstacles require significant modification of existing techniques and the creation of new C. elegans-based HTS platforms. In this review, we outline these challenges in detail and survey the novel technologies and methods that have been developed to address them.

Global, cell non-autonomous gene regulation drives individual lifespan among isogenic C. elegans.

Across species, lifespan is highly variable among individuals within a population. Even genetically identical Caenorhabditis elegans reared in homogeneous environments are as variable in lifespan as outbred human populations. We hypothesized that persistent inter-individual differences in expression of key regulatory genes drives this lifespan variability. As a test, we examined the relationship between future lifespan and the expression of 22 microRNA promoter::GFP constructs. Surprisingly, expression of nearly half of these reporters, well before death, could effectively predict lifespan. This indicates that prospectively long- vs. short-lived individuals have highly divergent patterns of transgene expression and transcriptional regulation. The gene-regulatory processes reported on by two of the most lifespan-predictive transgenes do not require DAF-16, the FOXO transcription factor that is a principal effector of insulin/insulin-like growth factor (IGF-1) signaling. Last, we demonstrate a hierarchy of redundancy in lifespan-predictive ability among three transgenes expressed in distinct tissues, suggesting that they collectively report on an organism-wide, cell non-autonomous process that acts to set each individual's lifespan.

A Simple Apparatus for Individual C. elegans Culture.

We miniaturized standard, solid-phase C. elegans culture conditions to produce a system in which many isolated, individual C. elegans can be housed throughout their lives. This system, the "worm corral," is compatible with high-resolution brightfield and fluorescent microscopy, allowing imaging of fluorescent transgenes and morphological phenotypes from hatch until death. These culture devices can be constructed on the benchtop with commercially available reagents and standard laboratory equipment, making this an attainable solution for most labs.

A simple culture system for long-term imaging of individual C. elegans.

We have miniaturized standard culture techniques to rear arrays of isolated, individual C. elegans throughout their lives on solid gel media. The resulting apparatus is compatible with brightfield and fluorescence microscopy, enabling longitudinal studies of morphology and fluorescent transgene expression. Our culture system exploits a novel crosslinking reaction between a polyethylene glycol hydrogel and a silicone elastomer to constrain animals to individual "corrals" on the gel surface. These devices are simple to construct on the benchtop with commercially available reagents, and, unlike microfluidic isolation methods, do not rely on micropatterned materials. We demonstrate that this new culture method has negligible effects on the physiology of C. elegans compared to standard culture on agar plates. In addition, RNAi techniques are effective in this system. Finally, the hydrogel-silicone binding chemistry that we developed also allows traditional microfluidic devices to be covalently attached to gel substrates instead of glass.

Domain-domain interactions determine the gating, permeation, pharmacology, and subunit modulation of the IKs ion channel.

Voltage-gated ion channels generate electrical currents that control muscle contraction, encode neuronal information, and trigger hormonal release. Tissue-specific expression of accessory (ß) subunits causes these channels to generate currents with distinct properties. In the heart, KCNQ1 voltage-gated potassium channels coassemble with KCNE1 ß-subunits to generate the IKs current (Barhanin et al., 1996; Sanguinetti et al., 1996), an important current for maintenance of stable heart rhythms. KCNE1 significantly modulates the gating, permeation, and pharmacology of KCNQ1 (Wrobel et al., 2012; Sun et al., 2012; Abbott, 2014). These changes are essential for the physiological role of IKs (Silva and Rudy, 2005); however, after 18 years of study, no coherent mechanism explaining how KCNE1 affects KCNQ1 has emerged. Here we provide evidence of such a mechanism, whereby, KCNE1 alters the state-dependent interactions that functionally couple the voltage-sensing domains (VSDs) to the pore.