A primordial target: Mitochondria mediate both primary and collateral anesthetic effects of volatile anesthetics.

One of the unsolved mysteries of medicine is how do volatile anesthetics (VAs) cause a patient to reversibly lose consciousness. In addition, identifying mechanisms for the collateral effects of VAs, including anesthetic-induced neurotoxicity (AiN) and anesthetic preconditioning (AP), has proven challenging. Multiple classes of molecules (lipids, proteins, and water) have been considered as potential VA targets, but recently proteins have received the most attention. Studies targeting neuronal receptors or ion channels had limited success in identifying the critical targets of VAs mediating either the phenotype of "anesthesia" or their collateral effects. Recent studies in both nematodes and fruit flies may provide a paradigm shift by suggesting that mitochondria may harbor the upstream molecular switch activating both primary and collateral effects. The disruption of a specific step of electron transfer within the mitochondrion causes hypersensitivity to VAs, from nematodes to Drosophila and to humans, while also modulating the sensitivity to collateral effects. The downstream effects from mitochondrial inhibition are potentially legion, but inhibition of presynaptic neurotransmitter cycling appears to be specifically sensitive to the mitochondrial effects. These findings are perhaps of even broader interest since two recent reports indicate that mitochondrial damage may well underlie neurotoxic and neuroprotective effects of VAs in the central nervous system (CNS). It is, therefore, important to understand how anesthetics interact with mitochondria to affect CNS function, not just for the desired facets of general anesthesia but also for significant collateral effects, both harmful and beneficial. A tantalizing possibility exists that both the primary (anesthesia) and secondary (AiN, AP) mechanisms may at least partially overlap in the mitochondrial electron transport chain (ETC).

The Serial Anesthesia Array for the High-Throughput Investigation of Volatile Agents Using Drosophila melanogaster.

Volatile general anesthetics (VGAs) are used worldwide on millions of people of all ages and medical conditions. High concentrations of VGAs (hundreds of micromolar to low millimolar) are necessary to achieve a profound and unphysiological suppression of brain function presenting as "anesthesia" to the observer. The full spectrum of the collateral effects triggered by such high concentrations of lipophilic agents is not known, but interactions with the immune-inflammatory system have been noted, although their biological significance is not understood. To investigate the biological effects of VGAs in animals, we developed a system termed the serial anesthesia array (SAA) to exploit the experimental advantages offered by the fruit fly (Drosophila melanogaster). The SAA consists of eight chambers arranged in series and connected to a common inflow. Some parts are available in the lab, and others can be easily fabricated or purchased. A vaporizer, which is necessary for the calibrated administration of VGAs, is the only commercially manufactured component. VGAs constitute only a small percentage of the atmosphere flowing through the SAA during operation, as the bulk (typically over 95%) is carrier gas; the default carrier is air. However, oxygen and any other gases can be investigated. The SAA's principal advantage over prior systems is that it allows the simultaneous exposure of multiple cohorts of flies to exactly titrable doses of VGAs. Identical concentrations of VGAs are achieved within minutes in all the chambers, thus providing indistinguishable experimental conditions. Each chamber can contain from a single fly to hundreds of flies. For example, the SAA can simultaneously examine eight different genotypes or four genotypes with different biological variables (e.g., male vs. female, old vs. young). We have used the SAA to investigate the pharmacodynamics of VGAs and their pharmacogenetic interactions in two experimental fly models associated with neuroinflammation-mitochondrial mutants and traumatic brain injury (TBI).

Anesthetic Preconditioning of Traumatic Brain Injury Is Ineffective in a Drosophila Model of Obesity.

We tested the hypothesis that obesity influences the pharmacodynamics of volatile general anesthetics (VGAs) by comparing effects of anesthetic exposure on mortality from traumatic brain injury (TBI) in lean and obese Drosophila melanogaster We induced TBI with a high-impact trauma device. Starvation-selection over multiple generations resulted in an obese phenotype (SS flies). Fed flies served as lean controls (FC flies). Adult (1-8-day-old) SS and FC flies were exposed to equianesthetic doses of isoflurane or sevoflurane either before or after TBI. The principal outcome was percent mortality 24 hours after injury, expressed as the Mortality Index at 24 hours (MI24). TBI resulted in a lower MI24 in FC than in SS flies [21 (2.35) and 57.8 (2.14), respectively n = 12, P = 0.0001]. Pre-exposure to isoflurane or sevoflurane preconditioned FC flies to TBI, reducing the risk of death to 0.53 (0.25 to 1.13) and 0.82 (0.43 to 1.58), respectively, but had no preconditioning effect in SS flies. Postexposure to isoflurane or sevoflurane increased the risk of death in SS flies, but only postexposure to isoflurane increased the risk in FC flies [1.39 (0.81 to 2.38)]. Thus, obesity affects the pharmacodynamics of VGAs, thwarting the preconditioning effect of isoflurane and sevoflurane in TBI. SIGNIFICANCE STATEMENT: Inadvertent preconditioning in models of traumatic brain injury (TBI) is a recognized confounder. The findings in a fruit fly (Drosophila melanogaster) model of closed-head TBI indicate that anesthetic pharmacodynamics are profoundly affected by obesity. Specifically, obesity thwarts the brain-protective effect of anesthetic preconditioning. This finding is important for experimental studies of TBI and supports the versatility of the fruit fly as a model for the exploration of anesthetic pharmacodynamics in a wide parameter space.

Acetylated α-tubulin K394 regulates microtubule stability to shape the growth of axon terminals.

Microtubules are essential to neuron shape and function. Acetylation of tubulin has the potential to directly tune the behavior and function of microtubules in cells. Although proteomic studies have identified several acetylation sites in α-tubulin, the effects of acetylation at these sites remains largely unknown. This includes the highly conserved residue lysine 394 (K394), which is located at the αß-tubulin dimer interface. Using a fly model, we show that α-tubulin K394 is acetylated in the nervous system and is an essential residue. We found that an acetylation-blocking mutation in endogenous α-tubulin, K394R, perturbs the synaptic morphogenesis of motoneurons and reduces microtubule stability. Intriguingly, the K394R mutation has opposite effects on the growth of two functionally and morphologically distinct motoneurons, revealing neuron-type-specific responses when microtubule stability is altered. Eliminating the deacetylase HDAC6 increases K394 acetylation, and the over-expression of HDAC6 reduces microtubule stability similar to the K394R mutant. Thus, our findings implicate α-tubulin K394 and its acetylation in the regulation of microtubule stability and suggest that HDAC6 regulates K394 acetylation during synaptic morphogenesis.

Non-enzymatic Activity of the α-Tubulin Acetyltransferase αTAT Limits Synaptic Bouton Growth in Neurons.

Neuronal axons terminate as synaptic boutons that form stable yet plastic connections with their targets. Synaptic bouton development relies on an underlying network of both long-lived and dynamic microtubules that provide structural stability for the boutons while also allowing for their growth and remodeling. However, a molecular-scale mechanism that explains how neurons appropriately balance these two microtubule populations remains a mystery. We hypothesized that α-tubulin acetyltransferase (αTAT), which both stabilizes long-lived microtubules against mechanical stress via acetylation and has been implicated in promoting microtubule dynamics, could play a role in this process. Using the Drosophila neuromuscular junction as a model, we found that non-enzymatic dαTAT activity limits the growth of synaptic boutons by affecting dynamic, but not stable, microtubules. Loss of dαTAT results in the formation of ectopic boutons. These ectopic boutons can be similarly suppressed by resupplying enzyme-inactive dαTAT or by treatment with a low concentration of the microtubule-targeting agent vinblastine, which acts to suppress microtubule dynamics. Biophysical reconstitution experiments revealed that non-enzymatic αTAT1 activity destabilizes dynamic microtubules but does not substantially impact the stability of long-lived microtubules. Further, during microtubule growth, non-enzymatic αTAT1 activity results in increasingly extended tip structures, consistent with an increased rate of acceleration of catastrophe frequency with microtubule age, perhaps via tip structure remodeling. Through these mechanisms, αTAT enriches for stable microtubules at the expense of dynamic ones. We propose that the specific suppression of dynamic microtubules by non-enzymatic αTAT activity regulates the remodeling of microtubule networks during synaptic bouton development.

Effects of mutating α-tubulin lysine 40 on sensory dendrite development.

Microtubules are essential for neuronal structure and function. Axonal and dendritic microtubules are enriched in post-translational modifications that impact microtubule dynamics, transport and microtubule-associated proteins. Acetylation of α-tubulin lysine 40 (K40) is a prominent and conserved modification of neuronal microtubules. However, the cellular role of microtubule acetylation remains controversial. To resolve how microtubule acetylation might affect neuronal morphogenesis, we mutated endogenous α-tubulin in vivo using a new Drosophila strain that facilitates the rapid knock-in of designer αTub84B alleles (the predominant α-tubulin-encoding gene in flies). Leveraging our new strain, we found that microtubule acetylation, as well as polyglutamylation and (de)tyrosination, is not essential for survival. However, we found that dendrite branch refinement in sensory neurons relies on α-tubulin K40. Mutagenesis of K40 reveals moderate yet significant changes in dendritic lysosome transport, microtubule polymerization and Futsch protein distribution in dendrites but not in axons. Our studies point to an unappreciated role for α-tubulin K40 and acetylation in dendrite morphogenesis. While our results are consistent with the idea that acetylation tunes microtubule function within neurons, they also suggest there may be an acetylation-independent requirement for α-tubulin K40.This article has an associated First Person interview with the first author of the paper.

Multiple-pathway analysis of double-strand break repair mutations in Drosophila.

The analysis of double-strand break (DSB) repair is complicated by the existence of several pathways utilizing a large number of genes. Moreover, many of these genes have been shown to have multiple roles in DSB repair. To address this complexity we used a repair reporter construct designed to measure multiple repair outcomes simultaneously. This approach provides estimates of the relative usage of several DSB repair pathways in the premeiotic male germline of Drosophila. We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes. Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others. Perhaps surprisingly, none of the single mutants suppressed more than one pathway, but they varied widely in how the suppression was compensated. We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated. Taken as a whole, the data suggest that the choice of which repair pathway is used for a given DSB occurs by a two-stage "decision circuit" in which the DSB is first placed into one of two pools from which a specific pathway is then selected.

A third link connecting aging with double strand break repair.

Until recently, the connection between aging and DNA repair has rested on two classes of observation. First, DNA damage and unrepaired double-strand breaks (DSBs) accumulate with age. Second, several defects in DNA repair genes are associated with early onset of age-related diseases and other signs of premature aging. Now, a third link has emerged: The mechanisms by which cells repair DSB damage can change dramatically with age, shifting from simpler end-joining processes in younger organisms to homologous mechanisms in which missing genetic information is restored through use of a template. So far this third link between aging and DNA repair has only been observed in a small number of experimental systems, and cannot yet claim the generality of the other two. Here we review the evidence for this phenomenon and present new data testing models for the underlying causes. If the generality of age-related changes in DSB repair pathway usage can be established, it will provide a new insight into the underlying molecular basis of aging and how evolution has shaped these processes.

Template disruptions and failure of double Holliday junction dissolution during double-strand break repair in Drosophila BLM mutants.

Previous biochemical studies of the BLM gene product have shown its ability in conjunction with topoisomerase IIIalpha to resolve double Holliday structures through a process called "dissolution." This process could prevent crossing over during repair of double-strand breaks. We report an analysis of the Drosophila BLM gene, DmBlm, in the repair of double-strand breaks in the premeiotic germ line of Drosophila males. With a repair reporter construct, Rr3, and other genetic tools, we show that DmBlm mutants are defective for homologous repair but show a compensating increase in single-strand annealing. Increases of 40- to 50-fold in crossing over and flanking deletions also were seen. Perhaps most significantly, the template used for homologous repair in DmBlm mutants is itself subject to deletions and complex rearrangements. These template disruptions are indicative of failure to resolve double Holliday junctions. These findings, along with the demonstration that a weak allele of topoisomerase IIIalpha has some of the same defects as DmBlm, support the dissolution model. Finally, an analysis of DmBlm mutants in conjunction with mus81 or spnA (Rad51) reveals a second function of BLM distinct from the repair of induced double-strand breaks and possibly related to maintenance of replication forks.

The effect of gap length on double-strand break repair in Drosophila.

When a double-strand break has a gap between the broken ends, the missing information can be restored through synthesis from a homologous template. Here we address the question of how long such a gap can be before this process fails. We measured the frequency of homologous repair in the Drosophila germ line following the creation of gaps of specific sizes ranging from 3.8 to 210 kb. We found that gaps of >11 kb.

The Drosophila Mre11/Rad50 complex is required to prevent both telomeric fusion and chromosome breakage.

The MRN complex consists of the two evolutionarily conserved components Mre11 and Rad50 and the third less-conserved component Nbs1/Xrs2. This complex mediates telomere maintenance in addition to a variety of functions in response to DNA double-strand breaks, including homologous recombination, nonhomologous end joining (NHEJ), and activation of DNA damage checkpoints. Mutations in the Mre11 gene cause the human ataxia-telangiectasia-like disorder (ATDL). Here, we show that null mutations in the Drosophila mre11 and rad50 genes cause both telomeric fusion and chromosome breakage. Moreover, we demonstrate that these mutations are in the same epistasis group required for telomere capping and mitotic chromosome integrity. Using an antibody against Rad50, we show that this protein is uniformly distributed along mitotic chromosomes, and that Rad50 is unstable in the absence of its binding partner Mre11. To define the roles of rad50 and mre11 in telomere protection, mutant chromosome preparations were immunostained for both HP1 and HOAP, two proteins that protect Drosophila telomeres from fusion. Cytological analysis revealed that mutations in rad50 and mre11 drastically reduce accumulation of HOAP and HP1 at telomeres. This suggests that the MRN complex protects Drosophila telomeres by facilitating recruitment of HOAP and HP1 at chromosome ends.