Effect of the P838L mutation on myosin function in Drosophila melanogaster

Includes bibliographical references (p. 33-39).Myosin, a motor protein, is composed of two heavy chains and four light chains. It hydrolyzes ATP to generate actin-based motility and the contractile force in muscles. A mutation in human cardiac beta myosin heavy chain that changes amino acid residue 838 from proline to leucine (P838L) results in pediatric restrictive cardiomyopathy. Restrictive cardiomyopathy (RCM) leads to rigidity of the ventricular wall and the heart is restricted from stretching and properly filling with blood. Therefore, blood flow is reduced and blood that would normally enter the heart is backed up in the circulatory system. Gradually, the patient loses the ability to pump blood efficiently, leading to heart failure. Drosophila has a rhythmically beating heart and serves as a powerful tool to study the genetic basis of heart development and disease in humans. To define the biochemical basis of myosin-based RCM and to test the hypothesis that the P838L mutation causes RCM in Drosophila, a gene encoding myosin with the P838L mutation was constructed and was expressed in place of wild-type myosin heavy chain by P element transformation. Jump tests indicated that the mutation did not have any effect on the jump muscles. However reduced flight ability was observed, indicating impairment in the indirect flight muscle expressing this mutant myosin. ATPase assays and in vitro motility assays were performed to determine the effect of the mutation at the molecular level. We found that there was a slight increase in the actin-sliding velocity and that the basal and actin-activated MgATPase activity was significantly higher for the mutants. Electron microscopy on 2 day-old mutants and the controls suggested that the myofibrils were intact but some of them were oblong in shape and had rough edges when compared to the control myofibrils. Preliminary video microscopy data suggest that the transgenic hearts display a restrictive phenotype. The P838L mutation affects an "invariant proline" located at the junction of the myosin S1 head and the S2 rod and might affect the movement of the myosin heads during the force generation step. This could lead to the observed increase in the ATPase rate. Also, the P838L mutation is located near to where the regulatory light chain (RLC) binds to the heavy chain. RLC has an important role in the ATPase cycle and the P838L mutation might alter the RLC conformation leading to the increased ATPase rate. The increased ATPase rate seen in this and another Drosophila myosin that causes a restrictive phenotype may initiate a cascade of events that result in the observed cardiac defects

Assessing Basal and Acute Autophagic Responses in the Adult Drosophila Nervous System: The Impact of Gender, Genetics and Diet on Endogenous Pathway Profiles.

The autophagy pathway is critical for the long-term homeostasis of cells and adult organisms and is often activated during periods of stress. Reduced pathway efficacy plays a central role in several progressive neurological disorders that are associated with the accumulation of cytotoxic peptides and protein aggregates. Previous studies have shown that genetic and transgenic alterations to the autophagy pathway impacts longevity and neural aggregate profiles of adult Drosophila. In this study, we have identified methods to measure the acute in vivo induction of the autophagy pathway in the adult fly CNS. Our findings indicate that the genotype, age, and gender of adult flies can influence pathway responses. Further, we demonstrate that middle-aged male flies exposed to intermittent fasting (IF) had improved neuronal autophagic profiles. IF-treated flies also had lower neural aggregate profiles, maintained more youthful behaviors and longer lifespans, when compared to ad libitum controls. In summary, we present methodology to detect dynamic in vivo changes that occur to the autophagic profiles in the adult Drosophila CNS and that a novel IF-treatment protocol improves pathway response in the aging nervous system

Aging and Intermittent Fasting Impact on Transcriptional Regulation and Physiological Responses of Adult Drosophila Neuronal and Muscle Tissues

The progressive decline of the nervous system, including protein aggregate formation, reflects the subtle dysregulation of multiple functional pathways. Our previous work has shown intermittent fasting (IF) enhances longevity, maintains adult behaviors and reduces aggregates, in part, by promoting autophagic function in the aging Drosophila brain. To clarify the impact that IF-treatment has upon aging, we used high throughput RNA-sequencing technology to examine the changing transcriptome in adult Drosophila tissues. Principle component analysis (PCA) and other analyses showed ~1200 age-related transcriptional differences in head and muscle tissues, with few genes having matching expression patterns. Pathway components showing age-dependent expression differences were involved with stress response, metabolic, neural and chromatin remodeling functions. Middle-aged tissues also showed a significant increase in transcriptional drift-variance (TD), which in the CNS included multiple proteolytic pathway components. Overall, IF-treatment had a demonstrably positive impact on aged transcriptomes, partly ameliorating both fold and variance changes. Consistent with these findings, aged IF-treated flies displayed more youthful metabolic, behavioral and basal proteolytic profiles that closely correlated with transcriptional alterations to key components. These results indicate that even modest dietary changes can have therapeutic consequences, slowing the progressive decline of multiple cellular systems, including proteostasis in the aging nervous system

Aging and Autophagic Function Influences the Progressive Decline of Adult Drosophila Behaviors

<div><p>Multiple neurological disorders are characterized by the abnormal accumulation of protein aggregates and the progressive impairment of complex behaviors. Our Drosophila studies demonstrate that middle-aged wild-type flies (WT, ~4-weeks) exhibit a marked accumulation of neural aggregates that is commensurate with the decline of the autophagy pathway. However, enhancing autophagy via neuronal over-expression of <i>Atg8a</i> (Atg8a-OE) reduces the age-dependent accumulation of aggregates. Here we assess basal locomotor activity profiles for single- and group-housed male and female WT flies and observed that only modest behavioral changes occurred by 4-weeks of age, with the noted exception of group-housed male flies. Male flies in same-sex social groups exhibit a progressive increase in nighttime activity. Infrared videos show aged group-housed males (4-weeks) are engaged in extensive bouts of courtship during periods of darkness, which is partly repressed during lighted conditions. Together, these nighttime courtship behaviors were nearly absent in young WT flies and aged Atg8a-OE flies. Previous studies have indicated a regulatory role for olfaction in male courtship partner choice. Coincidently, the mRNA expression profiles of several olfactory genes decline with age in WT flies; however, they are maintained in age-matched Atg8a-OE flies. Together, these results suggest that middle-aged male flies develop impairments in olfaction, which could contribute to the dysregulation of courtship behaviors during dark time periods. Combined, our results demonstrate that as Drosophila age, they develop early behavior defects that are coordinate with protein aggregate accumulation in the nervous system. In addition, the nighttime activity behavior is preserved when neuronal autophagy is maintained (Atg8a-OE flies). Thus, environmental or genetic factors that modify autophagic capacity could have a positive impact on neuronal aging and complex behaviors.</p></div

Aging and the negative geotaxis response (NGR) of adult Drosophila.

<p>The NGR of outcrossed wild-type control male and female flies (<i>w¹¹¹⁸/+</i>) was used to determining changes in average climbing index (CI, distance traveled within 5 seconds) between the ages of 1 and 4-weeks. *** P ≤ 0.001. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132768#pone.0132768.s001" target="_blank">S1 Fig</a></b> for the design of the NGR apparatus and additional details.</p

Distribution of Atg8a positive punctae in the adult <i>Drosophila</i> CNS.

<p>(<b>A</b>) Representative confocal image (1.0 μm optical section, top left) of adult male fly brains following a 4-hour fast. Adult brains were co-stained with the anti-Elav neuronal (green) and the anti-Atg8a autophagy (red) markers. (<b>B</b>) Higher magnification images (see <b>Fig 1A</b> inset) highlight areas enriched with Atg8a positive punctae, which primarily include neuronal soma (cell bodies) and regions of neuropil (blue arrows). Yellow boxes (20 μm²) show the location of regions in the CNS that primarily contain neuronal soma that were used to count and establish autophagosome punctae profiles that occur in the adult fly brain. Additional, higher magnification images are included in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164239#pone.0164239.s003" target="_blank">S1A and S1B Fig</a></b> (regions highlighted by yellow arrows). (<b>C</b>) Magnified images from similar brain locations of non-fasted adult male flies stained with anti-Elav and anti-Atg8a antibodies. Yellow boxes indicate regions containing neuronal soma (green) that were used to count Atg8a positive punctae (red). (<b>D</b>) Average number of Atg8a positive punctae or autophagosomes in control (n = 53 fields) neural tissues and following a brief 4-hour fast (n = 66 fields). P*** ≤ 0.001.</p

The influence of genetics on basal autophagy profiles occurring in the fly CNS.

<p>Age-matched WT (<i>w</i>^<i>1118</i>/+), <i>Atg8a</i>^<i>1</i> and <i>chico</i>^<i>1</i>/+ male flies (1-week) were collected and heads used to prepare total protein extracts. (<b>A</b>) Western blots were probed for Atg8a, Ref(2)P, ubiquitin (UB), and Actin proteins (n = 3). Quantification of total (<b>B</b>) Ref(2)P, (<b>C</b>) UB-proteins, (<b>D</b>) total Atg8a (I+II), and (<b>E</b>) Atg8a-II proteins, normalized using Actin. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.</p

Changes to the lifespan and autophagy profiles of <i>chico</i>^<i>1</i>/+ male flies.

<p>(<b>A</b>) The average lifespan profiles of <i>chico</i>^<i>1</i>/+ mutant male flies exposed to <i>ad libitum</i> or IF treatment conditions, starting at 1-week of age. (<b>B</b>) Total head protein extracts from control (0h) or fasted (4h) male flies at 1-week, 3-week or IF-treated 3-week of age (n = 3), were used for Western blot analysis of the Atg8a, Ref(2)P, and Actin proteins. (<b>C</b>) The relative ratio of Atg8a-II to Atg8a-I proteins. *P ≤ 0.05.</p

Enhanced neural autophagy rescues male nighttime wakefulness.

<p>1 and 4-week old group-housed <i>Appl-Gal4/+</i>, <i>UAS-GFP-Atg8a/+</i> and <i>Appl-Gal4/ UAS-GFP-Atg8a</i> (Atg8a-OE) transgenic male flies were assayed using standard LD conditions. Yellow and black bars indicate day (8:00am to 8:00pm) and night (8:00pm to 8:00am) time periods, respectively. Activity is presented as an average per individual fly. (<b>A-B</b>) The average activity profiles of group-housed male flies at 1-week. (<b>C-D</b>) The average activity profiles of group-housed male flies at 4-weeks. **P ≤ 0.01 and *** P ≤ 0.001.</p