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.

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.

A Restrictive Cardiomyopathy Mutation in an Invariant Proline at the Myosin Head/Rod Junction Enhances Head Flexibility and Function, Yielding Muscle Defects in Drosophila.

An "invariant proline" separates the myosin S1 head from its S2 tail and is proposed to be critical for orienting S1 during its interaction with actin, a process that leads to muscle contraction. Mutation of the invariant proline to leucine (P838L) caused dominant restrictive cardiomyopathy in a pediatric patient (Karam et al., Congenit. Heart Dis. 3:138-43, 2008). Here, we use Drosophila melanogaster to model this mutation and dissect its effects on the biochemical and biophysical properties of myosin, as well as on the structure and physiology of skeletal and cardiac muscles. P838L mutant myosin isolated from indirect flight muscles of transgenic Drosophila showed elevated ATPase and actin sliding velocity in vitro. Furthermore, the mutant heads exhibited increased rotational flexibility, and there was an increase in the average angle between the two heads. Indirect flight muscle myofibril assembly was minimally affected in mutant homozygotes, and isolated fibers displayed normal mechanical properties. However, myofibrils degraded during aging, correlating with reduced flight abilities. In contrast, hearts from homozygotes and heterozygotes showed normal morphology, myofibrillar arrays, and contractile parameters. When P838L was placed in trans to Mhc(5), an allele known to cause cardiac restriction in flies, it did not yield the constricted phenotype. Overall, our studies suggest that increased rotational flexibility of myosin S1 enhances myosin ATPase and actin sliding. Moreover, instability of P838L myofibrils leads to decreased function during aging of Drosophila skeletal muscle, but not cardiac muscle, despite the strong evolutionary conservation of the P838 residue.

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

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 Atg8a (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.