Continuous adult-specific hNTE expression improves climbing performance and endurance in <i>sws</i>^<i>5</i> flies at 2 ages.

<p>(A) Average climbing ability is increased in hNTE rescue flies during week 1 of adult life in comparison to age-matched flies without hNTE expression (p≤0.0001), but not to the level of the wild-type Berlin K flies (p = 0.0001). Flies were assessed daily on days 4 through 8 at 29°C. Climbing performance was assessed on a minimum of 2 cohorts. Data were analyzed using two-way ANOVA with Bonferroni post-test. (n≥112, p≤0.0001, genotype effect, p<0.0001, RU effect, p = 0.0233, RU-by-genotype effect, p = .0003). (B)Average climbing performance assessed on days 25–29 (week 4) of adulthood is improved in hNTE rescue flies in comparison to age-matched flies without hNTE expression (p≤0.0001), but not fully rescued to wild-type levels (p≤0.0001). Climbing was assessed on a minimum of 2 separate biological cohorts. Data were analyzed using two-way ANOVA with Bonferroni post-test. (n≥84, p≤0.0001, genotype effect, p<0.0001, RU effect, p = .0043,RU-by-genotype effect, p<0.0001). Data from both timepoints (2A and 2B) were combined to assess effects across age, age effect, p<0.0001, RU effect, p<0.0001, interaction, p = 0.8656. Thus, there is an age-independent effect of RU on climbing. (C) Time to fatigue is increased in hNTE rescue flies compared to <i>sws</i>^<i>5</i> flies and uninduced controls. Endurance experiments are analyzed statistically by log-rank (n≥150 p≤0.0001). Flies were aged at 29°C and assessed for time-to-fatigue on the 10^th day. (D) 24-day old control flies exhibit age-related decline in endurance relative to day 10. <i>sws</i>^<i>5</i> mutants and RU- control flies display marked reduction in time-to-fatigue- in comparison to Berlin K flies (log-rank, p≤0.0001). Time-to-fatigue is increased in hNTE rescue flies compared to <i>sws</i>^<i>5</i> and RU- controls (log rank, p≤0.0001), but not fully restored to wild-type (log rank with Pearson correlation, p = 0.0020). Endurance was assessed a minimum of 3 times to confirm reproducibility. n≥100 for all day-24 assessments.</p

Adult-specific hNTE expression restores NTE-like esterase activity and improves negative geotaxis in <i>sws</i>^<i>5</i> deficient flies.

<p>(A) hNTE protein is present in heads of adult hNTE rescue flies, but not controls or <i>sws</i>^<i>5</i> mutants. Loading control using anti-Tubulin is shown below. Representative image from 3 biological replicates. (n = 10 fly heads for each sample) (B) Esterase activity is reduced in <i>sws</i>^<i>5</i> mutants and control RU- flies in comparison to wild-type Berlin K (pairwise t-test, p = 0.006 and p = 0.0177, respectively). Flies expressing hNTE using elav-GAL4 increase esterase activity to the level of the wild type Berlin K (pairwise t-test, p = 0.9668). Data across groups were analyzed using two-way ANOVA with Bonferroni post-test and represent duplicate biological samples (n = 10, p≤0.0029, genotype effect, p<0.0001, RU effect, p = 0.0094, RU-by-genotype effect, p = 0.0038). (C) Age-matched <i>sws</i>^<i>5</i><i>;hNTE;elav GS</i> RU <i>+</i> flies display increased climbing vigor in comparison to flies without hNTE induction. Mifepristone (RU+) or vehicle (RU-) was fed to adult flies 3 days post-eclosion and maintained throughout life in order to induce transgene expression after development was complete. Photograph was taken 2 seconds after sharp rapping of vials onto a solid surface in order to induce negative geotaxis response.</p

Adult-specific hNTE expression arrests vacuole expansion in <i>sws</i>^<i>5</i> brains.

<p>Representative 40X images of 500 nm transverse sections (approximately 75μm caudal to first appearance of tissue) of <i>Drosophila</i> right hemisphere from 10-day old flies. Berlin K RU+/RU- and <i>sws</i>^<i>5</i> RU+/RU- were morphologically identical, so a single image is presented for clarity. (A) Background control flies have no vacuoles present. (B) <i>sws</i>^<i>5</i> and RU- control flies exhibit characteristic vacuolization. (C, D) <i>sws</i>^<i>5</i> flies expressing adult-specific hNTE have reduced percent vacuolization when compared to mutants (as quantified in E). (E) Quantification of percent vacuolization phenotype from sectioned <i>Drosophila</i> brains. Berlin K RU+/RU- and <i>sws</i>^<i>5</i> RU+/RU- cohorts did not display statistically significant differences in area of brain occupied by vacuoles and are pooled. Data represent total area of vacuoles divided by total sample area. Although overall RU effect is not significant, <i>sws</i>^<i>5</i> flies expressing pan-neuronal hNTE display significantly less vacuolization area in comparison to uninduced mutants (pairwise t-test, p≤0.0001) and uninduced controls (pairwise t-test, p≤0.0001) (two-way ANOVA with Bonferroni post-test, p≤0.0001, genotype effect, p<0.0001, RU effect, p = 0.8707, RU-by-genotype effect, p = 0.9324). n≥23 flies of each genotype.</p

Delayed hNTE expression rescues endurance in <i>sws</i>^<i>5</i> flies.

<p>Endurance tolerance was assessed before (A, B) and after (C, D) mifepristone (RU486) application and induction of hNTE expression. (A) Berlin K flies exhibit wild-type endurance on day 3 of adulthood, while both <i>sws</i>^<i>5</i> mutant and <i>sws</i>^<i>5</i><i>;hNTE;elav GS</i> show a marked reduction in time-to-fatigue (n = 200 for all groups, log-rank, p<0.0001). (B) 48 hours later <i>sws</i>^<i>5</i> mutant and <i>sws</i>^<i>5</i><i>;hNTE;elav GS</i> cohorts continue to exhibit much lower endurance than Berlin K controls (n = 200 for all groups, long-rank, p<0.0001). At the end of the day-5-test, cohorts are divided in half and split into induced (RU+) and control (RU-) groups. (C) The same flies from (A) and (B) are tested for endurance 48 hours after mifepristone induction. Berlin K flies begin to decline in performance compared to day 5, independent of RU486 feeding (log rank, p≤0.0001 for RU+ and RU-). <i>sws</i>^<i>5</i> mutant and <i>sws</i>^<i>5</i><i>;hNTE;elav GS</i> cohorts show reduced endurance relative to Berlin K whether RU486-fed or not (n = 100 for all groups, long-rank, p<0.0001). (D) On day 10, after 120 hours RU486 induction, hNTE rescue flies’ (<i>sws</i>^<i>5</i><i>;hNTE;elav</i> GS RU+) time-to-fatigue is fully rescued to wild-type Berlin K endurance capacity (log rank, p = 0.9436). <i>sws</i>^<i>5</i> mutant and RU- control groups remain deficient in time-to-fatigue (log rank, p<0.0001). n = 100 for all cohorts.</p

Adult-specific, pan-neuronal hNTE expression partially rescues reduced lifespan of <i>sws</i>^<i>5</i> mutants at 25°C, 29°C.

<p>(A) <i>sws</i>^<i>5</i> mutants have reduced lifespan at 29°C in comparison to both Berlin K RU+/RU- and <i>sws</i>^<i>5</i><i>;hNTE;elav GS</i> (RU+/RU-) flies (n≥248, log rank, p<0.0001). <i>sws</i>^<i>5</i><i>;hNTE;elav GS</i> RU- (RU- control) flies have slightly reduced lifespan relative to Berlin K RU+/RU- and <i>sws</i>^<i>5</i><i>;hNTE;elav GS</i> RU+ (hNTE rescue) flies (n≥248, log rank, p = 0.006) but are longer lived than <i>sws</i>^<i>5</i> RU+/RU- flies (n≥248, log rank, p<0.0001). (B) <i>sws</i>^<i>5</i> mutants (RU+/RU-) and RU- control flies have reduced lifespan at 25°C in comparison to Berlin K (RU+/RU-) and hNTE rescue flies (n≥243, log rank, p<0.0001). Survival of hNTE rescue flies is nearly fully rescued to wild-type Berlin K lifespan at 25°C but is still statistically significant (n≥247, log rank, p<0.0001.</p

The <em>Drosophila</em> PGC-1α Homolog <em>spargel</em> Modulates the Physiological Effects of Endurance Exercise

<div><p>Endurance exercise is an inexpensive intervention that is thought to provide substantial protection against several age-related pathologies, as well as inducing acute changes to endurance capacity and metabolism. Recently, it has been established that endurance exercise induces conserved alterations in physiological capacity in the invertebrate <em>Drosophila</em> model. If the genetic factors underlying these exercise-induced physiological alterations are widely conserved, then invertebrate genetic model systems will become a valuable tool for testing of genetic and pharmacological mimetics for endurance training. Here, we assess whether the <em>Drosophila</em> homolog of the vertebrate exercise response gene PGC-1α <em>spargel (srl)</em> is necessary or sufficient to induce exercise-dependent phenotypes. We find that reduction of <em>srl</em> expression levels acutely compromises negative geotaxis ability and reduces exercise-induced improvement in both negative geotaxis and time to exhaustion. Conversely, muscle/heart specific <em>srl</em> overexpression improves negative geotaxis and cardiac performance in unexercised flies. In addition, we find that <em>srl</em> overexpression mimics some, but not all, exercise-induced phenotypes, suggesting that other factors also act in parallel to <em>srl</em> to regulate exercise-induced physiological changes in muscle and heart.</p> </div

<i>srl</i> expression in muscle and heart acts synergistically with exercise to protect from cardiac failure.

<p>(<b>A</b>) External electrical pacing assays conducted on 4-day old <i>y¹w^67c23</i>, UAS-<i>srl</i>;<i>mef2</i>-Gal4, and <i>srl¹</i> flies show reduced stress-induced failure rates in UAS-<i>srl</i>;<i>mef2</i>-Gal4 flies when compared with <i>y¹w^67c23</i> controls (F-test, p = 0.0178). Pacing assays conducted on 25 day-old exercised and unexercised (<b>B</b>) <i>y¹w^67c23</i> and UAS-<i>srl</i>;<i>mef2</i>-Gal4 and (<b>C</b>) <i>y¹w^67c23</i> and <i>srl¹</i> flies show decreased stress-induced failure rates in exercised flies relative to unexercised age-matched siblings in all genotypes (F-test, p = 0.0054, 0.0044, and 0.0394 for control, <i>srl</i> overexperssing and <i>srl</i> mutant flies respectively). Exercised flies overexpressing <i>srl</i> in the muscle also showed a decreased stress-induced failure rate when compared to exercised <i>y¹w^67c23</i> flies (F-test, p = 0.0430). All <i>n</i> were between 56–122.</p

Exercise and <i>srl</i> expression in muscle act additively to increase survival and CO₂ production during treatment.

<p>CO₂ emission rates of 28 day old <i>srl¹</i>, +;<i>mef2</i>-Gal4 and UAS-<i>srl</i>;<i>mef2</i>-Gal4 (<b>A</b>) unexercised and (<b>B</b>) exercised flies following a three-week exercise regimen. Exercised flies with muscle-specific <i>spargel</i> overexpression emit an increased volume of CO₂ compared with both unexercised <i>spargel</i> overexpressing control flies and exercised +;<i>mef2-</i>Gal4 flies (<i>t</i>-test: p = 0.0217 and p = 0.0283 respectively). Activity levels of 27–29 day old +;<i>mef2</i>-Gal4, +;UAS-<i>srl</i>, UAS-<i>srl</i>;<i>mef2</i>-Gal4 and <i>srl¹</i> (<b>C</b>) unexercised and (<b>D</b>) exercised flies. Exercised flies have statistically similar activity levels to unexercised controls (one-way ANOVA: p = 0.1388). Within each treatment, genotype has no significant effect on activity (one-way ANOVA: p = 0.7563 for unexercised and p = 0.1146 for exercised). Lifespans of female <i>y¹w^67c23</i>, +;UAS-<i>srl</i>, <i>srl¹</i>, and UAS-<i>srl</i>;<i>mef2</i>-Gal4 flies (<b>E</b>). <i>srl¹</i> flies show a significantly a shorter lifespan (<b>F</b>) than control flies (log-rank (Mantel-Cox) test: p<0.0001 for all genotype comparisons).</p

<i>srl</i> is required, but not sufficient, for exercise-induced improvement to endurance capacity.

<p>(<b>A</b>) Time to exhaustion assays conducted on <i>y¹w^67c23</i>, UAS-<i>srl</i>;<i>mef2</i>-Gal4, and <i>srl¹</i> flies. Flies overexpressing <i>srl</i> in the muscle increase time to exhaustion as compared to <i>y¹w^67c23</i> control flies (<i>t</i>-test: p = 0.0031). (<b>B</b>) Change in time to exhaustion in exercised flies as compared to unexercised flies of the same genotype. Wild-type and <i>srl</i> overexpressing flies increase time to exhaustion as a result of exercise (<i>t</i>-test, exercised and unexercised flies: p = 0.0001 and 0.0004, respectively for each genotype) to the same extent, while <i>srl¹</i> mutant flies in fact decrease time to exhaustion after exercise training (<i>t</i>-test, exercised and unexercised <i>srl¹</i>: p = 0.0044, exercised <i>y¹w^67c23</i> and exercised <i>srl¹</i>: p = 0.0005). (<b>C</b>) Endurance climbing assays conducted on 25 day-old <i>y¹w^67c23</i>, UAS-<i>srl</i>;<i>mef2</i>-Gal4, and <i>srl¹</i> flies. Unexercised <i>srl¹</i> display a decrease in climbing speed compared to unexercised <i>y¹w^67c23</i> (multivariate regression, genotype effect: p = 0.0011), while unexercised <i>srl</i> overexpressers do not statistically differ from unexercised <i>y¹w^67c23</i> flies. (<b>D</b>) After training, both exercised <i>y¹w^67c23</i> and exercised UAS-<i>srl</i>;<i>mef2</i>-Gal4 flies show increased climbing speed when compared to unexercised control flies (multivariate regression, genotype effect: p = 0.0002 and p<0.0001, respectively), though <i>srl¹</i> flies show no significant improvement (multivariate regression, genotype effect: p = 0.817). Survival rates during treatment of (<b>E</b>) unexercised and (<b>F</b>) exercised <i>y¹w^67c23</i>, UAS-<i>srl</i>;<i>mef2</i>-Gal4, and <i>srl¹</i> flies. Unexercised flies were placed on the training machine but not allowed to run. Survival during treatment is statistically improved by exercise in all three genotypes (multivariate regression; treatment-by-age, all p values<0.012). Exercised UAS-<i>srl</i>;<i>mef2</i>-Gal4 flies also show increased survival compared to exercised <i>y¹w^67c23</i> flies (multivariate regression, genotype effect, p = 0.0027).</p

Nanothermometry Measure of Muscle Efficiency

Despite recent advances in thermometry, determination of temperature at the nanometer scale in single molecules to live cells remains a challenge that holds great promise in disease detection among others. In the present study, we use a new approach to nanometer scale thermometry with a spatial and thermal resolution of 80 nm and 1 mK respectively, by directly associating 2 nm cadmium telluride quantum dots (CdTe QDs) to the subject under study. The 2 nm CdTe QDs physically adhered to bovine cardiac and rabbit skeletal muscle myosin, enabling the determination of heat released when ATP is hydrolyzed by both myosin motors. Greater heat loss reflects less work performed by the motor, hence decreased efficiency. Surprisingly, we found rabbit skeletal myosin to be more efficient than bovine cardiac. We have further extended this approach to demonstrate the gain in efficiency of <i>Drosophila melanogaster</i> skeletal muscle overexpressing the PGC-1α homologue <i>spargel</i>, a known mediator of improved exercise performance in humans. Our results establish a novel approach to determine muscle efficiency with promise for early diagnosis and treatment of various metabolic disorders including cancer