Age-related changes in climbing behavior and neural circuit physiology in Drosophila

Identifying the cellular and molecular basis for functional decline remains key to understanding aging. To this end, we have characterized age-dependent changes in climbing and the electrophysiology of the giant fiber circuitry in wild type (Wt) and mutant flies with altered lifespan (methuselah and fragile-X). Our data demonstrate a gradual decline in climbing in Wt and methuselah flies aged 5-45 days. In contrast, fragile-X flies climbed poorly even at 5 days and failed completely at 45 days. We then examined whether synaptic transmission to indirect flight muscles along the giant fiber circuit was altered with aging. At 5 days, the dorsal longitudinal muscle (DLM) in Wt flies followed high frequency stimulation well (at 130 Hz or above). At 35 and 45 days, these flies only followed 60-80 Hz. Methuselah flies did not follow stimuli as well as the Wt flies did at 5 and 25 days, but they were similar to Wt flies at older ages. Fragile-X flies responded poorly even at 5 days (40 Hz) and worsened at 35 days (30 Hz). Unlike DLMs, the tergotrochanteral muscle followed high frequency stimuli relatively well in all genotypes, suggesting that the peripheral interneuron along the DLM pathway or the DLM muscular synapse is prone to age-dependent functional decline. These studies reveal subcellular structures as potential targets of aging, indicating that the giant fiber pathway can be used as a model circuit for quantitative studies of aging in flies as well as fly models of age-related human neurological disorders.     

Fine structure of flight muscles in different Notch mutants of Drosophila melanogaster reared at different temperatures

Summary The fine structure of the indirect flight muscles was studied by electron microscopy in the following Notch locus mutants of Drosophila melanogaster reared at 18° C or 29° C for 6 days after eclosion: Ax ¹⁶¹⁷²/Ax¹⁶¹⁷², Ax²⁸/ Ax²⁸, l(1)N^ts1/l(1)N^ts1,l(1)N^ts1/Y and in wild-type controls. The flies were raised up to eclosion at 25° C or 18° C. It was observed that the l(1)N^ts1 flies gradually became flightless within a few days if reared at 29° C as adults, and gross changes in the fine structure of the flight muscles were also observed in flies of this genotype. Peripheral myofilaments of myofibrils were disarranged and the mitochondria diminutive. At 18° C the flight muscles remained normal. In all of the Abruptex (Ax) combinations the flight muscles remained similar to the wild-type controls at both 18° C and 29° C, i.e. they were normal. The results suggest that the Notch gene is active in adult flies in addition to its activity during embryonic, larval and pupal stages, and is directly or indirectly involved in the adult development of the muscle tissue.     

Transformation and rescue of a flightless Drosophila tropomyosin mutant.

In the Drosophila flightless mutant Ifm(3)3, a transposable element inserted into the alternatively spliced fourth exon of the tropomyosin I (TmI) gene prevents proper expression of Ifm-TmI, the tropomyosin isoform found in indirect flight muscle. We have rescued the flightless phenotype of Ifm(3)3 flies using P-element-mediated transformation with a segment of the Drosophila genome containing the wild-type TmI gene plus 2.5 kb of 5' flanking and 2 kb of 3' flanking DNA. The inserted TmI gene is expressed with the proper developmental and tissue specificity, although its level of expression varies among the five transformed lines examined. These conclusions are based on analyses of flight, myofibrillar morphology, and TmI RNA and protein levels. A minimum of two copies of the inserted TmI gene per cell is necessary to restore flight to most of the flies in each line. We also show that the Ifm-TmI isoform is expressed in the leg muscle of wild-type flies and is decreased in Ifm(3)3 leg muscle. Homozygous Ifm(3)3 mutants do not jump. The ability to jump can be restored with a single copy of the wild-type TmI gene per cell.     

Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stress

Mutations in the gene parkin in humans (PARK2) are responsible for a large number of familial cases of autosomal-recessive Parkinson disease. We have isolated a Drosophila homolog of human PARK2 and characterized its expression and null phenotype. parkin null flies have 30% lower mass than wild-type controls which is in part accounted for by a reduced cell size and number. In addition, these flies are infertile, show significantly reduced longevity, and are unable to jump or fly. Rearing mutants on paraquat, which generates toxic free radicals in vivo, causes a further reduction in longevity. Furthermore, loss of parkin results in progressive degeneration of most indirect flight muscle (IFM) groups soon after eclosion, accompanied by apoptosis. However, parkin mutants have normal neuromuscular junction recordings during the third larval instar stage, suggesting that larval musculature is intact and that parkin is required only in pupal and adult muscle. parkin flies do not show an age-dependent dopaminergic neuron loss in the brain, even after aging adults for 3 weeks. Nevertheless, degeneration of IFMs demonstrates the importance of parkin in maintaining specific cell groups, perhaps those with a high-energy demand and the concomitant production of high levels of free radicals. parkin mutants will be a valuable model for future analysis of the mechanisms of cell and tissue degeneration.     

Phosphorylation-dependent power output of transgenic flies: an integrated study

We examine how the structure and function of indirect flight muscle (IFM) and the entire flight system of Drosophila melanogaster are affected by phosphorylation of the myosin regulatory light chain (MLC2). This integrated study uses site-directed mutagenesis to examine the relationship between removal of the myosin light chain kinase (MLCK) phosphorylation site, in vivo function of the flight system (flight tests, wing kinematics, metabolism, power output), isolated IFM fiber mechanics, MLC2 isoform pattern, and sarcomeric ultrastructure. The MLC2 mutants exhibit graded impairment of flight ability that correlates with a reduction in both IFM and flight system power output and a reduction in the constitutive level of MLC2 phosphorylation. The MLC2 mutants have wild-type IFM sarcomere and cross-bridge structures, ruling out obvious changes in the ultrastructure as the cause of the reduced performance. We describe a viscoelastic model of cross-bridge dynamics based on sinusoidal length perturbation analysis (Nyquist plots) of skinned IFM fibers. The sinusoidal analysis suggests the high power output of Drosophila IFM required for flight results from a phosphorylation-dependent recruitment of power-generating cross-bridges rather than a change in kinetics of the power generating step. The reduction in cross-bridge number appears to affect the way mutant flies generate flight forces of sufficient magnitude to keep them airborne. In two MLC2 mutant strains that exhibit a reduced IFM power output, flies appear to compensate by lowering wingbeat frequency and by elevating wingstroke amplitude (and presumably muscle strain). This behavioral alteration is not seen in another mutant strain in which the power output and estimated number of recruited cross-bridges is similar to that of wild type.     

Drosophila neural pathways

Summary TheDrosophila giant axon pathways cervical connective — thoracic indirect flight muscles were studied by a combined electrophysiological and genetic analysis. A functional coupling of the left and right giant axon pathways was revealed by intracellular recordings of electrical responses of the thoracic indirect flight muscles, when evoked by electrical stimulation of cervical connective (Fig. 2). This functional coupling was demonstrated in wild-type flies and in flies of the single gene, temperature-sensitive paralytic mutation,para ^ts . The functional coupling was evident also in selected bilateral gynandromorph flies, mosaics for thepara ^ts mutation (Fig. 1), even at restricted elevated ambient temperature (Tables 1–3). Analysis of neurally evoked electrogenic muscle responses of wild-type flies, following injection of picrotoxin, verifies the notion that both the dorsoventral and the dorsolongitudinal flight muscles share a common activating pathway (Fig. 3). Picrotoxin application to gynandromorph flies demonstrated the existence of neuronal elements additional to the giant axon pathways, that evoke the indirect flight muscles in response to cervical stimulation (Figs. 4, 5). An unexpected finding was the poor correlation between the mosaic external phenotype of the gynandromorph flies ofpara ^ts mutation and the genotype of neural pathways activating their thoracic flight muscles, as evidenced by the intracellular recordings.Abbreviations GA giant axon - DVM dorsoventral muscle - DLM dorsolongitudinal muscle - PSI peripherally synapsing interneuron - ts temperature sensitive     

Acquired temperature-sensitive paralysis as a biomarker of declining neuronal function in aging Drosophila

General locomotor activity decreases with normal aging in animals and could be partially explained by decreases in neuronal function. Voltage-gated Na⁺ channels are essential in initiating and propagating rapid electrical impulses underlying normal locomotor activity and behavior in animals. Isolation of mutations conferring temperature-sensitive (ts) paralysis has been an extremely powerful paradigm for identifying genes involved in neuronal functions, such as membrane excitability and synaptic transmission. For instance, decreased expression of wild-type Na⁺ channels in flies harboring the no-action-potential ( nap ) mutant allele ( mle^napts ) confers rapid and reversible ts paralysis, because of failure of action potential propagation. Here, we report that aging wild-type Drosophila gradually develops an acquired susceptibility to ts paralysis that is indistinguishable from that seen in young ts paralytic mle^napts mutants. Moreover, we show that this general age-dependent susceptibility is also present in mle^napts flies, although the effects are shifted to lower temperature regimes. The mle^napts flies also exhibit decreased lifespan and increased frailty. Paralysis and decreased lifespan of mle^napts flies were partially rescued by increasing the dosage of para , the structural gene for the major action potential Na⁺ channel in central nervous system of Drosophila . Lastly, we show a dramatic scaling of ts paralysis susceptibility with chronological age in short-lived and long-lived mutant flies, further demonstrating that this age-dependent risk is independent of genetic background. Thus, decreased neural transmission, a hallmark of which is ts paralysis, is a biomarker of aging.     

A comparative study of the inducibility of cardiac creatine phosphokinase by corticosteroids in rats of various ages

A comparative study of the activity and age-dependent inducibility of creatine phosphokinase of rat cardiac muscle has been presented. The specific activity of creatine phosphokinase (CPK) is higher in young (7-week) than in the adult (30-week) and old (80-week) rats. Adrenalectomy decreases the activity of CPK of young and old rats, but not of adult rats. Both cortisol and cortisone induce the enzyme significantly in young and old rats. The magnitude of induction is higher in the old than in the young. Both the hormones decrease the activity of CPK in the adult cardiac muscle. The induction by the hormones is repressed by actinomycin D. The above findings have been discussed in relation to deterioration of the cardiac function during aging of the animal.     

Small differences in Drosophila tropomyosin expression have significant effects on muscle function.

The effects of promoter deletions on Drosophila tropomyosin I (TmI) gene expression have been determined by measuring TmI RNA levels in transformed flies. Decreases in RNA levels have been correlated with rescue of flightless and jumpless mutant phenotypes in Ifm(3)3 mutant transformed flies and changes in muscle ultrastructure. The results of this analysis have allowed us to identify a region responsible for 20% of maximal TmI expression, estimate threshold levels of TmI RNA required for indirect flight and jump muscle function, and obtain evidence suggesting that sarcomere length may be an important determinant of flight muscle function.     

A saturation transfer phosphorus nuclear magnetic resonance study of arginine phosphokinase in the muscle of a marine mollusc

The forward and reverse fluxes of arginine phosphokinase were measured in vitro preparations of the phasic adductor muscle of the scallop Argopecten irradians concentricus using saturation transfer ³¹P-NMR techniques. The respective fluxes were 4.72 ± 1.46 and 6.98 ± 1.58 μmol/s per g wet weight. The forward-to-reverse flux ratio was near unity, indicating that the arginine phosphokinase reaction is near equilibrium under resting conditions. ³¹P-NMR spectra showed no evidence of ADP in scallop muscles. Using an experimentally determined apparent equilibrium constant for arginine phosphokinase, the free ADP level was estimated to be 51 nmol/g wet wt. It appears that at least 90% of the ADP pool is bound in this muscle.     

The effects of age on radiation resistance and oxidative stress in adult Drosophila melanogaster

Drosophila melanogaster (fruit fly) is a well-established model organism for genetic studies of development and aging. We examined the effects of lethal ionizing radiation on male and female adult Drosophila of different ages, using doses of radiation from 200 to 1500 Gy. Fifty percent lethality 2 days postirradiation (LD(50/2)) in wild-type 1-day-old adult fruit flies was approximately 1238 Gy for males and 1339 Gy for females. We observed a significant age-dependent decline in the radiation resistance of both males and females. Radiation damage is postulated to occur by the generation of oxygen radicals. An age-related decline in the ability of flies to resist an agent that induces oxygen radicals, paraquat, was observed when comparing 10- and 20-day adults. Female flies are more resistant to paraquat than male flies. Oxidative stress mediated by paraquat was additive with sublethal exposures to radiation in young adults. Therefore, the ability to repair the damage caused by oxygen radicals seems to decline with the age of the flies. Because Drosophila adults are largely post-mitotic, our data suggest that adult Drosophila melanogaster can serve as an excellent model to study the factors responsible for radiation resistance in post-mitotic tissue and age-dependent changes in this resistance.     

On the role of arginine kinase in insect flight muscle

In the flight muscle of Locusta migratoria L., arginine kinase activity increased 10-fold when 5th instar larvae and adult animals were compared. During the onset of flight, ATP decreased slightly with the amount of phospho-l-arginine remaining constant. Thus, high arginine kinase activity characterizes the adult muscle, giving rise to the speculation that the phospho-l-arginine/l-arginine kinase system does not act only as a buffer system for high-energy phosphate but also as a shuttle mechanism for high-energy phosphate between mitochondria and myofibrils. Judged from electrophoretic mobility, only one isoenzyme exists that is not bound to subcellular structures. Calculations of the diffusive fluxes of ATP, ADP, phosphate, phospho-l-arginine and l-arginine between the sites of ATP-consumption and production, respectively, can be interpreted in such a way, that the low concentration of ADP_free might limit ATP-turnover during flight. Judging from the high arginine kinase activity, the major acceptor for high-energy phosphate at the mitochondria could be l-arginine, while phospho-l-arginine is transphosphorylated to ATP at the myofibrils, thus presumably serving as an energy shuttle.     

Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines. The glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also localized along the sarcomere with a periodic pattern that is indistinguishable from that of GPDH localization. Furthermore, localization of aldolase and GAPDH requires simultaneous localization of GPDH, because aldolase and GAPDH are not localized along the sarcomere in muscles of strains that carry Gpdh null alleles. In an attempt to understand the process of glycolytic enzyme colocalization, we have explored in more detail the mechanism of GPDH localization. In flight muscle, there is only one GPDH isoform, GPDH-1, which is distinguished from isoforms found in other tissues by having three C-terminal amino acids: glutamine, asparagine, and leucine. Transgenic flies that can produce only GPDH-1 display enzyme colocalization similar to wild-type flies. However, transgenic flies that synthesize only GPDH-3, lacking the C-terminal tripeptide, do not show the periodic banding pattern of localization at Z-discs and M-lines for GPDH. In addition, neither GAPDH nor aldolase colocalize at Z-discs and M-lines in the sarcomeres of muscles from GPDH-3 transgenic flies. Failure of the glycolytic enzymes to colocalize in the sarcomere results in the inability to fly, even though the full complement of active glycolytic enzymes is present in flight muscles. Therefore, the presence of active enzymes in the cell is not sufficient for muscle function; colocalization of the enzymes is required. These results indicate that the mechanisms by which ATP is supplied to the myosin ATPase, for muscle contraction, requires a highly organized cellular system.     

Aging enhances indirect flight muscle fiber performance yet decreases flight ability in Drosophila

We investigated the effects of aging on Drosophila melanogaster indirect flight muscle from the whole organism to the actomyosin cross-bridge. Median-aged (49-day-old) flies were flight impaired, had normal myofilament number and packing, barely longer sarcomeres, and slight mitochondrial deterioration compared with young (3-day-old) flies. Old (56-day-old) flies were unable to beat their wings, had deteriorated ultrastructure with severe mitochondrial damage, and their skinned fibers failed to activate with calcium. Small-amplitude sinusoidal length perturbation analysis showed median-aged indirect flight muscle fibers developed greater than twice the isometric force and power output of young fibers, yet cross-bridge kinetics were similar. Large increases in elastic and viscous moduli amplitude under active, passive, and rigor conditions suggest that median-aged fibers become stiffer longitudinally. Small-angle x-ray diffraction indicates that myosin heads move increasingly toward the thin filament with age, accounting for the increased transverse stiffness via cross-bridge formation. We propose that the observed protein composition changes in the connecting filaments, which anchor the thick filaments to the Z-disk, produce compensatory increases in longitudinal stiffness, isometric tension, power and actomyosin interaction in aging indirect flight muscle. We also speculate that a lack of MgATP due to damaged mitochondria accounts for the decreased flight performance.     

Alternative exon-encoded regions of Drosophila myosin heavy chain modulate ATPase rates and actin sliding velocity

To investigate the molecular functions of the regions encoded by alternative exons from the single Drosophila myosin heavy chain gene, we made the first kinetic measurements of two muscle myosin isoforms that differ in all alternative regions. Myosin was purified from the indirect flight muscles of wild-type and transgenic flies expressing a major embryonic isoform. The in vitro actin sliding velocity on the flight muscle isoform (6.4 microm x s(-1) at 22 degrees C) is among the fastest reported for a type II myosin and was 9-fold faster than with the embryonic isoform. With smooth muscle tropomyosin bound to actin, the actin sliding velocity on the embryonic isoform increased 6-fold, whereas that on the flight muscle myosin slightly decreased. No difference in the step sizes of Drosophila and rabbit skeletal myosins were found using optical tweezers, suggesting that the slower in vitro velocity with the embryonic isoform is due to altered kinetics. Basal ATPase rates for flight muscle myosin are higher than those of embryonic and rabbit myosin. These differences explain why the embryonic myosin cannot functionally substitute in vivo for the native flight muscle isoform, and demonstrate that one or more of the five myosin heavy chain alternative exons must influence Drosophila myosin kinetics.     

The effect of removing the N-terminal extension of the Drosophila myosin regulatory light chain upon flight ability and the contractile dynamics of indirect flight muscle

The Drosophila myosin regulatory light chain (DMLC2) is homologous to MLC2s of vertebrate organisms, except for the presence of a unique 46-amino acid N-terminal extension. To study the role of the DMLC2 N-terminal extension in Drosophila flight muscle, we constructed a truncated form of the Dmlc2 gene lacking amino acids 2-46 (Dmlc2(Delta2-46)). The mutant gene was expressed in vivo, with no wild-type Dmlc2 gene expression, via P-element-mediated germline transformation. Expression of the truncated DMLC2 rescues the recessive lethality and dominant flightless phenotype of the Dmlc2 null, with no discernible effect on indirect flight muscle (IFM) sarcomere assembly. Homozygous Dmlc2(Delta2-46) flies have reduced IFM dynamic stiffness and elastic modulus at the frequency of maximum power output. The viscous modulus, a measure of the fly's ability to perform oscillatory work, was not significantly affected in Dmlc2(Delta2-46) IFM. In vivo flight performance measurements of Dmlc2(Delta2-46) flies using a visual closed-loop flight arena show deficits in maximum metabolic power (P(*)(CO(2))), mechanical power (P(*)(mech)), and flight force. However, mutant flies were capable of generating flight force levels comparable to body weight, thus enabling them to fly, albeit with diminished performance. The reduction in elastic modulus in Dmlc2(Delta2-46) skinned fibers is consistent with the N-terminal extension being a link between the thick and thin filaments that is parallel to the cross-bridges. Removal of this parallel link causes an unfavorable shift in the resonant properties of the flight system, thus leading to attenuated flight performance.     

Imaging mitophagy in the fruit fly

Loss-of-function mutations in the genes encoding PRKN/parkin and PINK1 cause autosomal recessive Parkinson disease (PD). Seminal work in Drosophila revealed that loss of park/parkin and Pink1 causes prominent mitochondrial pathology in flight muscle and, to a lesser extent, in dopaminergic neurons. Subsequent studies in cultured mammalian cells discovered a crucial role for PRKN/PARK2 and PINK1 in selective macroautophagic removal of mitochondria (mitophagy). However, direct evidence for the existence of a PINK1-PRKN/PARK2-mediated mitophagy pathway in vivo is still scarce. Recently, we engineered Drosophila that express the mitophagy reporter mt-Keima. We demonstrated that mitophagy occurs in flight muscle cells and dopaminergic neurons in vivo and increases with aging. Moreover, this age-dependent rise depends on park and Pink1. Our data also suggested that some aspects of the mitochondrial phenotype of park- and Pink1-deficient flies are independent of the mitophagy defect, and that park and Pink1 may have multiple functions in the regulation of the integrity of these organelles. Here, we discuss implications of these findings as well as possible future applications of the mt-Keima fly model.     

Circadian changes in Drosophila motor terminals

In Drosophila melanogaster, as in most other higher organisms, a circadian clock controls the rhythmic distribution of rest/sleep and locomotor activity. Here we report that the morphology of Drosophila flight neuromuscular terminals changes between day and night, with a rhythm in synaptic bouton size that continues in constant darkness, but is abolished during aging. Furthermore, arrhythmic mutations in the clock genes timeless and period also disrupt this circadian rhythm. Finally, these clock mutants also have an opposing effect on the nonrhythmic phenotype of neuronal branching, with tim mutants showing a dramatic hyperbranching morphology and per mutants having fewer branches than wild-type flies. These unexpected results reveal further circadian as well as nonclock related pleiotropic effects for these classic behavioral mutants.