Effect of sodium channel abundance on Drosophila development, reproductive capacity and aging

doi:10.4161/fly.18570

Fly (Austin). 2012 January 1; 6(1): 57–67.

PMCID: PMC3365838

Effect of sodium channel abundance on Drosophila development, reproductive capacity and aging

Graham Garber,¹ Lee Ann Smith,¹^† Robert A. Reenan,² and Blanka Rogina¹^*

¹Department of Genetics and Developmental Biology; School of Medicine; University of Connecticut Health Center; Farmington, CT USA

²Department of Molecular Biology; Cell Biology and Biochemistry; Brown University; Providence, RI USA

^†Current affiliation: Department of Biological Sciences; Benedictine University; Lisle, IL USA

* Corresponding author: Blanka Rogina; Email: rogina/at/neuron.uchc.edu

Abstract

The voltage-gated Na⁺ channels (VGSC) are complex membrane proteins responsible for generation and propagation of the electrical signals through the brain, the skeletal muscle and the heart. The levels of sodium channels affect behavior and physical activity. This is illustrated by the maleless mutant allele (mle^napts) in Drosophila, where the decreased levels of voltage-gated Na⁺ channels cause temperature-sensitive paralysis.

Here, we report that mle^napts mutant flies exhibit developmental lethality, decreased fecundity and increased neurodegeneration. The negative effect of decreased levels of Na⁺ channels on development and ts-paralysis was more pronounced at 18 and 29°C than at 25°C, suggesting particular sensitivity of the mle^napts flies to temperatures above and below normal environmental conditions. Similarly, longevity of mle^napts flies was unexpectedly short at 18 and 29°C compared with flies heterozygous for the mle^napts mutation. Developmental lethality and neurodegeneration of mle^napts flies was partially rescued by increasing the dosage of para, confirming a vital role of Na⁺ channels in development, longevity and neurodegeneration of flies and their adaptation to temperatures.

Keywords: aging, cold-sensitive development, heat-sensitive development, Na+ channels, neurodegeneration

Introduction

The voltage-gated Na⁺ channels (VGSC) are required for initiation and propagation of the action potentials in the central and peripheral nervous system. Action potentials regulate locomotor activity in animals. Mutations of Na⁺ channel encoding genes that either alter their expression or affect the function of the channels have been implicated in a variety of disorders ranging from epilepsy, myotomias, ataxia, weakness, migraine, paralysis, cardiac arrhythmia and several neurodegenerative disorders.¹^,² Drosophila melanogaster has been an excellent model to isolate Na⁺ channel mutations and use them to study the functions of sodium channels in the nervous system and their role in neurodegeneration.³ In Drosophila, mutations in ion channels, in synaptic transmission proteins and in other genes that result in lower levels of Na⁺ channels are associated with increased temperature sensitive paralysis due to a loss of action potential.⁴ Mutant flies are paralyzed immediately when exposed to the restrictive temperature, but recover completely after transfer to room temperature. One such mutation, mle^napts, is the no-action potential temperature sensitive mutation of the maleless (mle) gene. The mle gene encodes an ATP-dependent double-stranded RNA helicase necessary for X chromosome dosage compensation and male viability. All mle loss-of-function mutations are male-specific lethal and have normal levels of paralytic (para) encoded Na⁺ channel gene expression.⁵ In contrast, mle^napts is an allele of mle with a single amino acid substitution, which does not cause male lethality.⁶ mle^napts is a recessive gain-of-function mutation of mle that prevents proper resolving of the dsRNA secondary structure of the para transcript necessary for A-to-I RNA editing. This results in a splicing defect of the para encoded Na⁺ channel transcript, severe reduction of Na⁺channel RNA levels and channel activity in both male and female flies, corroborating that ts phenotype is independent from the dosage compensation role of the mle gene.⁶^,⁷ The ts-paralytic phenotype reflects a decreased abundance of Na⁺ channels in the brain because it was shown that ts-paralysis in mle^napts flies can be rescued by the addition of a single dose of the wild-type para⁺ the structural gene for the major action potential Na⁺ channel in Drosophila, which is only expressed in fly brains.⁸ In addition, electrophysiological and behavioral studies show that the phenotypes of mle^napts mutant flies are almost identical to the phenotypes of para^ts mutants.⁹ We have reported that decreased levels of voltage-gated Na⁺ channels found in the mle^napts mutant flies decrease longevity and enhance an age-dependent increase in temperature-sensitive paralysis, and we have shown that these phenotypes can be reversed by increasing the dosage of the para gene.¹⁰

Some mutations that affect the abundance of sodium channels have been reported to negatively affect fly development at high or low temperatures. Flies homozygous for alleles of para, para^sbl1 and para^sbl2, exhibit heat-sensitive developmental lethality.¹¹^,¹² For instance, when para^sbl1 and para^sbl2 homozygous mutant females laid eggs at 29°C only 2% developed to adulthood. Although, para^sbl mutant flies do not show the same cold induced developmental lethality, para^sbl1 and para^sbl2 mutant flies developed and aged at 18°C have a dramatically shorter adult life span.¹² This recessive adult coldsensitivity is more pronounced in females than males. This cold sensitivity in para^sbl1 begins sometime during embryogenesis and in para^sbl2 during metamorphosis.¹²

We wanted to extend our understanding of how a reduction of Na⁺ channels impacts fly physiology by investigating the effect of the mle^napts homozygous mutation on development, reproduction and neurodegeneration. We show here that lower abundance of Na⁺ channels in mle^napts homozygous mutant flies affects fecundity, causes cold- and heat-sensitive developmental lethality, and causes a dramatic cold- and heat-sensitive reduction in life spans compared with heterozygous mle^napts flies. Furthermore, we show that addition of extra copies of the para sodium channel gene rescue temperature dependent developmental lethality and neurodegeneration of mle^napts homozygous flies when introduced into the mle^napts homozygous flies, an effect that confirms the role of voltage-gated sodium channels in development, fecundity, neurodegeneration and longevity. However, overexpression of the para sodium channel gene in a non-sodium channel depleted strain has yet to show a further benefit to longevity.

Results

Lower levels of Na⁺ channels decrease eclosion rate of the mle^napts homozygous mutant flies

In order to determine if the decreased levels of Na⁺ channels of mle^napts/mle^napts flies affects development, we determined the number of flies that eclose from eggs that are homozygous and heterozygous for mle^napts, wild-type (CS) and rescue flies, which have three copies of Na⁺ channel in the mle^napts mutant background (f/f; mle^napts/mle^napts; Dp(1:4)r⁺f ⁺para /⁺). The parental flies were kept on corn food with addition of yeast at 25°C for 8 h, the parents were then removed and the number of eggs was counted. The number of adult flies eclosed in the vials was determined 14 d after initial egg laying. Only homozygous mle^napts flies had a significant decrease in the number of eclosed flies (mle^napts/mle^napts = 70.9%; mle^napts /+ = 89.7%; CS = 86.9%; rescue = 83.6%) (Fig. 1A), Statistical analysis is listed in the supplemental data. Homozygous mutant flies carrying an extra copy of the para gene had similar number of eggs and eclosion rates to heterozygous flies, showing that the fecundity and developmental defect is due to decreased sodium channels. These findings show that normal levels of Na⁺ channels in the brain of flies are required for development of fruit flies.

Figure 1. Eclosion rate of the flies is affected in the mle^napts homozygous mutant flies. Eclosion rate of adult flies from eggs produced by female flies homozygous (mle^napts/mle^napts) (magenta) and heterozygous for the mle^napts/+ (blue) mutation, (more ...)

Mutant flies are affected by temperature during development

mle^napts mutant flies have a temperature-sensitive paralysis phenotype due to the low levels of Na⁺ channels.⁶ We wanted to determine if the developmental defects of mle^napts mutant flies were also temperature sensitive. Development was allowed to proceed at low (18°C) or high (29°C) temperatures and eclosion rates were measured. Wild-type (CS) flies exhibited only small differences in eclosion at 25, 18 or 29°C (25°C = 86.9%; 18°C = 80.8%; 29°C = 76.7%), (Fig. 1A–CandTable 1) but heterozygous mutants and rescued flies had significantly lower rates of eclosion when their development occurred at 18°C suggesting that the mle^napts allele has an additional dominant cold-sensitive developmental effect that is not associated with decreased levels of sodium channels. The data presented in Figure 1C show that the rescue flies have lower rate of eclosion at 29°C (mle^napts/+ at 25°C = 89.7%; 18°C = 58.4%; 29°C = 83.1; rescue flies at 25°C = 83.6%; 18°C = 54.6%; 29°C = 50.7%). Intriguingly, no adult flies eclosed from eggs laid by mle^napts homozygous mutant flies at 18 or 29°C, revealing the detrimental effects of low and high temperatures on flies with low levels of Na⁺-channels (Fig. 1B and C).

Table 1. Eclosion rate of flies homozygous for the mle^napts mutation is drastically impaired at 29 and 18°C

Temperature shifts reveal temperature sensitive periods during fly development

The eclosion rate of mle^napts/mle^napts flies was 0% at 18 and 29°C revealing that high and low temperatures are lethal for development of the flies with low levels of Na⁺ channels. We wanted to determine the developmental stage at which temperature sensitive lethality occurs. In the first experiment, parents were kept for 8 h in vials at 25°C, and after removal of the parents, eggs were counted and kept at 25°C for embryonic development. First instar larvae were transferred to 18 or to 29°C and kept at that temperature for the rest of their development. In the second experiment, embryos were kept for 24 h at 29 or 18°C and then transferred them to 25°C. We found that when embryonic development of mle^napts homozygous flies occurred at 25°C and larval and pupal development occurred at 18°C the eclosion rate is ~78% of adult flies eclosed compared with mle^napts/+ = 83.1%, CS = 68.2% and rescue = 83.7% (Fig. 2A and Table 1). When embryonic development took place at 25°C and subsequent development at 29°C, there was an effect of mle^napts mutation on larval and/or pupal development and the total percent of eclosed flies was 50.5% compared with CS = 89.7%, mle^napts/+ = 87.5% and rescue = 79.4%, suggesting that heat-sensitive embryonic development in mle^napt mutant homozygous flies is associated with low levels of sodium channels (Fig. 2B). However, when embryonic development took place at 18 or 29°C and larvae were transfer to 25°C, the eclosion rate was 0.4 and 0% respectively (Fig. 2A and B). Rescue flies also have a lower eclosion rate when embryonic development occur at 29°C (rescue 18–25°C = 76.2%, 29–25°C = 69.8%, 25–18°C = 83.7%; 25–29°C = 79.4) (Fig. 2A and B; Table 1; Supplemental Data A?G).

Figure 2. Embryonic development of the mle^napts homozygous mutant flies is affected at 18 and 29°C. Eclosion rate of adult flies from eggs laid by female flies homozygous (mle^napts/mle^napts) (magenta) and heterozygous for the mle^napts (more ...)

Temperature of the environment affects the susceptibility of mutant flies to paralysis

We examined whether the environmental temperature affects ts-paralysis of the mle^napts mutant flies by determining ts-paralysis of flies that were kept at 18°C from the day of eclosion until testing. Flies living at the lower temperature (18°C) displayed a concomitant greatly increased sensitivity to paralysis at a high temperature (35°C) compared with flies raised at 25°C (Fig. 3A and B and data not shown). Data for ts-paralysis at 25°C were published previously.¹⁰ Whereas mle^napts mutants when aged at 25°C only become susceptible to paralysis at 35°C in extreme age, animals reared at a lower temperature show almost complete paralysis at 35°C as early as 10 d in males and 20 d in females (Fig. 3A and B). Moreover, this effect requires only a few days to take effect. For instance, even after only 5 d at lowered temperature there was a 245% (male) and 365% (female) increase in sensitivity to paralysis at 33°C. While only 15.9% of the 5 d old male flies homozygous for the mle^napts mutation experience paralysis at 33°C if they were kept at 25°C from eclosion, 54.8% of male flies were paralyzed at 33°C if they were kept at 18° from eclosion (Fig. 3A and C). Female flies show similar differences in ts-paralysis. 14.7% of females are paralyzed at 33°C if raised at 25°C but 68.5% if they were raised at 18°C (Fig. 3B). Conversely, male and female flies homozygous for the mle^napts mutation kept at 29°C from eclosion until testing adjust to environmental temperature better than flies aged at 18°C (Fig. 3C and D). However, they are still more sensitive to ts-paralysis compared with the siblings raised at 25°C.¹⁰ Similarly to our finding, Nelson and Wyman reported that mle^napts flies reared at 18°C adjust to environmental temperature and exhibit ts-paralysis at lower temperatures than flies reared at 25°C.³ However, while we found that mle^napts flies are more sensitive to ts-paralysis when kept at 29°C compared with 25°C, mle^napts flies were previously reported to adjust well to 30°C. The discrepancy could be due to a different protocol: we tested more than 3,000 flies at specific ages since our previous data showed that ts-paralysis is age-dependent. In addition, each fly was tested only once. Our data indicate that the temperature at which flies are raised can change their susceptibility to ts-paralysis and even accelerate the age-dependent increase in susceptibility to ts-paralysis seen in mle^napts mutant backgrounds.

Figure 3. Flies with low abundance of Na⁺ channels are more sensitive to ts-paralysis when aged at 18 or 29°C. Male (A) and female (B) flies homozygous for the mle^napts mutation that were aged at 18°C from the eclosion until testing (more ...)

Temperature affects the longevity of mle^napts mutant flies

We have reported that the lower abundance of Na⁺ channels associated with the mle^napts mutation has a negative effect on longevity in Drosophila kept at 25°C. That is, mle^napts homozygous male and female flies live significantly shorter at 25°C compared with flies that are heterozygous for the mle^napts mutation.¹⁰ Because mle^napts homozygous mutant flies raised at 18 or 29°C are much more sensitive to ts-paralysis than ones raised at 25°C, we wanted to explore the effects of different environmental temperatures on the life span of the mle^napts homozygous mutant flies. When aged at 29°C the mle^napts homozygous male flies have 53.1% and female 52% shorter life span compared with the control mle^napts heterozygous flies (Fig. 4A and Table 2). Similarly, mle^napts homozygous flies aged at 18°C have dramatically decreased average life span compared with the controls (Fig. 4D and Table 2). Homozygous male flies have 65.3% and female 68.1% decreased median life span compared with the controls. Our data point to the increased sensitivity of the mle^napts flies not only to temperatures above 35°C but also to lower environmental temperatures such as 18°C. Observation of the shape of the curves suggests that mle^napts flies do not exhibit a normal aging process. At 18°C mle^napts homozygous mutant flies show severely decreased early survival and this trend continues during their short life. A single gene mutation or environmental manipulations can decrease longevity by increasing the mortality rate or by increasing the frailty of flies, which could be determined by fitting the survivorship data to the Gompertz equation; changes in the slope of the mortality curve correspond to a change in the rate of aging, while shift in the mortality curve represent the change in the frailty of the flies.¹³^,¹⁴ We fitted the data from Figure 4 to the Gompertz equation and we found that decreased levels of Na⁺ channels affect survivorship of mle^napts mutant flies by increasing the frailty in both males and females aged at 18 and 29°C illustrated by the change in y-intercept. Similar findings were obtained for female flies at 25°C.¹⁰ The results suggest that both mle^napts homozygous and mle^napts/CS flies age at the same rate, but homozygous flies prematurely experience the negative physiologic effects of aging. In other words, these results reveal that decreased levels of Na⁺ channels in the mle^napts mutants increases the intrinsic vulnerability of the flies. Taken in sum, our results suggest that chronic disruption of the normal level of Na⁺ channel expression has no detectible impact on the rate of aging but can make flies prematurely experience some of the negative physiological effects of aging and increased frailty.

Figure 4. Life span of mle^napts flies is shorter at 29 and 18°C. Male and female flies homozygous for the mle^napts mutation have shorter life span compared with genetically matched heterozygous mle^napts /CS control flies at 29 (A) and (more ...)

Table 2. Life span of flies homozygous for the mle^napts mutation is decreased at 29 and 18°C

In order to examine the effects of increasing Na⁺ channels levels on fly longevity we used the para strain P{UAS-para13.5} and the conditional ELAV-geneSwitch driver to drive overexpression of the para splice variant 13.5 in the brain.¹⁵^-¹⁷ Flies that overexpress the para gene throughout their adult lives have little or no effect on longevity (effect on mean female longevity of 5.5% and no effect was seen in male flies (RU males = 55.08 d, females = 56.32 d; EtOH males = 53.17 d, females = 53.38 d) (Fig. 5A–D). Increasing para Na⁺ channels levels had no effect on fly mortality rate. Although, chronic overexpression of the para gene exhibits a marginal beneficial effect on longevity in female flies (5.5% life span extension of female longevity), it is still possible that selective increases in Na⁺ channel levels in older adults will have a more pronounced effect on Drosophila longevity. In addition, since the ELAV GeneSwitch drives overexpression only in some areas of the nervous system it is possible that overexpressing the para gene in the brain ubiquitously may have beneficial effects.¹⁷

Figure 5. Increased levels of Na⁺ channels have no beneficial effect on fly longevity. Survivorship (A and B) and mortality rate (C and D) of male (A and C) and female (B and D) ELAV&UASpara flies fed on food with 200 μM RU486 (more ...)

Lower abundance of the para Na⁺ channels is associated with decreased female fecundity

Flies homozygous for the mle^napts mutation have a shorter life span and increased frailty at 25, 18 and 29°C compared with genetic controls (see ref. ¹⁰ and Fig. 4). Since low fecundity may be one of the hallmarks of the frailty, we wanted to determine if lower levels of Na⁺ channels also affects fecundity of mle^napts/mle^napts mutant flies.¹⁸ We found that average egg production during the first 16 d of life is lower in mle^napts homozygous mutant flies compared with mle^napts/+ heterozygous or wild-type CS flies, (Fig. 6A and B) (average eggs/fly: CS = 41.25; mle^napts/CS = 32.125; mle^napts/mle^napts = 9). Even the heterozygous females appear to have considerably reduced fecundity. In a second experiment that followed egg laying until late in life, these results were confirmed for homozygous and heterozygous flies (Fig. 6C and D). Female flies homozygous for the mle^napts mutation laid on average 3.92 eggs/day, which is 60% fewer eggs than heterozygous females that produced on average 9.7 eggs/day (total life-long egg production number mle^napts /CS = 655.00; mle^napts/mle^napts = 184.43 Average number of eggs/day mle^napts /CS = 9.78; mle^napts/mle^napts = 3.92). Therefore, the mle^napts homozygous phenotype includes decreased female fecundity in addition to shorter life span and increased frailty.

Figure 6. Fecundity of the flies is affected in the mle^napts/ mle^napts homozygous mutant flies. The average (A) and cumulative (B) egg production for the first 16 d of flies homozygous for the mle^napts (black circles) mutation is lower compared (more ...)

Increased levels of Na⁺ channels do not affect fecundity

We used the UAS-GAL4 gene-switch system to determine whether increased levels of the para Na⁺ channel gene may also affect fecundity of females. We used the para strain P{UAS-para13.5} and the conditional ELAV-geneSwitch driver to pan-neuronally express the para splice variant 13.5.¹⁵ We determined the 24 h egg production of ELAVswitch-UAS-para flies fed on food with the addition of either RU486, which stimulates para expression, or EtOH as a control, every other day during their first 20 d of life. We found that both groups have very similar average fecundity and cumulative egg production suggesting that overexpression above wild-type levels did not affect egg production (average daily eggs/fly RU486 = 32.78, EtOH = 34.04; cumulative number of egg/20 d RU486 = 622.8, EtOH = 646.73), (Fig. 7A and B).

Figure 7. Increased levels of Na⁺ channels do not affect fecundity of female flies. Daily average (A) and cumulative (B) egg production of ELAV&UASpara flies fed on food with RU486 (black circles) or EtOH (open squares) during the first (more ...)

Decreased levels of Na⁺ channels is associated with increased neurodegeneration

Data presented here and in a previous publication, indicate that homozygous mle^napts mutant flies exhibit signs of premature aging characterized by accelerated increase in age-associated ts-paralysis, decreased longevity, and an increase in frailty. These data suggest that flies with decreased levels of Na⁺ channels exhibit a premature decline of neurological function, and we expected to see evidence of accelerated aging and degeneration in the nervous system. Fergestad et al. (2006) have characterized the neuropathology in Drosophila mutants harboring similar mutations in genes encoding sodium channels when flies were kept at 29°C.¹⁹ Therefore, we examined histological sections of brains from homozygous and heterozygous mutant flies aged at 25°C. In agreement with previous studies, we observed multiple regions of vacuolization present in our mutant strain long before any evidence of such pathology occurred in a heterozygous control strain (Fig. 8A–D and Table 3). We quantified neurodegeneration by calculating the percentage of brains with more than one vacuolar structure larger than 0.12 μM. Our analysis shows that at age 50 d 52.9% of brains dissected from mle^napts/mle^napts homozygous male flies have more then one vacuole per brain, while only 16.7% of heterozygous mle^napts/+ and 11.1% of rescue males, respectively, have the same phenotype. Similarly, 50% of brains from homozygous mle^napts/mle^napts females have vacuoles at 50 d compared with only 21.4 and 15.1% in heterozygous mle^napts/+ and rescue females of the same age (Fig. 8C and D; Table 3). Our data confirm that normal Na⁺ channel expression is a requirement for maintaining an intact nervous system.

Figure 8. Aging homozygous mle^napts/mle^napts mutant flies display premature neurodegeneration. Increased neurodegeneration is found in male (A–C) and female (D) flies homozygous for the mle^napts mutation. Frontal sections of male mle^napts (more ...)

Table 3.mle^napts mutation increases neurodegeneration in aging flies.

Discussion

Decreased levels of the para Na⁺ channel gene affect development of the flies

We found that the development of the mle^napts/mle^napts homozygous mutant flies is altered, which is illustrated by a significant reduction in the percentage of adult flies that eclose from eggs laid by homozygous mutant flies compared with control flies. In order to confirm that the decreased eclosion rate found in mle^napts/mle^napts homozygous mutant flies is due to the lower levels of Na⁺ channels, we genetically introduced extra copies of para, the structural gene for the major Na⁺ channel in the central nervous system of Drosophila into the mle^napts/mle^napts mutant flies. An extra copy of the para gene rescued the low eclosion rate of mle^napts/mle^napts mutant flies demonstrating that lower levels of Na⁺ schannels are specifically responsible for the reduced eclosion rate of the mle^napts/mle^napts mutant flies. A decrease in Na⁺ channels of mle^napts mutant could affect development and maintenance of the nervous system contributing to lethality and lower fecundity. Negative developmental effects in mle^napts flies may be due to structural defects in the nervous system. For instance, mle^napts mutant flies have a decrease in branching of neurons that could limit neuronal connectivity and decrease excitability.³^,²⁰

Flies with decreased levels of Na⁺ channels exhibit heat- and cold-sensitive developmental lethality

Developmental lethality was previously reported with other para mutations at 29 or 18°C.¹² We examined whether low or high temperatures also negatively affect development of mle^napts homozygous mutant flies, and if so, at which developmental stage this negative effect occurs. We found the mle^napt homozygous mutant flies exhibit 100% developmental lethality when eggs were laid at 29 or 18°C. Shifting experiments show that early embryonic development is the cold- and heat-sensitive period. In addition, if larvae are switched from 25–29°C near the time of hatching, only 50% of flies developed to adults suggesting that larval development and morphogenesis are also affected by high temperatures but less compared with the cold.

Decreased dosage of Na⁺ channels increases adult sensitivity to cold and high temperatures, when assayed by ts-paralysis or longevity studies

Decreased levels of Na⁺ channels in mle^napts homozygous mutant flies causes reversible ts- paralysis when exposed to temperatures above 38°C due to the failure of action potential propagation.²¹ Here, we show that aging at low or high temperature increases susceptibility of mle^napts mutant flies to ts-paralysis, however, heat-sensitivity is less pronounced than coldsensitivity. mle^napts homozygous mutant flies aged at 18°C are more sensitive to ts paralysis compared with mutant flies kept at 25°C, while mutants aged at 29°C are less sensitive compared with the controls. Similar increased susceptibility to heat induced seizures was found in mammals. For instance, mice heterozygous for loss-of-function mutations in the α subunit of the type I voltage-gated sodium channels Nav1.1. develop seizures and ataxia.²² Mice are also susceptible to seizures when exposed to high temperature. Similar to our finding of an age-associated increase in sensitivity to high temperature, these mice exhibit an age-dependent increase in temperature- induced seizures and at very old age seizures occur at standard temperatures. In humans, infants heterozygous for the Nav1.1 mutation in the α subunit of the type I voltage-gated Na⁺ channels experience severe myoclonic epilepsy in infancy (SMEI) and develop seizures around 6–9 mo of age.²³ The seizures are often initiated by fever or infections, and sometimes may be initiated by warm baths. In humans several mutations in the skeletal muscle Na⁺ channels are associated with paralysis of the affected individuals when exposed to cold. For instance, the Q270 mutation of the skeletal muscle Na⁺ channel α-subunit (Nav1.4) is associated with thermosensitive paramyotonia and sensitivity to cold.²⁴ Paralysis is observed only when individuals are exposed to cold, and the symptoms disappear when individuals are warmed. Similar cold sensitivity was found in two classes of Out-cold (Ocd) mutant flies with the single missense T15511 mutation (para^ocd2), and the double missense I11545M and G1571R mutation (para^ocd1, para^ocd3 and para^ocd5) in the para gene, which encodes the major voltage-gated sodium channel.¹⁵ Taken together, these data indicate that flies are similar to both mice and humans with regard to the fact that mutations that reduce Na⁺ channel function can cause sensitivity to low or high ambient temperature.

We found that the reduction in Na⁺ channels in mle^napts homozygous flies shortens their life span at all temperatures by increasing fly frailty. However, we show here that the most dramatic effect on longevity was seen in flies aged at 18°C. The high impact on longevity at 18°C is in agreement with our findings that mle^napts mutant flies are more sensitive to ts-paralysis if aged at 18°C compared with when they are kept at 29°C.

Decreased levels of the para Na⁺ channel gene affect fecundity

A mutation that increases frailty and shortens life span could potentially also affect reproduction of the flies.²⁵ In order to determine whether the shortened life span associated with the decreased abundance of para Na⁺ channels in mle^napts mutant flies could be associated with decreased fecundity of the mutants, we determined daily egg production of mle^napts/mle^napts, mle^napts/CS and CS wild-type flies. We found that mle^napts/mle^napts homozygous mutant flies produced significantly fewer eggs compared with heterozygous or wild-type females. Since low levels of Na⁺ channels negatively affect fly longevity and female fecundity, and the addition of an extra copy of para in mutant background rescues these phenotypes, we wanted to determine if overexpression of the para gene above wild-type levels in the brain of wild-type flies would have any effect on the longevity and fecundity of the females. We used the conditional ELAV-GeneSwitch system to overexpress the para gene pan-neuronally and found that longevity and the fecundity of the flies overexpressing the para genes was similar to the genetic controls showing that only a decreasing abundance of Na⁺ channels has a negative impact on fecundity.

Decreased levels of Na⁺ channels increase neurodegeneration of mle^napts homozygous mutant flies

Our published report indicates that wild-type flies develop an age-related increase in susceptibility to ts-paralysis, which could be caused by age-related changes in the levels or function of Na⁺ channels or other components of neuronal signaling. We also reported that the homozygous mle^napts mutant flies displayed an early increase in age-dependent ts-paralysis compared with controls.¹⁰ Thus, this mutant strain is likely to be a model for accelerated aging in the nervous system. Therefore, we examined histological sections from this mutant population and found that the mle^napts strain exhibits mild neuropathology illustrated by multiple regions of vacuolization present in our mutant strain long before any evidence of such pathology occurred in a heterozygote control strain. Others¹⁹ have characterized neuropathology in Drosophila mutants harboring similar ion channel defects when flies were aged at 29°C. The premature neurodegeneration observed in our mutant flies can be avoided when extra copies of the para Na⁺ channel gene are added into the homozygous mle^napts mutant strain.

mle^napts mutation affects development, longevity and neurodegeneration of Drosophila

The data presented here indicate that the decrease in the abundance in Na⁺ channels causes developmental lethality, sensitivity to cold, shorter life span at low and high temperature, increased frailty, lower fecundity, and premature neurodegeneration in mle^napts mutant homozygous flies. There are several possible explanations for these phenotypes. The processes mediated by Na⁺ channels are temperature sensitive, and normal levels of Na⁺ channels are required to maintain action potential conduction at higher and lower temperature. Synaptic transmission through motor axons requires an optimal number of Na⁺ channels for locomotor activity, development and maintenance of the nervous system, and any decrease in abundance of Na⁺ channels contributes to suboptimal conditions, which cause phenotypic consequences. For instance, mle^napts mutant flies have a decrease in branching of neurons that could limit neuronal connectivity and decrease excitability.³^,²⁰ In humans, changes in function of Na⁺ channels have been implicated as major contributors in axon degeneration and inflammation associated with multiple sclerosis.² Suboptimal functioning of Na⁺ channels caused by other mutations also causes phenotypic consequences similar to cold and heat sensitivity observed in mle^napts mutant. Alteration in fast inactivation of Na⁺ channels was observed in the T1313A mutation in the SCN4A gene, which encodes the pore-forming α subunit (hSkM1) of the skeletal muscle Na⁺ channel, causes paramyotonia congenital (PC).²⁶ These PC patients suffer from muscle stiffness when exposed to cold. The dominant cold-sensitive para^Ocd mutants in Drosophila melanogaster have a single or the double missense mutations in the para gene.¹⁵ para^Ocd mutants have altered expression of several mitochondrial proteins that may change the physiological function of mitochondria and the levels of energy production. The authors hypothesized that such a change in mitochondrial function is necessary to produce more energy required for dealing with excess intracellular sodium, which comes as a result of slow Na⁺ channel inactivation.¹⁵ However, changes in mitochondrial physiology may be secondary and caused by the decreased levels of sodium channels. Interestingly, the G1306V mutation found in the ortholog residue of the human skeletal muscle Na⁺ channel SCN4A, causes the human cold-sensitive muscle disorder paratomyotonia congenital.²⁷ Drosophila melanogaster has been a powerful genetic model to study the molecular mechanism underlying development, aging, neurodegeneration and human diseases. Our data highlight the important role that Na channels play in development, neurodegeneration and aging.

Material and Methods

Fly strains

Wild-type flies were Canton S (CS) (Stock #1). The mle^napts, para and para^1k5 f^3ba car; Dp(1:4)[para⁺r⁺f⁺]/Ci ^D.K lines were generously provided from the Ganetzky lab.⁵^,²⁸^,²⁹ The para1k5 f36a car; Dp(1:4)[para+r+f+]/Ci^D.K. flies have both mutation and duplication of the para gene: para^1k5 a recessive lethal para mutation and Dp(1:4)[para⁺r⁺f⁺] duplication of the entire para gene.⁸ P{UAS:para13.5} flies were generously provided by Dr. Richard Baines, and the strain was used to express para splice variant13.5 under control of the ELAV-GeneSwitch driver.¹⁵^,¹⁶ Flies w;Cyo/Sco and f¹ (Stock#36) used in crosses were from the Bloomington Stock Center.

Rescue

Crosses performed to introduce extra copies of the para gene into the mle^napts mutant background were described previously.¹⁰ We also used the binary ELAV-GAL4- geneswitch system to overexpress the para gene in wild-type flies. Virgin females with the conditional ELAV-GeneSwitch driver were crossed to the UAS-para⁺ males and their progeny were collected.

Life span

Vials were cleared of adult flies in the morning and the collection of newly eclosed flies occurred in the afternoon. ~20 male and ~20 female flies were kept together in plastic vials with approximately 5–10 mL of a standard cornmeal media.¹⁰ Flies were housed in humidity-controlled incubators, maintained at 29 or 18°C on a 12 h light:dark cycle. Vials of fresh food were supplied three times weekly (Monday, Wednesday and Friday) and the number of dead flies was recorded during each passage from old to new vials.

Temperature sensitive paralysis

Soon after eclosion flies were collected and kept in vials with standard cornmeal food in the temperature-controlled, humidified incubator at 29 or 18°C. Flies were passed twice a week into new vials. On the day of analysis males and females were separated on CO₂ 2–3 h before testing. Ten males or ten female flies were transferred into a glass vial mostly submerged into a water bath at the tested temperature. The number of paralyzed flies was recorded after 30 sec. Each fly was tested only once.

Fecundity studies

Virgin flies were collected on CO₂. A single female fly was kept with a single male fly in a plastic vial containing a standard cornmeal agar medium with several grains of yeast at 25°C in a temperature-controlled incubator. The flies were passed to new vials daily and the number of eggs was recorded. The number of eclosed flies from each vial was counted 14 d after eggs were laid onto the food. In experiments design to determine the effect of overexpression of the para gene on female fecundity, 20 vials with a single ELAVGeneSwitch/UAS- para⁺ male and a single female each were put on food with RU486 200 μM or with EtOH. The number of eggs laid by a single male/female pair was determined every 24 h.

Determination of the developmental lethality

Ten male and ten females flies at about 2 weeks of age were put in a fresh vial of corn food with several grains of yeast and kept at 25°C in a humidified incubator. After 8 h flies were removed and the number of eggs was counted. The number of adult flies eclosed from the eggs was counted after 2 weeks. A similar protocol was used to determine the number of flies eclosed at 29 and 18°C, with the difference that at 18°C the flies were allowed to develop for 3 weeks before the number of adult flies was counted.

Histology

Briefly, heads and bodies from adult flies were dissected and placed in freshly prepared 4% paraformaldehyde fixative at room temperature for 20 min and then washed with 70% ethanol and processed into paraffin. Heads were embedded to obtain frontal sections. Serial 4 μm sections were stained with hematoxylin and eosin and examined under a light microscope (number of brains examined for each genotype is listed in Table 3). The degree of neuropathology present for each experimental group (mle^napts, mle^napts/+ and rescue) was determined by the examination of the frequency of vacuolar pathology in serial sections, according to the following rating scale. A negative score was assigned to brains exhibiting no gross neuropathology or a single small vacuolar structure (0.12 μm in diameter). Brain tissue presenting with small sporadic individual vacuolar structures (0.12 μm in diameter) in multiple sections of a brain was given a positive score.

Statistical analysis

Statistical analysis of life span data was performed with Kaplan-Meier survival functions and log-rank tests (to assess statistical differences between median longevity statistics) using the Statview (SAS Institute, Inc.) software package. Age-specific mortality rates were determined using WinModest software.³⁰ Maximal survival data was calculated as the mean of the final surviving 10% for each population. For the statistical comparison of survivorship data, p = 0.0001 is used as the threshold for significance. Statistical analyses were performed by computing z-tests for independent proportions across groups (CS, HETERO, NAP and RESCUE) at each temperature or temperature shift (e.g., 18°C shift to 25°C). Due to the number of comparisons made, the significance level used for each comparison was 0.001, as to exercise some control over familywise Type I error. The null hypothesis for each comparison was that the given proportions in the population were of equal value. The alternative hypothesis was that they were of unequal value.

Supplementary Material

Additional material

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Acknowledgments

We thank Suzanne Kowalski, Cara Spagnoletti and Luke Piscitelli for technical support and Drs Joseph Jack and Stewart Frankel for critical reading of the manuscript. We thank Dr Daniel J. Denis for his expert statistical analysis. This work was supported by grants from the National Science Foundation (R.A.R.), the Ellison Medical Foundation (R.A.R) and National Institute of Health (AG023088 to B.R.).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Footnotes

Previously published online: www.landesbioscience.com/journals/fly/article/18570

References

1. Catterall WA, Dib-Hajj S, Meisler MH, Pietrobon D. Inherited neuronal ion channelopathies: new windows on complex neurological diseases. J Neurosci. 2008;28:11768–77. doi: 10.1523/JNEUROSCI.3901-08.2008. [PMC free article] [PubMed] [Cross Ref]

2. Waxman SG. Mechanisms of disease: sodium channels and neuroprotection in multiple sclerosis-current status. Nat Clin Pract Neurol. 2008;4:159–69. doi: 10.1038/ncpneuro0735. [PubMed] [Cross Ref]

3. Nelson JC, Wyman RJ. Examination of paralysis in Drosophila temperature-sensitive paralytic mutations affecting sodium channels; a proposed mechanism of paralysis. J Neurobiol. 1990;21:453–69. doi: 10.1002/neu.480210307. [PubMed] [Cross Ref]

4. Vijayakrishnan N, Broadie N. Temperature-sensitive paralytic mutants: Insights into the synaptic vesicle cycle. Biochem Soc Trans. 2006;34:81–7. doi: 10.1042/BST0340081. [PubMed] [Cross Ref]

5. Kuroda MI, Kernan MJ, Kreber R, Ganetzky B, Baker BS. The maleless protein associates with the X chromosome to regulate dosage compensation in Drosophila. Cell. 1991;66:935–47. doi: 10.1016/0092-8674(91)90439-6. [PubMed] [Cross Ref]

6. Kernan MJ, Kuroda MI, Kreber R, Baker BS, Ganetzky B. ^napts, a mutation affecting sodium channel activity in Drosophila, is an allele of mle, a regulator of X chromosome transcription. Cell. 1991;66:949–59. doi: 10.1016/0092-8674(91)90440-A. [PubMed] [Cross Ref]

7. Reenan RA, Hanrahan CJ, Ganetzky B. The mlenapts RNA helicase mutation in Drosophila results in a splicing catastrophe of the para Na+ channel transcript in a region of RNA editing. Neuron. 2000;25:139–49. doi: 10.1016/S0896-6273(00)80878-8. [PubMed] [Cross Ref]

8. Stern M, Kreber R, Ganetzky B. Dosage effects of a Drosophila sodium channel gene on behavior and axonal excitability. Genetics. 1990;124:133–43. [PubMed]

9. Hong CS, Ganetzky B. Spatial and temporal expression patterns of two sodium channel genes in Drosophila. J Neurosci. 1994;14:5160–9. [PubMed]

10. Reenan RA, Rogina B. Acquired temperature-sensitive paralysis as a biomarker of declining neuronal function in aging Drosophila. Aging Cell. 2008;7:179–86. doi: 10.1111/j.1474-9726.2008.00368.x. [PubMed] [Cross Ref]

11. Lilly M, Carlson J. smellblind: A gene required for Drosophila olfaction. Genetics. 1990;124:293–302. [PubMed]

12. Lilly M, Riesgo-Escobar J, Carlson J. Developmental analysis of the smellblind mutants: evidence for the role of sodium channels in Drosophila development. Dev Biol. 1994;162:1–8. doi: 10.1006/dbio.1994.1061. [PubMed] [Cross Ref]

13. Bronikowski AM, Alberts SC, Altmann J, Packer C, Carey KD, Tatar M. The aging baboon: comparative demography in a non-human primate. Proc Natl Acad Sci USA. 2002;99:9591–5. doi: 10.1073/pnas.142675599. [PubMed] [Cross Ref]

14. Jacobson J, Lambert AJ, Portero-Oti´n M, Pamplona R, Magwere T, Miwa S, et al. Biomarkers of aging in Drosophila. Aging Cell. 2010;9:466–77. doi: 10.1111/j.1474-9726.2010.00573.x. [PubMed] [Cross Ref]

15. Lindsay HA, Baines R. ffrench-Constant R, Lilley K, Jacons HT, O'Dell KMC. The dominant cold-sensitive Out-Cold mutants of Drosophila melanogaster have novel missense mutation in the voltage-gated sodium channel gene paralytic. Genetics. 2008;180:873–84. doi: 10.1534/genetics.108.090951. [PubMed] [Cross Ref]

16. Osterwalder T, Yoon KS, White BH, Keshishian H. A conditional tissue-specific transgene expression system using inducible GAL4. Proc Natl Acad Sci USA. 2001;98:12596–601. doi: 10.1073/pnas.221303298. [PubMed] [Cross Ref]

17. Poirier L, Shane A, Zheng J, Seroude L. Characterization of the Drosophila Gene-Switch system in aging studies: a cautionary tale. Aging Cell. 2008;7:758–70. doi: 10.1111/j.1474-9726.2008.00421.x. [PubMed] [Cross Ref]

18. Rogina B, Wolverton T, Bross TG, Chen K, Muller H-G, Carey JR. Distinct biological epochs in the reproductive life of female Drosophila melanogaster. Mech Ageing Dev. 2007;128:477–85. doi: 10.1016/j.mad.2007.06.004. [PMC free article] [PubMed] [Cross Ref]

19. Fergestad T, Ganetzky B, Palladino MJ. Neuropathology in Drosophila membrane excitability mutants. Genetics. 2006;172:1031–42. doi: 10.1534/genetics.105.050625. [PubMed] [Cross Ref]

20. Budnik V, Zhong Y, Wu CF. Morphological plasticity of motor axons in Drosophila mutants with altered excitability. J Neurosci. 1990;10:3754–68. [PubMed]

21. Wu CF, Ganetzky B, Jan LY, Jan YN. 1978 A Drosophila mutant with a temperature-sensitive block in nerve conduction. Proc Natl Acad Sci USA. 1978;75:4047–51. doi: 10.1073/pnas.75.8.4047. [PubMed] [Cross Ref]

22. Oakley JC, Kalume F, Yu FH, Scheuer T, Catterall WA. Temperature- and age-dependent seizures in a mouse model of severe myoclonic epilepsy in infancy. Proc Natl Acad Sci USA. 2009;106:3994–9. doi: 10.1073/pnas.0813330106. [PubMed] [Cross Ref]

23. Oguni H, Hayashi K, Osawa M, Awaya Y, Fukuyama Y, Fukuma G, et al. Severe myoclonic epilepsy in infancy: clinical analysis and relationship to SCN1A mutations in a Japanese cohort. Adv Neurol. 2005;95:103–17. [PubMed]

24. Carle T, Fournier E, Sternberg D, Fontaine B, Tabti N. Cold-induced disruption of Na+ channel slow inactivation underlies paralysis in highly thermosensitive paramyotonia. J Physiol. 2009;587:1705–14. doi: 10.1113/jphysiol.2008.165787. [PubMed] [Cross Ref]

25. Flatt T. Survival cost of reproduction in Drosophila. Exp Gerontol. 2011;46:369–75. doi: 10.1016/j.exger.2010.10.008. [PubMed] [Cross Ref]

26. Bouhours M, Sternberg D, Davoine CS, Ferrer X, Willer JC, Fontaine B, et al. Functional characterization and cold sensitivity of T1313A, a new mutation of the skeletal muscle sodium channel causing paramyotonia congenita in humans. J Physiol. 2004;554:635–47. doi: 10.1113/jphysiol.2003.053082. [PubMed] [Cross Ref]

27. McClatchey AIP, Van den Bergh P, Pericak-Vance MA, Raskind W, Verellen C, McKenna-Yasek D, et al. Temperatue-sensitive mutation in the III-IV cytoplasmic loop region of the skeletal muscle sodium channel gene in Paramyotinia congenital. Cell. 1992;68:769–74. doi: 10.1016/0092-8674(92)90151-2. [PubMed] [Cross Ref]

28. Ganetzky B. Genetic studies of membrane excitability in Drosophila: lethal interaction between two temperature-sensitive paralytic mutations. Genetics. 1984;108:897–911. [PubMed]

29. Stern M, Ganetzky B. Altered synaptic transmission in Drosophila hyperkinetic mutants. J Neurogenet. 1989;5:215–28. doi: 10.3109/01677068909066209. [PubMed] [Cross Ref]

30. Pletcher SD. Model fitting and hypothesis testing for age-specific mortality data. J Evol Biol. 1999;12:430–39. doi: 10.1046/j.1420-9101.1999.00058.x. [Cross Ref]

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