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Originally published as Genetics Published Articles Ahead of Print on June 3, 2005.

Genetics, Vol. 170, 1677-1689, August 2005, Copyright © 2005
doi:10.1534/genetics.105.043174

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The mei-P26 Gene Encodes a RING Finger B-box Coiled-Coil-NHL Protein That Regulates Seizure Susceptibility in Drosophilia

Edward Glasscock*,1, Ayush Singhania* and Mark A. Tanouye*,{dagger}

* Department of Molecular and Cell Biology, Division of Neurobiology
{dagger} Department of Environmental Science, Policy and Management, Division of Insect Biology, University of California, Berkeley, California 94720

1 Corresponding author: Department of Molecular and Cell Biology, Life Sciences Addition, Room 131, University of California, Berkeley, CA 94720.
E-mail: edwardg{at}uclink.berkeley.edu

Manuscript received March 10, 2005. Accepted for publication April 22, 2005.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Seizure-suppressor mutations provide unique insight into the genes and mechanisms involved in regulating nervous system excitability. Drosophila bang-sensitive (BS) mutants present a useful tool for identifying seizure suppressors since they are a well-characterized epilepsy model. Here we describe the isolation and characterization of a new Drosophila seizure-suppressor mutant that results from disruption of the meiotic gene mei-P26, which belongs to the RBCC-NHL family of proteins. The mei-P26 mutation reduces seizures in easily shocked (eas) and slamdance (sda) epileptic flies following mechanical stimulation and electroconvulsive shock. In addition, mutant mei-P26 flies exhibit seizure thresholds at least threefold greater than those of wild type. The mei-P26 phenotypes appear to result from missense mutation of a critical residue in the NHL protein-protein interaction domain of the protein. These results reveal a surprising role for mei-P26 outside of the germline as a regulator of seizure susceptibility, possibly by affecting synaptic development as a ubiquitin ligase.

HUMAN seizure disorders represent a serious health concern due to the large number of people affected as well as the inadequacy of available treatments. Seizures are defined as abnormal, synchronous, and rhythmic firing of neurons in the central nervous system. At least 10% of the population will experience a seizure during their lifetime and ~3% of people suffer persistent, spontaneous epileptic seizures (SHNEKER and FOUNTAIN 2003). This problem is amplified by the fact that one-third of epileptics cannot adequately control their seizures with medication (SHNEKER and FOUNTAIN 2003). Thus, seizure disorders represent a pervasive class of disease with unsatisfactory treatment options.

Seizures can result from a variety of brain insults including head trauma, fever, illness, and electroconvulsive shock, but a main source of seizure susceptibility appears to be genetic predisposition. More than 70 genes have been linked to epilepsy from work done on inherited disorders in humans, mice, and flies (NOEBELS 2003). These genes encode a wide variety of products ranging from ion channel proteins to tRNAs. The large number of disparate genes involved in epileptogeneis, as well as the frequent lack of obvious functional relationships between mutation and seizure susceptibility, complicates understanding epilepsy on a mechanistic level (JACOBS et al. 2001).

Identification of genetic seizure suppressors is an underutilized resource for understanding seizures. Seizure suppressors are second-site modifier mutations that when combined with seizure-prone mutants are capable of reverting their seizure susceptibility to wild-type levels. Seizure-suppressor mutations are valuable for two main reasons: (1) they provide insight into the mechanisms underlying seizure susceptibility and (2) they provide potential targets for novel therapeutic drugs. By giving insight into seizure mechanisms, seizure suppressors can reveal new relationships and novel roles for genes in the nervous system.

One organism that avails itself to seizure-suppressor screening is Drosophila. The fly has well-characterized genetics, behavior, and electrophysiology and is amenable to high-throughput mutagenesis screens. In addition, a Drosophila model of epilepsy exists in the bang-sensitive (BS) class of behavioral mutants. The BS mutants are so named because they exhibit increased susceptibility to seizures following mechanical stimulation and electroconvulsive shock. The BS mutant class includes among others bangsenseless (bss; gene unknown), easily shocked (eas; ethanolamine kinase gene), and slamdance (sda; aminopeptidase gene). BS mutants are well characterized on a behavioral and electrophysiological level and their seizure activity shows numerous similarities with seizure activity in humans, making them a useful tool for identifying new seizure suppressors (BENZER 1971; KUEBLER and TANOUYE 2000; HEKMAT-SCAFE et al. 2005).

Previous studies in Drosophila have identified seizure-suppressor mutations using two methods: reverse genetics and gain-of-function analysis. Using a reverse genetics approach, KUEBLER et al. (2001) combined previously characterized neural mutations with BS mutants to see if they could act as seizure suppressors. Several seizure suppressors were identified in these double-mutant combinations, including a connexin gap-junction protein (Shaking B), a potassium channel mutant (Shaker), and two mutants affecting sodium channel expression (paralytic and maleless no-action-potential temperature-sensitive). Using a gain-of-function approach, HEKMAT-SCAFE et al. (2005) utilized enhancer P elements (EP) to identify genes that when overexpressed in a BS genetic background could reduce seizure susceptibility. Overexpression analysis identified several weak suppressor genes and one strong suppressor gene, which encodes a transcription factor (escargot). Although these screening methods were useful for providing some of the first evidence that modifier mutations could ameliorate seizures in an animal model of epilepsy, they possessed some inherent limitations that prevented isolation of certain types of genes and mutations. The reverse genetics approach could identify only suppressors that corresponded to previously characterized neural mutants, while the gain-of-function approach could identify only autosomal suppressors that possessed EP insertions upstream of the gene.

This work describes the first extensive forward genetics screen to identify loss-of-function X chromosome seizure suppressors in Drosophila, which resulted in the surprising isolation of a new allele of mei-P26, a gene not previously associated with a role in the nervous system. In the screen, seizure-suppressor mutants were isolated by mutagenizing eas flies and looking for mutations that reduce seizure susceptibility. This work focuses on the suppressor that showed the strongest phenotype su(eas)16, which is a new allele of the gene mei-P26. Before this work, the mei-P26 gene was primarily associated with the germline. The mei-P26 gene was first identified in a screen for novel meiotic genes (SEKELSKY et al. 1999). Mutants of mei-P26 exhibit defects in meiotic recombination, chromosome segregation, and germline differentiation (SEKELSKY et al. 1999; PAGE et al. 2000). In addition, mei-P26 mutants frequently exhibit female sterility as a result of tumorous ovaries (PAGE et al. 2000). The mei-P26 gene encodes a member of the RING finger B-box coiled-coil (RBCC) family of proteins that is rare because it also contains an NHL (NCL-1, HT2A, and LIN-41) protein-protein interaction domain (PAGE et al. 2000). Proteins with RBCC domains have numerous well-characterized roles in processes as diverse as ubiquitination, oncogenesis, and gene regulation (XU et al. 2003; LALONDE et al. 2004; WANG et al. 2004). However, functions for proteins with both RBCC and NHL domains are less clear, but it is thought that they may participate in multiprotein complexes.

Here we report a surprising new role for mei-P26 in the nervous system as a seizure suppressor. The mei-P26 mutation su(eas)16 isolated in this study acts as a general seizure suppressor, drastically reducing the seizure susceptibility of eas and sda "epileptic" flies. The ability of the mei-P26 mutation to suppress seizures seems to result from its extremely high seizure threshold outside of a BS background. The su(eas)16 mutation also causes chromosome segregation defects similar to those seen in other mei-P26 mutants. Mapping, complementation, and sequencing analyses verify that su(eas)16 is a new allele of the mei-P26 gene resulting from two missense mutations, one of which disrupts an important conserved residue in the NHL protein-protein interaction domain. The ability of a mei-P26 mutation to suppress seizures, as well as its high seizure threshold, points to a novel role for the mei-P26 gene in regulating nervous system excitability.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Fly stocks:
The mei-P26 gene maps to 8D2 and encodes an RBCC-NHL protein involved in meiotic exchange and germline differentiation (PAGE et al. 2000). The su(eas)16 allele was isolated in this study and is caused by two missense mutations (see text). The mei-P26fs1 allele is caused by tandem insertion of two P elements in the first intron of mei-P26 (PAGE et al. 2000). The fs1 allele is associated with female sterility, reduced meiotic recombination, nondisjunction, and ovarian tumors (PAGE et al. 2000). The P{w+mC; N1} stock is a third chromosome insertion of a wild-type mei-P26 rescue construct described in PAGE et al. (2000) that rescues mei-P26 mutant phenotypes. The eas gene maps to 14B7 and encodes an ethanolamine kinase (PAVLIDIS et al. 1994). The eas allele utilized was easPC80, caused by a 2-bp deletion that introduces a frameshift and causes the protein to be truncated and lack the kinase domain (PAVLIDIS et al. 1994). The sda gene maps to 97D and encodes an aminopeptidase N (ZHANG et al. 2002). The sda allele utilized was sdaiso7.8, caused by a 2-bp insertion in the 5'-untranslated region. Both the eas and sda mutations cause seizure sensitivity and bang-sensitive paralysis (PAVLIDIS et al. 1994; ZHANG et al. 2002). Other deficiency, duplication, and P-element stocks utilized in this work for mapping purposes are described in the text and were obtained from the Bloomington Drosophila Stock Center at Indiana University unless otherwise stated.

EMS mutagenesis:
Ethyl methanesulfonate (EMS) mutagenesis was performed as described in GRIGLIATTI (1998). Bang-sensitive w easPC80 f male flies were starved overnight and then fed 25 mM EMS in a 1% sucrose solution for 4–8 hr. Flies were then allowed to rest and recover overnight before being mated to C(1)DX, y w f females. Flies were allowed to mate for 6 days and then the mutagenized w easPC80 f males were discarded to ensure that only postmeiotic chromosomes were tested. F1 male progeny were tested for bang sensitivity (see Behavioral testing). Exceptional bang-resistant F1 males are potential seizure suppressors and were individually mated again to C(1)DX, y w f females. The resulting F2 males were again tested for bang sensitivity. F2 males that still scored as bang resistant were mated to C(1)DX, y w f females to establish stocks.

Behavioral testing:
BS paralysis was assayed by vortexing flies in a food vial (Applied Scientific) on a VWR vortex at maximum setting for 10 sec. Any fly that lay motionless following the vortex was scored BS. Recovery times were calculated by measuring the time from the beginning of the vortex until the flies resumed an upright, standing position. Flies were always rested overnight following CO2 anesthesia prior to behavioral testing.

Electrophysiology:
Electrophysiology was performed on male flies using methods previously described to stimulate and record giant fiber (GF)-driven muscle potentials and seizures (KUEBLER and TANOUYE 2000). Briefly, the fly was removed from a food vial by sucking onto its head with a 23-gauge syringe needle attached to a vacuum line. Another syringe needle was then used to suck onto the abdomen, thereby completely immobilizing the fly. The fly was then mounted by gluing a tungsten wire across its neck. In experiments, the GF was driven by brain stimulation via bipolar tungsten electrodes inserted through the ventral antennal margin and into the brain. The preparations were grounded by placing an electrode into the abdomen. Stimulating, recording, and ground electrodes were all made from uninsulated tungsten wire (WPI 0.075 mm) electrolytically sharpened to the desired diameter, usually <1 µm.

Seizures were elicited by delivering an electroconvulsive shock (ECS) to the brain of the fly. The ECS was composed of a short wavetrain (0.5-msec pulses at 200 Hz for 300 msec) of high-frequency electrical stimuli (HFS) with sufficient intensity (i.e., above threshold) to induce seizure. In this work, seizures were monitored by dorsal longitudinal muscle (DLM) activity. For some seizure-resistant genotypes, an HFS of 300 msec was not effective at eliciting seizures so 400-msec wavetrains were used where noted. The GF firing threshold was determined before measuring the seizure threshold by administering single pulses of 0.2 msec. The GF threshold was defined as the lowest voltage that elicits a stable, short latency (~1.4 msec) DLM response.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
X . chromsome EMS screen for eas suppressors:
We performed a screen for X chromosome mutations that suppress seizures in the BS mutant eas. Flies with the eas mutation exhibit increased seizure susceptibility due to disruption of the ethanolamine kinase gene (PAVLIDIS et al. 1994). In the screen, bang-sensitive eas males were fed 25 mM EMS, a chemical mutagen that typically produces point mutations in DNA by inducing GC-to-AT substitutions. These mutagenized eas flies were mated to C(1)DX females and the F1 male progeny screened for exceptional bang-resistant flies (see MATERIALS AND METHODS). Normally, eas flies show 100% BS paralysis in behavioral tests so bang-resistant F1 flies represent potential seizure suppressors.

A screen of 6700 mutagenized X chromosomes identified 28 potential suppressor mutants in the F1 generation. Of these putative suppressors, most were eliminated as real suppressors or lost because they were sterile, lethal, or false positives. However, three new suppressor mutants, named su(eas)7, su(eas)13, and su(eas)16, that consistently reduced the seizure susceptibility of eas flies were isolated. Each of the mutations behaves as a viable, recessive seizure suppressor, reducing the bang sensitivity of eas flies by 26–99% and elevating the eas seizure threshold by three to four times, depending on genotype. Recombination and deficiency mapping assign the suppressors to regions of the X chromosome not previously associated with seizure suppression. The su(eas)7 mutation maps just proximal to the gene white (w) near the cytological location 3A/B. The su(eas)13 mutation maps about eight map units distal to the gene forked (f). The su(eas)16 mutation maps to the gene mei-P26 at the cytological location 8D2. Here we focus on su(eas)16, the strongest of the three suppressor mutations, which is shown in this work to be a new allele of mei-P26, a gene not previously associated with a nervous system function.

su(eas)16 reduces the bang sensitivity of eas flies in an age-dependent manner:
The su(eas)16 mutation reduces the bang sensitivity of eas flies the most of the three suppressors isolated in the screen (Figure 1A). Normally, 100% of eas flies show BS paralysis when administered a mechanical stimulus in the form of a brief 10-sec vortex. However, when the su(eas)16 mutation is combined with eas to form the genotype su(eas)16 eas/Y, only ~32% of flies paralyze when tested at 1–2 days old (Figure 1A). This percentage is reduced to 0% in eas flies that are heterozygous for su(eas)16 and a deficiency that fails to complement the mutation (su(eas)16/Df(1)18.1.15 eas/eas). Since a deficiency strengthens the su(eas)16 suppression phenotype, su(eas)16 is likely only a partial loss-of-function mutant. When eas flies are heterozygous for the su(eas)16 mutation (su(eas)16/+ eas/eas), they show 100% bang sensitivity, indicating that the suppressor is recessive (Figure 1B). Interestingly, the su(eas)16 suppression phenotype strengthens with age. By 2–3 days old, only 17% of su(eas)16 eas/Y flies paralyze and at 4–5 days old, su(eas)16 shows almost complete suppression of eas with only 1% of su(eas)16 eas/Y flies paralyzing (Figure 1C). The other two suppressor mutations show weaker suppression of eas than su(eas)16, reducing the number of eas flies that paralyze at 1–2 days old to ~74 and 51% for su(eas)7 and su(eas)13, respectively (Figure 1A). Neither of the other two suppressors shows a strong age-dependent suppression phenotype as observed in su(eas)16 flies (data not shown).



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  		FIGURE 1.— Bang-sensitive paralysis in eas suppressor strains. (A) Suppression of eas at 1–2 days by the three suppressors isolated in the screen. Each of the eas suppressors reduces the BS behavior of eas. The su(eas)16 mutation is the most effective at suppressing eas. (B) Characterization of the su(eas)16 mutation's ability to suppress eas at 1–2 days. The su(eas)16 mutation acts as a recessive eas suppressor with su(eas)16/+ eas/eas heterozygotes exhibiting 100% BS behavior (n = 42). In addition, the mei-P26EG16 mutation acts as a partial loss-of-function mutation as flies heterozygous for su(eas)16 and a deletion that removes mei-P26 suppresses eas completely (n = 27), unlike su(eas)16 homozygotes. (C) Age-dependent suppression of eas () by su(eas)16 ({blacksquare}). Flies with su(eas)16 exhibit an age-dependent eas suppression phenotype. As su(eas)16 eas double mutants age, their BS behavior decreases until only ~1% of su(eas)16 eas/Y flies show BS behavior at an age of 4–5 days (n ≥ 100 for each age tested).

 
The su(eas)16 mutation also reduces the recovery time from paralysis, a phenomenon previously seen in BS mutants treated with human anticonvulsant drugs (TAN et al. 2004). As shown in Table 1, mutants of eas normally require 44.6 ± 5.6 sec to recover from paralysis when young (1–2 days). As eas flies age, their paralysis recovery time lengthens to 46.2 ± 8.6 and 61.4 ± 16.3 sec at 2–3 days and 4–5 days of age, respectively. When 1–2 days old, the su(eas)16 mutation shortens the time needed for recovery in eas flies by about 12% to 39.4 ± 5.0 sec. By 2–3 days old, su(eas)16 shortens the recovery time in eas flies by ~19% to 37.4 ± 6.8 sec. Recovery times for 4–5 day-old su(eas)16 eas double mutants could not be determined because of the infrequency of BS paralysis in these flies. The su(eas)7 and su(eas)13 mutations also reduced recovery time in eas flies, as shown in Table 1. These reductions in paralysis time are reminiscent of the effects that human anticonvulsant drugs have on BS mutants. TAN et al. (2004) reported that feeding bang-sensitive bss flies the drug potassium bromide shortens their paralysis time by ~50%. Thus, in addition to reducing the occurrence of BS paralysis, the su(eas)16 mutation can also abbreviate the paralysis time of BS mutants.


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  		TABLE 1 Paralysis recovery times by age for eas suppressors

 
su(eas)16 increases the seizure threshold of eas flies:
All BS mutants, including eas, have been shown to have low seizure thresholds in electrophysiology experiments (KUEBLER and TANOUYE 2000; KUEBLER et al. 2001). Since the suppressors reduce bang sensitivity, we examined the seizure thresholds of the suppressors in an eas background to see if this reduction correlated with an increase in seizure threshold. The seizure threshold here is defined as the minimum voltage required to induce seizure activity in the dorsal longitudinal muscles (DLMs) by administration of an ECS to the brain of the fly. A seizure, which appears as intense, high-frequency muscle activity, is followed by a brief period of synaptic failure in the GF pathway during which time brain stimulation fails to elicit DLM muscle potentials (PAVLIDIS and TANOUYE 1995). Previous work has shown that seizures are not limited to the DLMs, but rather involve at least seven different muscle groups and >30 thoracic muscle fibers (KUEBLER and TANOUYE 2000). The seizure activity in a particular muscle corresponds to activity in the motoneuron that innervates it (KOENIG and IKEDA 1983; PAVLIDIS and TANOUYE 1995).

We measured the seizure thresholds for su(eas)16 mutants in an eas background and found that they raise the seizure threshold of eas flies by nearly threefold. Normally, eas flies exhibit low seizure thresholds of 3.0 ± 1.0 V. However, su(eas)16 raises this threshold to 8.3 ± 2.5 V (Table 2). The other suppressors showed similar increases in eas seizure threshold raising it to 9.4 ± 3.3 and 11.6 ± 3.3 V for su(eas)7 and su(eas)13, respectively (Table 2). For each of the suppressors, the increase in seizure threshold is not accompanied by an increase in GF firing threshold (Table 3). Interestingly, the other two suppressors raised the eas seizure threshold slightly more than su(eas)16 did despite being considerably less effective than su(eas)16 at reducing the bang sensitivity of eas. This observation may reflect differences in the mechanisms underlying seizures induced by mechanical and electrical means. However, the standard deviations in these experiments make it difficult to assess whether or not the small differences in seizure threshold between the suppressors are meaningful.


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  		TABLE 2 Seizure thresholds for various genotypes

 

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  		TABLE 3 GF thresholds for various genotypes

 
su(eas)16 mutants completely suppress seizures in sda flies:
The su(eas)16 mutation acts like a general seizure suppressor, completely restoring the seizure susceptibility of sda mutants to wild-type levels. Flies with the sda mutation exhibit bang sensitivity and low seizure thresholds due to disruption of the aminopeptidase N gene (ZHANG et al. 2002). In general, bss is regarded as the most difficult BS mutant to suppress, followed by eas, and then sda. This correlates with the seizure thresholds of the BS mutants. Mutants of bss have slightly lower seizure thresholds than eas flies, while sda flies have thresholds around 7 V, higher than those of both eas and bss (KUEBLER et al. 2001). Although we could not find any significant evidence that su(eas)16 could suppress bss flies (data not shown), we did find that su(eas)16 could completely suppress seizures in sda flies. In behavioral tests, the su(eas)16 mutation completely suppresses the sda BS phenotype. Normally, 100% of sda flies paralyze following a 10-sec vortex. However, only 1% of sda flies paralyze when combined with su(eas)16 to form the genotype su(eas)16 /Y; sda (Figure 2A). Heterozygous su(eas)16/ + flies show no suppression of sda, again indicating that the su(eas)16 suppression phenotype is recessive (Figure 2A).



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  		FIGURE 2.— The su(eas)16 mutation completely suppresses seizures in sda flies. (A) Behavioral suppression of seizures in sda flies by su(eas)16. The su(eas)16 mutation reduces BS paralysis in sda flies to 1% (n = 144). Suppression of the sda BS behavior by su(eas)16 is recessive as su(eas)16 heterozygotes are 100% BS (n = 64). (B) Electrophysiological suppression of seizures in sda flies by su(eas)16. The su(eas)16 mutation restores the sda seizure threshold to a wild-type level of 32.8 ± 5.5 V (n = 9).

 
In electrophysiology experiments, su(eas)16 restores the sda seizure threshold to a wild-type level. Normally, sda flies exhibit seizure thresholds of 6.8 ± 1.2 V. However, in combination with su(eas)16, the sda seizure threshold is restored to 32.8 ± 5.5 V, the same level as wild-type flies (Figure 2B; Figure 3). These data show that su(eas)16 acts as a general suppressor of seizure and does not interact solely with the eas mutation.



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  		FIGURE 3.— Suppression of seizures in sda flies by su(eas)16. (A) A representative seizure in a sda fly as recorded from the DLM following a high-frequency brain stimulus of 7 V (depicted as a cross-hatched box). The DLM shows aberrant high-frequency firing approaching 100 Hz during the seizure phase. These high-frequency responses then give way to synaptic failure (not shown). (B) Failure to elicit seizure in a su(eas)16; sda double-mutant fly following a 16-V high-frequency brain stimulus. When sda mutants are placed in double-mutant combination with su(eas)16, seizures are completely suppressed at voltage levels below the su(eas)16; sda seizure threshold of 33 V. Here, seizure activity is absent and only a few DLM responses can be seen following administration of a 16-V high-frequency brain stimulus. (C) A seizure is elicited in a su(eas)16; sda double mutant following a 32-V high-frequency brain stimulus. The seizure appears qualitatively similar to seizures in sda single mutants as shown in A. However, su(eas)16 restores the sda seizure threshold to wild-type levels.

 
su(eas)16 mutants have high seizure thresholds:
When analyzed in electrophysiology experiments outside of a BS background, su(eas)16 mutants exhibit extremely high seizure thresholds that exceed those exhibited by Na+ channel and gap junction mutants. Several previously characterized seizure suppressors have elevated seizure thresholds that are significantly higher than those of wild type. For example, the seizure-suppressor mutants shaking-B2 (shak-B2; connexin gap junction) and paralytic (para; voltage-gated Na+ channel {alpha}-subunit) exhibit seizure thresholds of 94.7 ± 10.2 and 65.0 ± 7.2 V, respectively, which are well above the wild-type threshold of ~30 V (KUEBLER et al. 2001). The su(eas)16 mutation causes similar increases, raising the seizure threshold to 91.4 ± 5.1 V. Unlike most genotypes, su(eas)16 flies do not exhibit seizure activity following 300-msec duration ECS, which is the typical stimulus duration we use in electrophysiology experiments. Instead, su(eas)16 flies require 400-msec duration ECS to show seizure activity. Only two other single-mutant genotypes have been found to require 400-msec ECS wavetrains to elicit seizures: maleless no-action-potential temperature-sensitive (mlenapts; RNA helicase-like protein affecting Na+ channel expression) and ShakerKS133 (ShKS133; voltage-gated K+ channel {alpha}-subunit). However, 400-msec ECS produced seizures in mlenapts (72.2 ± 7.3 V) and ShKS133 (83.8 ± 12.8 V) at voltage levels lower than the 91 V required in su(eas)16 flies (KUEBLER et al. 2001). The high seizure threshold of su(eas)16 flies indicates a critical role for mei-P26 in the nervous system as an important regulator of seizure susceptibility.

su(eas)16 phenotypes result from disruption of the mei-P26 gene:
Mapping, rescue, and complementation experiments show that the su(eas)16 phenotypes stem from disruption of the meiotic gene mei-P26 (Figure 4). We initially rough-mapped su(eas)16 by analyzing recombinants relative to visible marker mutations in an eas background. These recombination experiments placed su(eas)16 in a genetic interval between the wing mutations cut (ct; 7B1-2) and miniature (m; 10E1-2) on the X chromosome. To refine this interval we performed recombination experiments with su(eas)16 relative to P elements with molecularly mapped insertion sites in an eas background. Analysis of recombinants relative to P elements narrowed the possible location of su(eas)16 to a region between the insertions P[EPgy2]EY00577 and P[EPgy2]EY00880 at 8C16 and 8D12, respectively, on the cytological map (Figure 4A). Since the 8C16; 8D12 interval still contains numerous candidate genes, we sought to minimize the su(eas)16 gene region further by testing complementation with deficiency and duplication chromosomes in an eas background (Figure 4B). Using this approach, we reduced the distal boundary of su(eas)16 to 8D8-9. Deletion of 8C10; 8E1-2 in Df(1)18.1.15 fails to complement su(eas)16, while duplication of 8C; 8D8-9 in Dp(1;4)A17 does complement the su(eas)16 mutation. The combination of recombination mapping and complementation analysis confined the su(eas)16 mutation to a small 28-kb region on the X chromosome between 8C16 and 8D8-9 that contains only 10 candidate genes (Figure 4C). To identify which of these 10 genes is responsible for the su(eas)16 phenotypes, we sequenced several of these genes and analyzed complementation with available alleles. The only gene that corresponded to the su(eas)16 phenotypes was mei-P26. When we crossed su(eas)16 mutants to flies carrying a mei-P26+ rescue construct we found that it successfully rescues all of the su(eas)16 phenotypes (Table 4). The mei-P26+ transgene reverts su(eas)16 suppression of eas and sda on a behavioral and electrophysiological level, as well as restores the high seizure threshold of su(eas)16 flies to near wild type. The rescue of su(eas)16 phenotypes by the mei-P26+ transgene identifies mei-P26 as the mutated gene responsible for the observed phenotypes in su(eas)16 flies.



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  		FIGURE 4.— Mapping experiments show that su(eas)16 is an allele of mei-P26. (A) Cytological map of the su(eas)16 region showing important genetic markers used for mapping the su(eas)16 mutation. The su(eas)16 mutation was initially mapped using recombination to the interval between the visible markers ct (7B1-2) and m (10E1-2). This interval was then reduced to 8C16; 8E1-2 by recombination mapping relative to the molecularly mapped P elements EY00577 and EY00880. (B) Deficiency and duplication mapping of su(eas)16. Complementation testing using various deficiency and duplication chromosomes narrows the su(eas)16 mutation to a small region between 8C16; 8D8. Solid boxes indicate chromosomal regions that are present. Open boxes indicate deleted regions, while shaded boxes indicate ambiguities about the actual chromosomal breakpoints. (C) Genes located in the interval defined by complementation analysis. The su(eas)16 region indicated by complementation limits the identity of su(eas)16 to 10 possible genes. A mei-P26+ wild-type rescue construct restores su(eas)16 phenotypes to wild type revealing that su(eas)16 is a new allele of the mei-P26 gene. (D) Schematic of the MEI-P26 protein showing locations of the conserved domains as well as the two molecular lesions present in su(eas)16 flies. MEI-P26 belongs to the RBCC-NHL family of proteins. Beginning from the N-terminal end, MEI-P26 contains a RING finger, two B-boxes (type 1 and type 2), and a coiled-coil region composed of three individual sequences predicted to form coils. The C-terminal end of MEI-P26 contains an NHL ß-propeller domain that mediates protein-protein interactions.

 

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  		TABLE 4 Rescue of su(eas)16 phenotypes with P{w+mC; N1} rescue construct

 
We next sequenced the mei-P26 gene in su(eas)16 flies to determine the molecular lesion underlying the su(eas)16 phenotypes. Typically EMS induces GC -> AT base pair substitutions in DNA. Sequencing of the mei-P26 gene in su(eas)16 flies reveals two different missense mutations caused by two different single-nucleotide substitutions typical of the type produced by EMS (Figure 4D). The first mutation is a C -> T switch that results in a serine-to-leucine substitution (S627L) at a nonconserved amino acid in the coiled-coil domain of the protein. The second mutation is a C -> T switch that causes a proline-to-leucine substitution (P1071L) at an important conserved residue of the NHL protein-protein interaction domain. The P1071L mutation probably has an especially detrimental effect on the MEI-P26 protein since the proline 1071 residue is highly conserved and thought to be important for the ability of the NHL domain to adopt the proper three-dimensional ß-propeller conformation (SLACK and RUVKUN 1998; EDWARDS et al. 2003). To ensure that the identified molecular mutations were a result of EMS mutagenesis and not already present in the genetic background, we sequenced the corresponding regions in su(eas)7 flies as a control since they should share similar genetic backgrounds. We found neither of the two point mutations present in su(eas)7 flies, indicating that they are unique to su(eas)16. Thus, molecular analysis verifies that su(eas)16 is a mutant allele of mei-P26.

For further evidence that su(eas)16 phenotypes result from disruption of mei-P26, we examined whether a previously characterized hypomorphic allele of mei-P26, called fs1, could complement the su(eas)16 phenotypes. Prior to this work, the mei-P26 gene has been extensively characterized for its role in the germline (SEKELSKY et al. 1999; PAGE et al. 2000). Mutants of mei-P26 were originally isolated in a P-element mutagenesis screen for mutations that affect segregation of chromosomes during meiosis (SEKELSKY et al. 1999). Previously characterized mutants of mei-P26, such as fs1, exhibit numerous germline defects, including reduced meiotic recombination, nondisjunction, female sterility, and tumorous ovaries (PAGE et al. 2000). The fs1 allele is a tandem insertion of two P elements in the first intron of mei-P26 that results in mei-P26 meiotic phenotypes (PAGE et al. 2000). We analyzed the ability of fs1 to complement su(eas)16 and found that the fs1 allele fails to complement all of the su(eas)16 seizure suppression phenotypes (Table 5). In addition, we found that the fs1 allele by itself drastically reduces bang sensitivity in the BS mutants eas and sda to 2% (n = 129) and 0% (n = 59), respectively. Notably, in the course of this analysis, we observed that su(eas)16 flies exhibit X chromosome nondisjunction defects similar to those previously described for other alleles of mei-P26 (data not shown). The fs1 allele also fails to complement this nondisjunction phenotype in su(eas)16 flies (Table 5). Thus, the interaction of fs1 with su(eas)16, as well as the seizure-suppression phenotypes exhibited by fs1 on its own, confirm that the su(eas)16 phenotypes result from disruption of mei-P26.


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  		TABLE 5 Failure to complement su(eas)16 phenotypes by mei-P26fs1

 
MEI-P26 protein shows similarity to other neural proteins:
The MEI-P26 protein sequence exhibits a unique RBCC-NHL domain configuration that provides the potential for extensive interaction with other proteins. The N-terminal portion of MEI-P26 contains an RBCC domain, or tripartite motif (TRIM), composed of a RING (Really Interesting New Gene) finger followed by two B-box motifs (type 1 and type 2) and a coiled-coil region (Figure 4D). RBCC domains are thought to mediate protein-protein interactions and to contribute to the specification of cellular compartments (BORDEN and FREEMONT 1996; REYMOND et al. 2001). The coiled-coil motif of the RBCC domain enables the protein to homo-oligomerize (REYMOND et al. 2001). In addition to an RBCC domain, the C-terminal end of MEI-P26 contains six NHL repeats, which form a six-blade ß-propeller structure (SLACK and RUVKUN 1998). Similar to RBCC domains, NHL domains also mediate protein-protein interactions (EL-HUSSEINI and VINCENT 1999; OHKAWA et al. 2001; EDWARDS et al. 2003). Thus, the presence of both RBCC and NHL domains in MEI-P26 protein provides ample opportunity for interaction with other proteins.

We performed a database search (SIB BLAST Network Service) for proteins similar to MEI-P26 and identified only three homologous proteins with an RBCC-NHL domain configuration, as well as two additional homologous proteins that lack a RING finger, but retain the rest of the RBCC-NHL structure (Figure 5A). The protein most similar to MEI-P26 is the Caenorhabditis elegans protein NHL-2 that putatively encodes an E3 ubiquitin ligase. NHL-2 is the only RBCC-NHL protein identified in our database homology search that contains two B-box motifs like MEI-P26. The other two MEI-P26 homologs are mammalian proteins that function in the nervous system and possess only a single B-box. They include Tripartite motif protein 3 (TRIM3) in Homo sapiens, Mus musculus, and Rattus norvegicus and Tripartite motif protein 2 (TRIM2) in H. sapiens and M. musculus. Our homology search also identified two proteins that resemble MEI-P26, but lack a RING finger domain. These "RING-less" proteins include NCL-1 in C. elegans and Brain Tumor (BRAT) in D. melanogaster. Although they lack a RING finger, both NCL-1 and BRAT still retain two B-box motifs, a coiled-coil region, and an NHL domain similar to MEI-P26. Both NHL-2 and BRAT function to control cell growth as tumor suppressors. The similarity of these proteins to MEI-P26 suggests that they may share similar functions and act in similar pathways.



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  		FIGURE 5.— Comparison of MEI-P26 structure and sequence to homologous proteins. (A) Comparison of MEI-P26 to its homologs. The general structure of MEI-P26 homologs is shown along with their percentage amino acid identity/similarity to the MEI-P26 RING/B-box and NHL domains. Dm, Drosophila melanogaster; Ce, Caernorhabditis elegans; Rn, Rattus norvegicus; Mm, Mus musculus; Hs, Homo sapiens. (B) Alignment of the MEI-P26 NHL ß-propeller blade V with MEI-P26 homologs showing conservation of P1071. One of the missense mutations in su(eas)16 flies is a P1071L substitution (the affected P is indicated by an *) at a critical conserved residue in blade V of the ß-propeller protein-protein interaction domain. Only one of the MEI-P26 homologs lacks a proline at this position. Residues matching the MEI-P26 sequence identically are solid, while residues similar, but not identical to MEI-P26 are shaded.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
su(eas)16 phenotypes likely stem from disruption of the NHL ß-propeller:
The su(eas)16 seizure-suppression phenotypes likely result from impaired protein-protein interactions due to missense mutation of a critical residue in the NHL domain. Although two missense mutations were identified in the mei-P26 gene of su(eas)16 flies, one appears to be more deleterious. The first mutation is a serine-to-leucine substitution at residue 627. This mutation is probably relatively innocuous since the serine 627 residue is located between two regions predicted to form coils and is not conserved among the proteins that show similarity to MEI-P26. The second mutation is a proline-to-leucine substitution at residue 1071. This mutation is likely the most harmful of the two since the proline 1071 residue occurs at a critical portion of blade V of the NHL ß-propeller domain and is highly conserved among MEI-P26 homologous proteins, as well as all proteins with NHL domains (Figure 5B; SLACK and RUVKUN 1998). The crystal structure of the BRAT NHL domain, which shows the most similarity of any homologous proteins to the MEI-P26 NHL domain, also emphasizes the importance of this proline residue. The BRAT crystal structure reveals that this proline is critical for the ability of the NHL domain to adopt the tight turns required for formation of the ß-propeller structure (EDWARDS et al. 2003). In addition, the proline residue lies on the top surface of the ß-propeller that interacts with other binding partners to form protein complexes (EDWARDS et al. 2003). Analysis of brat mutants reveals that mutations disrupting the ß-propeller induce the brain overgrowth characteristic of brat mutants (ARAMA et al. 2000). Thus, the missense mutation in the MEI-P26 NHL domain is likely responsible for the observed mutant phenotypes since it is predicted to interfere with formation of the ß-propeller and to hinder the interaction of MEI-P26 with other proteins.

mei-P26 has a role in the nervous system as a modifier of seizure susceptibility:
The mei-P26 gene appears to play an important role in the nervous system as a key regulator of seizure susceptibility. The su(eas)16 mutation shows the ability to suppress seizures in multiple BS strains, as well as an extreme resistance to seizures following ECS. Prior to this work, mei-P26 was primarily known to function in germline differentiation and meiotic recombination (PAGE et al. 2000). However, other research has indicated that mei-P26 may be important in the nervous system. Recently, IVANOV et al. (2004) reported that RNA interference of mei-P26 causes breaks and disorganization of the ventral nerve cord in embryos, as well as disorganization of the peripheral nervous system. These data suggest that mei-P26 may be important for proper nervous system development. In addition, another group has found that mei-P26 is expressed in larval brains and imaginal discs of the eye-antennae and wings (L. KADYROVA and R. WHARTON, personal communication). When taken together, these observations indicate a critical role for mei-P26 in the development and proper functioning of the nervous system.

Functional analysis of mei-P26 mammalian homologs shows that RBCC-NHL proteins play critical roles in the nervous system. The two homologs that have been studied most extensively are mouse TRIM2 and rat TRIM3. OHKAWA et al. (2001) showed that mouse TRIM2 (NARF) is expressed in the hippocampus and upregulated following seizure-related neural activity. Rat TRIM3 (BERP) shows expression in neurites and growth cones and disruption of its NHL domain causes neurites to be unresponsive to nerve growth factor (EL-HUSSEINI and VINCENT 1999). Both NARF and BERP have been shown to bind myosin V molecules through their NHL domains (EL-HUSSEINI and VINCENT 1999; OHKAWA et al. 2001). Myosin V is an unconventional myosin motor protein involved in neurite extension and growth cone development (WANG et al. 1996). Myosin V transports cargo in nerve terminals by binding synaptic vesicles through interactions with synaptobrevin and synaptophysin (PREKERIS and TERRIAN 1997). Although no functional studies have been conducted, expression studies show that human TRIM3 is expressed at high levels in adult heart and brain (REYMOND et al. 2001). Thus, mammalian homologs suggest an important role for mei-P26 in the nervous system.

The mammalian gene EPM2B presents a particularly interesting link between mei-P26 and the regulation of seizure susceptibility in the nervous system. Although it appears to be only distantly related to mei-P26, the EPM2B gene encodes a putative E3 ubiquitin ligase protein called Malin that contains a RING finger and an NHL domain like MEI-P26 (CHAN et al. 2003). Mutations in mei-P26 and EPM2B both alter seizure susceptibility, albeit in opposite directions. Whereas mei-P26 mutation decreases seizure susceptibility, disruption of the EPM2B gene in humans and dogs increases seizure susceptibility leading to progressive myoclonus epilepsy (CHAN et al. 2003; LOHI et al. 2005). The effects of disrupting mei-P26 and EPM2B establish a direct link between seizure disorders and proteins that contain both a RING finger and an NHL domain. In addition, the connection between mei-P26 and EPM2B emphasizes the utility of using fly seizure-suppressor screens to identify human counterparts involved with regulating seizure susceptibility.

MEI-P26 shows sequence similarity to E3 ubiquitin ligases:
The presence of an RBCC domain in MEI-P26 protein suggests a possible role for MEI-P26 in the ubiquitin pathway. Proteins with a tripartite RBCC domain are involved in a variety of diverse cellular processes including ubiquitination, transcription regulation, microtubule stabilization, tumorigenesis, and apoptosis (XU et al. 2003; DHO and KWON 2003; BERTI et al. 2004; LALONDE et al. 2004; WANG et al. 2004). An interesting but unproven role for MEI-P26 is in mediating ubiquitination as an E3 ubiquitin ligase. RING fingers appear to be a hallmark of E3 ubiquitin ligases, which mediate the transfer of ubiquitin from E2 ubiquitin-conjugating enzymes onto a target protein to mark it for degradation (JOAZEIRO and WEISSMAN 2000). Several RBCC proteins have been shown to have ubiquitin ligase activity including TRIM5{delta}, Efp, and MID1 (TROCKENBACHER et al. 2001; URANO et al. 2002; XU et al. 2003).

Multiple E3 ubiquitin ligases have important roles in the nervous system. In addition to the previously mentioned mammalian epilepsy gene EPM2B, the human E3 ubiquitin ligase gene UBE3A also has an integral role in the nervous system as a regulator of seizure susceptibility. Disruption of UBE3A causes Angelman syndrome, a complex neurological disorder characterized by mental retardation and seizures (KISHINO et al. 1997). In Drosophila, the E3 ubiquitin ligase Nedd4 interacts with Commisureless to regulate Roundabout, which is essential for proper axonal pathfinding (MYAT et al. 2002). Another E3 ubiquitin ligase in Drosophila called Highwire has been shown to be a negative regulator of synaptic growth at the larval neuromuscular junction and required for proper synaptic function (DIANTONIO et al. 2001). Interestingly, computer sequence analysis predicts that the mei-P26 homolog nhl-2 in C. elegans acts as an E3 ubiquitin ligase. Depletion of nhl-2 mRNA using RNAi causes sterility defects in worms, reminiscent of the defects seen in mei-P26 mutants (MAEDA et al. 2001). Although a role for mei-P26 in ubiquitination is an enticing prospect, future experiments are required to sort out the function of mei-P26 on a biochemical level.

How does mei-P26 regulate seizure susceptibility?
The actual mechanism by which mei-P26 regulates seizure-susceptibility is not clear, but several clues about its possible function in the nervous system exist. The ability of su(eas)16 to suppress seizures appears to result from its extremely high seizure threshold. One explanation for this high seizure threshold could be a decrease in excitability of individual neurons required for seizures in su(eas)16 flies. However, at least in the case of the giant fiber, the excitability of individual neurons in mei-P26 flies does not appear to be altered. Mutant su(eas)16 flies exhibit high seizure thresholds without a concomitant increase in the firing threshold of the giant fiber. Evidence of neuronal hypoexcitability can also manifest itself as impaired synaptic transmission in the GF neural circuit, such as increased response latencies and decreased following frequencies of indirect flight muscles. However, the performance of the two major motoneuronal outputs of the GF system, the dorsal longitudinal muscle and tergotrochanteral muscle, do not show any obvious abnormalities in su(eas)16 flies (data not shown). Thus, individual neuronal excitability does not appear to be decreased in mei-P26 flies but deficits that we have not been able to measure may exist.

Another explanation for the seizure resistance of mei-P26 flies could be that they possess unidentified increases in inhibition or decreases in excitation, such as overgrowth of inhibitory synapses or defective performance of excitatory synapses. MEI-P26 appears to have a role as a tumor suppressor acting to negatively regulate growth. Disruption of mei-P26 expression leads to increased cell growth manifested as ovarian tumors (PAGE et al. 2000). In addition, the mei-P26 homologs ncl-1 in C. elegans and brat in D. melanogaster also act as negative regulators of growth. The ncl-1 gene functions as a repressor of ribosome synthesis and cell growth (FRANK and ROTH 1998). Loss-of-function ncl-1 mutants exhibit enlarged nucleoli and increased cell size, which result in larger worms (HEDGECOCK and HERMAN 1995). The brat gene also acts as a tumor suppressor: disruption of the NHL domain of brat causes brain overgrowth in optical centers, as well as sterility defects (ARAMA et al. 2000). Although NCL-1 and BRAT lack RING fingers, their NHL domains show a high degree of similarity to MEI-P26, indicating that they may interact with similar protein partners. One interesting possibility is that mei-P26 acts to negatively regulate growth of inhibitory interneurons and that disruption of mei-P26 results in overgrowth of these synapses facilitating seizure suppression. Interestingly, mutants of the E3 ubiquitin ligase highwire (hiw) show a synaptic overgrowth phenotype that leads to defects in synaptic transmission. However, in hiw mutants, synapses are normal ultrastructurally but exhibit increased bouton number and synaptic branching that results in weaker synapses with decreased quantal content physiology (WAN et al. 2000). Possibly mei-P26 loss-of-function decreases seizure susceptibility in a manner similar to the action of hiw by causing overgrowth of excitatory synapses, which leads to weaker synaptic transmission in neurons required for seizure. Although the role of mei-P26 in the nervous system is not yet clear, the extreme and unprecedented seizure resistance of mei-P26 flies indicates that mei-P26 is a critical regulator of excitability in the nervous system.

The fly genome contains numerous unidentified seizure suppressors:
This work identified three new seizure-suppressor mutations, but the fly genome likely contains many more that have not yet been identified. Previous work has identified at least three different loci on the X chromosome that are associated with seizure suppression including paralytic, Shaker, and shaking-B (KUEBLER et al. 2001). However, no alleles of these genes were isolated in this screen, indicating that the Drosophila X chromosome is likely not yet saturated for seizure suppressors. In addition, many seizure-suppressor mutations were likely not identified in the screen as a result of lethality or sterility since 28 putative suppressors were initially isolated in the screen, many of which produced no viable progeny. Since the design of the screen permitted isolation of only dominant and recessive X chromosome mutants and dominant autosomal mutants, the autosomes likely carry numerous seizure suppressors. The autosomal genes maleless and escargot have already shown the ability to suppress seizures in BS mutants (KUEBLER et al. 2001; HEKMAT-SCAFE et al. 2005). Therefore, future genetic screens show promise for isolating numerous new seizure-suppressor mutants, which could help elucidate the mechanisms underlying seizure susceptibility.

The mei-P26 gene holds promise as an important tool for understanding seizure mechanisms. The MEI-P26 protein has the potential for multiple protein interactions since it contains both RBCC and NHL protein-protein interaction domains. These protein-binding motifs, especially the NHL domain, which is disrupted in su(eas)16 flies, may be useful for identifying new seizure suppressors and for studying the interactions of proteins that regulate seizure susceptibility. Given the strong seizure-suppression phenotype of mei-P26 mutants, some of the proteins that interact with MEI-P26 to regulate seizure susceptibility may be seizure suppressors themselves. In addition, identification of these protein partners may provide valuable insight into some of the mechanisms underlying the ability of mei-P26 to regulate excitability, such as a possible role as a ubiquitin ligase. Future work to determine the biochemical nature of MEI-P26, as well as identification of its binding partners, will help elucidate the role of mei-P26 in the nervous system.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We thank Scott Page for giving us the mei-P26+ rescue stock and the Dp(1;4)A17 duplication stock. We are also grateful to Dan Evans and Tom Cline for providing us with additional mei-P26 alleles. Thanks go to Daria Hekmat-Scafe, Juan Song, and Jeff Tan for their intellectual contributions and support. This work was supported by a National Institutes of Health (NIH) research grant and an Epilepsy Foundation grant to M.T.


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Communicating editor: T. KAUFMAN




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