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Genome-wide screen for modifiers of Na (+) /K (+) ATPase alleles identifies critical genetic loci.

Talsma AD, Chaves JF, LaMonaca A, Wieczorek ED, Palladino MJ - Mol Brain (2014)

Bottom Line: We successfully identified 64 modifier loci and used classical mutations and RNAi to confirm 50 single gene interactions.The genes identified include those with known function, several with unknown function or that were otherwise uncharacterized, and many loci with no described association with locomotor or Na(+)/K(+) ATPase function.We have identified many critical regions and narrowed several of these to single genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, BST3 7042, Pittsburgh, PA, 15261, USA. adt40@pitt.edu.

ABSTRACT

Background: Mutations affecting the Na (+) / K (+) ATPase (a.k.a. the sodium-potassium pump) genes cause conditional locomotor phenotypes in flies and three distinct complex neurological diseases in humans. More than 50 mutations have been identified affecting the human ATP1A2 and ATP1A3 genes that are known to cause rapid-onset Dystonia Parkinsonism, familial hemiplegic migraine, alternating hemiplegia of childhood, and variants of familial hemiplegic migraine with neurological complications including seizures and various mood disorders. In flies, mutations affecting the ATPalpha gene have dramatic phenotypes including altered longevity, neural dysfunction, neurodegeneration, myodegeneration, and striking locomotor impairment. Locomotor defects can manifest as conditional bang-sensitive (BS) or temperature-sensitive (TS) paralysis: phenotypes well-suited for genetic screening.

Results: We performed a genome-wide deficiency screen using three distinct missense alleles of ATPalpha and conditional locomotor function assays to identify novel modifier loci. A secondary screen confirmed allele-specificity of the interactions and many of the interactions were mapped to single genes and subsequently validated. We successfully identified 64 modifier loci and used classical mutations and RNAi to confirm 50 single gene interactions. The genes identified include those with known function, several with unknown function or that were otherwise uncharacterized, and many loci with no described association with locomotor or Na(+)/K(+) ATPase function.

Conclusions: We used an unbiased genome-wide screen to find regions of the genome containing elements important for genetic modulation of ATPalpha dysfunction. We have identified many critical regions and narrowed several of these to single genes. These data demonstrate there are many loci capable of modifying ATPalpha dysfunction, which may provide the basis for modifying migraine, locomotor and seizure dysfunction in animals.

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Related in: MedlinePlus

Genetic interactions betweenFKBP59andATPalpha.FKBP59; ATPalpha double mutants and ATPalpha*, FKBP59 RNAi flies were assayed and compared to ATPalpha* heterozygous controls. The RNAi knockdown was driven with da-Gal4. The genotypes in each graph are: ATPalpha*/+ (green), FKBP59E03444/+; ATPalpha*/+ (red), daGal4,ATPalpha*/+ (blue), and FKBP59-RNAi/+;daGal4,ATPalpha*/+ (orange). FKBP59 mutants significantly enhanced the ATPalphaDTS1 phenotype. The ATPalphaCJ5 phenotype is suppressed by both the FKBP59 mutant and RNAi. *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig6: Genetic interactions betweenFKBP59andATPalpha.FKBP59; ATPalpha double mutants and ATPalpha*, FKBP59 RNAi flies were assayed and compared to ATPalpha* heterozygous controls. The RNAi knockdown was driven with da-Gal4. The genotypes in each graph are: ATPalpha*/+ (green), FKBP59E03444/+; ATPalpha*/+ (red), daGal4,ATPalpha*/+ (blue), and FKBP59-RNAi/+;daGal4,ATPalpha*/+ (orange). FKBP59 mutants significantly enhanced the ATPalphaDTS1 phenotype. The ATPalphaCJ5 phenotype is suppressed by both the FKBP59 mutant and RNAi. *p < 0.05, **p < 0.01, ***p < 0.001.

Mentions: Another interesting possibility is that loss-of-function ATPalpha mutations are disrupting neuronal development through alterations in NF-κB signaling. It has been shown that sub-inhibitory concentrations of ouabain activate NF-κB via an Na+/K+ ATPase dependent mechanism in rat kidney cells. The effect is mediated by slow, inositol triphosphate-dependent, calcium oscillations likely caused by shifting electrochemical gradients [86]. More recently, agrin, a protein involved in synapse formation at NMJs and in the CNS, has been shown to bind to and inhibit the mammalian Na+/K+ ATPase α3 isoform. Furthermore, agrin seems to bind at the same site as ouabain because a protein fragment can prevent ouabain inhibition of the Na+/K+ ATPase [87]. Thus it is possible that agrin exerts its effects through NF-κB. If a similar pathway exists in flies it would likely be constitutively active in our loss-of-function mutants and its dysregulation could cause developmental changes, which might increase seizure susceptibility. This is consistent with our finding that knockdown of proteins required for NF-κB activation suppresses seizures in our loss-of-function mutants. NF-κB activation may be caused by calcium oscillations [86], making it possible that some of the calcium channels we found also play a role in this pathway. FKBP59 (Figure 6) is particularly interesting because it inhibits an inositol triphosphate sensitive, non-specific calcium channel, TrpL [53]. Inhibition of calcium channels would likely be required in calcium oscillations. The preponderance of hits related to the NF-κB pathway suggests a possible role for this pathway in seizure pathogenesis.Figure 6


Genome-wide screen for modifiers of Na (+) /K (+) ATPase alleles identifies critical genetic loci.

Talsma AD, Chaves JF, LaMonaca A, Wieczorek ED, Palladino MJ - Mol Brain (2014)

Genetic interactions betweenFKBP59andATPalpha.FKBP59; ATPalpha double mutants and ATPalpha*, FKBP59 RNAi flies were assayed and compared to ATPalpha* heterozygous controls. The RNAi knockdown was driven with da-Gal4. The genotypes in each graph are: ATPalpha*/+ (green), FKBP59E03444/+; ATPalpha*/+ (red), daGal4,ATPalpha*/+ (blue), and FKBP59-RNAi/+;daGal4,ATPalpha*/+ (orange). FKBP59 mutants significantly enhanced the ATPalphaDTS1 phenotype. The ATPalphaCJ5 phenotype is suppressed by both the FKBP59 mutant and RNAi. *p < 0.05, **p < 0.01, ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4302446&req=5

Fig6: Genetic interactions betweenFKBP59andATPalpha.FKBP59; ATPalpha double mutants and ATPalpha*, FKBP59 RNAi flies were assayed and compared to ATPalpha* heterozygous controls. The RNAi knockdown was driven with da-Gal4. The genotypes in each graph are: ATPalpha*/+ (green), FKBP59E03444/+; ATPalpha*/+ (red), daGal4,ATPalpha*/+ (blue), and FKBP59-RNAi/+;daGal4,ATPalpha*/+ (orange). FKBP59 mutants significantly enhanced the ATPalphaDTS1 phenotype. The ATPalphaCJ5 phenotype is suppressed by both the FKBP59 mutant and RNAi. *p < 0.05, **p < 0.01, ***p < 0.001.
Mentions: Another interesting possibility is that loss-of-function ATPalpha mutations are disrupting neuronal development through alterations in NF-κB signaling. It has been shown that sub-inhibitory concentrations of ouabain activate NF-κB via an Na+/K+ ATPase dependent mechanism in rat kidney cells. The effect is mediated by slow, inositol triphosphate-dependent, calcium oscillations likely caused by shifting electrochemical gradients [86]. More recently, agrin, a protein involved in synapse formation at NMJs and in the CNS, has been shown to bind to and inhibit the mammalian Na+/K+ ATPase α3 isoform. Furthermore, agrin seems to bind at the same site as ouabain because a protein fragment can prevent ouabain inhibition of the Na+/K+ ATPase [87]. Thus it is possible that agrin exerts its effects through NF-κB. If a similar pathway exists in flies it would likely be constitutively active in our loss-of-function mutants and its dysregulation could cause developmental changes, which might increase seizure susceptibility. This is consistent with our finding that knockdown of proteins required for NF-κB activation suppresses seizures in our loss-of-function mutants. NF-κB activation may be caused by calcium oscillations [86], making it possible that some of the calcium channels we found also play a role in this pathway. FKBP59 (Figure 6) is particularly interesting because it inhibits an inositol triphosphate sensitive, non-specific calcium channel, TrpL [53]. Inhibition of calcium channels would likely be required in calcium oscillations. The preponderance of hits related to the NF-κB pathway suggests a possible role for this pathway in seizure pathogenesis.Figure 6

Bottom Line: We successfully identified 64 modifier loci and used classical mutations and RNAi to confirm 50 single gene interactions.The genes identified include those with known function, several with unknown function or that were otherwise uncharacterized, and many loci with no described association with locomotor or Na(+)/K(+) ATPase function.We have identified many critical regions and narrowed several of these to single genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, BST3 7042, Pittsburgh, PA, 15261, USA. adt40@pitt.edu.

ABSTRACT

Background: Mutations affecting the Na (+) / K (+) ATPase (a.k.a. the sodium-potassium pump) genes cause conditional locomotor phenotypes in flies and three distinct complex neurological diseases in humans. More than 50 mutations have been identified affecting the human ATP1A2 and ATP1A3 genes that are known to cause rapid-onset Dystonia Parkinsonism, familial hemiplegic migraine, alternating hemiplegia of childhood, and variants of familial hemiplegic migraine with neurological complications including seizures and various mood disorders. In flies, mutations affecting the ATPalpha gene have dramatic phenotypes including altered longevity, neural dysfunction, neurodegeneration, myodegeneration, and striking locomotor impairment. Locomotor defects can manifest as conditional bang-sensitive (BS) or temperature-sensitive (TS) paralysis: phenotypes well-suited for genetic screening.

Results: We performed a genome-wide deficiency screen using three distinct missense alleles of ATPalpha and conditional locomotor function assays to identify novel modifier loci. A secondary screen confirmed allele-specificity of the interactions and many of the interactions were mapped to single genes and subsequently validated. We successfully identified 64 modifier loci and used classical mutations and RNAi to confirm 50 single gene interactions. The genes identified include those with known function, several with unknown function or that were otherwise uncharacterized, and many loci with no described association with locomotor or Na(+)/K(+) ATPase function.

Conclusions: We used an unbiased genome-wide screen to find regions of the genome containing elements important for genetic modulation of ATPalpha dysfunction. We have identified many critical regions and narrowed several of these to single genes. These data demonstrate there are many loci capable of modifying ATPalpha dysfunction, which may provide the basis for modifying migraine, locomotor and seizure dysfunction in animals.

Show MeSH
Related in: MedlinePlus