Novel mutations affecting the Na, K ATPase alpha model complex neurological diseases and implicate the sodium pump in increased longevity

Abstract

Mutations affecting the Na(+), K(+) ATPase alpha subunit have been implicated in at least two distinct human diseases, rapid-onset dystonia Parkinsonism (RDP), and familial hemiplegic migraine (FHM). Over 40 mutations have been mapped to the human ATP1A2 and ATP1A3 genes and are known to result in RDP, FHM or a variant of FHM with neurological complications. To develop a genetically tractable model system for investigating the role of the Na(+), K(+) ATPase in neural pathologies we performed genetic screens in Drosophila melanogaster to isolate loss-of-function alleles affecting the Na(+), K(+) ATPase alpha subunit. Flies heterozygous for these mutations all exhibit reduced respiration, consistent with a loss-of-function in the major ATPase. However, these mutations do not affect all functions of the Na(+), K(+) ATPase alpha protein since embryos homozygous for these mutations have normal septate junction paracellular barrier function and tracheal morphology. Importantly, all of these mutations cause neurological phenotypes and, akin to the mutations that cause RDP and FHM, these new alleles are missense mutations. All of these alleles exhibit progressive stress-induced locomotor impairment suggesting neuromuscular dysfunction, yet neurodegeneration is observed in an allele-specific manner. Surprisingly, studies of longevity demonstrate that mild hypomorphic mutations in the sodium pump significantly improve longevity, which was verified using the Na(+), K(+) ATPase antagonist ouabain. The isolation and characterization of a series of new missense alleles of ATPalpha in Drosophila provides the foundation for further studies of these neurological diseases and the role of sodium pump impairment in animal longevity.

Fig. 1

Pathogenic mutations affecting Na, K ATPase alpha subunits. A 3.5 Å crystal structure of the Na, K ATPase as a dimer is available (Morth et al. 2007). Numbered amino acids indicate those known to be affected by pathogenic mutations in the human ATP1A2 (FHM), human ATP1A3 (RDP), or Drosophila ATPalpha genes. These residues have been mapped onto a common structure for clarity. Citations for human ATP1A2 mutations: Ambrosini et al. (2005), Castro et al. (2008), De Fusco et al. (2003), Fernandez et al. (2008), Gallanti et al. (2008), Jen et al. (2007), Jurkat-Rott et al. (2004), Kaunisto et al. (2004), Koenderink et al. (2005), Pierelli et al. (2006), Riant et al. (2005), Segall et al. (2004, 2005), Spadaro et al. (2004), Swoboda et al. (2004), Todt et al. (2005), Vanmolkot et al. (2003, 2006a, b, 2007). Citations for human ATP1A3 mutations: Brashear et al. (2007), de Carvalho Aguiar et al. (2004), Kamm et al. (2008), Zanotti-Fregonara et al. (2008)

Fig. 2

Adult ATPalpha protein expression in animals heterozygous for dominant alleles. a Previously described ATPalpha mutants demonstrate a significant decrease in expression in the revertant lines examined but not in the temperature-sensitive alleles, compared to wild-type controls. ATPalpha ^DTS2R2 and ATPalpha ^DTS2R3 express significantly more protein than the 50% expected for a heterozygous null (P < 0.05) suggesting the possibility of a compensatory mechanism. b Newly isolated missense mutants ATPalpha ^CJ6, ATPalpha ^CJ7, and ATPalpha ^CJ13 have significantly reduced ATPalpha protein relative to wild-type controls, whereas the remaining CJ alleles do not have significantly altered expression. In all cases: n = 3, ATPalpha protein was normalized to the internal control protein TPI. Error shown is SEM. Student t test reduction from control: * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 3

Locomotor impairment in ATPalpha alleles. Waking activity levels (measured in total activity counts/total minutes active) were determined for daytime baseline locomotor activity (a) and a 2-h period of repeated startle stimulation during a period of inactivity (b) for heterozygous populations at 25°C (see “Materials and methods”). At the age of testing (12–15 days) no gross locomotor deficits were observed in any line, and baseline waking activity levels were not significantly different from ATPalpha ^DTS1R1 or wild type (ve e) except for ATPalpha ^CJ12, which appeared to be significantly hyperactive (** P < 0.005). Upon stimulation, however, all ATPalpha mutant strains display significantly lower activity levels than wild type controls (* P < 0.05)

Fig. 4

Histopathology from ATPalpha alleles. a–g Brain histology from aged ATPalpha ^CJ alleles and control tissues. b ATPalpha ^CJ12 animals exhibit modest neuropathology akin to that observed in aged wild-type control brains (a). ATPalpha ^CJ13 and ATPalpha ^CJ10 exhibit marked neuropathology as is evident by vacuolar and spongiform-like neuropath throughout the neuropil and optic lobes (c and d, respectively). e ATPalpha ^CJ7 exhibits a fine vacuolar pathology that is more clearly observed at higher magnification as seen in g (compare to panel f from wild type). h–j Muscle histology from aged animals. Wild-type and ATPalpha ^CJ5 mutants do not exhibit significant myopathology in aged specimens (h and j, respectively). I ATPalpha ^CJ12 do exhibit pathology. Bars are all 100 µm. Histopathology was obtained from animals at the median age for their genotype

Fig. 5

Animals heterozygous for ATPalpha alleles have reduced respiration. Respiration was measured from the emergent carbon dioxide from individual ATPalpha mutants and age-matched wild-type controls. ATPalpha mutants uniformly have significantly lower metabolic rates than controls. Error is SEM, n = 4–10, per genotype. Animals were 5–6 days old adults. Statistical significance was determined by Student’s t test (* P < 0.05, ** P < 0.01, and *** P < 0.001)

Fig. 6

ATPalpha ^CJ alleles have normal septate junction function and tracheal morphogenesis. a–b Heterozygous null alleles have normal dorsal trunks and ganglionic branches. c–d ATPalpha ^CJ alleles in trans with null alleles are also normal. e–f Homozygous null alleles demonstrate a lengthened dorsal trunk and incomplete ganglionic branches. Scale bars are 15 µm

Fig. 7

Longevity and stress-sensitive locomotor impairment in ATPalpha alleles. a Progressive locomotor impairment in heterozygous ATPalpha mutants. Recovery from mechanical stress-induced paralysis (seconds) was measured in ATPalpha mutants and aged-matched wild type controls at 25°C. Paralysis was never observed in wild type controls, but all mutant lines showed some degree of progressive impairment by 20–30 days post-eclosion. Asterisks indicate significant differences from age-matched wild type controls using a Student’s t test (* P < 0.001). Time points examined are days 3, 10 and 30 post-eclosion. b Lifespans were performed on heterozygous ATPalpha mutant and control animals. Median lifespan was used to compare longevity between the genotypes. Asterisks indicate significant differences from wild type controls using a Student’s t test (* P < 0.05, ** P < 0.01, and *** P < 0.001). Error is SEM

Fig. 8

Ouabain improves longevity in wild type animals. Longevity assays were performed on wild-type animals administrated one of four test doses of ouabain or vehicle. a The presence of ouabain altered survivorship and resulted in a right or left shift to the survival curve relative to the control, representing an increase or decrease, respectively. b Median lifespan was used to compare the survival curves. Asterisks indicate significant differences from wild-type controls using a Student’s t test (* P < 0.05, ** P < 0.01)

Fig. 9

Drosophila feeding assays. Feeding was measured as 625 nm absorption owing to dye included in the test media. The ATPalpha ^CJ6 strain consumed significantly more media than controls and the positive control strain consumed significantly less. n = 2–3 groups per genotype, representing 60–90 animals. Error is standard deviation. * P < 0.01 and ** P < 0.001 (Student’s t test). All genotypes consumed a significant amount of media and their absorption was in the linear range of detection. Genotypes tested were young age-matched adults (days 3–5)