Karin Lykke-Hartmann

Distribution of Na/K‐ATPase alpha 3 isoform, a sodium‐potassium P‐type pump associated with rapid‐onset of dystonia parkinsonism (RDP) in the adult mouse brain

The Na+/K+‐ATPase1 alpha subunit 3 (ATP1α3) is one of many essential components that maintain the sodium and potassium gradients across the plasma membrane in animal cells. Mutations in the ATP1A3 gene cause rapid‐onset of dystonia parkinsonism (RDP), a rare movement disorder characterized by sudden onset of dystonic spasms and slowness of movement. To achieve a better understanding of the pathophysiology of the disease, we used immunohistochemical approaches to describe the regional and cellular distribution of ATP1α3 in the adult mouse brain. Our results show that localization of ATP1α3 is restricted to neurons, and it is expressed mostly in projections (fibers and punctuates), but cell body expression is also observed. We found high expression of ATP1α3 in GABAergic neurons in all nuclei of the basal ganglia (striatum, globus pallidus, subthalamic nucleus, and substantia nigra), which is a key circuitry in the fine movement control. Several thalamic nuclei structures harboring connections to and from the cortex expressed high levels of the ATP1α3 isoform. Other structures with high expression of ATP1α3 included cerebellum, red nucleus, and several areas of the pons (reticulotegmental nucleus of pons). We also found high expression of ATP1α3 in projections and cell bodies in hippocampus; most of these ATP1α3‐positive cell bodies showed colocalization to GABAergic neurons. ATP1α3 expression was not significant in the dopaminergic cells of substantia nigra. In conclusion, and based on our data, ATP1α3 is widely expressed in neuronal populations but mainly in GABAergic neurons in areas and nuclei related to movement control, in agreement with RDP symptoms. J. Comp. Neurol. 519:376‐404, 2011.

The chromosome passenger complex is required for fidelity of chromosome transmission and cytokinesis in meiosis of mouse oocytes

The existence of two forms of the chromosome passenger complex (CPC) in the mammalian oocyte has meant that its role in female meiosis has remained unclear. Here we use loss- and gain-of function approaches to assess the meiotic functions of one of the shared components of these complexes, INCENP, and of the variable kinase subunits, Aurora B or Aurora C. We show that either the depletion of INCENP or the combined inhibition of Aurora kinases B and C activates the anaphase-promoting complex or cyclosome (APC/C) before chromosomes have properly congressed in meiosis I and also prevents cytokinesis and hence extrusion of the first polar body. Overexpression of Aurora C also advances APC/C activation and results in cytokinesis failure in a high proportion of oocytes, indicative of a dominant effect on CPC function. Together, this points to roles for the meiotic CPC in functions similar to the mitotic roles of the complex: correcting chromosome attachment to microtubules, facilitating the spindle-assembly checkpoint (SAC) function and enabling cytokinesis. Surprisingly, overexpression of Aurora B leads to a failure of APC/C activation, stabilization of securin and consequently a failure of chiasmate chromosomes to resolve – a dominant phenotype that is completely suppressed by depletion of INCENP. Taken together with the differential distribution of Aurora proteins B and C on chiasmate chromosomes, this points to differential functions of the two forms of CPC in regulating the separation of homologous chromosomes in meiosis I.

Migraine- and dystonia-related disease-mutations of Na+/K+-ATPases: Relevance of behavioral studies in mice to disease symptoms and neurological manifestations in humans

The two autosomal dominantly inherited neurological diseases: familial hemiplegic migraine type 2 (FHM2) and familial rapid-onset of dystonia-parkinsonism (Familial RDP) are caused by in vivo mutations of specific alpha subunits of the sodium-potassium pump (Na(+)/K(+)-ATPase). Intriguingly, patients with classical FHM2 and RDP symptoms additionally suffer from other manifestations, such as epilepsy/seizures and developmental disabilities. Recent studies of FHM2 and RDP mouse models provide valuable tools for dissecting the vital roles of the Na(+)/K(+)-ATPases, and we discuss their relevance to the complex patient symptoms and manifestations. Thus, it is interesting that mouse models targeting a specific α-isoform cause different, although still comparable, phenotypes consistent with classical symptoms and other manifestations observed in FHM2 and RDP patients. This review highlights that use of mouse models have broad potentials for future research concerning migraine and dystonia-related diseases, which will contribute towards understanding the, yet unknown, pathophysiologies.

Glutamate-system defects behind psychiatric manifestations in a familial hemiplegic migraine type 2 disease-mutation mouse model.

Migraine is a complex brain disorder, and understanding the complexity of this prevalent disease could improve quality of life for millions of people. Familial Hemiplegic Migraine type 2 (FHM2) is a subtype of migraine with aura and co-morbidities like epilepsy/seizures, cognitive impairments and psychiatric manifestations, such as obsessive-compulsive disorder (OCD). FHM2 disease-mutations locate to the ATP1A2 gene encoding the astrocyte-located α2-isoform of the sodium-potassium pump (α2Na+/K+-ATPase). We show that knock-in mice heterozygous for the FHM2-associated G301R-mutation (α2+/G301R) phenocopy several FHM2-relevant disease traits e.g., by mimicking mood depression and OCD. In vitro studies showed impaired glutamate uptake in hippocampal mixed astrocyte-neuron cultures from α2G301R/G301R E17 embryonic mice, and moreover, induction of cortical spreading depression (CSD) resulted in reduced recovery in α2+/G301R male mice. Moreover, NMDA-type glutamate receptor antagonists or progestin-only treatment reverted specific α2+/G301R behavioral phenotypes. Our findings demonstrate that studies of an in vivo relevant FHM2 disease knock-in mouse model provide a link between the female sex hormone cycle and the glutamate system and a link to co-morbid psychiatric manifestations of FHM2.

The dormant and the fully competent oocyte: comparing the transcriptome of human oocytes from primordial follicles and in metaphase II

Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human oocyte in the quiescent state and at the pinnacle of maturity at ovulation. In silico bioinformatic comparisons were made between the transcriptome of human oocytes from dormant primordial follicles and that of human metaphase II (MII) oocytes and granulosa cells and unique gene expression profiles were identified as well as functional and pathway enrichments associated with the oocytes from the two developmental hallmarks. A total of 729 genes were highly enriched in oocytes from primodial follicles and 1456 genes were highly enriched in MII oocytes (>10-fold, P < 0.001) representing functional categories such as cell cycle regulation, DNA protection and epigenetics, with representative genes validated by qPCR analysis. Dominating canonical pathways in the oocytes from primordial follicles were androgen, estrogen receptor, glucocorticoid receptor and PI3K/AKT signaling (P < 0.001). In the MII, mitotic roles of polo-like kinases, estrogen receptor, JAK/Stat signaling (P < 0.001) and the ERK/MAPK (P < 0.01) signaling were enriched. Some of the highly differentially expressed genes were completely new in human reproduction (CDR1, TLC1A, UHRF2) while other genes [ABO, FOLR1 (folate receptor), CHRNA3 (nicotine receptor)] may relate to clinical observations as diverse as premature ovarian failure, folic acid deficiency and smoking affecting female fertility. The in silico analysis identified novel reproduction-associated genes and highlighted molecular mechanisms and pathways associated with the unique functions of the human oocyte in its two extremes during folliculogenesis. The data provides a fundamental basis for future functional studies in regulation of human oogenesis.

Insights into the Pathology of the α3 Na+/K+-ATPase Ion Pump in Neurological Disorders; Lessons from Animal Models

The transmembrane Na+-/K+ ATPase is located at the plasma membrane of all mammalian cells. The Na+-/K+ ATPase utilizes energy from ATP hydrolysis to extrude three Na+ cations and import two K+ cations into the cell. The minimum constellation for an active Na+-/K+ ATPase is one alpha (α) and one beta (β) subunit. Mammals express four α isoforms (α1−4), encoded by the ATP1A1-4 genes, respectively. The α1 isoform is ubiquitously expressed in the adult central nervous system (CNS) whereas α2 primarily is expressed in astrocytes and α3 in neurons. Na+ and K+ are the principal ions involved in action potential propagation during neuronal depolarization. The α1 and α3 Na+-/K+ ATPases are therefore prime candidates for restoring neuronal membrane potential after depolarization and for maintaining neuronal excitability. The α3 isoform has approximately four-fold lower Na+ affinity compared to α1 and is specifically required for rapid restoration of large transient increases in [Na+]i. Conditions associated with α3 deficiency are therefore likely aggravated by suprathreshold neuronal activity. The α3 isoform been suggested to support re-uptake of neurotransmitters. These processes are required for normal brain activity, and in fact autosomal dominant de novo mutations in ATP1A3 encoding the α3 isoform has been found to cause the three neurological diseases Rapid Onset Dystonia Parkinsonism (RDP), Alternating Hemiplegia of Childhood (AHC), and Cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS). All three diseases cause acute onset of neurological symptoms, but the predominant neurological manifestations differ with particularly early onset of hemiplegic/dystonic episodes and mental decline in AHC, ataxic encephalopathy and impairment of vision and hearing in CAPOS syndrome and late onset of dystonia/parkinsonism in RDP. Several mouse models have been generated to study the in vivo consequences of Atp1a3 modulation. The different mice show varying degrees of hyperactivity, gait problems, and learning disability as well as stress-induced seizures. With the advent of several Atp1a3-gene or chemically modified animal models that closely phenocopy many aspects of the human disorders, we will be able to reach a much better understanding of the etiology of RDP, AHC, and CAPOS syndrome.

Tuning of the Na,K-ATPase by the beta subunit.

The vital gradients of Na+ and K+ across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na+-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix correlates with its functional effect, suggesting that the relative orientation of β modulates ion binding at the α subunit. β2 is primarily expressed in granule neurons and glomeruli in the cerebellum, and we propose that its unique functional characteristics are important to respond appropriately to the cerebellar Na+ and K+ gradients.

TRPM7-like Channels are Functionally Expressed in Oocytes and Modulate Post-Fertilization Embryo Development in Mouse

The Transient Receptor Potential (TRP) channels are a family of cationic ion channels widely distributed in mammalian tissues. In general, the global genetic disruption of individual TRP channels result in phenotypes associated with impairment of a particular tissue and/or organ function. An exception is the genetic ablation of the TRP channel TRPM7, which results in early embryonic lethality. Nevertheless, the function of TRPM7 in oocytes, eggs and pre-implantation embryos remains unknown. Here, we described an outward rectifying non-selective current mediated by a TRP ion channel in immature oocytes (germinal vesicle stage), matured oocytes (metaphase II eggs) and 2-cell stage embryos. The current is activated by specific agonists and inhibited by distinct blockers consistent with the functional expression of TRPM7 channels. We demonstrated that the TRPM7-like channels are homo-tetramers and their activation mediates calcium influx in oocytes and eggs, which is fundamental to support fertilization and egg activation. Lastly, we showed that pharmacological inhibition of the channel function delays pre-implantation embryo development and reduces progression to the blastocyst stage. Our data demonstrate functional expression of TRPM7-like channels in mouse oocytes, eggs and embryos that may play an essential role in the initiation of embryo development.

The α2Na+/K+-ATPase is critical for skeletal and heart muscle function in zebrafish.

Summary The Na+/K+-ATPase generates ion gradients across the plasma membrane, essential for multiple cellular functions. In mammals, four different Na+/K+-ATPase &agr;-subunit isoforms are associated with characteristic cell-type expression profiles and kinetics. We found the zebrafish &agr;2Na+/K+-ATPase associated with striated muscles and that knockdown causes a significant depolarization of the resting membrane potential in slow-twitch fibers of skeletal muscles. Abrupt mechanosensory responses were observed in &agr;2Na+/K+-ATPase-deficient embryos, possibly linked to a postsynaptic defect. The &agr;2Na+/K+-ATPase deficiency reduced the heart rate and caused a loss of left-right asymmetry in the heart tube. Similar phenotypes from knockdown of the Na+/Ca2+ exchanger indicated a role for the interplay between these two proteins in the observed phenotypes. Furthermore, proteomics identified up- and downregulation of specific phenotype-related proteins, such as parvalbumin, CaM, GFAP and multiple kinases, thus highlighting a potential proteome change associated with the dynamics of &agr;2Na+/K+-ATPase. Taken together, our findings show that zebrafish &agr;2Na+/K+-ATPase is important for skeletal and heart muscle functions.

TRPV3 channels mediate Ca²⁺ influx induced by 2-APB in mouse eggs.

Fertilization in mammals is initiated when a sperm fuses with a mature MII oocyte, also known as egg, and triggers a plethora of finely controlled processes identified as egg activation. The completion of all events of egg activation is driven by and depends on a series of repetitive calcium (Ca(2+)) increases (Ca(2+) oscillations), which rely on Ca(2+) influx from the extracellular media. Ca(2+) channels on the egg plasma membrane (PM) are thought to mediate this influx. The TRP Ca(2+) channel TRPV3 is differentially expressed during oocyte maturation, being most active at the MII stage. Specific stimulation of TRPV3 channels promotes Ca(2+) influx sufficient to induce egg activation and parthenogenesis. Here, we explore the function and distribution dynamics of the TRPV3 channel protein during maturation. Using dsRNA, TrpV3 overexpression, and inhibitors of protein synthesis, we modified the expression levels of the channel and showed that the TRPV3 protein is synthesized and translocated to the PM during maturation. We demonstrated that 2-APB at the concentrations used here to promote Ca(2+) influx in eggs, specifically and reversibly targets TRPV3 channels without blocking IP3R1. Finally, we found that the activity of TRPV3 channels is dependent upon an intact actin cytoskeleton, suggesting an actin-based regulation of its expression and/or function on the PM. Collectively, our results show TRPV3 is a target of 2-APB in eggs, a condition that can be used to induce parthenogenesis. The need of an intact actin cytoskeleton for the function of TRPV3 channels in oocytes is a novel finding and suggests the rearrangements of actin that occur during maturation could regulate both the presence on the PM and/or the function of TRPV3 and of other Ca(2+) channels involved in oocyte maturation and fertilization.