Developmental origins of Parkinson disease: Improving the rodent models.

Numerous pesticides are inhibitors of the oxidative phosphorylation system. Oxidative phosphorylation dysfunction adversely affects neurogenesis and often accompanies Parkinson disease. Since brain development occurs mainly in the prenatal period, early exposure to pesticides could alter the development of the nervous system and increase the risk of Parkinson disease. Different rodent models have been used to confirm this hypothesis. However, more precise considerations of the selected strain, the xenobiotic, its mode of administration, and the timing of animal analysis, are necessary to resemble the model to the human clinical condition and obtain more reliable results.

How hot can mitochondria be? Incubation at temperatures above 43 °C induces the degradation of respiratory complexes and supercomplexes in intact cells and isolated mitochondria.

Mitochondrial function generates an important fraction of the heat that contributes to cellular and organismal temperature maintenance, but the actual values of this parameter reached in the organelles is a matter of debate. The studies addressing this issue have reported divergent results: from detecting in the organelles the same temperature as the cell average or the incubation temperature, to increasing differences of up to 10 degrees above the incubation value. Theoretical calculations based on physical laws exclude the possibility of relevant temperature gradients between mitochondria and their surroundings. These facts have given rise to a conundrum or paradox about hot mitochondria. We have examined by Blue-Native electrophoresis, both in intact cells and in isolated organelles, the stability of respiratory complexes and supercomplexes at different temperatures to obtain information about their tolerance to heat stress. We observe that, upon incubation at values above 43 °C and after relatively short periods, respiratory complexes, and especially complex I and its supercomplexes, are unstable even when the respiratory activity is inhibited. These results support the conclusion that high temperatures (>43 °C) cause damage to mitochondrial structure and function and question the proposal that these organelles can physiologically work at close to 50 °C.

Down syndrome is an oxidative phosphorylation disorder.

Down syndrome is the most common genomic disorder of intellectual disability and is caused by trisomy of chromosome 21. Several genes in this chromosome repress mitochondrial biogenesis. The goal of this study was to evaluate whether early overexpression of these genes may cause a prenatal impairment of oxidative phosphorylation negatively affecting neurogenesis. Reduction in the mitochondrial energy production and a lower mitochondrial function have been reported in diverse tissues or cell types, and also at any age, including early fetuses, suggesting that a defect in oxidative phosphorylation is an early and general event in Down syndrome individuals. Moreover, many of the medical conditions associated with Down syndrome are also frequently found in patients with oxidative phosphorylation disease. Several drugs that enhance mitochondrial biogenesis are nowadays available and some of them have been already tested in mouse models of Down syndrome restoring neurogenesis and cognitive defects. Because neurogenesis relies on a correct mitochondrial function and critical periods of brain development occur mainly in the prenatal and early neonatal stages, therapeutic approaches intended to improve oxidative phosphorylation should be provided in these periods.

Oxidative Phosphorylation Dysfunction Modifies the Cell Secretome.

Mitochondrial oxidative phosphorylation disorders are extremely heterogeneous conditions. Their clinical and genetic variability makes the identification of reliable and specific biomarkers very challenging. Until now, only a few studies have focused on the effect of a defective oxidative phosphorylation functioning on the cell's secretome, although it could be a promising approach for the identification and pre-selection of potential circulating biomarkers for mitochondrial diseases. Here, we review the insights obtained from secretome studies with regard to oxidative phosphorylation dysfunction, and the biomarkers that appear, so far, to be promising to identify mitochondrial diseases. We propose two new biomarkers to be taken into account in future diagnostic trials.

Uridine Prevents Negative Effects of OXPHOS Xenobiotics on Dopaminergic Neuronal Differentiation.

Neuronal differentiation appears to be dependent on oxidative phosphorylation capacity. Several drugs inhibit oxidative phosphorylation and might be detrimental for neuronal differentiation. Some pregnant women take these medications during their first weeks of gestation when fetal nervous system is being developed. These treatments might have later negative consequences on the offspring's health. To analyze a potential negative effect of three widely used medications, we studied in vitro dopaminergic neuronal differentiation of cells exposed to pharmacologic concentrations of azidothymidine for acquired immune deficiency syndrome; linezolid for multidrug-resistant tuberculosis; and atovaquone for malaria. We also analyzed the dopaminergic neuronal differentiation in brains of fetuses from pregnant mice exposed to linezolid. The drugs reduced the in vitro oxidative phosphorylation capacity and dopaminergic neuronal differentiation. This differentiation process does not appear to be affected in the prenatally exposed fetus brain. Nevertheless, the global DNA methylation in fetal brain was significantly altered, perhaps linking an early exposure to a negative effect in older life. Uridine was able to prevent the negative effects on in vitro dopaminergic neuronal differentiation and on in vivo global DNA methylation. Uridine could be used as a protective agent against oxidative phosphorylation-inhibiting pharmaceuticals provided during pregnancy when dopaminergic neuronal differentiation is taking place.

Brain pyrimidine nucleotide synthesis and Alzheimer disease.

Many patients suffering late-onset Alzheimer disease show a deficit in respiratory complex IV activity. The de novo pyrimidine biosynthesis pathway connects with the mitochondrial respiratory chain upstream from respiratory complex IV. We hypothesized that these patients would have decreased pyrimidine nucleotide levels. Then, different cell processes for which these compounds are essential, such as neuronal membrane generation and maintenance and synapses production, would be compromised. Using a cell model, we show that inhibiting oxidative phosphorylation function reduces neuronal differentiation. Linking these processes to pyrimidine nucleotides, uridine treatment recovers neuronal differentiation. To unmask the importance of these pathways in Alzheimer disease, we firstly confirm the existence of the de novo pyrimidine biosynthesis pathway in adult human brain. Then, we report altered mRNA levels for genes from both de novo pyrimidine biosynthesis and pyrimidine salvage pathways in brain from patients with Alzheimer disease. Thus, uridine supplementation might be used as a therapy for those Alzheimer disease patients with low respiratory complex IV activity.

Prenatal exposure to oxidative phosphorylation xenobiotics and late-onset Parkinson disease.

Late-onset Parkinson disease is a multifactorial and multietiological disorder, age being one of the factors implicated. Genetic and/or environmental factors, such as pesticides, can also be involved. Up to 80% of dopaminergic neurons of the substantia nigra are lost before motor features of the disorder begin to appear. In humans, these neurons are only formed a few weeks after fertilization. Therefore, prenatal exposure to pesticides or industrial chemicals during crucial steps of brain development might also alter their proliferation and differentiation. Oxidative phosphorylation is one of the metabolic pathways sensitive to environmental toxicants and it is crucial for neuronal differentiation. Many inhibitors of this biochemical pathway, frequently found as persistent organic pollutants, affect dopaminergic neurogenesis, promote the degeneration of these neurons and increase the risk of suffering late-onset Parkinson disease. Here, we discuss how an early, prenatal, exposure to these oxidative phosphorylation xenobiotics might trigger a late-onset, old age, Parkinson disease.

Kidney-specific genetic deletion of both AMPK α-subunits causes salt and water wasting.

AMP-activated kinase (AMPK) controls cell energy homeostasis by modulating ATP synthesis and expenditure. In vitro studies have suggested AMPK may also control key elements of renal epithelial electrolyte transport but in vivo physiological confirmation is still insufficient. We studied sodium renal handling and extracellular volume regulation in mice with genetic deletion of AMPK catalytic subunits. AMPKα1 knockout (KO) mice exhibit normal renal sodium handling and a moderate antidiuretic state. This is accompanied by higher urinary aldosterone excretion rates and reduced blood pressure. Plasma volume, however, was found to be increased compared with wild-type mice. Thus blood volume is preserved despite a significantly lower hematocrit. The lack of a defect in renal function in AMPKα1 KO mice could be explained by a compensatory upregulation in AMPK α2-subunit. Therefore, we used the Cre-loxP system to knock down AMPKα2 expression in renal epithelial cells. Combining this approach with the systemic deletion of AMPKα1 we achieved reduced renal AMPK activity, accompanied by a shift to a moderate water- and salt-wasting phenotype. Thus we confirm the physiologically relevant role of AMPK in the kidney. Furthermore, our results indicate that in vivo AMPK activity stimulates renal sodium and water reabsorption.

Ovarian hormones and prolactin increase renal NaCl cotransporter phosphorylation.

Unique situations in female physiology require volume retention. Accordingly, a dimorphic regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) has been reported, with a higher activity in females than in males. However, little is known about the hormones and mechanisms involved. Here, we present evidence that estrogens, progesterone, and prolactin stimulate NCC expression and phosphorylation. The sex difference in NCC abundance, however, is species dependent. In rats, NCC phosphorylation is higher in females than in males, while in mice both NCC expression and phosphorylation is higher in females, and this is associated with increased expression and phosphorylation of full-length STE-20 proline-alanine-rich kinase (SPAK). Higher expression/phosphorylation of NCC was corroborated in humans by urinary exosome analysis. Ovariectomy in rats resulted in decreased expression and phosphorylation of the cotransporter and promoted the shift of SPAK isoforms toward the short inhibitory variant SPAK2. Conversely, estradiol or progesterone administration to ovariectomized rats restored NCC phosphorylation levels and shifted SPAK expression and phosphorylation towards the full-length isoform. Estradiol administration to male rats induced a significant increase in NCC phosphorylation. NCC is also modulated by prolactin. Administration of this peptide hormone to male rats induced increased phosphorylation of NCC, an effect that was observed even using the ex vivo kidney perfusion strategy. Our results indicate that estradiol, progesterone, and prolactin, the hormones that are involved in sexual cycle, pregnancy and lactation, upregulate the activity of NCC.

WNK3-SPAK interaction is required for the modulation of NCC and other members of the SLC12 family.

The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl(-) influx and prevent Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation.

Similar effects of all WNK3 variants on SLC12 cotransporters.

With-no-lysine kinase 3 (WNK3) is a member of a subfamily of serine/threonine kinases that modulate the activity of the electroneutral cation-coupled chloride cotransporters. WNK3 activates NKCC1/2 and NCC and inhibits the KCCs. Four splice variants are generated from the WNK3 gene. Our previous studies focused on the WNK3-18a variant. However, it has been suggested that other variants could have different effects on the cotransporters. Thus, the present study was designed to define the effects of all WNK3 variants on members of the SLC12 family. By RT-PCR from a fetal brain library, exons 18b and 22 were separately amplified and subcloned into the original WNK3-18a or catalytically inactive WNK3-D294A to obtain all four potential combinations with and without catalytic activity (18a, 18a+22, 18b, and 18b+22). The basal activity of the cotransporters and the effects of WNK3 isoforms were assessed in Xenopus laevis oocytes coinjected with each of the WNK3 variant cRNAs. In isotonic conditions, the basal activity of NCC and NKCC1/2 were increased by coinjection with any of the WNK3. The positive effects occurred even in hypotonic conditions, in which the basal activity of NKCC1 is completely prevented. Consistent with these observations, when expressed in hypotonicity, all KCCs were active, but in the presence of any of the WNK3 variants, KCC activity was completely reduced. That is, NKCC1/2 and NCC were inhibited, even in hypertonicity, while KCCs were activated, even in isotonic conditions. We conclude that the effects of all WNK3 variants toward SLC12 proteins are similar.

The Na+:Cl- cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion.

The renal Na(+):Cl(-) cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl(-) influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl(-) efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr(53) and Thr(58). Elimination of these threonines together with serine residue Ser(71) completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation.

WNK3 modulates transport of Cl- in and out of cells: implications for control of cell volume and neuronal excitability.

The regulation of Cl(-) transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion cotransporters: Cl(-) influx is mediated by the Na-K-2Cl cotransporter NKCC1 and Cl(-) efflux via K-Cl cotransporters, KCC1 or KCC2. A Cl(-)/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl(-)-transporting epithelia and in neurons expressing ionotropic GABA(A) receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl(-) influx via NKCC1, and that it inhibits Cl(-) exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl(-) via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl(-)/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission.

WNK3 kinase is a positive regulator of NKCC2 and NCC, renal cation-Cl- cotransporters required for normal blood pressure homeostasis.

WNK1 and WNK4 [WNK, with no lysine (K)] are serine-threonine kinases that function as molecular switches, eliciting coordinated effects on diverse ion transport pathways to maintain homeostasis during physiological perturbation. Gain-of-function mutations in either of these genes cause an inherited syndrome featuring hypertension and hyperkalemia due to increased renal NaCl reabsorption and decreased K(+) secretion. Here, we reveal unique biochemical and functional properties of WNK3, a related member of the WNK kinase family. Unlike WNK1 and WNK4, WNK3 is expressed throughout the nephron, predominantly at intercellular junctions. Because WNK4 is a potent inhibitor of members of the cation-cotransporter SLC12A family, we used coexpression studies in Xenopus oocytes to investigate the effect of WNK3 on NCC and NKCC2, related kidney-specific transporters that mediate apical NaCl reabsorption in the thick ascending limb and distal convoluted tubule, respectively. In contrast to WNK4's inhibitory activity, kinase-active WNK3 is a potent activator of both NKCC2 and NCC-mediated transport. Conversely, in its kinase-inactive state, WNK3 is a potent inhibitor of NKCC2 and NCC activity. WNK3 regulates the activity of these transporters by altering their expression at the plasma membrane. Wild-type WNK3 increases and kinase-inactive WNK3 decreases NKCC2 phosphorylation at Thr-184 and Thr-189, sites required for the vasopressin-mediated plasmalemmal translocation and activation of NKCC2 in vivo. The effects of WNK3 on these transporters and their coexpression in renal epithelia implicate WNK3 in NaCl, water, and blood pressure homeostasis, perhaps via signaling downstream of vasopressin.

Pathophysiology of functional mutations of the thiazide-sensitive Na-Cl cotransporter in Gitelman disease.

Most of the missense mutations that have been described in the human SLC12A3 gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC, NCC, or NCCT), as the cause of Gitelman disease, block TSC function by interfering with normal protein processing and glycosylation. However, some mutations exhibit considerable activity. To investigate the pathogenesis of Gitelman disease mediated by such mutations and to gain insights into structure-function relationships on the cotransporter, five functional disease mutations were introduced into mouse TSC cDNA, and their expression was determined in Xenopus laevis oocytes. Western blot analysis revealed immunoreactive bands in all mutant TSCs that were undistinguishable from wild-type TSC. The activity profile was: wild-type TSC (100%) > G627V (66%) > R935Q (36%) = V995M (32%) > G610S (12%) > A585V (6%). Ion transport kinetics in all mutant clones were similar to wild-type TSC, except in G627V, in which a small but significant increase in affinity for extracellular Cl(-) was observed. In addition, G627V and G610S exhibited a small increase in metolazone affinity. The surface expression of wild-type and mutant TSCs was performed by laser-scanning confocal microscopy. All mutants exhibited a significant reduction in surface expression compared with wild-type TSC, with a profile similar to that observed in functional expression analysis. Our data show that biochemical and functional properties of the mutant TSCs are similar to wild-type TSC but that the surface expression is reduced, suggesting that these mutations impair the insertion of a functional protein into the plasma membrane. The small increase in Cl(-) and thiazide affinity in G610S and G627V suggests that the beginning of the COOH-terminal domain could be implicated in defining kinetic properties.

[Molecular physiopathology of Bartter's syndrome]. / Fisiopatología molecular del síndrome de Bartter.

Bartter's syndrome is an autosomic recessive disease characterized by hypokalemic metabolic alkalosis accompanied with hypercalciuria, polyuria and hypotension due to volume depletion. The pathophysiology of this hereditary disease was largely unknown until the last few years in which inactivating mutations in up to five different genes have been shown to produce or be associated with the development of this syndrome. All the involved proteins are expressed either in the apical or basolateral membrane of the thick ascending limb of Henle's loop. These clinical and molecular findings have increased our understanding of the Bartter's disease and also of the thick ascending limb physiology.

cAMP-dependent activation of the renal-specific Na+-K+-2Cl- cotransporter is mediated by regulation of cotransporter trafficking.

The murine apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter gene (mBSC1) exhibits two spliced isoform products that differ at the COOH-terminal domain. A long COOH-terminal isoform (L-mBSC1) encodes the Na(+)-K(+)-2Cl(-) cotransporter, and a short isoform (S-mBSC1) exerts a dominant-negative effect on L-mBSC1 cotransporter activity that is abrogated by cAMP. However, the mechanism of this dominant-negative effect was not clear. In this study, we used confocal microscopic analysis of an enhanced green fluorescent protein (EGFP) fusion construct (L-mBSC1-EGFP) expressed to characterize the surface expression of the L-BSC1 isoform in Xenopus laevis oocytes. Functional expression was also assessed in L-mBSC1-injected oocytes by measuring the bumetanide-sensitive (86)Rb(+) uptake. Oocytes injected with L-mBSC1-EGFP cRNA developed a distinct plasma membrane-associated fluorescence that colocalized with the fluorescent membrane dye FM 4-64. The fluorescence intensity in L-mBSC1-EGFP oocytes did not change after cAMP was added to the extracellular medium. In contrast, L-mBSC1-EGFP fluorescence intensity was reduced in a dose-dependent manner, with coexpression of S-mBSC1. The inhibitory effect of S-mBSC1 was abrogated by cAMP. Finally, the exocytosis inhibitor colchicine blocked the effect of cAMP on the L-mBSC1-EGFP/S-mBSC1-coinjected oocytes. All changes in L-mBSC1 surface expression correlated with modification of bumetanide-sensitive (86)Rb(+) uptake. Our data suggest that the dominant-negative effect of S-mBSC1 on L-mBSC1 transport function is due to the effects of the cotransporter on trafficking.

Functional properties of the apical Na+-K+-2Cl- cotransporter isoforms.

The bumetanide-sensitive Na(+):K(+):2Cl(-) cotransporter (BSC1) is the major pathway for salt reabsorption in the apical membrane of the mammalian thick ascending limb of Henle. Three isoforms of the cotransporter, known as A, B, and F, exhibit axial expression along the thick ascending limb. We report here a functional comparison of the three isoforms from mouse kidney. When expressed in Xenopus oocytes the mBSC1-A isoform showed higher capacity of transport, with no difference in the amount of surface expression. Kinetic characterization revealed divergent affinities for the three cotransported ions. The observed EC(50) values for Na(+), K(+), and Cl(-) were 5.0 +/- 3.9, 0.96 +/- 0.16, and 22.2 +/- 4.8 mm for mBSC1-A; 3.0 +/- 0.6, 0.76 +/- 0.07, and 11.6 +/- 0.7 mm for mBSC1-B; and 20.6 +/- 7.2, 1.54 +/- 0.16, and 29.2 +/- 2.1 mm for mBSC1-F, respectively. Bumetanide sensitivity was higher in mBSC1-B compared with the mBSC1-A and mBSC1-F isoforms. All three transporters were partially inhibited by hypotonicity but to different extents. The cell swelling-induced inhibition profile was mBSC1-F > mBSC1-B > mBSC1-A. The function of the Na(+):K(+):2Cl(-) cotransporter was not affected by extracellular pH or by the addition of metolazone, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), or R(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1-H-indenyl-5-yl)-oxy]acetic acid (DIOA) to the extracellular medium. In contrast, exposure of oocytes to HgCl(2) before the uptake period reduced the activity of the cotransporter. The effect of HgCl(2) was dose-dependent, and mBSC1-A and mBSC1-B exhibited higher affinity than mBSC1-F. Overall, the functional comparison of the murine apical renal-specific Na(+):K(+):2Cl(-) cotransporter isoforms A, B, and F reveals important functional, pharmacological, and kinetic differences, with both physiological and structural implications.