[show abstract][hide abstract] ABSTRACT: Glial cells in their plurality account for most of the mass of the human brain and impact on brain structure and function. A principal component of the emerging glial doctrine is the hypothesis that astrocytes, the most abundant type of glial cells, trigger major molecular processes leading to brain ageing. Astrocyte biology has been examined using three-dimensional (3D) atomic and molecular structural methods, as well as 3D brain imaging in live animals and humans. Exosomes are extracelluar membrane vesicles that facilitate communication between glia, and have significant potential for biomarker discovery and drug delivery. Polymorphisms in DNA repair genes may indirectly influence the structure and function of membrane proteins expressed in glial cells and predispose specific population subgroups to astro-and neurodegeneration. Physical exercise may reduce or retard age-related brain deterioration by a mechanism involving neuro-glial processes. It is most likely that more information about the distribution, structure and function of glial cells will yield novel insight into human brain ageing. Systematic studies of glia and their functions are expected to eventually lead to earlier detection of ageing-related brain dysfunction and to interventions that could delay, reduce or prevent brain dysfunction.
Mechanisms of ageing and development 10/2013; · 4.18 Impact Factor
[show abstract][hide abstract] ABSTRACT: Tragically common among children in sub-Saharan Africa, cerebral malaria is characterized by rapid progression to coma and death. In this study, we used a model of cerebral malaria appearing in C57BL/6 WT mice after infection with the rodent malaria parasite Plasmodium berghei ANKA. Expression and cellular localization of the brain water channel aquaporin-4 (AQP4) was investigated during the neurological syndrome. Semiquantitative real-time PCR comparing uninfected and infected mice showed a reduction of brain AQP4 transcript in cerebral malaria, and immunoblots revealed reduction of brain AQP4 protein. Reduction of brain AQP4 protein was confirmed in cerebral malaria by quantitative immunogold EM; however, polarized distribution of AQP4 at the perivascular and subpial astrocyte membranes was not altered. To further examine the role of AQP4 in cerebral malaria, WT mice and littermates genetically deficient in AQP4 were infected with P. berghei. Upon development of cerebral malaria, WT and AQP4-null mice exhibited similar increases in width of perivascular astroglial end-feet in brain. Nevertheless, the AQP4-null mice exhibited more severe signs of cerebral malaria with greater brain edema, although disruption of the blood-brain barrier was similar in both groups. In longitudinal studies, cerebral malaria appeared nearly 1 d earlier in the AQP4-null mice, and reduced survival was noted when chloroquine rescue was attempted. We conclude that the water channel AQP4 confers partial protection against cerebral malaria.
Proceedings of the National Academy of Sciences 12/2012; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: AIM: Aquaporin-1 (AQP1) is expressed in human and mouse hearts, but little is known about its cellular and subcellular localization and regulation. The aim of this study was to investigate the localization of AQP1 in the mouse heart and to determine effects of ischemia and hypoxia on its expression. METHODS: Mouse myocardial cells were freshly isolated and split into cardiomyocyte and non-cardiomyocyte fractions. Isolated, Langendorff-perfused C57Bl6 mouse hearts (n=46) were harvested with no intervention, subjected to 35minutes of ischemia or ischemia followed by 60minutes of reperfusion. Eleven mouse hearts were perfusion-fixed for electron microscopy. Forty C57Bl6 mice were exposed to normobaric hypoxia for one or two weeks (n=12). Needle biopsies of human left ventricular myocardium were sampled (n=30) during coronary artery bypass surgery before cardioplegia and after 30minutes of reperfusion. Human umbilical vein endothelial cells (HUVECs) were subjected to four hours of hypoxia with reoxygenation for either 4 or 24hours. AQP1 expression was studied by electron microscopy with immunogold labeling, Western blot, and qPCR. Expression of miR-214 and miR-320 in HUVECs with hypoxia was studied with qPCR. HUVECs were then transfected with precursors and inhibitors of miR-214. RESULTS: AQP1 expression was confined to cardiac endothelial cells, with no signal in cardiomyocytes or cardiac fibroblasts. Immunogold electron microscopy showed AQP1 expression in endothelial caveolae with equal distribution along the basal and apical membranes. Ischemia and reperfusion tended to decrease AQP1 mRNA expression in mouse hearts by 37±9% (p=0.06), while glycosylated AQP1 protein was reduced by 16±9% (p=0.03). No difference in expression was found between ischemia alone and ischemia-reperfusion. In human left ventricles AQP1 mRNA expression was reduced following cardioplegia and reperfusion (p=0.008). Hypoxia in mice reduced AQP1 mRNA expression by 20±7% (p<0.0001), as well as both glycosylated (-47±10%, p=0.03) and glycan-free protein (-34±16%, p=0.05). Hypoxia and reoxygenation in HUVECs downregulated glycan-free AQP1 protein (-34±24%, p=0.04) and upregulated miR-214 (+287±52%, p<0.05). HUVECs transfected with anti-miR-214 had increased glycosylated (1.5 fold) and glycan-free (2 fold) AQP1. CONCLUSION: AQP1 in mouse hearts is localized to endothelial cell membranes and caveolae. Cardioplegia, ischemia and hypoxia decrease AQP1 mRNA as well as total protein expression and glycosylation, possibly regulated by miR-214.
Journal of Molecular and Cellular Cardiology 12/2012; · 5.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aquaporins (AQPs) are channel-forming membrane proteins highly permeable to water. AQP4 is found in mammalian hearts; however, its expression sites, regulation and function are largely unknown. The aim was to investigate cardiac AQP4 expression in humans and mice, its regulation by ischemia and hypoxia, and in particular its role in cardiac ischemic injury using AQP4 knockout (KO) mice. Comparable levels of AQP4 were detected by Western blot and qPCR in biopsies from human donor hearts and wild type C57Bl6 mouse hearts. In mice, AQP4 was expressed on cardiomyocyte plasmalemma (qPCR, Western blot, immunogold), and its mRNA decreased following ischemia/reperfusion (isolated hearts, p = 0.02) and after normobaric hypoxia in vivo (oxygen fraction 10 % for 1 week, p < 0.001). Isolated hearts from AQP4 KO mice undergoing global ischemia and reperfusion had reduced infarct size (p = 0.05) and attenuated left ventricular end-diastolic pressure during reperfusion (p = 0.04). Infarct size was also reduced in AQP4 KO mice 24 h after left coronary artery ligation in vivo (p = 0.036). AQP4 KO hearts had no compensatory change in AQP1 protein expression. AQP4 KO cardiomyocytes were partially resisted to hypoosmotic stress in the presence of hypercontracture. AQP4 is expressed in human and mouse hearts, in the latter confined to the cardiomyocyte plasmalemma. AQP4 mRNA expression is downregulated by hypoxia and ischemia. Deletion of AQP4 is protective in acute myocardial ischemia-reperfusion, and this molecule might be a future target in the treatment of acute myocardial infarction.
Archiv für Kreislaufforschung 09/2012; 107(5):280. · 7.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Infants suffering from infection or hypoxia-ischemia around the time of birth can develop brain damage resulting in life-long impairment such as cerebral palsy, epilepsy and cognitive disability. Inflammation appears to be an important contributor irrespective of whether the primary event is infection or hypoxia-ischemia. Activation of the transcription factor NF-κB is a hallmark of inflammation. To study perinatal brain inflammation, we developed a transgenic reporter mouse for imaging NF-κB activity in live animals and tissue samples. The reporter genes firefly luciferase and a destabilized version of enhanced GFP (dEGFP) were regulated by common NF-κB sites using a bidirectional promoter. Luciferase activity was imaged in vivo, while dEGFP was detected at cellular level in tissue sections. In newborn mice subjected to experimental models of infections or hypoxia-ischemia; luciferase signal increased in brains of live animals. In brain sections dEGFP expression, revealing NF-κB activation was observed in the endothelial cells of the blood-brain barrier in all disease models. In meningitis and hypoxia-ischemia expression of dEGFP was also induced in perivascular astrocytes. In conclusion, by using this transgenic reporter mouse in experimental models of perinatal complications, we could assess NF-κB activity in vivo and subsequently determine the cellular origin in the tissues.
[show abstract][hide abstract] ABSTRACT: Aquaporin-4 (AQP4) is the predominant water channel in brain and is selectively expressed in astrocytes. Astrocytic endfoot membranes exhibit tenfold higher densities of AQP4 than non-endfoot membranes, making AQP4 an excellent marker of astrocyte polarization. Loss of astrocyte polarization is known to compromise astrocytic function and to be associated with impaired water and K+ homeostasis. Here we investigate by a combination of light and electron microscopic immunocytochemistry whether amyloid deposition is associated with a loss of astrocyte polarization, using AQP4 as a marker. We used the tg-ArcSwe mouse model of Alzheimer's disease, as this model displays perivascular plaques as well as plaques confined to the neuropil. 3D reconstructions were done to establish the spatial relation between plaques and astrocytic endfeet, the latter known to contain the perivascular pool of AQP4. Changes in AQP4 expression emerge just after the appearance of the first plaques. Typically, there is a loss of AQP4 from endfoot membranes at sites of perivascular amyloid deposits, combined with an upregulation of AQP4 in the neuropil surrounding plaques. By electron microscopy it could be verified that the upregulation reflects an increased concentration of AQP4 in those delicate astrocytic processes that abound in synaptic regions. Thus, astrocytes exhibit a redistribution of AQP4 from endfoot membranes to non-endfoot membrane domains. The present data suggest that the development of amyloid deposits is associated with a loss of astrocyte polarization. The possible perturbation of water and K+ homeostasis could contribute to cognitive decline and seizure propensity in patients with Alzheimer's disease.
[show abstract][hide abstract] ABSTRACT: Regulatory volume decrease (RVD) is a key mechanism for volume control that serves to prevent detrimental swelling in response to hypo-osmotic stress. The molecular basis of RVD is not understood. Here we show that a complex containing aquaporin-4 (AQP4) and transient receptor potential vanilloid 4 (TRPV4) is essential for RVD in astrocytes. Astrocytes from AQP4-KO mice and astrocytes treated with TRPV4 siRNA fail to respond to hypotonic stress by increased intracellular Ca(2+) and RVD. Coimmunoprecipitation and immunohistochemistry analyses show that AQP4 and TRPV4 interact and colocalize. Functional analysis of an astrocyte-derived cell line expressing TRPV4 but not AQP4 shows that RVD and intracellular Ca(2+) response can be reconstituted by transfection with AQP4 but not with aquaporin-1. Our data indicate that astrocytes contain a TRPV4/AQP4 complex that constitutes a key element in the brain's volume homeostasis by acting as an osmosensor that couples osmotic stress to downstream signaling cascades.
Proceedings of the National Academy of Sciences 02/2011; 108(6):2563-8. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Short peptides are important as lead compounds and molecular probes in drug discovery and chemical biology, but their well-known drawbacks, such as high conformational flexibility, protease lability, poor bioavailability and short half-lives in vivo, have prevented their potential from being fully realized. Side chain-to-side chain cyclization, e.g., by ring-closing olefin metathesis, known as stapling, is one approach to increase the biological activity of short peptides that has shown promise when applied to 3(10)- and α-helical peptides. However, atomic resolution structural information on the effect of side chain-to-side chain cyclization in 3(10)-helical peptides is scarce, and reported data suggest that there is significant potential for improvement of existing methodologies. Here, we report a novel stapling methodology for 3(10)-helical peptides using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction in a model aminoisobutyric acid (Aib) rich peptide and examine the structural effect of side chain-to-side chain cyclization by NMR, X-ray diffraction, linear IR and femtosecond 2D IR spectroscopy. Our data show that the resulting cyclic peptide represents a more ideal 3(10)-helix than its acyclic precursor and other stapled 3(10)-helical peptides reported to date. Side chain-to-side chain stapling by CuAAC should prove useful when applied to 3(10)-helical peptides and protein segments of interest in biomedicine.
The Journal of Organic Chemistry 01/2011; 76(5):1228-38. · 4.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Arachnoid cysts (AC) are filled with liquid very similar to cerebrospinal fluid (CSF). The mechanisms of fluid accumulation have remained unknown; previous studies have however indicated both fluid secretion and a one-way valve as a mechanism. If the filling was caused by fluid secretion, mechanisms similar to those underlying CSF production would be anticipated. We have investigated the expression levels of all genes known to be involved in mammalian CSF production in surgically removed AC. Based on mRNA microarray analysis of AC and normal arachnoid tissue, we extracted the RNA expression profiles of all genes known to code for proteins involved in CSF production. A selection of genes was further investigated with quantitative real-time polymerase chain reaction (qRT-PCR). For selected CSF production proteins, electron microscopic immunogold techniques (EM) and Western blots were performed. Seven genes were expressed in both cysts and controls. The gene encoding the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 was significantly up-regulated in AC. Gene expression data were supported by Western blot. EM demonstrated NKCC1 expressed at the plasma membranes of the cyst-lining cells. This result points at secretion as the main mechanism of cyst filling, and NKCC1 as the key candidate of fluid transport. Based on these findings, we hypothesize that selective NKCC1 inhibitors could be used in preventing expansion of temporal AC.
[show abstract][hide abstract] ABSTRACT: The Na(+)-K(+)-2Cl(-) cotransporter localized in the brain vascular endothelium has been shown to be important in the evolution of cerebral edema following experimental stroke. Previous in vivo studies have demonstrated that bumetanide, a selective Na(+)-K(+)-2Cl(-) cotransport inhibitor, attenuates ischemia-evoked cerebral edema. Recently, bumetanide has been shown to also inhibit water permeability via aquaporin-4 (AQP4) expressed in Xenopus laevis oocytes. We tested the hypothesis that the perivascular pool of AQP4 plays a significant role in the anti-edema effect of bumetanide by utilizing wild-type (WT) mice as well as mice with targeted disruption of alpha-syntrophin (alpha-Syn(-/-)) that lack the perivascular pool of AQP4.
Isoflurane-anesthetized adult male WT C57Bl6 and alpha-Syn(-/-) mice were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by 24 or 48 h of reperfusion. Adequacy of MCAO and reperfusion was monitored with laser-Doppler flowmetry over the ipsilateral parietal cortex. Infarct volume (tetrazolium staining), cerebral edema (wet-to-dry ratios), and AQP4 protein expression (immunoblotting) were determined in different treatment groups in separate sets of experiments.
Bumetanide significantly attenuated infarct volume and decreased ipsilateral hemispheric water content in WT mice compared to vehicle treatment. In alpha-Syn(-/-) mice, bumetanide treatment had no effect on infarct volume or ischemia-evoked cerebral edema. Bumetanide-treated WT mice had a significant attenuation of AQP4 protein expression at 48 h post-MCAO compared to vehicle-treated WT mice.
These data suggest that bumetanide exerts its neuroprotective and anti-edema effects partly via blockade of the perivascular pool of AQP4 and may have therapeutic potential for ischemic stroke in the clinical setting.
Neurocritical Care 05/2010; 13(1):123-31. · 3.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: Arachnoid cyst (AC) fluid has not previously been compared with cerebrospinal fluid (CSF) from the same patient. ACs are commonly referred to as containing "CSF-like fluid". The objective of this study was to characterize AC fluid by clinical chemistry and to compare AC fluid to CSF drawn from the same patient. Such comparative analysis can shed further light on the mechanisms for filling and sustaining of ACs.
Cyst fluid from 15 adult patients with unilateral temporal AC (9 female, 6 male, age 22-77y) was compared with CSF from the same patients by clinical chemical analysis.
AC fluid and CSF had the same osmolarity. There were no significant differences in the concentrations of sodium, potassium, chloride, calcium, magnesium or glucose. We found significant elevated concentration of phosphate in AC fluid (0.39 versus 0.35 mmol/L in CSF; p = 0.02), and significantly reduced concentrations of total protein (0.30 versus 0.41 g/L; p = 0.004), of ferritin (7.8 versus 25.5 ug/L; p = 0.001) and of lactate dehydrogenase (17.9 versus 35.6 U/L; p = 0.002) in AC fluid relative to CSF.
AC fluid is not identical to CSF. The differential composition of AC fluid relative to CSF supports secretion or active transport as the mechanism underlying cyst filling. Oncotic pressure gradients or slit-valves as mechanisms for generating fluid in temporal ACs are not supported by these results.
Cerebrospinal Fluid Research 01/2010; 7:8. · 1.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Four cyclic pentapeptides and two cyclic heptapeptides modelled on the 3(10) helical Pro138-Gly144 segment of the water channel aquaporin-4 (AQP4) postulated to mediate adhesive interactions between AQP4 tetramers were synthesised by olefin metathesis. Three related acyclic pentapeptides Boc-Ser(All)-Xaa1-Val-Ser(All)-Gly-OMe (Xaa1 = Val, Aib; Boc = tert-butoxycarbonyl; All = allyl) and Boc-Ser(Bn)-Val-Val-Gly-Gly-OMe (Bn = benzyl) and two acyclic heptapeptides Boc-Pro-Pro-Ser(All)-Val-Val-Ser(All)-Gly-OMe and Boc-Pro-Pro-Ser(Bn)-Val-Val-Gly-Gly-OMe were also prepared. NMR, CD and IR data provided evidence that the peptides can access a 3(10) helical structure in apolar solvents and pointed to a significant stabilising effect of the olefinic bridge on helicity in an aqueous environment. Thus we could demonstrate the viability of using ring closing olefin metathesis to stabilise short protein segments in the helical conformation that they adopt in their native protein environment. Our approach provides access to a set of peptides with potential binding affinity for AQP4.
[show abstract][hide abstract] ABSTRACT: AQP9 is an aquaglyceroporin that serves important functions in peripheral organs, including the liver. Reflecting the lack of AQP9 knockout mice, uncertainties still prevail regarding the localization and roles of AQP9 in the central nervous system. Here we present a comprehensive analysis of AQP9 gene expression in brain, based on a quantitative and multipronged approach that includes the use of animals with targeted deletion of the AQP9 gene. We show by real-time PCR that AQP9 mRNA concentration in rat and mouse brain is approximately 3% and approximately 0.5%, respectively, of that in rat and mouse liver, the organ with the highest level of AQP9. By blue native gel analysis it could be demonstrated that the brain contains tetrameric AQP9, corresponding to the functional form of AQP9. The band corresponding to the AQP9 tetramer was absent in AQP9 knockout brain and liver. Immunocytochemistry and in situ hybridization analyses with AQP9 knockout controls show that subpopulations of nigral neurons express AQP9 both at the mRNA and at the protein levels and that populations of cortical cells (including hilar neurons in the hippocampus) contain AQP9 mRNA but no detectable AQP9 immunosignal. The present data provide conclusive evidence for the presence of tetrameric AQP9 in brain and for the expression of AQP9 in neurons.
Journal of Neuroscience Research 01/2009; 87(6):1310-22. · 2.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aquaporin-4 (AQP4) has been shown to be important in the evolution of stroke-associated cerebral edema. However, the role of AQP4 in stroke-associated cerebral edema as it pertains to sex has not been previously studied. The perivascular pool of AQP4 is important in the influx and efflux of water during focal cerebral ischemia. We used mice with targeted disruption of the gene encoding alpha-syntrophin (alpha-Syn(-/-)) that lack the perivascular AQP4 pool but retain the endothelial pool of this protein. Infarct volume at 72 h after transient focal ischemia (90 mins) in isoflurane-anesthetized mice was attenuated in both sexes with alpha-Syn deletion as compared with their wild-type (WT) counterparts. There were no sex differences in hemispheric water content in WT and alpha-Syn(-/-) mice or regional AQP4 expression in WT mice. In neither sex did alpha-Syn deletion lead to alterations in end-ischemic regional cerebral blood flow (rCBF). These data suggest that after experimental stroke: (1) there is no difference in stroke-associated cerebral edema based on sex, (2) AQP4 does not involve in sex-based differences in stroke volume, and (3) perivascular pool of AQP4 has no significant role in end-ischemic rCBF.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 12/2008; 28(12):1898-906. · 5.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Osmotherapy with hypertonic saline ameliorates cerebral edema associated with experimental ischemic stroke. We tested the hypothesis that hypertonic saline exerts its antiedema effect by promoting an efflux of water from brain via the perivascular aquaporin-4 pool. We used mice with targeted disruption of the gene encoding alpha-syntrophin (alpha-Syn(-/-)) that lack the perivascular aquaporin-4 pool but retain the endothelial pool of this protein.
Prospective laboratory animal study.
Research laboratory in a university teaching hospital.
Halothane-anesthetized adult male wildtype C57B/6 and alpha-Syn(-/-) mice were subjected to 90 min of transient middle cerebral artery occlusion and treated with either a continuous intravenous infusion of 0.9% saline or 3% hypertonic saline (1.5 mL/kg/hr) for 48 hr. In the first series of experiments (n = 59), increased brain water content analyzed by wet-to-dry ratios in the ischemic hemisphere of wildtype mice was attenuated after hypertonic saline (79.9% +/- 0.5%; mean +/- SEM) but not after 0.9% saline (82.3% +/- 1.0%) treatment. In contrast in alpha-Syn(-/-) mice, hypertonic saline had no effect on the postischemic edema (hypertonic saline: 80.3% +/- 0.7%; 0.9% saline: 80.3% +/- 0.4%). In the second series of experiments (n = 32), treatment with hypertonic saline attenuated postischemic blood-brain barrier disruption at 48 hr in wildtype mice but not in alpha-Syn(-/-) mice; alpha-Syn(-/-) deletion alone had no effect on blood-brain barrier integrity. In the third series of experiments (n = 34), alpha-Syn(-/-) mice treated with either hypertonic saline or 0.9% saline had smaller infarct volume as compared with their wildtype counterparts.
These data demonstrate that 1) osmotherapy with hypertonic saline exerts antiedema effects via the perivascular pool of aquaporin-4, 2) hypertonic saline attenuates blood-brain barrier disruption depending on the presence of perivascular aquaporin-4, and 3) deletion of the perivascular pool of aquaporin-4 alleviates tissue damage after stroke, in mice subjected to osmotherapy and in nontreated mice.
Critical care medicine 10/2008; 36(9):2634-40. · 6.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Taurine is an abundant organic osmolyte with antioxidant and immunomodulatory properties. Its role in the pathogenesis of chronic liver disease is unknown. The liver phenotype was studied in taurine transporter knockout (taut-/-) mice. Hepatic taurine levels were ~21, 15 and 6 mumol/g liver wet weight in adult wild-type, heterozygous (taut+/-) and homozygous (taut-/-) mice, respectively. Immunoelectronmicroscopy revealed an almost complete depletion of taurine in Kupffer and sinusoidal endothelial cells, but not in parenchymal cells of (taut-/-) mice. Compared with wild-type mice, (taut-/-) and (taut+/-) mice developed moderate unspecific hepatitis and liver fibrosis with increased frequency of neoplastic lesions beyond 1 year of age. Liver disease in (taut-/-) mice was characterized by hepatocyte apoptosis, activation of the CD95 system, elevated plasma TNF-alpha levels, hepatic stellate cell and oval cell proliferation, and severe mitochondrial abnormalities in liver parenchymal cells. Mitochondrial dysfunction was suggested by a significantly lower respiratory control ratio in isolated mitochondria from (taut-/-) mice. Taut knockout had no effect on taurine-conjugated bile acids in bile; however, the relative amount of cholate-conjugates acid was decreased at the expense of 7-keto-cholate-conjugates. In conclusion, taurine deficiency due to defective taurine transport triggers chronic liver disease, which may involve mitochondrial dysfunction.
The FASEB Journal 04/2006; 20(3):574-6. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aquaporins are a family of water channels found in animals, plants, and microorganisms. A subfamily of aquaporins, the aquaglyceroporins, are permeable for water as well as certain solutes such as glycerol, lactate, and urea. Here we show that the brain contains two isoforms of AQP9--an aquaglyceroporin with a particularly broad substrate specificity--and that the more prevalent of these isoforms is expressed in brain mitochondria. The mitochondrial AQP9 isoform is detected as an approximately 25 kDa band in immunoblots. This isoform is likely to correspond to a new AQP9 mRNA that is obtained by alternative splicing and has a shorter ORF than the liver isoform. Subfractionation experiments and high-resolution immunogold analyses revealed that this novel AQP9 isoform is enriched in mitochondrial inner membranes. AQP9 immunopositive mitochondria occurred in astrocytes throughout the brain and in a subpopulation of neurons in the substantia nigra, ventral tegmental area, and arcuate nucleus. In the latter structures, the AQP9 immunopositive mitochondria were located in neurons that were also immunopositive for tyrosine hydroxylase, as demonstrated by double labeling immunogold electron microscopy. Our findings suggest that mitochondrial AQP9 is a hallmark of astrocytes and midbrain dopaminergic neurons. In physiological conditions, the flux of lactate and other metabolites through AQP9 may confer an advantage by allowing the mitochondria to adjust to the metabolic status of the extramitochondrial cytoplasm. We hypothesize that the complement of mitochondrial AQP9 in dopaminergic neurons may relate to the vulnerability of these neurons in Parkinson's disease.
The FASEB Journal 10/2005; 19(11):1459-67. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: During chronic metabolic acidosis (CMA), the plasma levels of glutamine are increased and so is glutamine metabolism in the kidney tubule cells. Degradation of glutamine results in the formation of ammonium (NH(4)(+)) and bicarbonate (HCO(3)(-)) ions, which are excreted in the pre-urine and transported to the peritubular blood, respectively. This process contributes to counteract acidosis and to restore normal pH, but the molecular mechanism, the localization of the proteins involved and the regulation of glutamine transport into the renal tubular cells, remains unknown. SN1, a Na(+)- and H(+)-dependent glutamine transporter has previously been identified molecularly, and its mRNA has been detected in tubule cells in the medulla of the kidney. Now shown is the selective targeting of the protein to the basolateral membranes of the renal tubule cells of the S3 segment throughout development of the normal rat kidney. During CMA, SN1 expression increases five- to six-fold and appears also in cortical tubule cells in parallel with the increased expression and activity of phosphate-activated glutaminase, a mitochondrial enzyme involved in ammoniagenesis. However, SN1 remains sorted to the basolateral membranes. The unique ability of SN1 to change transport direction according to physiologic changes in transmembrane gradients of [glutamine] and pH and its sorting to the basolateral membranes and the presence of a putative pH responsive element in the 3' untranslated region (UTR) of the gene (supported here by the demonstration in CMA kidney of a protein that binds SN1 mRNA) are conducive to the function of this transporter in pH regulation.
Journal of the American Society of Nephrology 05/2005; 16(4):869-77. · 8.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: The formation of brain edema, commonly occurring as a potentially lethal complication of acute hyponatremia, is delayed following knockout of the water channel aquaporin-4 (AQP4). Here we show by high-resolution immunogold analysis of the blood-brain-barrier that AQP4 is expressed in brain endothelial cells as well as in the perivascular membranes of astrocyte endfeet. A selective removal of perivascular AQP4 by alpha-syntrophin deletion delays the buildup of brain edema (assessed by Diffusion-weighted MRI) following water intoxication, despite the presence of a normal complement of endothelial AQP4. This indicates that the perivascular membrane domain, which is peripheral to the endothelial blood-brain barrier, may control the rate of osmotically driven water entry. This study is also the first to demonstrate that the time course of edema development differs among brain regions, probably reflecting differences in aquaporin-4 distribution. The resolution of the molecular basis and subcellular site of osmotically driven brain water uptake should help design new therapies for acute brain edema.
The FASEB Journal 04/2004; 18(3):542-4. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Brain function is inextricably coupled to water homeostasis. The fact that most of the volume between neurons is occupied by glial cells, leaving only a narrow extracellular space, represents an important challenge, as even small extracellular volume changes will affect ion concentrations and therefore neuronal excitability. Further, the ionic transmembrane shifts that are required to maintain ion homeostasis during neuronal activity must be accompanied by water. It follows that the mechanisms for water transport across plasma membranes must have a central part in brain physiology. These mechanisms are also likely to be of pathophysiological importance in brain oedema, which represents a net accumulation of water in brain tissue. Recent studies have shed light on the molecular basis for brain water transport and have identified a class of specialized water channels in the brain that might be crucial to the physiological and pathophysiological handling of water.