Maria Svelto

Aquaporin-4 deficiency in skeletal muscle and brain of dystrophic mdx mice

We report a detailed study of AQP4 expression in the neuromuscular system of mdx mice. Immunocytochemical analysis performed by double immunostaining revealed that mdx mice manifest a progressive reduction in AQP4 at the sarcolemmal level of skeletal muscle fast fibers and that type IIB fibers are the first to manifest this reduction in AQP4 expression. No labeling was observed in the cytoplasm of muscle fibers, indicating that the reduction in sarcolemma staining is not associated with an intracellular compartmentalization of mistargeted protein. By Western blot and RT‐PCR analysis, we found that whereas the total content of AQP4 protein decreased (by 90% in adult mdx mice), mRNA levels for AQP4 remained unchanged. A similar age related reduction in AQP4 expression was found in brain astrocytic end‐feet surrounding capillaries of mdx mice. Morphometric analysis performed after immunogold electron microscopy indicated a reduction of ~85% in gold particles (32±2/μm vs. 4.7±0.61/μm). Western blot experiments conducted using membrane fractions from brain cortex revealed a strong reduction (of 70%) in AQP4 protein in adult mdx mice, and RT‐PCR experiments demonstrated that the reduction was not at transcription level. More interesting was the finding that AQP4 reduction was associated with swelling of astrocytic perivascular processes whose ultrastructural modifications are commonly indicated as an important and early event in the development of brain edema. No apparent reduction in AQP4 was found in mdx stomach and kidney. Our data provide evidence that dystrophin deficiency in mdx mice leads to disturbances in AQP4 assembly in the plasma membrane of fast skeletal muscle fibers and brain astrocytic end‐feet, suggesting that changes in the osmotic equilibrium of the neuromuscular apparatus may be involved in the pathology of muscular dystrophy.—Frigeri, A., Nicchia, G. P., Nico, B., Quondamatteo, F., Herken, R., Roncali, L., Svelto, M. Aquaporin‐4 deficiency in skeletal muscle and brain of dystrophic mdx mice. FASEB J. 15, 90–98 (2001)

The role of aquaporin-4 in the blood-brain barrier development and integrity: studies in animal and cell culture models.

Aquaporin-4 (AQP4) is the major water channel expressed in brain perivascular astrocyte processes. Although the role of AQP4 in brain edema has been extensively investigated, little information exists regarding its functional role at the blood-brain barrier (BBB). The purpose of this work is to integrate previous and recent data regarding AQP4 expression during BBB formation and depending on BBB integrity, using several experimental models. Results from studies on the chick optic tectum, a well-established model of BBB development, and the effect of lipopolysaccharide on the BBB integrity and on perivascular AQP4 expression have been analyzed and discussed. Moreover, data on the BBB structure and AQP4 expression in murine models of Duchenne muscular dystrophy are reviewed. In particular, published results obtained from mdx(3cv) mice have been analyzed together with new data obtained from mdx mice in which all the dystrophin isoforms including DP71 are strongly reduced. Finally, the role of the endothelial component on AQP4 cellular expression and distribution has been investigated using rat primary astrocytes and brain capillary endothelial cell co-cultures as an in vitro model of BBB.

Severe Alterations of Endothelial and Glial Cells in the Blood-Brain Barrier of Dystrophic mdx Mice

In this study, we investigated the involvement of the blood‐brain barrier (BBB) in the brain of the dystrophin‐deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO‐1) and claudin‐1 proteins, and a glial marker, the aquaporin‐4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron‐dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO‐1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO‐1 immunocytochemistry and claudin‐1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO‐1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO‐1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of α‐actin and ZO‐1 revealed colocalization of these proteins. As in ZO‐1 staining, the pattern of immunolabeling with anti–α‐actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. α‐actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas α‐actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti‐AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4‐, CD8‐, CD20‐, and CD68‐positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in α‐actin cytoskeleton, ZO‐1, claudin‐1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD. GLIA 42:235–251, 2003.

cAMP-induced AQP2 translocation is associated with RhoA inhibition through RhoA phosphorylation and interaction with RhoGDI

We have recently demonstrated that inhibition of Rho GTPase with Clostridium difficile toxin B, or with Clostridium botulinum C3 toxin, causes actin depolymerization and translocation of aquaporin 2 (AQP2) in renal CD8 cells in the absence of hormonal stimulation. Here we demonstrate that Rho inhibition is part of the signal transduction cascade activated by vasopressin leading to AQP2 insertion into the apical membrane. Quantitation of active RhoA (GTP-bound) by selective pull down experiments demonstrated that the amount of active RhoA decreased upon stimulation of CD8 cells with the cAMP-elevating agent forskolin. Consistent with this observation, forskolin treatment resulted in a decreased expression of membrane-associated (active) Rho, as assessed by cell fractionation followed by western blotting analysis. In addition, the abundance of the endogenous Rho GDP dissociation inhibitor (Rho-GDI) was found to have decreased in the membrane fraction after forskolin stimulation. Co-immunoprecipitation experiments revealed that, after forskolin stimulation, the amount of Rho-GDI complexed with RhoA increased, suggesting that Rho GTPase inhibition occurs through association of RhoA with Rho-GDI. Finally, forskolin stimulation was associated with an increase in Rho phosphorylation on a serine residue, a protein modification known to stabilize the inactive form of RhoA and to increase its interaction with Rho-GDI. Taken together, these data demonstrate that RhoA inhibition through Rho phosphorylation and interaction with Rho-GDI is a key event for cytoskeletal dynamics controlling cAMP-induced AQP2 translocation.

Aquaporin-4 orthogonal arrays of particles are the target for neuromyelitis optica autoantibodies

Neuromyelitis optica (NMO) is an inflammatory autoimmune demyelinating disease of the central nervous system (CNS) which in autoantibodies produced by patients with NMO (NMO‐IgG) recognize a glial water channel protein, Aquaporin‐4 (AQP4) expressed as two major isoforms, M1‐ and M23‐AQP4, in which the plasma membrane form orthogonal arrays of particles (OAPs). AQP4‐M23 is the OAP‐forming isoform, whereas AQP4‐M1 alone is unable to form OAPs. The function of AQP4 organization into OAPs in normal physiology is unknown; however, alteration in OAP assemblies is reported for several CNS pathological states. In this study, we demonstrate that in the CNS, NMO‐IgG is able to pull down both M1‐ and M23‐AQP4 but experiments performed using cells selectively transfected with M1‐ or M23‐AQP4 and native tissues show NMO‐IgG epitope to be intrinsic in AQP4 assemblies into OAPs. Other OAP‐forming water‐channel proteins, such as the lens Aquaporin‐0 and the insect Aquaporin‐cic, were not recognized by NMO‐IgG, indicating an epitope characteristic of AQP4‐OAPs. Finally, water transport measurements show that NMO‐IgG treatment does not significantly affect AQP4 function. In conclusion, our results suggest for the first time that OAP assemblies are required for NMO‐IgG to recognize AQP4.

A Multidisciplinary Evaluation of the Effectiveness of Cyclosporine A in Dystrophic Mdx Mice

Chronic inflammation is a secondary reaction of Duchenne muscular dystrophy and may contribute to disease progression. To examine whether immunosuppressant therapies could benefit dystrophic patients, we analyzed the effects of cyclosporine A (CsA) on a dystrophic mouse model. Mdx mice were treated with 10 mg/kg of CsA for 4 to 8 weeks throughout a period of exercise on treadmill, a protocol that worsens the dystrophic condition. The CsA treatment fully prevented the 60% drop of forelimb strength induced by exercise. A significant amelioration (P < 0.05) was observed in histological profile of CsA-treated gastrocnemius muscle with reductions of nonmuscle area (20%), centronucleated fibers (12%), and degenerating area (50%) compared to untreated exercised mdx mice. Consequently, the percentage of normal fibers increased from 26 to 35% in CsA-treated mice. Decreases in creatine kinase and markers of fibrosis were also observed. By electrophysiological recordings ex vivo, we found that CsA counteracted the decrease in chloride conductance (gCl), a functional index of degeneration in diaphragm and extensor digitorum longus muscle fibers. However, electrophysiology and fura-2 calcium imaging did not show any amelioration of calcium homeostasis in extensor digitorum longus muscle fibers. No significant effect was observed on utrophin levels in diaphragm muscle. Our data show that the CsA treatment significantly normalized many functional, histological, and biochemical endpoints by acting on events that are independent or downstream of calcium homeostasis. The beneficial effect of CsA may involve different targets, reinforcing the usefulness of immunosuppressant drugs in muscular dystrophy.

Expression and immunolocalization of the aquaporin-8 water channel in rat gastrointestinal tract

A remarkable amount, of water is transported in the gastrointestinal (GI) organs to fulfil the secretory and absorptive functions of the GI tract. However, the molecular basis of water movement in the GI epithelial barriers is still poorly known. Important clues about the mechanisms by which water is transported in the GI tract were provided by the recent identification of multiple aquaporin water channels expressed in GI tissues. Here we define the mRNA and protein expression and the cellular and subcellular distribution of aquaporin-8 (AQP8) in the rat GI tract. By semi-quantitative RT-PCR the AQP8 mRNA was detected in duodenum, proximal jejunum, proximal colon, rectum, pancreas and liver and, to a lesser extent, in stomach and distal colon. Immunohistochemistry using affinity-purified antibodies revealed AQP8 staining in the absorptive epithelial cells of duodenum, proximal jejunum, proximal colon and rectum where labeling was largely intracellular and confined to the subapical cytoplasm. Confirming previous results, AQP8 staining was seen at the apical pole of pancreatic acinar cells. Interestingly, both light and immunoelectron microscopy analyses showed AQP8 reactivity in liver where labeling was associated to hepatocyte intracellular vesicles and over the plasma membrane delimiting the bile canaliculi. A complex pattern was observed by immunoblotting with total membranes of the above GI organs incubated with affinity-purified anti-AQP8 antibodies which revealed multiple bands with molecular masses ranging between 28 and 45 kDa. This immunoblotting pattern was not modified after deglycosylation with N-glycosidase F except the 34-kDa band of liver that, as already reported, was partially down-shifted to 28 kDa. No bands were detected after preadsorption of the anti-AQP8 antibodies with the immunizing peptide. The cellular and subcellular distribution of AQP8 suggest physiological roles for this aquaporin in the absorption of water in the intestine and the secretion of bile and pancreatic juice in liver and pancreas, respectively. The large intracellular expression of AQP8 may indicate its recycling between the cytoplasmic compartment and the plasma membrane. The cytoplasmic localization observed may also relate to the involvement of AQP8 in processes of intracellular osmoregulation.

Inhibition of aquaporin-4 expression in astrocytes by RNAi determines alteration in cell morphology, growth, and water transport and induces changes in ischemia-related genes

Recent studies indicate a key role of aquaporin (AQP) 4 in astrocyte swelling and brain edema and suggest that AQP4 inhibition may be a new therapeutic way for reducing cerebral water accumulation. To understand the physiological role of AQP4‐mediated astroglial swelling, we used 21‐nucleotide small interfering RNA duplexes (siRNA) to specifically suppress AQP4 expression in astrocyte primary cultures. Semiquantitative RT‐PCR experiments and Western blot analysis showed that AQP4 silencing determined a progressive and parallel reduction in AQP4 mRNA and protein. AQP4 gene suppression determined the appearance of a new morphological cell phenotype associated with a strong reduction in cell growth. Water transport measurements showed that the rate of shrinkage of AQP4 knockdown astrocytes was one‐half of that of controls. Finally, cDNA microarray analysis revealed that the gene expression pattern perturbed by AQP4 gene silencing concerned ischemia‐related genes, such as GLUT1 and hexokinase. Taken together, these results indicate that 1) AQP4 seems to be the major factor responsible for the fast water transport of cultured astrocytes; 2) as in skeletal muscle, AQP4 is a protein involved in cell plasticity; 3) AQP4 alteration may be a primary factor in ischemia‐induced cerebral edema; and 4) RNA interference could be a new potent tool for studying AQP pathophysiology in those organs and tissues where they are expressed.

Tissue Distribution and Membrane Localization of Aquaporin-9 Water Channel Evidence for Sex-linked Differences in Liver

Aquaporin-9 (AQP9) is a water channel membrane protein also permeable to small solutes such as urea, glycerol, and 5-fluorouracil, a chemotherapeutic agent. With the aim of understanding the pathophysiological role of AQP9, we performed an extensive analysis by Western blotting, RT-PCR, and immunolocalization in rat tissues. Western blotting analysis revealed a major band of approximately 32 kD in testis, liver, and brain. Immunofluorescence showed strong expression of AQP9 in the plasma membrane of testis Leydig cells. In liver, AQP9 expression was found to be sex-linked. Male rats had higher levels of AQP9 than female in terms of both protein and mRNA. Moreover, in female livers the expression of AQP9 was mostly confined to perivascular hepatocytes, whereas males showed a more homogeneous hepatocyte staining. No differences in AQP9 expression level related to the age or to protein content of the diet were found, indicating that differences in the liver may be gender-dependent. In the brain, AQP9 expression was found in tanycytes mainly localized in the areas lacking a blood-brain barrier (BBB), such as the circumventricular organs (CVOs) of the third ventricles, the subfornical organ, the hypothalamic regions, and the glial processes of the pineal gland. AQP9 expression in the osmosensitive region of the brain suggests a role in the mechanism of central osmoreception. All these findings show a unique tissue distribution of AQP9 compared to the other known aquaporins.

Microvessel overexpression of aquaporin 1 parallels bone marrow angiogenesis in patients with active multiple myeloma

The erythrocyte water channel aquaporin 1 (AQP1) is expressed in multiple absorptive and secretory epithelia including the capillary endothelia. Immunoblot analysis showed that bone marrow biopsies of patients with active multiple myeloma (MM) display significantly higher levels of AQP1 than those from patients with non‐active MM, whose values are higher, but to a lesser extent, than those of patients with monoclonal gammopathies of undetermined significance (MGUS). Values of MGUS overlapped those of patients with anaemia as a result of iron or vitamin B12 deficiencies (called ‘benign anaemias’). Immunohistochemistry and computerized image analysis of AQP1 highlighted bone marrow microvessels whose area per microscopic field was significantly greater in patients with active MM, and always larger than and closely correlated with the microvessel area when assessed with factor VIII‐related antigen/von Willebrands factor (FVIII–VWF). The intensity of AQP1 expression by microvessels evaluated using image analysis was significantly greater in active than non‐active MM and in the latter over MGUS or benign anaemias. It is suggested that, among plasma cell tumours, AQP1 expression is preferentially associated with microvessels of MM and that the highest degree of expression occurs in active MM in step with enhanced angiogenesis, in which AQP1 recognizes more immature neovessels than FVIII–VWF. It may, perhaps, favour angiogenesis in a positive loop and, hence, MM progression, and thus be applied for therapeutic vascular targeting.