Effect of Dp71 deficiency on the oxytocin hypothalamic axis in osmoregulation function in mice.

Dp71 is the major form of dystrophins (Dp) in the supraoptic nucleus (SON) and in the neural lobe of hypophysis (NL/HP). Dp71-null mice exhibit a hypo-osmolar status attributed to an altered osmosensitivity of the SON and to a perturbed vasopressinergic axis. Because oxytocin (OT) is implicated in osmoregulation via natriuresis, this study explored the oxytocinergic axis in Dp71-null mice after salt-loading (SL). Under normosmolar conditions, OT-mRNA expression was higher in the Dp71-null SON compared to wild-type (wt) and the OT peptide level has not changed. Dp-immunostaining was localized in astrocytes end-feet surrounding vessels in wt SON. This distribution changed in Dp71-null SON, Dp being detected in OT-soma of MCNs. nNOS and NADPH-diaphorase levels increased in the OT area of the Dp71-null SON compared to wt. In the NL/HP, OT level reduced in Dp71-null mice and Dp localization changed from pituicytes end-feet in wt SON to OT terminals in Dp71-null SON. Salt-Loading resulted in an increase of OT-mRNA and peptide levels in wt SON but had no effect in Dp71-null SON. In the NL/HP, OT content was reduced after SL. For Dp71-null mice, OT level, already low in control, was not modified by SL. Dp level was not affected by SL in the SON nor in the NL/HP. Our data confirmed the importance of Dp71 for the SON functionality in osmoregulation. The localization of Dp71 at the glial-vascular interface could be associated with SON osmosensitivity, leading to an adequate OT synthesis in the SON and release from the NL/HP upon plasmatic hyperosmolality.

Expression and localization of dystrophins and ß-dystroglycan in the hypothalamic supraoptic nuclei of rat from birth to adulthood.

Dystrophins (Dps) are the sub-membranous proteins that work via the dystrophin-associated proteins complex, which comprises ß-dystroglycan (ß-DG), a cell surface receptor for extracellular matrix. Recently, we have revealed ß-DG decrease and central function impairment of supraoptic nucleus (SON) in Dp71 deficient adult mice, opening the question on the profiles of Dps and ß-DG during SON development. At birth and the age of 10, 20 and 60 days, we examined the expression by RT-PCR and Western-blotting, and the distribution by immunohistochemistry of Dps and ß-DG. Also, we analyzed, by immunohistochemistry and Western-blotting, the neuropeptide, arginine vasopressin (AVP), in the SON at the different ages. At birth, Dp71 and to a lesser extends, Dp140 and Dp427, and also ß-DG are revealed in the SON. They are localized in the magnocellular neurons (MCNs), astrocytes and vessels. From birth to adulthood, the AVP raise in the SON coincides with the progressive increase of Dp71 level while the level of Dp140 and Dp427 increased only at D20, D10 post-natal development, respectively, and ß-DG expression did not change. Moreover, the location of Dps or/and ß-DG in the cell compartments was modified during development: at D10, Dps appeared in the astrocytes end-feet surrounding MCNs, and at D20, Dps and ß-DG codistributed in the astrocytes end-feet, surrounding MCNs and vessels. Such a distribution marks the first steps of post-natal SON development and may be considered essential in the establishment of structural plasticity mechanisms in SON, where astrocyte end-feet, vessels, magnocellular neurons, are physiologically associated. The disappearance of ß-DG in the MCNs nucleus marks the adulthood SON and suggests that the complex of Dps associating ß-DG is required for the nucleoskeleton function in the post-natal development.

Dystrophin 71 and α1syntrophin in morpho-functional plasticity of rat supraoptic nuclei: Effect of saline surcharge and reversibly normal hydration.

Dystrophin (Dp) is a multidomain protein that links the actin cytoskeleton to the extracellular matrix through the dystrophin associated proteins complex (DAPC). Dp of 71 kDa (Dp71), corresponding to the COOH-terminal domain of dystrophin, and α1-syntrophin (α1Syn) as the principal component of the DAPC, are strongly expressed in the brain. To clarify their involvement in the central control of osmotic homeostasis, we investigated the effect of 14 days of salt loading (with drinking water containing 2% NaCl) and then reversibly to 30 days of normal hydration (with drinking water without salt), first on the expression by western-blotting and the distribution by immunochemistry of Dp71 and α1Syn in the SON of the rat and, second, on the level of some physiological parameters, as the plasma osmolality, natremia and hematocrit. Dp71 is the most abundant form of dystrophin revealed in the supraoptic nucleu (SON) of control rat. Dp71 was localized in magnocellular neurons (MCNs) and astrocytes, when α1Syn was observed essentially in astrocytes end feet. After 14 days of salt-loading, Dp71 and α1Syn signals decreased and a dual signal for these two proteins was revealed in the astrocytes processes SON surrounding blood capillaries. In addition, salt loading leads to an increase in plasma osmolality, natremia and hematocrit. Reversibly, after 30 days of normal hydration, the intensity of the signal for the two proteins, Dp71 and α1Syn, increased and approached that of control. Furtheremore, the levels of the physiological parameters decreased and approximated those of control. This suggests that Dp71 and α1Syn may be involved in the functional activity of the SON. Their localization in astrocyte end feet emphasizes their importance in neuronal-vascular-astrocyte interactions for the central detection of osmolality. In the SON, Dp71 and α1Syn may be involved in osmosensitivity.

Dp71 gene disruption alters the composition of the dystrophin-associated protein complex and neuronal nitric oxide synthase expression in the hypothalamic supraoptic and paraventricular nuclei.

DP71 is the major cerebral dystrophin isoform and exerts its multiple functions via the dystrophin-associated protein complex (DAPC), also comprised of ß-dystroglycan (ß-DG) and α1-syntrophin (α1-Syn). Since DP71 disruption leads to impairment in the central control of the osmoregulatory axis, we investigated: 1) the DAPC composition in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) of Dp71-null mice; and 2) the expression and activity of neuronal nitric oxide synthase (nNOS), because it is a potential partner of the DAPC and a functional index of osmoregulatory axis activity. In wild-type mice, dystrophins and their autosomal homologs the utrophins, ß-DG, and α1-Syn were localized in astrocyte end feet. In Dp71-null mice, the levels of ß-DG and α1-Syn were lower and utrophin expression did not change. The location of the DAPC in astrocytic end feet suggests that it could be involved in hypothalamic osmosensitivity, which adapts the osmotic response. The altered composition of the DAPC in Dp71-null mice could thus explain why these mice manifest an hypo-osmolar status. In the SON and PVN neurons of Dp71-null mice, nNOS expression and activity were increased. Although we previously established that DP140 is expressed de novo in these neurons, the DAPC remained incomplete due to the low levels of ß-DG and α1-Syn produced in these cells. Our data reveal the importance of DP71 for the constitution of a functional DAPC in the hypothalamus. Such DAPC disorganization may lead to modification of the microenvironment of the SON and PVN neurons and thus may result in a perturbed osmoregulation.

Expression of dystrophins and the dystrophin-associated-protein complex by pituicytes in culture.

The dystrophin-associated-protein complex (DAPC) has been extensively characterized in the central nervous system where it is localized both in neuronal and glial cells. Few studies have characterized this complex in the neurohypophysis. To further study this complex in pituicytes, the resident astroglia of the neurophypophysis, we used adult pituicyte cultures and determined the expression and localization of dystrophins/utrophins and the DAPC by RT-PCR, western blotting and immunofluorescence. Our data show that the pituicytes express dystrophins, utrophins and several members of the DAPC including dystroglycans, δ-, γ-sarcoglycans, α-dystrobrevin-1 and α1-syntrophin. Double immunofluorescence analysis shows that laminin colocalizes with dystroglycan, suggesting that similarly to muscle and astrocytes, the DAPC interacts with the extracellular matrix in pituicytes. Collectively these findings show that dystrophins/utrophins and members of the DAPC are expressed in pituicytes where they may form multiprotein complexes and play a role in the retraction-reinsertion of pituicyte endfeet during specific physiological conditions.

Cellular and subcellular aquaporin-4 distribution in the mouse neurohypophysis and the effects of osmotic stimulation.

Water channel aquaporin-4 (AQP4) is the most abundant water channel in the rodent brain and is mainly expressed in cerebral areas involved in central osmoreception and osmoregulation. The neurohypophysis is the release site of hypothalamic neurohormones vasopressin and oxytocin, which are involved in the regulation of the water balance. The authors investigated the cellular and subcellular distribution of AQP4 in the mouse neurohypophysis before and after chronic osmotic stimulation, using immunofluorescence microscopy and immunoperoxidase electron microscopy. They showed that AQP4 was abundant in the mouse hypophysis, mainly in the neural lobe. AQP4 was discontinuously distributed along pituicytes plasma membranes, in the dense neurosecretory granules and microvesicles of nerve endings and fibers, and along the luminal and abluminal membranes of fenestrated capillary endothelial cells. After chronic osmotic stimulation, AQP4 immunolabeling was enhanced. Taken together, these results suggest that AQP4 could be involved in the pituicyte sensor effect during osmoregulation, the modification and/or maturation mechanism of neurosecretory granules during neurohormone release, and the blood perfusion of the hypophysis.

A deficit of brain dystrophin 71 impairs hypothalamic osmostat.

Patients with Duchenne muscular dystrophy (DMD) and mdx mice, devoid of dystrophin proteins, show altered ionic homeostasis. To clarify dystrophin's involvement in the central control of osmotic stimuli, we investigated the effect of the disruption of Dp71, the major form of dystrophin in the brain, on the hypothalamoneurohypophysis system (HNHS) osmoregulatory response. Dp71 and Dp140 are the principal DMD gene products in the supraoptic nucleus (SON) and neurohypophysis (NH). They are present in astrocyte and pituicyte end-feet, suggesting involvement in both intrinsic osmosensitivity of the SON and vasopressin (AVP) release from the NH. In Dp71-null mice, the cellular distribution of Dp140 was modified, this protein being detected on the membrane of magnocellular soma. The plasma osmolality of Dp71-null mice was lower than that of wild-type mice under normal conditions, and this difference was maintained after salt loading, indicating a change in the set point for osmoregulation in the absence of Dp71. The increase in AVP levels detected in the SON and NH of the wild-type was not observed in Dp71-null mice following salt loading, and the increase in AVP mRNA levels in the SON was smaller in Dp71-null than in wild-type mice. This suggests that Dp71 may be involved in the functional activity of the HNHS. Its astrocyte end-feet localization emphasizes the importance of neuronal-vascular-glial interactions for the central detection of osmolality. In the SON, Dp71 may be involved in osmosensitivity and definition of the "osmostat," whereas, in the neurohypophysis, it may be involved in fine-tuning AVP release.