Stress-induced changes in neuronal Aquaporin-9 (AQP9) in a retinal ganglion cell-line
Introduction
Abnormalities in water balance play an important role in the pathophysiology of a variety of neurological disorders. The discovery of aquaporins (AQPs) has provided a molecular basis for understanding water transport in a number of tissues, including the ocular system [1]. The AQPs are a family of homologous water channel proteins (numbering at least 12 in mammals) that provide the major route for water movement across plasma membranes in a variety of cell types. AQPs are small hydrophobic membrane proteins (∼30 KDa monomer) that assemble in homotetramers and facilitate bi-directional water transport across the plasma membrane in response to osmotic gradients created by solute movement. AQPs 1, 2, 4, 5 and 8 function primarily as water selective transporters; AQPs 3, 7, 9 and 10 (referred to as aquaglyceroporins) also transport small solutes such as glycerol [1].
A number of AQPs are expressed in the eye: AQP0 (MIP) in lens fiber, AQP1 in cornea endothelium, ciliary and lens epithelia, trabecular meshwork and retinal photoreceptor cells, AQP3 in conjunctiva, AQP4 in ciliary epithelium and retinal Muller cells, and AQP5 in corneal and lacrimal gland epithelia [2]. Aquaporins have received attention after reports showing that mice lacking AQP1 have reduced IOP [3] and impaired corneal transparency after swelling [4]. Mice lacking AQP4 have reduced light-evoked potentials by electroretinography. Retinal function and cell survival were significantly improved in AQP4-deficient mice in both inbred (C57/bl6) and outbred (CD1) genetic backgrounds [5]. Therefore, it is suggested that AQP4 deletion in mice is neuroprotective in a transient ischaemia model of retinal injury. There is evidence for impaired cellular processing of AQP5 in lacrimal glands of humans with Sjogren's syndrome [6].
APQ9 was first isolated from adipose tissue [7]. AQP9 mRNA was also detected in leukocytes, testis, liver (highest expression), brain, epididymis duct cells and Leydig cells of rat testes [8] and goblet cells in small intestine [9]. In the rodent brain, AQP9 expression was observed in glial cells (tanycytes and astrocytes) [10], [11], [12], endothelial cells [11] and neurons [11], [13]. Neuronal AQP9 expression was found predominantly in the catecholaminergic neurons [11].
AQP9 possesses general features of a water channel, but in addition is permeable to lactate [14] and a wide variety of non-charged solutes such as: β-hydroxybutyrate, glycerol, carbamides, purines (adenine), pyrimidines (uracil and chemotherapeutic agent 5-fluorouracil), urea, mannitol, and sorbitol but impermeable to cyclic sugars (d-glucose, d-mannose and myo-inositol), the nucleoside uridine, glutamine and glycine [14]. AQP9 shows high homology to AQP3 and AQP7, but less homology with AQP1 [15]. AQP9 has a cysteine at position 213, three residues prior to the second NPA sequence that may explain inhibition by mercury similar to AQP1 [15]. AQP9 protein has protein kinase C and casein kinase II phosphorylation sites [15]. AQP9 mRNA and protein were down-regulated by stimulation of the PKC pathway [16]. By contrast, activation of cAMP-dependent protein kinase (PKA) by dibutyryl cAMP induces an increase in AQP9 mRNA and protein expression in astrocytic cultures [17]. P38 MAP-kinase has been also shown to be involved in increasing AQP4 and AQP9 expression after an osmotic stress [18].
The rodent AQP9 gene contains a negative insulin response element [19] which explains its down regulation by increased insulin levels while up-regulation in diabetic rats [19], [20]. In the liver, AQP9 functions as a glycerol channel allowing glycerol entry then participation in the neoglucogenesis during fasting periods [20]. Glycerol-mediated hepatic glucogenesis accounts for 90% of glucose production in the prolonged fasting state in rodents [21]. In human, it is estimated that ∼20% of the glucogenesis is mediated by glycerol after 60 h fasting [22]. Neuronal AQP9 also plays an important role in osmoregulation as well as energy balance by functioning as a glycerol–lactate channel. Both glycerol and lactate can serve as fuel for neurons and enhanced the recovery of neurons after ischaemic insults [23], [24], [25], [26], [27], [28].
A number of studies have implicated changes in AQP9 in the pathology of neurological diseases. Hypoxia, ischaemia and hyper-osmotic stress are associated with changes in the densities of AQP9 expression. Interestingly, such insults are key risk factors in the development of glaucomatous optic nerve neuropathy. The presence of neuronal AQP9 in the retinal ganglion cells was therefore investigated. AQP9 is present in RGC-5 cells, primary cultures of RGC cells as well as the retinal ganglion cell layer in rats. It is suggested that AQP9 acting as a water channel or a channel for glycerol and lactate (or a neuronal energy sensor) plays a key role following insults or stress that leads to optic nerve degeneration as seen in glaucoma or other diseases associated with optic nerve neuropathy.
Section snippets
Tissue culture
RGC-5 cells, a rat retinal ganglion cell-line, were maintained in DMEM low glucose in T-150 culture flasks (Gibco, Grand Island, NY) supplemented with 44 mM NaHCO3, 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) and antibiotics (Gibco) as described [29]. Cell passages 7–20 were used in this study. Primary RGC cultures were isolated by papain digestion and cell populations were plated on collagen-coated coverslips in neurobasal media (Gibco 10888-022) containing BDNF (50 ng ml−1), CNTF (10
Results
The simultaneous recording of cell volume changes in single cells technique is based on measurements of changes in the concentration of intracellularly fluorescent-trapped dyes such as fura 2 at its isosbestic point. Since dye concentration is inversely proportional to cell volume, measurement of changes in fluorescence intensity at the isosbestic point should report the dynamic changes in cell volume [33]. Cells bathed in isotonic solution then switched a hypotonic solution (20%) exhibited
Conclusion
The current study is first to report the existence of neuronal AQP9 in a retinal ganglion cell-line RGC-5 cells, primary RGC cultures, as well as the retinal ganglion cell layer in rats. AQP9 importance in retinal ganglion cells is suggested by at least two reactive mechanisms. The first is by acting as a lactate-glycerol channel allowing the uptake of excess lactate produced by astrocytes following stress (e.g., ischaemia) and thus providing an alternative fuel for neurons. Second, AQP9 plays
Acknowledgments
This research was supported in part by a grant from the glaucoma foundation to A.D. and an NEI 11179 grant to T.Y.
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2019, Medical HypothesesCitation Excerpt :Fourteen putative HRE sites have been identified in hAQP9 gene, but in cells treated with CoCl2 AQP9 expression decreased [62]. In contrast, Dibas et al. [63] showed an increase in AQP9 mRNA and protein levels in rat retinal ganglion cells (RGC-5) exposed to hypoxia. Taken together, there is a significant amount of experimental evidence supporting the hypothesis that the expression of AQPs is negatively correlated to oxygen levels and that HIF-1 participates in the stimulation of transcription.
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2014, Biochimica et Biophysica Acta - General SubjectsLoss of aquaporin 9 expression adversely affects the survival of retinal ganglion cells
2013, American Journal of PathologyCitation Excerpt :These findings suggest that AQP9 loss did not simply coincide with but rather led to RGC death in our experimental models. Immunoreactivity for neuronal AQP9 on the somata and cell processes has been demonstrated in several studies,10,16,17 and further studies scrutinizing the subcellular localization of this protein have identified that AQP9 is expressed both in mitochondria and plasma membranes.29 Amiry-Moghaddam et al29 reported that a shorter isoform of AQP9 is enriched in the mitochondrial inner membranes of astrocytes throughout the brain and in midbrain dopaminergic neurons.
Age-related changes of aquaporin expression patterns in the postnatal rat retina
2013, Acta HistochemicaCitation Excerpt :Decreased expression of AQP6 was observed at week 12 and 40. The presence of neuronal AQP9 in the retinal ganglion cells was investigated and suggested a role in optic nerve degeneration as seen in glaucoma or other diseases associated with optic nerve neuropathy (Dibas et al., 2007; Naka et al., 2010). In the present study, AQP9 was only observed in the RPE, GCL, OLM and ILM in adult rat retinas.
Diabetes induces expression of aquaporin-0 in the retinal nerve fibers of spontaneously diabetic Torii rats
2011, Experimental Eye ResearchCitation Excerpt :The AQP family is composed of aquaporin, which is selective for water, and aquaglyceroporin, which is also permeable to a wide variety of non-charged solutes, such as lactate and glycerol (Agre and Kozono, 2003; Tait et al., 2008; Verkman et al., 2008). Among the 13 isoforms of the mammalian AQP protein family identified so far, at least four AQPs (AQP-0, -1, -4, and -9) are reportedly expressed in the retina (Fukuda et al., 2010; Nagelhus et al., 1998; Iandiev et al., 2005, 2006, 2007; Kim et al., 1998; Dibas et al., 2007; Naka et al., 2010), and two (AQP-4 and -9) are expressed in the optic nerve (Nagelhus et al., 1998; Li et al., 2002; Naka et al., 2010). Although AQP-0, known also as major intrinsic protein, is the most abundant membrane protein in lens fiber cells (Gorin et al., 1984), two recent papers have reported that AQP-0 is predominantly constitutively expressed in subpopulations of bipolar cells and is expressed less prominently by amacrine cells and retinal ganglion cells (RGCs) in control rats (Iandiev et al., 2007; Farjo et al., 2008).
Altered expression of aquaporins 1 and 4 coincides with neurodegenerative events in retinas of spontaneously diabetic Torii rats
2010, Experimental Eye ResearchCitation Excerpt :AQP1 is normally expressed in the outer retina in photoreceptors and in distinct amacrine cells (Kim et al., 1998; Nagelhus et al., 1998; Iandiev et al., 2005), while AQP4 is expressed predominantly in the perivascular and vitreal end feet of Müller cells and in astrocytes in the inner retina (Nagelhus et al., 1998; Verkman et al., 2008; Goodyear et al., 2009). AQP9 is expressed in catecholaminergic amacinre cells (Iandiev et al., 2006a) and RGCs (Dibas et al., 2007). Among these, AQPs 1 and 4 received much attention, since it has been demonstrated that in the CNS, glial AQPs not only facilitate water flux into and out of the brain parenchyma (Manley et al., 2000) but also modulate neuronal excitability and enhance astrocyte migration (Kong et al., 2008; Saadoun et al., 2005a,b; Tait et al., 2008).