Drosophila glia use a conserved cotransporter mechanism to regulate extracellular volume

Abstract

The nervous system is protected by blood barriers that use multiple systems to control extracellular solute composition, osmotic pressure, and fluid volume. In the human nervous system, misregulation of the extracellular volume poses serious health threats. Here, we show that the glial cells that form the Drosophila blood-nerve barrier have a conserved molecular mechanism that regulates extracellular volume: the Serine/Threonine kinase Fray, which we previously showed is an ortholog of mammalian PASK/SPAK; and the Na-K-Cl cotransporter Ncc69, which we show is an ortholog of human NKCC1. In mammals, PASK/SPAK binds to NKCC1 and regulates its activity. In Drosophila, larvae mutant for Ncc69 develop a peripheral neuropathy, where fluid accumulates between glia and axons. The accumulation of fluid has no detectable impact on action potential conduction, suggesting that the role of Ncc69 is to maintain volume or osmotic homeostasis. Drosophila Ncc69 has kinetics similar to human NKCC1, and NKCC1 can rescue Ncc69, suggesting that they function in a conserved physiological mechanism. We show that fray and Ncc69 are coexpressed in nerve glia, interact in a yeast-two-hybrid assay, and have an essentially identical bulging nerve phenotype. We propose that normally functioning nerves generate extracellular solutes that are removed by Ncc69 under the control of Fray. This mechanism may perform a similar role in humans, given that NKCC1 is expressed at the blood-brain barrier.

Figure 1

NCC69 is a member of the Drosophila N(K)CC family. A: A cladogram showing the relationship between the Drosophila (red) and human (black) cation chloride cotransporters (CCCs). Humans have three Na-(K)-Cl cotransporters: one Na-Cl and two Na-K-Cl cotransporters, denoted NCC and NKCC, respectively. Drosophila has two related cotransporters, denoted NCC69 and NCC83, but the phylogenetic analysis fails to reveal to which class (NCC or NKCC) these proteins belong. B: A model of human NKCC1 showing homology to NCC69. The color-coding shows the similarity to NCC69. The residues annotated with P’s are conserved phosphorylation sites that are important for NKCC1 activation in the shark (Darman and Forbush 2002; Vitari et al. 2006). C: Map of the NCC69 genomic region. Two transcripts are predicted, which encode the same protein of 1,171 amino acid residues. The deleted regions are shown of r1 and r2, two alleles that were used in this study, which span 537 and 8,743 bp, respectively.

Figure 2

NCC69 is expressed and required in subperineurial glia. A: Drosophila whole embryo, stage 16, labeled with anti-HRP to reveal neurons and neuronal projections (green) and anti-Repo to label glial nuclei (magenta). At this magnification, the nerves (arrowheads) can be easily identified linking the CNS to the periphery in a segmentally repeated and bilaterally symmetric pattern. Bar: 50 microns. B, C: Higher magnification of region boxed in A showing the double label B and the anti-Repo (magenta) channel C. At this magnification, the labeling reveals individual nuclei (arrows) of glial cells that are closely associated with the axons. These glial cells have been shown to express fray and Gliotactin, and will give rise to the subperineurial glia in larval nerves. Bar: 20 microns. D, E: Drosophila whole embryo, stage 16, labeled with anti-HRP (green) and probed with NCC69 anti-sense DNA (magenta). The nerve glia express the NCC69 mRNA (arrowheads), visible in the double-labeled image D and the NCC69 (magenta) channel E. Bar: 20 microns. F, G: Fillet preps from 3^rd instar larvae, stained with DAB anti-HRP to label the nervous system. Wild type F nerves appear uniform in width (arrowheads). By contrast, NCC69 G mutant nerves have localized bulges and swellings (arrowheads). Fillet preps from 3^rd instar larvae expressing an RNAi construct targeted against NCC69 in neurons H or subperineurial glia I. When the RNAi construct is expressed in neurons, the nerves appear wild type (compare with F). Expression of the RNAi construct in nerve glia causes bulges in the nerves G. Genotypes: F, CS. G, NCC69[r1]. H, w, C155-GAL4/+; UAS-RNAi(NCC69)/+. I, w; Gli-GAL4/+; UAS-RNAi(NCC69). F–I are to the same scale (Bar = 20 microns).

Figure 3. Evoked action potential activity is unaffected in NCC69 mutants

Excitatory junctional currents (EJCs) were recorded from specific ventral longitudinal muscle fibers of a wild type (CS) and an NCC69^r2 larva, in response to trains of stimuli at 10 and 50 Hz evoked by shocking the nerve at a distance of at least 500 micrometers from the innervated segment. This ensured that in NCC69 mutants, the resulting action potentials would encounter bulges as they traveled toward the nerve terminal. The failure of an action potential to arrive at the nerve terminal results in a missing EJC (asterisks above the wild type trace at 50 Hz). The all-or-nothing response indicates that the EJCs are a reliable indicator of action potential propagation. The shock artifacts, which appeared as upward deflections on the traces, have been clipped for easier viewing.

Figure 4

In vitro analysis of NCC69 cotransporter function. A: Anti-HA staining of 1B4, a stable SL2 cell line transfected with a construct to express HA:NCC69. A high proportion (ca. 85%) of the cells express HA:NCC69 at varying levels. The construct is detected at the cell membrane (inset), as well as in intracellular compartments. Bar: 10 microns (inset, 5 microns). B: Assays of ⁸⁶Rb flux in SL2 transfected cell lines expressing HA:NCC69 versus IZT (vector alone). Cell lines were subjected to four different “activation” conditions: isotonic (control), hypertonic, hypotonic, and 0 Cl isotonic media. In the three activation conditions, the HA:NCC69-transfected cell line responded with a greater ⁸⁶Rb flux than the control. The flux was inhibited by bumetanide, a compound that inhibits cotransporters from humans and other species. C–F: Dependence of ⁸⁶Rb flux in HA:NCC69 cells on bumetanide C, Na D, Rb E, and Cl F concentrations. The results are shown for a representative experiment with individual data points and a curve fit using a non-linear least squares algorithm. For Na and Rb, the data best fit using a model that assumes a single ligand-binding site; for Cl, the best fit is consistent with two ligand-binding sites. The data points shown as black diamonds with error bars represent the average of four replicates with the standard error about the mean. Where the error bars are not visible, the standard error is smaller than the symbols.

Figure 5

Human NKCC1 can substitute for NCC69 in vivo. 3^rd instar fillets stained with anti-HRP, A–C or anti-HA D–F. A, D: NCC69. Many nerve bulges are visible (arrowheads in A), corresponding to the accumulation of extracellular fluid between glia and axons. Staining of this prep with anti-HA D serves as a negative control for the rescue experiments. Genotype: w; NCC69^r2. B, E: Fly rescue. Expressing the NCC69 cDNA in subperineurial glia in NCC69 mutants reduces the nerve swelling considerably (arrowheads in B). Staining with anti-HA E reveals ample expression of the HA:NCC69 transgene in the nerve. Genotype: w; Gli-GAL4; NCC69^r2, UAS-HA:NCC69. C, F: Human rescue. Expressing the human NKCC1 cDNA in subperineurial glia in NCC69 mutants also reduces nerve swelling (arrowheads in C), demonstrating that these two genes encode functional orthologs. Genotype: w; Gli-GAL4; UAS-HA:NKCC1 NCC69^r1. Bar: 50 microns; A–F are all to the same scale.

Figure 6

Quantitation of the nerve phenotype. A: Cumulative frequency graphs of nerve widths. Curves from animals with a higher proportion of large nerve widths are shifted to the right; those with a lower proportion are shifted to the left. The rightmost curve is for NCC69. The fly rescue (expressing HA:NCC69 in the nerve glia in NCC69 mutants) shifts the curve leftward to overlap the wild type curve. The human rescue (expressing HA:NKCC1 in the nerve glia of NCC69 mutants) shifts the curve leftward as well, but for the most part does not overlap the wild type curve. The NCC69 distribution is significantly different from the others (p<0.005; see Results and Table S6). B: Percentiles from the distributions shown in A. The 50^th, 75^th, 80^th, 85^th and 90^th percentiles are shown. In each case, NCC69 has a value that shows a statistically significant difference from the values of the other genotypes (p<10⁻⁴; see Results and Table S7). C: Average cross-section area of the nerves from the genotypes shown in A. The average cross-section area from NCC69 is significantly larger than the other genotypes (one-tail t test p<0.005; Table 3).

Figure 7

NCC69 and Fray interact by yeast-two-hybrid assay and have strikingly similar mutant nerve phenotypes. A: Yeast-two-hybrid interactions between NCC69 and Fray. B–D: Transmission electron micrographs of nerves from 3^rd instar larvae. In wild type B, axons (“A”) and glial cell processes (“G” and arrowheads) are closely associated, with little extracellular space. By contrast, in NCC69 C and fray D mutant nerve bulges, axons and glia may be separated by a large amount of extracellular space (“E”). Bar in D: 1 micron; B–D are to the same scale. Genotypes: B, CS. C, w; NCC69^r2. D, ry fray^r1.

Figure 8

A model of NCC69 function in larval nerves, showing how the Na/K ATPase could create the need for solute removal from the extracellular space in larval nerves. The diagram shows a simplified view of the nerve, consisting of an axon and the subperineurial glia, whose septate junctions (SJ) restrict paracellular flow. The model leaves out two classes of glia, the wrapping glia, which are located inside the nerve, and the perineurial cells which are located on the outside, because they do not form septate junctions, and would not pose much of a barrier to paracellular ion flow. The ion flows in the diagram are represented by arrows. Several known ion flows (e.g., I_h and I_Na) have been omitted for the sake of simplicity. 1: The flow of ions is initiated by the action potential which leaves increased extracellular K in its wake through voltage-gated K channels. 2: In each cycle, the Na/K ATPase removes 2 K ions from the extracellular space, and replaces them with 3 Na ions. 3: Cl ions move to balance the gain in positive charge. These Cl ions could flow from other parts of the extracellular space and/or from intracellular sources, e.g., through Cl channels or transporters. The net effect of the Na/K ATPase is to accumulate NaCl in the extracellular space. If left unchecked (as in NCC69 mutants), the accumulation of NaCl draws water into the extracellular space through osmosis, causing swelling. 4: NCC69 relieves the pressure by transporting solutes into the subperineurial glia, causing it to swell. 5: The subperineurial cell exports solutes, presumably into the hemolymph, to maintain volume homeostasis. 6: K flows down the axoplasm to replace the K that is lost.