The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood-brain barrier formation and maturation

The blood-brain barrier (BBB) of Drosophila is comprised of a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previously, we identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. Here, we identify cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. We show that PKA is enriched at the basal side of the SPG cell and that this polarized Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity, by precisely regulating the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA’s molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments and not their total length is crucial for generating a proper paracellular seal. Based on these findings, we propose a model that polarized Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity during the continuous morphogenesis of the SPG secondary epithelium, which is critical for maintain tissue size and brain homeostasis during organogenesis.


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The blood-brain barrier (BBB) is a complex physical barrier between the nervous 54 system and the peripheral circulatory system that regulate CNS homeostasis to ensure 55 proper neuronal function. The Drosophila BBB is established by a thin epithelium of 56 subperineural glia (SPG), which ensheath and insulate the nervous system against the 57 potassium-rich hemolymph by forming intercellular septate junctions (SJs) (Bainton et 143 To identify molecules that act downstream of Moody signaling in BBB formation, we 144 examined genes known to be involved in GPCR signaling, such as PkaC1, PI3K, PTEN, 145 PLC, and Rap1. We tested BBB permeability in genomic mutants or transgenic RNAi 146 knockdowns of these GPCR effectors, by injecting a fluorescent dye into the body cavity 147 and determining its penetration into the CNS using confocal imaging. We found that 148 zygotic mutants of the PKA catalytic subunit PkaC1 (originally named DC0 in 149 Drosophila), namely the two null alleles PkaC1 B3 and PkaC1 H2 as well as the 150 hypomorphic allele PkaC1 A13 (Kalderon and Rubin, 1988) show severe CNS insulation 151 defects ( Figure 1A-B), similar in strength to zygotic mutants of the negative regulator 152 loco. By contrast, the removal of the other candidates had no effect (data not shown).

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PkaC1 has both maternal and zygotic components, and its maternal contribution 154 perdures until late embryogenesis (Lane and Kalderon, 1993). The BBB defect we 155 observe could explain the morphologically inconspicuous embryonic lethality of PkaC1 156 zygotic null mutants (Lane and Kalderon, 1993). To rule out the possibility that the 157 observed BBB defects are caused by glial cell fate or migration defects, we examined 158 the presence and position of SPG using an antibody against the pan-glial, nuclear 159 protein Reversed polarity (Repo) (Halter et al., 1995). In PkaC1 zygotic mutants, the full 160 set of SPG is present on the surface of the nerve cord, although the position of the 161 nuclei is more variable than in WT ( Figure 1C), an effect that is also observed in known 162 mutants of the Moody signaling pathway (Granderath et al., 1999;Schwabe et al., 2005). 163 Since SJs are the principal structure providing BBB insulation and are disrupted in  Figure 1D). In contrast, in PkaC1 H2 zygotic mutants, the overall organization of SJs 170 appears perturbed, and their length, as measured in random single sections, is 1F, movies S1 and S2). In WT embryos, SPG are relatively uniform in cell size and 181 shape, and grow to form cell-cell contacts in a highly synchronized manner. By 15.5 h of 182 development, the glial sheet is closed ( Figure 1F). By contrast, SPG in PkaC1 H2 zygotic 183 mutants show increased variability in size and shape, and their spreading and contact 184 formation is less well coordinated. This results in patchy cell-cell contacts with gaps of 185 variable sizes ( Figure 1F). Moreover, the complete closure of the SPG epithelium is 186 delayed compared to WT ( Figure 1F)   and S1); removal of one genomic copy of Gβ13F or loco on their own have no effect. non-significant or no effect in a PkaC1 B3 background, suggesting that they are less 204 dosage-sensitive ( Figure S1).   Procedures). We indeed observed the dye penetrated the blood-eye barrier ( Figure 2F), 234 indicating that PKA is also required for BBB integrity function in the adult.   Figure 4F), confirming the observations from confocal microscopy ( Fig 3A-D), but the 316 SJ belt is discontinuous and appears patchy ( Figure 4B-E). This suggests that it is the 317 continuity of the belt, rather than the total area covered by SJs, that is essential for 318 generating the intercellular sealing capacity. To examine this question directly, we 319 measured SJ length in randomly selected sections. Compared with WT, the mean 320 length of individual SJ segments (0.69 ± 0.08 µm vs. 2.16 ± 0.14 µm, p<0.0001) is 321 12 indeed significantly decreased, while the mean total length of SJs (4.28 ± 0.43 µm vs. 322 2.16 ± 0.14 µm, p= 0.000523) is significantly increased ( Figure 4G).

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Upon reducing PKA activity, the cell contacts and SJ area between neighboring 324 SPGs were reduced, and the SJ belt became patchy as well ( Figure 4A-E). In this case, 325 both the mean total length of SJs (1.49 ± 0.08 µm vs. 2.16 ± 0.14 µm, p= 0.000878) and 326 the mean length of individual SJ segments (0.67 ± 0.14 µm vs. 2.16 ± 0.14 µm, 327 p<0.0001) were significantly shorter than in WT ( Figure 4G). Intriguingly, the ratio of    decreased signaling and appears to cause the leakiness of the BBB in both conditions.

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Our ssTEM-based 3D reconstruction thus demonstrates that the total area covered by 464 SJs and the length of individual contiguous SJ segments are independent parameters.

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The latter appears to be critical for the paracellular seal, consistent with the idea that 466 Moody plays a role in the formation of continuous SJ stands (Babatz et al., 2018).

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The asymmetric localization of PKA that we observed sheds further light on the