Supplementary Materials Supplemental Textiles (PDF) JCB_201907138_sm

Supplementary Materials Supplemental Textiles (PDF) JCB_201907138_sm. sensitivity and provide a stylish readout for determining molecules that must maintain ionic and drinking water homeostasis and a wholesome degree of neuronal excitability. While very much is well known about the main element transporters and stations that mediate the flux of drinking water and ions (Djukic et al., 2007; Jayakumar et al., 2011; Larsen et al., 2014; Leiserson et al., 2011, 2000; MacVicar et al., 2002; Papadopoulos et al., 2004; Rusan et al., 2014; Stenesen et al., 2019; Wu et al., 2014), the mechanisms by which glial cells regulate expression of the relevant transporters are not well understood. Delineating such regulatory mechanisms could identify approaches to leverage glial K+ and water buffering as a therapeutic strategy for neuroprotection against K+ stress-related damage. We conducted a glial-specific screen in and identified a signal transduction pathway required for glial regulation of water and ion homeostasis. This screen uncovered a central role for salt-inducible kinase 3 (SIK3), a highly conserved AMP-activated protein kinase (AMPK)Cfamily kinase that links signal sensing to changes in cellular response (Choi et Fumagillin al., 2015; Sasagawa et al., 2012; Uebi et al., 2012; Wang et al., 2011). Loss of SIK3 in glia results in nerve edema, neuronal hyperexcitability, and increased seizure susceptibility, all phenotypes that are commonly associated with human genetic disorders disrupting glial water and ion homeostasis (Min and van der Knaap, 2018). This swelling phenotype is critically and selectively sensitive to K+ stress. Moreover, we Fumagillin demonstrate that SIK3 functions via regulation of a downstream HDAC4/Mef2 transcriptional program that controls expression of relevant ion and water transporters. HDAC4 is a critical negative regulator in the pathway, and pharmacological inhibition of HDAC4 potently suppresses the edema, hyperexcitability, and seizure phenotypes of mutants. Hence, this study identifies a druggable pathway controlling the glial capacity to buffer K+ and water and a candidate therapeutic approach to achieve the long-standing goal of targeting glia for the control of hyperexcitability (Devinsky et al., 2013; Heuser et al., 2014). Results and discussion SIK3 in glia restricts extracellular fluid accumulation in the peripheral nervous system Glia remove K+ from the extracellular space to maintain ionic and water homeostasis in the nervous system. Disrupting K+ buffering leads to nerve edema, neuronal hyperexcitability, and increased seizure susceptibility. In = 10; Fig. 1 A). Glial knockdown of SIK3 using a second, non-overlapping RNAi transgene provides same phenotype (Fig. S1 B). Null alleles of are lethal; nevertheless, there’s a solid hypomorphic allele (larvae show an identical but even more pronounced nerve bloating phenotype, with bigger (W: 68 3.2 m, L: 140 9.5 m) and more regular swellings (44 3.4 per larva, = 12). Our results through the RNAi screen indicate an important function of SIK3 in glia. To Nr2f1 check whether the even more dramatic phenotype in the real mutant demonstrates a glial function for SIK3, we examined rescue having a SIK3 transgene indicated from the glial (Repo-Gal4) or neuronal (Elav-Gal4) promoter. Nerve swellings in mutants are rescued by glial-specific manifestation of SIK3 completely, while neuronal manifestation of SIK3 does not have any effects for the bloating phenotype (Fig. 1 B). Furthermore, knockdown of SIK3 selectively in neurons will not induce nerve swellings Fumagillin (Fig. S1 C). Therefore, SIK3 is necessary in glia however, not neurons to suppress nerve swellings. Open up in another window Shape 1. Glial SIK3 helps prevent extracellular nerve edema. (A) Consultant pictures of peripheral nerves in third instar larvae stained for the nerve membrane marker HRP. The pan-glial drivers Repo-GAL4 was utilized expressing a UAS-GFP transgene (control; abbreviated mainly because mutants, and mutants with SIK3 manifestation in neurons or glia. Fumagillin Control larvae (mutants ( 30. ANOVA with Tukeys multiple evaluations One-way; ****, P < 0.0001; NS, P > 0.05. Data are mean SEM. (C) Quantification of nerve Fumagillin swellings in mutant with UAS-SIK3 either in the lack of a GAL4 drivers (larvae are suppressed when SIK3 can be indicated in wrapping glia or subperineurial glia. 20. One-way ANOVA with Tukeys multiple evaluations; ****, P < 0.0001; NS, P > 0.05. (DCG) Consultant EM areas through peripheral nerves. The axons (good examples tagged A) are firmly ensheathed by encircling glia (good examples tagged G) in crazy type (D), but axons and glia are separated by huge extracellular swellings (asterisks) in larvae.