InteractiveFly: GeneBrief

Wnk kinase: Biological Overview | References

Glial cells play a critical role in maintaining homeostatic ion concentration gradients. Salt-inducible kinase 3 (SIK3) regulates a gene expression program that controls K+ buffering in glia, and upregulation of this pathway suppresses seizure behavior in the eag, Shaker hyperexcitability mutant. This study show that boosting the glial SIK3 K+ buffering pathway suppresses seizures in three additional molecularly diverse hyperexcitable mutants, highlighting the therapeutic potential of upregulating glial K+ buffering. Additional mechanisms regulating glial K+ buffering were explored. Frayed (Fray), a transcriptional target of the SIK3 K+ buffering program, is a kinase that promotes K+ uptake by activating the Na+/K+/Cl- co-transporter, Ncc69. The Wnk kinase phosphorylates Fray in Drosophila glia; this activity is required to promote K+ buffering. This identifies Fray as a convergence point between the SIK3-dependent transcriptional program and Wnk-dependent post-translational regulation. Bypassing both regulatory mechanisms via overexpression of a constitutively active Fray in glia is sufficient to robustly suppress seizure behavior in multiple Drosophila models of hyperexcitability. Finally, cortex glia were identified as a critical cell type for regulation of seizure susceptibility, as boosting K+ buffering via expression of activated Fray exclusively in these cells is sufficient to suppress seizure behavior. These findings highlight Fray as a key convergence point for distinct K+ buffering regulatory mechanisms and cortex glia as an important locus for control of neuronal excitability (Lones, 2023).

This study demonstrates that enhancing glial ionic buffering is sufficient to suppress seizure behavior across a variety of Drosophila hyperexcitability models. Fray, an important upstream regulator of glial K+ transporters and a transcriptional target of SIK3-HDAC4 signaling, is also a substrate for the kinase Wnk in glia, and Wnk-dependent regulation of Fray is required for proper ionic buffering in glia. Overexpressing a constitutively active Fray, bypassing both SIK3- and Wnk-dependent regulation, potently suppressed seizure behavior in molecularly distinct hyperexcitability mutants. These findings suggest that Fray is a central convergence point between two major glial ion homeostasis regulatory pathways. Finally, this study identified cortex glia as central for the control of neuronal excitability, as enhancing K+ buffering specifically in this glial subtype is sufficient to suppress seizure behavior across diverse hyperexcitability mutants. Taken together, these findings emphasize the centrality of glial ionic buffering in the control of neuronal excitability, deepen understanding of the molecular mechanisms controlling glial ionic homeostasis, and highlight the potential therapeutic utility of bolstering glial K+ buffering as a new paradigm for the treatment of seizure disorders (Lones, 2023).

In a previous study, the surprising observation was made that the SIK3 glial ionic buffering program is inactivated in eag Shaker, a classic seizure mutant. Evidence was provided that the program is turned off by glia for self-protection, as re-activating this program induces glial cell swelling and activates JNK, a central cell stress signaling pathway. However, turning off the glial SIK3 program comes at the cost of exacerbating neuronal hyperexcitability. Indeed, reactivation of the SIK3 glial ionic buffering program is sufficient to suppress seizure behavior and dramatically extend lifespan in the eag Shaker mutant. This study investigated whether the glioprotective inactivation of K+ buffering was a peculiarity of the eag Shaker mutant, or a more general feature of hyperexcitability mutants. Evidence was found that the pathway is turned off in two additional mutants, a model of human Dup15q syndrome in which the ubiquitin ligase ../genebrief/ube3a_angelman.htm">Ube3A is overexpressed in glia, and the NCKXzyd, in which a glial K+ -dependent Na+/Ca2+ exchanger is mutant. In addition, this study found that all tested mutants could be suppressed by activation of the SIK3 K+ buffering program in glia. It has been previously demonstrated that a decrease in glial K+ buffering can promote seizure behavior. The current findings indicate that hyperexcitable mutants may often inhibit glial K+ buffering, and that reactivation of the glial K+ buffering program may be a general mechanism to inhibit seizure behavior and so could have potential as a treatment for seizure disorders (Lones, 2023).

Fray was the first protein demonstrated to be required for glial ion homeostasis in Drosophila, where it phosphorylates and activates the cation-chloride transporter Ncc69 to promote proper ion and water buffering in peripheral nerve. In addition, Fray was demonstrated to be one of the downstream genes activated by the SIK3-HDAC4-Mef2 glial transcriptional pathway. This study demonstrates that Wnk is also required for proper ion and water homeostasis in Drosophila glia, and that Wnk functions through Fray. Hence, both the transcriptional SIK3 pathway and post-translational Wnk pathway converge on Fray to regulate ion homeostasis. Moreover, transgenic overexpression of a constitutively active Fray showed that this single protein, when active, is sufficient to promote glial ion and water homeostasis and suppress neuronal excitability in a variety of seizure mutants. The identification of Fray as a convergence node for these two pathways has interesting implications for regulatory mechanisms controlling the balance between glial uptake of ions, which can be neuroprotective but lead to glial swelling and damage, and glial cell volume regulatory mechanisms that preserve glial health at the expense of extracellular ion accumulation and increased neuronal excitability. Previous work showed that with hyperexcitability the monoamine octopamine signaled to glia to turn down the SIK3 transcriptional program and, presumably, decrease the expression of Fray. While such a mechanism would be a powerful approach to reduce glial ion uptake, it is also expected to be quite slow. With the identification of Wnk as a required activator of Fray, there is now the likelihood of a second, faster mechanism to inhibit glial ion uptake. Wnk is a potassium-sensitive kinase—high levels of intracellular K+ inhibit Wnk activity, providing a direct mechanism for excess K+ uptake to rapidly blunt glial ion uptake and protect the glia from osmotic stress. Future studies will address whether hyperexcitability mutants inhibit Wnk activity in glia and whether there may be additional cross talk between the SIK3 and Wnk pathways (Lones, 2023).

Much like their mammalian counterparts, Drosophila glial cells have specialized functions including phagocytosis, maintenance of the blood-brain barrier, neurotransmitter reuptake, and ion homeostasis. In previous work, it was demonstrated that SIK3-HDAC4 K+ buffering pathway was particularly critical in wrapping glia for suppressing extracellular edema in peripheral nerves, and that the SIK3-HDAC4 K+ buffering program was turned off in peripheral glia in the eag, Shaker mutant. However, this study showed that enhancing the K+ buffering program in wrapping glia failed to suppress seizures in the eag, Shaker mutant. It was reasoned that seizures start in the central nervous system and propagate to the periphery, and so there must be a different glial subtype in the central nervous system contributing to seizure behavior. This study focused on cortex glia, which wrap neuronal cell bodies and can participate in K+ buffering, and which was found also to turn off the SIK3 K+ buffering program in eag, Shaker. As was predicted, activation of the glial ion buffering program exclusively in cortex glia robustly suppressed seizures in this mutant. Moreover, these findings are not specific for eag, Shaker, as the NCKXzyd mutant also inhibits the SIK3-HDAC4 K+ buffering pathway in cortex glia, and reactivation of this program in these cells again dramatically suppresses seizure behavior. Cortex glia are likely important for regulating excitability because they control the extracellular ionic milieu around the site of action potential initiation. Taken together these finding indicate that multiple glial subtypes contribute to ionic buffering around distinct portions of the neuron, and these studies do not exclude the possibility that additional glial subtypes may also contribute to ion homeostasis. Nonetheless, it is suggested that methods to selectively target mammalian astrocytes at the axon initial segment could be a powerful approach for novel therapies to inhibit neuronal hyperexcitability (Lones, 2023).

Unanticipated domain requirements for Drosophila Wnk kinase in vivo

WNK (With no Lysine [K]) kinases have critical roles in the maintenance of ion homeostasis and the regulation of cell volume. Their overactivation leads to pseudohypoaldosteronism type II (Gordon syndrome) characterized by hyperkalemia and high blood pressure. More recently, WNK family members have been shown to be required for the development of the nervous system in mice, zebrafish, and flies, and the cardiovascular system of mice and fish. Furthermore, human WNK2 and Drosophila Wnk modulate canonical Wnt signaling. In addition to a well-conserved kinase domain, animal WNKs have a large, poorly conserved C-terminal domain whose function has been largely mysterious. In most but not all cases, WNKs bind and activate downstream kinases OSR1/SPAK, which in turn regulate the activity of various ion transporters and channels. This study shows that Drosophila Wnk regulates Wnt signaling and cell size during the development of the wing in a manner dependent on Fray, the fly homolog of OSR1/SPAK. The only canonical RF(X)V/I motif of Wnk, thought to be essential for WNK interactions with OSR1/SPAK, is required to interact with Fray in vitro. However, this motif is unexpectedly dispensable for Fray-dependent Wnk functions in vivo during fly development and fluid secretion in the Malpighian (renal) tubules. In contrast, a structure function analysis of Wnk revealed that the less-conserved C-terminus of Wnk, that recently has been shown to promote phase transitions in cell culture, is required for viability in vivo. These data thus provide novel insights into unexpected in vivo roles of specific WNK domains (Yarikipati, 2023).

This study demonstrates that enhancing glial ionic buffering is sufficient to suppress seizure behavior across a variety of Drosophila hyperexcitability models. Fray, an important upstream regulator of glial K+ transporters and a transcriptional target of SIK3-HDAC4 signaling, is also a substrate for the kinase Wnk in glia, and Wnk-dependent regulation of Fray is required for proper ionic buffering in glia. Overexpressing a constitutively active Fray, bypassing both SIK3- and Wnk-dependent regulation, potently suppressed seizure behavior in molecularly distinct hyperexcitability mutants. These findings suggest that Fray is a central convergence point between two major glial ion homeostasis regulatory pathways. Finally, cortex glia were identified as central for the control of neuronal excitability, since enhancing K+ buffering specifically in this glial subtype is sufficient to suppress seizure behavior across diverse hyperexcitability mutants. Taken together, these findings emphasize the centrality of glial ionic buffering in the control of neuronal excitability, deepen understanding of the molecular mechanisms controlling glial ionic homeostasis, and highlight the potential therapeutic utility of bolstering glial K+ buffering as a new paradigm for the treatment of seizure disorders (Yarikipati, 2023).

In a previous study, the surprising observation was made that the SIK3 glial ionic buffering program is inactivated in eag Shaker, a classic seizure mutant. This study provides evidence that the program is turned off by glia for self-protection, as re-activating this program induces glial cell swelling and activates JNK, a central cell stress signaling pathway. However, turning off the glial SIK3 program comes at the cost of exacerbating neuronal hyperexcitability. Indeed, reactivation of the SIK3 glial ionic buffering program is sufficient to suppress seizure behavior and dramatically extend lifespan in the eag Shaker mutant. Wether this glioprotective inactivation of K+ buffering was a peculiarity of the eag Shaker mutant, or a more general feature of hyperexcitability mutants. Evidence was found that the pathway is turned off in two additional mutants; a model of human Dup15q syndrome in which the ubiquitin ligase Ube3A is overexpressed in glia and the NCKXzyd, in which a glial K+ -dependent Na+/Ca2+ exchanger is mutant. In addition, all tested mutants could be suppressed by activation of the SIK3 K+ buffering program in glia. It has been demonstrated that a decrease in glial K+ buffering can promote seizure behavior. The current findings indicate that hyperexcitable mutants may often inhibit glial K+ buffering, and that reactivation of the glial K+ buffering program may be a general mechanism to inhibit seizure behavior and so could have potential as a treatment for seizure disorders (Yarikipati, 2023).

Fray was the first protein demonstrated to be required for glial ion homeostasis in Drosophila, where it phosphorylates and activates the cation-chloride transporter Ncc69 to promote proper ion and water buffering in peripheral nerves. In addition, this study demonstrated that Fray is one of the downstream genes activated by the SIK3-HDAC4-Mef2 glial transcriptional pathway. Wnk was demonstrated to be also required for proper ion and water homeostasis in Drosophila glia and that Wnk functions through Fray. Hence, both the transcriptional SIK3 pathway and post-translational Wnk pathway converge on Fray to regulate ion homeostasis. Moreover, transgenic overexpression of a constitutively active Fray demonstrated that this single protein, when active, is sufficient to promote glial ion and water homeostasis and suppress neuronal excitability in a variety of seizure mutants. The identification of Fray as a convergence node for these two pathways has interesting implications for regulatory mechanisms controlling the balance between glial uptake of ions, which can be neuroprotective but lead to glial swelling and damage, and glial cell volume regulatory mechanisms that preserve glial health at the expense of extracellular ion accumulation and increased neuronal excitability. It was previously shown that with hyperexcitability the monoamine octopamine signaled to glia to turn down the SIK3 transcriptional program and, presumably, decrease the expression of Fray. While such a mechanism would be a powerful approach to reduce glial ion uptake, it is also expected to be quite slow. With the identification of Wnk as a required activator of Fray, there is now the likelihood of a second, faster mechanism to inhibit glial ion uptake. Wnk is a potassium-sensitive kinase—high levels of intracellular K+ inhibit Wnk activity [28], providing a direct mechanism for excess K+ uptake to rapidly blunt glial ion uptake and protect the glia from osmotic stress. Future studies will address whether hyperexcitability mutants inhibit Wnk activity in glia and whether there may be additional cross talk between the SIK3 and Wnk pathways (Yarikipati, 2023).

Much like their mammalian counterparts, Drosophila glial cells have specialized functions including phagocytosis, maintenance of the blood-brain barrier, neurotransmitter reuptake, and ion homeostasis> demonstrated that SIK3-HDAC4 K+ buffering pathway was particularly critical in wrapping glia for suppressing extracellular edema in peripheral nerves and that the SIK3-HDAC4 K+ buffering program was turned off in peripheral glia in the eag, Shaker mutant. However,this study shows enhancing the K+ buffering program in wrapping glia failed to suppress seizures in the eag, Shaker mutant. It was reasoned that seizures start in the central nervous system and propagate to the periphery, and so there must be a different glial subtype in the central nervous system contributing to seizure behavior. Focus was placed on cortex glia, which wrap neuronal cell bodies and can participate in K+ buffering, and which was found also to turn off the SIK3 K+ buffering program in eag, Shaker. As was predicted, activation of the glial ion buffering program exclusively in cortex glia robustly suppressed seizures in this mutant. Moreover, these findings are not specific for , as the NCKXzyd mutant also inhibits the SIK3-HDAC4 K+ buffering pathway in cortex glia, and reactivation of this program in these cells again dramatically suppresses seizure behavior. Cortex glia are likely important for regulating excitability because they control the extracellular ionic milieu around the site of action potential initiation. Taken together these finding indicate that multiple glial subtypes contribute to ionic buffering around distinct portions of the neuron, and these studies do not exclude the possibility that additional glial subtypes may also contribute to ion homeostasis. Nonetheless, it is suggested that methods to selectively target mammalian astrocytes at the axon initial segment could be a powerful approach for novel therapies to inhibit neuronal hyperexcitability (Yarikipati, 2023).

Chloride oscillation in pacemaker neurons regulates circadian rhythms through a chloride-sensing WNK kinase signaling cascade

Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies. Whether and how intracellular chloride regulates circadian rhythms remains controversial. This study demonstrates a signaling role for intracellular chloride in the Drosophila small ventral lateral (sLN(v)) pacemaker neurons. In control flies, intracellular chloride increases in sLN(v)s over the course of the morning. Chloride transport through sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations. Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride 6 h after lights on, loss of morning anticipation, and a prolonged circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of WNK (with no lysine [K]) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila sLN(v) pacemakers restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length (Schellinger, 2022).

Central pacemaker neurons are master regulators of organismal circadian rhythms, which govern physiological processes throughout the body in anticipation of daily changes in light and temperature. In mammals, the central pacemaker neurons reside in the suprachiasmatic nucleus (SCN) of the hypothalamus. Neuronal activity and electrophysiological parameters, such as the resting membrane potential, change in a circadian manner in these neurons. Most SCN neurons are GABA (γ-amino butyric acid)-ergic. One mechanism for GABAergic neurotransmission is through the GABAA receptor, a ligand-gated chloride channel. Because intracellular chloride in neurons is typically low, GABA is usually hyperpolarizing, due to chloride influx. However, it has been shown that GABA had excitatory effects on SCN neurons during the day, and inhibitory effects at night, which was proposed to be due to higher intracellular chloride concentrations during daytime compared to night. Since then, several papers have inferred oscillating intracellular chloride concentrations, as estimated by the GABA reversal potential. However, SCN neuron responses to GABA in the day vs. night have differed across studies. More recently, using a transgenic chloride sensor to directly measure intracellular chloride concentrations in two specific SCN neuronal subpopulations, increased intracellular chloride during the day and decreased intracellular chloride at night, has been demonstrated. However, GABA application consistently resulted in chloride influx, suggesting hyperpolarizing (inhibitory) effects of GABA in both day and night. Thus, the physiological role of intracellular chloride oscillations in pacemaker neurons remains unclear (Schellinger, 2022).

The SLC12 cation-chloride cotransporters are major determinants of intracellular chloride. These electroneutral cotransporters couple transmembrane chloride transport to sodium and/or potassium, with chloride influx through the sodium-potassium-2-chloride (NKCC) cotransporters and efflux through the potassium-chloride (KCC) cotransporters. Prior studies have demonstrated SCN neuron expression of these transporters, and physiological roles in determining intracellular chloride and the GABA reversal potential in the SCN (Schellinger, 2022).

This study used Drosophila melanogaster, in which mechanisms regulating circadian rhythms have been extensively studied, to understand the role of intracellular chloride in pacemaker neurons. Intracellular chloride was found to increase over the course of the morning in the ventral lateral (LNv) pacemaker neurons, in an NKCC-dependent manner. Intracellular chloride has a signaling role in these neurons, restraining excess activity of the chloride-sensitive WNK (With No Lysine (K)) kinase, an upstream activator of an inwardly-rectifying potassium channel. Dysregulation of this pathway prolongs the circadian period of diurnal locomotor activity patterns (Schellinger, 2022).

Intracellular chloride oscillates in the central pacemaker neurons of the mammalian SCN, but the functional significance of this oscillation has remained unclear. This study demonstrates an NKCC-dependent increase in intracellular chloride in Drosophila LNv pacemaker neurons over the course of the morning, which constrains activity of the chloride-sensitive WNK kinase, its downstream substrate, Fray, and an inwardly rectifying potassium channel, Irk1, to maintain normal circadian periodicity (Schellinger, 2022).

Loss of the Ncc69 NKCC in the LNv pacemaker neurons has two consequences: the failure of intracellular chloride to increase over the course of the morning, and lengthening of the circadian period. These observations are consistent with studies in the SCN implicating NKCC in the determination of intracellular chloride in mammalian pacemaker neurons, and connects intracellular chloride to a behavioral circadian phenotype. Another SLC12 transporter has been linked to Drosophila behavioral rhythmicity in constant light conditions and the electrophysiological response to GABA in lLNv neurons, which have GABAA receptor chloride channels. This transporter was named NKCC and was proposed to regulate intracellular chloride. However, its transport activity has not been characterized, and it aligns to a distinct clade of SLC12 cotransporters whose sequences predict differences in ion selectivity compared to the NKCCs. Consistent with this idea, an Aedes aegypti homolog has a transport profile distinct from the NKCCs, with an electrogenic, chloride-independent lithium/sodium conductance. Thus, the functions of the Ncc69-encoded NKCC and the NKCC-encoded transporter are likely distinct (Schellinger, 2022).

Diurnal variations in intracellular chloride have been proposed to influence the effect of GABAergic neurotransmission on clock neurons in mammals, but a recent study questioned this idea. The current study demonstrates a signaling role for intracellular chloride in the LNv neurons, in which the rise in intracellular chloride inhibits WNK-Fray signaling. As chloride-sensitive kinases, WNKs are perfectly poised to interpret changes in intracellular chloride and initiate downstream signal transduction cascades, and the concept of chloride signaling has gained increasing traction in recent years. This has been studied in the context of transepithelial ion transport in Drosophila and mammalian renal epithelia, as well as in the clearance of apoptotic corpses. The finding that chloride signals via the WNK-Fray pathway in circadian pacemaker neurons further extends this concept (Schellinger, 2022).

Fray was also shown to activate the Irk1 inwardly rectifying potassium channel, in a manner dependent on a putative Fray-binding motif in Irk1 that is required for the lengthened circadian period of flies with activated Fray in LNv neurons. Pacemaker neurons in both flies and mammals undergo molecular clock-controlled circadian variation in electrical activity, and altering the excitability of the LNv pacemaker neurons disrupts circadian rhythms. Variation in pacemaker neuron resting membrane potential is thought to be an important determinant of the circadian pattern of electrical activity. Two voltage-gated potassium channels have been implicated in LNv neuron electrical oscillations, and a sodium leak current and potassium channels contribute to the day/night cycling of resting membrane potential in Drosophila dorsal clock neurons. Because inwardly rectifying potassium channels play an important role in determining cellular membrane potential, chloride regulation of Irk1 activity could also contribute to the diurnal variation in LNv neuron excitability, theraby influencing circadian period (Schellinger, 2022).

In sLNv neurons, resting membrane potential is most depolarized at lights on (ZT0), hyperpolarizes between ZT0 and ZT6, is variable between ZT6 and ZT18, and depolarizes between ZT18 and ZT24. Thus, the increased intracellular chloride observed in sLNv neurons at ZT6, which is predicted to inhibit Irk1 through inhibition of WNK-Fray signaling, coincides with the period in which there is loss of hyperpolarization in the sLNv neurons. Persistent Irk1 activity, e.g. in Ncc69 mutants, could maintain sLNv neurons in a hyperpolarized state, delaying subsequent depolarization over the course of the night and prolonging the circadian period. In contrast, the loss of Irk1 throughout the day-night cycle, as seen with Irk1 knockdown in the PDF-expressing neurons, could interfere with sLNv neuron hyperpolarization after lights-on, explaining the long-period phenotype of those mutants (Schellinger, 2022).

These results imply that intracellular chloride may regulate pacemaker neuron excitability in a cell autonomous fashion. This is consistent with the observation that circadian rhythms of pacemaker neuron electrophysiology persist in the absence of fast neurotransmission and in isolated neurons in invertebrates and vertebrates. This raises the question of how NKCC activity may be increasing during morning hours in the LNv neurons. Although the WNK-SPAK/OSR1/Fray kinase cascade is a well-understood upstream regulator of NKCCs, this study shows that in the LNv neurons, NKCC is acting upstream of WNK, and loss of WNK and Fray in the pacemaker neurons does not recapitulate the long-period phenotype of loss of Ncc69. This suggests other regulatory mechanisms (Schellinger, 2022).

Could intracellular chloride play a signaling role in SCN pacemaker neurons? NKCC1, KCCs and WNK3 are expressed in the rat SCN. The repertoire of ion channels regulating pacemaker neuron excitability is complex and incompletely understood, but large-conductance Ca2+-activated potassium channels (also known as BK or maxi-K) have been implicated, and are regulated by mammalian WNKs. Whether the oscillating intracellular chloride observed in SCN neurons regulates these or other ion channels underlying SCN neuron electrical properties will be of interest (Schellinger, 2022).

The opposing chloride cotransporters KCC and NKCC control locomotor activity in constant light and during long days

Cation chloride cotransporters (CCCs) regulate intracellular chloride ion concentration ([Cl(-)](i)) within neurons, which can reverse the direction of the neuronal response to the neurotransmitter GABA. Na(+) K(+) Cl(-) (NKCC) and K(+) Cl(-) (KCC) cotransporters transport Cl(-) into or out of the cell, respectively. When NKCC activity dominates, the resulting high [Cl(-)](i) can lead to an excitatory and depolarizing response of the neuron upon GABA(A) receptor opening, while KCC dominance has the opposite effect. This inhibitory-to-excitatory GABA switch has been linked to seasonal adaption of circadian clock function to changing day length, and its dysregulation is associated with neurodevelopmental disorders such as epilepsy. In Drosophila melanogaster, constant light normally disrupts circadian clock function and leads to arrhythmic behavior. This study demonstrates a function for CCCs in regulating Drosophila locomotor activity and GABA responses in circadian clock neurons because alteration of CCC expression in circadian clock neurons elicits rhythmic behavior in constant light. The same effects were observed after downregulation of the Wnk and Fray kinases, which modulate CCC activity in a [Cl(-)](i)-dependent manner. Patch-clamp recordings from the large LNv clock neurons show that downregulation of KCC results in a more positive GABA reversal potential, while KCC overexpression has the opposite effect. Finally, KCC and NKCC downregulation reduces or increases morning behavioral activity during long photoperiods, respectively. In summary, these results support a model in which the regulation of [Cl(-)](i) by a KCC/NKCC/Wnk/Fray feedback loop determines the response of clock neurons to GABA, which is important for adjusting behavioral activity to constant light and long-day conditions (Eick, 2022).

Axonal self-destruction and neurodegeneration

The molecular and cellular mechanisms underlying complex axon morphogenesis are still poorly understood. This study reports a novel, evolutionary conserved function for the Drosophila Wnk kinase (dWnk) and its mammalian orthologs, WNK1 and 2, in axon branching. This study uncovered that dWnk, together with the neuroprotective factor Nmnat, antagonizes the axon-destabilizing factors D-Sarm and Axundead (Axed) during axon branch growth, revealing a developmental function for these proteins. Overexpression of D-Sarm or Axed results in axon branching defects, which can be blocked by overexpression of dWnk or Nmnat. Surprisingly, Wnk kinases are also required for axon maintenance of adult Drosophila and mouse cortical pyramidal neurons. Requirement of Wnk for axon maintenance is independent of its developmental function. Inactivation of dWnk or mouse Wnk1/2 in mature neurons leads to axon degeneration in the adult brain. Therefore, Wnk kinases are novel signaling components that provide a safeguard function in both developing and adult axons (Izadifar, 2021).

A hallmark in the generation of neuronal cell type diversity is the acquisition of diverse morphologies, which requires the formation of axonal and dendritic compartments ranging from simple to highly complex, depending on the degree of neurite branching. Specifying the degree and pattern of neurite branching is crucial in brain development, as it directly impacts the total number and spatial distribution of synaptic contacts of each circuit element. However, the identity of the molecular effectors determining how diverse, cell-type-specific patterns of axon arborization are established and how they are stabilized as well as maintained throughout the life of an organism remains a major challenge (Izadifar, 2021).

A reverse genetic screen was performed to identify novel regulators of axon branching by utilizing an experimental system in Drosophila that combines efficient single-neuron labeling and simultaneous knockdown of candidate genes. Clear orthologs of selective candidates were then further examined in vertebrates. Using this approach, it was found that loss of the Drosophila dWnk kinase specifically disrupts axon growth and branch patterning of mechanosensory neurons. Surprisingly, unlike other essential regulators of axon branching, this study discovered that dWnk is also continuously required in mature neurons for axon maintenance. Moreover, comparative studies in mouse cortical pyramidal neurons (PNs) provide strong evidence that both of these functions of Wnk kinases are conserved and required in PNs, i.e., long-range projecting mammalian neurons of the central nervous system (CNS) (Izadifar, 2021).

Wnk kinases are present in most multicellular organisms, including plants and some unicellular organisms, but not in yeast. Mammals have four Wnk kinases (WNK1-4) and Drosophila one (dWnk). The WNK ('with no K(lysine)') kinases are catalytically active but are referred to as 'atypical kinases' because a catalytically important lysine residue is swapped from subdomain II to subdomain I. WNK proteins are involved in a broad spectrum of diseases (e.g., hypertension, sensory and autonomic neuropathy, osteoporosis, and many different cancers) (Izadifar, 2021).

The majority of studies on human WNK kinases have been conducted in the context of blood pressure regulation, due to the identification of mutations in human patients with hereditary hypertension (familial hyperkalemia and hypertension [FHHt] or Gordon's syndrome. For this reason, a major focus in dissecting WNK function has been on studying renal regulation of ion transport. However, regulation of ion homeostasis is only one of multiple functions of WNK kinases, and they are broadly expressed, including in the developing as well as mature brain. In rare cases, WNK function has been linked to a severe form of peripheral sensory neuropathy (hereditary sensory and autonomic neuropathy type 2, HSNA2); however, their developmental, cellular, and molecular mechanisms are poorly understood in neurons. The fact, however, that most identified mutations cluster in a neuron-specific alternatively spliced exon (HSN2) of human Wnk1 supports the notion that Wnk1 kinase plays an important role in sensory neurons (Izadifar, 2021).

In the process of studying the role of dWnk kinase in fly sensory neurons, this study identified novel interactors of Wnk kinases. Specifically, it was found that nicotinamide mononucleotide adenylyltransferase (Nmnat), Sarm, and Axundead (Axed) are molecular interactors of dWnk. While Nmnat is broadly required for axon maintenance, Sarm and Axed are primarily studied for their roles as effectors in active axon degeneration (e.g., in Wallerian degeneration) in response to axon injury. This study provides evidence that dWnk and Nmnat have synergistic functions in axon growth and branching but are also required in post-developmental processes to continuously support axon maintenance. The function of dWnk is evolutionarily conserved, as their mouse orthologs WNK1 and WNK2 are both required in cortical PNs during axon morphogenesis as well as maintenance. Genetic epistasis analysis demonstrates that both dWnk and Nmnat functions during axon development and axon maintenance are mediated by antagonizing the axon destruction function of Sarm and Axed. Depletion of axon-protective factors (e.g., dWnk/WNK1/2 and Nmnat) during development leads to axon branching defects, whereas their depletion in mature neurons eliminates their safeguard function and initiates spontaneous axon degeneration in the absence of axon injury (Izadifar, 2021).

This study reports that the function of the Wnk-family kinases (Drosophila dWnk and mammalian WNK1/2) are required in developmental axonal branch patterning as well as axon maintenance. Phenotypic defects observed in mechanosensory axons of dWnk mutant neurons are indistinguishable from defects observed in Nmnat mutant neurons. Both dWnk and Nmnat mutant mechanosensory axons can grow from the periphery to the VNC but require dWnk as well as Nmnat function during axonal branch growth and patterning. Moreover, a post-developmental knockdown of dWnk triggered progressive degeneration in mature and fully developed mechanosensory axons. Such an adult-specific function is also analogous to the well-documented role of Nmnat in axon maintenance. Although Nmnat has been studied most extensively for its role in axon maintenance in injury models or neurodegenerative disease models, it has been reported previously that LOF mutants are lethal and show axonal defects. Furthermore, this study shows that the dual developmental and maintenance functions of dWnk are evolutionary conserved, as knockdown of WNK1 and WNK2 results in remarkably similar axon morphogenesis defects and trigger axon degeneration in mouse cortical neurons. It is concluded that, in neurons, Wnk kinases exert novel functions that are analogous and synergistic to conserved Nmnat functions. The discovery of Wnk kinases having important neuroprotective roles analogous to Nmnat offers new tools and insights to further dissect the complex regulatory network underlying active axon degeneration (Izadifar, 2021).

Formally, the adult maintenance defects that were observed upon Wnk depletion could be a consequence of developmental defects. However, several reasons argue strongly against this possibility: first, the in vivo knockdown of WNK1 and WNK2 after P30 in mouse cortical layer 2/3 PNs triggered degeneration of axons of mature neurons. Second, knockdown of dWnk at post-developmental (late pupal) stages does not alter the axonal branching or targeting of mechanosensory neurons but triggered spontaneous degeneration of adult mechanosensory axons after 3 days post-eclosion. Third, a single-copy RNAi knockdown of dWnk resulted in strong axon branching defects. Fourth, in previous studies, several mutants were identified and characterized that lead to severe axon branching defects of mechanosensory axons. For example, in Dscam1-null mutant clones, the axon branching defects are even more severe than in dWnk mutant clones, yet no sign of axon degeneration was found even in 1 week or older flies. Fifth, even in cases of very short axon branches in dWnk or other mutants with a complete absence of contralateral projecting axon collaterals, the distal part of the mutant axons still reaches the corresponding ipsilateral target area. Given that putative trophic signals would have to support axons on both sides of the VNC, it seems highly unlikely that target-derived trophic signals would only support contralateral projecting axon arbors. In summary, the data provide strong evidence that Wnk kinases have dual roles: first, during developmental axon morphogenesis and, second and independently, during continuous axon maintenance in mature neurons (Izadifar, 2021).

A key question raised by these results is: how similar are the molecular processes in developmental axon growth and branching and adult maintenance? A related question has been discussed in a hallmark review (Raff, 2002). This perspective article discussed the discovery that neurons can activate a self-destructive program independent of general apoptosis. The authors noted that neurons 'apparently have a second, molecularly distinct self-destruct program in their axon.' And the authors raise the incisive question: 'Do neurons also use this second program to prune their axonal tree during development and to conserve resources in response to chronic insults?' (Izadifar, 2021).

It is assumed that 'pruning of axonal branches during development' could-as a cellular mechanism-also be involved in axonal branch patterning as analyzed in this study. Specifically, the developmental axon branching of mechanosensory axons is viewed as a process where continuous competitive interactions among nascent branches select for stabilization or retraction (timescale minutes or few hours). In contrast, the well-characterized pruning of axons during metamorphosis (e.g., mushroom body remodeling) is initiated when axon branches and connectivity have been already established. This type of pruning (remodeling) requires primarily a destabilization of a fully established axon projection and is likely different from axonal branch selection as investigated in this study. Consistent with this idea is the finding that Nmnat is not required in axon pruning of mushroom body neurons (Izadifar, 2021).

The results now provide genetic and molecular data that support the notion that components of a 'distinct self-destruct program' are involved in axon branch stabilization and destabilization. It is further suggested that molecular control mechanisms of axon branching and axon destruction (or preventing axon destruction, i.e., axon maintenance) are mechanistically related. Specifically, the dynamics of axon branching requires the selection of an exuberant number of nascent axon branches by either stabilizing or destabilizing nascent branches. During axon morphogenesis, the majority of filopodia and nascent branches are retracted to maintain just a few that are consolidated into axon collaterals. It seems plausible, therefore, that axon branch retraction in developing neurons might involve molecular effectors that are also required for a distinct type of axon branch pruning. Based on in vitro studies, it is speculated that regulation of de novo protein synthesis could be the molecular process that is targeted in both axon branching and axon maintenance (Izadifar, 2021).

Based on new findings, it is suggested that Wnk as well as Nmnat are necessary components of axon branching as well as axon maintenance. Support for this model comes from the results of genetic epistasis analysis. The loss of dWnk during axon branching is only a problem if Sarm or Axed are present: in the absence of Axed (the most downstream effector of the destruction program in Drosophila known so far), loss of dWnk does not cause defects in axonal branching or maintenance. Implicit to this model is that dWnk is unlikely instructing axon branching but rather provides a safeguard function curbing destructive effectors such that retraction, pruning, or branch destruction can be restricted and constrained in a spatially restricted manner. A further implication of this model is the notion that loss of dWnk effectively represents a gain of function (on switch) of an axonal destruction program in developing axons (Izadifar, 2021).

In this context, it is also interesting to note that expression of Nmnat can compensate (i.e., rescue) for loss of dWnk in both axon branching and maintenance. It is well established that local depletion of Nmnat in adult neurons does directly lead to axon degeneration, i.e., has a role in axon maintenance independent of its developmental role. This is consistent with the new finding that post-developmental inactivation of dWnk or mammalian Wnk1/2 triggers axon degeneration (Izadifar, 2021).

Nmnat has been previously shown to protect from axon degeneration following axotomy by counter-acting Sarm-induced NAD+ depletion and Axed activity. This study shows that, even in the absence of axon injury, loss of Nmnat as well as loss of dWnk or overexpression of dSarm and Axed leads to progressive degeneration of adult mechanosensory axons without injury. This is consistent with previous reports showing that Nmnat loss in sensory neurons of the wing leads to spontaneous axon degeneration in adult flies or that loss of NMNAT2 leads to truncation of peripheral nerve and CNS axon tracts in mice. A role of Wnk1 in axon maintenance is also consistent with the finding that WNK function has been linked to a severe form of peripheral neuropathy (Izadifar, 2021).

The results, therefore, support the notion that both axon morphogenesis and maintenance require a constitutive involvement of Wnk and Nmnat (Izadifar, 2021).

A link between dWnk, Nmnat, and axon destructive factors is further corroborated by biochemical experiments co-expressing these factors and using co-immunoprecipitations to analyze protein-protein interactions of dWnk or mammalian WNK1/2. First, the results suggest the possibility that dWnk, Nmnat, Sarm1, and Axed are able to form mixed complexes. Moreover, mammalian WNK1 can interact with WNK2 and Nmnat2 as well as SARM1. Although previous work has not identified Nmnat proteins as potential Wnk kinase substrates, the current results suggest that this possibility is worthwhile to examine in future experiments. Second, whereas a vertebrate ortholog of Axed has not been described, it was found that mammalian SARM1 overexpression strongly downregulates levels of WNK1, WNK2, NMNAT2, and NMNAT1. However, future experiments will have to confirm that this downregulation of proteins by Sarm1 is also occurring in neurons in vivo (Izadifar, 2021).

It has been reported that inhibition of axon degeneration can be accomplished by increasing or stabilizing levels of Nmnat protein. Moreover, Highwire/Phr1, which is an additional conserved factor functioning in Wallerian degeneration, directly promotes the downregulation of Nmnat, and axon degeneration is strongly inhibited in Highwire/Phr1 mutants. This previously described regulation of Nmnat levels is mediated via mitogen-activated protein kinase (MAPK) signaling and ubiquitin-dependent proteolysis. The findings of this study suggest that Sarm1 may rather inhibit de novo protein synthesis in order to deplete Nmnat and other axon protective factors. Future studies will have to investigate in detail how the SARM1-dependent depletion of NAD+ also leads to a Wnk/Nmnat protein depletion as described in this study. Particularly interesting will be to determine how the destructive activity of Sarm protein can be limited during development to selective axon branch compartments in order to enable local axon branch pruning but prevent progressive axon degeneration (Izadifar, 2021).

Finally, Nmnat function has not only been involved in neuroprotection as a response to injury, such as axotomy, but also in a diverse range of neurodegenerative diseases, such as spinocerebellar ataxia, fronto-temporal dementia (FTD) and Parkinsonism, or glaucomatous optic neuropathy, or following growth factor deprivation. Future studies will need to consider the possibility that the newly identified dWnk and WNK1 and WNK2 kinases may also play similar neuroprotective roles in diverse types of neurodegenerative conditions (Izadifar, 2021).

>Massive excretion of calcium oxalate from late prepupal salivary glands of Drosophila melanogaster demonstrates active nephridial-like anion transport

The Drosophila salivary glands (SGs) were well known for the puffing patterns of their polytene chromosomes and so became a tissue of choice to study sequential gene activation by the steroid hormone ecdysone. One well-documented function of these glands is to produce a secretory glue, which is released during pupariation to fix the freshly formed puparia to the substrate. Over the past two decades SGs have been used to address specific aspects of developmentally-regulated programmed cell death (PCD), as it was thought that they are doomed for histolysis and after pupariation are just awaiting their fate. More recently, however, it has been shown that for the first 3-4 h after pupariation SGs undergo tremendous endocytosis and vacuolation followed by vacuole neutralization and membrane consolidation. Furthermore, from 8 to 10 h after puparium formation (APF) SGs display massive apocrine secretion of a diverse set of cellular proteins. This study shows that during the period from 11 to 12 h APF, the prepupal glands are very active in calcium oxalate (CaOx) extrusion that resembles renal or nephridial excretory activity. Genetic evidence that Prestin, a Drosophila homologue of the mammalian electrogenic anion exchange carrier SLC26A5, is responsible for the instantaneous production of CaOx by the late prepupal SGs. Its positive regulation by the protein kinases encoded by fray and wnk lead to increased production of CaOx. The formation of CaOx appears to be dependent on the cooperation between Prestin and the vATPase complex as treatment with bafilomycin A1 or concanamycin A abolishes the production of detectable CaOx. These data demonstrate that prepupal SGs remain fully viable, physiologically active and engaged in various cellular activities at least until early pupal period, that is, until moments prior to the execution of PCD (Farkas, 2016).

WNK signaling is involved in neural development via Lhx8/Awh expression

WNK kinase family is conserved among many species and regulates SPAK/OSR1 and ion co-transporters. Some mutations in human WNK1 or WNK4 are associated with Pseudohypoaldosteronism type II, a form of hypertension. WNK is also involved in developmental and cellular processes, but the molecular mechanisms underlying its regulation in these processes remain unknown. This study identified a new target gene in WNK signaling, Arrowhead and Lhx8, which is a mammalian homologue of Drosophila Arrowhead. In Drosophila, WNK was shown to genetically interact with Arrowhead. In Wnk1 knockout mice, levels of Lhx8 expression were reduced. Ectopic expression of WNK1, WNK4 or Osr1 in mammalian cells induced the expression of the Lhx8. Moreover, neural specification was inhibited by the knockdown of both Wnk1 and Wnk4 or Lhx8. Drosophila WNK mutant caused defects in axon guidance during embryogenesis. These results suggest that WNK signaling is involved in the morphological and neural development via Lhx8/Arrowhead (Sato, 2013).

The WNK-SPAK/OSR1 pathway is known to regulate various ion co-transporters and is widely conserved among many species. Wnk1 knockout mice die before embryonic day 13, and display defects in cardiac development. WNK1 is also required for cell division in cultured cells. Furthermore, PHAII patients display a number of other clinical features, such as an intellectual impairment, dental abnormalities and impaired growth in addition to hypertension. Accordingly, the new role of the WNK signaling pathway described in this study may provide further insight into the development and pathogenesis of PHAII. In this study, Lhx8/Awh was identifed as a new downstream molecule in the WNK-SPAK/OSR1 pathway, and a novel function was discovered for the WNK-Lhx8 pathway in neural development (Sato, 2013).

There are four mammalian WNK family members, and WNK1 and WNK4 genes are linked to a hereditary form of human hypertension known as Pseudohypoaldosteronism type II (PHAII). In Drosophila, only one WNK gene, DWNK, has been identified. This study found that both the wild-type and kinase-dead forms of WNK1 or WNK4 caused the up-regulation of Lhx8 gene expression in NIH3T3 cells. Similarly, a previous study showed that SPAK, a substrate of WNK1, was weakly phosphorylated by the kinase-dead form of WNK1 following a long incubation (Moriguchi, 2005). These results are inconsistent with the idea that the kinase-dead form of DWNK functions as a dominant-negative mutant in Drosophila. Studies of WNK1 and WNK4 suggest that these molecules phosphorylate each other and coordinated to regulate NaCl cotransport. Therefore, these results raised the possibility that the kinase-dead forms of WNK1 and WNK4 coordinate with their respective endogenous WNK1 and WNK4 counterparts in mammalian cells. In fact, this study found that co-expression of both kinase-dead forms of WNK1 and WNK4 did not cause either induction of Lhx8 gene expression or phosphorylation of mOsr1. These results suggest that the kinase activity of WNKs is required for induction of Lhx8 gene expression and the activation of SPAK/OSR1, and that the kinase-dead form of WNK acts as an actual dominant-negative form in the signaling pathway. Furthermore, the expression of Lhx8 by either hypertonic or RA stimulation was required for the expression of both WNK1 and WNK4. Taken together, these results suggest that WNK1 and WNK4 function coordinately and redundantly in mammalian cells (Sato, 2013).

A previous report demonstrated that WNK1 might control the formation of microtubules in developing neurons. Other studies suggested that Lhx8 plays an important role in the development of basal forebrain cholinergic neurons, that Fray is required for axonal ensheathment, and that Awh is expressed in neuroblasts in stage 9 embryos in Drosophila. This study showed that the WNK-OSR1 pathway regulates Lhx8 gene expression, that knockdown of both Wnk1 and Wnk4 in Neuro2A cells caused a shortening of neurites, as well as reduced Lhx8 expression, and that the expression of the constitutively active form of mOsr1, mOsr1^S325D, could rescue the phenotype caused by the knockdown of both Wnk1 and Wnk4. In addition, mutation of DWNK or expression of a dominant-negative form of DWNK in fly embryos caused defects in axon guidance in the peripheral nervous system, and the constitutively active form of fray, fray^S347D, expression could rescue the phenotypes by the expression of the dominant negative form of DWNK. Furthermore, ubiquitous expression of Awh by da-Gal4 showed severe defects of axon guidance as similar to DWNK^D420A expression by da-Gal4, although neural specific expression of Awh did not showed any phenotype. Taken together, these findings clearly indicate that the WNK-OSR1/Fray-Lhx8/Awh pathway is involved in neural development. However, the phenotypes caused by knockdown of both Wnk1 and Wnk4, such as the shortening of neurites and the reduction in ChAT expression, were not rescued by the expression of Lhx8 in Neuro2A cells. In addition, the expression of Awh could not rescue the defects in the peripheral nervous system by the expression of the dominant-negative form of DWNK. Previous reports showed that Lhx8 might work with other factors, such as Lhx6 or Isl1. However, this study also found that the expression of Lhx6 and/or Isl1 with Lhx8 could not rescue the defects by knockdown of both Wnk1 and Wnk4 in Neuro2A cell. These results suggest that other molecule(s) are involved in neural differentiation induced by WNK signaling. The current studies may provide the first evidence identifying a target gene that acts downstream in the WNK-SPAK/OSR1 pathway, and demonstrate the significance of the WNK-OSR1-Lhx8 pathway in neural development. However, the details of how other unknown molecules controlled by WNK signaling specifically contribute to neural developmental remain to be determined and will require additional study (Sato, 2013).

Genetic mutations of WNK1 or WNK4 in PHAII patients result in abnormal expression of the WNK1 gene or WNK4 kinase activity, respectively. Abnormal activation of the WNK signaling pathway caused by these mutations result in the misregulation of NCCs in the kidney, which in turn causes hypertension. However, PHAII patients display other clinical features, such as an intellectual impairment, dental abnormalities and impaired growth. Although these features are also thought to be caused by WNK1 or WNK4 mutations, the details of how these pathologies occur are unknown except for hypertension. This study identified Lhx8 as a downstream target of the WNK signaling pathway. Evidence was also found that the WNK-Lhx8 pathway is involved in neural development. Previous studies have shown that knockdown of Lhx8 using antisense oligodeoxynucleotides caused the loss of tooth germ, and Lhx8 and Lhx6 are key regulators of mammalian dentitio. Furthermore, Lhx8 knockout mice show a reduction in the number of cholinergic neurons in the ventral forebrain and exhibit a severe deficit in spatial learning and memory . These observations indicate that Lhx8 has essential functions in the formation of the tooth development, the specification of the cholinergic neurons and the processing of the spatial information in mice. Therefore, the similarities between the clinical features of PHAII and the phenotypes of Lhx8 knockdown or knockout mice strongly suggest that the WNK-Lhx8 pathway is involved in the pathogenesis of PHAII, aside from hypertension. Further investigation will be needed to prove this hypothesis (Sato, 2013).

Hydrostatic Pressure Sensing by WNK kinases

Previous work has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N(2) gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N(2) gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed (Humphreys, 2023).

WNK1-dependent water influx is required for CD4(+) T cell activation and T cell-dependent antibody responses

Signaling from the T cell antigen receptor (TCR) on CD4(+) T cells plays a critical role in adaptive immune responses by inducing T cell activation, proliferation, and differentiation. This study demonstrates that WNK1, a kinase implicated in osmoregulation in the kidney, is required in T cells to support T-dependent antibody responses. The canonical WNK1-OXSR1-STK39 kinase signaling pathway is required for TCR signaling in CD4(+) T cells, their subsequent entry into the cell cycle, and suppression of the ATR-mediated G2/M cell cycle checkpoint. The WNK1 pathway regulates ion influx leading to water influx, potentially through AQP3, and water influx is required for TCR-induced signaling and cell cycle entry. Thus, TCR signaling via WNK1, OXSR1, STK39 and AQP3 leads to water entry that is essential for CD4(+) T cell proliferation and hence T cell-dependent antibody responses (O'May, 2025).

Zebrafish model for functional screening of flow-responsive genes controlling endothelial cell proliferation

Local haemodynamics control arterial homeostasis and dysfunction by generating wall shear stress (WSS) which regulates endothelial cell (EC) physiology. This study used a zebrafish model to identify genes that regulate EC proliferation in response to flow. Suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) reduced EC proliferation in the intersegmental vessels (ISVs) compared to controls exposed to flow. The expression of candidate regulators of proliferation was analysed in EC isolated from zebrafish embryos by qRT-PCR. Genes shown to be expressed in EC were analysed for the ability to regulate proliferation in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. Wk1 negatively regulated proliferation in no-flow conditions, whereas fzd5, gsk3beta, trpm7 and bmp2a promoted proliferation in EC exposed to flow. Immunofluorescent staining of mammalian arteries revealed that WNK1 is expressed at sites of low WSS in the murine aorta, and in EC overlying human atherosclerotic plaques. It is concluded that WNK1 is expressed in EC at sites of low WSS and in diseased arteries and may influence vascular homeostasis by reducing EC proliferation.

Delayed vacuolation in mammalian cells caused by hypotonicity and ion loss

Prolonged exposure of mammalian cells to hypotonic environments stimulates the development of sometimes large and numerous vacuoles of unknown origin. This study investigated the nature and formation of these vacuoles, which was termed LateVacs. Vacuolation starts after osmotic cell swelling has subsided and continues for many hours thereafter. Most of the vacuoles are positive for the lysosomal marker LAMP-1 but not for the autophagosomal marker LC3. Vacuoles do not appear to have acidic pH, as they exclude LysoTracker and acridine orange; inhibiting the V-ATPase with bafilomycin A1 has no effect on their formation. No LateVacs were formed in cells with a knockout of the essential LRRC8A subunit of the volume-regulated anion channel (VRAC). Since the main feature of cells recovered from hypotonic swelling should be reduced chloride concentration, tests were performed to see if chloride depletion can act as a signal for vacuolation. Indeed, four different low-chloride buffers resulted in the development of similar vacuoles. Moreover, vacuolation was suppressed in WNK1/WNK3 double knockouts or by the inhibition of WNK kinase, which is activated by low chloride; in hypotonic media, the WNK inhibitor had a similar effect. However, exposing cells to a low-sodium, high-potassium medium also resulted in vacuoles, which were insensitive to WNK. It is concluded that vacuole development can be triggered either by the loss of chloride or by the loss of sodium (Zook, 2024).

Water and chloride as allosteric inhibitors in WNK kinase osmosensing

Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to as conserved water networks 1 and 2 (CWN1 and CWN2). This study shows that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride-binding site are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. These charges were mutenized in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand (Teixeira, 2024).

Chemokine-mediated F-actin dynamics, polarity, and migration in B lymphocytes depend on WNK1 signaling

Ligand-engaged chemokine receptors trigger nucleotide exchange in heterotrimeric Galpha(i) proteins, which stimulates cytoskeletal reorganization and cell polarity changes. To better understand the signaling events responsible for these cellular changes, this study focused on early changes in F-actin dynamics after engagement of the chemokine receptor CXCR5 in murine splenic B cells. Within 10 seconds of exposure to the CXCR5 ligand CXCL13, three-dimensional lamellar-like pseudopods and F-actin-rich ridges appeared. The transient F-actin increase depended on Galpha(i2/3) signaling, the PI3K/AKT pathway, ERK activation, phospholipase C activity, and Rac1/2 activation mediated by Dock2 (dedicator of cytokinesis 2). Immunoblot analyses identified the kinase WNK1 (with no lysine kinase 1) as a potential early AKT effector. Treating B cells with specific WNK inhibitors disrupted F-actin dynamics and impaired B cell polarity, motility, and chemotaxis. These changes were mimicked in a murine B cell line by CRISPR-Cas9 gene editing of Wnk1, which also suggested that WNK1 contributed to B cell proliferation. Administration of a single dose of a WNK inhibitor transiently reduced B cell motility and polarity in the lymph nodes of live mice. These results indicate that WNK1 signaling maintains B cell responsiveness to CXCL13 and suggest that pharmacological inhibition of WNK1, which is involved in cancer progression and blood pressure regulation, may affect humoral immunity (Hwang, 2024).

Identification of WNK1 as a therapeutic target to suppress IgH/MYC expression in multiple myeloma

Multiple myeloma (MM) remains an incurable hematological malignancy demanding innovative therapeutic strategies. Targeting MYC, the notorious yet traditionally undruggable oncogene, presents an appealing avenue. Using a genome-scale CRISPR-Cas9 screen, this study identified the WNK lysine-deficient protein kinase 1 (WNK1) as a regulator of MYC expression in MM cells. Genetic and pharmacological inhibition of WNK1 reduces MYC expression and, further, disrupts the MYC-dependent transcriptional program. Mechanistically, WNK1 inhibition attenuates the activity of the immunoglobulin heavy chain (IgH) enhancer, thus reducing MYC transcription when this locus is translocated near the MYC locus. WNK1 inhibition profoundly impacts MM cell behaviors, leading to growth inhibition, cell-cycle arrest, senescence, and apoptosis. Importantly, the WNK inhibitor WNK463 inhibits MM growth in primary patient samples as well as xenograft mouse models and exhibits synergistic effects with various anti-MM compounds. Collectively, this study uncovers WNK1 as a potential therapeutic target in MM (Ye, 2024).

Search PubMed for articles about Drosophila Wnk

Bowley, G., Irving, S., Hoefer, I., Wilkinson, R., Pasterkamp, G., Darwish, H. M. S., White, S., Francis, S. E., Chico, T., Noel, E., Serbanovic-Canic, J., Evans, P. C. (2024). Zebrafish model for functional screening of flow-responsive genes controlling endothelial cell proliferation. Sci Rep, 14(1):30130 PubMed ID: 39627337

Eick, A. K., Ogueta, M., Buhl, E., Hodge, J. J. L. and Stanewsky, R. (2022). The opposing chloride cotransporters KCC and NKCC control locomotor activity in constant light and during long days. Curr Biol 32(6): 1420-1428. PubMed ID: 35303416

Farkas, R., Pecenova, L., Mentelova, L., Beno, M., Benova-Liszekova, D., Mahmoodova, S., Tejnecky, V., Raska, O., Juda, P., Svidenska, S., Hornacek, M., Chase, B. A. and Raska, I. (2016). Massive excretion of calcium oxalate from late prepupal salivary glands of Drosophila melanogaster demonstrates active nephridial-like anion transport. Dev Growth Differ 58: 562-574. PubMed ID: 27397870

Hwang, I. Y., Kim, J. S., Harrison, K. A., Park, C., Shi, C. S., Kehrl, J. H. (2024). Chemokine-mediated F-actin dynamics, polarity, and migration in B lymphocytes depend on WNK1 signaling. Sci Signal, 17(851):eade1119 PubMed ID: 39190707

Humphreys, J. M., Teixeira, L. R., Akella, R., He, H., Kannangara, A. R., Sekulski, K., Pleinis, J., Liwocha, J., Jiou, J., Servage, K. A., Orth, K., Joachimiak, L., Rizo, J., Cobb, M. H., Brautigam, C. A., Rodan, A. R. and Goldsmith, E. J. (2023). Hydrostatic Pressure Sensing by WNK kinases. Mol Biol Cell: mbcE23030113. PubMed ID: 37585288

Izadifar, A., Courchet, J., Virga, D. M., Verreet, T., Hamilton, S., Ayaz, D., Misbaer, A., Vandenbogaerde, S., Monteiro, L., Petrovic, M., Sachse, S., Yan, B., Erfurth, M. L., Dascenco, D., Kise, Y., Yan, J., Edwards-Faret, G., Lewis, T., Polleux, F. and Schmucker, D. (2021). Axon morphogenesis and maintenance require an evolutionary conserved safeguard function of Wnk kinases antagonizing Sarm and Axed. Neuron. PubMed ID: 34384519

Lones, L. and DiAntonio, A. (2023). SIK3 and Wnk converge on Fray to regulate glial K+ buffering and seizure susceptibility. PLoS Genet 19(1): e1010581. PubMed ID: 36626385

O'May, J. B., Vanes, L., de Boer, L. L., Lewis, D. A., Hartweger, H., Kunzelmann, S., Hayward, D., Llorian, M., Kochl, R., Tybulewicz, V. L. J. (2025). WNK1-dependent water influx is required for CD4(+) T cell activation and T cell-dependent antibody responses. Nat Commun, 16(1):1857 PubMed ID: 39984435

Sato, A. and Shibuya, H. (2013). WNK signaling is involved in neural development via Lhx8/Awh expression. PLoS One 8: e55301. PubMed ID: 23383144

Schellinger, J. N., Sun, Q., Pleinis, J. M., An, S. W., Hu, J., Mercenne, G., Titos, I., Huang, C. L., Rothenfluh, A. and Rodan, A. R. (2022). Chloride oscillation in pacemaker neurons regulates circadian rhythms through a chloride-sensing WNK kinase signaling cascade. Curr Biol 32(6): 1429-1438. PubMed ID: 35303418

Teixeira, L. R., Akella, R., Humphreys, J. M., He, H., Goldsmith, E. J. (2024). Water and chloride as allosteric inhibitors in WNK kinase osmosensing. Elife, 12 PubMed ID: 39584807

Yarikipati, P., Jonusaite, S., Pleinis, J. M., Dominicci Cotto, C., Sanchez-Hernandez, D., Morrison, D. E., Goyal, S., Schellinger, J., Penalva, C., Curtiss, J., Rodan, A. R., Jenny, A. (2023). Unanticipated domain requirements for Drosophila Wnk kinase in vivo. PLoS Genet, 19(10):e1010975 PubMed ID: 37819975

Ye, T., Mishra, A. K., Banday, S., Li, R., Hu, K., Coleman, M. M., Shan, Y., Chowdhury, S. R., Zhou, L., Pak, M. L., Simone, T. M., Malonia, S. K., Zhu, L. J., Kelliher, M. A., Green, M. R. (2024). Identification of WNK1 as a therapeutic target to suppress IgH/MYC expression in multiple myeloma. Cell Rep, 43(5):114211 PubMed ID: 38722741

Zook, E., Pan, Y. E., Wipplinger, A., Kerschbaum, H. H., Clements, R. J., Ritter, M., Stauber, T., Model, M. A. (2024). Delayed vacuolation in mammalian cells caused by hypotonicity and ion loss. Sci Rep, 14(1):29354 PubMed ID: 39592718

date revised: 14 May 2025

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