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Epilepsy is a neurological condition associated to significant brain damage produced by status epilepticus (SE) including neurodegeneration, gliosis and ectopic neurogenesis. Reduction of these processes constitutes a useful strategy to improve recovery and ameliorate negative outcomes after an initial insult. SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), has been shown to increase M-current density in neurons, leading to reduced excitability and protection against seizures. For this study, we used 4-5 months old male transgenic C57BL/6 J and FVB/NJ mice expressing near physiological levels of a constitutively active form of the kinase controlled by its endogenous promoter. Here we show that SGK1.1 activation potently reduces levels of neuronal death (assessed using Fluoro-Jade C staining) and reactive glial activation (reported by GFAP and Iba-1 markers) in limbic regions and cortex, 72 h after SE induced by kainate, even in the context of high seizure activity. This neuroprotective effect is not exclusively through M-current activation but is also directly linked to decreased apoptosis levels assessed by TUNEL assays and quantification of Bim and Bcl-x L by western blot of hippocampal protein extracts. Our results demonstrate that this newly described antiapoptotic role of SGK1.1 activation acts synergistically with the regulation of cellular excitability, resulting in a significant reduction of SE-induced brain damage in areas relevant to epileptogenesis., (Copyright © 2021. Published by Elsevier Inc.)

Approaches to control epilepsy, one of the most important idiopathic brain disorders, are of great importance for public health. We have previously shown that in sympathetic neurons the neuronal isoform of the serum and glucocorticoid-regulated kinase (SGK1.1) increases the M-current, a well-known target for seizure control. The effect of SGK1.1 activation on kainate-induced seizures and neuronal excitability was studied in transgenic mice that express a permanently active form of the kinase, using electroencephalogram recordings and electrophysiological measurements in hippocampal brain slices. Our results demonstrate that SGK1.1 activation leads to reduced seizure severity and lower mortality rates following status epilepticus, in an M-current-dependent manner. EEG is characterized by reduced number, shorter duration, and early termination of kainate-induced seizures in the hippocampus and cortex. Hippocampal neurons show decreased excitability associated to increased M-current, without altering basal synaptic transmission or other neuronal properties. Altogether, our results reveal a novel and selective anticonvulsant pathway that promptly terminates seizures, suggesting that SGK1.1 activation can be a potent factor to secure the brain against permanent neuronal damage associated to epilepsy., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The M-current formed by tetramerization of Kv7.2 and Kv7.3 subunits is a neuronal voltage-gated K(+) conductance that controls resting membrane potential and cell excitability. In Xenopus laevis oocytes, an increase in Kv7.2/3 function by the serum- and glucocorticoid-regulated kinase 1 (SGK1) has been reported previously (Schuetz et al., 2008). We now show that the neuronal isoform of this kinase (SGK1.1), with distinct subcellular localization and modulation, upregulates the Kv7.2/3 current in Xenopus oocytes and mammalian human embryonic kidney HEK293 cells. In contrast to the ubiquitously expressed SGK1, the neuronal isoform SGK1.1 interacts with phosphoinositide-phosphatidylinositol 4,5-bisphosphate (PIP(2)) and is distinctly localized to the plasma membrane (Arteaga et al., 2008). An SGK1.1 mutant with disrupted PIP(2) binding sites produced no effect on Kv7.2/3 current amplitude. SGK1.1 failed to modify the voltage dependence of activation and did not change activation or deactivation kinetics of Kv7.2/3 channels. These results suggest that the kinase increases channel membrane abundance, which was confirmed with flow cytometry assays. To evaluate the effect of the kinase in neuronal excitability, we generated a transgenic mouse (Tg.sgk) expressing a constitutively active form of SGK1.1 (S515D). Superior cervical ganglion (SCG) neurons isolated from Tg.sgk mice showed a significant increase in M-current levels, paralleled by reduced excitability and more negative resting potentials. SGK1.1 effect on M-current in Tg.sgk-SCG neurons was counteracted by muscarinic receptor activation. Transgenic mice with increased SGK1.1 activity also showed diminished sensitivity to kainic acid-induced seizures. Altogether, our results unveil a novel role of SGK1.1 as a physiological regulator of the M-current and neuronal excitability.

The epithelial Na(+) channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na(+) reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, α, β, and γ. A fourth ENaC subunit, named δ, is expressed in the nervous system of primates, where its role is unknown. The human δ-ENaC gene generates at least two splice isoforms, δ(1) and δ(2) , differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either δ(1) or δ(2) , with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of δ(1) in Xenopus oocytes and HEK293 cells produces higher current levels than δ(2) . Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of β- and γ-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in δ(2)-N terminus. Analysis of δ(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The δ(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that δ-ENaC undergoes dynamin-independent endocytosis as opposed to αβγ-channels.

The δ-subunit of the epithelial Na(+) channel (ENaC) is expressed in neurons of the human and monkey central nervous system and forms voltage-independent, amiloride-sensitive Na(+) channels when expressed in heterologous systems. It has been proposed that δ-ENaC could affect neuronal excitability and participate in the transduction of ischemic signals during hypoxia or inflammation. The regulation of δ-ENaC activity is poorly understood. ENaC channels in kidney epithelial cells are regulated by the serum- and glucocorticoid-induced kinase 1 (SGK1). Recently, a new isoform of this kinase (SGK1.1) has been described in the central nervous system. Here we show that δ-ENaC isoforms and SGK1.1 are coexpressed in pyramidal neurons of the human and monkey (Macaca fascicularis) cerebral cortex. Coexpression of δβγ-ENaC and SGK1.1 in Xenopus oocytes increases amiloride-sensitive current and channel plasma membrane abundance. The kinase also exerts its effect when δ-subunits are expressed alone, indicating that the process is not dependent on accessory subunits or the presence of PY motifs in the channel. Furthermore, SGK1.1 action depends on its enzymatic activity and binding to phosphatidylinositol(4,5)-bisphosphate. Physiological or pharmacological activation of phospholipase C abrogates SGK1.1 interaction with the plasma membrane and modulation of δ-ENaC. Our data support a physiological role for SGK1.1 in the regulation of δ-ENaC through a pathway that differs from the classical one and suggest that the kinase could serve as an integrator of different signaling pathways converging on the channel.

The dopamine transporter (DAT) is a membrane glycoprotein responsible for dopamine (DA) uptake, which has been involved in the degeneration of DA cells in Parkinson's disease (PD). Given that DAT activity depends on its glycosylation status and membrane expression, and that not all midbrain DA cells show the same susceptibility to degeneration in PD, we have investigated a possible relationship between DAT glycosylation and function and the differential vulnerability of DA cells. Glycosylated DAT expression, DA uptake, and DAT V(max) were significantly higher in terminals of nigrostriatal neurons than in those of mesolimbic neurons. No differences were found in non-glycosylated DAT expression and DAT K(m), and DA uptake differences disappeared after deglycosylation of nigrostriatal synaptosomes. The expression pattern of glycosylated DAT in the human midbrain and striatum showed a close anatomical relationship with DA degeneration in parkinsonian patients. This relationship was confirmed in rodent and monkey models of PD, and in HEK cells expressing the wild-type and a partially deglycosylated DAT form. These results strongly suggest that DAT glycosylation is involved in the differential vulnerability of midbrain DA cells in PD.

Epithelial sodium channel (ENaC) is a member of the ENaC/degenerin family of amiloride-sensitive, non-voltage gated sodium ion channels. ENaC alpha, beta and gamma subunits are abundantly expressed in epithelial tissues, where they have been well characterized. An ENaC delta subunit has also been described in the human nervous system, although its histological distribution pattern remains unexplored. We have now isolated a novel ENaC delta isoform (delta2) from human brain and studied the expression pattern of both the known (delta1) and the new (delta2) isoforms in the human and monkey telencephalon. ENaC delta2 is produced by a combination of alternative transcription start sites, a frame shift in exon 3 and alternative splicing of exon 4. It forms functional amiloride-sensitive sodium channels when co-expressed with ENaC beta and gamma accessory subunits. Comparison with the classical ENaC channel (alphabetagamma) indicates that the interaction between delta2, beta and gamma is functionally inefficient. Both ENaC delta isoforms are widely expressed in pyramidal cells of the human and monkey cerebral cortex and in different neuronal populations of telencephalic subcortical nuclei, but double-labelling experiments demonstrated a low level of co-localization between isoforms (5-8%), suggesting specific functional roles for each of them.

An understanding of the composition of the various nerves of the pelvic plexus is essential in the design of studies to explore the autonomic control of pelvic visceral tissues. As a correlate of this interest, the present study was designed to determine the composition of the main penile nerve in the pelvic plexus of the laboratory rat, an animal commonly used for studies of reproductive physiology. Retrograde tracing studies indicate that the main penile nerve contains neurons which project to the penile crura, the corpus spongiosum, and the bulbourethral glands. The main penile nerve is the major source of neurons which innervate the corpus spongiosum and bulbourethral gland and contains about one-third of all parasympathetic neurons which project to the penile crura. Dye placed on the proximal cut end of the main penile nerve indicates that neurons in the parasympathetic region of the spinal cord (L6-S1) and to a lesser extent a sympathetic region of the cord, L1-L2, provide preganglionic innervation to ganglion cells in the main pelvic nerve. Processes of neurons in dorsal root ganglia L6-S1 and of neurons in the abdominopelvic sympathetic chain course in the main penile nerve to unknown destinations. In many respects this presumed postganglionic fiber tract is essentially a region of the pelvic plexus which subserves extrapelvic visceral tissues.