Department of Physiology, David Geffen School of Medicine, UCLA, Los Angeles, California, USA.
Address correspondence to: Stephen Cannon, Department of Physiology, David Geffen School of Medicine, 10833 Le Conte Ave., Los Angeles, California 90095, USA. Phone: 310.825.5882; Email: email@example.com.
Published April 15, 2021 - More info
The excitability of interneurons requires Nav1.1, the α subunit of the voltage-gated sodium channel. Nav1.1 deficiency and mutations reduce interneuron excitability, a major pathological mechanism for epilepsy syndromes. However, the regulatory mechanisms of Nav1.1 expression remain unclear. Here, we provide evidence that neddylation is critical to Nav1.1 stability. Mutant mice lacking Nae1, an obligatory component of the E1 ligase for neddylation, in parvalbumin-positive interneurons (PVINs) exhibited spontaneous epileptic seizures and premature death. Electrophysiological studies indicate that Nae1 deletion reduced PVIN excitability and GABA release and consequently increased the network excitability of pyramidal neurons (PyNs). Further analysis revealed a reduction in sodium-current density, not a change in channel property, in mutant PVINs and decreased Nav1.1 protein levels. These results suggest that insufficient neddylation in PVINs reduces Nav1.1 stability and thus the excitability of PVINs; the ensuing increased PyN activity causes seizures in mice. Consistently, Nav1.1 was found reduced by proteomic analysis that revealed abnormality in synapses and metabolic pathways. Our findings describe a role of neddylation in maintaining Nav1.1 stability for PVIN excitability and reveal what we believe is a new mechanism in the pathogenesis of epilepsy.
Wenbing Chen, Bin Luo, Nannan Gao, Haiwen Li, Hongsheng Wang, Lei Li, Wanpeng Cui, Lei Zhang, Dong Sun, Fang Liu, Zhaoqi Dong, Xiao Ren, Hongsheng Zhang, Huabo Su, Wen-Cheng Xiong, Lin Mei
Loss-of-function mutations of SCN1A encoding the pore-forming α subunit of the NaV1.1 neuronal sodium channel cause a severe developmental epileptic encephalopathy, Dravet syndrome (DS). In this issue of the JCI, Chen, Luo, Gao, et al. describe a phenocopy for DS in mice deficient for posttranslational conjugation with neural precursor cell expressed, developmentally downregulated 8 (NEDD8) (neddylation), selectively engineered in inhibitory interneurons. Pursuing the possibility that this phenotype is also caused by loss of NaV1.1, Chen, Luo, Gao, and colleagues show that interneuron excitability and GABA release are impaired, NaV1.1 degradation rate is increased with a commensurate decrease of NaV1.1 protein, and NaV1.1 is a substrate for neddylation. These findings establish neddylation as a mechanism for stabilizing NaV1.1 subunits and suggest another pathomechanism for epileptic sodium channelopathy.
Epilepsy is a disorder of brain function of which the cardinal feature is an enduring propensity for generating seizures. These anomalous paroxysms of hypersynchronous network activity may arise from a variety of brain insults, including developmental defects, acquired lesions (trauma, stroke, infection), and a myriad of associated gene defects. Mutations that disrupt ion channel genes, so-called channelopathies, are often identified in familial epilepsy syndromes or as de novo lesions in developmental epileptic encephalopathy.
Not surprisingly, epilepsy mutations often occur in genes coding for the pore-forming α subunits of voltage-gated sodium channels that generate action potentials in excitable cells (1–3). Mutant α subunits may have altered function or may fail to form a functional channel, and these discoveries have served as the foundation for understanding epileptogenesis at a molecular level. Accessory subunits of the channel complex and channel-interacting proteins have also emerged as culprits in epileptic channelopathies (4). In this issue of the JCI, Chen, Luo, Gao, et al. (5) report on uncovering a new potential mechanism for sodium-channel epilepsy, wherein disrupted neddylation of the channel α subunit increases the protein degradation rate and results in lower Na+ current density with reduced GABAergic inhibition by interneurons.
The human genome contains nine distinct α subunit genes (SCN1A, SCN2A, etc.) coding for the main pore-forming subunits NaV1.1, NaV1.2, etc. and four accessory β subunit genes (SCN1B, etc.). Epilepsy syndromes have been associated with mutations of NaV1.1, NaV1.2, NaV1.3, NaV1.6, and the β1 subunit. The number of identified epilepsy mutations for NaV1.1 greatly exceeds that for all the other sodium-channel subunits, and mutations of NaV1.1 cause a wide variety of clinical phenotypes (Figure 1) ranging from mild to severe epilepsy with seizures refractory to drug treatment, developmental delay, and cognitive impairment (i.e., epileptic encephalopathy). A genotype/phenotype pattern is emerging wherein missense mutations of NaV1.1 with modest alterations of channel function are found in mild syndromes (generalized epilepsy with febrile seizures [GEFS] and generalized epilepsy with febrile seizures plus [GEFS+]). In contrast, haploinsufficiency from a single null allele (e.g., frameshift, nonsense with premature truncation) causes a severe developmental epileptic encephalopathy with onset at six months of age (Dravet syndrome [DS]) (3). Over 700 pathogenic or likely pathogenic variants of SCN1A are listed for DS on ClinVar (https://www.ncbi.nlm.nih.gov/clinvar) as of March 2021.
Spectrum of epilepsy syndromes from NaV1.1 loss of function. NaV1.1 loss of function caused by SCN1A mutations or NEDD8 deficiency induces a wide variety of clinical phenotypes with varying severity.
The association of a severe epilepsy phenotype with a sodium-channel loss of function was initially puzzling because epilepsy is a disorder of anomalously enhanced excitability of neuronal activity. A major clue was provided by the NaV1.1 knockout mouse (6). Global haploinsufficiency from heterozygous deletion of exon 26 (Scn1a+/–) leads to early onset seizures (spontaneous and temperature induced), mild ataxia, and sudden unexplained death in epilepsy (SUDEP), which is similar to what occurs in individuals with DS. NaV1.1-deficient mice had a reduction of Na+ current density and decreased firing rates of parvalbumin expressing inhibitory GABAergic neurons (PVINs), but not excitatory neurons, in the cerebral cortex, hippocampus, cerebellar Purkinje cells, and reticular nucleus of the thalamus. A similar epilepsy phenotype occurred with selective haploinsufficiency (PV-Scn1a+/–) restricted to PV-positive interneurons, while the epilepsy phenotype was milder with Scn1a+/– selective for somatostatin-positive inhibitory interneurons (7, 8). Conversely, haploinsufficiency of Scn1a in excitatory neurons ameliorates the DS phenotype (9). Exceptions to this genotype/phenotype paradigm indicate more work needs to be done. For example, about half of the SCN1A variants reported in DS cases are missense mutations, some of which caused gain-of-function changes in expression studies (10); the reduced excitability of PVINs in Scn1a+/– mice at P14 later resolved at P18–P21, whereas seizures persisted (11). Others reported hyperexcitability and increased Na+ currents in P21–P24 hippocampal pyramidal neurons, suggesting overexpression of other sodium-channel genes (12). Nevertheless, the effects of murine NaV1.1 loss of function coupled to severe early onset seizures and SUDEP are robust and were the basis for implicating NaV1.1 in the neddylation studies by Chen, Luo, Gao, et al. reported in this issue (5).
Posttranslational modification by the ubiquitin-like protein neural precursor cell expressed, developmentally downregulated 8 (NEDD8) regulates many cellular functions (transcription, proliferation, differentiation, apoptosis), is essential for dendritic spine maturation and stability (13), and has been implicated in disorders of the central nervous system (14). To interrogate the role of NEDD8 in parvalbumin-positive inhibitory interneurons (PVINs), Chen, Luo, Gao, et al. (5) generated a conditional knockout of an obligatory subunit for the NEDD8-specific E1 (NAE1). Unexpectedly, the mice had severe epilepsy, ataxia, and a median survival of only 60 days. The abundance of PVINs was unchanged in PV-Nae1–/– mice, but inhibitory GABAergic neurotransmission was reduced and intrinsic excitability was reduced for PVINs, but not pyramidal neurons. Further investigation for the cause of reduced excitability in PVINs revealed a Na+ current density of 62% of WT. When measured by immunoblot, NaV1.1 protein amounts were decreased, whereas the abundance of the other major α subunit (NaV1.6) remained unchanged. By comparison, Na+ current density for PVINs in the DS Scn1a+/– mice was 47% of WT and 38% of WT for homozygous Scna1–/– (6). Epileptic seizures began around P30 for PV-Nae1–/– mice, whereas for DS Scna1+/– mice, seizure onset was earlier (P21), consistent with a more severe loss of Na+ current for Scna1+/–.
The observed reduction in Na+ current density is sufficient to explain the reduced excitability of PVINs, but why does impaired neddylation lead to lower amounts of NaV1.1? There is no previously established role for regulating the abundance of voltage-gated ion channel proteins by neddylation, although for the epithelial sodium channel (α-ENaC), NEDD8 conjugated to cullin-1 increases ubiquitination and proteolytic degradation of the channel (15). Chen, Luo, Gao, and colleagues excluded a NEDD8 effect on NaV1.1 transcript levels in PV-Nae1–/– mice (5). Instead, expression studies in a human cell line (tsA-201 cells) showed an increased rate of proteasome-dependent NaV1.1 degradation when neddylation was inhibited using MLN4924. The interpretation is that neddylation stabilizes NaV1.1 by preventing ubiquitination, just the inverse of how NEDD8/cullin-1 regulates α-ENaC.
What is the evidence NaV1.1 is a substrate for neddylation? NEDD8 immunoreactivity was detected after immunoprecipitation of tagged NaV1.1 expressed in a human cell line (HEK cells) or for native NaV1.1 from brain. Moreover, the NEDD8 signal was reduced in the presence of the NAE inhibitor MLN-4924 and for brain homogenates from PV-Nae1–/– mice, consistent with neddylation of NaV1.1. To search for potential neddylation sites at lysines in NaV1.1, the authors screened for variants of Lys residues in ClinVar and an epilepsy database. Thirteen epilepsy variants were identified, two of which were studied further because they are located in cytoplasmic loops. The NaV1.1 K1936E variant, 74 residues upstream from the C-terminal Lys, was expressed in HEK cells and found to have increased rates of NaV1.1 degradation, reduced NEDD8 immunostaining, and lower Na+ current density, all of which are consistent with impaired neddylation and destabilized NaV1.1 protein. NaV1.1 K1936 is not conserved in NaV1.6 (established using the multiple sequence alignment program CLUSTAL 1.2.4), which may account for the lack of a detectable change for NaV1.6 expression in Nae1–/– mice. Unfortunately, the clinical annotation for NaV1.1 K1936E was insufficient to ascertain the confidence level showing that this is indeed a pathogenic mutation for epilepsy.
Many substrates for neddylation are surely present in PVINs, and the downstream effects of this posttranslational modification may be diverse and extensive. The authors acknowledge that effects beyond the reduction of NaV1.1 may contribute to the ataxia and severe epilepsy in the PV-Nae1–/– mouse. A proteome screen identified 5167 proteins with 169 downregulated and 279 upregulated for PV-Nae1–/– mice compared with WT. The former group is implicated in neural development, synaptic plasticity, and glutamatergic neurotransmission, whereas the upregulated set is involved with ubiquitin-regulated catabolism and the metabolic pathway. Interestingly, of the 53 ion channel genes identified in this analysis, only the reduction of NaV1.1 could readily account for the decreased excitability observed in PVINs.
The study by Chen, Luo, Gao, et al. in this issue of the JCI identifies a mechanism for fine-tuning neuronal excitability by neddylation-dependent stabilization of ion channel proteins and also reveals that selective disruption of this regulatory pathway in PVINs leads to ataxia, severe epilepsy, and SUDEP in mice (5). This phenotype highly resembles that of DS models in which the coding potential of Scn1a is destroyed and lends credence to the notion that loss-of-function defects for NaV1.1 in PVINs produces susceptibility to severe epilepsy. It remains to be established that epilepsy in humans is attributable to impaired neddylation of NaV1.1. One question is whether haploinsufficiency of Nae1 or some other critical component for NEDD8 conjugation will be sufficient to cause epilepsy. A global null of Nae1 is embryonic lethal (16). If neddylation is also deficient in excitatory pyramidal neurons, will the balance between excitation and inhibition be restored to ameliorate the risk of seizures as occurs in Scn1a+/– mice (9)? Similarly, it is unknown whether a single NaV1.1 K1936E allele is sufficient to cause an epilepsy phenotype in mice or humans. The probability of homozygous NaV1.1 K1936E would be exceedingly small, except for cases of consanguinity. More studies in murine models may shed light on whether the heterozygous cases are sufficient to cause epilepsy.
SCC is supported by NIH grant R01 AR 063182 and the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at the UCLA Research Award Program.
Conflict of interest: The author has declared that no conflict of interest exists.
Copyright: © 2021, American Society for Clinical Investigation.
Reference information: J Clin Invest. 2021;131(8):e148370. https://doi.org/10.1172/JCI148370.