Function And Mechanism Of Neddylation Of Na_v1.1 In The Excitability Of Interneurons And Seizure Prevention

In the central nervous system,proper neural activities between excitatory pyramidal neurons and inhibitory interneurons is critical to brain functions.In particular,parvalbumin-positive(PV+)interneurons(PVINs)are a major source of inhibitory signal in maintaining the excitation-inhibition(E-I)balance.PVINs fire at high frequency and,via feedback and feedforward inhibitions,are necessary for generation of local circuit oscillations and connectivities or synchronization between brain regions.Functional changes in PVINs have been found in several neurological and psychiatric disorders,such as epilepsy,schizophrenia,autism spectrum disorder,depression,and Alzheimer's disease.The excitability of PVINs requires Na_v1.1,the?subunit of a voltage-gated sodium channels which is encoded by the SCN1A gene and is specifically expressed in PVINs.Na_v1.1 deficiency reduces the PVIN excitability,increases the threshold of action potentials(APs),and decreases AP amplitudes.Mutations in Na_v1.1 channels have been identified in patients with mild as well as severe forms of epilepsy including Severe Myoclonic Epilepsy of Infancy(SMEI or Dravet Syndrome)as well as those with GEFS+(Generalized Epilepsy with Febrile Seizures Plus).Studies of mouse models with Na_v1.1mutations or deletions suggest that epilepsy may be caused by impaired firing of GABAergic interneurons.A unified loss-of-function hypothesis posits that mild impairment of Na_v1.1 functions causes febrile seizures,intermediate loss-of-function gives rise to GEFS+epilepsy,and severe impairment causes the intractable seizures and co-morbidities of SMEI.However,in contrast to the impact of Na_v1.1 mutations on channel properties,less is known about mechanisms in regulating its stability or expression.Neddylation is a chemical reaction in which a ubiquitin-like protein Nedd8(Neural Precursor Cell Expressed,Developmentally Down-regulated 8)is conjugated to the lysine of substrate proteins by its C-terminal glycine.Like ubiquitination,neddylation requires Nedd8-specific E1,a heterodimer of NAE1(also known as APP-BP1)and UBA3,which together activate NEDD8 in an ATP-dependent manner;UBC12(a Nedd8E2 enzyme);and substrate-specific E3 ligases.Neddylated proteins could be deneddylated by NEDD8-specific proteases such as the COP9 signalosome(CSN)and NEDP1(also known as SENP8).Nedd8 is evolutionarily highly conserved and ubiquitously expressed in many cell types.Neddylation regulates a variety of cellular processes including gene transcription,cell division and differentiation,ribosome biogenesis,apoptosis and proteolysis.However,the functions of neddylation in the brain is less clear although it has been recently implicated in the development of the neuromuscular junction and excitatory synapses onto pyramidal neurons.In this study,we determined whether the development and function of interneurons is regulated by neddylation.To this end,we mutated Nae1,an obligatory subunit of the only identified Nedd8 E1 enzyme,specifically in PVINs.Immunostaining indicated that NAE1 signal was specifically reduced in PVINs.Nae1 mutation mice displayed spontaneous epileptic seizures and premature death.Morphological studies indicated that the mutation had no apparent effect on the number of PVINs in the cortex and hippocampus.Remarkably,the release of GABA,but not glutamate,was reduced in mutant mice,associated with reduced excitability of PVINs,suggesting that neddylation is critical to controlling the excitability of interneurons.Furthermore,we found that Nae1mutation impaired action potential and reduced Na_v channel current of PVINs,suggesting that Nae1 mutation causes a deficiency of Na_v in PVINs.Further molecular mechanistic studies identified Na_v1.1 as a target of Nae1 mutation,which becomes unstable in the absence of neddylation.In addition,we found that a mutation(K1936E)identified in patients with epilepsy was largely reduced surface expression and Na_v1.1 neddylation,suggesting that neddylation at Lys1936 is important in maintaining the stability of Na_v1.1.Finally,we found that restoring h Nav1.1 in neddylation-deficient PVINs was sufficient to recover the excitability,suggesting that Na_v1.1 deficiency is a major mechanism of Nae1 mutation in regulating PVIN excitability.Together,the results demonstrate that Na_v1.1 stability in PVINs requires neddylation,revealing a novel function of neddylation and identifying neddylation as a target for potential therapeutic intervention for epilepsy.