The intramembrane COOH-terminal domain of PRRT2 regulates voltage-dependent Na+ channels.

Proline-rich transmembrane protein 2 (PRRT2) is the single causative gene for pleiotropic paroxysmal syndromes, including epilepsy, kinesigenic dyskinesia, episodic ataxia, and migraine. PRRT2 is a neuron-specific type-2 membrane protein with a COOH-terminal intramembrane domain and a long proline-rich NH2-terminal cytoplasmic region. A large array of experimental data indicates that PRRT2 is a neuron stability gene that negatively controls intrinsic excitability by regulating surface membrane localization and biophysical properties of voltage-dependent Na+ channels Nav1.2 and Nav1.6, but not Nav1.1. To further investigate the regulatory role of PRRT2, we studied the structural features of this membrane protein with molecular dynamics simulations, and its structure-function relationships with Nav1.2 channels by biochemical and electrophysiological techniques. We found that the intramembrane COOH-terminal region maintains a stable conformation over time, with the first transmembrane domain forming a helix-loop-helix motif within the bilayer. The unstructured NH2-terminal cytoplasmic region bound to the Nav1.2 better than the isolated COOH-terminal intramembrane domain, mimicking full-length PRRT2, while the COOH-terminal intramembrane domain was able to modulate Na+ current and channel biophysical properties, still maintaining the striking specificity for Nav1.2 versus Nav1.1. channels. The results identify PRRT2 as a dual-domain protein in which the NH2-terminal cytoplasmic region acts as a binding antenna for Na+ channels, while the COOH-terminal membrane domain regulates channel exposure on the membrane and its biophysical properties.

Missense mutations in the membrane domain of PRRT2 affect its interaction with Nav1.2 voltage-gated sodium channels.

PRRT2 is a neuronal protein that controls neuronal excitability and network stability by modulating voltage-gated Na+ channel (Nav). PRRT2 pathogenic variants cause pleiotropic syndromes including epilepsy, paroxysmal kinesigenic dyskinesia and episodic ataxia attributable to loss-of-function pathogenetic mechanism. Based on the evidence that the transmembrane domain of PRRT2 interacts with Nav1.2/1.6, we focused on eight missense mutations located within the domain that show expression and membrane localization similar to the wild-type protein. Molecular dynamics simulations showed that the mutants do not alter the structural stability of the PRRT2 membrane domain and preserve its conformation. Using affinity assays, we found that the A320V and V286M mutants displayed respectively decreased and increased binding to Nav1.2. Accordingly, surface biotinylation showed an increased Nav1.2 surface exposure induced by the A320V mutant. Electrophysiological analysis confirmed the lack of modulation of Nav1.2 biophysical properties by the A320V mutant with a loss-of-function phenotype, while the V286M mutant displayed a gain-of-function with respect to wild-type PRRT2 with a more pronounced left-shift of the inactivation kinetics and delayed recovery from inactivation. The data confirm the key role played by the PRRT2-Nav interaction in the pathogenesis of the PRRT2-linked disorders and suggest an involvement of the A320 and V286 residues in the interaction site. Given the similar clinical phenotype caused by the two mutations, we speculate that circuit instability and paroxysmal manifestations may arise when PRRT2 function is outside the physiological range.

Vesicular Glutamate Release from Feeder-FreehiPSC-Derived Neurons.

Human-induced pluripotent stem cells (hiPSCs) represent one of the main and powerful tools for the in vitro modeling of neurological diseases. Standard hiPSC-based protocols make use of animal-derived feeder systems to better support the neuronal differentiation process. Despite their efficiency, such protocols may not be appropriate to dissect neuronal specific properties or to avoid interspecies contaminations, hindering their future translation into clinical and drug discovery approaches. In this work, we focused on the optimization of a reproducible protocol in feeder-free conditions able to generate functional glutamatergic neurons. This protocol is based on a generation of neuroprecursor cells differentiated into human neurons with the administration in the culture medium of specific neurotrophins in a Geltrex-coated substrate. We confirmed the efficiency of this protocol through molecular analysis (upregulation of neuronal markers and neurotransmitter receptors assessed by gene expression profiling and expression of the neuronal markers at the protein level), morphological analysis, and immunfluorescence detection of pre-synaptic and post-synaptic markers at synaptic boutons. The hiPSC-derived neurons acquired Ca2+-dependent glutamate release properties as a hallmark of neuronal maturation. In conclusion, our study describes a new methodological approach to achieve feeder-free neuronal differentiation from hiPSC and adds a new tool for functional characterization of hiPSC-derived neurons.

Constitutive Inactivation of the PRRT2 Gene Alters Short-Term Synaptic Plasticity and Promotes Network Hyperexcitability in Hippocampal Neurons.

Mutations in PRoline-Rich Transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders including epilepsy, kinesigenic dyskinesia and migraine. Most of the mutations lead to impaired PRRT2 expression and/or function, emphasizing the pathogenic role of the PRRT2 deficiency. In this work, we investigated the phenotype of primary hippocampal neurons obtained from mouse embryos in which the PRRT2 gene was constitutively inactivated. Although PRRT2 is expressed by both excitatory and inhibitory neurons, its deletion decreases the number of excitatory synapses without significantly affecting the number of inhibitory synapses or the nerve terminal ultrastructure. Analysis of synaptic function in primary PRRT2 knockout excitatory neurons by live imaging and electrophysiology showed slowdown of the kinetics of exocytosis, weakened spontaneous and evoked synaptic transmission and markedly increased facilitation. Inhibitory neurons showed strengthening of basal synaptic transmission, accompanied by faster depression. At the network level these complex synaptic effects resulted in a state of heightened spontaneous and evoked activity that was associated with increased excitability of excitatory neurons in both PRRT2 knockout primary cultures and acute hippocampal slices. The data indicate the existence of network instability/hyperexcitability as the possible basis of the paroxysmal phenotypes associated with PRRT2 mutations.

Progress of Induced Pluripotent Stem Cell Technologies to Understand Genetic Epilepsy.

The study of the pathomechanisms by which gene mutations lead to neurological diseases has benefit from several cellular and animal models. Recently, induced Pluripotent Stem Cell (iPSC) technologies have made possible the access to human neurons to study nervous system disease-related mechanisms, and are at the forefront of the research into neurological diseases. In this review, we will focalize upon genetic epilepsy, and summarize the most recent studies in which iPSC-based technologies were used to gain insight on the molecular bases of epilepsies. Moreover, we discuss the latest advancements in epilepsy cell modeling. At the two dimensional (2D) level, single-cell models of iPSC-derived neurons lead to a mature neuronal phenotype, and now allow a reliable investigation of synaptic transmission and plasticity. In addition, functional characterization of cerebral organoids enlightens neuronal network dynamics in a three-dimensional (3D) structure. Finally, we discuss the use of iPSCs as the cutting-edge technology for cell therapy in epilepsy.

PRRT2 controls neuronal excitability by negatively modulating Na+ channel 1.2/1.6 activity.

See Lerche (doi:10.1093/brain/awy073) for a scientific commentary on this article.Proline-rich transmembrane protein 2 (PRRT2) is the causative gene for a heterogeneous group of familial paroxysmal neurological disorders that include seizures with onset in the first year of life (benign familial infantile seizures), paroxysmal kinesigenic dyskinesia or a combination of both. Most of the PRRT2 mutations are loss-of-function leading to haploinsufficiency and 80% of the patients carry the same frameshift mutation (c.649dupC; p.Arg217Profs*8), which leads to a premature stop codon. To model the disease and dissect the physiological role of PRRT2, we studied the phenotype of neurons differentiated from induced pluripotent stem cells from previously described heterozygous and homozygous siblings carrying the c.649dupC mutation. Single-cell patch-clamp experiments on induced pluripotent stem cell-derived neurons from homozygous patients showed increased Na+ currents that were fully rescued by expression of wild-type PRRT2. Closely similar electrophysiological features were observed in primary neurons obtained from the recently characterized PRRT2 knockout mouse. This phenotype was associated with an increased length of the axon initial segment and with markedly augmented spontaneous and evoked firing and bursting activities evaluated, at the network level, by multi-electrode array electrophysiology. Using HEK-293 cells stably expressing Nav channel subtypes, we demonstrated that the expression of PRRT2 decreases the membrane exposure and Na+ current of Nav1.2/Nav1.6, but not Nav1.1, channels. Moreover, PRRT2 directly interacted with Nav1.2/Nav1.6 channels and induced a negative shift in the voltage-dependence of inactivation and a slow-down in the recovery from inactivation. In addition, by co-immunoprecipitation assays, we showed that the PRRT2-Nav interaction also occurs in brain tissue. The study demonstrates that the lack of PRRT2 leads to a hyperactivity of voltage-dependent Na+ channels in homozygous PRRT2 knockout human and mouse neurons and that, in addition to the reported synaptic functions, PRRT2 is an important negative modulator of Nav1.2 and Nav1.6 channels. Given the predominant paroxysmal character of PRRT2-linked diseases, the disturbance in cellular excitability by lack of negative modulation of Na+ channels appears as the key pathogenetic mechanism.

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE.

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N cyt/C exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders.

The PRRT2 knockout mouse recapitulates the neurological diseases associated with PRRT2 mutations.

Heterozygous and rare homozygous mutations in PRoline-Rich Transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders including epilepsy, kinesigenic dyskinesia episodic ataxia and migraine. Most of the mutations lead to impaired PRRT2 expression and/or function. Recently, an important role for PRTT2 in the neurotransmitter release machinery, brain development and synapse formation has been uncovered. In this work, we have characterized the phenotype of a mouse in which the PRRT2 gene has been constitutively inactivated (PRRT2 KO). ß-galactosidase staining allowed to map the regional expression of PRRT2 that was more intense in the cerebellum, hindbrain and spinal cord, while it was localized to restricted areas in the forebrain. PRRT2 KO mice are normal at birth, but display paroxysmal movements at the onset of locomotion that persist in the adulthood. In addition, adult PRRT2 KO mice present abnormal motor behaviors characterized by wild running and jumping in response to audiogenic stimuli that are ineffective in wild type mice and an increased sensitivity to the convulsive effects of pentylentetrazol. Patch-clamp electrophysiology in hippocampal and cerebellar slices revealed specific effects in the cerebellum, where PRRT2 is highly expressed, consisting in a higher excitatory strength at parallel fiber-Purkinje cell synapses during high frequency stimulation. The results show that the PRRT2 KO mouse reproduces the motor paroxysms present in the human PRRT2-linked pathology and can be proposed as an experimental model for the study of the pathogenesis of the disease as well as for testing personalized therapeutic approaches.

SYN2 is an autism predisposing gene: loss-of-function mutations alter synaptic vesicle cycling and axon outgrowth.

An increasing number of genes predisposing to autism spectrum disorders (ASDs) has been identified, many of which are implicated in synaptic function. This 'synaptic autism pathway' notably includes disruption of SYN1 that is associated with epilepsy, autism and abnormal behavior in both human and mice models. Synapsins constitute a multigene family of neuron-specific phosphoproteins (SYN1-3) present in the majority of synapses where they are implicated in the regulation of neurotransmitter release and synaptogenesis. Synapsins I and II, the major Syn isoforms in the adult brain, display partially overlapping functions and defects in both isoforms are associated with epilepsy and autistic-like behavior in mice. In this study, we show that nonsense (A94fs199X) and missense (Y236S and G464R) mutations in SYN2 are associated with ASD in humans. The phenotype is apparent in males. Female carriers of SYN2 mutations are unaffected, suggesting that SYN2 is another example of autosomal sex-limited expression in ASD. When expressed in SYN2  knockout neurons, wild-type human Syn II fully rescues the SYN2 knockout phenotype, whereas the nonsense mutant is not expressed and the missense mutants are virtually unable to modify the SYN2 knockout phenotype. These results identify for the first time SYN2  as a novel predisposing gene for ASD and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies ASD.

Phosphorylation of synapsin I by cyclin-dependent kinase-5 sets the ratio between the resting and recycling pools of synaptic vesicles at hippocampal synapses.

Cyclin-dependent kinase-5 (Cdk5) was reported to downscale neurotransmission by sequestering synaptic vesicles (SVs) in the release-reluctant resting pool, but the molecular targets mediating this activity remain unknown. Synapsin I (SynI), a major SV phosphoprotein involved in the regulation of SV trafficking and neurotransmitter release, is one of the presynaptic substrates of Cdk5, which phosphorylates it in its C-terminal region at Ser(549) (site 6) and Ser(551) (site 7). Here we demonstrate that Cdk5 phosphorylation of SynI fine tunes the recruitment of SVs to the active recycling pool and contributes to the Cdk5-mediated homeostatic responses. Phosphorylation of SynI by Cdk5 is physiologically regulated and enhances its binding to F-actin. The effects of Cdk5 inhibition on the size and depletion kinetics of the recycling pool, as well as on SV distribution within the nerve terminal, are virtually abolished in mouse SynI knock-out (KO) neurons or in KO neurons expressing the dephosphomimetic SynI mutants at sites 6,7 or site 7 only. The observation that the single site-7 mutant phenocopies the effects of the deletion of SynI identifies this site as the central switch in mediating the synaptic effects of Cdk5 and demonstrates that SynI is necessary and sufficient for achieving the effects of the kinase on SV trafficking. The phosphorylation state of SynI by Cdk5 at site 7 is regulated during chronic modification of neuronal activity and is an essential downstream effector for the Cdk5-mediated homeostatic scaling.

Synapsin knockdown is associated with decreased neurite outgrowth, functional synaptogenesis impairment, and fast high-frequency neurotransmitter release.

Synapsins (Syns) are an evolutionarily conserved family of synaptic vesicle-associated proteins related to fine tuning of synaptic transmission. Studies with mammals have partially clarified the different roles of Syns; however, the presence of different genes and isoforms and the development of compensatory mechanisms hinder accurate data interpretation. Here, we use a simple in vitro monosynaptic Helix neuron connection, reproducing an in vivo physiological connection as a reliable experimental model to investigate the effects of Syn knockdown. Cells overexpressing an antisense construct against Helix Syn showed a time-dependent decrease of Syn immunostaining, confirming protein loss. At the morphological level, Syn-silenced cells showed a reduction in neurite linear outgrowth and branching and in the size and number of synaptic varicosities. Functionally, Syn-silenced cells presented a reduced ability to form synaptic connections; however, functional chemical synapses showed similar basal excitatory postsynaptic potentials and similar short-term plasticity paradigms. In addition, Syn-silenced cells presented faster neurotransmitter release and decreased postsynaptic response toward the end of long tetanic presynaptic stimulations, probably related to an impairment of the synaptic vesicle trafficking resulting from a different vesicle handling, with an increased readily releasable pool and a compromised reserve pool.

SYN1 loss-of-function mutations in autism and partial epilepsy cause impaired synaptic function.

Several genes predisposing to autism spectrum disorders (ASDs) with or without epilepsy have been identified, many of which are implicated in synaptic function. Here we report a Q555X mutation in synapsin 1 (SYN1), an X-linked gene encoding for a neuron-specific phosphoprotein implicated in the regulation of neurotransmitter release and synaptogenesis. This nonsense mutation was found in all affected individuals from a large French-Canadian family segregating epilepsy and ASDs. Additional mutations in SYN1 (A51G, A550T and T567A) were found in 1.0 and 3.5% of French-Canadian individuals with autism and epilepsy, respectively. The majority of these SYN1 mutations were clustered in the proline-rich D-domain which is substrate of multiple protein kinases. When expressed in synapsin I (SynI) knockout (KO) neurons, all the D-domain mutants failed in rescuing the impairment in the size and trafficking of synaptic vesicle pools, whereas the wild-type human SynI fully reverted the KO phenotype. Moreover, the nonsense Q555X mutation had a dramatic impact on phosphorylation by MAPK/Erk and neurite outgrowth, whereas the missense A550T and T567A mutants displayed impaired targeting to nerve terminals. These results demonstrate that SYN1 is a novel predisposing gene to ASDs, in addition to epilepsy, and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies the pathogenesis of both diseases.

A Push-Pull Mechanism Between PRRT2 and ß4-subunit Differentially Regulates Membrane Exposure and Biophysical Properties of NaV1.2 Sodium Channels.

Proline-rich transmembrane protein 2 (PRRT2) is a neuron-specific protein implicated in the control of neurotransmitter release and neural network stability. Accordingly, PRRT2 loss-of-function mutations associate with pleiotropic paroxysmal neurological disorders, including paroxysmal kinesigenic dyskinesia, episodic ataxia, benign familial infantile seizures, and hemiplegic migraine. PRRT2 is a negative modulator of the membrane exposure and biophysical properties of Na+ channels NaV1.2/NaV1.6 predominantly expressed in brain glutamatergic neurons. NaV channels form complexes with ß-subunits that facilitate the membrane targeting and the activation of the α-subunits. The opposite effects of PRRT2 and ß-subunits on NaV channels raises the question of whether PRRT2 and ß-subunits interact or compete for common binding sites on the α-subunit, generating Na+ channel complexes with distinct functional properties. Using a heterologous expression system, we have observed that ß-subunits and PRRT2 do not interact with each other and act as independent non-competitive modulators of NaV1.2 channel trafficking and biophysical properties. PRRT2 antagonizes the ß4-induced increase in expression and functional activation of the transient and persistent NaV1.2 currents, without affecting resurgent current. The data indicate that ß4-subunit and PRRT2 form a push-pull system that finely tunes the membrane expression and function of NaV channels and the intrinsic neuronal excitability.

Modeling PCDH19 clustering epilepsy by Neurogenin 2 induction of patient-derived induced pluripotent stem cells.

BACKGROUND: Loss of function mutations in PCDH19 gene causes an X-linked, infant-onset clustering epilepsy, associated with intellectual disability and autistic features. The unique pattern of inheritance includes random X-chromosome inactivation, which leads to pathological tissue mosaicism. Females carrying PCDH19 mutations are affected, while males have a normal phenotype. No cure is presently available for this disease. METHODS: Fibroblasts from a female patient carrying frameshift mutation were reprogrammed into human induced pluripotent stem cells (hiPSCs). To create a cell model of PCDH19-clustering epilepsy (PCDH19-CE) where both cell populations co-exist, we created mosaic neurons by mixing wild-type (WT) and mutated (mut) hiPSC clones, and differentiated them into mature neurons with overexpression of the transcriptional factor Neurogenin 2. RESULTS: We generated functional neurons from patient-derived iPSC using a rapid and efficient method of differentiation through overexpression of Neurogenin 2. Was revealed an accelerated maturation and higher arborisation in the mutated neurons, while the mosaic neurons showed the highest frequency of action potential firing and hyperexcitability features, compared to mutated and WT neurons. CONCLUSIONS: Our findings provide evidence that PCDH19 c.2133delG mutation affects proper metaphases with increased numbers of centrosomes in stem cells and accelerates neuronal maturation in premature cells. PCDH19 mosaic neurons showed elevated excitability, representing the situation in PCDH19-CE brain. We suggest Ngn2 hiPSC-derived PCDH19 neurons as an informative experimental tool for understanding the pathogenesis of PCDH19-CE and a suitable approach for use in targeted drug screening strategies.

MAPK/Erk-dependent phosphorylation of synapsin mediates formation of functional synapses and short-term homosynaptic plasticity.

MAPK/Erk is a protein kinase activated by neurotrophic factors involved in synapse formation and plasticity, which acts at both the nuclear and cytoplasmic level. Synapsin proteins are synaptic-vesicle-associated proteins that are well known to be MAPK/Erk substrates at phylogenetically conserved sites. However, the physiological role of MAPK/Erk-dependent synapsin phosphorylation in regulating synaptic formation and function is poorly understood. Here, we examined whether synapsin acts as a physiological effector of MAPK/Erk in synaptogenesis and plasticity. To this aim, we developed an in vitro model of soma-to-soma paired Helix B2 neurons, that establish bidirectional excitatory synapses. We found that the formation and activity-dependent short-term plasticity of these synapses is dependent on the MAPK/Erk pathway. To address the role of synapsin in this pathway, we generated non-phosphorylatable and pseudo-phosphorylated Helix synapsin mutants at the MAPK/Erk sites. Overexpression experiments revealed that both mutants interfere with presynaptic differentiation, synapsin clustering, and severely impair post-tetanic potentiation, a form of short-term homosynaptic plasticity. Our findings show that MAPK/Erk-dependent synapsin phosphorylation has a dual role both in the establishment of functional synaptic connections and their short-term plasticity, indicating that some of the multiple extranuclear functions of MAPK/Erk in neurons can be mediated by the same multifunctional presynaptic target.

Both Schwann cell and axonal defects cause motor peripheral neuropathy in Ebf2-/- mice.

Charcot-Marie-Tooth neuropathies are frequent hereditary disorders of the nervous system and most cases remain without a molecular definition. Mutations in transcription factors have been previously associated to various types of this disease. Mice carrying a null mutation in Ebf2 transcription factor present peripheral nerve abnormalities. To get insight into Ebf2 function in peripheral nervous system, here we characterize the peripheral neuropathy affecting these mice. We first show that Ebf2 is largely expressed in peripheral nerve throughout postnatal development, its expression being not only restricted to non-myelin forming Schwann cells, but also involving myelin forming Schwann cells and the perineurium. As a consequence, the onset of myelination is delayed and Schwann cell differentiation markers are downregulated in Ebf2-/- mice. Later in development, myelin pathology appears less severe and characterized by isolated clusters of hypomyelinated fibers. However, we find defects in the nerve architecture, such as abnormalities of the nodal region and shorter internodal length. Furthermore, we demonstrate a significant decrease in axonal calibre, with a lack of large calibre axons, and a severe impairment of motor nerve conduction velocity and amplitude, whereas the sensory nerve parameters are less affected. Interestingly, a clinical case with peripheral motor neuropathy and clinical features similar to Ebf2-/- mice phenotype was associated with a deletion encompassing EBF2 human genomic locus. These findings demonstrate that Ebf2 is a new molecule implicated in peripheral nerve development and a potential candidate gene for peripheral nerve disorders.

An Emerging Role of PRRT2 in Regulating Growth Cone Morphology.

Mutations in the PRRT2 gene are the main cause for a group of paroxysmal neurological diseases including paroxysmal kinesigenic dyskinesia, episodic ataxia, benign familial infantile seizures, and hemiplegic migraine. In the mature central nervous system, the protein has both a functional and a structural role at the synapse. Indeed, PRRT2 participates in the regulation of neurotransmitter release, as well as of actin cytoskeleton dynamics during synaptogenesis. Here, we show a role of the protein also during early stages of neuronal development. We found that PRRT2 accumulates at the growth cone in cultured hippocampal neurons. Overexpression of the protein causes an increase in the size and the morphological complexity of growth cones. In contrast, the growth cones of neurons derived from PRRT2 KO mice are smaller and less elaborated. Finally, we demonstrated that the aberrant shape of PRRT2 KO growth cones is associated with a selective alteration of the growth cone actin cytoskeleton. Our data support a key role of PRRT2 in the regulation of growth cone morphology during neuronal development.

PRRT2 modulates presynaptic Ca2+ influx by interacting with P/Q-type channels.

Loss-of-function mutations in proline-rich transmembrane protein-2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We find that either acute or constitutive PRRT2 deletion induces a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that is insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype parallels a decrease in somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation, pull-down assays, and proteomics reveal a specific and direct interaction of PRRT2 with P/Q-type Ca2+ channels. At presynaptic terminals lacking PRRT2, P/Q-type Ca2+ channels reduce their clustering at the active zone, with a corresponding decrease in the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in ensuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses.

An interaction between PRRT2 and Na+/K+ ATPase contributes to the control of neuronal excitability.

Mutations in PRoline Rich Transmembrane protein 2 (PRRT2) cause pleiotropic syndromes including benign infantile epilepsy, paroxysmal kinesigenic dyskinesia, episodic ataxia, that share the paroxysmal character of the clinical manifestations. PRRT2 is a neuronal protein that plays multiple roles in the regulation of neuronal development, excitability, and neurotransmitter release. To better understand the physiopathology of these clinical phenotypes, we investigated PRRT2 interactome in mouse brain by a pulldown-based proteomic approach and identified α1 and α3 Na+/K+ ATPase (NKA) pumps as major PRRT2-binding proteins. We confirmed PRRT2 and NKA interaction by biochemical approaches and showed their colocalization at neuronal plasma membrane. The acute or constitutive inactivation of PRRT2 had a functional impact on NKA. While PRRT2-deficiency did not modify NKA expression and surface exposure, it caused an increased clustering of α3-NKA on the plasma membrane. Electrophysiological recordings showed that PRRT2-deficiency in primary neurons impaired NKA function during neuronal stimulation without affecting pump activity under resting conditions. Both phenotypes were fully normalized by re-expression of PRRT2 in PRRT2-deficient neurons. In addition, the NKA-dependent afterhyperpolarization that follows high-frequency firing was also reduced in PRRT2-silenced neurons. Taken together, these results demonstrate that PRRT2 is a physiological modulator of NKA function and suggest that an impaired NKA activity contributes to the hyperexcitability phenotype caused by PRRT2 deficiency.