The Experts below are selected from a list of 2313 Experts worldwide ranked by ideXlab platform
Bruno Sterlini - One of the best experts on this subject based on the ideXlab platform.
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PRRT2 modulates presynaptic ca2 influx by interacting with p q type channels
Cell Reports, 2021Co-Authors: Daniele Ferrante, Bruno Sterlini, Cosimo Prestigio, Antonella Marte, Franco Onofri, Anna Corradi, Andrea Petretto, Giorgio Tortarolo, Giuseppe Vicidomini, Jessica MuiaAbstract: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.
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an interaction between PRRT2 and na k atpase contributes to the control of neuronal excitability
Cell Death and Disease, 2021Co-Authors: Bruno Sterlini, Pierluigi Valente, Alessandra Romei, Flavia Valtorta, Chiara Parodi, Davide Aprile, Michele Oneto, Anita Aperia, Anna Fassio, Pietro BaldelliAbstract: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.
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PRRT2 modulates presynaptic ca 2 influx by interacting with p q type channels
Social Science Research Network, 2020Co-Authors: Daniele Ferrante, Bruno Sterlini, Cosimo Prestigio, Antonella Marte, Franco Onofri, Anna Corradi, Andrea Petretto, Jessica Muja, Agnes Thalhammer, Pierluigi ValenteAbstract:Loss-of-function mutations in proline-rich transmembrane protein 2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We found that either acute or constitutive PRRT2 deletion induced a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that was insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype was paralleled by a decrease of somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation assays and proteomic screens revealed an interaction between PRRT2 and 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 of the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in assuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses.
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constitutive inactivation of the PRRT2 gene alters short term synaptic plasticity and promotes network hyperexcitability in hippocampal neurons
Cerebral Cortex, 2019Co-Authors: Pierluigi Valente, Floriana Fruscione, Bruno Sterlini, Alessandra Romei, Manuela Fadda, Enrico Castroflorio, Caterina Michetti, Nicola Forte, Davide Lonardoni, Giorgia GiansanteAbstract: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.
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PRRT2 controls neuronal excitability by negatively modulating na channel 1 2 1 6 activity
Brain, 2018Co-Authors: Floriana Fruscione, Pierluigi Valente, Bruno Sterlini, Alessandra Romei, Simona Baldassari, Manuela Fadda, Cosimo Prestigio, Giorgia Giansante, Jacopo Sartorelli, Pia RossiAbstract: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.
Pierluigi Valente - One of the best experts on this subject based on the ideXlab platform.
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increased responsiveness at the cerebellar input stage in the PRRT2 knockout model of paroxysmal kinesigenic dyskinesia
Neurobiology of Disease, 2021Co-Authors: Francesca Binda, Pierluigi Valente, Antonella Marte, Pietro Baldelli, Fabio BenfenatiAbstract:PRoline-Rich Transmembrane protein-2 (PRRT2) is a recently described neuron-specific type-2 integral membrane protein with a large cytosolic N-terminal domain that distributes in presynaptic and axonal domains where it interacts with several presynaptic proteins and voltage-gated Na+ channels. Several PRRT2 mutations are the main cause of a wide and heterogeneous spectrum of paroxysmal disorders with a loss-of-function pathomechanism. The highest expression levels of PRRT2 in brain occurs in cerebellar granule cells (GCs) and cerebellar dysfunctions participate in the dyskinetic phenotype of PRRT2 knockout (KO) mice. We have investigated the effects of PRRT2 deficiency on the intrinsic excitability of GCs and the input-output relationships at the mossy fiber-GC synapses. We show that PRRT2 KO primary GCs display increased expression of Na+ channels, increased amplitude of Na+ currents and increased length of the axon initial segment, leading to an overall enhancement of intrinsic excitability. In acute PRRT2 KO cerebellar slices, GCs were more prone to action potential discharge in response to mossy fiber activation and exhibited an enhancement of transient and persistent Na+ currents, in the absence of changes at the mossy fiber-GC synapses. The results support a key role of PRRT2 expressed in GCs in the physiological regulation of the excitatory input to the cerebellum and are consistent with a major role of a cerebellar dysfunction in the pathogenesis of the PRRT2-linked paroxysmal pathologies.
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an interaction between PRRT2 and na k atpase contributes to the control of neuronal excitability
Cell Death and Disease, 2021Co-Authors: Bruno Sterlini, Pierluigi Valente, Alessandra Romei, Flavia Valtorta, Chiara Parodi, Davide Aprile, Michele Oneto, Anita Aperia, Anna Fassio, Pietro BaldelliAbstract: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.
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proline rich transmembrane protein 2 PRRT2 regulates the actin cytoskeleton during synaptogenesis
Cell Death and Disease, 2020Co-Authors: Elisa Savino, Pierluigi Valente, Anna Corradi, Fabio Benfenati, Romina Ines Cervigni, Fabrizia C Guarnieri, Miriana Povolo, Alessandra Stefanetti, Daniele Ferrante, Flavia ValtortaAbstract:Mutations in proline-rich transmembrane protein 2 (PRRT2) have been recently identified as the leading cause of a clinically heterogeneous group of neurological disorders sharing a paroxysmal nature, including paroxysmal kinesigenic dyskinesia and benign familial infantile seizures. To date, studies aimed at understanding its physiological functions in neurons have mainly focused on its ability to regulate neurotransmitter release and neuronal excitability. Here, we show that PRRT2 expression in non-neuronal cell lines inhibits cell motility and focal adhesion turnover, increases cell aggregation propensity, and promotes the protrusion of filopodia, all processes impinging on the actin cytoskeleton. In primary hippocampal neurons, PRRT2 silencing affects the synaptic content of filamentous actin and perturbs actin dynamics. This is accompanied by defects in the density and maturation of dendritic spines. We identified cofilin, an actin-binding protein abundantly expressed at the synaptic level, as the ultimate effector of PRRT2. Indeed, PRRT2 silencing unbalances cofilin activity leading to the formation of cofilin-actin rods along neurites. The expression of a cofilin phospho-mimetic mutant (cof-S3E) is able to rescue PRRT2-dependent defects in synapse density, spine number and morphology, but not the alterations observed in neurotransmitter release. Our data support a novel function of PRRT2 in the regulation of the synaptic actin cytoskeleton and in the formation of synaptic contacts.
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PRRT2 modulates presynaptic ca 2 influx by interacting with p q type channels
Social Science Research Network, 2020Co-Authors: Daniele Ferrante, Bruno Sterlini, Cosimo Prestigio, Antonella Marte, Franco Onofri, Anna Corradi, Andrea Petretto, Jessica Muja, Agnes Thalhammer, Pierluigi ValenteAbstract:Loss-of-function mutations in proline-rich transmembrane protein 2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We found that either acute or constitutive PRRT2 deletion induced a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that was insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype was paralleled by a decrease of somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation assays and proteomic screens revealed an interaction between PRRT2 and 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 of the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in assuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses.
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constitutive inactivation of the PRRT2 gene alters short term synaptic plasticity and promotes network hyperexcitability in hippocampal neurons
Cerebral Cortex, 2019Co-Authors: Pierluigi Valente, Floriana Fruscione, Bruno Sterlini, Alessandra Romei, Manuela Fadda, Enrico Castroflorio, Caterina Michetti, Nicola Forte, Davide Lonardoni, Giorgia GiansanteAbstract: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.
Fabio Benfenati - One of the best experts on this subject based on the ideXlab platform.
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an emerging role of PRRT2 in regulating growth cone morphology
Cells, 2021Co-Authors: Elisa Savino, Anna Corradi, Fabio Benfenati, Jinwu Tsai, Fabrizia C Guarnieri, Flavia ValtortaAbstract: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.
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increased responsiveness at the cerebellar input stage in the PRRT2 knockout model of paroxysmal kinesigenic dyskinesia
Neurobiology of Disease, 2021Co-Authors: Francesca Binda, Pierluigi Valente, Antonella Marte, Pietro Baldelli, Fabio BenfenatiAbstract:PRoline-Rich Transmembrane protein-2 (PRRT2) is a recently described neuron-specific type-2 integral membrane protein with a large cytosolic N-terminal domain that distributes in presynaptic and axonal domains where it interacts with several presynaptic proteins and voltage-gated Na+ channels. Several PRRT2 mutations are the main cause of a wide and heterogeneous spectrum of paroxysmal disorders with a loss-of-function pathomechanism. The highest expression levels of PRRT2 in brain occurs in cerebellar granule cells (GCs) and cerebellar dysfunctions participate in the dyskinetic phenotype of PRRT2 knockout (KO) mice. We have investigated the effects of PRRT2 deficiency on the intrinsic excitability of GCs and the input-output relationships at the mossy fiber-GC synapses. We show that PRRT2 KO primary GCs display increased expression of Na+ channels, increased amplitude of Na+ currents and increased length of the axon initial segment, leading to an overall enhancement of intrinsic excitability. In acute PRRT2 KO cerebellar slices, GCs were more prone to action potential discharge in response to mossy fiber activation and exhibited an enhancement of transient and persistent Na+ currents, in the absence of changes at the mossy fiber-GC synapses. The results support a key role of PRRT2 expressed in GCs in the physiological regulation of the excitatory input to the cerebellum and are consistent with a major role of a cerebellar dysfunction in the pathogenesis of the PRRT2-linked paroxysmal pathologies.
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proline rich transmembrane protein 2 PRRT2 regulates the actin cytoskeleton during synaptogenesis
Cell Death and Disease, 2020Co-Authors: Elisa Savino, Pierluigi Valente, Anna Corradi, Fabio Benfenati, Romina Ines Cervigni, Fabrizia C Guarnieri, Miriana Povolo, Alessandra Stefanetti, Daniele Ferrante, Flavia ValtortaAbstract:Mutations in proline-rich transmembrane protein 2 (PRRT2) have been recently identified as the leading cause of a clinically heterogeneous group of neurological disorders sharing a paroxysmal nature, including paroxysmal kinesigenic dyskinesia and benign familial infantile seizures. To date, studies aimed at understanding its physiological functions in neurons have mainly focused on its ability to regulate neurotransmitter release and neuronal excitability. Here, we show that PRRT2 expression in non-neuronal cell lines inhibits cell motility and focal adhesion turnover, increases cell aggregation propensity, and promotes the protrusion of filopodia, all processes impinging on the actin cytoskeleton. In primary hippocampal neurons, PRRT2 silencing affects the synaptic content of filamentous actin and perturbs actin dynamics. This is accompanied by defects in the density and maturation of dendritic spines. We identified cofilin, an actin-binding protein abundantly expressed at the synaptic level, as the ultimate effector of PRRT2. Indeed, PRRT2 silencing unbalances cofilin activity leading to the formation of cofilin-actin rods along neurites. The expression of a cofilin phospho-mimetic mutant (cof-S3E) is able to rescue PRRT2-dependent defects in synapse density, spine number and morphology, but not the alterations observed in neurotransmitter release. Our data support a novel function of PRRT2 in the regulation of the synaptic actin cytoskeleton and in the formation of synaptic contacts.
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PRRT2 from paroxysmal disorders to regulation of synaptic function
Trends in Neurosciences, 2016Co-Authors: Flavia Valtorta, Fabio Benfenati, Federico Zara, Jacopo MeldolesiAbstract:In the past few years, proline-rich transmembrane protein (PRRT)2 has been identified as the causative gene for several paroxysmal neurological disorders. Recently, an important role of PRRT2 in synapse development and function has emerged. Knock down of the protein strongly impairs the formation of synaptic contacts and neurotransmitter release. At the nerve terminal, PRRT2 endows synaptic vesicle exocytosis with Ca2+ sensitivity by interacting with proteins of the fusion complex and with the Ca2+ sensors synaptotagmins (Syts). In the postsynaptic compartment, PRRT2 interacts with glutamate receptors. The study of PRRT2 and of its mutations may help in refining our knowledge of the process of synaptic transmission and elucidating the pathogenetic mechanisms leading to derangement of network function in paroxysmal disorders.
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a novel topology of proline rich transmembrane protein 2 PRRT2 hints for an intracellular function at the synapse
Journal of Biological Chemistry, 2016Co-Authors: Pia Rossi, Bruno Sterlini, Enrico Castroflorio, Antonella Marte, Franco Onofri, Flavia Valtorta, Luca Maragliano, Anna Corradi, Fabio BenfenatiAbstract: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.
Flavia Valtorta - One of the best experts on this subject based on the ideXlab platform.
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an emerging role of PRRT2 in regulating growth cone morphology
Cells, 2021Co-Authors: Elisa Savino, Anna Corradi, Fabio Benfenati, Jinwu Tsai, Fabrizia C Guarnieri, Flavia ValtortaAbstract: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.
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an interaction between PRRT2 and na k atpase contributes to the control of neuronal excitability
Cell Death and Disease, 2021Co-Authors: Bruno Sterlini, Pierluigi Valente, Alessandra Romei, Flavia Valtorta, Chiara Parodi, Davide Aprile, Michele Oneto, Anita Aperia, Anna Fassio, Pietro BaldelliAbstract: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.
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proline rich transmembrane protein 2 PRRT2 regulates the actin cytoskeleton during synaptogenesis
Cell Death and Disease, 2020Co-Authors: Elisa Savino, Pierluigi Valente, Anna Corradi, Fabio Benfenati, Romina Ines Cervigni, Fabrizia C Guarnieri, Miriana Povolo, Alessandra Stefanetti, Daniele Ferrante, Flavia ValtortaAbstract:Mutations in proline-rich transmembrane protein 2 (PRRT2) have been recently identified as the leading cause of a clinically heterogeneous group of neurological disorders sharing a paroxysmal nature, including paroxysmal kinesigenic dyskinesia and benign familial infantile seizures. To date, studies aimed at understanding its physiological functions in neurons have mainly focused on its ability to regulate neurotransmitter release and neuronal excitability. Here, we show that PRRT2 expression in non-neuronal cell lines inhibits cell motility and focal adhesion turnover, increases cell aggregation propensity, and promotes the protrusion of filopodia, all processes impinging on the actin cytoskeleton. In primary hippocampal neurons, PRRT2 silencing affects the synaptic content of filamentous actin and perturbs actin dynamics. This is accompanied by defects in the density and maturation of dendritic spines. We identified cofilin, an actin-binding protein abundantly expressed at the synaptic level, as the ultimate effector of PRRT2. Indeed, PRRT2 silencing unbalances cofilin activity leading to the formation of cofilin-actin rods along neurites. The expression of a cofilin phospho-mimetic mutant (cof-S3E) is able to rescue PRRT2-dependent defects in synapse density, spine number and morphology, but not the alterations observed in neurotransmitter release. Our data support a novel function of PRRT2 in the regulation of the synaptic actin cytoskeleton and in the formation of synaptic contacts.
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the PRRT2 knockout mouse recapitulates the neurological diseases associated with PRRT2 mutations
Neurobiology of Disease, 2017Co-Authors: Caterina Michetti, Floriana Fruscione, Bruno Sterlini, Enrico Castroflorio, Flavia Valtorta, Francesca Binda, Pietro Baldelli, Ivan Marchionni, Nicola Forte, Federico ZaraAbstract: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). β-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.
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PRRT2 from paroxysmal disorders to regulation of synaptic function
Trends in Neurosciences, 2016Co-Authors: Flavia Valtorta, Fabio Benfenati, Federico Zara, Jacopo MeldolesiAbstract:In the past few years, proline-rich transmembrane protein (PRRT)2 has been identified as the causative gene for several paroxysmal neurological disorders. Recently, an important role of PRRT2 in synapse development and function has emerged. Knock down of the protein strongly impairs the formation of synaptic contacts and neurotransmitter release. At the nerve terminal, PRRT2 endows synaptic vesicle exocytosis with Ca2+ sensitivity by interacting with proteins of the fusion complex and with the Ca2+ sensors synaptotagmins (Syts). In the postsynaptic compartment, PRRT2 interacts with glutamate receptors. The study of PRRT2 and of its mutations may help in refining our knowledge of the process of synaptic transmission and elucidating the pathogenetic mechanisms leading to derangement of network function in paroxysmal disorders.
Anna Corradi - One of the best experts on this subject based on the ideXlab platform.
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an emerging role of PRRT2 in regulating growth cone morphology
Cells, 2021Co-Authors: Elisa Savino, Anna Corradi, Fabio Benfenati, Jinwu Tsai, Fabrizia C Guarnieri, Flavia ValtortaAbstract: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.
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PRRT2 modulates presynaptic ca2 influx by interacting with p q type channels
Cell Reports, 2021Co-Authors: Daniele Ferrante, Bruno Sterlini, Cosimo Prestigio, Antonella Marte, Franco Onofri, Anna Corradi, Andrea Petretto, Giorgio Tortarolo, Giuseppe Vicidomini, Jessica MuiaAbstract: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.
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proline rich transmembrane protein 2 PRRT2 regulates the actin cytoskeleton during synaptogenesis
Cell Death and Disease, 2020Co-Authors: Elisa Savino, Pierluigi Valente, Anna Corradi, Fabio Benfenati, Romina Ines Cervigni, Fabrizia C Guarnieri, Miriana Povolo, Alessandra Stefanetti, Daniele Ferrante, Flavia ValtortaAbstract:Mutations in proline-rich transmembrane protein 2 (PRRT2) have been recently identified as the leading cause of a clinically heterogeneous group of neurological disorders sharing a paroxysmal nature, including paroxysmal kinesigenic dyskinesia and benign familial infantile seizures. To date, studies aimed at understanding its physiological functions in neurons have mainly focused on its ability to regulate neurotransmitter release and neuronal excitability. Here, we show that PRRT2 expression in non-neuronal cell lines inhibits cell motility and focal adhesion turnover, increases cell aggregation propensity, and promotes the protrusion of filopodia, all processes impinging on the actin cytoskeleton. In primary hippocampal neurons, PRRT2 silencing affects the synaptic content of filamentous actin and perturbs actin dynamics. This is accompanied by defects in the density and maturation of dendritic spines. We identified cofilin, an actin-binding protein abundantly expressed at the synaptic level, as the ultimate effector of PRRT2. Indeed, PRRT2 silencing unbalances cofilin activity leading to the formation of cofilin-actin rods along neurites. The expression of a cofilin phospho-mimetic mutant (cof-S3E) is able to rescue PRRT2-dependent defects in synapse density, spine number and morphology, but not the alterations observed in neurotransmitter release. Our data support a novel function of PRRT2 in the regulation of the synaptic actin cytoskeleton and in the formation of synaptic contacts.
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PRRT2 modulates presynaptic ca 2 influx by interacting with p q type channels
Social Science Research Network, 2020Co-Authors: Daniele Ferrante, Bruno Sterlini, Cosimo Prestigio, Antonella Marte, Franco Onofri, Anna Corradi, Andrea Petretto, Jessica Muja, Agnes Thalhammer, Pierluigi ValenteAbstract:Loss-of-function mutations in proline-rich transmembrane protein 2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We found that either acute or constitutive PRRT2 deletion induced a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that was insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype was paralleled by a decrease of somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation assays and proteomic screens revealed an interaction between PRRT2 and 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 of the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in assuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses.
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a novel topology of proline rich transmembrane protein 2 PRRT2 hints for an intracellular function at the synapse
Journal of Biological Chemistry, 2016Co-Authors: Pia Rossi, Bruno Sterlini, Enrico Castroflorio, Antonella Marte, Franco Onofri, Flavia Valtorta, Luca Maragliano, Anna Corradi, Fabio BenfenatiAbstract: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.
