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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•PRRT2 KO granule cells express higher levels of functional Na+ channels.•they have longer and more proximal axon initial segments.•they experience enhanced transient and persistent Na+ currents in cerebellar slices.•they are more prone to action potential firing in response to mossy fiber activation.
The repressor-element 1-silencing transcription/neuron-restrictive silencer factor (REST/NRSF) controls hundreds of neuron-specific genes. We showed that REST/NRSF downregulates glutamatergic ...transmission in response to hyperactivity, thus contributing to neuronal homeostasis. However, whether GABAergic transmission is also implicated in the homeostatic action of REST/NRSF is unknown. Here, we show that hyperactivity-induced REST/NRSF activation, triggers a homeostatic rearrangement of GABAergic inhibition, with increased frequency of miniature inhibitory postsynaptic currents (IPSCs) and amplitude of evoked IPSCs in mouse cultured hippocampal neurons. Notably, this effect is limited to inhibitory-onto-excitatory neuron synapses, whose density increases at somatic level and decreases in dendritic regions, demonstrating a complex target- and area-selectivity. The upscaling of perisomatic inhibition was occluded by TrkB receptor inhibition and resulted from a coordinated and sequential activation of the
and
gene programs. On the opposite, the downscaling of dendritic inhibition was REST-dependent, but BDNF-independent. The findings highlight the central role of REST/NRSF in the complex transcriptional responses aimed at rescuing physiological levels of network activity in front of the ever-changing environment.
DEP-domain containing 5 (DEPDC5) is part of the GATOR1 complex that functions as key inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1). Loss-of-function mutations in DEPDC5 leading ...to mTOR hyperactivation have been identified as the most common cause of either lesional or non-lesional focal epilepsy. However, the precise mechanisms by which DEPDC5 loss-of-function triggers neuronal and network hyperexcitability are still unclear. In this study, we investigated the cellular mechanisms of hyperexcitability by comparing the constitutive heterozygous Depdc5 knockout mouse versus different levels of acute Depdc5 deletion (≈40% and ≈80% neuronal knockdown of Depdc5 protein) by RNA interference in primary cortical cultures. While heterozygous Depdc5+/− neurons have only a subtle phenotype, acutely knocked-down neurons exhibit a strong dose-dependent phenotype characterized by mTOR hyperactivation, increased soma size, dendritic arborization, excitatory synaptic transmission and intrinsic excitability. The robust synaptic phenotype resulting from the acute knockdown Depdc5 deficiency highlights the importance of the temporal dynamics of Depdc5 knockdown in triggering the phenotypic changes, reminiscent of the somatic second-hit mechanism in patients with focal cortical dysplasia. These findings uncover a novel synaptic phenotype that is causally linked to Depdc5 knockdown, highlighting the developmental role of Depdc5. Interestingly, the synaptic defect appears to affect only excitatory synapses, while inhibitory synapses develop normally. The increased frequency and amplitude of mEPSCs, paralleled by increased density of excitatory synapses and expression of glutamate receptors, may generate an excitation/inhibition imbalance that triggers epileptogenesis.
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•Acute Depdc5 knockdown leads to a robust neuronal and synaptic phenotype•Depdc5 knockdown increases neuronal soma size and dendritic arborization•Depdc5 knockdown alters synaptic connectivity•Depdc5 knockdown induces an excitation/inhibition imbalance at the synaptic level•The hyperexcitability of excitatory neurons further increases excitatory strength
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.
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•Point mutations in the PRRT2 transmembrane region alter Nav1.2 channel function.•The PRRT2 elicits an increased surface exposure of Nav1.2 channels.•The PRRT2 A320V and V286M mutants have opposite effects on Nav1.2 biophysics.•The PRRT2 transmembrane region plays a key role in Nav interaction.
Heterozygous 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, suggesting that loss of PRRT2 function may contribute to pathogenesis. We show that PRRT2 is enriched in presynaptic terminals and that its silencing decreases the number of synapses and increases the number of docked synaptic vesicles at rest. PRRT2-silenced neurons exhibit a severe impairment of synchronous release, attributable to a sharp decrease in release probability and Ca2+ sensitivity and associated with a marked increase of the asynchronous/synchronous release ratio. PRRT2 interacts with the synaptic proteins SNAP-25 and synaptotagmin 1/2. The results indicate that PRRT2 is intimately connected with the Ca2+-sensing machinery and that it plays an important role in the final steps of neurotransmitter release.
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•PRRT2 is a presynaptic protein•PRRT2 is required for synchronous neurotransmitter release•PRRT2 silencing decreases synaptic density and release probability•PRRT2 interacts with the fast Ca2+ sensors synaptotagmin 1/2
Valente et al. show that PRRT2, a single causative gene for a group of paroxysmal neurological diseases, is a key component of regulated exocytosis. Silencing PRRT2 dramatically impairs neurotransmitter release by markedly reducing release probability. PRRT2 interacts with the fast Ca2+ sensors synaptotagmin 1/2 and endows the SNARE complex with Ca2+ sensitivity.
High-pressure and temperature extraction (HPTE) can effectively recover bioactive compounds from olive pomace (OP). HPTE extract obtained by extracting OP with ethanol and water (50:50
/
) at 180 °C ...for 90 min demonstrated a pronounced ability to preserve intracellular calcium homeostasis, shielding neurons from the harmful effects induced by N-methyl-d-aspartate (NMDA) receptor (NMDAR) overactivation, such as aberrant calpain activation. In this study, the extraction temperature was changed from 37 to 180 °C, and the extracts were evaluated for their antioxidant potency and ability to preserve crucial intracellular Ca
-homeostasis necessary for neuronal survival. Additionally, to verify the temperature-induced activity of the extract, further extractions on the exhausted olive pomace were conducted, aiming to identify variations in the quality and quantity of extracted phenolic molecules through HPLC analysis. The results revealed a significant increase in bioactive compounds as a function of temperature variation, reaching 6.31 ± 0.09 mgCAE/mL extract for the extraction performed at 180 °C. Subsequent extraction of the exhausted residues yielded extracts that remained active in preventing calcium-induced cell death. Moreover, despite increased antiradical power, extracts re-treated at 180 °C did not display cell protection activity. Our results indicate that the molecules able to maintain physiological Ca
-homeostasis in murine cortical neurons in conditions of cytotoxic stimulation of NMDAR are wholly recovered from olive pomace only following extraction performed at 180 °C.
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.
Loss-of-function mutations in proline-rich transmembrane protein-2 (PRRT2) cause paroxysmal disorders associated with defective Ca
2+
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 Ca
2+
and associated with a reduced contribution of P/Q-type Ca
2+
channels to the EPSC amplitude. This synaptic phenotype parallels a decrease in somatic P/Q-type Ca
2+
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 Ca
2+
channels. At presynaptic terminals lacking PRRT2, P/Q-type Ca
2+
channels reduce their clustering at the active zone, with a corresponding decrease in the P/Q-dependent presynaptic Ca
2+
signal. The data highlight the central role of PRRT2 in ensuring the physiological Ca
2+
sensitivity of the release machinery at glutamatergic synapses.
•
PRRT2 deficiency reduces the contribution of P/Q-type Ca
2+
channels to the EPSC
•
PRRT2 deficiency decreases P/Q-type currents and membrane targeting of the channels
•
PRRT2 directly interacts with P/Q-type Ca
2+
channels
•
PRRT2 deficiency reduces P/Q-channel clustering and Ca
2+
signals in nerve terminals
PRRT2 deficiency causes paroxysmal disorders associated with defective Ca
2+
dependence of glutamatergic transmission. Ferrante et al. now find that, in the absence of PRRT2, the membrane targeting of P/Q-type Ca
2+
channels is reduced, and the channels fail to concentrate at the nanodomain where the machinery for synchronous release is assembled.
Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including ...predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function.
Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial Parkinson's disease (PD). LRRK2 protein contains several functional domains, including protein-protein ...interaction domains at its N- and C-termini. In this study, we analyzed the functional features attributed to LRRK2 by its N- and C-terminal domains. We combined TIRF microscopy and synaptopHluorin assay to visualize synaptic vesicle trafficking. We found that N- and C-terminal domains have opposite impact on synaptic vesicle dynamics. Biochemical analysis demonstrated that different proteins are bound at the two extremities, namely β3-Cav2.1 at N-terminus part and β-Actin and Synapsin I at C-terminus domain. A sequence variant (G2385R) harboured within the C-terminal WD40 domain increases the risk for PD. Complementary biochemical and imaging approaches revealed that the G2385R variant alters strength and quality of LRRK2 interactions and increases fusion of synaptic vesicles. Our data suggest that the G2385R variant behaves like a loss-of-function mutation that mimics activity-driven events. Impaired scaffolding capabilities of mutant LRRK2 resulting in perturbed vesicular trafficking may arise as a common pathophysiological denominator through which different LRRK2 pathological mutations cause disease.
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