ATP6V1A is required for synaptic rearrangements and plasticity in murine hippocampal neurons.

AIM: Understanding the physiological role of ATP6V1A, a component of the cytosolic V1 domain of the proton pump vacuolar ATPase, in regulating neuronal development and function. METHODS: Modeling loss of function of Atp6v1a in primary murine hippocampal neurons and studying neuronal morphology and function by immunoimaging, electrophysiological recordings and electron microscopy. RESULTS: Atp6v1a depletion affects neurite elongation, stabilization, and function of excitatory synapses and prevents synaptic rearrangement upon induction of plasticity. These phenotypes are due to an overall decreased expression of the V1 subunits, that leads to impairment of lysosomal pH-regulation and autophagy progression with accumulation of aberrant lysosomes at neuronal soma and of enlarged vacuoles at synaptic boutons. CONCLUSIONS: These data suggest a physiological role of ATP6V1A in the surveillance of synaptic integrity and plasticity and highlight the pathophysiological significance of ATP6V1A loss in the alteration of synaptic function that is associated with neurodevelopmental and neurodegenerative diseases. The data further support the pivotal involvement of lysosomal function and autophagy flux in maintaining proper synaptic connectivity and adaptive neuronal properties.

A micropatterned thermoplasmonic substrate for neuromodulation of in vitro neuronal networks.

Understanding how the spatial organization of a neural network affects its activity represents a leading issue in neuroscience. Thanks to their accessibility and easy handling, in vitro studies remain an essential tool to investigate the relationship between the structure and function of a neuronal network. Among all the patterning techniques, ink-jet printing acquired great interest thanks to its direct-write approach, which allows the patterned substrate realization without mold, leading to a considerable saving of both cost and time. However, the inks commonly used give the possibility to control only the structure of a neuronal network, leaving aside the functional aspect. In this work, we synthesize a photosensitive ink combining the rheological and bioadhesive properties of chitosan with the plasmonic properties of gold nanorods, obtaining an ink able to control both the spatial organization of a two-dimensional neuronal network and its activity through photothermal effect. After the ink characterization, we demonstrate that it is possible to print, with high precision, different geometries on a microelectrode array. In this way, it is possible obtaining a patterned device to control the structure of a neuronal network, to record its activity and to modulate it via photothermal effect. Finally, to our knowledge, we report the first evidence of photothermal inhibition of human neurons activity. STATEMENT OF SIGNIFICANCE: Patterned cell cultures remain the most efficient and simple tool for linking structural and functional studies, especially in the neuronal field. Ink-jet printing is the technique with which it is possible to realize patterned structures in the fastest, simple, versatile and low-cost way. However, the inks currently used permit the control only of the neuronal network structure but do not allow the control-modulation of the network activity. In this study, we realize and characterize a photosensitive bioink with which it is possible to drive both the structure and the activity of a neuronal network. Moreover, we report the first evidence of activity inhibition by the photothermal effect on human neurons as far as we know.

Ca2+ binding to synapsin I regulates resting Ca2+ and recovery from synaptic depression in nerve terminals.

Synapsin I (SynI) is a synaptic vesicle (SV)-associated phosphoprotein that modulates neurotransmission by controlling SV trafficking. The SynI C-domain contains a highly conserved ATP binding site mediating SynI oligomerization and SV clustering and an adjacent main Ca2+ binding site, whose physiological role is unexplored. Molecular dynamics simulations revealed that the E373K point mutation irreversibly deletes Ca2+ binding to SynI, still allowing ATP binding, but inducing a destabilization of the SynI oligomerization interface. Here, we analyzed the effects of this mutation on neurotransmitter release and short-term plasticity in excitatory and inhibitory synapses from primary hippocampal neurons. Patch-clamp recordings showed an increase in the frequency of miniature excitatory postsynaptic currents (EPSCs) that was totally occluded by exogenous Ca2+ chelators and associated with a constitutive increase in resting terminal Ca2+ concentrations. Evoked EPSC amplitude was also reduced, due to a decreased readily releasable pool (RRP) size. Moreover, in both excitatory and inhibitory synapses, we observed a marked impaired recovery from synaptic depression, associated with impaired RRP refilling and depletion of the recycling pool of SVs. Our study identifies SynI as a novel Ca2+ buffer in excitatory terminals. Blocking Ca2+ binding to SynI results in higher constitutive Ca2+ levels that increase the probability of spontaneous release and disperse SVs. This causes a decreased size of the RRP and an impaired recovery from depression due to the failure of SV reclustering after sustained high-frequency stimulation. The results indicate a physiological role of Ca2+ binding to SynI in the regulation of SV clustering and trafficking in nerve terminals.

On the way back from 3D to 2D: Chitosan promotes adhesion and development of neuronal networks onto culture supports.

Most in vitro functional and morphological studies for developing nervous system have been performed using traditional monolayer cultures onto supports modified by extracellular matrix components or synthetic biopolymers. These biomolecules act as adhesion factors essential for neuronal growth and differentiation. In this study, the use of chitosan as adhesion factor was investigated. Primary rat neurons and neurons differentiated from human induced pluripotent stem cells were cultured onto chitosan and standard adhesion factors modified supports. The initiation, elongation and branching of neuritic processes, synaptogenesis and electrophysiological behavior were studied. The biopolymers affected neurites outgrowth in a time dependent manner; in particular, chitosan promoted neuronal polarity in both cell cultures. These results indicate chitosan as a valid adhesion factor alternative to the standard ones, with the advantage that it can be used both in 2D and 3D cultures, acting as a bridge between these in vitro models.

Synaptic genes and neurodevelopmental disorders: From molecular mechanisms to developmental strategies of behavioral testing.

Synaptopathies are a class of neurodevelopmental disorders caused by modification in genes coding for synaptic proteins. These proteins oversee the process of neurotransmission, mainly controlling the fusion and recycling of synaptic vesicles at the presynaptic terminal, the expression and localization of receptors at the postsynapse and the coupling between the pre- and the postsynaptic compartments. Murine models, with homozygous or heterozygous deletion for several synaptic genes or knock-in for specific pathogenic mutations, have been developed. They have proved to be extremely informative for understanding synaptic physiology, as well as for clarifying the patho-mechanisms leading to developmental delay, epilepsy and motor, cognitive and social impairments that are the most common clinical manifestations of neurodevelopmental disorders. However, the onset of these disorders emerges during infancy and adolescence while the behavioral phenotyping is often conducted in adult mice, missing important information about the impact of synaptic development and maturation on the manifestation of the behavioral phenotype. Here, we review the main achievements obtained by behavioral testing in murine models of synaptopathies and propose a battery of behavioral tests to improve classification, diagnosis and efficacy of potential therapeutic treatments. Our aim is to underlie the importance of studying behavioral development and better focusing on disease onset and phenotypes.

Phenotypic and genetic spectrum of ATP6V1A encephalopathy: a disorder of lysosomal homeostasis.

Vacuolar-type H+-ATPase (V-ATPase) is a multimeric complex present in a variety of cellular membranes that acts as an ATP-dependent proton pump and plays a key role in pH homeostasis and intracellular signalling pathways. In humans, 22 autosomal genes encode for a redundant set of subunits allowing the composition of diverse V-ATPase complexes with specific properties and expression. Sixteen subunits have been linked to human disease. Here we describe 26 patients harbouring 20 distinct pathogenic de novo missense ATP6V1A variants, mainly clustering within the ATP synthase α/ß family-nucleotide-binding domain. At a mean age of 7 years (extremes: 6 weeks, youngest deceased patient to 22 years, oldest patient) clinical pictures included early lethal encephalopathies with rapidly progressive massive brain atrophy, severe developmental epileptic encephalopathies and static intellectual disability with epilepsy. The first clinical manifestation was early hypotonia, in 70%; 81% developed epilepsy, manifested as developmental epileptic encephalopathies in 58% of the cohort and with infantile spasms in 62%; 63% of developmental epileptic encephalopathies failed to achieve any developmental, communicative or motor skills. Less severe outcomes were observed in 23% of patients who, at a mean age of 10 years and 6 months, exhibited moderate intellectual disability, with independent walking and variable epilepsy. None of the patients developed communicative language. Microcephaly (38%) and amelogenesis imperfecta/enamel dysplasia (42%) were additional clinical features. Brain MRI demonstrated hypomyelination and generalized atrophy in 68%. Atrophy was progressive in all eight individuals undergoing repeated MRIs. Fibroblasts of two patients with developmental epileptic encephalopathies showed decreased LAMP1 expression, Lysotracker staining and increased organelle pH, consistent with lysosomal impairment and loss of V-ATPase function. Fibroblasts of two patients with milder disease, exhibited a different phenotype with increased Lysotracker staining, decreased organelle pH and no significant modification in LAMP1 expression. Quantification of substrates for lysosomal enzymes in cellular extracts from four patients revealed discrete accumulation. Transmission electron microscopy of fibroblasts of four patients with variable severity and of induced pluripotent stem cell-derived neurons from two patients with developmental epileptic encephalopathies showed electron-dense inclusions, lipid droplets, osmiophilic material and lamellated membrane structures resembling phospholipids. Quantitative assessment in induced pluripotent stem cell-derived neurons identified significantly smaller lysosomes. ATP6V1A-related encephalopathy represents a new paradigm among lysosomal disorders. It results from a dysfunctional endo-lysosomal membrane protein causing altered pH homeostasis. Its pathophysiology implies intracellular accumulation of substrates whose composition remains unclear, and a combination of developmental brain abnormalities and neurodegenerative changes established during prenatal and early postanal development, whose severity is variably determined by specific pathogenic variants.

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.

Emerging Role of the Autophagy/Lysosomal Degradative Pathway in Neurodevelopmental Disorders With Epilepsy.

Autophagy is a highly conserved degradative process that conveys dysfunctional proteins, lipids, and organelles to lysosomes for degradation. The post-mitotic nature, complex and highly polarized morphology, and high degree of specialization of neurons make an efficient autophagy essential for their homeostasis and survival. Dysfunctional autophagy occurs in aging and neurodegenerative diseases, and autophagy at synaptic sites seems to play a crucial role in neurodegeneration. Moreover, a role of autophagy is emerging for neural development, synaptogenesis, and the establishment of a correct connectivity. Thus, it is not surprising that defective autophagy has been demonstrated in a spectrum of neurodevelopmental disorders, often associated with early-onset epilepsy. Here, we discuss the multiple roles of autophagy in neurons and the recent experimental evidence linking neurodevelopmental disorders with epilepsy to genes coding for autophagic/lysosomal system-related proteins and envisage possible pathophysiological mechanisms ranging from synaptic dysfunction to neuronal death.

Lesional and non-lesional epilepsies: A blurring genetic boundary.

There has been a traditional conceptual partition between the so-called non-lesional genetic epilepsies and the genetically determined interposed epileptogenic structural abnormalities. In this review, we summarise how growing evidence acquired through neuroimaging and neurobiology modelling is demonstrating that a distinction between lesional and functional (or non-lesional) epileptogenesis is less obvious than previously thought, particularly for epileptogenic neurodevelopmental disorders, but also for most genetically determined epilepsies.

Biallelic DMXL2 mutations impair autophagy and cause Ohtahara syndrome with progressive course.

Ohtahara syndrome, early infantile epileptic encephalopathy with a suppression burst EEG pattern, is an aetiologically heterogeneous condition starting in the first weeks or months of life with intractable seizures and profound developmental disability. Using whole exome sequencing, we identified biallelic DMXL2 mutations in three sibling pairs with Ohtahara syndrome, belonging to three unrelated families. Siblings in Family 1 were compound heterozygous for the c.5135C>T (p.Ala1712Val) missense substitution and the c.4478C>G (p.Ser1493*) nonsense substitution; in Family 2 were homozygous for the c.4478C>A (p.Ser1493*) nonsense substitution and in Family 3 were homozygous for the c.7518-1G>A (p.Trp2507Argfs*4) substitution. The severe developmental and epileptic encephalopathy manifested from the first day of life and was associated with deafness, mild peripheral polyneuropathy and dysmorphic features. Early brain MRI investigations in the first months of life revealed thin corpus callosum with brain hypomyelination in all. Follow-up MRI scans in three patients revealed progressive moderate brain shrinkage with leukoencephalopathy. Five patients died within the first 9 years of life and none achieved developmental, communicative or motor skills following birth. These clinical findings are consistent with a developmental brain disorder that begins in the prenatal brain, prevents neural connections from reaching the expected stages at birth, and follows a progressive course. DMXL2 is highly expressed in the brain and at synaptic terminals, regulates v-ATPase assembly and activity and participates in intracellular signalling pathways; however, its functional role is far from complete elucidation. Expression analysis in patient-derived skin fibroblasts demonstrated absence of the DMXL2 protein, revealing a loss of function phenotype. Patients' fibroblasts also exhibited an increased LysoTracker® signal associated with decreased endolysosomal markers and degradative processes. Defective endolysosomal homeostasis was accompanied by impaired autophagy, revealed by lower LC3II signal, accumulation of polyubiquitinated proteins, and autophagy receptor p62, with morphological alterations of the autolysosomal structures on electron microscopy. Altered lysosomal homeostasis and defective autophagy were recapitulated in Dmxl2-silenced mouse hippocampal neurons, which exhibited impaired neurite elongation and synaptic loss. Impaired lysosomal function and autophagy caused by biallelic DMXL2 mutations affect neuronal development and synapse formation and result in Ohtahara syndrome with profound developmental impairment and reduced life expectancy.

TBC1D24 regulates axonal outgrowth and membrane trafficking at the growth cone in rodent and human neurons.

Mutations in TBC1D24 are described in patients with a spectrum of neurological diseases, including mild and severe epilepsies and complex syndromic phenotypes such as Deafness, Onycodystrophy, Osteodystrophy, Mental Retardation and Seizure (DOORS) syndrome. The product of TBC1D24 is a multifunctional protein involved in neuronal development, regulation of synaptic vesicle trafficking, and protection from oxidative stress. Although pathogenic mutations in TBC1D24 span the entire coding sequence, no clear genotype/phenotype correlations have emerged. However most patients bearing predicted loss of function mutations exhibit a severe neurodevelopmental disorder. Aim of the study is to investigate the impact of TBC1D24 knockdown during the first stages of neuronal differentiation when axonal specification and outgrowth take place. In rat cortical primary neurons silenced for TBC1D24, we found defects in axonal specification, the maturation of axonal initial segment and action potential firing. The axonal phenotype was accompanied by an impairment of endocytosis at the growth cone and an altered activation of the TBC1D24 molecular partner ADP ribosylation factor 6. Accordingly, acute knockdown of TBC1D24 in cerebrocortical neurons in vivo analogously impairs callosal projections. The axonal defect was also investigated in human induced pluripotent stem cell-derived neurons from patients carrying TBC1D24 mutations. Reprogrammed neurons from a patient with severe developmental encephalopathy show significant axon formation defect that were absent from reprogrammed neurons of a patient with mild early onset epilepsy. Our data reveal that alterations of membrane trafficking at the growth cone induced by TBC1D24 loss of function cause axonal and excitability defects. The axonal phenotype correlates with the disease severity and highlight an important role for TBC1D24 in connectivity during brain development.

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.

The epilepsy-associated protein TBC1D24 is required for normal development, survival and vesicle trafficking in mammalian neurons.

Mutations in the Tre2/Bub2/Cdc16 (TBC)1 domain family member 24 (TBC1D24) gene are associated with a range of inherited neurological disorders, from drug-refractory lethal epileptic encephalopathy and DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, seizures) to non-syndromic hearing loss. TBC1D24 has been implicated in neuronal transmission and maturation, although the molecular function of the gene and the cause of the apparently complex disease spectrum remain unclear. Importantly, heterozygous TBC1D24 mutation carriers have also been reported with seizures, suggesting that haploinsufficiency for TBC1D24 is significant clinically. Here we have systematically investigated an allelic series of disease-associated mutations in neurons alongside a new mouse model to investigate the consequences of TBC1D24 haploinsufficiency to mammalian neurodevelopment and synaptic physiology. The cellular studies reveal that disease-causing mutations that disrupt either of the conserved protein domains in TBC1D24 are implicated in neuronal development and survival and are likely acting as loss-of-function alleles. We then further investigated TBC1D24 haploinsufficiency in vivo and demonstrate that TBC1D24 is also crucial for normal presynaptic function: genetic disruption of Tbc1d24 expression in the mouse leads to an impairment of endocytosis and an enlarged endosomal compartment in neurons with a decrease in spontaneous neurotransmission. These data reveal the essential role for TBC1D24 at the mammalian synapse and help to define common synaptic mechanisms that could underlie the varied effects of TBC1D24 mutations in neurological disease.

De novo mutations of the ATP6V1A gene cause developmental encephalopathy with epilepsy.

V-type proton (H+) ATPase (v-ATPase) is a multi-subunit proton pump that regulates pH homeostasis in all eukaryotic cells; in neurons, v-ATPase plays additional and unique roles in synapse function. Through whole exome sequencing, we identified de novo heterozygous mutations (p.Pro27Arg, p.Asp100Tyr, p.Asp349Asn, p.Asp371Gly) in ATP6V1A, encoding the A subunit of v-ATPase, in four patients with developmental encephalopathy with epilepsy. Early manifestations, observed in all patients, were developmental delay and febrile seizures, evolving to encephalopathy with profound delay, hypotonic/dyskinetic quadriparesis and intractable multiple seizure types in two patients (p.Pro27Arg, p.Asp100Tyr), and to moderate delay with milder epilepsy in the other two (p.Asp349Asn, p.Asp371Gly). Modelling performed on the available prokaryotic and eukaryotic structures of v-ATPase predicted p.Pro27Arg to perturb subunit interaction, p.Asp100Tyr to cause steric hindrance and destabilize protein folding, p.Asp349Asn to affect the catalytic function and p.Asp371Gly to impair the rotation process, necessary for proton transport. We addressed the impact of p.Asp349Asn and p.Asp100Tyr mutations on ATP6V1A expression and function by analysing ATP6V1A-overexpressing HEK293T cells and patients' lymphoblasts. The p.Asp100Tyr mutant was characterized by reduced expression due to increased degradation. Conversely, no decrease in expression and clearance was observed for p.Asp349Asn. In HEK293T cells overexpressing either pathogenic or control variants, p.Asp349Asn significantly increased LysoTracker® fluorescence with no effects on EEA1 and LAMP1 expression. Conversely, p.Asp100Tyr decreased both LysoTracker® fluorescence and LAMP1 levels, leaving EEA1 expression unaffected. Both mutations decreased v-ATPase recruitment to autophagosomes, with no major impact on autophagy. Experiments performed on patients' lymphoblasts using the LysoSensor™ probe revealed lower pH of endocytic organelles for p.Asp349Asn and a reduced expression of LAMP1 with no effect on the pH for p.Asp100Tyr. These data demonstrate gain of function for p.Asp349Asn characterized by an increased proton pumping in intracellular organelles, and loss of function for p.Asp100Tyr with decreased expression of ATP6V1A and reduced levels of lysosomal markers. We expressed p.Asp349Asn and p.Asp100Tyr in rat hippocampal neurons and confirmed significant and opposite effects in lysosomal labelling. However, both mutations caused a similar defect in neurite elongation accompanied by loss of excitatory inputs, revealing that altered lysosomal homeostasis markedly affects neurite development and synaptic connectivity. This study provides evidence that de novo heterozygous ATP6V1A mutations cause a developmental encephalopathy with a pathomechanism that involves perturbations of lysosomal homeostasis and neuronal connectivity, uncovering a novel role for v-ATPase in neuronal development.

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.

APache Is an AP2-Interacting Protein Involved in Synaptic Vesicle Trafficking and Neuronal Development.

Synaptic transmission is critically dependent on synaptic vesicle (SV) recycling. Although the precise mechanisms of SV retrieval are still debated, it is widely accepted that a fundamental role is played by clathrin-mediated endocytosis, a form of endocytosis that capitalizes on the clathrin/adaptor protein complex 2 (AP2) coat and several accessory factors. Here, we show that the previously uncharacterized protein KIAA1107, predicted by bioinformatics analysis to be involved in the SV cycle, is an AP2-interacting clathrin-endocytosis protein (APache). We found that APache is highly enriched in the CNS and is associated with clathrin-coated vesicles via interaction with AP2. APache-silenced neurons exhibit a severe impairment of maturation at early developmental stages, reduced SV density, enlarged endosome-like structures, and defects in synaptic transmission, consistent with an impaired clathrin/AP2-mediated SV recycling. Our data implicate APache as an actor in the complex regulation of SV trafficking, neuronal development, and synaptic plasticity.

Neuronal hyperactivity causes Na+/H+ exchanger-induced extracellular acidification at active synapses.

Extracellular pH impacts on neuronal activity, which is in turn an important determinant of extracellular H+ concentration. The aim of this study was to describe the spatio-temporal dynamics of extracellular pH at synaptic sites during neuronal hyperexcitability. To address this issue we created ex.E2GFP, a membrane-targeted extracellular ratiometric pH indicator that is exquisitely sensitive to acidic shifts. By monitoring ex.E2GFP fluorescence in real time in primary cortical neurons, we were able to quantify pH fluctuations during network hyperexcitability induced by convulsant drugs or high-frequency electrical stimulation. Sustained hyperactivity caused a pH decrease that was reversible upon silencing of neuronal activity and located at active synapses. This acidic shift was not attributable to the outflow of synaptic vesicle H+ into the cleft nor to the activity of membrane-exposed H+ V-ATPase, but rather to the activity of the Na+/H+-exchanger. Our data demonstrate that extracellular synaptic pH shifts take place during epileptic-like activity of neural cultures, emphasizing the strict links existing between synaptic activity and synaptic pH. This evidence may contribute to the understanding of the physio-pathological mechanisms associated with hyperexcitability in the epileptic brain.

Clinical intrafamilial variability in lethal familial neonatal seizure disorder caused by TBC1D24 mutations.

TBC1D24-related disorders include a wide phenotypic ranging from mild to lethal seizure disorders, non-syndromic deafness, and composite syndromes such as DOORS (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures). The TBC1D24 gene has a role in cerebral cortex development and in presynaptic neurotransmission. Here, we present a familial case of a lethal early-onset epileptic encephalopathy, associated with two novel compound heterozygous missense variants on the TBC1D24 gene, which were detected by exome sequencing. The detailed clinical data of the three siblings is summarized in order to support the variability of the phenotype, severity, and progression of this disorder among these family members. Functional studies demonstrated that the identified novel missense mutations result in a loss of expression of the protein, suggesting a correlation between residual expression, and the disease severity. This indicates that protein expression analysis is important for interpreting genetic results when novel variants are found, as well as for complementing clinical assessment by predicting the functional impact. Further analysis is necessary to delineate the clinical presentation of individuals with TBC1D24 pathogenic variants, as well as to develop markers for diagnosis, prognosis, and potential targeted treatments. © 2016 Wiley Periodicals, Inc.

Molecular Machines Determining the Fate of Endocytosed Synaptic Vesicles in Nerve Terminals.

The cycle of a synaptic vesicle (SV) within the nerve terminal is a step-by-step journey with the final goal of ensuring the proper synaptic strength under changing environmental conditions. The SV cycle is a precisely regulated membrane traffic event in cells and, because of this, a plethora of membrane-bound and cytosolic proteins are devoted to assist SVs in each step of the journey. The cycling fate of endocytosed SVs determines both the availability for subsequent rounds of release and the lifetime of SVs in the terminal and is therefore crucial for synaptic function and plasticity. Molecular players that determine the destiny of SVs in nerve terminals after a round of exo-endocytosis are largely unknown. Here we review the functional role in SV fate of phosphorylation/dephosphorylation of SV proteins and of small GTPases acting on membrane trafficking at the synapse, as they are emerging as key molecules in determining the recycling route of SVs within the nerve terminal. In particular, we focus on: (i) the cyclin-dependent kinase-5 (cdk5) and calcineurin (CN) control of the recycling pool of SVs; (ii) the role of small GTPases of the Rab and ADP-ribosylation factor (Arf) families in defining the route followed by SV in their nerve terminal cycle. These regulatory proteins together with their synaptic regulators and effectors, are molecular nanomachines mediating homeostatic responses in synaptic plasticity and potential targets of drugs modulating the efficiency of synaptic transmission.

TBC1D24 genotype-phenotype correlation: Epilepsies and other neurologic features.

OBJECTIVE: To evaluate the phenotypic spectrum associated with mutations in TBC1D24. METHODS: We acquired new clinical, EEG, and neuroimaging data of 11 previously unreported and 37 published patients. TBC1D24 mutations, identified through various sequencing methods, can be found online (http://lovd.nl/TBC1D24). RESULTS: Forty-eight patients were included (28 men, 20 women, average age 21 years) from 30 independent families. Eighteen patients (38%) had myoclonic epilepsies. The other patients carried diagnoses of focal (25%), multifocal (2%), generalized (4%), and unclassified epilepsy (6%), and early-onset epileptic encephalopathy (25%). Most patients had drug-resistant epilepsy. We detail EEG, neuroimaging, developmental, and cognitive features, treatment responsiveness, and physical examination. In silico evaluation revealed 7 different highly conserved motifs, with the most common pathogenic mutation located in the first. Neuronal outgrowth assays showed that some TBC1D24 mutations, associated with the most severe TBC1D24-associated disorders, are not necessarily the most disruptive to this gene function. CONCLUSIONS: TBC1D24-related epilepsy syndromes show marked phenotypic pleiotropy, with multisystem involvement and severity spectrum ranging from isolated deafness (not studied here), benign myoclonic epilepsy restricted to childhood with complete seizure control and normal intellect, to early-onset epileptic encephalopathy with severe developmental delay and early death. There is no distinct correlation with mutation type or location yet, but patterns are emerging. Given the phenotypic breadth observed, TBC1D24 mutation screening is indicated in a wide variety of epilepsies. A TBC1D24 consortium was formed to develop further research on this gene and its associated phenotypes.