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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.
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Hipocampo , Plasticidad Neuronal , Neuronas , Sinapsis , ATPasas de Translocación de Protón Vacuolares , Animales , Hipocampo/metabolismo , Hipocampo/citología , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Ratones , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Sinapsis/metabolismo , Sinapsis/fisiología , Células Cultivadas , Autofagia/fisiología , Lisosomas/metabolismoRESUMEN
Dietary restriction, such as low glycemic index diet (LGID), have been successfully used to treat drug-resistant epilepsy. However, if such diet could also counteract antiepileptogenesis is still unclear. Here, we investigated whether the administration of LGID during the latent pre-epileptic period, prevents or delays the appearance of the overt epileptic phenotype. To this aim, we used the Synapsin II knockout (SynIIKO) mouse, a model of temporal lobe epilepsy in which seizures manifest 2-3 months after birth, offering a temporal window in which LGID may affect epileptogenesis. Pregnant SynIIKO mice were fed with either LGID or standard diet during gestation and lactation. Both diets were maintained in weaned mice up to 5 months of age. LGID delayed the seizure onset and induced a reduction of seizures severity only in female SynIIKO mice. In parallel with the epileptic phenotype, high-density multielectrode array recordings revealed a reduction of frequency, amplitude, duration, velocity of propagation and spread of interictal events by LGID in the hippocampus of SynIIKO females, but not mutant males, confirming the gender-specific effect. ELISA-based analysis revealed that LGID increased cortico-hippocampal allopregnanolone (ALLO) levels only in females, while it was unable to affect ALLO plasma concentrations in either sex. The results indicate that the gender-specific interference of LGID with the epileptogenic process can be ascribed to a gender-specific increase in cortical ALLO, a neurosteroid known to strengthen GABAergic transmission. The study highlights the possibility of developing a personalized gender-based therapy for temporal lobe epilepsy.
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Epilepsia del Lóbulo Temporal , Epilepsia , Masculino , Embarazo , Femenino , Ratones , Animales , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/inducido químicamente , Índice Glucémico , Convulsiones , Hipocampo , Epilepsia/genética , DietaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Autophagy and endolysosomal trafficking are crucial in neuronal development, function and survival. These processes ensure efficient removal of misfolded aggregation-prone proteins and damaged organelles, such as dysfunctional mitochondria, thus allowing the maintenance of proper cellular homeostasis. Beside this, emerging evidence has pointed to their involvement in the regulation of the synaptic proteome needed to guarantee an efficient neurotransmitter release and synaptic plasticity. Along this line, an intimate interplay between the molecular machinery regulating synaptic vesicle endocytosis and synaptic autophagy is emerging, suggesting that synaptic quality control mechanisms need to be tightly coupled to neurosecretion to secure release accuracy. Defects in autophagy and endolysosomal pathway have been associated with neuronal dysfunction and extensively reported in Alzheimer's, Parkinson's, Huntington's and amyotrophic lateral sclerosis among other neurodegenerative diseases, with common features and emerging genetic bases. In this review, we focus on the multiple roles of autophagy and endolysosomal system in neuronal homeostasis and highlight how their defects probably contribute to synaptic default and neurodegeneration in the above-mentioned diseases, discussing the most recent options explored for therapeutic interventions.
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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.
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Proteínas Adaptadoras Transductoras de Señales/genética , Autofagia/genética , Encéfalo/fisiopatología , Proteínas del Tejido Nervioso/genética , Espasmos Infantiles/genética , Encéfalo/diagnóstico por imagen , Niño , Preescolar , Progresión de la Enfermedad , Electroencefalografía , Femenino , Humanos , Lactante , Lisosomas/fisiología , Imagen por Resonancia Magnética , Masculino , Mutación , Linaje , Espasmos Infantiles/diagnóstico por imagen , Espasmos Infantiles/fisiopatología , Secuenciación del ExomaRESUMEN
Synapsin I is a phosphoprotein that coats the cytoplasmic side of synaptic vesicles and regulates their trafficking within nerve terminals. Autoantibodies against Syn I have been described in sera and cerebrospinal fluids of patients with numerous neurological diseases, including limbic encephalitis and clinically isolated syndrome; however, the effects and fate of autoantibodies in neurons are still unexplored. We found that in vitro exposure of primary hippocampal neurons to patient's autoantibodies to SynI decreased the density of excitatory and inhibitory synapses and impaired both glutamatergic and GABAergic synaptic transmission. These effects were reproduced with a purified SynI antibody and completely absent in SynI knockout neurons. Autoantibodies to SynI are internalized by FcγII/III-mediated endocytosis, interact with endogenous SynI, and promote its sequestration and intracellular aggregation. Neurons exposed to human autoantibodies to SynI display a reduced density of SVs, mimicking the SynI loss-of-function phenotype. Our data indicate that autoantibodies to intracellular antigens such as SynI can reach and inactivate their targets and suggest that an antibody-mediated synaptic dysfunction may contribute to the evolution and progression of autoimmune-mediated neurological diseases positive for SynI autoantibodies.
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Autoanticuerpos/inmunología , Enfermedades del Sistema Nervioso/inmunología , Sinapsis/inmunología , Sinapsinas/genética , Animales , Autoanticuerpos/genética , Citoplasma/genética , Citoplasma/inmunología , Neuronas GABAérgicas/inmunología , Neuronas GABAérgicas/metabolismo , Humanos , Encefalitis Límbica/genética , Encefalitis Límbica/inmunología , Ratones , Enfermedades del Sistema Nervioso/genética , Neuronas , Transporte de Proteínas/genética , Sinapsis/genética , Sinapsinas/inmunología , Transmisión Sináptica/genética , Transmisión Sináptica/inmunología , Vesículas Sinápticas/genética , Vesículas Sinápticas/inmunologíaRESUMEN
Here, we describe a purification protocol for isolating clathrin-coated vesicles (CCVs) from adult rat brain by using differential centrifugation coupled with Ficoll-sucrose and D2O-sucrose density gradient centrifugation and an additional linear sucrose step gradient at the end to separate CCVs from contaminating membranes present in the crude microsomal fraction.
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Encéfalo/metabolismo , Fraccionamiento Celular , Vesículas Cubiertas por Clatrina/metabolismo , Animales , Fraccionamiento Celular/métodos , Centrifugación por Gradiente de Densidad/métodos , Ratas , Fracciones SubcelularesRESUMEN
The striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase involved in synaptic transmission. The current hypothesis on STEP function holds that it opposes synaptic strengthening by dephosphorylating and inactivating key neuronal proteins involved in synaptic plasticity and intracellular signaling, such as the MAP kinases ERK1/2 and p38, as well as the tyrosine kinase Fyn. Although STEP has a predominant role at the post-synaptic level, it is also expressed in nerve terminals. To better investigate its physiological role at the presynaptic level, we functionally investigated brain synaptosomes and autaptic hippocampal neurons from STEP knockout (KO) mice. Synaptosomes purified from mutant mice were characterized by an increased basal and evoked glutamate release compared with wild-type animals. Under resting conditions, STEP KO synaptosomes displayed increased cytosolic Ca2+ levels accompanied by an enhanced basal activity of Ca2+/calmodulin-dependent protein kinase type II (CaMKII) and hyperphosphorylation of synapsin I at CaMKII sites. Moreover, STEP KO hippocampal neurons exhibit an increase of excitatory synaptic strength attributable to an increased size of the readily releasable pool of synaptic vesicles. These results provide new evidence that STEP plays an important role at nerve terminals in the regulation of Ca2+ homeostasis and neurotransmitter release.
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Calcio/metabolismo , Ácido Glutámico/metabolismo , Homeostasis , Espacio Intracelular/metabolismo , Neostriado/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/deficiencia , Transmisión Sináptica , Animales , Calcineurina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Citosol/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones Noqueados , Modelos Biológicos , Mutación/genética , Fosforilación , Terminales Presinápticos/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Sinapsis/metabolismo , Sinapsinas/metabolismo , Sinaptosomas/metabolismoRESUMEN
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.
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Complejo 2 de Proteína Adaptadora , Endocitosis , Neurogénesis , Vesículas Sinápticas/metabolismo , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Células Cultivadas , Vesículas Cubiertas por Clatrina/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/metabolismo , Neuronas/fisiología , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
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 Ca(2+) 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 Ca(2+)-sensing machinery and that it plays an important role in the final steps of neurotransmitter release.
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Señalización del Calcio , Exocitosis , Proteínas de la Membrana/metabolismo , Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Animales , Células Cultivadas , Hipocampo/citología , Hipocampo/metabolismo , Ratones , Ratones Endogámicos C57BL , Terminales Presinápticos/fisiología , Ratas , Ratas Sprague-Dawley , Potenciales Sinápticos , Vesículas Sinápticas/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagminas/metabolismoRESUMEN
L1 (also known as L1CAM) is a trans-membrane glycoprotein mediating neuron-neuron adhesion through homophilic and heterophilic interactions. Although experimental evidence has implicated L1 in axonal outgrowth, fasciculation and pathfinding, its contribution to voltage-gated Na(+) channel function and membrane excitability has remained unknown. Here, we show that firing rate, single cell spiking frequency and Na(+) current density are all reduced in hippocampal excitatory neurons from L1-deficient mice both in culture and in slices owing to an overall reduced membrane expression of Na(+) channels. Remarkably, normal firing activity was restored when L1 was reintroduced into L1-deficient excitatory neurons, indicating that abnormal firing patterns are not related to developmental abnormalities, but are a direct consequence of L1 deletion. Moreover, L1 deficiency leads to impairment of action potential initiation, most likely due to the loss of the interaction of L1 with ankyrin G that produces the delocalization of Na(+) channels at the axonal initial segment. We conclude that L1 contributes to functional expression and localization of Na(+) channels to the neuronal plasma membrane, ensuring correct initiation of action potential and normal firing activity.
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Membrana Celular/metabolismo , Regulación de la Expresión Génica/fisiología , Hipocampo/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Canales de Sodio Activados por Voltaje/biosíntesis , Animales , Membrana Celular/genética , Hipocampo/citología , Ratones , Ratones Noqueados , Molécula L1 de Adhesión de Célula Nerviosa/genética , Neuronas/citología , Canales de Sodio Activados por Voltaje/genéticaRESUMEN
Synapsin III (SynIII) is a neuron-specific phosphoprotein that plays a unique role in neuronal development. SynIII is phosphorylated by cAMP-dependent protein kinase (PKA) at a highly conserved phosphorylation site and by cyclin-dependent kinase-5 (Cdk5) at a newly described site. Although SynIII is known to be involved in axon elongation in vitro, the role of its phosphorylation by PKA and Cdk5 in the modulation of this process is unknown. We expressed either wild-type (WT) or phosphorylation-site mutants of SynIII in primary SynIII knock-out (KO) mouse neurons at early stages of in vitro development. Whereas the neurite elongation phenotype of SynIII KO neurons was fully rescued by the expression of WT SynIII, the expression of nonphosphorylatable and pseudo-phosphorylated PKA mutants was ineffective. Also, the nonphosphorylatable Cdk5 mutant was unable to rescue the neurite elongation phenotype of SynIII KO neurons. By contrast, the pseudo-phosphorylated mutant rescued the delay in neuronal maturation and axonal elongation, revealing a Cdk5-dependent regulation of SynIII function. Interestingly, SynIII KO neurons also exhibited decreased survival that was fully rescued by the expression of WT SynIII, but not by its phosphorylation mutants, and was associated with increased activated caspase3 and altered tropomyosin receptor kinase B isoform expression. These results indicate that PKA and Cdk5 phosphorylation is required for the physiological action of SynIII on axon specification and neurite outgrowth and that the expression of a functional SynIII is crucial for cell survival. Significance statement: Synapsin III is an atypical member of the synapsin family of synaptic vesicle-associated phosphoproteins that is precociously expressed in neurons and is downregulated afterward. Although experimental evidence suggests a specific role for Synapsin III in neuronal development, the molecular mechanisms are still largely unknown. We found that Synapsin III plays a central role in early stages of neuronal development involving neuronal survival, polarization, and neuritic growth and that these effects are dependent on phosphorylation by cAMP-dependent protein kinase and cyclin-dependent protein kinase-5. These results explain the recently described neurodevelopmental defects in the migration and orientation of Synapsin III-depleted cortical neurons and support the potential association of Synapsin III with neurodevelopmental disorders such as schizophrenia.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Neuronas/fisiología , Sinapsinas/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Quinasa 5 Dependiente de la Ciclina/genética , Embrión de Mamíferos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sinapsinas/genética , Tubulina (Proteína)/metabolismoRESUMEN
Synapsin III (SynIII) is a phosphoprotein that is highly expressed at early stages of neuronal development. Whereas in vitro evidence suggests a role for SynIII in neuronal differentiation, in vivo evidence is lacking. Here, we demonstrate that in vivo downregulation of SynIII expression affects neuronal migration and orientation. By contrast, SynIII overexpression affects neuronal migration, but not orientation. We identify a cyclin-dependent kinase-5 (CDK5) phosphorylation site on SynIII and use phosphomutant rescue experiments to demonstrate its role in SynIII function. Finally, we show that SynIII phosphorylation at the CDK5 site is induced by activation of the semaphorin-3A (Sema3A) pathway, which is implicated in migration and orientation of cortical pyramidal neurons (PNs) and is known to activate CDK5. Thus, fine-tuning of SynIII expression and phosphorylation by CDK5 activation through Sema3A activity is essential for proper neuronal migration and orientation.
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Corteza Cerebral/crecimiento & desarrollo , Quinasa 5 Dependiente de la Ciclina/genética , Semaforina-3A/biosíntesis , Sinapsinas/genética , Animales , Proteína C-Reactiva/genética , Células COS , Movimiento Celular/genética , Chlorocebus aethiops , Quinasa 5 Dependiente de la Ciclina/biosíntesis , Dendritas/genética , Dendritas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Proteínas del Tejido Nervioso/genética , Fosforilación , Cultivo Primario de Células , Células Piramidales/citología , Células Piramidales/metabolismo , Ratas , Semaforina-3A/genética , Transducción de Señal , Sinapsinas/biosíntesisRESUMEN
During cortical development, N-methyl-D-aspartate (NMDA) receptors (NMDARs) facilitate presynaptic terminal formation, enhance neurotransmitter release and are required in presynaptic neurons for spike-timing-dependent long-term depression (tLTD). However, the extent to which NMDARs are found within cortical presynaptic terminals has remained controversial, and the sub-synaptic localization and dynamics of axonal NMDARs are unknown. Here, using live confocal imaging and biochemical purification of presynaptic membranes, we provide strong evidence that NMDARs localize to presynaptic terminals in vitro and in vivo in a developmentally regulated manner. The NR1 and NR2B subunits (also known as GRIN1 and GRIN2B, respectively) were found within the active zone membrane, where they could respond to synaptic glutamate release. Surprisingly, NR1 also appeared in glutamatergic and GABAergic synaptic vesicles. During synaptogenesis, NR1 was mobile throughout axons - including growth cones and filopodia, structures that are involved in synaptogenesis. Upon synaptogenic contact, NMDA receptors were quickly recruited to terminals by neuroligin-1 signaling. Unlike dendrites, the trafficking and distribution of axonal NR1 were insensitive to activity changes, including NMDA exposure, local glutamate uncaging or action potential blockade. These results support the idea that presynaptic NMDARs play an early role in presynaptic development.
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Terminales Presinápticos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Vesículas Sinápticas/metabolismo , Corteza Visual/embriología , Animales , Axones/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Células Cultivadas , Dendritas/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Células HEK293 , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Neurotransmisores/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Presinapticos/metabolismo , Transducción de Señal , Transmisión Sináptica/fisiologíaRESUMEN
Several proteins encoded by PD genes are implicated in synaptic vesicle traffic. Endophilin, a key factor in the endocytosis of synaptic vesicles, was shown to bind to, and be ubiquitinated by, the PD-linked E3 ubiquitin ligase Parkin. Here we report that Parkin's level is specifically upregulated in brain and fibroblasts of endophilin mutant mice due to increased transcriptional regulation. Studies of transfected HEK293T cells show that Parkin ubiquitinates not only endophilin, but also its major binding partners, dynamin and synaptojanin 1. These results converge with the recently reported functional relationship of endophilin to the PD gene LRRK2 and with the identification of a PD-linked synaptojanin 1 mutation, in providing evidence for a link between PD and endocytosis genes.
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Aciltransferasas/deficiencia , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Ubiquitina-Proteína Ligasas/metabolismo , Regulación hacia Arriba , Aciltransferasas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Encéfalo/metabolismo , Dinaminas/metabolismo , Endocitosis/genética , Endocitosis/fisiología , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Transcripción Genética , Ubiquitinación/fisiologíaRESUMEN
Synapsins (Syns) are synaptic vesicle (SV)-associated proteins involved in the regulation of synaptic transmission and plasticity, which display a highly conserved ATP binding site in the central C-domain, whose functional role is unknown. Using molecular dynamics simulations, we demonstrated that ATP binding to SynI is mediated by a conformational transition of a flexible loop that opens to make the binding site accessible; such transition, prevented in the K269Q mutant, is not significantly affected in the absence of Ca(2+) or by the E373K mutation that abolishes Ca(2+)-binding. Indeed, the ATP binding to SynI also occurred under Ca(2+)-free conditions and increased its association with purified rat SVs regardless of the presence of Ca(2+) and promoted SynI oligomerization. However, although under Ca(2+)-free conditions, SynI dimerization and SV clustering were enhanced, Ca(2+) favored the formation of tetramers at the expense of dimers and did not affect SV clustering, indicating a role of Ca(2+)-dependent dimer/tetramer transitions in the regulation of ATP-dependent SV clustering. To elucidate the role of ATP/SynI binding in synaptic physiology, mouse SynI knock-out hippocampal neurons were transduced with either wild-type or K269Q mutant SynI and inhibitory transmission was studied by patch-clamp and electron microscopy. K269Q-SynI expressing inhibitory synapses showed increased synaptic strength due to an increase in the release probability, an increased vulnerability to synaptic depression and a dysregulation of SV trafficking, when compared with wild-type SynI-expressing terminals. The results suggest that the ATP-SynI binding plays predocking and postdocking roles in the modulation of SV clustering and plasticity of inhibitory synapses.
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Adenosina Trifosfato/metabolismo , Exocitosis/fisiología , Neuronas/metabolismo , Sinapsis/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Femenino , Hipocampo/citología , Hipocampo/metabolismo , Hipocampo/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Neuronas/ultraestructura , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Sinapsis/ultraestructura , Sinapsinas/genética , Transmisión Sináptica/fisiología , Vesículas Sinápticas/ultraestructuraRESUMEN
Autism spectrum disorders (ASDs) are heterogeneous neurodevelopmental disorders characterized by deficits in social interaction and social communication, restricted interests, and repetitive behaviors. Many synaptic protein genes are linked to the pathogenesis of ASDs, making them prototypical synaptopathies. An array of mutations in the synapsin (Syn) genes in humans has been recently associated with ASD and epilepsy, diseases that display a frequent comorbidity. Syns are pre-synaptic proteins regulating synaptic vesicle traffic, neurotransmitter release, and short-term synaptic plasticity. In doing so, Syn isoforms control the tone of activity of neural circuits and the balance between excitation and inhibition. As ASD pathogenesis is believed to result from dysfunctions in the balance between excitatory and inhibitory transmissions in neocortical areas, Syns are novel ASD candidate genes. Accordingly, deletion of single Syn genes in mice, in addition to epilepsy, causes core symptoms of ASD by affecting social behavior, social communication, and repetitive behaviors. Thus, Syn knockout mice represent a good experimental model to define synaptic alterations involved in the pathogenesis of ASD and epilepsy.
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Analysis of presynaptic protein expression in glutamatergic and GABAergic central synapses performed in several laboratories and with different techniques is unveiling a complex scenario, largely because each presynaptic protein exists in several isoforms. The interpretation of these findings is generally based on the notion that each synapse and each synaptic vesicle contains one of the isoforms of each family of presynaptic proteins. We verified whether this interpretation is tenable by performing triple labeling and immunoisolation studies with the aim of detecting two isoforms of a given presynaptic protein in glutamatergic or GABAergic axon terminals and/or synaptic vesicles (SVs). Here, we show that: (1) the possibility that not all families of presynaptic proteins are expressed in all terminals must be taken into serious account; (2) the expression of a given protein isoform in a terminal does not exclude the expression of other isoforms of the same protein in the same terminal and in the same vesicle. These conclusions open new and interesting problems; their experimental analysis might improve our understanding of the physiology and pathophysiology of central synapses.
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Podocytes are specialized cells that play an integral role in the renal glomerular filtration barrier via their foot processes. The foot processes form a highly organized structure, the disruption of which causes nephrotic syndrome. Interestingly, several similarities have been observed between mechanisms that govern podocyte organization and mechanisms that mediate neuronal synapse development. Dynamin, synaptojanin, and endophilin are functional partners in synaptic vesicle recycling via interconnected actions in clathrin-mediated endocytosis and actin dynamics in neurons. A role of dynamin in the maintenance of the kidney filtration barrier via an action on the actin cytoskeleton of podocytes was suggested. Here we used a conditional double-KO of dynamin 1 (Dnm1) and Dnm2 in mouse podocytes to confirm dynamin's role in podocyte foot process maintenance. In addition, we demonstrated that while synaptojanin 1 (Synj1) KO mice and endophilin 1 (Sh3gl2), endophilin 2 (Sh3gl1), and endophilin 3 (Sh3gl3) triple-KO mice had grossly normal embryonic development, these mutants failed to establish a normal filtration barrier and exhibited severe proteinuria due to abnormal podocyte foot process formation. These results strongly implicate a protein network that functions at the interface between endocytosis and actin at neuronal synapses in the formation and maintenance of the kidney glomerular filtration barrier.
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Proteínas Adaptadoras Transductoras de Señales/genética , Dinamina II/genética , Dinamina I/genética , Barrera de Filtración Glomerular/patología , Proteínas del Tejido Nervioso/genética , Monoéster Fosfórico Hidrolasas/genética , Podocitos/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Dinamina I/metabolismo , Dinamina II/metabolismo , Endocitosis , Barrera de Filtración Glomerular/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Microscopía Fluorescente , Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteinuria/genética , Proteinuria/patología , Insuficiencia Renal/genética , Insuficiencia Renal/patología , Insuficiencia Renal/orinaRESUMEN
Endophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.