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Cytoplasmic dynein is essential for intracellular transport. Despite extensive in vitro characterizations, how the dynein motors transport vesicles by processive steps in live cells remains unclear. To dissect the molecular mechanisms of dynein, we develop optical probes that enable long-term single-particle tracking in live cells with high spatiotemporal resolution. We find that the number of active dynein motors transporting cargo switches stochastically between one and five dynein motors during long-range transport in neuronal axons. Our very bright optical probes allow the observation of individual molecular steps. Strikingly, these measurements reveal that the dwell times between steps are controlled by two temperature-dependent rate constants in which two ATP molecules are hydrolyzed sequentially during each dynein step. Thus, our observations uncover a previously unknown chemomechanical cycle of dynein-mediated cargo transport in living cells.
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Neuroligin-3 (Nlgn3) is an autism-associated cell-adhesion molecule that interacts with neurexins and is robustly expressed in both neurons and astrocytes. Neuronal Nlgn3 is an essential regulator of synaptic transmission but the function of astrocytic Nlgn3 is largely unknown. Given the high penetrance of Nlgn3 mutations in autism and the emerging role of astrocytes in neuropsychiatric disorders, we here asked whether astrocytic Nlgn3 might shape neural circuit properties in the cerebellum similar to neuronal Nlgn3. Imaging of tagged Nlgn3 protein produced by CRISPR/Cas9-mediated genome editing showed that Nlgn3 is enriched in the cell body but not the fine processes of cerebellar astrocytes (Bergmann glia). Astrocyte-specific knockout of Nlgn3 did not detectably alter the number of synapses, synaptic transmission, or astrocyte morphology in mouse cerebellum. However, spatial transcriptomic analyses revealed a significant shift in gene expression among multiple cerebellar cell types after the deletion of astrocytic Nlgn3. Hence, in contrast to neuronal Nlgn3, astrocytic Nlgn3 in the cerebellum is not involved in shaping synapses but may modulate gene expression in specific brain areas.
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Social communication guides decision-making, which is essential for survival. Social transmission of food preference (STFP) is an ecologically relevant memory paradigm in which an animal learns a desirable food odour from another animal in a social context, creating a long-term memory1,2. How food-preference memory is acquired, consolidated and stored is unclear. Here we show that the posteromedial nucleus of the cortical amygdala (COApm) serves as a computational centre in long-term STFP memory consolidation by integrating social and sensory olfactory inputs. Blocking synaptic signalling by the COApm-based circuit selectively abolished STFP memory consolidation without impairing memory acquisition, storage or recall. COApm-mediated STFP memory consolidation depends on synaptic inputs from the accessory olfactory bulb and on synaptic outputs to the anterior olfactory nucleus. STFP memory consolidation requires protein synthesis, suggesting a gene-expression mechanism. Deep single-cell and spatially resolved transcriptomics revealed robust but distinct gene-expression signatures induced by STFP memory formation in the COApm that are consistent with synapse restructuring. Our data thus define a neural circuit for the consolidation of a socially communicated long-term memory, thereby mechanistically distinguishing protein-synthesis-dependent memory consolidation from memory acquisition, storage or retrieval.
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Amígdala del Cerebelo , Preferencias Alimentarias , Consolidación de la Memoria , Memoria a Largo Plazo , Conducta Social , Animales , Masculino , Ratones , Amígdala del Cerebelo/fisiología , Amígdala del Cerebelo/citología , Consolidación de la Memoria/fisiología , Memoria a Largo Plazo/fisiología , Ratones Endogámicos C57BL , Odorantes/análisis , Bulbo Olfatorio/fisiología , Bulbo Olfatorio/citología , Análisis de la Célula Individual , Sinapsis/metabolismo , Transcriptoma , Preferencias Alimentarias/fisiología , Preferencias Alimentarias/psicologíaRESUMEN
Synaptic adhesion molecules (SAMs) are evolutionarily conserved proteins that play an important role in the form and function of neuronal synapses. Teneurins (Tenms) and latrophilins (Lphns) are well-known cell adhesion molecules that form a transsynaptic complex. Recent studies suggest that Tenm3 and Lphn2 (gene symbol Adgrl2) are involved in hippocampal circuit assembly via their topographical expression. However, it is not known whether other teneurins and latrophilins display similar topographically restricted expression patterns during embryonic and postnatal development. Here, we reveal the cartography of all teneurin (Tenm1-4) and latrophilin (Lphn1-3 [Adgrl1-3]) paralog expression in the mouse hippocampus across prenatal and postnatal development as monitored by large-scale single-molecule RNA in situ hybridization mapping. Our results identify a striking heterogeneity in teneurin and latrophilin expression along the spatiotemporal axis of the hippocampus. Tenm2 and Tenm4 expression levels peak at the neonatal stage when compared to Tenm1 and Tenm3, while Tenm1 expression is restricted to the postnatal pyramidal cell layer. Tenm4 expression in the dentate gyrus (DG) exhibits an opposing topographical expression pattern in the embryonic and neonatal hippocampus. Our findings were validated by analyses of multiple RNA-seq datasets at bulk, single-cell, and spatial levels. Thus, our study presents a comprehensive spatiotemporal map of Tenm and Lphn expression in the hippocampus, showcasing their diverse expression patterns across developmental stages in distinct spatial axes.
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Regulación del Desarrollo de la Expresión Génica , Hipocampo , Proteínas del Tejido Nervioso , Receptores de Péptidos , Animales , Hipocampo/metabolismo , Hipocampo/embriología , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Receptores de Péptidos/metabolismo , Receptores de Péptidos/genética , Femenino , Ratones Endogámicos C57BL , Masculino , Tenascina , Receptores Acoplados a Proteínas GRESUMEN
Latrophilin-1 (Lphn1, aka CIRL1 and CL1; gene symbol Adgrl1) is an adhesion GPCR that has been implicated in excitatory synaptic transmission as a candidate receptor for α-latrotoxin. Here we analyzed conditional knock-in/knock-out mice for Lphn1 that contain an extracellular myc epitope tag. Mice of both sexes were used in all experiments. Surprisingly, we found that Lphn1 is localized in cultured neurons to synaptic nanoclusters that are present in both excitatory and inhibitory synapses. Conditional deletion of Lphn1 in cultured neurons failed to elicit a detectable impairment in excitatory synapses but produced a decrease in inhibitory synapse numbers and synaptic transmission that was most pronounced for synapses close to the neuronal soma. No changes in axonal or dendritic outgrowth or branching were observed. Our data indicate that Lphn1 is among the few postsynaptic adhesion molecules that are present in both excitatory and inhibitory synapses and that Lphn1 by itself is not essential for excitatory synaptic transmission but is required for some inhibitory synaptic connections.
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Ratones Noqueados , Receptores de Péptidos , Sinapsis , Animales , Femenino , Masculino , Ratones , Células Cultivadas , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/metabolismo , Hipocampo/citología , Potenciales Postsinápticos Inhibidores/fisiología , Ratones Endogámicos C57BL , Inhibición Neural/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Sinapsis/metabolismo , Sinapsis/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0158295.].
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Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory.
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Astrocitos , Comunicación Celular , Perfilación de la Expresión Génica , Memoria a Largo Plazo , Neuronas , Astrocitos/citología , Astrocitos/metabolismo , Astrocitos/fisiología , Complejo Nuclear Basolateral/citología , Complejo Nuclear Basolateral/metabolismo , Complejo Nuclear Basolateral/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Memoria a Largo Plazo/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neuronas/fisiología , Análisis de Secuencia de ARN , Imagen Individual de Molécula , Análisis de Expresión Génica de una Sola Célula , UbiquitinaciónRESUMEN
The assembly and specification of synapses in the brain is incompletely understood1-3. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion G-protein-coupled receptor-mediates synapse formation in the hippocampus4 but the mechanisms involved remain unclear. Here we show in mice that LPHN3 organizes synapses through a convergent dual-pathway mechanism: activation of Gαs signalling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell-type-specific alternative splicing of Lphn3 controls the LPHN3 G-protein-coupling mode, resulting in LPHN3 variants that predominantly signal through Gαs or Gα12/13. CRISPR-mediated manipulation of Lphn3 alternative splicing that shifts LPHN3 from a Gαs- to a Gα12/13-coupled mode impaired synaptic connectivity as severely as the overall deletion of Lphn3, suggesting that Gαs signalling by LPHN3 splice variants mediates synapse formation. Notably, Gαs-coupled, but not Gα12/13-coupled, splice variants of LPHN3 also recruit phase-transitioned postsynaptic protein scaffold condensates, such that these condensates are clustered by binding of presynaptic teneurin and FLRT ligands to LPHN3. Moreover, neuronal activity promotes alternative splicing of the synaptogenic Gαs-coupled variant of LPHN3. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways: Gαs signalling and clustered phase separation of postsynaptic protein scaffolds.
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Empalme Alternativo , Receptores Acoplados a Proteínas G , Receptores de Péptidos , Sinapsis , Animales , Ratones , Empalme Alternativo/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Subunidades alfa de la Proteína de Unión al GTP Gs , Ligandos , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/deficiencia , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Sinapsis/metabolismo , Transducción de SeñalRESUMEN
Rapid delivery of glutamate receptors to the postsynaptic membrane via vesicle fusion is a central component of synaptic plasticity. However, it is unknown how this process supports specific neural computations during behavior. To bridge this gap, we combined conditional genetic deletion of a component of the postsynaptic membrane fusion machinery, Syntaxin3 (Stx3), in hippocampal CA1 neurons of mice with population in vivo calcium imaging. This approach revealed that Stx3 is necessary for forming the neural dynamics that support novelty processing, spatial reward memory and offline memory consolidation. In contrast, CA1 Stx3 was dispensable for maintaining aspects of the neural code that exist presynaptic to CA1 such as representations of context and space. Thus, manipulating postsynaptic membrane fusion identified computations that specifically require synaptic restructuring via membrane trafficking in CA1 and distinguished them from neural representation that could be inherited from upstream brain regions or learned through other mechanisms.
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α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function.
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Enfermedad de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Vesículas Sinápticas/metabolismo , Lisofosfatidilcolinas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Fosfolípidos/metabolismoRESUMEN
Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. Most aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-autoproteolysis inducing (GAIN) domain, but the two resulting fragments remain noncovalently associated on the cell surface. Force-mediated dissociation of the fragments is thought to activate G-protein signaling, but how this mechanosensitivity arises is poorly understood. Here, we use magnetic tweezer assays to show that physiologically relevant forces in the 1-10 pN range lead to dissociation of the latrophilin-3 GAIN domain on the seconds-to-minutes time scale, compared to days in the absence of force. In addition, we find that the GAIN domain undergoes large changes in length in response to increasing mechanical load. These data are consistent with a model in which a force-sensitive equilibrium between compact and extended GAIN domain states precedes dissociation, suggesting a mechanism by which latrophilins and other aGPCRs may mediate mechanically induced signal transduction.
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Receptores Acoplados a Proteínas G , Receptores de Péptidos , Adhesión Celular , Receptores Acoplados a Proteínas G/metabolismo , Membrana Celular/metabolismoRESUMEN
Synaptic adhesion molecules (SAMs) shape the structural and functional properties of synapses and thereby control the information processing power of neural circuits. SAMs are broadly expressed in the brain, suggesting that they may instruct synapse formation and specification via a combinatorial logic. Here, we generate sextuple conditional knockout mice targeting all members of the two major families of presynaptic SAMs, Neurexins and leukocyte common antigen-related-type receptor phospho-tyrosine phosphatases (LAR-PTPRs), which together account for the majority of known trans-synaptic complexes. Using synapses formed by cerebellar Purkinje cells onto deep cerebellar nuclei as a model system, we confirm that Neurexins and LAR-PTPRs themselves are not essential for synapse assembly. The combinatorial deletion of both neurexins and LAR-PTPRs, however, decreases Purkinje-cell synapses on deep cerebellar nuclei, the major output pathway of cerebellar circuits. Consistent with this finding, combined but not separate deletions of neurexins and LAR-PTPRs impair motor behaviors. Thus, Neurexins and LAR-PTPRs are together required for the assembly of a functional cerebellar circuit.
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Cerebelo , Células de Purkinje , Animales , Ratones , Encéfalo , Cognición , Ratones Noqueados , Fosfotirosina , Proteínas Tirosina Fosfatasas , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genéticaRESUMEN
The production of induced neuronal (iN) cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells by the forced expression of proneural transcription factors is rapid, efficient and reproducible. The ability to generate large numbers of human neurons in such a robust manner enables large-scale studies of human neural differentiation and neuropsychiatric diseases. Surprisingly, similar transcription factor-based approaches for converting mouse ESCs into iN cells have been challenging, primarily because of low cell survival. Here, we provide a detailed approach for the efficient and reproducible generation of functional iN cells from mouse ESC cultures by the genetically induced expression of neurogenin-2. The resulting iN cells display mature pre- and postsynaptic specializations and form synaptic networks. Our method provides the basis for studying neuronal development and enables the direct comparison of cellular phenotypes in mouse and human neurons generated in an equivalent way. The procedure requires 14 d and can be carried out by users with expertise in stem cell culture.
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Presenilin mutations that alter γ-secretase activity cause familial Alzheimer's disease (AD), whereas ApoE4, an apolipoprotein for cholesterol transport, predisposes to sporadic AD. Both sporadic and familial AD feature synaptic dysfunction. Whether γ-secretase is involved in cholesterol metabolism and whether such involvement impacts synaptic function remains unknown. Here, we show that in human neurons, chronic pharmacological or genetic suppression of γ-secretase increases synapse numbers but decreases synaptic transmission by lowering the presynaptic release probability without altering dendritic or axonal arborizations. In search of a mechanism underlying these synaptic impairments, we discovered that chronic γ-secretase suppression robustly decreases cholesterol levels in neurons but not in glia, which in turn stimulates neuron-specific cholesterol-synthesis gene expression. Suppression of cholesterol levels by HMG-CoA reductase inhibitors (statins) impaired synaptic function similar to γ-secretase inhibition. Thus, γ-secretase enables synaptic function by maintaining cholesterol levels, whereas the chronic suppression of γ-secretase impairs synapses by lowering cholesterol levels.