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1.
Proc Natl Acad Sci U S A ; 119(45): e2203499119, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36322761

RESUMO

Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules that fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin in neurons both in vitro and in vivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers like guanosine triphosphate (GTP)-tubulin. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin velocity and density on microtubules consisting of elongated dimers add to the increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin contributes to the regulation of mitochondrial motility and as such to the local distribution of mitochondria along axons.


Assuntos
Transporte Axonal , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Cinesinas , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Axônios/metabolismo , Conformação Molecular
2.
Neurobiol Dis ; 202: 106696, 2024 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-39389154

RESUMO

There is now compelling evidence for the presence of pathological forms of Tau in tissues of both patients and animal models of Huntington's disease (HD). While the root cause of this illness is a mutation within the huntingtin gene, a number of studies now suggest that HD could also be considered a secondary tauopathy. However, the contributory role of Tau in the pathogenesis and pathophysiology of this condition, as well as its implications in cellular toxicity and consequent behavioral impairments are largely unknown. We therefore performed intracerebral stereotaxic injections of recombinant human Tau monomers and fibrils into the knock-in zQ175 mouse model of HD. Tau fibrils induced cognitive and anxiety-like phenotypes predominantly in zQ175 mice and increased the number and size of insoluble mutant huntingtin (mHTT) aggregates in the brains of treated animals. To better understand the putative mechanisms through which Tau could initiate and/or contribute to pathology, we incubated StHdh striatal cells, an in vitro model of HD, with the different Tau forms and evaluated the effects on cell functionality and heat shock proteins Hsp70 and Hsp90. Calcium imaging experiments showed functional impairments of HD StHdh cells following treatment with Tau fibrils, as well as significant changes to the levels of both heat shock proteins which were found trapped within mHTT aggregates. The accumulation of Hsp70 and 90 within aggregates was also present in mouse tissue which suggests that alteration of molecular chaperone-dependent protein quality control may influence aggregation, implicating proteostasis in the mHTT-Tau interplay.

3.
J Neurochem ; 156(1): 88-105, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-31886886

RESUMO

Insulin signaling through the insulin receptor has long been studied in classic target organs, such as adipose tissue and skeletal muscle, where one of its effects is to increase glucose uptake. Insulin and insulin receptor are present in many areas of the brain, but the functions of brain insulin signaling outside feeding circuits are not well defined. It has been proposed that hippocampal insulin signaling is important for memory, that brain insulin signaling is deficient in Alzheimer's disease, and that intranasal insulin treatment improves cognition, but the mechanisms remain unclear and do not seem to involve increased glucose uptake by neurons. The molecular behavior of the insulin receptor itself is not well known in living neurons; therefore, we investigated the spatial dynamics of the insulin receptor on somatodendritic membranes of live rat hippocampal neurons in culture. Using single-molecule tracking of quantum dot-tagged insulin receptors and single-particle tracking photoactivation localization microscopy, we show that the insulin receptor is distributed over both dendritic shafts and spines. Using colocalization with synaptic markers, we also show that in contrast to the glutamate receptor subunit glutamate receptor subunit A1, the dynamics of the insulin receptor are not affected by association with excitatory synapses; however, the insulin receptor is immobilized by components of inhibitory synapses. The mobility of the insulin receptor is reduced both by low concentrations of the pro-inflammatory cytokine tumor necrosis factor α and by cholesterol depletion, suggesting an association with sphingolipid-rich membrane domains. On the other hand, the insulin receptor dynamics in hippocampal neurons are not affected by increased excitatory signaling. Finally, using real-time single-event quantification, we find evidence of strong insulin receptor exocytosis on dendritic shafts. Our results suggest an association of the neuronal insulin receptor with specific elements of the dendritic shaft, rather than excitatory synapses.


Assuntos
Dendritos/metabolismo , Hipocampo/metabolismo , Receptor de Insulina/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley
4.
Nat Methods ; 13(8): 673-8, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27271196

RESUMO

Although neuronal activity can be modulated using a variety of techniques, there are currently few methods for controlling neuronal connectivity. We introduce a tool (GFE3) that mediates the fast, specific and reversible elimination of inhibitory synaptic inputs onto genetically determined neurons. GFE3 is a fusion between an E3 ligase, which mediates the ubiquitination and rapid degradation of proteins, and a recombinant, antibody-like protein (FingR) that binds to gephyrin. Expression of GFE3 leads to a strong and specific reduction of gephyrin in culture or in vivo and to a substantial decrease in phasic inhibition onto cells that express GFE3. By temporarily expressing GFE3 we showed that inhibitory synapses regrow following ablation. Thus, we have created a simple, reversible method for modulating inhibitory synaptic input onto genetically determined cells.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp/métodos , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Hipocampo , Masculino , Transtornos Motores/metabolismo , Transtornos Motores/patologia , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Coluna Vertebral/citologia , Coluna Vertebral/metabolismo , Ubiquitinação , Peixe-Zebra
5.
PLoS Genet ; 12(7): e1006192, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27462983

RESUMO

Local translation at the synapse plays key roles in neuron development and activity-dependent synaptic plasticity. mRNAs are translocated from the neuronal soma to the distant synapses as compacted ribonucleoparticles referred to as RNA granules. These contain many RNA-binding proteins, including the Fragile X Mental Retardation Protein (FMRP), the absence of which results in Fragile X Syndrome, the most common inherited form of intellectual disability and the leading genetic cause of autism. Using FMRP as a tracer, we purified a specific population of RNA granules from mouse brain homogenates. Protein composition analyses revealed a strong relationship between polyribosomes and RNA granules. However, the latter have distinct architectural and structural properties, since they are detected as close compact structures as observed by electron microscopy, and converging evidence point to the possibility that these structures emerge from stalled polyribosomes. Time-lapse video microscopy indicated that single granules merge to form cargoes that are transported from the soma to distal locations. Transcriptomic analyses showed that a subset of mRNAs involved in cytoskeleton remodelling and neural development is selectively enriched in RNA granules. One third of the putative mRNA targets described for FMRP appear to be transported in granules and FMRP is more abundant in granules than in polyribosomes. This observation supports a primary role for FMRP in granules biology. Our findings open new avenues for the study of RNA granule dysfunctions in animal models of nervous system disorders, such as Fragile X syndrome.


Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Proteínas de Ligação a RNA/genética , Sinapses/genética , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Plasticidade Neuronal/genética , Neurônios/metabolismo , Polirribossomos/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/biossíntese , Sinapses/metabolismo
6.
PLoS Genet ; 9(10): e1003890, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24204304

RESUMO

Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.


Assuntos
Corpos Enovelados/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Isoformas de Proteínas/biossíntese , Animais , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Corpos Enovelados/ultraestrutura , Proteína do X Frágil da Deficiência Intelectual/biossíntese , Síndrome do Cromossomo X Frágil/patologia , Regulação da Expressão Gênica , Humanos , Camundongos , Neurônios/metabolismo , Isoformas de Proteínas/ultraestrutura , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética
7.
Neurophotonics ; 11(1): 014401, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38550388

RESUMO

The editorial presents the two-part Special Section on Frontiers in Neurophotonics.

8.
Neurophotonics ; 11(1): 014415, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38545127

RESUMO

The Frontiers in Neurophotonics Symposium is a biennial event that brings together neurobiologists and physicists/engineers who share interest in the development of leading-edge photonics-based approaches to understand and manipulate the nervous system, from its individual molecular components to complex networks in the intact brain. In this Community paper, we highlight several topics that have been featured at the symposium that took place in October 2022 in Québec City, Canada.

9.
J Neurosci ; 32(31): 10767-79, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22855824

RESUMO

Understanding how brief synaptic events can lead to sustained changes in synaptic structure and strength is a necessary step in solving the rules governing learning and memory. Activation of ERK1/2 (extracellular signal regulated protein kinase 1/2) plays a key role in the control of functional and structural synaptic plasticity. One of the triggering events that activates ERK1/2 cascade is an NMDA receptor (NMDAR)-dependent rise in free intracellular Ca(2+) concentration. However the mechanism by which a short-lasting rise in Ca(2+) concentration is transduced into long-lasting ERK1/2-dependent plasticity remains unknown. Here we demonstrate that although synaptic activation in mouse cultured cortical neurons induces intracellular Ca(2+) elevation via both GluN2A and GluN2B-containing NMDARs, only GluN2B-containing NMDAR activation leads to a long-lasting ERK1/2 phosphorylation. We show that αCaMKII, but not ßCaMKII, is critically involved in this GluN2B-dependent activation of ERK1/2 signaling, through a direct interaction between GluN2B and αCaMKII. We then show that interfering with GluN2B/αCaMKII interaction prevents synaptic activity from inducing ERK-dependent increases in synaptic AMPA receptors and spine volume. Thus, in a developing circuit model, the brief activity of synaptic GluN2B-containing receptors and the interaction between GluN2B and αCaMKII have a role in long-term plasticity via the control of ERK1/2 signaling. Our findings suggest that the roles that these major molecular elements have in learning and memory may operate through a common pathway.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , 4-Aminopiridina/farmacologia , Análise de Variância , Animais , Bicuculina/farmacologia , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Córtex Cerebral/citologia , Espinhas Dendríticas/metabolismo , Proteína 4 Homóloga a Disks-Large , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Guanilato Quinases/metabolismo , Imunoprecipitação , Técnicas In Vitro , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fotodegradação , Bloqueadores dos Canais de Potássio/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/genética , Transfecção
10.
Proc Natl Acad Sci U S A ; 107(32): 14437-42, 2010 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-20660727

RESUMO

CaMKII is an abundant synaptic protein strongly implicated in plasticity. Overexpression of autonomous (T286D) CaMKII in CA1 hippocampal cells enhances synaptic strength if T305/T306 sites are not phosphorylated, but decreases synaptic strength if they are phosphorylated. It has generally been thought that spine size and synaptic strength covary; however, the ability of CaMKII and its various phosphorylation states to control spine size has not been previously examined. Using a unique method that allows the effects of overexpressed protein to be monitored over time, we found that all autonomous forms of CaMKII increase spine size. Thus, for instance, the T286D/T305D/T306D form increases spine size but decreases synaptic strength. Further evidence for such dissociation is provided by experiments with the T286D form that has been made catalytically dead. This form fails to enhance synaptic strength but increases spine size, presumably by a structural process. Thus very different mechanisms govern how CaMKII affects spine structure and synaptic function.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Espinhas Dendríticas/fisiologia , Sinapses/fisiologia , Animais , Espinhas Dendríticas/ultraestrutura , Eletrofisiologia , Hipocampo/citologia , Microscopia Confocal , Plasticidade Neuronal , Fosforilação , Ratos , Ratos Sprague-Dawley , Sinapses/ultraestrutura
11.
Neurophotonics ; 10(4): 044409, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37786400

RESUMO

In the past two decades, digital brain atlases have emerged as essential tools for sharing and integrating complex neuroscience datasets. Concurrently, the larval zebrafish has become a prominent vertebrate model offering a strategic compromise for brain size, complexity, transparency, optogenetic access, and behavior. We provide a brief overview of digital atlases recently developed for the larval zebrafish brain, intersecting neuroanatomical information, gene expression patterns, and connectivity. These atlases are becoming pivotal by centralizing large datasets while supporting the generation of circuit hypotheses as functional measurements can be registered into an atlas' standard coordinate system to interrogate its structural database. As challenges persist in mapping neural circuits and incorporating functional measurements into zebrafish atlases, we emphasize the importance of collaborative efforts and standardized protocols to expand these resources to crack the complex codes of neuronal activity guiding behavior in this tiny vertebrate brain.

12.
Neurophotonics ; 10(4): 044406, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37766924

RESUMO

Despite decades of research on the noradrenergic system, our understanding of its impact on brain function and behavior remains incomplete. Traditional recording techniques are challenging to implement for investigating in vivo noradrenergic activity, due to the relatively small size and the position in the brain of the locus coeruleus (LC), the primary location for noradrenergic neurons. However, recent advances in optical and fluorescent methods have enabled researchers to study the LC more effectively. Use of genetically encoded calcium indicators to image the activity of noradrenergic neurons and biosensors that monitor noradrenaline release with fluorescence can be an indispensable tool for studying noradrenergic activity. In this review, we examine how these methods are being applied to record the noradrenergic system in the rodent brain during behavior.

13.
Neurophotonics ; 9(3): 032211, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35434179

RESUMO

Brain functional connectivity based on the measure of blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) signals has become one of the most widely used measurements in human neuroimaging. However, the nature of the functional networks revealed by BOLD fMRI can be ambiguous, as highlighted by a recent series of experiments that have suggested that typical resting-state networks can be replicated from purely vascular or physiologically driven BOLD signals. After going through a brief review of the key concepts of brain network analysis, we explore how the vascular and neuronal systems interact to give rise to the brain functional networks measured with BOLD fMRI. This leads us to emphasize a view of the vascular network not only as a confounding element in fMRI but also as a functionally relevant system that is entangled with the neuronal network. To study the vascular and neuronal underpinnings of BOLD functional connectivity, we consider a combination of methodological avenues based on multiscale and multimodal optical imaging in mice, used in combination with computational models that allow the integration of vascular information to explain functional connectivity.

14.
Curr Opin Biotechnol ; 73: 308-313, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34653834

RESUMO

The importance of the gut microbiota in host health is now well established, but the underlying mechanisms remain poorly understood. Among the animal models used to investigate microbiota-host interactions, the zebrafish (Danio renio) is gaining attention. Several factors contribute to the recent interest in this model, including its low cost, the ability to assess large cohorts, the possibility to obtain germ-free larvae from non-axenic parents, and the availability of optical methodologies to probe the transparent larvae and adults from various genetic lines. We review recent findings on the zebrafish gut microbiota and its modulation by exogenous microbes, nutrition, and environmental factors. We also highlight the potential of this model for assessing the impact of the gut microbiota on brain development.


Assuntos
Microbioma Gastrointestinal , Animais , Larva , Modelos Animais , Peixe-Zebra
15.
J Neurosci ; 30(26): 8704-9, 2010 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-20592192

RESUMO

Ca(2+)/calmodulin-dependent kinase II (CaMKII) is a key mediator of long-term potentiation (LTP). Whereas acute intracellular injection of catalytically active CaMKII fragments saturates LTP (Lledo et al., 1995), an autonomously active form (T286D) of CaMKII holoenzyme expressed in transgenic mice did not saturate potentiation (Mayford et al., 1995). To better understand the role of the holoenzyme in the control of synaptic strength, we transfected hippocampal neurons with constructs encoding forms of CaMKII mimicking different phosphorylation states. Surprisingly, T286D not only failed to potentiate synaptic strength, but produced synaptic depression through an long-term depression (LTD)-like process. T305/T306 phosphorylation was critical for this depression because overexpression of the pseudophosphorylated form (T286D/T305D/T306D) caused depression that occluded LTD, and overexpression of an autonomous form in which T305/T306 could not be phosphorylated (T286D/T305A/T306A) prevented LTD (instead producing potentiation). Therefore, autonomous CaMKII can lead to either LTP or LTD, depending on the phosphorylation state of the control point, T305/T306.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Neurônios/fisiologia , Sequência de Aminoácidos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/efeitos dos fármacos , Holoenzimas/genética , Holoenzimas/metabolismo , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Mutação , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosforilação , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Fatores de Tempo , Transfecção
16.
Nature ; 424(6951): 938-42, 2003 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-12931188

RESUMO

Modern pain-control theory predicts that a loss of inhibition (disinhibition) in the dorsal horn of the spinal cord is a crucial substrate for chronic pain syndromes. However, the nature of the mechanisms that underlie such disinhibition has remained controversial. Here we present evidence for a novel mechanism of disinhibition following peripheral nerve injury. It involves a trans-synaptic reduction in the expression of the potassium-chloride exporter KCC2, and the consequent disruption of anion homeostasis in neurons of lamina I of the superficial dorsal horn, one of the main spinal nociceptive output pathways. In our experiments, the resulting shift in the transmembrane anion gradient caused normally inhibitory anionic synaptic currents to be excitatory, substantially driving up the net excitability of lamina I neurons. Local blockade or knock-down of the spinal KCC2 exporter in intact rats markedly reduced the nociceptive threshold, confirming that the reported disruption of anion homeostasis in lamina I neurons was sufficient to cause neuropathic pain.


Assuntos
Neurônios/metabolismo , Dor/fisiopatologia , Medula Espinal/citologia , Medula Espinal/fisiopatologia , Simportadores/metabolismo , Sinapses/metabolismo , Animais , Ânions/metabolismo , Doença Crônica , Homeostase , Técnicas In Vitro , Masculino , Modelos Neurológicos , Limiar da Dor , Traumatismos dos Nervos Periféricos , Nervos Periféricos/fisiopatologia , Ratos , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Simportadores/antagonistas & inibidores , Simportadores/genética , Cotransportadores de K e Cl-
17.
Sci Rep ; 10(1): 11960, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32686703

RESUMO

The nanoscale organization of the F-actin cytoskeleton in neurons comprises membrane-associated periodical rings, bundles, and longitudinal fibers. The F-actin rings have been observed predominantly in axons but only sporadically in dendrites, where fluorescence nanoscopy reveals various patterns of F-actin arranged in mixed patches. These complex dendritic F-actin patterns pose a challenge for investigating quantitatively their regulatory mechanisms. We developed here a weakly supervised deep learning segmentation approach of fluorescence nanoscopy images of F-actin in cultured hippocampal neurons. This approach enabled the quantitative assessment of F-actin remodeling, revealing the disappearance of the rings during neuronal activity in dendrites, but not in axons. The dendritic F-actin cytoskeleton of activated neurons remodeled into longitudinal fibers. We show that this activity-dependent remodeling involves [Formula: see text] and NMDA receptor-dependent mechanisms. This highly dynamic restructuring of dendritic F-actin based submembrane lattice into longitudinal fibers may serve to support activity-dependent membrane remodeling, protein trafficking and neuronal plasticity.


Assuntos
Actinas/metabolismo , Axônios/metabolismo , Membrana Celular/metabolismo , Dendritos/metabolismo , Hipocampo/citologia , Citoesqueleto de Actina/metabolismo , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Aprendizado Profundo , Modelos Neurológicos , Nanoestruturas/química , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo
18.
Front Neural Circuits ; 14: 57, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33177994

RESUMO

The organization of proteins in the apposed nanodomains of pre- and postsynaptic compartments is thought to play a pivotal role in synaptic strength and plasticity. As such, the alignment between pre- and postsynaptic proteins may regulate, for example, the rate of presynaptic release or the strength of postsynaptic signaling. However, the analysis of these structures has mainly been restricted to subsets of synapses, providing a limited view of the diversity of synaptic protein cluster remodeling during synaptic plasticity. To characterize changes in the organization of synaptic nanodomains during synaptic plasticity over a large population of synapses, we combined STimulated Emission Depletion (STED) nanoscopy with a Python-based statistical object distance analysis (pySODA), in dissociated cultured hippocampal circuits exposed to treatments driving different forms of synaptic plasticity. The nanoscale organization, characterized in terms of coupling properties, of presynaptic (Bassoon, RIM1/2) and postsynaptic (PSD95, Homer1c) scaffold proteins was differently altered in response to plasticity-inducing stimuli. For the Bassoon - PSD95 pair, treatments driving synaptic potentiation caused an increase in their coupling probability, whereas a stimulus driving synaptic depression had an opposite effect. To enrich the characterization of the synaptic cluster remodeling at the population level, we applied unsupervised machine learning approaches to include selected morphological features into a multidimensional analysis. This combined analysis revealed a large diversity of synaptic protein cluster subtypes exhibiting differential activity-dependent remodeling, yet with common features depending on the expected direction of plasticity. The expanded palette of synaptic features revealed by our unbiased approach should provide a basis to further explore the widely diverse molecular mechanisms of synaptic plasticity.


Assuntos
Espinhas Dendríticas/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Sinapses/metabolismo , Animais , Espinhas Dendríticas/patologia , Hipocampo/citologia , Processamento de Imagem Assistida por Computador , Microscopia , Neurônios/citologia , Terminações Pré-Sinápticas/patologia , Ratos , Sinapses/patologia , Aprendizado de Máquina não Supervisionado
19.
Elife ; 92020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985978

RESUMO

Cell migration is a dynamic process that entails extensive protein synthesis and recycling, structural remodeling, and considerable bioenergetic demand. Autophagy is one of the pathways that maintain cellular homeostasis. Time-lapse imaging of autophagosomes and ATP/ADP levels in migrating cells in the rostral migratory stream of mouse revealed that decreases in ATP levels force cells into the stationary phase and induce autophagy. Pharmacological or genetic impairments of autophagy in neuroblasts using either bafilomycin, inducible conditional mice, or CRISPR/Cas9 gene editing decreased cell migration due to the longer duration of the stationary phase. Autophagy is modulated in response to migration-promoting and inhibiting molecular cues and is required for the recycling of focal adhesions. Our results show that autophagy and energy consumption act in concert in migrating cells to dynamically regulate the pace and periodicity of the migratory and stationary phases to sustain neuronal migration.


Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Autofagia/fisiologia , Movimento Celular/fisiologia , Neurônios/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL
20.
Cell Rep ; 30(7): 2374-2386.e5, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32075770

RESUMO

The neurodevelopmental origin of hyperactivity disorder has been suggested to involve the dopaminergic system, but the underlying mechanisms are still unknown. Here, transcription factors Lmx1a and Lmx1b are shown to be essential for midbrain dopaminergic (mDA) neuron excitatory synaptic inputs and dendritic development. Strikingly, conditional knockout (cKO) of Lmx1a/b in postmitotic mDA neurons results in marked hyperactivity. In seeking Lmx1a/b target genes, we identify positively regulated Slitrk2 and negatively regulated Slitrk5. These two synaptic adhesion proteins promote excitatory and inhibitory synapses on mDA neurons, respectively. Knocking down Slitrk2 reproduces some of the Lmx1a/b cKO cellular and behavioral phenotypes, whereas Slitrk5 knockdown has opposite effects. The hyperactivity caused by this imbalance in excitatory/inhibitory synaptic inputs on dopamine neurons is reproduced by chronically inhibiting the ventral tegmental area during development using pharmacogenetics. Our study shows that alterations in developing dopaminergic circuits strongly impact locomotor activity, shedding light on mechanisms causing hyperactivity behaviors.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Agitação Psicomotora/metabolismo , Sinapses/metabolismo , Animais , Neurônios Dopaminérgicos/patologia , Potenciais Pós-Sinápticos Excitadores , Feminino , Humanos , Potenciais Pós-Sinápticos Inibidores , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Knockout , Gravidez , Cultura Primária de Células , Agitação Psicomotora/patologia , Sinapses/patologia , Fatores de Transcrição/metabolismo , Transfecção
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