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1.
PLoS Biol ; 18(9): e3000873, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32966273

RESUMO

The inhibitory axonless olfactory bulb granule cells form reciprocal dendrodendritic synapses with mitral and tufted cells via large spines, mediating recurrent and lateral inhibition. As a case in point for dendritic transmitter release, rat granule cell dendrites are highly excitable, featuring local Na+ spine spikes and global Ca2+- and Na+-spikes. To investigate the transition from local to global signaling, we performed holographic, simultaneous 2-photon uncaging of glutamate at up to 12 granule cell spines, along with whole-cell recording and dendritic 2-photon Ca2+ imaging in acute juvenile rat brain slices. Coactivation of less than 10 reciprocal spines was sufficient to generate diverse regenerative signals that included regional dendritic Ca2+-spikes and dendritic Na+-spikes (D-spikes). Global Na+-spikes could be triggered in one third of granule cells. Individual spines and dendritic segments sensed the respective signal transitions as increments in Ca2+ entry. Dendritic integration as monitored by the somatic membrane potential was mostly linear until a threshold number of spines was activated, at which often D-spikes along with supralinear summation set in. As to the mechanisms supporting active integration, NMDA receptors (NMDARs) strongly contributed to all aspects of supralinearity, followed by dendritic voltage-gated Na+- and Ca2+-channels, whereas local Na+ spine spikes, as well as morphological variables, barely mattered. Because of the low numbers of coactive spines required to trigger dendritic Ca2+ signals and thus possibly lateral release of GABA onto mitral and tufted cells, we predict that thresholds for granule cell-mediated bulbar lateral inhibition are low. Moreover, D-spikes could provide a plausible substrate for granule cell-mediated gamma oscillations.


Assuntos
Potenciais de Ação , Sinalização do Cálcio , Dendritos/metabolismo , Bulbo Olfatório/metabolismo , Sódio/metabolismo , Animais , Cálcio/metabolismo , Feminino , Holografia , Técnicas In Vitro , Masculino , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(37): 23073-23084, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32873638

RESUMO

The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Culina/metabolismo , Hipocampo/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Morfogênese/fisiologia , Animais , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Dendritos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Neurogênese/fisiologia , Células Piramidais/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo
3.
J Vis Exp ; (159)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32478717

RESUMO

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology.  To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.


Assuntos
Dendritos/metabolismo , Drosophila melanogaster/metabolismo , Imageamento Tridimensional , Microtúbulos/metabolismo , Junção Neuromuscular/metabolismo , Imagem Individual de Molécula , Sinapses/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Larva/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA , Software
4.
PLoS Genet ; 16(5): e1008767, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32357156

RESUMO

Despite the importance of dendritic targeting in neural circuit assembly, the mechanisms by which it is controlled still remain incompletely understood. We previously showed that in the developing Drosophila antennal lobe, the Wnt5 protein forms a gradient that directs the ~45˚ rotation of a cluster of projection neuron (PN) dendrites, including the adjacent DA1 and VA1d dendrites. We report here that the Van Gogh (Vang) transmembrane planar cell polarity (PCP) protein is required for the rotation of the DA1/VA1d dendritic pair. Cell type-specific rescue and mosaic analyses showed that Vang functions in the olfactory receptor neurons (ORNs), suggesting a codependence of ORN axonal and PN dendritic targeting. Loss of Vang suppressed the repulsion of the VA1d dendrites by Wnt5, indicating that Wnt5 signals through Vang to direct the rotation of the DA1 and VA1d glomeruli. We observed that the Derailed (Drl)/Ryk atypical receptor tyrosine kinase is also required for the rotation of the DA1/VA1d dendritic pair. Antibody staining showed that Drl/Ryk is much more highly expressed by the DA1 dendrites than the adjacent VA1d dendrites. Mosaic and epistatic analyses showed that Drl/Ryk specifically functions in the DA1 dendrites in which it antagonizes the Wnt5-Vang repulsion and mediates the migration of the DA1 glomerulus towards Wnt5. Thus, the nascent DA1 and VA1d glomeruli appear to exhibit Drl/Ryk-dependent biphasic responses to Wnt5. Our work shows that the final patterning of the fly olfactory map is the result of an interplay between ORN axons and PN dendrites, wherein converging pre- and postsynaptic processes contribute key Wnt5 signaling components, allowing Wnt5 to orient the rotation of nascent synapses through a PCP mechanism.


Assuntos
Antenas de Artrópodes/crescimento & desenvolvimento , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Wnt/metabolismo , Animais , Antenas de Artrópodes/metabolismo , Axônios/metabolismo , Padronização Corporal , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Neurônios Receptores Olfatórios/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , Proteínas Wnt/genética
5.
Proc Natl Acad Sci U S A ; 117(21): 11760-11769, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32393629

RESUMO

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative diseases that present with similar TDP-43 pathology in patient tissue. TDP-43 is an RNA-binding protein which forms aggregates in neurons of ALS and FTD patients as well as in a subset of patients diagnosed with other neurodegenerative diseases. Despite our understanding that TDP-43 is essential for many aspects of RNA metabolism, it remains obscure how TDP-43 dysfunction contributes to neurodegeneration. Interestingly, altered neuronal dendritic morphology is a common theme among several neurological disorders and is thought to precede neurodegeneration. We previously found that both TDP-43 overexpression (OE) and knockdown (KD) result in reduced dendritic branching of cortical neurons. In this study, we used TRIBE (targets of RNA-binding proteins identified by editing) as an approach to identify signaling pathways that regulate dendritic branching downstream of TDP-43. We found that TDP-43 RNA targets are enriched for pathways that signal to the CREB transcription factor. We further found that TDP-43 dysfunction inhibits CREB activation and CREB transcriptional output, and restoring CREB signaling rescues defects in dendritic branching. Finally, we demonstrate, using RNA sequencing, that TDP-43 OE and KD cause similar changes in the abundance of specific messenger RNAs, consistent with their ability to produce similar morphological defects. Our data therefore provide a mechanism by which TDP-43 dysfunction interferes with dendritic branching, and may define pathways for therapeutic intervention in neurodegenerative diseases.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Proteínas de Ligação a DNA , Dendritos , Regulação da Expressão Gênica/genética , Transdução de Sinais , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dendritos/metabolismo , Dendritos/patologia , Células HEK293 , Humanos , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteinopatias TDP-43
6.
Nat Commun ; 11(1): 2248, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382046

RESUMO

During high-frequency network activities, fast-spiking, parvalbumin-expressing basket cells (PV+-BCs) generate barrages of fast synaptic inhibition to control the probability and precise timing of action potential (AP) initiation in principal neurons. Here we describe a subcellular specialization that contributes to the high speed of synaptic inhibition mediated by PV+-BCs. Mapping of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel distribution in rat hippocampal PV+-BCs with subcellular patch-clamp methods revealed that functional HCN channels are exclusively expressed in axons and completely absent from somata and dendrites. HCN channels not only enhance AP initiation during sustained high-frequency firing but also speed up the propagation of AP trains in PV+-BC axons by dynamically opposing the hyperpolarization produced by Na+-K+ ATPases. Since axonal AP signaling determines the timing of synaptic communication, the axon-specific expression of HCN channels represents a specialization for PV+-BCs to operate at high speed.


Assuntos
Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Potenciais de Ação/fisiologia , Animais , Axônios , Dendritos/metabolismo , Masculino , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo
7.
PLoS One ; 15(4): e0230787, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32275724

RESUMO

The mechanisms of epileptic discharge generation and spread are not yet fully known. A recently proposed simple biophysical model of interictal and ictal discharges, Epileptor-2, reproduces well the main features of neuronal excitation and ionic dynamics during discharge generation. In order to distinguish between two hypothesized mechanisms of discharge propagation, we extend the model to the case of two-dimensional propagation along the cortical neural tissue. The first mechanism is based on extracellular potassium diffusion, and the second is the propagation of spikes and postsynaptic signals along axons and dendrites. Our simulations show that potassium diffusion is too slow to reproduce an experimentally observed speed of ictal wavefront propagation (tenths of mm/s). By contrast, the synaptic mechanism predicts well the speed and synchronization of the pre-ictal bursts before the ictal front and the afterdischarges in the ictal core. Though this fact diminishes the role of diffusion and electrodiffusion, the model nevertheless highlights the role of potassium extrusion during neuronal excitation, which provides a positive feedback that changes at the ictal wavefront the balance of excitation versus inhibition in favor of excitation. This finding may help to find a target for a treatment to prevent seizure propagation.


Assuntos
Dendritos/metabolismo , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Potássio/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Potenciais de Ação/fisiologia , Axônios/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Pareamento Cromossômico/fisiologia , Modelos Neurológicos , Neurônios/metabolismo , Potenciais Sinápticos/fisiologia
8.
Am J Hum Genet ; 106(5): 623-631, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32275884

RESUMO

Nucleoporins (NUPs) are an essential component of the nuclear-pore complex, which regulates nucleocytoplasmic transport of macromolecules. Pathogenic variants in NUP genes have been linked to several inherited human diseases, including a number with progressive neurological degeneration. We present six affected individuals with bi-allelic truncating variants in NUP188 and strikingly similar phenotypes and clinical courses, representing a recognizable genetic syndrome; the individuals are from four unrelated families. Key clinical features include congenital cataracts, hypotonia, prenatal-onset ventriculomegaly, white-matter abnormalities, hypoplastic corpus callosum, congenital heart defects, and central hypoventilation. Characteristic dysmorphic features include small palpebral fissures, a wide nasal bridge and nose, micrognathia, and digital anomalies. All affected individuals died as a result of respiratory failure, and five of them died within the first year of life. Nuclear import of proteins was decreased in affected individuals' fibroblasts, supporting a possible disease mechanism. CRISPR-mediated knockout of NUP188 in Drosophila revealed motor deficits and seizure susceptibility, partially recapitulating the neurological phenotype seen in affected individuals. Removal of NUP188 also resulted in aberrant dendrite tiling, suggesting a potential role of NUP188 in dendritic development. Two of the NUP188 pathogenic variants are enriched in the Ashkenazi Jewish population in gnomAD, a finding we confirmed with a separate targeted population screen of an international sampling of 3,225 healthy Ashkenazi Jewish individuals. Taken together, our results implicate bi-allelic loss-of-function NUP188 variants in a recessive syndrome characterized by a distinct neurologic, ophthalmologic, and facial phenotype.


Assuntos
Alelos , Encéfalo/anormalidades , Proteínas de Drosophila/genética , Anormalidades do Olho/genética , Cardiopatias Congênitas/genética , Mutação com Perda de Função/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Pré-Escolar , Dendritos/metabolismo , Dendritos/patologia , Drosophila melanogaster , Anormalidades do Olho/mortalidade , Feminino , Fibroblastos , Genes Recessivos , Cardiopatias Congênitas/mortalidade , Humanos , Lactente , Recém-Nascido , Judeus/genética , Masculino , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Convulsões/metabolismo , Síndrome , beta Carioferinas/metabolismo
9.
PLoS Biol ; 18(3): e3000647, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163403

RESUMO

Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, we found that canonical Wnt signaling proteins regulate localization of the core nucleation protein γTubulin (γTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Iγ, G proteins, and Axin reduced γTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic γTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with fz, dsh, Axin, and Rab5. We propose that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points.


Assuntos
Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Microtúbulos/metabolismo , Proteínas Wnt/metabolismo , Animais , Complexo de Sinalização da Axina/genética , Complexo de Sinalização da Axina/metabolismo , Axônios/metabolismo , Polaridade Celular , Dendritos/genética , Drosophila , Proteínas de Drosophila/genética , Endossomos/genética , Microtúbulos/genética , Mutação , Receptores Wnt/genética , Receptores Wnt/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
10.
J Vis Exp ; (156)2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32176208

RESUMO

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.


Assuntos
Drosophila/citologia , Drosophila/crescimento & desenvolvimento , Imobilização/métodos , Imagem com Lapso de Tempo/métodos , Animais , Dendritos/metabolismo , Dendritos/patologia , Dimetilpolisiloxanos , Larva/citologia , Larva/crescimento & desenvolvimento , Neurônios/citologia
11.
Nat Neurosci ; 23(4): 533-543, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203497

RESUMO

Learning disabilities are hallmarks of congenital conditions caused by prenatal exposure to harmful agents. These include fetal alcohol spectrum disorders (FASDs) with a wide range of cognitive deficiencies, including impaired motor skill development. Although these effects have been well characterized, the molecular effects that bring about these behavioral consequences remain to be determined. We previously found that the acute molecular responses to alcohol in the embryonic brain are stochastic, varying among neural progenitor cells. However, the pathophysiological consequences stemming from these heterogeneous responses remain unknown. Here we show that acute responses to alcohol in progenitor cells altered gene expression in their descendant neurons. Among the altered genes, an increase of the calcium-activated potassium channel Kcnn2 in the motor cortex correlated with motor learning deficits in a mouse model of FASD. Pharmacologic blockade of Kcnn2 improves these learning deficits, suggesting Kcnn2 blockers as a new intervention for learning disabilities in FASD.


Assuntos
Comportamento Animal/efeitos dos fármacos , Transtornos do Espectro Alcoólico Fetal/tratamento farmacológico , Deficiências da Aprendizagem/tratamento farmacológico , Aprendizagem/efeitos dos fármacos , Córtex Motor/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Animais , Forma Celular/efeitos dos fármacos , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Modelos Animais de Doenças , Deficiências da Aprendizagem/metabolismo , Camundongos , Atividade Motora/efeitos dos fármacos , Córtex Motor/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Venenos de Escorpião/uso terapêutico , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
12.
Neuron ; 106(3): 452-467.e8, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32155441

RESUMO

Dendrite arbor pattern determines the functional characteristics of a neuron. It is founded on primary branch structure, defined through cell intrinsic and transcription-factor-encoded mechanisms. Developing arbors have extensive acentrosomal microtubule dynamics, and here, we report an unexpected role for the atypical actin motor Myo6 in creating primary branch structure by specifying the position, polarity, and targeting of these events. We carried out in vivo time-lapse imaging of Drosophila adult sensory neuron differentiation, integrating machine-learning-based quantification of arbor patterning with molecular-level tracking of cytoskeletal remodeling. This revealed that Myo6 and the transcription factor Knot regulate transient surges of microtubule polymerization at dendrite tips; they drive retrograde extension of an actin filament array that specifies anterograde microtubule polymerization and guides these microtubules to subdivide the tip into multiple branches. Primary branches delineate functional compartments; this tunable branching mechanism is key to define and diversify dendrite arbor compartmentalization.


Assuntos
Dendritos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Neurogênese , Animais , Linhagem Celular , Células Cultivadas , Dendritos/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Microtúbulos/metabolismo , Cadeias Pesadas de Miosina/genética , Fatores de Transcrição/metabolismo
13.
J Neurosci ; 40(16): 3231-3249, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32144180

RESUMO

Endogenous neuropeptide Y (NPY) and corticotrophin-releasing factor (CRF) modulate the responses of the basolateral amygdala (BLA) to stress and are associated with the development of stress resilience and vulnerability, respectively. We characterized persistent effects of repeated NPY and CRF treatment on the structure and function of BLA principal neurons in a novel organotypic slice culture (OTC) model of male rat BLA, and examined the contributions of specific NPY receptor subtypes to these neural and behavioral effects. In BLA principal neurons within the OTCs, repeated NPY treatment caused persistent attenuation of excitatory input and induced dendritic hypotrophy via Y5 receptor activation; conversely, CRF increased excitatory input and induced hypertrophy of BLA principal neurons. Repeated treatment of OTCs with NPY followed by an identical treatment with CRF, or vice versa, inhibited or reversed all structural changes in OTCs. These structural responses to NPY or CRF required calcineurin or CaMKII, respectively. Finally, repeated intra-BLA injections of NPY or a Y5 receptor agonist increased social interaction, a validated behavior for anxiety, and recapitulated structural changes in BLA neurons seen in OTCs, while a Y5 receptor antagonist prevented NPY's effects both on behavior and on structure. These results implicate the Y5 receptor in the long-term, anxiolytic-like effects of NPY in the BLA, consistent with an intrinsic role in stress buffering, and highlight a remarkable mechanism by which BLA neurons may adapt to different levels of stress. Moreover, BLA OTCs offer a robust model to study mechanisms associated with resilience and vulnerability to stress in BLA.SIGNIFICANCE STATEMENT Within the basolateral amygdala (BLA), neuropeptide Y (NPY) is associated with buffering the neural stress response induced by corticotropin releasing factor, and promoting stress resilience. We used a novel organotypic slice culture model of BLA, complemented with in vivo studies, to examine the cellular mechanisms associated with the actions of NPY. In organotypic slice cultures, repeated NPY treatment reduces the complexity of the dendritic extent of anxiogenic BLA principal neurons, making them less excitable. NPY, via activation of Y5 receptors, additionally inhibits and reverses the increases in dendritic extent and excitability induced by the stress hormone, corticotropin releasing factor. This NPY-mediated neuroplasticity indicates that resilience or vulnerability to stress may thus involve neuropeptide-mediated dendritic remodeling in BLA principal neurons.


Assuntos
Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Hormônio Liberador da Corticotropina/farmacologia , Dendritos/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Neuropeptídeo Y/farmacologia , Receptores de Neuropeptídeo Y/agonistas , Comportamento Social , Animais , Complexo Nuclear Basolateral da Amígdala/metabolismo , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Calcineurina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Dendritos/metabolismo , Masculino , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Neuropeptídeo Y/metabolismo , Resiliência Psicológica
14.
Gene ; 738: 144473, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32057929

RESUMO

Cytoplasmic polyadenylation element binding proteins (CPEBs) are widely conserved proteins that regulate the length of poly(A) tails in the cytoplasm, regulate translation, and regulate mRNA transport. While CPEBs are best known for regulating maternal messages in oocytes, CPEBs also have roles in many other cell types including neurons. Here we extend our knowledge of the roles of CPEBs in neurons by showing that the Drosophila CPEB-encoding gene, orb, is required for proper dendrite development in larval sensory dendritic arborization neurons. Furthermore, we provide evidence that orb is important for neuron cell fate specification.


Assuntos
Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem da Célula/fisiologia , Citoplasma/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Larva/genética , Neurogênese/fisiologia , Proteínas de Ligação a Poli(A)/metabolismo , Poliadenilação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Fatores de Transcrição/genética
15.
PLoS Pathog ; 16(2): e1008319, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32106265

RESUMO

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in children worldwide. Sirtuin 1 (SIRT1), a NAD+ dependent deacetylase, has been associated with induction of autophagy, reprogramming cellular metabolism, and regulating immune mediators. In this study, we investigated the role of SIRT1 in bone marrow dendritic cell (BMDC) function during RSV infection. SIRT1 deficient (SIRT1 -/-) BMDC showed a defect in mitochondrial membrane potential (Δ⍦m) that worsens during RSV infection. This defect in Δ⍦m caused the generation of elevated levels of reactive oxygen species (ROS). Furthermore, the oxygen consumption rate (OCR) was reduced as assessed in Seahorse assays, coupled with lower levels of ATP in SIRT1-/- DC. These altered responses corresponded to altered innate cytokine responses in the SIRT1-/- DC in response to RSV infection. Reverse Phase Protein Array (RPPA) functional proteomics analyses of SIRT1-/- and WT BMDC during RSV infection identified a range of differentially regulated proteins involved in pathways that play a critical role in mitochondrial metabolism, autophagy, oxidative and ER stress, and DNA damage. We identified an essential enzyme, acetyl CoA carboxylase (ACC1), which plays a central role in fatty acid synthesis and had significantly increased expression in SIRT1-/- DC. Blockade of ACC1 resulted in metabolic reprogramming of BMDC that ameliorated mitochondrial dysfunction and reduced pathologic innate immune cytokines in DC. The altered DC responses attenuated Th2 and Th17 immunity allowing the appropriate generation of anti-viral Th1 responses both in vitro and in vivo during RSV infection thus reducing the enhanced pathogenic responses. Together, these studies identify pathways critical for appropriate DC function and innate immunity that depend on SIRT1-mediated regulation of metabolic processes.


Assuntos
Dendritos/metabolismo , Infecções por Vírus Respiratório Sincicial/imunologia , Sirtuína 1/metabolismo , Animais , Autofagia/imunologia , Citocinas/metabolismo , Dendritos/virologia , Células Dendríticas/imunologia , Feminino , Homeostase/imunologia , Imunidade Inata/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sinciciais Respiratórios/patogenicidade , Sirtuína 1/fisiologia , Células Th17/imunologia , Células Th2/imunologia
16.
J Vis Exp ; (155)2020 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-31984960

RESUMO

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.


Assuntos
Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Corantes Fluorescentes/metabolismo , Lipídeos/química , Neurônios Motores/citologia , Animais , Dendritos/metabolismo , Dissecação , Feminino , Injeções , Masculino , Neurogênese
17.
ACS Appl Mater Interfaces ; 12(3): 4163-4173, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31891476

RESUMO

Engineering of cell surfaces holds promise in manipulating cellular activities in a physicochemical route as a complement to the biological approach. Mediated by Ca2+, a quick and convenient yet cytocompatible method is used to achieve surface engineering, by which polydopamine nanostructures can be in situ grown onto dendritic cell (DC) surfaces within 10 min. Ca2+, as the physical bridge between the negative cell surface and polydopamine, avoids the direct chemical polymerization of polydopamine onto the cell surface, critically important to maintain the cell viability. As a proof of concept in potential applications, this cell surface engineering shows a good control toward DC maturation. Upon surface polydopamine engineering, bone-marrow-derived DC exhibits a unique bidirectional control of maturation. The polydopamine structure enables effective suppression of DC activation by acting as an efficient scavenger of reactive oxygen species, a key signal during maturation. Conversely, an 808 nm laser irradiation can remotely relieve the suppressed state and effectively activate DC maturation by the photoheat effect of polydopamine (39 °C). The work provides an easily implemented, straightforward approach to achieve cell surface engineering, through which the DC maturation can be controlled.


Assuntos
Cálcio/metabolismo , Engenharia Celular/métodos , Células Dendríticas/citologia , Indóis/química , Polímeros/química , Animais , Diferenciação Celular , Engenharia Celular/instrumentação , Sobrevivência Celular , Dendritos/metabolismo , Células Dendríticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Polimerização , Espécies Reativas de Oxigênio/metabolismo
18.
Cell ; 180(2): 373-386.e15, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31955847

RESUMO

Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.


Assuntos
Condutos Olfatórios/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Proteômica/métodos , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/metabolismo , Neurogênese/fisiologia , Nervo Olfatório/metabolismo , Condutos Olfatórios/citologia , Condutos Olfatórios/fisiologia , Receptores de Lipoproteínas/metabolismo , Olfato/fisiologia
19.
Nucleic Acids Res ; 48(6): e32, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-31974573

RESUMO

In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. Here, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and transport of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA mobility in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of long-term potentiation (LTP) in culture revealed up-regulation of mRNA translation with a similar effect in dendrites and somata, which appeared to be GluR-dependent 6 h post-activation. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Neurônios/metabolismo , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Animais , Compartimento Celular , Células Cultivadas , Dendritos/metabolismo , Corantes Fluorescentes/metabolismo , Potenciação de Longa Duração , Masculino , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
20.
Neuron ; 105(1): 106-121.e10, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31757604

RESUMO

The extent to which behavior is shaped by experience varies between individuals. Genetic differences contribute to this variation, but the neural mechanisms are not understood. Here, we dissect natural variation in the behavioral flexibility of two Caenorhabditis elegans wild strains. In one strain, a memory of exposure to 21% O2 suppresses CO2-evoked locomotory arousal; in the other, CO2 evokes arousal regardless of previous O2 experience. We map that variation to a polymorphic dendritic scaffold protein, ARCP-1, expressed in sensory neurons. ARCP-1 binds the Ca2+-dependent phosphodiesterase PDE-1 and co-localizes PDE-1 with molecular sensors for CO2 at dendritic ends. Reducing ARCP-1 or PDE-1 activity promotes CO2 escape by altering neuropeptide expression in the BAG CO2 sensors. Variation in ARCP-1 alters behavioral plasticity in multiple paradigms. Our findings are reminiscent of genetic accommodation, an evolutionary process by which phenotypic flexibility in response to environmental variation is reset by genetic change.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Dendritos/metabolismo , Plasticidade Neuronal/fisiologia , Neuropeptídeos/biossíntese , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Nível de Alerta/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Dióxido de Carbono/farmacologia , Feminino , Individualidade , Diester Fosfórico Hidrolases/metabolismo , Polimorfismo Genético , Células Receptoras Sensoriais/metabolismo , Especificidade da Espécie
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