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1.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33526652

RESUMO

Identifying molecular mediators of neural circuit development and/or function that contribute to circuit dysfunction when aberrantly reengaged in neurological disorders is of high importance. The role of the TWEAK/Fn14 pathway, which was recently reported to be a microglial/neuronal axis mediating synaptic refinement in experience-dependent visual development, has not been explored in synaptic function within the mature central nervous system. By combining electrophysiological and phosphoproteomic approaches, we show that TWEAK acutely dampens basal synaptic transmission and plasticity through neuronal Fn14 and impacts the phosphorylation state of pre- and postsynaptic proteins in adult mouse hippocampal slices. Importantly, this is relevant in two models featuring synaptic deficits. Blocking TWEAK/Fn14 signaling augments synaptic function in hippocampal slices from amyloid-beta-overexpressing mice. After stroke, genetic or pharmacological inhibition of TWEAK/Fn14 signaling augments basal synaptic transmission and normalizes plasticity. Our data support a glial/neuronal axis that critically modifies synaptic physiology and pathophysiology in different contexts in the mature brain and may be a therapeutic target for improving neurophysiological outcomes.


Assuntos
Degeneração Neural/metabolismo , Transdução de Sinais , Acidente Vascular Cerebral/metabolismo , Sinapses/metabolismo , Receptor de TWEAK/metabolismo , Animais , Citocina TWEAK/metabolismo , Modelos Animais de Doenças , Feminino , Hipocampo/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Degeneração Neural/fisiopatologia , Plasticidade Neuronal/fisiologia , Terminações Pré-Sinápticas/metabolismo , Acidente Vascular Cerebral/fisiopatologia , Transmissão Sináptica/fisiologia
2.
Neuron ; 108(3): 451-468.e9, 2020 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-32931754

RESUMO

Sensory experience remodels neural circuits in the early postnatal brain through mechanisms that remain to be elucidated. Applying a new method of ultrastructural analysis to the retinogeniculate circuit, we find that visual experience alters the number and structure of synapses between the retina and the thalamus. These changes require vision-dependent transcription of the receptor Fn14 in thalamic relay neurons and the induction of its ligand TWEAK in microglia. Fn14 functions to increase the number of bulbous spine-associated synapses at retinogeniculate connections, likely contributing to the strengthening of the circuit that occurs in response to visual experience. However, at retinogeniculate connections near TWEAK-expressing microglia, TWEAK signals via Fn14 to restrict the number of bulbous spines on relay neurons, leading to the elimination of a subset of connections. Thus, TWEAK and Fn14 represent an intercellular signaling axis through which microglia shape retinogeniculate connectivity in response to sensory experience.


Assuntos
Microglia/fisiologia , Microglia/ultraestrutura , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Sinapses/ultraestrutura , Animais , Citocina TWEAK/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão/métodos , Neurônios/metabolismo , Neurônios/ultraestrutura , Estimulação Luminosa , Receptor de TWEAK/metabolismo , Vias Visuais/fisiologia , Vias Visuais/ultraestrutura
3.
Front Cell Neurosci ; 14: 592005, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33473245

RESUMO

Microglia are central nervous system (CNS) resident immune cells that have been implicated in neuroinflammatory pathogenesis of a variety of neurological conditions. Their manifold context-dependent contributions to neuroinflammation are only beginning to be elucidated, which can be attributed in part to the challenges of studying microglia in vivo and the lack of tractable in vitro systems to study microglia function. Organotypic brain slice cultures offer a tissue-relevant context that enables the study of CNS resident cells and the analysis of brain slice microglial phenotypes has provided important insights, in particular into neuroprotective functions. Here we use RNA sequencing, direct digital quantification of gene expression with nCounter® technology and targeted analysis of individual microglial signature genes, to characterize brain slice microglia relative to acutely-isolated counterparts and 2-dimensional (2D) primary microglia cultures, a widely used in vitro surrogate. Analysis using single cell and population-based methods found brain slice microglia exhibited better preservation of canonical microglia markers and overall gene expression with stronger fidelity to acutely-isolated adult microglia, relative to in vitro cells. We characterized the dynamic phenotypic changes of brain slice microglia over time, after plating in culture. Mechanical damage associated with slice preparation prompted an initial period of inflammation, which resolved over time. Based on flow cytometry and gene expression profiling we identified the 2-week timepoint as optimal for investigation of microglia responses to exogenously-applied stimuli as exemplified by treatment-induced neuroinflammatory changes observed in microglia following LPS, TNF and GM-CSF addition to the culture medium. Altogether these findings indicate that brain slice cultures provide an experimental system superior to in vitro culture of microglia as a surrogate to investigate microglia functions, and the impact of soluble factors and cellular context on their physiology.

4.
Sci Signal ; 11(541)2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30065029

RESUMO

Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB-inducing kinase (NIK)-dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin ß receptor (LTßR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)-dependent nuclear translocation of p52, but not of the TNF-α receptor-mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTßR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.


Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/metabolismo , Regulação Neoplásica da Expressão Gênica , NF-kappa B/metabolismo , Osteossarcoma/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/metabolismo , Perfilação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Indóis/farmacologia , Receptor beta de Linfotoxina/antagonistas & inibidores , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Propionatos/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma , Transdução de Sinais , Receptor de TWEAK/antagonistas & inibidores , Receptor de TWEAK/genética , Receptor de TWEAK/metabolismo , Células Tumorais Cultivadas , Quinase Induzida por NF-kappaB
5.
Neuron ; 91(6): 1330-1341, 2016 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-27593180

RESUMO

Inhibition of granule cells plays a key role in gating the flow of signals into the cerebellum, and it is thought that Golgi cells are the only interneurons that inhibit granule cells. Here we show that Purkinje cells, the sole output neurons of the cerebellar cortex, also directly inhibit granule cells via their axon collaterals. Anatomical and optogenetic studies indicate that this non-canonical feedback is region specific: it is most prominent in lobules that regulate eye movement and process vestibular information. Collaterals provide fast, slow, and tonic inhibition to granule cells, and thus allow Purkinje cells to regulate granule cell excitability on multiple timescales. We propose that this feedback mechanism could regulate excitability of the input layer, contribute to sparse coding, and mediate temporal integration.


Assuntos
Córtex Cerebelar/citologia , Inibição Neural , Neurônios/fisiologia , Células de Purkinje/fisiologia , Animais , Córtex Cerebelar/metabolismo , Camundongos , Sinapses/fisiologia , Ácido gama-Aminobutírico/metabolismo
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