Formation of functional synaptic connections between cultured cortical neurons from agrin-deficient mice

SummaryHomeostatic synaptic scaling allows for bi-directional adjustment of the strength of synaptic connections in response to changes in their input. Protein phosphorylation modulates many neuronal and synaptic processes, but it has not been studied on a global, proteome-wide scale during synaptic scaling. To examine this, we used LC-MS/MS analyses to measure changes in the phosphoproteome in response to up- or down-scaling in cultured cortical neurons over minutes to 24 hours. Out of 45,000 phosphorylation events measured, ~3,300 (associated with 1,280 phospho-proteins) were regulated by homeostatic scaling. The activity-sensitive phosphoproteins were predominantly located at synapses and involved in cytoskeletal reorganization. We identified many early transient phosphorylation events which could serve as sensors for the activity offset as well as late and/or persistent phosphoregulation that could represent effector mechanisms driving the homeostatic response. Much of the persistent phosphorylation was reciprocally regulated by up- or down-scaling, suggesting that the mechanisms underlying these two poles of synaptic regulation make use of a common signaling axis.

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Folate andS-adenosylmethionine modulate synaptic activity in cultured cortical neurons: acute differential impact on normal and apolipoprotein-deficient mice

Physical Biology ◽

10.1088/1478-3975/5/4/044002 ◽

2008 ◽

Vol 5 (4) ◽

pp. 044002 ◽

Cited By ~ 6

Author(s):

Michael Serra ◽

Amy Chan ◽

Maya Dubey ◽

Vladimir Gilman ◽

Thomas B Shea

Keyword(s):

Cortical Neurons ◽

Synaptic Activity ◽

Differential Impact ◽

Cultured Cortical Neurons ◽

Deficient Mice

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The Bacterial Enzyme Cas13 Interferes with Neurite Outgrowth from Cultured Cortical Neurons

Toxins ◽

10.3390/toxins13040262 ◽

2021 ◽

Vol 13 (4) ◽

pp. 262

Author(s):

Qin-Wei Wu ◽

Josef P. Kapfhammer

Keyword(s):

Rna Editing ◽

Cortical Neurons ◽

High Efficiency ◽

Primary Cultures ◽

Cultured Neurons ◽

Therapeutic Tool ◽

Bacterial Enzyme ◽

Rna Targeting ◽

Cultured Cortical Neurons ◽

New Generation

The CRISPR-Cas13 system based on a bacterial enzyme has been explored as a powerful new method for RNA manipulation. Due to the high efficiency and specificity of RNA editing/interference achieved by this system, it is currently being developed as a new therapeutic tool for the treatment of neurological and other diseases. However, the safety of this new generation of RNA therapies is still unclear. In this study, we constructed a vector expressing CRISPR-Cas13 under a constitutive neuron-specific promoter. CRISPR-Cas13 from Leptotrichia wadei was expressed in primary cultures of mouse cortical neurons. We found that the presence of CRISPR-Cas13 impedes the development of cultured neurons. These results show a neurotoxic action of Cas13 and call for more studies to test for and possibly mitigate the toxic effects of Cas13 enzymes in order to improve CRISPR-Cas13-based tools for RNA targeting.

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Role of Satb1 and Satb2 Transcription Factors in the Glutamate Receptors Expression and Ca2+ Signaling in the Cortical Neurons In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms22115968 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5968

Author(s):

Egor A. Turovsky ◽

Maria V. Turovskaya ◽

Evgeniya I. Fedotova ◽

Alexey A. Babaev ◽

Viktor S. Tarabykin ◽

...

Keyword(s):

Transcription Factors ◽

Nmda Receptor ◽

Cortical Neurons ◽

Target Genes ◽

Cortical Cell ◽

Inhibitory System ◽

Selective Agonist ◽

Genes Encoding ◽

Deficient Mice

Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.

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The effect of DS16570511, a new inhibitor of mitochondrial calcium uniporter, on calcium homeostasis, metabolism, and functional state of cultured cortical neurons and isolated brain mitochondria

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/j.bbagen.2021.129847 ◽

2021 ◽

Vol 1865 (5) ◽

pp. 129847

Author(s):

Konstantin N. Belosludtsev ◽

Rinat R. Sharipov ◽

Dmitry P. Boyarkin ◽

Natalia V. Belosludtseva ◽

Mikhail V. Dubinin ◽

...

Keyword(s):

Functional State ◽

Calcium Homeostasis ◽

Cortical Neurons ◽

Brain Mitochondria ◽

Mitochondrial Calcium ◽

Mitochondrial Calcium Uniporter ◽

Calcium Uniporter ◽

Cultured Cortical Neurons

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Characterization of the Growth of Cultured Cortical Neurons on Sections of Adult Rat Cortex

Cerebral Cortex ◽

10.1093/cercor/1.2.134 ◽

1991 ◽

Vol 1 (2) ◽

pp. 134-142 ◽

Cited By ~ 5

Author(s):

Eldon E. Geisert

Keyword(s):

Cortical Neurons ◽

Rat Cortex ◽

Adult Rat ◽

Cultured Cortical Neurons

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Abstract 91: Remodeling After Stroke: The Recovering Brain Is Vulnerable To Lipoxygenase-dependent Semaphorin Signaling

Stroke ◽

10.1161/str.44.suppl_1.a91 ◽

2013 ◽

Vol 44 (suppl_1) ◽

Author(s):

Anton Pekcec ◽

Kazim Yigitkanli ◽

Joo Eun Jung ◽

Hulya Karatas ◽

Eng H Lo ◽

...

Keyword(s):

Knockout Mice ◽

Cortical Neurons ◽

Second Messengers ◽

Artery Occlusion ◽

Tube Formation ◽

Stroke Recovery ◽

Experimental Stroke ◽

Axon Extension ◽

Cultured Cortical Neurons ◽

Cerebral Artery Occlusion

Background and Purpose— Recovery from stroke is limited in part by an inhibitory environment in the post-ischemic brain, but factors preventing successful remodeling are not well known. We sought to investigate if signaling from the axon guidance molecule semaphorin 3A (Sema3A) via eicosanoid second messengers can contribute to this inhibitory environment, and if blocking the Sema3A pathway can provide a benefit following experimental stroke. Methods— Cultured cortical neurons from mice were treated with recombinant Sema3A, or with the eicosanoids 12-HETE and 12-HPETE. Neurons from ALOX15 knockout mice, and a human brain endothelial cell line, were treated similarly. The filament model of MCAO was used to induce experimental stroke in mice, in some of which Sema3A was injected stereotactically into the striatum. The 12/15-LOX inhibitor LOXBlock-1 was injected intraperitoneally one week after MCAO. Results— Expression levels of 12/15-lipoxygenase (12/15-LOX) were increased within two hours after exposure of primary neurons to 90nM recombinant Sema3A. Either Sema3A, or the 12/15-lipoxygenase (12/15-LOX) metabolites 12-HETE and 12-HPETE at 300nM, blocked axon extension in neurons compared to solvent controls, and decreased tube formation in endothelial cells. The Sema3A effect was reversed by inhibiting 12/15-LOX, and neurons derived from 12/15-LOX knockout mice were insensitive to Sema3A. Following middle cerebral artery occlusion to induce stroke in mice, immunohistochemistry showed both Sema3A and 12/15-LOX are increased in the cortex up to two weeks. To determine if a Sema3A-dependent damage pathway is activated following ischemia, we injected recombinant Sema3A into the striatum. Sema3A alone did not cause injury in normal brains. But when injected into post-ischemic brains, Sema3A increased cortical damage by 79%, and again this effect was reversed by 12/15-LOX inhibition. Administration of the 12/15-LOX inhibitor LOXBlock-1 7 days after transient MCAO increased vascularization in the infarcted and peri-infarct area one week later. Conclusions— Our findings suggest that blocking the semaphorin pathway may provide a novel therapeutic strategy to improve stroke recovery.

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