scholarly journals 14-3-3 proteins promote synaptic localization of N-methyl d-aspartate receptors (NMDARs) in mouse hippocampal and cortical neurons

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0261791
Author(s):  
Gloria S. Lee ◽  
Jiajing Zhang ◽  
Yuying Wu ◽  
Yi Zhou
Keyword(s):  
Cortical Neurons ◽  
Hippocampal Neurons ◽  
Critical Role ◽  
Surface Expression ◽  
Primary Neurons ◽  
Protein Inhibitor ◽  
Synaptic Localization ◽  
Convergence Point ◽  
Enhanced Surface ◽  
Nmdar Subunits

One of the core pathogenic mechanisms for schizophrenia is believed to be dysfunction in glutamatergic synaptic transmissions, particularly hypofunction of N-methyl d-aspartate receptors (NMDARs). Previously we showed that 14-3-3 functional knockout mice exhibit schizophrenia-associated behaviors accompanied by reduced synaptic NMDARs in forebrain excitatory neurons. To investigate how 14-3-3 proteins regulate synaptic localization of NMDARs, here we examined changes in levels of synaptic NMDARs upon 14-3-3 inhibition in primary neurons. Expression of 14-3-3 protein inhibitor (difopein) in primary glutamatergic cortical and hippocampal neurons resulted in lower number of synaptic puncta containing NMDARs, including the GluN1, GluN2A, or GluN2B subunits. In heterologous cells, 14-3-3 proteins enhanced surface expression of these NMDAR subunits. Furthermore, we identified that 14-3-3ζ and ε isoforms interact with NMDARs via binding to GluN2A and GluN2B subunits. Taken together, our results demonstrate that 14-3-3 proteins play a critical role in NMDAR synaptic trafficking by promoting surface delivery of NMDAR subunits GluN1, GluN2A, and GluN2B. As NMDAR hypofunctionality is known to act as a convergence point for progression of symptoms of schizophrenia, further studies on these signaling pathways may help understand how dysfunction of 14-3-3 proteins can cause NMDAR hypofunctionality and lead to schizophrenia-associated behaviors.

scholarly journals Development of a High-Throughput AlphaScreen Assay for Modulators of Synapsin I Phosphorylation in Primary Neurons

2013 ◽  
Vol 19 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Betty Chan ◽  
Jeffrey R. Cottrell ◽  
Bing Li ◽  
Kelley C. Larson ◽  
Crystle J. Ashford ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
High Throughput ◽  
Cortical Neurons ◽  
High Throughput Screening ◽  
Critical Role ◽  
Neuronal Cultures ◽  
Synapsin I ◽  
Primary Neurons ◽  
Primary Neuronal Cultures ◽  
Small Molecule Modulators

Alterations in synaptic transmission have been implicated in a number of psychiatric and neurological disorders. The discovery of small-molecule modulators of proteins that regulate neurotransmission represents a novel therapeutic strategy for these diseases. However, high-throughput screening (HTS) approaches in primary neurons have been limited by challenges in preparing and applying primary neuronal cultures under conditions required for generating sufficiently robust and sensitive HTS assays. Synapsin I is an abundant presynaptic protein that plays a critical role in neurotransmission through tethering synaptic vesicles to the actin cytoskeleton. It has several phosphorylation sites that regulate its modulation of synaptic vesicle trafficking and, therefore, the efficacy of synaptic transmission. Here, we describe the development of a rapid, sensitive, and homogeneous assay to detect phospho-synapsin I (pSYN1) in primary cortical neurons in 384-well plates using AlphaScreen technology. From results of a pilot screening campaign, we show that the assay can identify compounds that modulate synapsin I phosphorylation via multiple signaling pathways. The implementation of the AlphaScreen pSYN1 assay and future development of additional primary neuronal HTS assays provides an attractive approach for discovery of novel classes of therapeutic candidates for a variety of CNS disorders.

Combined β-adrenergic and corticosteroid receptor activation regulates AMPA receptor function in hippocampal neurons

2011 ◽  
Vol 26 (4) ◽  
pp. 516-524 ◽  
Author(s):  
Ming Zhou ◽  
Casper C Hoogenraad ◽  
Marian Joëls ◽  
Harm J Krugers
Keyword(s):  
Adrenergic Receptors ◽  
Hippocampal Neurons ◽  
Hippocampal Slices ◽  
Primary Cultures ◽  
Surface Expression ◽  
Receptor Activation ◽  
Behavioural Adaptation ◽  
Corticosteroid Receptor ◽  
Β Adrenergic Receptors ◽  
Enhanced Surface

Shortly after stress, limbic neurons are exposed to high levels of noradrenaline and corticosterone. These hormones are necessary for optimal behavioural adaptation. Behavioural effects critically depend on noradrenaline acting via β-adrenergic receptors, but these effects are strongly modulated by corticosterone, indicating putative interactions between the two hormones. Since both noradrenaline and corticosterone are known to quickly affect properties of AMPA-type glutamate receptors (AMPAR), we here examined – in hippocampal neurons – three parameters which give insight in the functionality of AMPARs: phosphorylation, surface expression and spontaneous synaptic transmission. In homogenates of adult hippocampal slices, application of corticosterone (30 nM for 15 min) by itself did not affect phosphorylation of the AMPAR GluA1 subunit at S845 or S831. Co-application of the β-adrenergic receptor agonist isoproterenol (10 µM) largely increased S845 (but not S831) phosphorylation. Corticosterone also did not change GluA1 and GluA2 surface expression in hippocampal primary cultures. However, combined administration of corticosterone and 1 µM isoproterenol – which by itself was ineffective – enhanced surface expression. Interestingly, 10 µM isoproterenol alone enhanced GluA1 surface expression, but this was decreased by corticosterone. Finally, in hippocampal primary cultures, the inter-event interval of miniature excitatory postsynaptic currents (mEPSCs) was decreased by the combination of 1 µM isoproterenol and corticosterone (which were ineffective by themselves) while the same combination did not affect the amplitude. We conclude that AMPAR phosphorylation, surface expression and mEPSC inter-event interval respond most strongly to a combination of corticosterone and β-adrenergic receptors. These combined hormonal effects on glutamate transmission might contribute to their memory-enhancing effects.

scholarly journals Shisa7 phosphorylation regulates GABAergic transmission and neurodevelopmental behaviors

2021 ◽  
Author(s):  
Kunwei Wu ◽  
Ryan David Shepard ◽  
David Castellano ◽  
Qingjun Tian ◽  
Lijin Dong ◽  
...  
Keyword(s):  
Neurodevelopmental Disorders ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Long Term Potentiation ◽  
Surface Expression ◽  
Tonic Inhibition ◽  
Gaba A

GABA-A receptors (GABAARs) are crucial for development and regulation of the central nervous system. Altered GABAergic signaling is hypothesized to be involved in the pathophysiology of neurodevelopmental disorders. Nevertheless, how aberrant cellular and molecular mechanisms affect GABAARs in these diseases remain elusive. Recently, we identified Shisa7 as a GABAAR auxiliary subunit that modulates GABAAR trafficking, kinetics, and pharmacology, and discovered a phosphorylation site in Shisa7 (S405) critical for extrasynaptic a5-GABAAR trafficking and tonic inhibition. However, the role of S405 phosphorylation in the regulation of synaptic inhibition, plasticity, and behavior remains unknown. Here, we found that expression of a phospho-null mutant (Shisa7 S405A) in heterologous cells and neurons diminishes a2-GABAAR trafficking. Subsequently, we generate a Shisa7 S405A knock-in (KI) mouse line that displays reduced surface expression of GABAARs in hippocampal neurons. Importantly, both synaptic and tonic inhibition are decreased in KI mice. Moreover, chemically induced inhibitory long-term potentiation is impaired, highlighting a critical role of Shisa7 S405 in GABAergic plasticity. Lastly, KI mice exhibit enhanced locomotor activity and grooming associated with neurodevelopmental disorders. Collectively, our study reveals a phosphorylation site critical for Shisa7-dependent trafficking of synaptic and extrasynaptic GABAARs which contributes to behavioral endophenotypes displayed in neurodevelopmental disorders.

scholarly journals A Super-Resolved View of the Alzheimer’s Disease-Related Amyloidogenic Pathway in Hippocampal Neurons

2021 ◽  
pp. 1-20
Author(s):  
Yang Yu ◽  
Yang Gao ◽  
Bengt Winblad ◽  
Lars Tjernberg ◽  
Sophia Schedin Weiss
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Hippocampal Neurons ◽  
Three Dimensional ◽  
Amyloid Β ◽  
Stimulated Emission ◽  
Amyloid Β Peptide ◽  
Primary Neurons ◽  
Amyloid Β Protein ◽  
Early Endosomes

Background: Processing of the amyloid-β protein precursor (AβPP) is neurophysiologically important due to the resulting fragments that regulate synapse biology, as well as potentially harmful due to generation of the 42 amino acid long amyloid β-peptide (Aβ 42), which is a key player in Alzheimer’s disease. Objective: Our aim was to clarify the subcellular locations of the amyloidogenic AβPP processing in primary neurons, including the intracellular pools of the immediate substrate, AβPP C-terminal fragment (APP-CTF) and the product (Aβ 42). To overcome the difficulties of resolving these compartments due to their small size, we used super-resolution microscopy. Methods: Mouse primary hippocampal neurons were immunolabelled and imaged by stimulated emission depletion (STED) microscopy, including three-dimensional, three-channel imaging and image analyses. Results: The first (β-secretase) and second (γ-secretase) cleavages of AβPP were localized to functionally and distally distinct compartments. The β-secretase cleavage was observed in early endosomes, where we were able to show that the liberated N- and C-terminal fragments were sorted into distinct vesicles budding from the early endosomes in soma. Lack of colocalization of Aβ 42 and APP-CTF in soma suggested that γ-secretase cleavage occurs in neurites. Indeed, APP-CTF was, in line with Aβ 42 in our previous study, enriched in the presynapse but absent from the postsynapse. In contrast, full-length AβPP was not detected in either the pre- or the postsynaptic side of the synapse. Furthermore, we observed that endogenously produced and endocytosed Aβ 42 were localized in different compartments. Conclusion: These findings provide critical super-resolved insight into amyloidogenic AβPP processing in primary neurons.

scholarly journals Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Anthony R. Anzell ◽  
Garrett M. Fogo ◽  
Zoya Gurm ◽  
Sarita Raghunayakula ◽  
Joseph M. Wider ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Ischemia Reperfusion Injury ◽  
Mitochondrial Dynamics ◽  
Ischemia Reperfusion ◽  
Mitochondrial Fission ◽  
Conditional Knockout ◽  
Mitochondrial Morphology ◽  
Primary Neurons ◽  
Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

scholarly journals Reelin Signals through Phosphatidylinositol 3-Kinase and Akt To Control Cortical Development and through mTor To Regulate Dendritic Growth

2007 ◽  
Vol 27 (20) ◽  
pp. 7113-7124 ◽  
Author(s):  
Yves Jossin ◽  
André M. Goffinet
Keyword(s):  
Cortical Neurons ◽  
Hippocampal Neurons ◽  
Matrix Protein ◽  
Glycogen Synthase ◽  
Extracellular Matrix Protein ◽  
Cortical Plate ◽  
Target Cells ◽  
Neuronal Cultures ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

ABSTRACT Reelin is an extracellular matrix protein with various functions during development and in the mature brain. It activates different signaling cascades in target cells, one of which is the phosphatidylinositol 3-kinase (PI3K) pathway, which we investigated further using pathway inhibitors and in vitro brain slice and neuronal cultures. We show that the mTor (mammalian target of rapamycin)-S6K1 (S6 kinase 1) pathway is activated by Reelin and that this depends on Dab1 (Disabled-1) phosphorylation and activation of PI3K and Akt (protein kinase B). PI3K and Akt are required for the effects of Reelin on the organization of the cortical plate, but their downstream partners mTor and glycogen synthase kinase 3β (GSK3β) are not. On the other hand, mTor, but not GSK3β, mediates the effects of Reelin on the growth and branching of dendrites of hippocampal neurons. In addition, PI3K fosters radial migration of cortical neurons through the intermediate zone, an effect that is independent of Reelin and Akt.

Blockage of IL-17 Ameliorates Aβ-Induced Neurotoxicity and Cognitive Decline

2021 ◽  
Author(s):  
Yulan Liu ◽  
Yang Meng ◽  
Chenliang Zhou ◽  
Wenfang Xia ◽  
Lu Wang ◽  
...  
Keyword(s):  
Cognitive Decline ◽  
Lateral Ventricle ◽  
Hippocampal Neurons ◽  
Cognitive Impairments ◽  
Critical Role ◽  
Synaptic Dysfunction ◽  
Neural Damage ◽  
Primary Hippocampal Neurons ◽  
The Impact ◽  
Aβ Production

Abstract BackgroundNeuroinflammation plays a critical role in the pathophysiology of Alzheimer’s disease (AD), particularly in amyloid-β (Aβ) production. But the impact of the cytokine, interleukin-17A (IL-17) on the course of AD has not been well defined. The goal was to determine the effect of IL-17 on neural damage and whether IL-17 inhibitor (Y-320) could ameliorate Aβ-induced neurotoxicity and cognitive decline.MethodsThe expression level of IL-17 was analyzed in APP/PS1 mice. Then IL-17 was injected into the lateral ventricle of C57BL WT mice and roles on synaptic dysfunction and cognitive impairments were examined. Aβ42 was injected into the lateral ventricle of to mimic Aβ42 model mice. The effects of IL-17 inhibitor by oral gavage on Aβ42-induced neurotoxicity and cognitive decline were examined. ResultsWe found that IL-17 was increased in the hippocampus of APP/PS1 transgenic mouse, which has a fundamental role in mediating brain damage in neuroinflammatory processes. Furthermore, we reported that IL-17 was administrated in primary hippocampal neurons, leading to neural damage and synaptic dysfunction. At the same time, IL-17 caused synaptic dysfunction and cognitive impairments accompanying with increased of Aβ levels in mice. In addition, we found that Y-320 could rescue Aβ42–induced neural damage in primary hippocampal neurons, and ameliorate neuronal damage and cognitive impairments in Aβ42 model mice. Interestingly, we observed that IL-17 upregulated the production of soluble amyloid precursor protein β (sAPPβ) and phosphorylation of APP at T668 (pT668), moreover, Y-320 inhibited the Aβ production by down-regulation the sAPPβ and pT668. Conclusions Blockage of IL-17 might ameliorate Aβ-induced neurotoxicity and cognitive decline. These results strongly demonstrate a potential therapeutic role for IL-17 inhibitor in AD.

Abstract 3115: Low-Level Laser Therapy Protects Against OGD-induced Neurotoxicity Of Primary Cultured Mouse Cortical Neurons

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
Zhanyang Yu ◽  
Ning Liu ◽  
Eng H Lo ◽  
Thomas J McCarthy ◽  
Xiaoying Wang
Keyword(s):  
Laser Therapy ◽  
Mitochondrial Function ◽  
Cortical Neurons ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Low Level Laser Therapy ◽  
Primary Neurons ◽  
Novel Therapy ◽  
Low Level ◽  
Mtt Reduction

Background: Low level light (or laser) therapy (LLLT) has been studied and practiced for promoting wound healing, reducing pain, inflammation, and ischemic tissue damage. Recently, a series of experimental and clinical investigations have suggested that LLLT may be a novel therapy against hypoxic/ischemic brain damage. A clinical trial of LLLT therapy for ischemic stroke is now on going. However, the molecular mechanism of LLLT-conferred neuroprotection remains poorly defined. In this study, we tested our hypothesis that LLLT may attenuate impairments of mitochondrial function induced by hypoxic/ischemic insults in primary cultured mouse cortical neurons. Method: At day 9 of culture, primary neurons were subjected to 4 hr OGD followed by reoxygenation. One 810-nm LLLT treatment was applied for 2 minutes at 2 hr after reoxygenation. Neurotoxicity was measured after 20 hr after reoxygenation by LDH release assay. We also measured MTT reduction and mitochondria membrane potential (MMP) at 2 hr after LLLT treatment as markers of mitochondrial function. Results: The neurotoxicity study showed that 4 hr OGD plus 20 hr reoxygenation caused 33.8± 3.4% neuronal cell death, while LLLT treatment significantly reduced the neuronal death rate to 23.6± 2.9% (30.2% reduction, n=6, p smaller than 0.05). Mitochondrial functional assays showed OGD decreased MTT reduction to 75.9± 2.68%, but LLLT treatment significantly rescued MTT reduction to 87.6±4.55% (15.4% improvement, n=6, p smaller than 0.05). Furthermore, after OGD, MMP was reduced to 48.9±4.39%, while LLLT treatment significantly ameliorated this reduction to 89.6± 13.9% (83% improvement, n=4, p smaller than 0.05) compared to normoxic controls. Conclusion: The present study suggests that LLLT treatment is protective against OGD-induced neurotoxicity of primary neurons and that this protection may be conferred through preservation or rescue of mitochondrial function.

Sign in / Sign up

Export Citation Format

Share Document