Distortion of protein analysis in primary neuronal cultures by serum albumin from culture medium: A methodological approach to improve target protein quantification

Willis, Ashleigh; Pratt, Judith A.; Morris, Brian J.

doi:10.1016/j.jneumeth.2018.07.002

Cited by 3 publications

(3 citation statements)

References 18 publications

(20 reference statements)

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“…Protein from primary cortical neuronal cultures was extracted in line with our previously published protocols [ 61 ]. In brief, culture medium was removed, plates were placed on ice and 1000 µl of ice-cold PBS (pH 7.5) was added for 15–30 s. Upon wash buffer removal, 80 µl of RIPA buffer (10 nM Tris–HCl, 150 mM NaCl, 1 mM EGTA, 0.5% NP40, 0.1% SDS, 0.1% sodium deoxycholate) was added.…”

Section: Methodsmentioning

confidence: 99%

Enzymatic Degradation of Cortical Perineuronal Nets Reverses GABAergic Interneuron Maturation

2022

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Perineuronal nets (PNNs) are specialised extracellular matrix structures which preferentially enwrap fast-spiking (FS) parvalbumin interneurons and have diverse roles in the cortex. PNN maturation coincides with closure of the critical period of cortical plasticity. We have previously demonstrated that BDNF accelerates interneuron development in a c-Jun-NH2-terminal kinase (JNK)–dependent manner, which may involve upstream thousand-and-one amino acid kinase 2 (TAOK2). Chondroitinase-ABC (ChABC) enzymatic digestion of PNNs reportedly reactivates ‘juvenile-like’ plasticity in the adult CNS. However, the mechanisms involved are unclear. We show that ChABC produces an immature molecular phenotype in cultured cortical neurons, corresponding to the phenotype prior to critical period closure. ChABC produced different patterns of PNN-related, GABAergic and immediate early (IE) gene expression than well-characterised modulators of mature plasticity and network activity (GABAA-R antagonist, bicuculline, and sodium-channel blocker, tetrodotoxin (TTX)). ChABC downregulated JNK activity, while this was upregulated by bicuculline. Bicuculline, but not ChABC, upregulated Bdnf expression and ERK activity. Furthermore, we found that BDNF upregulation of semaphorin-3A and IE genes was TAOK mediated. Our data suggest that ChABC heightens structural flexibility and network disinhibition, potentially contributing to ‘juvenile-like’ plasticity. The molecular phenotype appears to be distinct from heightened mature synaptic plasticity and could relate to JNK signalling. Finally, we highlight that BDNF regulation of plasticity and PNNs involves TAOK signalling.

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Section: Methodsmentioning

confidence: 99%

Enzymatic Degradation of Cortical Perineuronal Nets Reverses GABAergic Interneuron Maturation

2022

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“…Primary cerebrocortical neuronal cultures were prepared using brain tissue from wild-type C57BL/6J mouse embryos (E17) as previously described [17][18][19], and maintained for 12-14 dayd in vitro (DIV). For primary astrocyte cultures [20], the medium was changed every 2 to 3 days, and then, to deplete as many microglia as possible, the flask was shaken at 100 rpm for 30 min on an orbital shaker.…”

Section: Culture Of Primary Cells -Preparation Of Neurons and Astrocytesmentioning

confidence: 99%

“…The quality and purity of neuronal and astrocyte cultures has been repeatedly checked -cultures are in good health and are around 90% specific for the defined cell type [17][18][19], as is standard for these techniques.…”

Section: Culture Of Primary Cells -Preparation Of Neurons and Astrocytesmentioning

confidence: 99%

Novel alternatively-spliced exons of the VRK2 gene in mouse brain and microglial cells

et al. 2020

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Common sequence variations in the VRK2 gene contribute to genetic risk for various psychiatric diseases including schizophrenia and major depressive disorder. Despite the clear importance of studying the regulation and function of VRK2 for understanding the causes of these diseases, the organisation and expression of the gene remain poorly characterised. Using reverse-transcriptase-PCR, we have amplifed exons of Vrk2 mRNA from regions of mouse brain, and from different cell classes comprising neurones, astrocytes and microglial cells. We find that Vrk2 mRNA is expressed in all cell types, and that the splicing of the mouse Vrk2 gene is much more complex than previously appreciated. In addition to the predicted alternative splicing (absence/presence) of the penultimate 3 prime exon, we also detected a variety of 5 prime structures, including two novel exons spanning the first characterised exon (exon 1), which we term exons 1a and 1b. While expressed in neurones and astrocytes, exon 1b was not expressed in microglial cells. Expression of transcripts containing exon 1a in microglia was increased by immune stimulation. An additional truncated transcript lacking 7 central exons was also identified. As with the human gene, the results confirm complex patterns of alternative splicing which are likely to be relevant for understanding the physiological and pathological function of the gene in the CNS. Electronic supplementary material The online version of this article (10.1007/s11033-020-05584-3) contains supplementary material, which is available to authorized users.

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BDNF and JNK Signaling Modulate Cortical Interneuron and Perineuronal Net Development: Implications for Schizophrenia-Linked 16p11.2 Duplication Syndrome

Willis

Pratt

Morris

2020

Schizophrenia Bulletin

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Schizophrenia (SZ) is a neurodevelopmental disorder caused by the interaction of genetic and environmental risk factors. One of the strongest genetic risk variants is duplication (DUP) of chr.16p11.2. SZ is characterized by cortical gamma-amino-butyric acid (GABA)ergic interneuron dysfunction and disruption to surrounding extracellular matrix structures, perineuronal nets (PNNs). Developmental maturation of GABAergic interneurons, and also the resulting closure of the critical period of cortical plasticity, is regulated by brain-derived neurotrophic factor (BDNF), although the mechanisms involved are unknown. Here, we show that BDNF promotes GABAergic interneuron and PNN maturation through JNK signaling. In mice reproducing the 16p11.2 DUP, where the JNK upstream activator Taok2 is overexpressed, we find that JNK is overactive and there are developmental abnormalities in PNNs, which persist into adulthood. Prefrontal cortex parvalbumin (PVB) expression is reduced, while PNN intensity is increased. Additionally, we report a unique role for TAOK2 signaling in the regulation of PVB interneurons. Our work implicates TAOK2-JNK signaling in cortical interneuron and PNN development, and in the responses to BDNF. It also demonstrates that over-activation of this pathway in conditions associated with SZ risk causes long-lasting disruption in cortical interneurons.

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Distortion of protein analysis in primary neuronal cultures by serum albumin from culture medium: A methodological approach to improve target protein quantification

Cited by 3 publications

References 18 publications

Enzymatic Degradation of Cortical Perineuronal Nets Reverses GABAergic Interneuron Maturation

Enzymatic Degradation of Cortical Perineuronal Nets Reverses GABAergic Interneuron Maturation

Novel alternatively-spliced exons of the VRK2 gene in mouse brain and microglial cells

BDNF and JNK Signaling Modulate Cortical Interneuron and Perineuronal Net Development: Implications for Schizophrenia-Linked 16p11.2 Duplication Syndrome

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