An Optimized Direct Lysis Gene Expression Microplate Assay and Applications for Disease, Differentiation, and Pharmacological Cell-Based Studies

Ng, Neville; Maksour, Simon; Lum, Jeremy S.; Newbery, Michelle; Shephard, V K; Ooi, Lezanne

doi:10.3390/bios12060364

Cited by 5 publications

(5 citation statements)

References 23 publications

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“…Gene expression assays were performed with a direct lysis microplate assay 25 . Cells were washed with 0.9% w/v saline before lysis in 5 mM Tris, 75 mM saline, and 0.05% Triton X‐100 for 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…Gene expression assays were performed with a direct lysis microplate assay. 25 Cells were washed with 0.9% w/v saline before lysis in 5 mM Tris, 75 mM saline, and 0.05% Triton X-100 for 5 min. RT-qPCR reactions were performed with MicroAmp 96-Well Reaction Plates (4366932, Thermo Fisher Scientific).…”

Section: Reverse Transcription Quantitative Pcr (Rt-qpcr)mentioning

confidence: 99%

“…Vanishing white matter disease astrocytes were generated using an adaptation of a previously published protocol (Figure S1). 20,25 Gene expression characterization indicated significantly decreased GFAP and EAAT1 gene expression in VWMD astrocytes compared to control astrocytes (Figure 1A). 20 VWMD astrocytes also showed decreased reduction activity, increased reactive oxygen species, and a minor decrease in mitochondrial membrane potential (ΔΨm) (Figure 1B).…”

Section: Altered Gene Expression and Function Of Vwmd Astrocytesmentioning

confidence: 99%

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Edaravone and mitochondrial transfer as potential therapeutics for vanishing white matter disease astrocyte dysfunction

Newbery

Touffu

et al. 2023

CNS Neurosci Ther

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Introduction Previous research has suggested that vanishing white matter disease (VWMD) astrocytes fail to fully differentiate and respond differently to cellular stresses compared to healthy astrocytes. However, few studies have investigated potential VWMD therapeutics in monoculture patient‐derived cell‐based models. Methods To investigate the impact of alterations in astrocyte expression and function in VWMD, astrocytes were differentiated from patient and control induced pluripotent stem cells and analyzed by proteomics, pathway analysis, and functional assays, in the absence and presence of stressors or potential therapeutics. Results Vanishing white matter disease astrocytes demonstrated significantly reduced expression of astrocyte markers and markers of inflammatory activation or cellular stress relative to control astrocytes. These alterations were identified both in the presence and absence of polyinosinic:polycytidylic acid stimuli, which is used to simulate viral infections. Pathway analysis highlighted differential signaling in multiple pathways in VWMD astrocytes, including eukaryotic initiation factor 2 (EIF2) signaling, oxidative stress, oxidative phosphorylation (OXPHOS), mitochondrial function, the unfolded protein response (UPR), phagosome regulation, autophagy, ER stress, tricarboxylic acid cycle (TCA) cycle, glycolysis, tRNA signaling, and senescence pathways. Since oxidative stress and mitochondrial function were two of the key pathways affected, we investigated whether two independent therapeutic strategies could ameliorate astrocyte dysfunction: edaravone treatment and mitochondrial transfer. Edaravone treatment reduced differential VWMD protein expression of the UPR, phagosome regulation, ubiquitination, autophagy, ER stress, senescence, and TCA cycle pathways. Meanwhile, mitochondrial transfer decreased VWMD differential expression of the UPR, glycolysis, calcium transport, phagosome formation, and ER stress pathways, while further modulating EIF2 signaling, tRNA signaling, TCA cycle, and OXPHOS pathways. Mitochondrial transfer also increased the gene and protein expression of the astrocyte marker, glial fibrillary acidic protein (GFAP) in VWMD astrocytes. Conclusion This study provides further insight into the etiology of VWMD astrocytic failure and suggests edaravone and mitochondrial transfer as potential candidate VWMD therapeutics that can ameliorate disease pathways in astrocytes related to oxidative stress, mitochondrial dysfunction, and proteostasis.

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Section: Methodsmentioning

confidence: 99%

Section: Reverse Transcription Quantitative Pcr (Rt-qpcr)mentioning

confidence: 99%

Section: Altered Gene Expression and Function Of Vwmd Astrocytesmentioning

confidence: 99%

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Edaravone and mitochondrial transfer as potential therapeutics for vanishing white matter disease astrocyte dysfunction

Newbery

Touffu

et al. 2023

CNS Neurosci Ther

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“…One-step RT-qPCR was performed as described in [36]. RNA was extracted by adding TBST (5 mM Tris, 75 mM NaCl and 0.05% Triton X-100) directly to cells for 5 min.…”

Section: One-step Rt-qpcrmentioning

confidence: 99%

RESTandRCORgenes display distinct expression profiles in neurons and astrocytes using 2D and 3D human pluripotent stem cell models

Maksour,

Ng,

Hulme

et al. 2024

Preprint

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Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors, CoREST1, CoREST2, or CoREST3 (encoded by RCOR1, RCOR2, and RCOR3, respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST, in various neural and glial cell types during different developmental stages. However, there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap, we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons, as well as mature functional NGN2-induced neurons. Additionally, a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids, along with RCOR2 in the latter. In summary, our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically, RCOR3 expression increases in neurons, while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans.

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“…HEK293T cells greater than 80% confluency were transfected with plasmid complex for 2 h at 37˚C and lentiviral particles collected as in previous description. 87…”

Section: Lentiviral Productionmentioning

confidence: 99%

Astrocytic glutamate regulation is implicated in the development of stress-related psychiatric disorders

Kaul,

Curry,

Gerstner

et al. 2024

Preprint

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Severe psychological stress is one of the most potent risk factors for developing a mood or psychotic disorder, yet the underlying molecular mechanisms are poorly understood. Astrocytes are a key brain cell type associated with stress and psychiatric phenotypes in animals, but how this translates to humans is largely unknown. Here, we show that cortical astrocytes are persistently changed both physically and molecularly in humans with psychiatric disorders exposed to profound stress before diagnosis. By profiling the diversity of human astrocytes with single nucleus and spatial transcriptomics, we identified distinct alterations to glutamate-related synaptic functions, supported by histological quantification of >20,000 astrocytes. Alterations were pronounced in females compared to males and in cases exposed to profound stress during childhood. The use of human pluripotent stem cell-derived astrocytes confirmed that glutamate signalling is directly impacted by glucocorticoid activation. Our findings suggest that astrocytes are strategic pharmacological targets for future intervention strategies.

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An Optimized Direct Lysis Gene Expression Microplate Assay and Applications for Disease, Differentiation, and Pharmacological Cell-Based Studies

Cited by 5 publications

References 23 publications

Edaravone and mitochondrial transfer as potential therapeutics for vanishing white matter disease astrocyte dysfunction

Edaravone and mitochondrial transfer as potential therapeutics for vanishing white matter disease astrocyte dysfunction

RESTandRCORgenes display distinct expression profiles in neurons and astrocytes using 2D and 3D human pluripotent stem cell models

Astrocytic glutamate regulation is implicated in the development of stress-related psychiatric disorders

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