Study of MeCP2 in LUHMES-derived neurons
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA517116 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8487260_1.fastq.gz | Fastqsanger.gz | |||
| SRR8487260_2.fastq.gz | Fastqsanger.gz | |||
| SRR8487270_1.fastq.gz | Fastqsanger.gz | |||
| SRR8487270_2.fastq.gz | Fastqsanger.gz | |||
| SRR8487280_1.fastq.gz | Fastqsanger.gz |
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Project description:we performed lentiviral CRISPR interference (CRISPRi) by recruiting dCas9 fused with the KRAB domain to the CSMD1 enhancer (fam3) in the neuronal precursor cell line – Lund human mesencephalic (LUHMES). Given that the expression of CSMD1 was not detectable in LUHMES cells we differentiated these cells into neurons. Differentiated neurons with CRISPRi of CSMD1 enhancer showed significantly higher expression of CSMD1 than control.
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Project description:This dataset comprises 15 raw AP-MS files, each with an associated peak list, captured using a Vanquish Neo nanoLC system in tandem with an Orbitrap Eclipse mass spectrometer. To generate MECP2 hESC-reporter lines for wild type (WT) and various mutations of MECP2, including R133C, R168X, and R270X, we first used CRISPR/Cas9 to create MECP2 alleles carrying the green fluorescent protein (GFP) sequences in the endogenous gene. The R133C mutation was then introduced into the WT MECP2-GFP reporter line. Mutations R133C, R168X, and R270X are recognized as loss-of-function variants in MECP2 and are also identified as primary Rett syndrome-causing mutations. For efficient neuronal differentiation, a doxycycline (DOX)-responsive NGN2 construct was incorporated at their AAVS1 safe harbor locus. Upon the addition of DOX, homogenous populations of neurons were generated within three weeks from those four MECP2 hESC-reporter lines. Subsequently, GFP-pull down assay and AP-MS were performed using these WT MECP2-GFP neurons along with R133C-, R168X-, and R270X-mutant MECP2-GFP reporter neurons. Neurons expressing only the GFP tag served as a negative control. AP-MS analysis identified proteins interacting differently between WT and mutant MECP2 within human neurons.
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Project description:Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by loss-of-function heterozygous mutations of MECP2. Reactivation of the silent wild-type MECP2 allele on the inactive X chromosome (Xi) represents a promising therapeutic opportunity for female RTT patients. Here, we applied a multiplex epigenome editing approach to reactivate MECP2 on Xi. Demethylation of the MECP2 promoter by dCas9-Tet1 with target sgRNA reactivated MECP2 on Xi in RTT hESCs without detectable off-target effects at the transcriptome level. Neurons derived from methylation edited RTT hESCs reversed the smaller soma size and electrophysiological abnormalities. Insulation of the methylation edited MECP2 locus in RTT neurons by dCpf1-CTCF with target crRNA stabilized MECP2 reactivation and rescued the RTT-related neuronal defects, providing a proof-of-concept study for multiplex epigenome editing to treat RTT.
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Project description:To study mechanisms of neurodegenerative diseases, neuronal cell lines are important model systems and are often differentiated into postmitotic neuron-like cells to resemble more closely primary neurons obtained from brains. One such cell line is the Lund Human Mesencephalic (LUHMES) cell line which can be differentiated into dopamine-like neurons and is frequently used to study mechanisms of Parkinson’s disease (PD) and neurotoxicity. Neuronal differentiation of LUHMES cells is commonly verified by measurement of selected neuronal markers, but little is known about proteome-wide protein abundance changes during differentiation. Using mass spectrometry and label-free quantification (LFQ) we compared the proteome of differentiated and undifferentiated LUHMES cells as well as of cultured primary murine midbrain neurons, which are mainly dopaminergic. Neuronal differentiation induced substantial changes of the LUHMES cell proteome (18.4% reveal protein abundance changes of more than 4-fold), with proliferation-related proteins (e.g. MCMs) being strongly down-regulated and neuronal and dopaminergic proteins being up to 1000-fold upregulated, such as L1CAM and SNCA. Several of these proteins, including MAPT and SYN1, may be useful new markers to experimentally validate neuronal differentiation of cultured LUHMES cells. Primary midbrain neurons were more closely related to differentiated than to undifferentiated LUHMES cells with respect to the abundance of proteins related to neurodegeneration or to genetic forms of PD. In summary, our comparative proteomic analysis demonstrates that differentiated LUHMES cells are a suitable model for studies on PD and neurodegeneration and provides a resource of the proteome-wide changes during neuronal differentiation.
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