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.
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.
Project description:RNA-seq analysis on a human neuronal cell line derived from fetal mesencephalon (LUHMES) wild type and RFX2 knockout from day 0 to day 6.
Project description:LUHMES cells share many characteritics with human dopamingeric neurons in the substantia nigra, the cells whose demise is responsible for the motor symptoms in Parkinson’s disease (PD). LUHMES cells can therefore be used bona fide as a model to study pathophysiological processes involved in PD. Previously, we showed that LUHMES cell degenerate after six days upon overexpression of wild type alpha-synuclein. In the present study we performed a transcriptome and proteome expression analysis in alpha-synuclein-overexpressing cells and GFP-expressing control cells in order to identify genes and proteins that are differentially regulated upon overexpression of alpha-synuclein. The analysis was performed four days after the initiation of alpha-synuclein or GFP overexpression, before the cells died in order to identify processes that preceded cell death.
Project description:Genome-wide patterns of DNA methylation were quantified using the Illumina Infinium HumanMethylationEPIC BeadChip in DNA samples extracted from LUHMES cell line cells repeatedly treated with bortezomib (BTZ).
Project description:LUHMES cells are non-tumorigenic model of human dopaminergic neurons. Their physiology and phenotypic changes upon differentiation can provide clues into the pathology of neurological diseases such as dyslexia, autism, schizophrenia.
