scholarly journals Direct induction of human neurons from fibroblasts carrying the neuropsychiatric 22q11.2 microdeletion reveals transcriptome- and epigenome-wide alterations

2021 ◽  
Author(s):  
Carolin Purmann ◽  
Cheen Euong Ang ◽  
Koji Tanabe ◽  
Yue Zhang ◽  
Soumya Kundu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Model ◽  
Neuronal Cells ◽  
Deletion Syndrome ◽  
Patient Specific ◽  
Neuronal Phenotype ◽  
Genome Wide ◽  
Human Neurons ◽  
Direct Induction ◽  
In Cells

Standard methods for the creation of neuronal cells via direct induction from primary tissue use perinatal fibroblasts, which hinders the important study of patient specific genetic lesions such as those underlying neuropsychiatric disorders. To address this we developed a novel method for the direct induction of neuronal cells (induced neuronal cells, iN cells) from adult human fibroblast cells. Reprogramming fibroblasts into iN cells via recombinant virus resulted in cells that stain for markers such as MAP2 and PSA-NCAM and exhibit electrophysiological properties such as action potentials and voltage dependent sodium- and potassium currents that reveal a neuronal phenotype. Transcriptome and chromatin analysis using RNA-Seq, microRNA-Seq and ATAC-Seq, respectively, further confirm neuronal character. 22q11.2 Deletion-Syndrome (22q11DS) is caused by a large 3 million base-pair heterozygous deletion on human chromosome 22 and is strongly associated with neurodevelopmental, neuropsychiatric phenotypes such as schizophrenia and autism. We leverage the direct-iN cell model for the study of genetic neurodevelopmental conditions by presenting gene-by-gene as well as network-wide effects of the 22q11DS deletion on gene expression in human neuronal cells, on several levels of functional genomics analysis. Some of the genes within the 22q11DS deletion boundary exhibit unexpected cell-type-specific changes in transcript levels, and genome-wide we can detect dysregulation of calcium channel subunit genes and other genes known to be involved in autism or schizophrenia, such as NRXN1, as well synaptic pathways. This genome-wide effect on gene expression can also be observed at the microRNA and chromatin levels, showing that the iN cells have indeed converted to a neuronal phenotype at several regulatory levels: chromatin, protein-coding RNAs and microRNAs, revealing relevant disease pathways and genes. We present this model of inducing neurons from fibroblasts as a useful general resource to study the genetic and molecular basis of normal and abnormal brain development and brain function.

scholarly journals Genome-wide molecular effects of the neuropsychiatric 16p11 CNVs in an iPSC-to-iN neuronal model

2020 ◽  
Author(s):  
Thomas R. Ward ◽  
Xianglong Zhang ◽  
Louis C. Leung ◽  
Bo Zhou ◽  
Kristin Muench ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cell Model ◽  
Copy Number Variants ◽  
Induced Pluripotent Stem Cell ◽  
Neuronal Model ◽  
Autism Spectrum ◽  
Genome Wide ◽  
Induced Pluripotent ◽  
Methylation Patterns

AbstractCopy number variants (CNVs), either deletions or duplications, at the 16p11.2 locus in the human genome are known to increase the risk for autism spectrum disorders (ASD), schizophrenia, and for several other developmental conditions. Here, we investigate the global effects on gene expression and DNA methylation using a 16p11.2 CNV patient-derived induced pluripotent stem cell (iPSC) to induced neuron (iN) cell model system. This approach revealed genome-wide and cell-type specific alterations to both gene expression and DNA methylation patterns and also yielded specific leads on genes potentially contributing to some of the known 16p11.2 patient phenotypes. PCSK9 is identified as a possible contributing factor to the symptoms seen in carriers of the 16p11.2 CNVs. The protocadherin (PCDH) gene family is found to have altered DNA methylation patterns in the CNV patient samples. The iPSC lines used for this study are available through a repository as a resource for research into the molecular etiology of the clinical phenotypes of 16p11.2 CNVs and into that of neuropsychiatric and neurodevelopmental disorders in general.

scholarly journals The Effects of Serum Removal on Gene Expression and Morphological Plasticity Markers in Differentiated SH-SY5Y Cells

2021 ◽  
Author(s):  
Alix C. Thomson ◽  
Teresa Schuhmann ◽  
Tom A. de Graaf ◽  
Alexander T. Sack ◽  
Bart P. F. Rutten ◽  
...  
Keyword(s):  
Gene Expression ◽  
Culture Media ◽  
Cell Model ◽  
Neuroblastoma Cell ◽  
Morphological Plasticity ◽  
Growth Cycle ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Human Neurons

AbstractDespite the widespread use of the SH-SY5Y human neuroblastoma cell line in modeling human neurons in vitro, protocols for growth, differentiation and experimentation differ considerably across the literature. Many studies fully differentiate SH-SY5Y cells before experimentation, to investigate plasticity measures in a mature, human neuronal-like cell model. Prior to experimentation, serum is often removed from cell culture media, to arrest the cell growth cycle and synchronize cells. However, the exact effect of this serum removal before experimentation on mature, differentiated SH-SY5Y cells has not yet been described. In studies using differentiated SH-SY5Y cells, any effect of serum removal on plasticity markers may influence results. The aim of the current study was to systematically characterize, in differentiated, neuronal-like SH-SY5Y cells, the potentially confounding effects of complete serum removal in terms of morphological and gene expression markers of plasticity. We measured changes in commonly used morphological markers and in genes related to neuroplasticity and synaptogenesis, particularly in the BDNF-TrkB signaling pathway. We found that complete serum removal from already differentiated SH-SY5Y cells increases neurite length, neurite branching, and the proportion of cells with a primary neurite, as well as proportion of βIII-Tubulin and MAP2 expressing cells. Gene expression results also indicate increased expression of PSD95 and NTRK2 expression 24 h after serum removal. We conclude that serum deprivation in differentiated SH-SY5Y cells affects morphology and gene expression and can potentially confound plasticity-related outcome measures, having significant implications for experimental design in studies using differentiated SH-SY5Y cells as a model of human neurons.

scholarly journals Addiction associated N40D mu-opioid receptor variant modulates synaptic function in human neurons

2018 ◽  
Author(s):  
Apoorva Halikere ◽  
Dina Popova ◽  
Aula Hamod ◽  
Mavis R. Swerdel ◽  
Jennifer C. Moore ◽  
...  
Keyword(s):  
Gene Expression ◽  
Synaptic Transmission ◽  
Opioid Receptor ◽  
Cell Lines ◽  
Synaptic Function ◽  
Ips Cell ◽  
Neuronal Marker ◽  
Human Neurons ◽  
Regulatory Effects ◽  
In Cells

AbstractBackgroundThe OPRM1 A118G gene variant (N40D) encoding the µ-opioid receptor (MOR) has been associated with dependence on opiates and other abused drugs but its mechanism is unknown. With opioid abuse-related deaths rising at unprecedented rates, understanding these mechanisms may provide a path to therapy.MethodsSeven human induced pluripotent stem (iPS) cell lines from homozygous N40D subjects (4 with N40 and 3 with D40 variants) were generated and human induced neuronal cells (iNs) were derived from these iPS cell lines. Morphological, gene expression as well as synaptic physiology analyses were conducted in human iN cells carrying N40D MOR variants; Two pairs of isogenic pluripotent stem cells carrying N40D were generated using CRISPR/Cas9 genome-editing and iN cells derived from them were analyzed.ResultsInhibitory human neurons generated from subjects carrying N40D MOR gene variants show mature properties in morphological and functional analyses. Gene expression revealed that they express mature neuronal marker and MORs. Activation of MORs suppressed inhibitory synaptic transmission in human neurons carrying both N40 or D40 MOR variants but D40 show stronger effects. To mitigate the confounding effects of background genetic variation on neuronal function, the regulatory effects of MORs on synaptic transmission were validated in two sets of independently generated isogenic N40D iNs.ConclusionsActivations of N40D MOR variants show different regulatory effects on synaptic transmission in inhibitory human neurons. This study identifies neurophysiological differences between human MOR variants that may predict altered opioid responsivity and/or dependence in this subset of individuals.

scholarly journals Genome-wide analyses in neuronal cells reveal that upstream transcription factors regulate lysosomal gene expression

FEBS Journal ◽  
2016 ◽  
Vol 283 (6) ◽  
pp. 1077-1087 ◽  
Author(s):  
Tomoyuki Yamanaka ◽  
Asako Tosaki ◽  
Masaru Kurosawa ◽  
Tomomi Shimogori ◽  
Nobutaka Hattori ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Neuronal Cells ◽  
Genome Wide ◽  
Lysosomal Gene

scholarly journals ChIP-seq of plasma cell-free nucleosomes identifies cell-of-origin gene expression programs

2019 ◽  
Author(s):  
Ronen Sadeh ◽  
Israa Sharkia ◽  
Gavriel Fialkoff ◽  
Ayelet Rahat ◽  
Jenia Gutin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromatin Immunoprecipitation ◽  
Blood Test ◽  
Patient Specific ◽  
Specific Information ◽  
Cell Of Origin ◽  
Genome Wide ◽  
Cells Of Origin ◽  
Transcriptional Changes ◽  
Dying Cells

Abstract:Genomic DNA is packed by histone proteins that carry a multitude of post-translational modifications that reflect cellular transcriptional state. Cell-free DNA (cfDNA) is derived from fragmented chromatin in dying cells, and as such it retains the histones markings present in the cells of origin. Here, we pioneer chromatin immunoprecipitation followed by sequencing of cell-free nucleosomes (cfChIP-seq) carrying active chromatin marks. Our results show that cfChIP-seq provides multidimensional epigenetic information that recapitulates the epigenetic and transcriptional landscape in the cells of origin. We applied cfChIP-seq to 268 samples including samples from patients with heart and liver pathologies, and 135 samples from 56 metastatic CRC patients. We show that cfChIP-seq can detect pathology-related transcriptional changes at the site of the disease, beyond the information on tissue of origin. In CRC patients we detect clinically-relevant, and patient-specific information, including transcriptionally active HER2 amplifications. cfChIP-seq provides genome-wide information and requires low sequencing depth. Altogether, we establish cell-free chromatin immunoprecipitation as an exciting modality with potential for diagnosis and interrogation of physiological and pathological processes using a simple blood test.One Sentence SummaryChIP-seq of plasma-circulating nucleosomes (cfChIP-seq) from a simple blood test provides detailed information about gene expression programs in human organs, and cancer.

scholarly journals Inhibition of mitochondrial complex II in neuronal cells triggers unique pathways culminating in autophagy with implications for neurodegeneration

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sathyanarayanan Ranganayaki ◽  
Neema Jamshidi ◽  
Mohamad Aiyaz ◽  
Santhosh-Kumar Rashmi ◽  
Narayanappa Gayathri ◽  
...  
Keyword(s):  
Movement Disorders ◽  
Cell Model ◽  
Neuronal Cells ◽  
Mitochondrial Complex ◽  
Complex Ii ◽  
Mechanistic Target Of Rapamycin ◽  
Nitropropionic Acid ◽  
Genome Wide ◽  
Biochemical Validation ◽  
3 Nitropropionic Acid

AbstractMitochondrial dysfunction and neurodegeneration underlie movement disorders such as Parkinson’s disease, Huntington’s disease and Manganism among others. As a corollary, inhibition of mitochondrial complex I (CI) and complex II (CII) by toxins 1-methyl-4-phenylpyridinium (MPP+) and 3-nitropropionic acid (3-NPA) respectively, induced degenerative changes noted in such neurodegenerative diseases. We aimed to unravel the down-stream pathways associated with CII inhibition and compared with CI inhibition and the Manganese (Mn) neurotoxicity. Genome-wide transcriptomics of N27 neuronal cells exposed to 3-NPA, compared with MPP+ and Mn revealed varied transcriptomic profile. Along with mitochondrial and synaptic pathways, Autophagy was the predominant pathway differentially regulated in the 3-NPA model with implications for neuronal survival. This pathway was unique to 3-NPA, as substantiated by in silico modelling of the three toxins. Morphological and biochemical validation of autophagy markers in the cell model of 3-NPA revealed incomplete autophagy mediated by mechanistic Target of Rapamycin Complex 2 (mTORC2) pathway. Interestingly, Brain Derived Neurotrophic Factor (BDNF), which was elevated in the 3-NPA model could confer neuroprotection against 3-NPA. We propose that, different downstream events are activated upon neurotoxin-dependent CII inhibition compared to other neurotoxins, with implications for movement disorders and regulation of autophagy could potentially offer neuroprotection.

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