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Loss of the Chr16p11.2 ASD candidate gene QPRT leads to aberrant neuronal differentiation in the SH-SY5Y neuronal cell model.

ABSTRACT: Background:Altered neuronal development is discussed as the underlying pathogenic mechanism of autism spectrum disorders (ASD). Copy number variations of 16p11.2 have recurrently been identified in individuals with ASD. Of the 29 genes within this region, quinolinate phosphoribosyltransferase (QPRT) showed the strongest regulation during neuronal differentiation of SH-SY5Y neuroblastoma cells. We hypothesized a causal relation between this tryptophan metabolism-related enzyme and neuronal differentiation. We thus analyzed the effect of QPRT on the differentiation of SH-SY5Y and specifically focused on neuronal morphology, metabolites of the tryptophan pathway, and the neurodevelopmental transcriptome. Methods:The gene dosage-dependent change of QPRT expression following Chr16p11.2 deletion was investigated in a lymphoblastoid cell line (LCL) of a deletion carrier and compared to his non-carrier parents. Expression of QPRT was tested for correlation with neuromorphology in SH-SY5Y cells. QPRT function was inhibited in SH-SY5Y neuroblastoma cells using (i) siRNA knockdown (KD), (ii) chemical mimicking of loss of QPRT, and (iii) complete CRISPR/Cas9-mediated knock out (KO). QPRT-KD cells underwent morphological analysis. Chemically inhibited and QPRT-KO cells were characterized using viability assays. Additionally, QPRT-KO cells underwent metabolite and whole transcriptome analyses. Genes differentially expressed upon KO of QPRT were tested for enrichment in biological processes and co-regulated gene-networks of the human brain. Results:QPRT expression was reduced in the LCL of the deletion carrier and significantly correlated with the neuritic complexity of SH-SY5Y. The reduction of QPRT altered neuronal morphology of differentiated SH-SY5Y cells. Chemical inhibition as well as complete KO of the gene were lethal upon induction of neuronal differentiation, but not proliferation. The QPRT-associated tryptophan pathway was not affected by KO. At the transcriptome level, genes linked to neurodevelopmental processes and synaptic structures were affected. Differentially regulated genes were enriched for ASD candidates, and co-regulated gene networks were implicated in the development of the dorsolateral prefrontal cortex, the hippocampus, and the amygdala. Conclusions:In this study, QPRT was causally related to in vitro neuronal differentiation of SH-SY5Y cells and affected the regulation of genes and gene networks previously implicated in ASD. Thus, our data suggest that QPRT may play an important role in the pathogenesis of ASD in Chr16p11.2 deletion carriers.

SUBMITTER: Haslinger D

PROVIDER: S-EPMC6220561 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Loss of the Chr16p11.2 ASD candidate gene <i>QPRT</i> leads to aberrant neuronal differentiation in the SH-SY5Y neuronal cell model.

Haslinger Denise D Waltes Regina R Yousaf Afsheen A Lindlar Silvia S Schneider Ines I Lim Chai K CK Tsai Meng-Miao MM Garvalov Boyan K BK Acker-Palmer Amparo A Krezdorn Nicolas N Rotter Björn B Acker Till T Guillemin Gilles J GJ Fulda Simone S Freitag Christine M CM Chiocchetti Andreas G AG

Molecular autism 20181106

<h4>Background</h4>Altered neuronal development is discussed as the underlying pathogenic mechanism of autism spectrum disorders (ASD). Copy number variations of 16p11.2 have recurrently been identified in individuals with ASD. Of the 29 genes within this region, <i>quinolinate phosphoribosyltransferase</i> (<i>QPRT</i>) showed the strongest regulation during neuronal differentiation of SH-SY5Y neuroblastoma cells. We hypothesized a causal relation between this tryptophan metabolism-related enzy ...[more]

PMID: 30443311

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Project description:Background: Altered neuronal development is discussed as underlying pathogenic mechanism of Autism Spectrum Disorders (ASD). Copy number variations of 16p11.2 have recurrently been identified in individuals with ASD. Of the 29 genes within this region quinolinate phosphoribosyltransferase (QPRT) showed the strongest regulation during in-vitro neuronal differentiation. We hypothesized a causal relation between this tryptophan related enzyme and neuronal differentiation. We thus analyzed the effect of QPRT on differentiation of a neuronal cell line and specifically focused on neuronal morphology, metabolites of the tryptophan pathway and the neurodevelopmental transcriptome. Methods: The gene dosage dependent change of QPRT expression following Chr16p11.2 deletion was investigated in a lymphoblastoid cell line (LCL) of a deletion carrier and compared to his non-carrier parents. Expression of QPRT was tested for correlation with neuromorphology in SH-SY5Y cells. QPRT function was inhibited in SH-SY5Y neuroblastoma cells using (i) siRNA knockdown (KD), (ii) chemical inhibition, and (iii) complete CRISPR/Cas9 induced knockout (KO). QPRT-KD cells underwent morphological analysis. Chemically inhibited and QPRT-KO cells were characterized using viability assays. Additionally, QPRT-KO cells underwent metabolite and whole transcriptome analyses. Genes differentially expressed upon KO of QPRT were tested for enrichment in biological processes and co-regulated gene-networks of the human brain. Results: QPRT expression was reduced in the LCL of the deletion carrier and significantly correlated with neurite maturation in SH-SY5Y cells. The reduction of QPRT altered neuronal morphology of differentiated SH-SY5Y cells. Chemical inhibition as well as complete KO of the gene were lethal upon induction of neuronal differentiation, but not proliferation. The QPRT associated tryptophan pathway was not affected by KO. At the transcriptome level neuronal processes like “synapse organization” were affected. Differentially regulated genes were enriched for ASD-candidates and co-regulated gene networks were implicated in the development of the dorsolateral prefrontal cortex, the hippocampus, and the amygdala. Conclusions: In this study QPRT was causally related to in vitro neuronal differentiation and affected the regulation of genes and gene-networks that were previously implicated in ASD. Thus, our data suggest that QPRT may play an important role in the pathogenesis of ASD in Chr16p11.2 deletion carriers.

Project description:Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors; however, the mechanisms remain unclear. We hypothesized that because the regulation of glycolysis and oxidative phosphorylation is modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis 2-deoxy-d-glucose. Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by the reactive lipid species 4-hydroxynonenal or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone. We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits, which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur upon differentiation of neuroblastoma cells to a neuron-like phenotype.

Dataset Information

Loss of the Chr16p11.2 ASD candidate gene QPRT leads to aberrant neuronal differentiation in the SH-SY5Y neuronal cell model.

Publications

Loss of the Chr16p11.2 ASD candidate gene <i>QPRT</i> leads to aberrant neuronal differentiation in the SH-SY5Y neuronal cell model.

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