Transcriptomic and Proteomic Analysis of CRISPR/Cas9-Mediated ARC-Knockout HEK293 Cells.

Arc/Arg3.1 (activity-regulated cytoskeletal-associated protein (ARC)) is a critical regulator of long-term synaptic plasticity and is involved in the pathophysiology of schizophrenia. The functions and mechanisms of human ARC action are poorly understood and worthy of further investigation. To investigate the function of the ARC gene in vitro, we generated an ARC-knockout (KO) HEK293 cell line via CRISPR/Cas9-mediated gene editing and conducted RNA sequencing and label-free LC-MS/MS analysis to identify the differentially expressed genes and proteins in isogenic ARC-KO HEK293 cells. Furthermore, we used bioluminescence resonance energy transfer (BRET) assays to detect interactions between the ARC protein and differentially expressed proteins. Genetic deletion of ARC disturbed multiple genes involved in the extracellular matrix and synaptic membrane. Seven proteins (HSPA1A, ENO1, VCP, HMGCS1, ALDH1B1, FSCN1, and HINT2) were found to be differentially expressed between ARC-KO cells and ARC wild-type cells. BRET assay results showed that ARC interacted with PSD95 and HSPA1A. Overall, we found that ARC regulates the differential expression of genes involved in the extracellular matrix, synaptic membrane, and heat shock protein family. The transcriptomic and proteomic profiles of ARC-KO HEK293 cells presented here provide new evidence for the mechanisms underlying the effects of ARC and molecular pathways involved in schizophrenia pathophysiology.

Ultrarare Loss-of-Function Mutations in the Genes Encoding the Ionotropic Glutamate Receptors of Kainate Subtypes Associated with Schizophrenia Disrupt the Interaction with PSD95.

Schizophrenia is a complex mental disorder with a genetic component. The GRIK gene family encodes ionotropic glutamate receptors of the kainate subtype, which are considered candidate genes for schizophrenia. We screened for rare and pathogenic mutations in the protein-coding sequences of the GRIK gene family in 516 unrelated patients with schizophrenia using the ion semiconductor sequencing method. We identified 44 protein-altered variants, and in silico analysis indicated that 36 of these mutations were rare and damaging or pathological based on putative protein function. Notably, we identified four truncating mutations, including two frameshift deletion mutations (GRIK1p.Phe24fs and GRIK1p.Thr882fs) and two nonsense mutations (GRIK2p.Arg300Ter and GRIK4p.Gln342Ter) in four unrelated patients with schizophrenia. They exhibited minor allele frequencies of less than 0.01% and were absent in 1517 healthy controls from Taiwan Biobank. Functional analysis identified these four truncating mutants as loss-of-function (LoF) mutants in HEK-293 cells. We also showed that three mutations (GRIK1p.Phe24fs, GRIK1p.Thr882fs, and GRIK2p.Arg300Ter) weakened the interaction with the PSD95 protein. The results suggest that the GRIK gene family harbors ultrarare LoF mutations in some patients with schizophrenia. The identification of proteins that interact with the kainate receptors will be essential to determine kainate receptor-mediated signaling in the brain.

Value of DSA in diagnosing ischemic cerebrovascular disease and evaluating therapeutic effect after in-tervention / 心血管康复医学杂志

Objective:To explore value of digital subtraction angiography(DSA)in diagnosing ischemic cerebrovascu-lar disease(ICD)and evaluating therapeutic effect after intervention.Methods:Clinical data of 134 ICD patients treated in our hospital from Jan 2014 to Jun 2015 were retrospectively analyzed.All patients received magnetic reso-nance angiography(MRA),CT angiography(CTA)and DSA before intervention,and diagnostic value of three methods for ischemic cerebrovascular lesion were compared;stenotic degree of vessels and score of United States National Institutes of Health Stroke Score(NIHSS)were compared between before and after intervention.Results:A total of 1340 vessels were checked in 134 patients.DSA discovered 341 lesioned vessels(25.45%),25 completely occluded vessels(1.87%),153 vessels of 70% ~99% stenosis(11.42%),61 vessels of 50% ~69% stenosis(4.55%) and 102 vessels of <50% stenosis(7.61%);compared with CTA and MRA,there were significant rise in detection rates of lesioned vessels(21.19%,19.40% vs.25.45%),50% ~69% stenosis vessels(3.06%,2.54% vs.4.55%) and <50% stenosis vessels(5.67%,4.93% vs.7.61%)in DSA,P<0.05 or <0.01. A total of 109 patients with 120 vessels received intervention,compared with before operation,there were significant reductions in stenotic rates of extracranial artery[(83.51 ± 8.74)% vs.(22.87 ± 7.90)%]and intracranial artery[(81.63 ± 9.02)% vs. (19.64 ± 6.18)%]after intervention,P=0.001 both.Compared with before intervention,there was significant re-duction in mean NIHSS score[(14.68 ± 3.18)scores vs.(3.16 ± 1.94)scores]on three months after intervention, P=0.001.Conclusion:Detection rate of vascular lesion by DSA is significantly higher than those of CTA and MRA in ICD patients.Postoperative therapeutic effect assessment using DSA possesses high value,which is worth exten-ding.