Gene Editing Used to Mimic Dysfunctional Neurodegenerative Calcium Transport

A team of neurodegenerative disease researchers working with various Alzheimer's disease model systems has found that expression levels of the STIM1 gene in brain tissues decreased with the progression of neurodegeneration.

STIM1 (Stromal interaction molecule 1) is an endoplasmic reticulum protein with a role in calcium ion mobilization and signaling. As a sensor of intraluminal Ca2+ levels, STIM1 modulates plasma membrane Ca2+ channels to regulate Ca2+ entry. In neuroblastoma SH-SY5Y cells and in familial Alzheimer’s disease patient skin fibroblasts, STIM1 is cleaved at the transmembrane domain by the enzyme presenilin-1-associated gamma-secretase, leading to dysregulation of calcium transport.

To study the role of STIM1 in neurodegeneration, investigators the University of Extremadura (Badajoz, Spain) designed an in vitro model to examine the phenotype of STIM1-deficient neuronal cells using the CRISPR/Cas9 genome-editing tool to knockout the expression of the STIM1 gene in the SH-SY5Y neuroblastoma cell line.

CRISPR/Cas9 is regarded as the cutting edge of molecular biology technology. CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. Since 2013, the CRISPR/Cas9 system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 enzyme and appropriate guide RNAs (sgRNAs) into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR/Cas9 system is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides that shepherd the Cas9 protein to the target gene on a DNA strand.

The investigators reported in the October 2018 issue of the Journal of Molecular Medicine that they had used a CRISPR-based method to generate STIM1-deficient SH-SY5Y cells by editing the STIM1 gene locus. Using these STIM1-KO cells, they found that STIM1 was not required for differentiation but was absolutely essential for cell survival in differentiating cells. In these cells, STIM1 loss triggered mitochondrial depolarization and senescence, two features of cell death not previously reported for STIM1-deficient cells.

"We use the CRISPR technique on a regular basis in the Faculty of Sciences at the University of Extremadura, as it has the great advantage that the genome remains stable despite the cuts in the DNA. In this specific case, the procedure CRISPR silences the gene, which permits the expression of the protein STIM1 in the neuron. Thus, we were able to simulate what occurs in the neuron without this protein, and we observed alterations very similar to those seen in tissues with Alzheimer's," said senior author Dr. Francisco Javier Martin-Romero, associate professor of biochemistry and molecular biology at the University of Extremadura. "This calcium is necessary for the cell to be fully viable, and an alteration in this process affects its entire physiology, finally causing its death. The cell is unable to halt the transport of calcium, which becomes unregulated and out of control."

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