by Ciaran Farley
What’s your idea of Staten Island? A landfill wasteland, deplete of intelligent life? And what’s your view of research? A mad scientist mixing test tubes in a lab? If one looks carefully, both are untrue. The Institute of Basic Research in Developmental Disabilities is located in the heart of Staten Island, and its scientists have made breakthroughs in autism, down syndrome, and epilepsy. One lab is now taking on Batten Disease, a terrible disease that is criminally under-studied.
What is Batten Disease?
The group of diseases known as Batten Disease, also called Neuronal Ceroid Lipofuscinoses (NCL), destroys the brain. They cause loss of motor function, epilepsy, blindness, and, eventually, death (Kousi, Lehesjoki, & Mole, 2011). Mutations in fourteen CLN family genes can each cause a different NCL (Mole & Cotman, 2015). Although this set of diseases is devastating, biomedical research has neglected NCL because of its rarity. Dr. Marius Walus is trying to change that. He studies a NCL called CLN2 disease, otherwise known as Late Infantile NCL. His research has the potential to revolutionize the analysis of this NCL and others.
As suggested by Late Infantile, this version of the disease usually appears between two and four years of age. There have been a few cases reported in children six to ten years old and even fewer cases in those less than one year old. In all cases, death usually arrives by the ill’s fifteenth birthdays (Kousi, Lehesjoki, & Mole, 2011).There are 116 known mutations of the CLN2 gene today, but two alone account for about 57% of Late Infantile NCL case. 55% of Late Infantile NCL patients are heterozygous for mutations in the CLN2 gene, and the rest are homozygous (Mole & Cotman, 2015). These changes in the gene affect the structure and functionality of TPP1, causing symptoms (Kousi, Lehesjoki, & Mole, 2011). This 563 amino acid protein degrades peptides in lysosomes. Defunct TPP1 leads to the buildup of ATP synthase’s subunit c; this problem, which can happen in several ways, as seen in other variants of NCL (Cooper, Tarczyluk, & Nelvagal, 2015). Over time, this accumulation brings the travesties associated with NCL (Kousi, Lehesjoki, & Mole, 2011).
What’s being done about this at the IBR?
Dr. Marius Walus’s work aims to analyze Late Infantile NCL in a whole new way. Scientists currently use genetically engineered mice to study the holistic effects of the disease. For Late Infantile NCL, this may be insufficient because mice with CLN2 mutations exhibit less severe symptoms than expected (Cooper, Tarczyluk, & Nelvagal, 2015). This and the results of some researchers studying the macroautophagy, the breakdown of cellular parts, of cells with CLN mutations led him to his current project. In a paper 2013 paper, researchers found that the cells with CLN causing mutations they studied recorded a ten-fold increase in ERK phosphorylation (Vidal-Donet, Cárcel-Trullols, Casanova, Aguado, & Knecht, 2013). ERK is a key pathway that relays signals outside the cell into directions for what DNA is made into proteins (Roux & Blenis, 2004). A 1000% increase is extremely significant and suggests that there might be a correlation between increased ERK phosphorylation and Late Infantile NCL. After making this observation, Dr. Walus decided to undertake his current project.
Figure from the eye-catching paper (Vidal-Donet, Cárcel-Trullols, Casanova, Aguado, & Knecht, 2013).
Dr. Walus’s goal is to create a line of cells that resemble the nerve cells of Late Infantile NCL patients to see if the results of the previously mentioned paper were reproducible. He selected SH-SY5Y as his cell line. These cells are “immortal;” they will divide in culture until they run out of room or nutrients. Since they also resemble nerve cells, they are perfect for this project (“SH-SY5Y (ATCC® CRL-2266™),” n.d.). To make them similar to Late Infantile NCL cells, Dr. Walus chose to knock out the CLN2 gene.
Picture of SH-SY5Y cells in culture (Ruiz, 2010)
He utilized two plasmids to accomplish this. One has CRISPR, a relatively new method for editing genes. The CRISPR genes on this plasmid code for proteins that take genes from the other plasmid and replace the CLN2 gene with its DNA. This second plasmid has the same DNA as what sandwiches the CLN2 gene, except an antibiotic resistance gene is located in between instead. Since CRISPR inserts the resistance gene where the CLN2 gene is, cells with successful transfections are similar to cells found in Late Infantile NCL patients and can be used for analysis.
Diagram of gene knockout
Dr. Walus uses electroporation to insert the plasmids. In this procedure, cells are shocked to create holes for the vectors to enter through. These holes close soon after the shock. Although this procedure is quick, cells can be killed or harmed beyond usefulness by the shocks (“Electroporation,” n.d.). It is important to note that after electroporation only the antibiotic resistance gene is inserted into the genome, so the CRISPR genes are not passed on. The resistance is useful because the cells with successful transfections will survive when the toxic chemical is added to the media, while the cells with no plasmids will die. This clever set up holds promise, but the results so far have been challenging.
Diagram of Plasmids
As with many endeavors in life, obstacles emerged in this project. Dr. Walus spent several months trying to find the best method of inserting the plasmids. Most chemical agents he used had success rates around 5%. He found that electroporation had a rate of about 50%. After choosing this procedure, he was able collect a decent amount of transfected SH-SY5Y through their resistance to hygromycine.
He then ran assays to determine whether or not the resistance genes had replaced the CLN2 gene or merely inserted somewhere else in the genome. He carried out Western blots to look for presence of the TPP1 protein that CLN2 is responsible for and inserted fluorescent antibodies that attach to TPP1. Both showed that the cells still expressed TPP1; the gene was not knocked out.
Although those results aren’t what was hoped for, Dr. Walus is still optimistic about the project’s future. He is working on creating a new plasmid that has a different resistance gene and plans to perform electroporation again. He feels that there are many chance events, like the resistance gene inserting into the wrong place, that have prevented success so far. He is confident that he will create his desired cell if he is persistent. If he is successful, he could lead a huge breakthrough for Late Infantile NCL and add to IBR’s list of accomplishments. Regardless of the outcome of this project, Dr. Walus and all of the other scientists’ hard work at IBR continue to better lives for those with developmental disabilities and give Staten Island something to be proud of.