PAX5‐Induced Expression of Endogenous Mucolipin‐2 (MCOLN2) Gene in Human Glial and Neuronal Cell Lines: A Potential Gene Complementation Therapy Approach for Mucolipidosis IV

Abstract

The loss of function in the Transient Receptor Potential Mucolipin‐1 (TRPML1) protein, encoded by the Mucolipin‐1 (MCOLN1) gene, is known to cause the neurodegenerative disease Mucolipidosis type IV (MLIV). Characteristics of MLIV are motor problems, cognitive dysfunction, achlorhydria, cataracts, and blindness caused by retinal cell death. The Ashkenazi Jew population is most afflicted by the disease, and MLIV has no known cure. The Mucolipin ion channels consist of TRPML1, ‐2, and ‐3 proteins, which are encoded by MCOLN1, ‐2, and ‐3 genes, respectively. They are known to serve as non‐selective cation channels located within endosomes and lysosomes and play a role in endosome‐lysosome fusion. Previous research indicates that TRPML1 and TRPML2 proteins share a high degree of homology, and we hypothesize that TRPML2, which has no known association to clinical disease, could substitute for the loss of functional TRPML1 in MLIV, resulting in a rescue of the disease phenotype. In order to study the potential therapeutic effects of TRPML2 in MLIV, we must first show that it is expressed in the brain, one of the tissues most affected by MLIV. We have previously discovered PAX5 as the transcriptional activator for MCOLN2 and have shown that MCOLN2 expression could be heterologously induced in both human neuroglioma (H4) cells and human embryonic kidney 293 (HEK‐293) cell lines. Previous reports demonstrated that MCOLN2 has tissue‐specific expression with little to no detectable transcripts in brain tissue. However, our preliminary research has detected endogenous levels of MCOLN2 transcripts in human H4 and neuroblastoma (SH‐SY5Y) cells. Moreover, to induce the expression of endogenous MCOLN2, we transduced SH‐SY5Y cells with adenovirus and lentivirus vectors containing the PAX5 cDNA. PAX5 serves as a B cell‐specific activator protein with tissue‐specific expression pattern as well. Standard reverse‐transcription polymerase chain reaction (RT‐PCR) and real‐time quantitative RT‐PCR both revealed MCOLN2 expression in SH‐SY5Y cells at 48 and 72 hours post‐transduction. We show that PAX5 transcriptionally activates and increases endogenous MCOLN2 expression in neuronal cells that are central to studying MLIV. Future studies include In‐Cell Western assay with an anti‐TRPML2 antibody and transcriptome analysis of PAX5‐treated cells to determine global gene expression. These results and future studies will further explore the therapeutic potential of gene complementation to rescue the cellular phenotype in MLIV disease.Support or Funding InformationThis work was partly funded by NIH R15 NS101594.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.