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Rescue of mitochondrial dysfunction in Mucopolysaccharidosis VIA Disease by treatment with an antioxidant
combination
Mucopolysaccharidosis IVA (MPSIVA) (Morquio A syndrome) is a type of lysosomal storage disorder caused by
an autosomal recessive mutation in lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene. GALNS
aids in the breakdown of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). The lysosomal accumulation of
undegraded glycosaminoglycans (GAGs), specifically KS and C6S, in MPSIVA could result in defective fusion between
autophagosomes and lysosomes and therefore the blockage of autophagic pathways. This block in autophagy causes a
build-up of dysfunctional mitochondria and cellular stress, which may trigger an inflammatory response and contribute
to apoptosis1. The antioxidant lipoic acid (LA) enhances mitochondrial function3 in chronic oxidative diseases such as
diabetes. Our first approach was to gain insight on the oxidative and inflammatory nature of MPSIVA by quantifying the
effects of KS or C6S exposure on normal THP-1 macrophages in the presence or absence of intracellular toll-like receptor
(TLR)-4 inhibitor TAK-242. Although TLR-4 receptors serve a critical role in innate immunity by sensing pathogenassociated molecular patterns, TLR-4 receptors have been observed to be activated by various endogenous damageassociated molecular patterns (DAMPs), such as hyaluronan, which induces cytokine production2. KS/C6S loading were
measured using the 1,9-dimethylmethylene blue (DMMB) and also by visualized using light microscopy. TLR-4 receptor
expression, reactive oxygen species (ROS) and mitochondrial ROS (mtROS) production, and cell viability were measured
by flow cytometry. We predict our MPSIVA model of THP-1 cell exposure to KS and CS will show mitochondrial
dysfunction, inflammation and apoptosis, as similarly observed in MPSVI3.
Light microscopy revealed an increased buildup of large dark inclusions near the cell surface membrane in THP-1
macrophages with increasing concentrations of KS/C6S exposure; that was confirmed to be KS or CS by the DMMB assay.
It is possible that these large dark inclusions are KS/C6S-filled autophagosomes, that have failed to fuse with overloaded
lysosomes, although future studies could utilize vacuole-specific tags to verify this1.
Interestingly, we observed an upregulation of TLR-4 receptors in KS treated cells, but not in C6S treated cells,
suggesting a possible transient pro-inflammatory role of KS in the pathophysiology of MPSIVA. We suspect the
inflammatory nature of MPSIVA is attributed largely to KS (or partially digested fragments of KS/C6S) acting as DAMPs.
Both KS and C6S decreased THP-1 cells ROS production at low concentrations, but less so at higher
concentrations. The presence of TLR-4 inhibitor decreased the percentage of ROS positive cells at high concentration of
both KS and C6S exposed cells, suggesting a possible decrease in cytokine mediated ROS production. It is possible that
higher concentrations of KS and C6S act as DAMPs and trigger the production of pro-inflammatory cytokines, which
results in an increase in mitochondrial ROS (mtROS), dampening any anti-oxidative effects. Furthermore, KS exposed
cells displayed more mtROS and cell death compared to C6S exposed or control cells, which has not been shown before.
This further supports our notion of KS being responsible for the overall inflammatory nature of MPSIVA.
Thus far, exposure of LA to our THP-1 macrophages has not shown protection to KS induced ROS production, but
these experiments are ongoing. Our next goal is to start a long-term culture of cells exposed to slightly elevated levels of
KS and C6S and periodically monitor autophagy, ROS, TLR-4, and cell viability in these macrophages. Although
verification of certain aspects of this exploratory study are still needed to affirm the significance of these findings, we
hope our preliminary results contribute to the current understanding of the molecular basis of MPSIVA and assist in the
search for new adjunct treatments.
Acknowledgements: I would like to extend my heartfelt gratitude to the following people for making this project a reality:
Dr. Patrick Walter, Nick Slater, Michael Minkley, Johnny Luo, Daniel Williams, Brandon Gosselin, Markand Patel, Marlise
Chan, Tyler Woods, Leah Hohman, Dr. Perry Howard, Dr. Julian Lum, Dr. Robert Linhardt, and the kind donors of The
Canadian MPS Society.
References
1.
Settembre, C. et al. A block of autophagy in lysosomal storage disorders. Hum. Mol. Genet. 17, 119–129 (2008).
2.
Frey, H., Schroeder, N., Manon-Jensen, T., Iozzo, R. V & Schaefer, L. Biological interplay between proteoglycans
and their innate immune receptors in inflammation. FEBS J. 280, 2165–79 (2013).
3.
Tessitore, A., Pirozzi, M. & Auricchio, A. Abnormal autophagy, ubiquitination, inflammation and apoptosis are
dependent upon lysosomal storage and are useful biomarkers of mucopolysaccharidosis VI. Pathogenetics 2, 4
(2009).