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Drug screening lysosomal storage diseases with a focus on CLN8 disease

Badell Grau, Rafael Andres 2020. Drug screening lysosomal storage diseases with a focus on CLN8 disease. PhD Thesis, Cardiff University.
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The neuronal ceroid lipofuscinoses (NCLs) are a group of childhood-onset neurodegenerative lysosomal storage diseases (LSDs) characterised by the accumulation of autofluorescent lipopigment in a variety of cells and tissue, particularly neurons. LSDs are inherited rare diseases that arise from mutations in genes encoding endo-lysosomal proteins, causing the intra-lysosomal accumulation of material such as lipids, proteins and polysaccharides. To develop therapies to mitigate these debilitating diseases, robust cell-based drug screens using cellular phenotypes can be developed. These phenotypes can also provide therapeutic targets. This project explores cellular phenotypes in CLN8 patient cells by first investigating common LSD phenotypes such as lysosomal storage, autophagy defects, ER stress, Ca2+ signalling and defects in lysosomal enzyme activities. CLN8 disease had a high degree of unexplained phenotypic variability between and within patient cell lines which was not observed with other LSD patient cells. Of the four CLN8 patient cell lines explored, one displayed some robust non-variable phenotypes, resulting in deeper phenotypic exploration of this cell line which identified defects in Ca2+ homeostasis such as decreased lysosomal Ca2+. Due to the high phenotypic variability in CLN8 patient cells, another method for compound screening was required. Niemann-Pick C1 (NPC1) disease was used as a model for all LSDs for the screening of potential therapeutic compounds given its extent of characterisation. Despite this, however, approved small molecules or those in clinical testing for NPC disease are often neglected by high-throughput screening (HTS) approaches. This project therefore aimed to uncover the reasons behind these failings by first validating a fast-growing NPC1 cell model with robust phenotypes to facilitate rapid drug screening and 2) exploring a whole range of variables that might explain why approved drugs are being missed. The ultimate aim is to deliver an improved approach for an HTS platform applicable to all LSDs. This project then explored alternative drug screens using control cell material to identify drugs of benefit to CLN8 and possibly other LSDs. Previous studies have reported defects in galactosylceramidase (GALC) activity in CLN8 disease, mutations in which are known to cause Krabbe disease, another LSD. A high-throughput enzymatic drug screen to identify compounds that modulate GALC activity was iv therefore prioritised. This identified ambroxol, ibudilast and mycophenolate mofetil as potential GALC chaperones and thus may be of benefit to LDSs such as Krabbe disease and CLN8 disease. Finally, currently approved small molecule modulators of other LSDs or neutraceutical compounds that could be repurposed and used to treat CLN8 were tested. Miglustat, which reduces lysosomal storage of glycosphingolipids, was tested in CLN8, CLN2 and CLN7 patient cells. The potential effects of miglustat were explored using cellular phenotypes such as lysosomal volume, autophagic compartment, ganglioside GM1 storage and mis-localisation, mitochondrial Ca2+ and ER Ca2+. Potential benefits to CLN7, CLN8 and particularly CLN2 patient cells was identified which merits further investigation in these diseases. Curcumin is a neutraceutical that increases cytosolic Ca2+. The potential benefit of two common curcumin nanoformulations (BCM95s and SLNL) on LSDs such as Niemann-Pick A, CLN3, CLN7, CLN8 and CLN10 were explored by investigating their effect on lysosomal volume and cholesterol accumulation. BCM95s may be of benefit to LSDs by overcoming reduced lysosomal Ca2+ levels in CLN7, CLN8 and possibly CLN10. SLNL was conversely found to be cytotoxic due to a combination of the nanoparticle fatty acid lipid mix in conjunction with curcumin, detrimentally affecting the mitochondria. This highlights the importance of exploring the interaction between active components and their excipient formulations as the excipient itself may be detrimental to some diseases and reduce the benefits conferred by the active compound in isolation. This is, to our knowledge, the first report of a detrimental effect of an excipient on LSDs. Overall, this study increased the understanding of the cellular pathologies underlying CLN8 disease, validated and characterised a fast-growing cellular model that may be used for high-throughput drug screening, identified compounds that may act as possible GALC chaperones, found that miglustat may be of benefit to certain NCLs and determined that curcumin may be beneficial to LSDs with decreased lysosomal Ca2+, but that care should be taken with certain drug formulations. Together, this provides an advance in the identification of therapies for these debilitating diseases, and the screening of these compounds highlighted herein to progress toward the improvement of treatment and patient lives.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Biosciences
Subjects: Q Science > Q Science (General)
Date of First Compliant Deposit: 21 January 2021
Last Modified: 22 Jan 2021 14:59

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