Biochemistry and Pharmacology - Research Publications

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    Gene dysregulation is restored in the Parkinson's disease MPTP neurotoxic mice model upon treatment of the therapeutic drug Cu-II(atsm)
    Cheng, L ; Quek, CYJ ; Hung, LW ; Sharples, RA ; Sherratt, NA ; Barnham, KJ ; Hill, AF (NATURE PUBLISHING GROUP, 2016-03-01)
    The administration of MPTP selectively targets the dopaminergic system resulting in Parkinsonism-like symptoms and is commonly used as a mice model of Parkinson's disease. We previously demonstrated that the neuroprotective compound Cu(II)(atsm) rescues nigral cell loss and improves dopamine metabolism in the MPTP model. The mechanism of action of Cu(II)(atsm) needs to be further defined to understand how the compound promotes neuronal survival. Whole genome transcriptomic profiling has become a popular method to examine the relationship between gene expression and function. Substantia nigra samples from MPTP-lesioned mice were evaluated using whole transcriptome sequencing to investigate the genes altered upon Cu(II)(atsm) treatment. We identified 143 genes affected by MPTP lesioning that are associated with biological processes related to brain and cognitive development, dopamine synthesis and perturbed synaptic neurotransmission. Upon Cu(II)(atsm) treatment, the expression of 40 genes involved in promoting dopamine synthesis, calcium signaling and synaptic plasticity were restored which were validated by qRT-PCR. The study provides the first detailed whole transcriptomic analysis of pathways involved in MPTP-induced Parkinsonism. In addition, we identify key therapeutic pathways targeted by a potentially new class of neuroprotective agents which may provide therapeutic benefits for other neurodegenerative disorders.
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    Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery
    Quek, C ; Bellingham, SA ; Jung, C-H ; Scicluna, BJ ; Shambrook, MC ; Sharples, RA ; Cheng, L ; Hill, AF (TAYLOR & FRANCIS INC, 2017-01-01)
    Small non-coding RNAs (ncRNA), including microRNAs (miRNA), enclosed in exosomes are being utilised for biomarker discovery in disease. Two common exosome isolation methods involve differential ultracentrifugation or differential ultracentrifugation coupled with Optiprep gradient fractionation. Generally, the incorporation of an Optiprep gradient provides better separation and increased purity of exosomes. The question of whether increased purity of exosomes is required for small ncRNA profiling, particularly in diagnostic and biomarker purposes, has not been addressed and highly debated. Utilizing an established neuronal cell system, we used next-generation sequencing to comprehensively profile ncRNA in cells and exosomes isolated by these 2 isolation methods. By comparing ncRNA content in exosomes from these two methods, we found that exosomes from both isolation methods were enriched with miRNAs and contained a diverse range of rRNA, small nuclear RNA, small nucleolar RNA and piwi-interacting RNA as compared with their cellular counterparts. Additionally, tRNA fragments (30-55 nucleotides in length) were identified in exosomes and may act as potential modulators for repressing protein translation. Overall, the outcome of this study confirms that ultracentrifugation-based method as a feasible approach to identify ncRNA biomarkers in exosomes.