Biochemistry and Pharmacology - Research Publications
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ItemStimulating the Release of Exosomes Increases the Intercellular Transfer of Prions(ELSEVIER, 2016-03-04)Exosomes are small extracellular vesicles released by cells and play important roles in intercellular communication and pathogen transfer. Exosomes have been implicated in several neurodegenerative diseases, including prion disease and Alzheimer disease. Prion disease arises upon misfolding of the normal cellular prion protein, PrP(C), into the disease-associated isoform, PrP(Sc). The disease has a unique transmissible etiology, and exosomes represent a novel and efficient method for prion transmission. The precise mechanism by which prions are transmitted from cell to cell remains to be fully elucidated, although three hypotheses have been proposed: direct cell-cell contact, tunneling nanotubes, and exosomes. Given the reported presence of exosomes in biological fluids and in the lipid and nucleic acid contents of exosomes, these vesicles represent an ideal mechanism for encapsulating prions and potential cofactors to facilitate prion transmission. This study investigates the relationship between exosome release and intercellular prion dissemination. Stimulation of exosome release through treatment with an ionophore, monensin, revealed a corresponding increase in intercellular transfer of prion infectivity. Conversely, inhibition of exosome release using GW4869 to target the neutral sphingomyelinase pathway induced a decrease in intercellular prion transmission. Further examination of the effect of monensin on PrP conversion revealed that monensin also alters the conformational stability of PrP(C), leading to increased generation of proteinase K-resistant prion protein. The findings presented here provide support for a positive relationship between exosome release and intercellular transfer of prion infectivity, highlighting an integral role for exosomes in facilitating the unique transmissible nature of prions.
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ItemiSRAP - a one-touch research tool for rapid profiling of small RNA-seq data(TAYLOR & FRANCIS LTD, 2015)Small non-coding RNAs have been significantly recognized as the key modulators in many biological processes, and are emerging as promising biomarkers for several diseases. These RNA species are transcribed in cells and can be packaged in extracellular vesicles, which are small vesicles released from many biotypes, and are involved in intercellular communication. Currently, the advent of next-generation sequencing (NGS) technology for high-throughput profiling has further advanced the biological insights of non-coding RNA on a genome-wide scale and has become the preferred approach for the discovery and quantification of non-coding RNA species. Despite the routine practice of NGS, the processing of large data sets poses difficulty for analysis before conducting downstream experiments. Often, the current analysis tools are designed for specific RNA species, such as microRNA, and are limited in flexibility for modifying parameters for optimization. An analysis tool that allows for maximum control of different software is essential for drawing concrete conclusions for differentially expressed transcripts. Here, we developed a one-touch integrated small RNA analysis pipeline (iSRAP) research tool that is composed of widely used tools for rapid profiling of small RNAs. The performance test of iSRAP using publicly and in-house available data sets shows its ability of comprehensive profiling of small RNAs of various classes, and analysis of differentially expressed small RNAs. iSRAP offers comprehensive analysis of small RNA sequencing data that leverage informed decisions on the downstream analyses of small RNA studies, including extracellular vesicles such as exosomes.
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ItemDefining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery(TAYLOR & FRANCIS INC, 2017)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.
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ItemExosomes: vehicles for the transfer of toxic proteins associated with neurodegenerative diseases?(FRONTIERS MEDIA SA, 2012)Exosomes are small membranous vesicles secreted by a number of cell types including neurons and can be isolated from conditioned cell media or bodily fluids such as urine and plasma. Exosome biogenesis involves the inward budding of endosomes to form multivesicular bodies (MVB). When fused with the plasma membrane, the MVB releases the vesicles into the extracellular environment as exosomes. Proposed functions of these vesicles include roles in cell-cell signaling, removal of unwanted proteins, and the transfer of pathogens between cells. One such pathogen which exploits this pathway is the prion, the infectious particle responsible for the transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) of humans or bovine spongiform encephalopathy (BSE) of cattle. Similarly, exosomes are also involved in the processing of the amyloid precursor protein (APP) which is associated with Alzheimer's disease. Exosomes have been shown to contain full-length APP and several distinct proteolytically cleaved products of APP, including Aβ. In addition, these fragments can be modulated using inhibitors of the proteases involved in APP cleavage. These observations provide further evidence for a novel pathway in which PrP and APP fragments are released from cells. Other proteins such as superoxide dismutase I and alpha-synuclein (involved in amyotrophic lateral sclerosis and Parkinson's disease, respectively) are also found associated with exosomes. This review will focus on the role of exosomes in neurodegenerative disorders and discuss the potential of these vesicles for the spread of neurotoxicity, therapeutics, and diagnostics for these diseases.
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ItemThe detection of microRNA associated with Alzheimer's disease in biological fluids using next-generation sequencing technologies.(Frontiers Media SA, 2013)Diagnostic tools for neurodegenerative diseases such as Alzheimer's disease (AD) currently involve subjective neuropsychological testing and specialized brain imaging techniques. While definitive diagnosis requires a pathological brain evaluation at autopsy, neurodegenerative changes are believed to begin years before the clinical presentation of cognitive decline. Therefore, there is an essential need for reliable biomarkers to aid in the early detection of disease in order to implement preventative strategies. microRNAs (miRNA) are small non-coding RNA species that are involved in post-transcriptional gene regulation. Expression levels of miRNAs have potential as diagnostic biomarkers as they are known to circulate and tissue specific profiles can be identified in a number of bodily fluids such as plasma, CSF and urine. Recent developments in deep sequencing technology present a viable approach to develop biomarker discovery pipelines in order to profile miRNA signatures in bodily fluids specific to neurodegenerative diseases. Here we review the potential use of miRNA deep sequencing in biomarker identification from biological fluids and its translation into clinical practice.
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ItemSmall RNA deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells(OXFORD UNIV PRESS, 2012-11)Prion diseases are transmissible neurodegenerative disorders affecting both humans and animals. The cellular prion protein, PrP(C), and the abnormal infectious form, PrP(Sc), are found associated with exosomes, which are small 50-130 nm vesicles released from cells. Exosomes also contain microRNAs (miRNAs), a class of non-coding RNA, and have been utilized to identify miRNA signatures for diagnosis of disease. While some miRNAs are deregulated in prion-infected brain tissue, the role of miRNA in circulating exosomes released during prion disease is unknown. Here, we investigated the miRNA profile in exosomes released from prion-infected neuronal cells. We performed the first small RNA deep sequencing study of exosomes and demonstrated that neuronal exosomes contain a diverse range of RNA species including retroviral RNA repeat regions, messenger RNA fragments, transfer RNA fragments, non-coding RNA, small nuclear RNA, small nucleolar RNA, small cytoplasmic RNA, silencing RNA as well as known and novel candidate miRNA. Significantly, we show that exosomes released by prion-infected neuronal cells have increased let-7b, let-7i, miR-128a, miR-21, miR-222, miR-29b, miR-342-3p and miR-424 levels with decreased miR-146 a levels compared to non-infected exosomes. Overall, these results demonstrate that circulating exosomes released during prion infection have a distinct miRNA signature that can be utilized for diagnosis and understanding pathogenic mechanisms in prion disease.