IntroductionmicroRNA (miRNA) are small non-coding RNA species that are transcriptionally processed in the host cell and released extracellularly into the bloodstream. Normally involved in post-transcriptional gene silencing, the deregulation of miRNA has been shown to influence pathogenesis of a number of diseases.BackgroundNext-generation deep sequencing (NGS) has provided the ability to profile miRNA in biological fluids making this approach a viable screening tool to detect miRNA biomarkers. However, collection and handling procedures of blood needs to be greatly improved for miRNA analysis in order to reliably detect differences between healthy and disease patients. Furthermore, ribonucleases present in blood can degrade RNA upon collection rendering extracellular miRNA at risk of degradation. These factors have consequently decreased sensitivity and specificity of miRNA biomarker assays.MethodsHere, we use NGS to profile miRNA in various blood components and identify differences in profiles within peripheral blood compared to cell-free plasma or serum and extracellular vesicles known as exosomes. We also analyse and compare the miRNA content in exosomes prepared by ultracentrifugation methods and commercial exosome isolation kits including treating samples with RNaseA.ConclusionThis study demonstrates that exosomal RNA is protected by RNaseA treatment and that exosomes provide a consistent source of miRNA for disease biomarker detection.

Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid ␤-peptide (A␤) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu 2؉ or Zn 2؉ resulted in an ϳ85-90% reduction of secreted A␤-(1-40) and A␤-(1-42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted A␤ was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu 2؉ . MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu 2؉ also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of A␤-(1-40). Our findings identify an alternative mechanism of action for CQ in the reduction of A␤ deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients. Alzheimer disease (AD)4 is characterized by progressive neuronal dysfunction, reactive gliosis, and the formation of amyloid plaques in the brain. The major constituent of AD plaques is the amyloid ␤-peptide (A␤), which is cleaved from the membrane-bound amyloid precursor protein (APP) (1). Aggregated or oligomeric A␤ can induce neurotoxicity through pathways involving free radical production and increased neuronal oxidative stress (2). Among the factors capable of promoting A␤ aggregation in vivo, recent evidence supports a central role for biometals such as Cu 2ϩ and Zn 2ϩ in this process (3). An important factor in controlling A␤ accumulation in AD patients is the activity of A␤-degrading enzymes. Recent studies have identified several candidate proteases that may contribute to catabolism of A␤ in the brain. Neprilysin, insulin-degrading enzyme, angiotensin-converting enzyme, and matrix metalloproteases (MMPs) have all demonstrated A␤-degrading activity in vitro and/or in vivo (4 -6). Reduced activity of these or other A␤-degrading proteases with age may play a role in promoting accumulation and deposition of A␤ in AD patients. Development of strategies to enhance clearance of A␤ may lead to novel therapeutic treatments for AD patients.Promoting A␤ clearance may be achieved through modulating metal sequestration or metal-protein interactions. 5-Chloro-7-iodo-8-hydroxyquinoline or clioquinol (CQ), a disused antibiotic, has received considerable attention as a potential metal ligand in AD and Parkinson disease patients (7-9). Preliminary studies revealed that CQ rapidly and potently dissolved aggregates of synthetic or AD brain-derived A␤ in vitro (10). In subsequent animal studies, a 9-week oral treatment with CQ resulted in a 49% reduction of...

Dopamine (DA) and alpha-synuclein (alpha-SN) are two key molecules associated with Parkinson's disease (PD). We have identified a novel action of DA in the initial phase of alpha-SN aggregation and demonstrate that DA induces alpha-SN to form soluble, SDS-resistant oligomers. The DA:alpha-SN oligomeric species are not amyloidogenic as they do not react with thioflavin T and lack the typical amyloid fibril structures as visualized with electron microscopy. Circular dichroism studies indicate that in the presence of lipid membranes DA interacts with alpha-SN, causing an alteration to the structure of the protein. Furthermore, DA inhibited the formation of iron-induced alpha-SN amyloidogenic aggregates, suggesting that DA acts as a dominant modulator of alpha-SN aggregation. These observations support the paradigm emerging for other neurodegenerative diseases that the toxic species is represented by a soluble oligomer and not the insoluble fibril.

There is no consensus for a blood-based test for the early diagnosis of Alzheimer's disease (AD). Expression profiling of small non-coding RNA's, microRNA (miRNA), has revealed diagnostic potential in human diseases. Circulating miRNA are found in small vesicles known as exosomes within biological fluids such as human serum. The aim of this work was to determine a set of differential exosomal miRNA biomarkers between healthy and AD patients, which may aid in diagnosis. Using next-generation deep sequencing, we profiled exosomal miRNA from serum (N=49) collected from the Australian Imaging, Biomarkers and Lifestyle Flagship Study (AIBL). Sequencing results were validated using quantitative reverse transcription PCR (qRT-PCR; N=60), with predictions performed using the Random Forest method. Additional risk factors collected during the 4.5-year AIBL Study including clinical, medical and cognitive assessments, and amyloid neuroimaging with positron emission tomography were assessed. An AD-specific 16-miRNA signature was selected and adding established risk factors including age, sex and apolipoprotein ɛ4 (APOE ɛ4) allele status to the panel of deregulated miRNA resulted in a sensitivity and specificity of 87% and 77%, respectively, for predicting AD. Furthermore, amyloid neuroimaging information for those healthy control subjects incorrectly classified with AD-suggested progression in these participants towards AD. These data suggest that an exosomal miRNA signature may have potential to be developed as a suitable peripheral screening tool for AD.

STING is an innate immune cytosolic adaptor for DNA sensors that engage malaria parasite (Plasmodium falciparum) or other pathogen DNA. As P. falciparum infects red blood cells and not leukocytes, how parasite DNA reaches such host cytosolic DNA sensors in immune cells is unclear. Here we show that malaria parasites inside red blood cells can engage host cytosolic innate immune cell receptors from a distance by secreting extracellular vesicles (EV) containing parasitic small RNA and genomic DNA. Upon internalization of DNA-harboring EVs by human monocytes, P. falciparum DNA is released within the host cell cytosol, leading to STING-dependent DNA sensing. STING subsequently activates the kinase TBK1, which phosphorylates the transcription factor IRF3, causing IRF3 to translocate to the nucleus and induce STING-dependent gene expression. This DNA-sensing pathway may be an important decoy mechanism to promote P. falciparum virulence and thereby may affect future strategies to treat malaria.