Extracellular microRNAs in human circulation are associated with miRISC complexes that are accessible to anti-AGO2 antibody and can bind target mimic oligonucleotides.

MicroRNAs (miRNAs) function cell-intrinsically to regulate gene expression by base-pairing to complementary mRNA targets while in association with Argonaute, the effector protein of the miRNA-mediated silencing complex (miRISC). A relatively dilute population of miRNAs can be found extracellularly in body fluids such as human blood plasma and cerebrospinal fluid (CSF). The remarkable stability of circulating miRNAs in such harsh extracellular environments can be attributed to their association with protective macromolecular complexes, including extracellular vesicles (EVs), proteins such as Argonaut 2 (AGO2), or high-density lipoproteins. The precise origins and the potential biological significance of various forms of miRNA-containing extracellular complexes are poorly understood. It is also not known whether extracellular miRNAs in their native state may retain the capacity for miRISC-mediated target RNA binding. To explore the potential functionality of circulating extracellular miRNAs, we comprehensively investigated the association between circulating miRNAs and the miRISC Argonaute AGO2. Using AGO2 immunoprecipitation (IP) followed by small-RNA sequencing, we find that miRNAs in circulation are primarily associated with antibody-accessible miRISC/AGO2 complexes. Moreover, we show that circulating miRNAs can base-pair with a target mimic in a seed-based manner, and that the target-bound AGO2 can be recovered from blood plasma in an ∼1:1 ratio with the respective miRNA. Our findings suggest that miRNAs in circulation are largely contained in functional miRISC/AGO2 complexes under normal physiological conditions. However, we find that, in human CSF, the assortment of certain extracellular miRNAs into free miRISC/AGO2 complexes can be affected by pathological conditions such as amyotrophic lateral sclerosis.

MicroRNA-137/181c regulates serine palmitoyltransferase and in turn amyloid ß, novel targets in sporadic Alzheimer's disease.

The contribution of mutations in amyloid precursor protein (APP) and presenilin (PSEN) to familial Alzheimer's disease (AD) is well established. However, little is known about the molecular mechanisms leading to amyloid ß (Aß) generation in sporadic AD. Increased brain ceramide levels have been associated with sporadic AD, and are a suggested risk factor. Serine palmitoyltransferase (SPT) is the first rate-limiting enzyme in the de novo ceramide synthesis. However, the regulation of SPT is not yet understood. Evidence suggests that it may be posttranscriptionally regulated. Therefore, we investigated the role of miRNAs in the regulation of SPT and amyloid ß (Aß) generation. We show that SPT is upregulated in a subgroup of sporadic AD patient brains. This is further confirmed in mouse model studies of risk factors associated with AD. We identified that the loss of miR-137, -181c, -9, and 29a/b-1 increases SPT and in turn Aß levels, and provides a mechanism for the elevated risk of AD associated with age, high-saturated-fat diet, and gender. Finally, these results suggest SPT and the respective miRNAs may be potential therapeutic targets for sporadic AD.

Immunovirotherapy with measles virus strains in combination with anti-PD-1 antibody blockade enhances antitumor activity in glioblastoma treatment.

Background: Glioblastoma (GBM) is the most common primary malignant brain tumor and has a dismal prognosis. Measles virus (MV) therapy of GBM is a promising strategy due to preclinical efficacy, excellent clinical safety, and its ability to evoke antitumor pro-inflammatory responses. We hypothesized that combining anti- programmed cell death protein 1 (anti-PD-1) blockade and MV therapy can overcome immunosuppression and enhance immune effector cell responses against GBM, thus improving therapeutic outcome. Methods: In vitro assays of MV infection of glioma cells and infected glioma cells with mouse microglia ± aPD-1 blockade were established to assess damage associated molecular pattern (DAMP) molecule production, migration, and pro-inflammatory effects. C57BL/6 or athymic mice bearing syngeneic orthotopic GL261 gliomas were treated with MV, aPD-1, and combination treatment. T2* weighted immune cell-specific MRI and fluorescence activated cell sorting (FACS) analysis of treated mouse brains was used to examine adaptive immune responses following therapy. Results: In vitro, MV infection induced human GBM cell secretion of DAMP (high-mobility group protein 1, heat shock protein 90) and upregulated programmed cell death ligand 1 (PD-L1). MV infection of GL261 murine glioma cells resulted in a pro-inflammatory response and increased migration of BV2 microglia. In vivo, MV+aPD-1 therapy synergistically enhanced survival of C57BL/6 mice bearing syngeneic orthotopic GL261 gliomas. MRI showed increased inflammatory cell influx into the brains of mice treated with MV+aPD-1; FACS analysis confirmed increased T-cell influx predominantly consisting of activated CD8+ T cells. Conclusions: This report demonstrates that oncolytic measles virotherapy in combination with aPD-1 blockade significantly improves survival outcome in a syngeneic GBM model and supports the potential of clinical/translational strategies combining MV with αPD-1 therapy in GBM treatment.

MiR-31 and miR-128 regulates poliovirus receptor-related 4 mediated measles virus infectivity in tumors.

Oncolytic measles virus strains are currently being evaluated in several clinical trials, as a promising novel oncolytic platform. Poliovirus receptor-related 4 (PVRL4) was recently identified as a potent measles virus (MV) receptor; however, its regulation is not yet understood. Increased levels of PVRL4 protein were observed in cell membrane, cytoplasm and nuclei of glioblastoma, breast and ovarian tumor clinical samples with no significant change in PVRL4 mRNA levels in glioblastoma and breast cancer compared with their corresponding control samples, suggesting that PVRL4 is likely post-transcriptionally regulated. Therefore, we sought to investigate the potential role of miRNAs in PVRL4 regulation and thus MV infectivity. We demonstrated that miR-31 and miR-128 can bind to the 3'UTR of PVRL4 and decrease PVRL4 levels while anti-miR-31/128 increase PVRL4 levels suggesting that PVRL4 is miRNA targeted. Furthermore, miR-31/128 expression levels were down-regulated in glioblastoma and breast tumor samples and showed significant negative correlations with PVRL4 levels. Infection with an MV strain that exclusively utilizes PVRL4 as its receptor showed that over-expression of miR-31/128 decreases MV infectivity while inhibition of the respective miRNAs via anti-miRs increase MV infectivity and reduce tumor size in mouse xenograft models of glioblastoma, breast and ovarian cancer. Additionally, miR-128 levels showed significant correlations with MV infection and in vivo anti-tumor effect, while MV infection increased miR-31 expression and thereby contributed to the observed decrease in PVRL4 levels. This study suggests that PVRL4 is post-transcriptionally regulated by miR-128 and miR-31 and harbors possible miRNA targets that could modulate MV infectivity and in turn enhance MV based oncolytic therapeutic strategies.

Inhibition of serine palmitoyltransferase reduces Aß and tau hyperphosphorylation in a murine model: a safe therapeutic strategy for Alzheimer's disease.

The contribution of the autosomal dominant mutations to the etiology of familial Alzheimer's disease (AD) is well characterized. However, the molecular mechanisms contributing to sporadic AD are less well understood. Increased ceramide levels have been evident in AD patients. We previously reported that increased ceramide levels, regulated by increased serine palmitoyltransferase (SPT), directly mediate amyloid ß (Aß) levels. Therefore, we inhibited SPT in an AD mouse model (TgCRND8) through subcutaneous administration of L-cylcoserine. The cortical Aß42 and hyperphosphorylated tau levels were down-regulated with the inhibition of SPT/ceramide. Positive correlations were observed among cortical SPT, ceramide, and Aß42 levels. With no evident toxic effects observed, inhibition of SPT could be a safe therapeutic strategy to ameliorate the AD pathology. We previously observed that miR-137, -181c, -9, and 29a/b post-transcriptionally regulate SPT levels, and the corresponding miRNA levels in the blood sera are potential diagnostic biomarkers for AD. Here, we observe a negative correlation between cortical Aß42 and sera Aß42, and a positive correlation between cortical miRNA levels and sera miRNA levels suggesting their potential as noninvasive diagnostic biomarkers.

Curcumin-induced upregulation of the anti-tau cochaperone BAG2 in primary rat cortical neurons.

Alzheimer's disease (AD) is a progressive, neurodegenerative disease characterized by extracellular deposits of amyloid beta (Aß) protein and intracellular neurofibrillary tangles of hyperphosphorylated tau protein. Various studies suggest that the tau tangle pathology, which lies downstream to Aß pathology, is essential to produce AD-associated clinical phenotype and thus treatments targeting tau pathology may prevent or delay disease progression effectively. In this context, our present study examined three polyphenol compounds (curcumin, EGCG and resveratrol) for their possible activity against two endogenous proteins (BAG2 and LAMP1) that are shown to play a vital role in clearing tau tangles from neurons. Human epidemiological and animal data suggest potential positive effects of these polyphenols against AD. Here, primary rat cortical neurons treated with these polyphenols significantly up-regulated BAG2 levels at different concentrations, while only EGCG upregulated LAMP1 levels, although at higher concentrations. Importantly, curcumin doubled BAG2 levels at low micromolar concentrations that are clinically relevant. In addition, curcumin also downregulated levels of phosphorylated tau, which may be potentially attributed to the curcumin-induced upregulation in BAG2 levels in the neurons. The present results demonstrate novel activity of polyphenol curcumin in up-regulating an anti-tau cochaperone BAG2 and thus, suggest probable benefit of curcumin against AD-associated tauopathy.

Blood serum miRNA: non-invasive biomarkers for Alzheimer's disease.

There is an urgent need to identify non-invasive biomarkers for the detection of sporadic Alzheimer's disease (AD). We previously studied microRNAs (miRNAs) in AD autopsy brain samples and reported a connection between miR-137, -181c, -9, -29a/b and AD, through the regulation of ceramides. In this study, the potential role of these miRNAs as diagnostic markers for AD was investigated. We identified that these miRNAs were down-regulated in the blood serum of probable AD patients. The levels of these miRNAs were also reduced in the serum of AD risk factor models. Although the ability of these miRNAs to conclusively diagnose for AD is currently unknown, our findings suggest a potential use for circulating miRNAs, along with other markers, as non-invasive and relatively inexpensive biomarkers for the early diagnosis of AD, however, with further research and validation.