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Tauopathies are a class of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia and progressive supranuclear palsy, which are associated with the pathological aggregation of tau protein into neurofibrillary tangles (NFT). Studies have characterized tau as a "prion-like" protein given its ability to form distinct, stable amyloid conformations capable of transcellular and multigenerational propagation in clonal fashion. It has been proposed that progression of tauopathy could be due to the prion-like propagation of tau, suggesting the possibility that end-stage pathologies, like NFT formation, may require an instigating event such as tau seeding. To investigate this, we applied a novel human induced pluripotent stem cell (hiPSC) system we have developed to serve as a human neuronal model. We introduced the tau repeat domain (tau-RD) with P301L and V337M (tau-RD-LM) mutations into hiPSC-derived neurons and observed expression of tau-RD at levels similar to total tau in postmortem AD brains. Tau aggregation occurred without the addition of recombinant tau fibrils. The conditioned media from tau-RD cultures contained tau-RD seeds, which were capable of inducing aggregate formation in homotypic mode in non-transduced recipient neuronal cultures. The resultant NFTs were thioflavin-positive, silver stain-positive, and assumed fibrillary appearance on transmission electron microscopy (TEM) with immunogold, which revealed paired helical filament 1 (PHF1)-positive NFTs, representing possible recruitment of endogenous tau in the aggregates. Functionally, expression of tau-RD caused neurotoxicity that manifested as axon retraction, synaptic density reduction, and enlargement of lysosomes. The results of our hiPSC study were reinforced by the observation that Tau-RD-LM is excreted in exosomes, which mediated the transfer of human tau to wild-type mouse neurons in vivo. Our hiPSC human neuronal system provides a model for further studies of tau aggregation and pathology as well as a means to study transcellular propagation and related neurodegenerative mechanisms.

To develop a cerebrospinal fluid (CSF) miRNA diagnostic biomarker for glioblastoma.Glioblastoma tissue and matched CSF from the same patient (obtained prior to tumor manipulation) were profiled by TaqMan OpenArray® Human MicroRNA Panel. CSF miRNA profiles from glioblastoma patients and controls were created from three discovery cohorts and confirmed in two validation cohorts.miRNA profiles from clinical CSF correlated with those found in glioblastoma tissues. Comparison of CSF miRNA profiles between glioblastoma patients and non-brain tumor patients yielded a tumor "signature" consisting of nine miRNAs. The "signature" correlated with glioblastoma tumor volume (p=0.008). When prospectively applied to cisternal CSF, the sensitivity and specificity of the 'signature' for glioblastoma detection were 67% and 80%, respectively. For lumbar CSF, the sensitivity and specificity of the signature were 28% and 95%, respectively. Comparable results were obtained from analyses of CSF extracellular vesicles (EVs) and crude CSF.We report a CSF miRNA signature as a "liquid biopsy" diagnostic platform for glioblastoma.

We examined the extracellular vesicle (EV) and RNA composition of pooled normal cerebrospinal fluid (CSF) samples and CSF from five major neurological disorders: Alzheimer's disease (AD), Parkinson's disease (PD), low-grade glioma (LGG), glioblastoma multiforme (GBM), and subarachnoid haemorrhage (SAH), representing neurodegenerative disease, cancer, and severe acute brain injury. We evaluated: (I) size and quantity of EVs by nanoparticle tracking analysis (NTA) and vesicle flow cytometry (VFC), (II) RNA yield and purity using four RNA isolation kits, (III) replication of RNA yields within and between laboratories, and (IV) composition of total and EV RNAs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNASeq). The CSF contained ~106 EVs/μL by NTA and VFC. Brain tumour and SAH CSF contained more EVs and RNA relative to normal, AD, and PD. RT-qPCR and RNASeq identified disease-related populations of microRNAs and messenger RNAs (mRNAs) relative to normal CSF, in both total and EV fractions. This work presents relevant measures selected to inform the design of subsequent replicative CSF studies. The range of neurological diseases highlights variations in total and EV RNA content due to disease or collection site, revealing critical considerations guiding the selection of appropriate approaches and controls for CSF studies.

BackgroundTumor specific genetic material can be detected in extracellular vesicles (EVs) isolated from blood, cerebrospinal fluid (CSF), and other biofluids of glioblastoma patients. As such, EVs have emerged as a promising platform for biomarker discovery. However, the optimal procedure to transport clinical EV samples remains poorly characterized.MethodsWe examined the stability of EVs isolated from CSF of glioblastoma patients that were stored under different conditions. EV recovery was determined by Nanoparticle tracking analysis, and qRT-PCR was performed to determine the levels of miRNAs.ResultsCSF EVs that were lyophilized and stored at room temperature (RT) for seven days exhibited a 37-43% reduction in EV number. This reduction was further associated with decreased abundance of representative miRNAs. In contrast, the EV number and morphology remained largely unchanged if CSF were stored at RT. Total RNA and representative miRNA levels were well-preserved under this condition for up to seven days. A single cycle of freezing and thawing did not significantly alter EV number, morphology, RNA content, or miRNA levels. However, incremental decreases in these parameters were observed after two cycles of freezing and thawing.ConclusionsThese results suggest that EVs in CSF are stable at RT for at least seven days. Repeated cycles of freezing/thawing should be avoided to minimize experimental artifacts.

IntroductionLevels of Alzheimer's disease (AD)-related proteins in plasma neuronal derived exosomes (NDEs) were quantified to identify biomarkers for prediction and staging of mild cognitive impairment (MCI) and AD.MethodsPlasma exosomes were extracted, precipitated, and enriched for neuronal source by anti-L1CAM antibody absorption. NDEs were characterized by size (Nanosight) and shape (TEM) and extracted NDE protein biomarkers were quantified by ELISAs. Plasma NDE cargo was injected into normal mice, and results were characterized by immunohistochemistry to determine pathogenic potential.ResultsPlasma NDE levels of P-T181-tau, P-S396-tau, and Aβ1-42 were significantly higher, whereas those of neurogranin (NRGN) and the repressor element 1-silencing transcription factor (REST) were significantly lower in AD and MCI converting to AD (ADC) patients compared to cognitively normal controls (CNC) subjects and stable MCI patients. Mice injected with plasma NDEs from ADC patients displayed increased P-tau (PHF-1 antibody)-positive cells in the CA1 region of the hippocampus compared to plasma NDEs from CNC and stable MCI patients.ConclusionsAbnormal plasma NDE levels of P-tau, Aβ1-42, NRGN, and REST accurately predict conversion of MCI to AD dementia. Plasma NDEs from demented patients seeded tau aggregation and induced AD-like neuropathology in normal mouse CNS.

Analysis of extracellular vesicles (EVs) derived from plasma or cerebrospinal fluid (CSF) has emerged as a promising biomarker platform for therapeutic monitoring in glioblastoma patients. However, the contents of the various subpopulations of EVs in these clinical specimens remain poorly defined. Here we characterize the relative abundance of miRNA species in EVs derived from the serum and cerebrospinal fluid of glioblastoma patients. EVs were isolated from glioblastoma cell lines as well as the plasma and CSF of glioblastoma patients. The microvesicle subpopulation was isolated by pelleting at 10,000×g for 30 min after cellular debris was cleared by a 2000×g (20 min) spin. The exosome subpopulation was isolated by pelleting the microvesicle supernatant at 120,000×g (120 min). qRT-PCR was performed to examine the distribution of miR-21, miR-103, miR-24, and miR-125. Global miRNA profiling was performed in select glioblastoma CSF samples. In plasma and cell line derived EVs, the relative abundance of miRNAs in exosome and microvesicles were highly variable. In some specimens, the majority of the miRNA species were found in exosomes while in other, they were found in microvesicles. In contrast, CSF exosomes were enriched for miRNAs relative to CSF microvesicles. In CSF, there is an average of one molecule of miRNA per 150-25,000 EVs. Most EVs derived from clinical biofluids are devoid of miRNA content. The relative distribution of miRNA species in plasma exosomes or microvesicles is unpredictable. In contrast, CSF exosomes are the major EV compartment that harbor miRNAs.

Tryptophan is an essential amino acid that is required for normal development in Chlamydia species, and tryptophan metabolism has been implicated in chlamydial persistence and tissue tropism. The ability to synthesize tryptophan is not universal among the Chlamydiaceae, but species that have a predicted tryptophan biosynthetic pathway also encode an ortholog of TrpR, a regulator of tryptophan metabolism in many gram-negative bacteria. We show that in Chlamydia trachomatis serovar D, TrpR regulates its own gene and trpB and trpA, the genes for the two subunits of tryptophan synthase. These three genes form an operon that is transcribed by the major form of chlamydial RNA polymerase. TrpR acts as a tryptophan-dependent aporepressor that binds specifically to operator sequences upstream of the trpRBA operon. We also found that TrpR repressed in vitro transcription of trpRBA in a promoter-specific manner, and the level of repression was dependent upon the concentrations of TrpR and tryptophan. Our findings provide a mechanism for chlamydiae to sense changes in tryptophan levels and to respond by modulating expression of the tryptophan biosynthesis genes, and we present a unified model that shows how C trachomatis can combine transcriptional repression and attenuation to regulate intrachlamydial tryptophan levels. In the face of host defense mechanisms that limit tryptophan availability from the infected cell, the ability to maintain homeostatic control of intrachlamydial tryptophan levels is likely to play an important role in chlamydial pathogenesis.

Chlamydia trachomatis is an obligate intracellular bacterium that is dependent on its host cell for nucleotides. Chlamydia imports ribonucleotide triphosphates (NTPs) but not deoxyribonucleotide triphosphates (dNTPs) and instead uses ribonucleotide reductase to convert imported ribonucleotides into deoxyribonucleotides for DNA synthesis. The genes encoding ribonucleotide reductase have been recently shown to be negatively controlled by a conserved regulator called NrdR. In this study, we provide direct evidence that Escherichia coli NrdR is a transcriptional repressor and that C. trachomatis CT406 encodes its chlamydial ortholog. We showed that CT406 binds specifically to two NrdR boxes upstream of the nrdAB operon in C. trachomatis. Using an in vitro transcription assay, we confirmed that these NrdR boxes function as an operator since they were necessary and sufficient for CT406-mediated repression. We validated our in vitro findings with reporter studies in E. coli showing that both E. coli NrdR and CT406 repressed transcription from the E. coli nrdH and C. trachomatis nrdAB promoters in vivo. This in vivo repression was reversed by hydroxyurea treatment. Since hydroxyurea inhibits ribonucleotide reductase and reduces intracellular deoxyribonucleotide levels, these results suggest that NrdR activity is modulated by a deoxyribonucleotide corepressor. [ABSTRACT FROM AUTHOR]

Introduction: Levels of Alzheimer's disease (AD)-related proteins in plasma neuronal derived exosomes (NDEs) were quantified to identify biomarkers for prediction and staging of mild cognitive impairment (MCI) and AD., Methods: Plasma exosomes were extracted, precipitated, and enriched for neuronal source by anti-L1CAM antibody absorption. NDEs were characterized by size (Nanosight) and shape (TEM) and extracted NDE protein biomarkers were quantified by ELISAs. Plasma NDE cargo was injected into normal mice, and results were characterized by immunohistochemistry to determine pathogenic potential., Results: Plasma NDE levels of P-T181-tau, P-S396-tau, and Aβ1-42 were significantly higher, whereas those of neurogranin (NRGN) and the repressor element 1-silencing transcription factor (REST) were significantly lower in AD and MCI converting to AD (ADC) patients compared to cognitively normal controls (CNC) subjects and stable MCI patients. Mice injected with plasma NDEs from ADC patients displayed increased P-tau (PHF-1 antibody)-positive cells in the CA1 region of the hippocampus compared to plasma NDEs from CNC and stable MCI patients., Conclusions: Abnormal plasma NDE levels of P-tau, Aβ1-42, NRGN, and REST accurately predict conversion of MCI to AD dementia. Plasma NDEs from demented patients seeded tau aggregation and induced AD-like neuropathology in normal mouse CNS.