miRNAs In Cerebrospinal Fluid Research Articles

4564 Nilotinib alters microRNAs that regulate specific autophagy and ubiquitination genes in the cerebrospinal fluid of Parkinson’s patients

OBJECTIVES/GOALS: Our preclinical data demonstrate that the principal effects of nilotinib, a multi-tyrosine kinase inhibitor, in models of neurodegeneration is clearance of misfolded proteins via autophagy. Here we aimed to evaluate the effects of nilotinib on microRNAs in the cerebrospinal fluid of Parkinson’s disease patients. METHODS/STUDY POPULATION: Cerebrospinal fluid (CSF) was collected as part of an open label phase I (NCT02281474) (n = 12, 300 mg nilotinib taken orally once daily for 6 months), and a phase II randomized, double-blind, placebo-controlled study (NCT02954978) (n = 75, randomized 1:1:1 into placebo, 150 mg or 300 mg nilotinib taken orally once daily for 12 months). RNA was isolated from CSF and Indexed sequencing libraries were prepared from total RNA plus miRNA. Next generation whole-genome sequencing (single-end 1x75 bp, 25 million raw reads per sample) was performed to identify miRNAs significantly differentially expressed (fold-change ≥ 2, Benjamini-Hochberg FDR p-value ≤ 0.05 or Empirical Bayes FDR ≤ 0.05) with treatment compared to baseline. RESULTS/ANTICIPATED RESULTS: Next generation whole-genome sequencing of microRNAs in the CSF demonstrated that nilotinib significantly increases microRNAs that specifically regulate expression of autophagy and ubiquitination genes in individuals with Parkinson’s disease. In the open label phase I, samples, 28 microRNAs found to regulate autophagy and ubiquitination genes, were significantly altered with treatment (Benjamini-Hochberg FDR p-value ≤ 0.05). In the phase II randomized, double-blind, placebo-controlled study samples, we verified several of those 28 candidate microRNAs had been significantly deferentially expressed with treatment (Empirical Bayes FDR p-value ≤ 0.05). DISCUSSION/SIGNIFICANCE OF IMPACT: Our data provide robust evidence that nilotinib’s effects on misfolded protein clearance is via autophagy and CSF miRNA sequencing is a valid biomarker of nilotinib’s effects in a definitive phase III study to investigate nilotinib in Parkinson’s and other neurodegenerative diseases. CONFLICT OF INTEREST DESCRIPTION: Charbel Moussa is listed as an inventor on several Georgetown University patents for the use of tyrosine kinase inhibitors as a treatment for neurodegenerative diseases

Identification of Cerebrospinal Fluid MicroRNAs Associated With Leptomeningeal Metastasis From Lung Adenocarcinoma.

Background: Leptomeningeal metastasis (LM) has frequently been observed in patients with lung adenocarcinoma. So far, its diagnosis and disease course monitoring are still extremely difficult. Moreover, there is no effective treatment regimen for LM due to a lack knowledge on the molecular mechanism of LM. This study aimed to identify LM-related cerebrospinal fluid (CSF) miRNAs, which have potential value for diagnosing and monitoring LM and exploring the molecular mechanism.Methods: CSF miRNAs were screened and verified by microarray analysis and quantitative real-time PCR (qRT-PCR) in LM patients with lung adenocarcinoma and non-LM controls, and the diagnostic performance of candidate miRNAs was evaluated. Then, candidate miRNAs in matched CSF samples from LM patients at diagnosis, after initial therapy, at relapse, and after salvage therapy, were analyzed to assess the relationship between CSF miRNAs and LM disease course. The effect of candidate miRNAs on proliferation, invasion, and migration of lung adenocarcinoma cell lines was assessed. The targeted genes of the candidate miRNA were predicted by TargetScan, miRDB, and miRTarbase online analysis tools. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the functional categories of predicted target genes.Results: CSF miR-7975, miR-7977, and miR-7641 were screened and verified to be statistically significantly up-regulated in LM patients compared to non-LM controls. The three miRNAs, when combined, exhibited optimal diagnostic performance. Longitudinal data of CSF miR-7975 and miR-7977 correlated well with clinical courses of LM. Overexpression of miR-7977 promoted proliferation, migration, and invasion of lung adenocarcinoma cells. Moreover, 385 targeted genes of miR-7977 were predicted and were involved in various pathways related to cancer metastasis.Conclusions: This study offers insights for future research of CSF miRNAs as robust tools for diagnosing and monitoring LM. It also reveals a novel pathway for exploration of underlying mechanisms of LM.

MicroRNA expression in the cerebrospinal fluid of dogs with and without cervical spondylomyelopathy.

BackgroundOsseous‐associated cervical spondylomyelopathy (OA‐CSM) is a common condition of the cervical vertebral column that affects giant dog breeds. MicroRNAs (miRNAs) are small RNAs that regulate gene expression, and recent data suggest that circulating miRNAs present in biological fluids may serve as potential biomarkers for disease. The miRNA profiles of cerebrospinal fluid (CSF) from healthy dogs and dogs clinically affected by OA‐CSM have not been described.ObjectiveTo characterize the expression levels of miRNAs present in the CSF of normal Great Danes and identify differentially expressed miRNAs in the CSF of Great Danes clinically affected with OA‐CSM.AnimalsClient‐owned dogs: 12 control, 12 OA‐CSM affected.MethodsCerebrospinal fluid samples were collected prospectively. MicroRNA expression was evaluated using the NanoString nCounter platform and quantitative real‐time PCR.ResultsWe identified 8 miRNAs with significant differential expression. MiR‐299‐5p and miR‐765 had increased expression levels in the CSF of OA‐CSM‐affected dogs, whereas miR‐494, miR‐612, miR‐302‐d, miR‐4531, miR‐4455, and miR‐6721‐5p had decreased expression levels in OA‐CSM affected dogs compared to clinically normal dogs. Quantitative real‐time PCR was performed to validate the expression levels of 2 miRNAs (miR‐494 and miR‐612), and we found a 1.5‐fold increase in miR‐494 expression and a 1.2‐fold decrease in miR‐612 in the CSF of the OA‐CSM affected group (P = .41 and .89, respectively).Conclusions and Clinical ImportanceData generated from our study represent an initial characterization of the miRNA profile of normal canine CSF and suggest that a distinct CSF miRNA expression profile is associated with OA‐CSM.

Cerebrospinal Fluid MicroRNA Signatures as Diagnostic Biomarkers in Brain Tumors.

Central nervous system (CNS) malignancies include primary tumors that originate within the CNS as well as secondary tumors that develop as a result of metastatic spread. Circulating microRNAs (miRNAs) were found in almost all human body fluids including cerebrospinal fluid (CSF), and they seem to be highly stable and resistant to even extreme conditions. The overall aim of our study was to identify specific CSF miRNA patterns that could differentiate among brain tumors. These new biomarkers could potentially aid borderline or uncertain imaging results onto diagnosis of CNS malignancies, avoiding most invasive procedures such as stereotactic biopsy or biopsy. In total, 175 brain tumor patients (glioblastomas, low-grade gliomas, meningiomas and brain metastases), and 40 non-tumor patients with hydrocephalus as controls were included in this prospective monocentric study. Firstly, we performed high-throughput miRNA profiling (Illumina small RNA sequencing) on a discovery cohort of 70 patients and 19 controls and identified specific miRNA signatures of all brain tumor types tested. Secondly, validation of 9 candidate miRNAs was carried out on an independent cohort of 105 brain tumor patients and 21 controls using qRT-PCR. Based on the successful results of validation and various combination patterns of only 5 miRNA levels (miR-30e, miR-140, let-7b, mR-10a and miR-21-3p) we proposed CSF-diagnostic scores for each tumor type which enabled to distinguish them from healthy donors and other tumor types tested. In addition to this primary diagnostic tool, we described the prognostic potential of the combination of miR-10b and miR-196b levels in CSF of glioblastoma patients. In conclusion, we performed the largest study so far focused on CSF miRNA profiling in patients with brain tumors, and we believe that this new class of biomarkers have a strong potential as a diagnostic and prognostic tool in these patients.

P.372CSF miRNAs as biomarkers to indicate the clinical response to Spinraza treatment for patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality, due to Survival Motor Neuron gene 1 (SMN1) gene mutatinos. Spinraza (Nusinersen, an antisense-oligonucleotide (AON) drug, is the only approved treatment for SMA. Although the majority of patients show a significant clinical improvement in response to Spinraza, some show minimal or no improvement. We here aim to determine if micro-RNAs (miRNAs) in the cerebrospinal fluid (CSF) can be used as biomarkers to predict the clinical outcomes of type I SMA patients in response to Spinraza treatment. 13 miRNAs were identified in a discovery profiling performed in CSF samples of type I SMA patients at pre- (n=10) and 2 months (n=10) post-Spinraza treatment. Further validation was performed in CSF samples from clinical responders (n=8) and non-responding (n=5) type I SMA patients, based on the improvement in CHOP-INTEND motor scores at 10 months post- treatment. 5 miRNAs were significantly changed during the course of treatment between responder and non-responder groups. A miRNA-target gene protein interaction network was created. The shortest paths between candidate miRNAs and SMN1 and SMN2 genes were identified, the central targets identified using network centrality algorithms. 3 miRNAs were isolated with 10 central target proteins linking them to SMN with less than 2 intermediary nodes. Interestingly, some of the target proteins have been previously linked to SMA pathogenesis. Our data suggest that CSF miRNAs could play a role as biomarkers to monitor SMA patients' responses to Spinraza treatment. Encouragingly, the link between candidate miRNAs and the SMA-related target proteins suggests a functional relevance to the disease. This may allow the identification of novel pathways for therapeutic intervention.

Abstract 1806: Global analysis and validation of miRNA levels in cerebrospinal fluid of brain tumor patients

Abstract Introduction. Incidence rate of primary brain tumors and brain metastases counts yearly around 40 patients per 100 000 persons in the world and is still growing. Prognosis and therapy differs between brain tumor types and, therefore, accurate and early diagnosis might improve survival and life quality of patients. However, current diagnostic approaches are limited by localization and tissue heterogeneity of brain tumors. Cerebrospinal fluid (CSF) bathes central nervous system (CNS) and thus is supposed to reflect all pathological conditions. From this perspective, CSF looks as ideal source of diagnostic biomarkers of brain tumors. MicroRNAs, short non-coding RNAs involved in the pathogenesis of many cancers including brain tumors, might represent group of new biomarkers. In addition, dysregulated levels of brain tumor specific miRNAs have been already observed also in CSF. Following these facts, analysis of CSF miRNAs in brain tumor patients promises a new diagnostic approach enabling more accurate diagnosis. Material and methods. Next-generation sequencing was performed for analysis of small RNAs in 89 CSF samples taken from 32 GBM, 14 low-grade glioma (LGG), 11 meningioma, 13 brain metastasis patients and 19 non-tumor donors. CleanTag Small RNA Library Prep Kit (TriLink BioTechnologies) was used for cDNA library preparation. NextSeq 500 instrument together with Next 500/550 High Output v2 Kit - 75 cycles (both Illumina) were used for final sequencing analysis. Subsequently, according to NGS results we measured levels of 13 miRNAs in independent set of CSF samples (35 GBM, 42 meningiomas, 8 metastasis patients and 19 control) using TaqMan Advanced miRNA Assays (ThermoFisher Scientific). Results. NGS analysis revealed 25, 3, 2 and 14 CSF miRNAs significantly differently expressed in GBM, meningiomas, LGG and metastasis patients (p < 0.001, for LGG p < 0.05), respectively, in comparison with control CSF samples. In addition, 6 miRNAs showed different levels between GBM and LGG (p < 0.05). Subsequent validation of selected CSF miRNAs has confirmed different levels of 7 miRNAs in GBM (miR-10a, miR-196a, miR-196b, miR-30e, miR-30c, miR-7b, miR-7c), 3 in meningioma (miR-140, miR-21, miR-30e) and 2 in brain metastasis (miR-30e and miR-7c) compared to control CSF samples (p < 0.05). Validation of miRNA levels in LGG CSF samples is in progress and will be part of the poster presentation. Conclusion. We have observed that CSF from patients with various brain tumors (GBM, LGG, meningioma, and brain metastasis) is characterized by specific miRNA signature. Our results suggest potential of CSF miRNAs to be useful biomarkers in brain tumors. This work was supported by Ministry of Health of the Czech Republic grant nr. NV18-03-00398, grant of Czech Grant Agency nr. 17-17636S and by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601). Citation Format: Alena Kopkova, Jiri Sana, Marek Vecera, Tana Machackova, Pavel Fadrus, Julia Kovacova, Ondrej Slaby. Global analysis and validation of miRNA levels in cerebrospinal fluid of brain tumor patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1806.

MicroRNAs in Cerebrospinal Fluid as Biomarkers in Brain Tumor Patients.

Backround: Although central nervous system (CNS) tumors are not the most common cancers, their incidence rate is constantly growing. Unfortunately, this group of cancers is characterized by a very poor prognosis with a very short average patient survival. Appropriate therapy depends on early and accurate diagnosis. However, this is often limited by brain tumor localization and heterogeneity. Therefore, new diagnostic approaches and biomarkers that are robust, sensitive, specific, and also without need of invasive biopsy, are still being sought. Cerebrospinal fluid (CSF) comes into direct contact with the CNS and becomes a suitable source of biological material that could reflect actual state of CNS. Suitable molecules in this regard appear to be microRNAs (miRNAs), short non-coding RNAs, that have been already detected in CSF and whose dysregulated levels are associated with various types of brain tumors. Purpose: Unfortunately, the methodical approaches used for CSF miRNA analysis have not been sufficiently standardized yet. For this reason, we summarize and evaluate methodical approaches which were previously used for miRNA analysis from CSF in order to find the most appropriate ones. Subsequently, we review studies focused on miRNA with potential to become biomarkers of CNS tumors in the future. Supported by Ministry of Health of the Czech Republic, grants No. 15-34553A and 15-33158A. All rights reserved. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 3. 1. 2019 Accepted: 3. 1. 2019.

Validation of MicroRNA Biomarkers for Alzheimer's Disease in Human Cerebrospinal Fluid.

We previously discovered microRNAs (miRNAs) in cerebrospinal fluid (CSF) that differentiate Alzheimer's disease (AD) patients from Controls. Here we examined the performance of 37 candidate AD miRNA biomarkers in a new and independent cohort of CSF from 47 AD patients and 71 Controls on custom TaqMan arrays. We employed a consensus ranking approach to provide an overall priority score for each miRNA, then used multimarker models to assess the relative contributions of the top-ranking miRNAs to differentiate AD from Controls. We assessed classification performance of the top-ranking miRNAs when combined with apolipoprotein E4 (APOE4) genotype status or CSF amyloid-β42 (Aβ42):total tau (T-tau) measures. We also assessed whether miRNAs that ranked higher as AD markers correlate with Mini-Mental State Examination (MMSE) scores. We show that of 37 miRNAs brought forth from the discovery study, 26 miRNAs remained viable as candidate biomarkers for AD in the validation study. We found that combinations of 6-7 miRNAs work better to identify AD than subsets of fewer miRNAs. Of 26 miRNAs that contribute most to the multimarker models, 14 have higher potential than the others to predict AD. Addition of these 14 miRNAs to APOE4 status or CSF Aβ42:T-tau measures significantly improved classification performance for AD. We further show that individual miRNAs that ranked higher as AD markers correlate more strongly with changes in MMSE scores. Our studies validate that a set of CSF miRNAs serve as biomarkers for AD, and support their advancement toward development as biomarkers in the clinical setting.

MicroRNAs as potential biomarkers for the diagnosis of glioma: A systematic review and meta-analysis.

Glioma is the most common central nervous system tumor and associated with poor prognosis. Identifying effective diagnostic biomarkers for glioma is particularly important in order to guide optimizing treatment. MicroRNAs (miRNAs) have drawn much attention because of their diagnostic value in diverse cancers, including glioma. We summarized studies to identify the potential diagnostic values of miRNAs in glioma patients. We included articles reporting miRNAs for differentiation of glioma patients from controls. We calculated sensitivities, specificities, and area under the curves (AUC) of individual miRNA and miRNA panels. We found that overall sensitivity, specificity, and AUC of miRNAs in diagnosis of glioma were 85% (95% confidence interval [CI]: 0.81‐0.89), 90% (95% CI 0.85‐0.93), and 93% (95% CI 0.91‐0.95), respectively. Meta‐regression analysis showed that the detection of miRNAs expression in cerebrospinal fluid (CSF) and brain tissue largely improved the diagnostic accuracy. Likewise, panels of multiple miRNAs could enhance the pooled sensitivity. Moreover, AUC of miR‐21 was 0.88, with 86% sensitivity and 94% specificity. This study demonstrated that miRNAs could function as potential diagnosis markers in glioma. Detection of miRNAs in CSF and brain tissue displays high accuracy in the diagnosis of glioma.

Abstract 3615: Analysis of microRNAs in cerebrospinal fluid of brain tumor patients

Abstract Introduction: Cerebrospinal fluid (CSF) is a body fluid with many important functions that is in direct contact with the extracellular environment of the central nervous system (CSN). Therefore, CSF serves inter alia as a both communication channel allowing the distribution of various substances among CNS cells as well as a reservoir of waste products that these cells released. For these reasons, CSF is a potential source of diagnostic biomarkers of many neurological diseases including brain tumors. Recent studies have revealed that CSF contains also circulating microRNAs (miRNAs), short non-coding RNAs, which have been described as biofluid diagnostic markers in many cancers. The analysis of CSF miRNAs in patients affected by brain tumors could enable more precise diagnosis and patient stratification.Material and methods: We performed next-generation sequencing analysis of small RNAs (small RNAseq) in 89 CSF samples taken from 35 GBM, 14 low-grade glioma (LGG), 14 meningioma, and 8 brain metastasis patients as well as 18 non-tumor donors. Informed consent approved by the local Ethical Commission was obtained from each patient before the lumbar puncture. cDNA libraries were prepared using CleanTaq Small RNA Library Prep Kit (TriLink BioTechnologies) and, subsequently, purified by Agencourt AMPure XP (Beckman Coulter). The final sequencing analysis was performed by Next 500/550 High Output v2 Kit - 75 cycles using the NextSeq 500 instrument (both Illumina). For miRNA mapping and analysis, an online tool Chimira was used. Obtained data were subsequently statistically evaluated in the environment of statistical language R using the Bioconductor edgeR and DESeq2 package.Results: We observed 211 miRNAs to be expressed in more than 60 CSF samples with 1 395 normalized counts in average (min 3, max 77 284; miR-486-5p was excluded from analysis due to its artificial expression). From these miRNAs, 63 showed different levels in CSF of GBM, 16 in LGG, 24 in meningioma, and 31 in brain metastases patients in comparison with CSF of non-tumor donors. MiR-10a-5p and miR-196a-5p have significantly highest levels in CSF of GBM samples. Moreover, 6 miRNAs (miR-30c-5p, miR-30e-5p, miR-145-5p, miR-320c, miR-3960, and miR-6131) indicated significantly different levels between GBM and LGG (P<0.01).Conclusion: We have demonstrated that CSF of various brain tumors (GBM, LGG, meningioma, and brain metastasis) is characterized by specific miRNA signature. Moreover, we found miRNA signature with ability to differentiate LGG and GBM from CSF. Our results suggest, that after independent validations, CSF miRNAs could serve as promising biomarkers in brain tumors. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-34553A, 15-33158A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S, 17-17636S. Citation Format: Jiri Sana, Alena Kopkova, Marek Vecera, Jaroslav Juracek, Tana Machackova, Parwez Ahmad, Natalia Anna Gablo, Pavel Fadrus, Marek Svoboda, Ondrej Slaby. Analysis of microRNAs in cerebrospinal fluid of brain tumor patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3615.

Recognition of Alzheimer’s Disease Related miRNAs Based on Genome-Wide Association Study Data

To analyze the mi RNA expression profile of patients with Alzheimer's disease (AD) in order to intensively study the relationship between mi RNA and the occurrence and development of AD, and to lay the foundations for the finding of new molecular markers. Use mi RNA chip to detect the mi RNA expression profiles of cerebrospinal fluid in AD patients and elderly people with normal cognition, and perform preliminary bioinformatics analysis. Real-time PCR was used to verify the results of mi RNA chip detection. The results of mi RNA chip analysis showed that there were many differentially expressed mi RNA in cerebrospinal fluid of AD patients and elderly people with normal cognition. Cerebrospinal fluid in AD patients has a specific mi RNA expression profile, suggesting that a new molecular marker for AD may be found in the expression of mi RNA.

F10. DIFFERENTIAL EXPRESSION OF MICRORNAS IN CEREBROSPINAL FLUID AND PLASMA SAMPLES IN SCHIZOPHRENIA

BackgroundGenetic variants in miRNA genes and abnormalities in the concentration of microRNAs (miRNAs) in tissues and biological fluids have recently been associated with a diagnosis of schizophrenia. Most of these studies used post-mortem brain tissue or whole blood as the source of RNA. However, examination of microRNAs in cerebrospinal fluid (CSF) might provide an in vivo biomarker, more accurately reflecting expression level changes in the brain. To date, there are no studies that have investigated miRNA expression in CSF in patients with schizophrenia using small RNA-seq. In the past, our group had the opportunity of investigating the correlation between miRNA profiles in CSF and blood measured using microarray technology. Therefore, to expand our findings and use current cutting-edge technology, we measured miRNA profiles in CSF and plasma using small RNA-seq in a sample of patients with schizophrenia-spectrum disorder (SSD) diagnosis and healthy volunteers.MethodsTwenty-two SSD patients and 17 healthy volunteers underwent a lumbar puncture and a blood draw. 15–25 cc of CSF and 5–10 cc of peripheral blood were obtained from each subject. CSF and peripheral blood samples were centrifuged. CSF and plasma samples were aliquoted into 1 mL cryovials, and stored at -80C degrees. Vesicular RNA was extracted from 1 mL of CSF and plasma samples following the protocol from the Qiagen exoRNA easy kit. The BioScientific NextFlex RNA sequencing kit was used for library construction. Sequencing was done on HiSeq2500. Samples that had at least 50,000 reads going to mature miRNA sequences were included in the analysis. Differential expression analyses were conducted in R using the DESeq2 package in Bioconductor.ResultsIn the overall sample cohort, most subjects were male (66.7%), not Hispanic (81.0%) and black (48.7%). Mean age was 36.8 years (SD=12.3), There were no differences in age, sex, ethnicity or race between the patient and healthy control groups. In the patient group, 16 (72.7%) had schizophrenia, 5 (22.7%) had schizoaffective disorder and 1 (4.5%) had psychosis not otherwise specified. Differential expression (DE) analyses were conducted for 144 miRNAs in CSF and 354 miRNAs in plasma. After adjusting for multiple comparisons, DE analysis between patients and controls in CSF showed statistically significant higher levels in patients of miR-769-5p, miR-99b-3p, miR-107, miR-451a and miR-708-5. Similar analysis in plasma showed statistically significant higher levels in patients for miR-375, miR-204-5p, miR-942-5p, miR-6734-5p, miR-423-5p and miR-144-5p. Principal component analysis showed a clear separation between CSF and peripheral blood samples. Out of 443 miRNAs used to examine the relationship between CSF and plasma, 205 (46.3%) were detected in both plasma and CSF samples, 88 (19.9%) were detected only in CSF samples while 150 (33.9%) were detected only in plasma samples.DiscussionFive miRNAs were upregulated in CSF samples and six were upregulated in plasma samples of SSD patients compared to healthy volunteers. There was no overlap in the statistically significant upregulated miRNAs between CSF and plasma samples. Therefore, miRNA profiles in CSF and plasma have important quantitative and qualitative differences that may make them excellent, but different, candidate biofluids for biomarker discovery.

Small RNA Sequencing of Sporadic Amyotrophic Lateral Sclerosis Cerebrospinal Fluid Reveals Differentially Expressed miRNAs Related to Neural and Glial Activity.

Amyotrophic lateral sclerosis (ALS) is a clinical subtype of motor neurone disease (MND), a fatal neurodegenerative disease involving the loss of both the upper and lower motor neurones from the motor cortex, brainstem, and spinal cord. Identifying specific disease biomarkers would help to not only improve diagnostic delay but also to classify disease subtypes, monitor response to therapeutic drugs and track disease progression. miRNAs are small non-coding RNA responsible for regulating gene expression and ultimately protein expression and have been used as biomarkers for many cancers and neurodegenerative disorders. Investigating the detection of miRNAs in cerebrospinal fluid (CSF), the fluid that bathes the central nervous system (CNS) is a prime target for identifying potential biomarkers for ALS. This is the first study to investigate the expression of miRNAs in the CSF of ALS patients using small RNA sequencing. We detected 11 differentially expressed miRNAs in the CSF of sporadic ALS (sALS) patients related to neural and glial activity. Additionally, miRNAs involved in glucose metabolism and the regulation of oxidative stress were also identified. Detecting the presence of potential CSF derived miRNA biomarkers in sALS could open up a whole new area of knowledge to help gain a better understanding of disease pathophysiology. Additionally, with further investigation, the tracking of CSF miRNA over the disease course could be used to follow the disease progression and monitor the effect of novel therapeutics that could be personalized to an individual disease phenotype.

Exercise \u2013 induced changes in cerebrospinal fluid miRNAs in Gulf War Illness, Chronic Fatigue Syndrome and sedentary control subjects

Gulf War Illness (GWI) and Chronic Fatigue Syndrome (CFS) have similar profiles of pain, fatigue, cognitive dysfunction and exertional exhaustion. Post-exertional malaise suggests exercise alters central nervous system functions. Lumbar punctures were performed in GWI, CFS and control subjects after (i) overnight rest (nonexercise) or (ii) submaximal bicycle exercise. Exercise induced postural tachycardia in one third of GWI subjects (Stress Test Activated Reversible Tachycardia, START). The remainder were Stress Test Originated Phantom Perception (STOPP) subjects. MicroRNAs (miRNA) in cerebrospinal fluid were amplified by quantitative PCR. Levels were equivalent between nonexercise GWI (n = 22), CFS (n = 43) and control (n = 22) groups. After exercise, START (n = 22) had significantly lower miR-22-3p than control (n = 15) and STOPP (n = 42), but higher miR-9-3p than STOPP. All post-exercise groups had significantly reduced miR-328 and miR-608 compared to nonexercise groups; these may be markers of exercise effects on the brain. Six miRNAs were significantly elevated and 12 diminished in post-exercise START, STOPP and control compared to nonexercise groups. CFS had 12 diminished miRNAs after exercise. Despite symptom overlap of CFS, GWI and other illnesses in their differential diagnosis, exercise-induced miRNA patterns in cerebrospinal fluid indicated distinct mechanisms for post-exertional malaise in CFS and START and STOPP phenotypes of GWI.

Elevation of brain-enriched miRNAs in cerebrospinal fluid of patients with acute ischemic stroke

BackgroundThe purpose of this study was to investigate the potential of cerebrospinal fluid miRNAs as diagnostic biomarkers of acute ischemic stroke using three different profiling techniques in order to identify and bypass any influence from technical variation.MethodsCerebrospinal fluid (CSF) from patients with acute ischemic stroke (n = 21) and controls (n = 21) was collected by lumbar puncture. miRNA analysis was performed with three different methods: 1) Trizol RNA extraction followed by Illumina Next Generation Sequencing (NGS) on all small RNAs, 2) Exiqon RNA extraction protocol and miRNA qPCR assays, and 3) validation of 24 selected miRNAs with Norgen Biotek RNA extraction protocol and Applied Biosystems qPCR assays.ResultsNGS detected 71 frequently expressed miRNAs in CSF of which brain-enriched miR-9-5p and miR-128-3p were significantly higher in CSF of stroke patients compared to controls. When dividing stroke patients into groups according to infarct size several brain-enriched miRNAs (miR-9-5p, miR-9-3p, miR-124-3p, and miR-128-3p) were elevated in patients with infarcts >2 cm3. This trend appeared in data from both NGS, qPCR (Exiqon), and qPCR (Applied Biosystems) but was only statistically significant in some of the measurement platforms.ConclusionsSeveral brain-enriched miRNAs are elevated in the CSF three days after stroke onset, suggesting that these miRNAs reflect the brain damage caused by ischemia. The expression differences seem, however, limited to patients with larger ischemic brain injury, which argues against the use of CSF miRNAs as diagnostic biomarkers of stroke based on current methods.

A cerebrospinal fluid microRNA signature as biomarker for glioblastoma.

PurposeTo develop a cerebrospinal fluid (CSF) miRNA diagnostic biomarker for glioblastoma.Experimental DesignGlioblastoma 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.ResultsmiRNA 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.ConclusionWe report a CSF miRNA signature as a “liquid biopsy” diagnostic platform for glioblastoma.

Abstract TP430: Analyses of CSF and Serum miRNA Expression in Subarachnoid Hemorrhage Patients Suggest Important Roles in Restoration of Impaired Vascular Homeostasis

Backgrounds: MicroRNAs (miRNAs) play important roles in the regulation of gene expression at the post-transcriptional level. Furthermore, in various human disease, miRNAs have been identified as potential biomarkers. However, it remains to be elucidated whether and how miRNAs play roles in the development of cerebral vasospasm (CVS) or delayed cerebral ischemia (DCI) after subarachnoid hemorrhage (SAH). The purposes of this study are to detect differential miRNAs in cerebrospinal fluid (CSF) and serum in SAH patients and to identify possible miRNAs involved in the mechanisms of the vascular proliferation or phenotypic changes after SAH. Methods: The CSF and serum samples were taken every other day for 2 weeks after SAH in patients with endovascular aneurysm repair. Microarray analysis of exosomal miRNA from samples was performed using 3D-Gene miRNA labeling kit (Toray). The temporal changes in the expression of differentially expressed miRNAs in early phase after SAH and its associated molecules were evaluated using quantitative RT-PCR. Results: Among over 2500 miRNAs screened out, microarray analysis revealed 12 diffrentially expressed genes which were up- and/or down-regulated (FC>2-fold) both in the CSF and serum during early phase after SAH. RT-PCR analysis revealed that miRNAs involving vascular angiogenesis were up-regulated on day3 in serum and day5 in CSF. Contrarily, RT-PCR also revealed mRNAs of those associated molecules which exert anti-angiogenic and anti-proliferative effects were down-regulated on day1 in CSF and day3 in serum. These results suggest that these miRNAs expressions are controlled by negative-feedback regulation of associated molecules participating in maintaining vascular homeostasis. Conclusion: Our present study indicates that some miRNAs and molecules may be involved in vascular proliferation or angiogenesis by interacting each other after SAH.

MiRNAs in cerebrospinal fluid identify patients with MS and specifically those with lipid-specific oligoclonal IgM bands

Background: MicroRNAs (miRNAs) are non-coding RNAs that regulate cellular processes by controlling protein translation and mRNA degradation. Objective: We aimed to explore the miRNA signature of multiple sclerosis (MS) patients versus controls and the possibility that patients with lipid-specific oligolconal IgM bands (LS_OCMB), a predictor of a more severe disease course, may have a distinct profile. Methods: An extensive profile of 754 miRNAs was evaluated in the cerebrospinal fluid (CSF) of 14 women using TaqMan low-density arrays. Differentially expressed miRNAs together with others previously identified in the literature were validated in an extended sample of 86 MS patients (39 LS_OCMB+) and 55 controls. Results: We detected higher levels of miR-150 in MS patients and especially in those with LS_OCMB+. Other miRNAs (miR-328, miR-30a-5p and miR-645) were up-regulated in MS patients compared to controls while miR-21, miR-199a-3p, miR-191, miR-365, miR-106a and miR-146a showed down-regulated expression. Considering only patients with LS_OCMB+, we also detected up-regulation of miR-30a-5p, miR-150 and miR-645 and down-regulation of miR-191 compared to controls. Conclusion: Our study confirms the recent findings regarding the deregulated expression of miR-150 not only with MS but also with the presence of LS_OCMB. This study highlights the potential utility of miRNAs in CSF as biomarkers for MS.

MicroRNA expressing profiles in A53T mutant alpha-synuclein transgenic mice and Parkinsonian.

α-synuclein gene mutations can cause α-synuclein protein aggregation in the midbrain of Parkinson's disease (PD) patients. MicroRNAs (miRNAs) play a key role in the metabolism of α-synuclein but the mechanism involved in synucleinopathy remains unclear. In this study, we investigated the miRNA profiles in A53T-α-synuclein transgenic mice and analyzed the candidate miRNAs in the cerebrospinal fluid (CSF) of PD patients. The 12-month A53T-transgenic mouse displayed hyperactive movement and anxiolytic-like behaviors with α-synuclein aggregation in midbrain. A total of 317,759 total and 289,207 unique small RNA sequences in the midbrain of mice were identified by high-throughput deep sequencing. We found 644 miRNAs were significantly changed in the transgenic mice. Based on the conserved characteristic of miRNAs, we selected 11 candidates from the 40 remarkably expressed miRNAs and explored their expression in 44 CSF samples collected from PD patients. The results revealed that 11 microRNAs were differently expressed in CSF, emphatically as miR-144-5p, miR-200a-3p and miR-542-3p, which were dramatically up-regulated in both A53T-transgenic mice and PD patients, and had a helpful accuracy for the PD prediction. The ordered logistic regression analysis showed that the severity of PD has strong correlation with an up-expression of miR-144-5p, miR-200a-3p and miR-542-3p in CSF. Taken together, our data suggested that miRNAs in CSF, such as miR-144-5p, miR-200a-3p and miR-542-3p, may be useful to the PD diagnosis as potential biomarkers.

MicroRNAs in Human Cerebrospinal Fluid as Biomarkers for Alzheimer's Disease.

Currently available biomarkers of Alzheimer's disease (AD) include cerebrospinal fluid (CSF) protein analysis and amyloid PET imaging, each of which has limitations. The discovery of extracellular microRNAs (miRNAs) in CSF raises the possibility that miRNA may serve as novel biomarkers of AD. Investigate miRNAs in CSF obtained from living donors as biomarkers for AD. We profiled miRNAs in CSF from 50 AD patients and 49 controls using TaqMan® arrays. Replicate studies performed on a subset of 32 of the original CSF samples verified 20 high confidence miRNAs. Stringent data analysis using a four-step statistical selection process including log-rank and receiver operating characteristic (ROC) tests, followed by random forest tests, identified 16 additional miRNAs that discriminate AD from controls. Multimarker modeling evaluated linear combinations of these miRNAs via best-subsets logistic regression, and computed area under the ROC (AUC) curve ascertained classification performance. The influence of ApoE genotype on miRNA biomarker performance was also evaluated. We discovered 36 miRNAs that discriminate AD from control CSF. 20 of these retested in replicate studies verified differential expression between AD and controls. Stringent statistical analysis also identified these 20 miRNAs, and 16 additional miRNA candidates. Top-performing linear combinations of 3 and 4 miRNAs have AUC of 0.80-0.82. Addition of ApoE genotype to the model improved performance, i.e., AUC of 3 miRNA plus ApoE4 improves to 0.84. CSF miRNAs can discriminate AD from controls. Combining miRNAs improves sensitivity and specificity of biomarker performance, and adding ApoE genotype improves classification.