Tear Proteins Altered in Patients with Persistent Eye Pain after Refractive Surgery: Biomarker Candidate Discovery.

Some patients develop persistent eye pain after refractive surgery, but factors that cause or sustain pain are unknown. We tested whether tear proteins of patients with pain 3 months after surgery differ from those of patients without pain. Patients undergoing refractive surgery (laser in situ keratomileusis or photorefractive keratectomy ) were recruited from 2 clinics, and tears were collected 3 months after surgery. Participants rated their eye pain using a numerical rating scale (NRS, 0-10; no pain-worst pain) at baseline, 1 day, and 3 months after surgery. Using tandem mass tag proteomic analysis, we examined tears from patients with pain [NRS ≥ 3 at 3 months (n = 16)] and patients with no pain [NRS ≤ 1 at 3 months (n = 32)] after surgery. A subset of proteins (83 of 2748 detected, 3.0%) were associated with pain 3 months after surgery. High-dimensional statistical models showed that the magnitude of differential expression was not the only important factor in classifying tear samples from pain patients. Models utilizing 3 or 4 proteins had better classification performance than single proteins and represented differences in both directions (higher or lower in pain). Thus, patterns of protein differences may serve as biomarkers of postsurgical eye pain as well as potential therapeutic targets.

A clathrin mediated endocytosis scaffolding protein, Intersectin 1, changes in an isoform, brain region, and sex specific manner in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia and is characterized by the accumulation of amyloid-beta (Aß) plaques and neurofibrillary Tau tangles in the brain. We previously identified a set of candidate AD microRNAs (miRNAs) in human cerebrospinal fluid (CSF) and used a target prediction pipeline to identify mRNAs and pathways that could potentially be regulated by the miRNAs. Of these pathways, clathrin mediated endocytosis (CME) was selected for further investigation. CME is altered in multiple brain cell types in AD and is implicated in early cellular phenotypes such as enlarged early endosomes and pathogenic processing of Aß. However, a comprehensive evaluation of major CME hub proteins in humans with AD across multiple brain regions is lacking. Thus, we used immunoblots to evaluate human post-mortem AD and control (CTL) frontal cortex (FC; AD n = 22, CTL n = 23) and hippocampus (HP; AD n = 34, CTL n = 22) for changes in Intersectin 1 (ITSN1), Phosphatidylinositol Binding Clathrin Assembly Protein gene (PICALM), Clathrin Light Chain (CLT), FCH and Mu Domain Containing Endocytic Adaptor 1 (FCHO1), Adaptor Related Protein Complex 2 (AP2) Subunit Alpha 1 (AP2A1), and Dynamin 2 (DNM2). Of these, we found that in AD, ITSN1-long (ITSN1-L) was decreased in the FC of males and HP of females, while ITSN1-short was increased in the HP of both males and females. We further evaluated ITSN1-L levels in cortex (CTX) and HP of the 5xFAD mouse model of Aß pathology at different timepoints during aging and disease progression by immunoblot (n = 5-8 per group). At 3 months, female 5xFAD exhibited an increase of ITSN1-L in CTX but a decrease at 6 and 9 months. Additionally, immunofluorescent staining of 5xFAD primary HP neurons showed an increase of ITSN1-L in matured 5xFAD neurons at 21 and 28 days in vitro. Together, our studies show that in AD, isoforms of ITSN1 change in a brain region-and sex-dependent manner. Further, changes in ITSN1-L are transient with levels increasing during early Aß accumulation and decreasing during later progression. These findings suggest that ITSN1 expression, and consequently CME activity, may change depending on the stage of disease progression.

Clathrin mediated endocytosis in Alzheimer's disease: cell type specific involvement in amyloid beta pathology.

This review provides a comprehensive examination of the role of clathrin-mediated endocytosis (CME) in Alzheimer's disease (AD) pathogenesis, emphasizing its impact across various cellular contexts beyond neuronal dysfunction. In neurons, dysregulated CME contributes to synaptic dysfunction, amyloid beta (Aß) processing, and Tau pathology, highlighting its involvement in early AD pathogenesis. Furthermore, CME alterations extend to non-neuronal cell types, including astrocytes and microglia, which play crucial roles in Aß clearance and neuroinflammation. Dysregulated CME in these cells underscores its broader implications in AD pathophysiology. Despite significant progress, further research is needed to elucidate the precise mechanisms underlying CME dysregulation in AD and its therapeutic implications. Overall, understanding the complex interplay between CME and AD across diverse cell types holds promise for identifying novel therapeutic targets and interventions.

Prenatal delta-9-tetrahydrocannabinol exposure alters fetal neurodevelopment in rhesus macaques.

Prenatal cannabis use is associated with adverse offspring neurodevelopmental outcomes, however the underlying mechanisms are relatively unknown. We sought to determine the impact of chronic delta-9-tetrahydrocannabinol (THC) exposure on fetal neurodevelopment in a rhesus macaque model using advanced imaging combined with molecular and tissue studies. Animals were divided into two groups, control (n = 5) and THC-exposed (n = 5), which received a daily THC edible pre-conception and throughout pregnancy. Fetal T2-weighted MRI was performed at gestational days 85 (G85), G110, G135 and G155 to assess volumetric brain development. At G155, animals underwent cesarean delivery with collection of fetal cerebrospinal fluid (CSF) for microRNA (miRNA) studies and fetal tissue for histologic analysis. THC exposure was associated with significant age by sex interactions in brain growth, and differences in fetal brain histology suggestive of brain dysregulation. Two extracellular vesicle associated-miRNAs were identified in THC-exposed fetal CSF; pathway analysis suggests that these miRNAs are associated with dysregulated axonal guidance and netrin signaling. This data is indicative of subtle molecular changes consistent with the observed histological data, suggesting a potential role for fetal miRNA regulation by THC. Further studies are needed to determine whether these adverse findings correlate with long-term offspring neurodevelopmental health.

Analysis of the longitudinal stability of human plasma miRNAs and implications for disease biomarkers.

There is great interest in developing clinical biomarker assays that can aid in non-invasive diagnosis and/or monitoring of human diseases, such as cancer, cardiovascular disease, and neurological diseases. Yet little is known about the longitudinal stability of miRNAs in human plasma. Here we assessed the intraindividual longitudinal stability of miRNAs in plasma from healthy human adults, and the impact of common factors (e.g., hemolysis, age) that may confound miRNA data. We collected blood by venipuncture biweekly over a 3-month period from 22 research participants who had fasted overnight, isolated total RNA, then performed miRNA qPCR. Filtering and normalization of the qPCR data revealed amplification of 134 miRNAs, 74 of which had high test-retest reliability and low percentage level drift, meaning they were stable in an individual over the 3-month time period. We also determined that, of nuisance factors, hemolysis and tobacco use have the greatest impact on miRNA levels and variance. These findings support that many miRNAs show intraindividual longitudinal stability in plasma from healthy human adults, including some reported as candidate biomarkers for Alzheimer's disease.

Recommendations for reproducibility of cerebrospinal fluid extracellular vesicle studies.

Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world-wide survey to ISEV members to assess methods considered 'best practices' for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.

Experimental design considerations for studies of human tear proteins.

PURPOSE: Human tears contain abundant, diverse sets of proteins that may serve as biomarkers of ocular surface health. There is a need for reproducible methods that consider multiple factors influencing the tear proteome, in addition to the variable of interest. Here we examined a workflow for proteomic analysis of tear proteins without the need to pool tear samples from multiple individuals, thus allowing for analyses based on individual factors, and increasing opportunities for protein biomarker discovery. METHODS: Tears were collected by Schirmer strip following topical ocular anesthetic application then individually stored at -80 °C prior to processing for proteomics. Tear proteins were extracted from Schirmer strips, digested using suspension trapping spin columns (S-Trap), and labeled with high multiplicity tandem mass tags (TMT). Peptide digests were then extensively fractionated by two-dimensional chromatography and analyzed by mass spectrometry to identify and measure changes in protein abundance in each sample. Analysis of select samples was performed to test protocols and to compare the impact of clinically relevant parameters. To facilitate comparison of separate TMT experiments, common pool samples were included in each TMT instrument run and internal reference scaling (IRS) was performed. RESULTS: Differences in subsets of tear proteins were noted for: geographic site of tear collection, contact lens use, and differences in tear fluid volume among individuals. CONCLUSION: These findings demonstrate that proteomic analysis of human tear proteins can be performed without the need to pool samples, and that development of analytic workflows must consider factors that may affect outcomes in studies focused on diverse clinical samples.

Differential Effects of APOE Genotype on MicroRNA Cargo of Cerebrospinal Fluid Extracellular Vesicles in Females With Alzheimer's Disease Compared to Males.

Multiple biological factors, including age, sex, and genetics, influence Alzheimer's disease (AD) risk. Of the 6.2 million Americans living with Alzheimer's dementia in 2021, 3.8 million are women and 2.4 million are men. The strongest genetic risk factor for sporadic AD is apolipoprotein E-e4 (APOE-e4). Female APOE-e4 carriers develop AD more frequently than age-matched males and have more brain atrophy and memory loss. Consequently, biomarkers that are sensitive to biological risk factors may improve AD diagnostics and may provide insight into underlying mechanistic changes that could drive disease progression. Here, we have assessed the effects of sex and APOE-e4 on the miRNA cargo of cerebrospinal fluid (CSF) extracellular vesicles (EVs) in AD. We used ultrafiltration (UF) combined with size exclusion chromatography (SEC) to enrich CSF EVs (e.g., Flotillin+). CSF EVs were isolated from female and male AD or controls (CTLs) that were either APOE-e3,4 or -e3,3 positive (n = 7/group, 56 total). MiRNA expression levels were quantified using a custom TaqMan™ array that assayed 190 miRNAs previously found in CSF, including 25 miRNAs that we previously validated as candidate AD biomarkers. We identified changes in the EV miRNA cargo that were affected by both AD and sex. In total, four miRNAs (miR-16-5p, -331-3p, -409-3p, and -454-3p) were significantly increased in AD vs. CTL, independent of sex and APOE-e4 status. Pathway analysis of the predicted gene targets of these four miRNAs with identified pathways was highly relevant to neurodegeneration (e.g., senescence and autophagy). There were also three miRNAs (miR-146b-5p, -150-5p, and -342-3p) that were significantly increased in females vs. males, independent of disease state and APOE-e4 status. We then performed a statistical analysis to assess the effect of APOE genotype in AD within each sex and found that APOE-e4 status affects different subsets of CSF EV miRNAs in females vs. males. Together, this study demonstrates the complexity of the biological factors associated with AD risk and the impact on EV miRNAs, which may contribute to AD pathophysiology.

Identification of miRNAs That Mediate Protective Functions of Anti-Cancer Drugs During White Matter Ischemic Injury.

We have previously shown that two anti-cancer drugs, CX-4945 and MS-275, protect and preserve white matter (WM) architecture and improve functional recovery in a model of WM ischemic injury. While both compounds promote recovery, CX-4945 is a selective Casein kinase 2 (CK2) inhibitor and MS-275 is a selective Class I histone deacetylase (HDAC) inhibitor. Alterations in microRNAs (miRNAs) mediate some of the protective actions of these drugs. In this study, we aimed to (1) identify miRNAs expressed in mouse optic nerves (MONs); (2) determine which miRNAs are regulated by oxygen glucose deprivation (OGD); and (3) determine the effects of CX-4945 and MS-275 treatment on miRNA expression. RNA isolated from MONs from control and OGD-treated animals with and without CX-4945 or MS-275 treatment were quantified using NanoString nCounter® miRNA expression profiling. Comparative analysis of experimental groups revealed that 12 miRNAs were expressed at high levels in MONs. OGD upregulated five miRNAs (miR-1959, miR-501-3p, miR-146b, miR-201, and miR-335-3p) and downregulated two miRNAs (miR-1937a and miR-1937b) compared to controls. OGD with CX-4945 upregulated miR-1937a and miR-1937b, and downregulated miR-501-3p, miR-200a, miR-1959, and miR-654-3p compared to OGD alone. OGD with MS-275 upregulated miR-2134, miR-2141, miR-2133, miR-34b-5p, miR-153, miR-487b, miR-376b, and downregulated miR-717, miR-190, miR-27a, miR-1959, miR-200a, miR-501-3p, and miR-200c compared to OGD alone. Interestingly, miR-501-3p and miR-1959 were the only miRNAs upregulated by OGD, and downregulated by OGD plus CX-4945 and MS-275. Therefore, we suggest that protective functions of CX-4945 or MS-275 against WM injury maybe mediated, in part, through miRNA expression.

Cerebrospinal Fluid MicroRNA Changes in Cognitively Normal Veterans With a History of Deployment-Associated Mild Traumatic Brain Injury.

A history of traumatic brain injury (TBI) increases the odds of developing Alzheimer's disease (AD). The long latent period between injury and dementia makes it difficult to study molecular changes initiated by TBI that may increase the risk of developing AD. MicroRNA (miRNA) levels are altered in TBI at acute times post-injury (<4 weeks), and in AD. We hypothesized that miRNA levels in cerebrospinal fluid (CSF) following TBI in veterans may be indicative of increased risk for developing AD. Our population of interest is cognitively normal veterans with a history of one or more mild TBI (mTBI) at a chronic time following TBI. We measured miRNA levels in CSF from three groups of participants: (1) community controls with no lifetime history of TBI (ComC); (2) deployed Iraq/Afghanistan veterans with no lifetime history of TBI (DepC), and (3) deployed Iraq/Afghanistan veterans with a history of repetitive blast mTBI (DepTBI). CSF samples were collected at the baseline visit in a longitudinal, multimodal assessment of Gulf War veterans, and represent a heterogenous group of male veterans and community controls. The average time since the last blast mTBI experienced was 4.7 ± 2.2 years [1.5 - 11.5]. Statistical analysis of TaqManTM miRNA array data revealed 18 miRNAs with significant differential expression in the group comparisons: 10 between DepTBI and ComC, 7 between DepC and ComC, and 8 between DepTBI and DepC. We also identified 8 miRNAs with significant differential detection in the group comparisons: 5 in DepTBI vs. ComC, 3 in DepC vs. ComC, and 2 in DepTBI vs. DepC. When we applied our previously developed multivariable dependence analysis, we found 13 miRNAs (6 of which are altered in levels or detection) that show dependencies with participant phenotypes, e.g., ApoE. Target prediction and pathway analysis with miRNAs differentially expressed in DepTBI vs. either DepC or ComC identified canonical pathways highly relevant to TBI including senescence and ephrin receptor signaling, respectively. This study shows that both TBI and deployment result in persistent changes in CSF miRNA levels that are relevant to known miRNA-mediated AD pathology, and which may reflect early events in AD.

Longitudinal Course of Traumatic Brain Injury Biomarkers for the Prediction of Clinical Outcomes: A Review.

Protein biomarkers are often measured at hospital presentation to diagnose traumatic brain injury (TBI) and predict patient outcomes. However, a biomarker measurement at this single time point is no more accurate at predicting patient outcomes than less invasive and more cost-effective methods. Here, we review evidence that TBI biomarkers provide greater prognostic value when measured repeatedly over time, such that a trajectory of biomarker concentrations can be evaluated. PubMed, Google Scholar, and Cochrane Central Register were searched to identify studies from the last decade in which established TBI biomarkers had been measured at more than one time point following acute TBI, and which related their findings to patient outcomes. Twenty-two studies were identified, 18 of which focused on adults and 4 of which focused on children. Three general biomarker trajectories were identified: persistently high, persistently low, and reversal of decreasing concentrations. Downtrend reversal was highly specific to predicting poor patient outcomes. Four studies demonstrated that biomarker trajectories can be affected by therapeutic interventions. Additional studies demonstrated that biomarkers measured at a later time point offered superior prognostic value than a single measurement obtained at initial hospital presentation. Among other details, longitudinal biomarker trajectory assessments may identify ongoing injury and predict patient deterioration before clinical symptoms develop and thus help guide therapeutic interventions.

Performance of Validated MicroRNA Biomarkers for Alzheimer's Disease in Mild Cognitive Impairment.

BACKGROUND: Cerebrospinal fluid (CSF) microRNA (miRNA) biomarkers of Alzheimer's disease (AD) have been identified, but have not been evaluated in prodromal AD, including mild cognitive impairment (MCI). OBJECTIVE: To assess whether a set of validated AD miRNA biomarkers in CSF are also sensitive to early-stage pathology as exemplified by MCI diagnosis. METHODS: We measured the expression of 17 miRNA biomarkers for AD in CSF samples from AD, MCI, and cognitively normal controls (NC). We then examined classification performance of the miRNAs individually and in combination. For each miRNA, we assessed median expression in each diagnostic group and classified markers as trending linearly, nonlinearly, or lacking any trend across the three groups. For trending miRNAs, we assessed multimarker classification performance alone and in combination with apolipoprotein E ɛ4 allele (APOEɛ4) genotype and amyloid-ß42 to total tau ratio (Aß42:T-Tau). We identified predicted targets of trending miRNAs using pathway analysis. RESULTS: Five miRNAs showed a linear trend of decreasing median expression across the ordered diagnoses (control to MCI to AD). The trending miRNAs jointly predicted AD with area under the curve (AUC) of 0.770, and MCI with AUC of 0.705. Aß42:T-Tau alone predicted MCI with AUC of 0.758 and the AUC improved to 0.813 (p = 0.051) after adding the trending miRNAs. Multivariate correlation of the five trending miRNAs with Aß42:T-Tau was weak. CONCLUSION: Selected miRNAs combined with Aß42:T-Tau improved classification performance (relative to protein biomarkers alone) for MCI, despite a weak correlation with Aß42:T-Tau. Together these data suggest that that these miRNAs carry novel information relevant to AD, even at the MCI stage. Preliminary target prediction analysis suggests novel roles for these biomarkers.

Methamphetamine use alters human plasma extracellular vesicles and their microRNA cargo: An exploratory study.

Methamphetamine (MA) is the largest drug threat across the globe, with health effects including neurotoxicity and cardiovascular disease. Recent studies have begun to link microRNAs (miRNAs) to the processes related to MA use and addiction. Our studies are the first to analyse plasma EVs and their miRNA cargo in humans actively using MA (MA-ACT) and control participants (CTL). In this cohort we also assessed the effects of tobacco use on plasma EVs. We used vesicle flow cytometry to show that the MA-ACT group had an increased abundance of EV tetraspanin markers (CD9, CD63, CD81), but not pro-coagulant, platelet-, and red blood cell-derived EVs. We also found that of the 169 plasma EV miRNAs, eight were of interest in MA-ACT based on multiple statistical criteria. In smokers, we identified 15 miRNAs of interest, two that overlapped with the eight MA-ACT miRNAs. Three of the MA-ACT miRNAs significantly correlated with clinical features of MA use and target prediction with these miRNAs identified pathways implicated in MA use, including cardiovascular disease and neuroinflammation. Together our findings indicate that MA use regulates EVs and their miRNA cargo, and support that further studies are warranted to investigate their mechanistic role in addiction, recovery, and recidivism.

The Extracellular RNA Communication Consortium: Establishing Foundational Knowledge and Technologies for Extracellular RNA Research.

The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.

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-ß42 (Aß42):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 Aß42: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.

Analytics of Cerebrospinal Fluid MicroRNA Quantitative PCR Studies.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate post-transcriptional gene expression. Recent studies have shown that human disease states correlate with measurable differences in the level of circulating miRNAs relative to healthy controls. Thus, there is great interest in developing clinical miRNA assays as diagnostic or prognostic biomarkers for diseases, and as surrogate measures for therapeutic outcomes. Our studies have focused on miRNAs in human cerebral spinal fluid (CSF) as biomarkers for central nervous system (CNS) diseases. Our objective here was to examine factors that may affect the outcome of quantitative PCR (qPCR) studies on CSF miRNAs, in order to guide planning and interpretation of future CSF miRNA TaqMan® low-density array (TLDA) studies. We obtained CSF from neurologically normal (control) donors and used TLDAs to measure miRNA expression. We examined sources of error in the TLDA outcomes due to (1) nonspecific amplification of products in total RNA, (2) variations in RNA isolations performed on different days, (3) miRNA primer probe efficiency, and (4) variations in individual TLDA cards. We also examined the utility of card-to-card TLDA corrections and use of an unchanged "reference standard" to remove batch processing effects in large-scale studies.

Analysis of extracellular RNA in cerebrospinal fluid.

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.

Comparison of MicroRNA Expression in Aqueous Humor of Normal and Primary Open-Angle Glaucoma Patients Using PCR Arrays: A Pilot Study.

Purpose: MicroRNAs (miRNAs) are small, endogenous noncoding RNAs that have been detected in human aqueous humor (AH). Prior studies have pooled samples to obtain sufficient quantities for analysis or used next-generation sequencing. Here, we used PCR arrays with preamplification to identify and compare miRNAs from individual AH samples between patients with primary open-angle glaucoma (POAG) and normal controls. Methods: AH was collected before cataract surgery from six stable, medically treated POAG patients and eight age-matched controls. Following reverse transcription and preamplification, individual patient samples were profiled on Taqman Low Density MicroRNA Array Cards. Differentially expressed miRNAs were stratified for fold changes larger than ±2 and for significance of P < 0.05. Significant Kyoto Encyclopedia of Genes and Genomes pathways influenced by the differentially expressed miRNAs were identified using the predicted target module of the miRWalk 2.0 database. Results: This approach detected 181 discrete miRNAs, which were consistently expressed across all samples of both experimental groups. Significant up-regulation of miR-518d and miR-143, and significant down-regulation of miR-660, was observed in the AH of POAG patients compared with controls. These miRNAs were predicted to reduce cell proliferation and extracellular matrix remodeling, endocytosis, Wnt signaling, ubiquitin-mediated proteolysis, and adherens junction function. Conclusions: This pilot study demonstrates that miRNA expression within the AH of POAG patients differs from age-matched controls. AH miRNAs exhibit potential as biomarkers of POAG, which merits further investigation in a larger case-controlled study. This technique provides a cost-effective and sensitive approach to assay miRNAs in individual patient samples without the need for pooling.

Sex differences in regulatory cells in experimental stroke.

Stroke is the leading cause of disability in the United States. Sex differences, including smaller infarcts in females and greater involvement of immune-mediated inflammation in males may affect the efficacy of immune-modulating interventions. To address these differences, we sought to identify distinct stroke-modifying mechanisms in female vs. male mice. The current study demonstrated smaller infarcts and increased levels of regulatory CD19+CD5+CD1dhi B10 cells as well as anti-inflammatory CD11b+CD206+ microglia/macrophages in the ipsilateral vs. contralateral hemisphere of female but not male mice undergoing 60min middle cerebral artery occlusion followed by 96h of reperfusion. Moreover, female mice with MCAO had increased total spleen cell numbers but lower B10 levels in spleens. These results elucidate differing sex-dependent regulatory mechanisms that account for diminished stroke severity in females and underscore the need to test immune-modulating therapies for stroke in both males and females.