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.

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.

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.

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

BACKGROUND: 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. OBJECTIVE: Investigate miRNAs in CSF obtained from living donors as biomarkers for AD. METHODS: 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. RESULTS: 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. CONCLUSIONS: CSF miRNAs can discriminate AD from controls. Combining miRNAs improves sensitivity and specificity of biomarker performance, and adding ApoE genotype improves classification.

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.

Gender effects on plasma and brain copper.

The effect of gender on systemic and brain levels of copper is relatively understudied. We examined gender effects in mice and human subjects. We observed a trend to higher serum copper levels in female compared to male LaFerla "triple transgenic" (1399 ± 233 versus 804 ± 436 ng/mL, P = 0.06) mice, and significantly higher brain copper levels in female- versus male wild-type mice (5.2 ± 0.2 versus 4.18 ± 0.3 ng/mg wet wt, P = 0.03). Plasma copper was significantly correlated with brain copper in mice (R2 = 0.218; P = 0.038). Among human subjects with AD, both plasma copper (1284 ± 118 versus 853 ± 81 ng/mL, P = 0.005) and cerebrospinal fluid copper (12.8 ± 1 versus 10.4 ± 0.7 ng/mL, P = 0.01) were elevated in women compared to men. Among healthy control subjects, plasma copper (1008 ± 51 versus 836 ± 41 ng/mL; P = 0.01) was higher in women than in men, but there was no difference in cerebrospinal fluid copper. We conclude that gender differences in copper status may influence copper-mediated pathological events in the brain.