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Plasma Exosomal miRNAs in Persons with and without Alzheimer Disease: Altered Expression and Prospects for Biomarkers.

Lugli G, Cohen AM, Bennett DA, Shah RC, Fields CJ, Hernandez AG, Smalheiser NR - PLoS ONE (2015)

Bottom Line: Although these samples contained less than 0.1 microgram of total RNA, deep sequencing gave reliable and informative results.Twenty miRNAs showed significant differences in the AD group in initial screening (miR-23b-3p, miR-24-3p, miR-29b-3p, miR-125b-5p, miR-138-5p, miR-139-5p, miR-141-3p, miR-150-5p, miR-152-3p, miR-185-5p, miR-338-3p, miR-342-3p, miR-342-5p, miR-548at-5p, miR-659-5p, miR-3065-5p, miR-3613-3p, miR-3916, miR-4772-3p, miR-5001-3p), many of which satisfied additional biological and statistical criteria, and among which a panel of seven miRNAs were highly informative in a machine learning model for predicting AD status of individual samples with 83-89% accuracy.Perhaps the most interesting single miRNA was miR-342-3p, which was a) expressed in the AD group at about 60% of control levels, b) highly correlated with several of the other miRNAs that were significantly down-regulated in AD, and c) was also reported to be down-regulated in AD in two previous studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
To assess the value of exosomal miRNAs as biomarkers for Alzheimer disease (AD), the expression of microRNAs was measured in a plasma fraction enriched in exosomes by differential centrifugation, using Illumina deep sequencing. Samples from 35 persons with a clinical diagnosis of AD dementia were compared to 35 age and sex matched controls. Although these samples contained less than 0.1 microgram of total RNA, deep sequencing gave reliable and informative results. Twenty miRNAs showed significant differences in the AD group in initial screening (miR-23b-3p, miR-24-3p, miR-29b-3p, miR-125b-5p, miR-138-5p, miR-139-5p, miR-141-3p, miR-150-5p, miR-152-3p, miR-185-5p, miR-338-3p, miR-342-3p, miR-342-5p, miR-548at-5p, miR-659-5p, miR-3065-5p, miR-3613-3p, miR-3916, miR-4772-3p, miR-5001-3p), many of which satisfied additional biological and statistical criteria, and among which a panel of seven miRNAs were highly informative in a machine learning model for predicting AD status of individual samples with 83-89% accuracy. This performance is not due to over-fitting, because a) we used separate samples for training and testing, and b) similar performance was achieved when tested on technical replicate data. Perhaps the most interesting single miRNA was miR-342-3p, which was a) expressed in the AD group at about 60% of control levels, b) highly correlated with several of the other miRNAs that were significantly down-regulated in AD, and c) was also reported to be down-regulated in AD in two previous studies. The findings warrant replication and follow-up with a larger cohort of patients and controls who have been carefully characterized in terms of cognitive and imaging data, other biomarkers (e.g., CSF amyloid and tau levels) and risk factors (e.g., apoE4 status), and who are sampled repeatedly over time. Integrating miRNA expression data with other data is likely to provide informative and robust biomarkers in Alzheimer disease.

No MeSH data available.


Related in: MedlinePlus

Expression of Alix in whole plasma vs. P3 fraction.Fractions were prepared from normal mouse and human plasma, and equal amounts of protein were loaded for immunoblotting using anti-Alix antibody (see Methods). The P3 fractions were positive for Alix and were enriched relative to whole plasma.
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pone.0139233.g001: Expression of Alix in whole plasma vs. P3 fraction.Fractions were prepared from normal mouse and human plasma, and equal amounts of protein were loaded for immunoblotting using anti-Alix antibody (see Methods). The P3 fractions were positive for Alix and were enriched relative to whole plasma.

Mentions: The exosomal-enriched fraction of plasma was obtained by thawing 4 ml of frozen plasma (thawing was carried out at 4C); a cocktail of protease and RNase inhibitors were added in a volume of 70 μL/mL to reach the final concentration desired (10 mM EDTA, 10mM NEM, 1 mM PMSF, 1 μg/mL Pepstatin A, 10 μg/mL Leupeptin, 2 μg/mL Aprotinin, 10mM NaF, 1mM Na3VO4, 160 U/mL SuperaseIN (Life Technologies, Grand Island, NY), 160 U/mL RNase OUT, (Life Technologies). Samples were spun down at 1,500g x 10 min at 4C; the S1 supernatant was collected and diluted 1:3 with RNase free PBS and spun down at 20,000xg for 20 min. The dilution was performed to improve yield and to minimize variability in osmolarity that might be associated with clinical samples. The S2 supernatant was collected and further spun down at 200,000xg for 2 h at 4C using a 41Ti rotor. The final P3 pellet was used as the exosomal-enriched fraction. The P3 fraction was enriched in Alix, a marker of exosomes, relative to total plasma (Fig 1). Although the total amount of RNA present in a pellet was too small to allow accurate quantification by the nanodrop method, plasma miRNAs such as let-7a and mir-16 were readily measured by manual RT-qPCR (see below).


Plasma Exosomal miRNAs in Persons with and without Alzheimer Disease: Altered Expression and Prospects for Biomarkers.

Lugli G, Cohen AM, Bennett DA, Shah RC, Fields CJ, Hernandez AG, Smalheiser NR - PLoS ONE (2015)

Expression of Alix in whole plasma vs. P3 fraction.Fractions were prepared from normal mouse and human plasma, and equal amounts of protein were loaded for immunoblotting using anti-Alix antibody (see Methods). The P3 fractions were positive for Alix and were enriched relative to whole plasma.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591334&req=5

pone.0139233.g001: Expression of Alix in whole plasma vs. P3 fraction.Fractions were prepared from normal mouse and human plasma, and equal amounts of protein were loaded for immunoblotting using anti-Alix antibody (see Methods). The P3 fractions were positive for Alix and were enriched relative to whole plasma.
Mentions: The exosomal-enriched fraction of plasma was obtained by thawing 4 ml of frozen plasma (thawing was carried out at 4C); a cocktail of protease and RNase inhibitors were added in a volume of 70 μL/mL to reach the final concentration desired (10 mM EDTA, 10mM NEM, 1 mM PMSF, 1 μg/mL Pepstatin A, 10 μg/mL Leupeptin, 2 μg/mL Aprotinin, 10mM NaF, 1mM Na3VO4, 160 U/mL SuperaseIN (Life Technologies, Grand Island, NY), 160 U/mL RNase OUT, (Life Technologies). Samples were spun down at 1,500g x 10 min at 4C; the S1 supernatant was collected and diluted 1:3 with RNase free PBS and spun down at 20,000xg for 20 min. The dilution was performed to improve yield and to minimize variability in osmolarity that might be associated with clinical samples. The S2 supernatant was collected and further spun down at 200,000xg for 2 h at 4C using a 41Ti rotor. The final P3 pellet was used as the exosomal-enriched fraction. The P3 fraction was enriched in Alix, a marker of exosomes, relative to total plasma (Fig 1). Although the total amount of RNA present in a pellet was too small to allow accurate quantification by the nanodrop method, plasma miRNAs such as let-7a and mir-16 were readily measured by manual RT-qPCR (see below).

Bottom Line: Although these samples contained less than 0.1 microgram of total RNA, deep sequencing gave reliable and informative results.Twenty miRNAs showed significant differences in the AD group in initial screening (miR-23b-3p, miR-24-3p, miR-29b-3p, miR-125b-5p, miR-138-5p, miR-139-5p, miR-141-3p, miR-150-5p, miR-152-3p, miR-185-5p, miR-338-3p, miR-342-3p, miR-342-5p, miR-548at-5p, miR-659-5p, miR-3065-5p, miR-3613-3p, miR-3916, miR-4772-3p, miR-5001-3p), many of which satisfied additional biological and statistical criteria, and among which a panel of seven miRNAs were highly informative in a machine learning model for predicting AD status of individual samples with 83-89% accuracy.Perhaps the most interesting single miRNA was miR-342-3p, which was a) expressed in the AD group at about 60% of control levels, b) highly correlated with several of the other miRNAs that were significantly down-regulated in AD, and c) was also reported to be down-regulated in AD in two previous studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
To assess the value of exosomal miRNAs as biomarkers for Alzheimer disease (AD), the expression of microRNAs was measured in a plasma fraction enriched in exosomes by differential centrifugation, using Illumina deep sequencing. Samples from 35 persons with a clinical diagnosis of AD dementia were compared to 35 age and sex matched controls. Although these samples contained less than 0.1 microgram of total RNA, deep sequencing gave reliable and informative results. Twenty miRNAs showed significant differences in the AD group in initial screening (miR-23b-3p, miR-24-3p, miR-29b-3p, miR-125b-5p, miR-138-5p, miR-139-5p, miR-141-3p, miR-150-5p, miR-152-3p, miR-185-5p, miR-338-3p, miR-342-3p, miR-342-5p, miR-548at-5p, miR-659-5p, miR-3065-5p, miR-3613-3p, miR-3916, miR-4772-3p, miR-5001-3p), many of which satisfied additional biological and statistical criteria, and among which a panel of seven miRNAs were highly informative in a machine learning model for predicting AD status of individual samples with 83-89% accuracy. This performance is not due to over-fitting, because a) we used separate samples for training and testing, and b) similar performance was achieved when tested on technical replicate data. Perhaps the most interesting single miRNA was miR-342-3p, which was a) expressed in the AD group at about 60% of control levels, b) highly correlated with several of the other miRNAs that were significantly down-regulated in AD, and c) was also reported to be down-regulated in AD in two previous studies. The findings warrant replication and follow-up with a larger cohort of patients and controls who have been carefully characterized in terms of cognitive and imaging data, other biomarkers (e.g., CSF amyloid and tau levels) and risk factors (e.g., apoE4 status), and who are sampled repeatedly over time. Integrating miRNA expression data with other data is likely to provide informative and robust biomarkers in Alzheimer disease.

No MeSH data available.


Related in: MedlinePlus