Plasma apolipoprotein E levels, isoform composition, and dimer profile in relation to plasma lipids in racially diverse patients with Alzheimer's disease and mild cognitive impairment.

BACKGROUND: The APOEε4-promoted risk of Alzheimer's disease (AD) is lower in Black/African-Americans (B/AAs), compared to non-Hispanic whites (NHWs). Previous studies reported lower plasma apolipoprotein E (apoE) levels in NHW APOEε4-carriers compared to non-carriers, and low plasma apoE levels were directly associated with an increased risk of AD and all dementia. We further showed that APOEε3/ε3 AD patients exhibited reduced plasma apoE dimers compared to corresponding control subjects. Whether plasma apoE levels and apoE dimer formation differ between races/ethnicities and therefore may help explain AD risk racial disparity remains to be elucidated. METHODS: Using mass spectrometry, we determined total plasma apoE and apoE isoform levels in a cohort of B/AAs (n = 58) and NHWs (n = 67) including subjects with normal cognition (B/AA: n = 25, NHW: n = 28), mild cognitive impairment (MCI) (B/AA: n = 24, NHW: n = 24), or AD dementia (B/AA: n = 9, NHW: n = 15). Additionally, we used non-reducing western blot analysis to assess the distribution of plasma apoE into monomers/disulfide-linked dimers. Plasma total apoE, apoE isoform levels, and % apoE monomers/dimers were assessed for correlations with cognition, cerebrospinal fluid (CSF) AD biomarkers, sTREM2, neurofilament light protein (NfL), and plasma lipids. RESULTS: Plasma apoE was predominantly monomeric in both racial groups and the monomer/dimer distribution was not affected by disease status, or correlated with CSF AD biomarkers, but associated with plasma lipids. Plasma total apoE levels were not related to disease status and only in the NHW subjects we observed lower plasma apoE levels in the APOEε4/ε4-carriers. Total plasma apoE levels were 13% higher in B/AA compared to NHW APOEε4/ε4 subjects and associated with plasma high-density lipoprotein (HDL) in NHW subjects but with low-density lipoprotein levels (LDL) in the B/AA subjects. Higher plasma apoE4 levels, exclusively in APOEε3/ε4 B/AA subjects, were linked to higher plasma total cholesterol and LDL levels. In the controls, NHWs and B/AAs exhibited opposite associations between plasma apoE and CSF t-tau. CONCLUSIONS: The previously reported lower APOEε4-promoted risk of AD in B/AA subjects may be associated with differences in plasma apoE levels and lipoprotein association. Whether differences in plasma apoE levels between races/ethnicities result from altered APOEε4 expression or turnover, needs further elucidation.

YKL-40 changes are not detected in post-mortem brain of patients with Alzheimer's disease and frontotemporal lobar degeneration.

BACKGROUND: YKL-40 (Chitinase 3-like I) is increased in CSF of Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD) patients and is therefore considered a potential neuroinflammatory biomarker. Whether changed YKL-40 levels in the CSF reflect dysregulation of YKL-40 in the brain is not completely understood yet. We aimed to extensively analyze YKL-40 levels in the brain of AD and different FTLD pathological subtypes. The direct relationship between YKL-40 levels in post-mortem brain and ante-mortem CSF was examined in a small set of paired brain-CSF samples. METHOD: YKL-40 was analyzed in post-mortem temporal and frontal cortex of non-demented controls and patients with AD and FTLD (including FTLD-Tau and FTLD-TDP) pathology by immunohistochemistry (temporal cortex: 51 controls and 56 AD and frontal cortex: 7 controls and 24 FTLD patients), western blot (frontal cortex: 14 controls, 5 AD and 67 FTLD patients), or ELISA (temporal cortex: 11 controls and 7 AD and frontal cortex: 14 controls, 5 AD and 67 FTLD patients). YKL-40 levels were also measured in paired post-mortem brain and ante-mortem CSF samples from dementia patients (n = 9, time-interval collection: 1.4 years) by ELISA. RESULTS: We observed that YKL-40 post-mortem brain levels were similar between AD, FTLD, and controls as shown by immunohistochemistry, western blot, and ELISA. Interestingly, strong YKL-40 immunoreactivity was observed in AD cases with cerebral amyloid angiopathy (CAA; n = 6). In paired CSF-brain samples, YKL-40 concentration was 8-times higher in CSF compared to brain. CONCLUSION: Our data suggest that CSF YKL-40 changes may not reflect YKL-40 changes within AD and FTLD pathological brain areas. The YKL-40 reactivity associated with classical CAA hallmarks indicates a possible relationship between YKL-40, neuroinflammation, and vascular pathology.

Automation vs. Experience: Measuring Alzheimer's Beta-Amyloid 1-42 Peptide in the CSF.

Cerebrospinal fluid (CSF) biomarkers can enhance the early and accurate etiologic detection of Alzheimer's disease (AD) even when symptoms are very mild, but are not yet widely available for clinical testing. There are a number of reasons for this, including the need for an experienced operator, the use of instruments mostly reserved for research, and low cost-effectiveness when patient samples do not completely fill each assay plate. Newer technology can overcome some of these issues through automated assays of a single patient sample on existing clinical laboratory platforms, but it is not known how these newer automated assays compare with previous research-based measurements. This is a critical issue in the clinical translation of CSF AD biomarkers because most cohort and clinicopathologic studies have been analyzed on older assays. To determine the correlation of CSF beta-amyloid 1-42 (Aß42) measures derived from the automated chemiluminescent enzyme immunoassay (CLEIA, on Lumipulse® G1200), a bead-based Luminex immunoassay, and a plate-based enzyme-linked immunoassay enzyme-linked immunosorbent assay (ELISA), we analyzed 30 CSF samples weekly on each platforms over 3 weeks. We found that, while CSF Aß42 levels were numerically closer between CLEIA and ELISA measurements, levels differed between all three assays. CLEIA-based measures correlated linearly with the two other assays in the low and intermediate Aß42 concentrations, while there was a linear correlation between Luminex assay and ELISA throughout all concentrations. For repeatability, the average intra-assay coefficient of variation (CV) was 2.0%. For intermediate precision, the inter-assay CV was lower in CLEIA (7.1%) than Luminex (10.7%, p = 0.009) and ELISA (10.8%, p = 0.009), primarily due to improved intermediate precision in the higher CSF Aß42 concentrations. We conclude that the automated CLEIA generated reproducible CSF Aß42 measures with improved intermediate precision over experienced operators using Luminex assays and ELISA, and are highly correlated with the manual Aß42 measures.

Race modifies the relationship between cognition and Alzheimer's disease cerebrospinal fluid biomarkers.

BACKGROUND: African Americans have been reported to have a higher prevalence of Alzheimer's disease (AD) than Caucasians, but etiology-specific AD biomarkers have not been systematically analyzed in older African Americans. Coexisting cerebrovascular disease may also contribute to this increased prevalence. We hypothesized that cerebrospinal fluid (CSF) biomarkers of amyloid, neurodegeneration, and endothelial dysfunction would differ between older African Americans and Caucasians with normal cognition and cognitive impairment associated with AD. METHODS: We prospectively recruited 135 older Americans to undergo detailed clinical, neuropsychological, genetic, magnetic resonance imaging (MRI), and CSF analysis from 2013 to 2015 at Emory University (Atlanta, GA, USA). We compared levels of CSF markers for ß-amyloid (Aß42, Aß40), total and phosphorylated tau (t-tau and p-tau181, respectively), endothelial dysfunction (soluble vascular cell adhesion molecule 1, soluble intercellular adhesion molecule 1), α-synuclein, and neurodegeneration (neurofilament light chain [NfL]), as well as MRI markers, for hippocampal atrophy and cerebrovascular disease (white matter hyperintensity [WMH] volume). RESULTS: Sixty-five older African Americans (average age, 69.1 years) and 70 older Caucasians (average age, 70.8 years) were included. After adjusting for demographic variables, AD risk alleles, and cognitive function, older African Americans had lower CSF levels of p-tau181 (difference of 7.4 pg/ml; 95% CI, 3.7-11.2 pg/ml; p < 0.001), t-tau (difference of 23.6 pg/ml; 95% CI, 9.5-37.7; p = 0.001), and Aß40 (difference of 1.35 ng/ml; 95% CI, 0.29-2.42 ng/ml; p = 0.013) despite similar levels of Aß42, NfL, WMH volume, and hippocampal volume. Cognitively impaired African Americans also had lower CSF t-tau/Aß42 (difference of 0.255 per 1-SD change in composite cognition; 95% CI, 0.100-0.409; p = 0.001) and p-tau181/Aß42 (difference of 0.076 per 1-SD change in composite cognition; 95% CI, 0.031-0.122; p = 0.001). These could not be explained by measured biomarkers of non-AD processes, but African Americans may be more susceptible than Caucasians to the cognitive effects of WMH. CONCLUSIONS: Despite comparable levels of CSF Aß42 and Aß42/Aß40, cognitive impairment in African Americans is associated with smaller changes in CSF tau markers but greater impact from similar WMH burden than Caucasians. Race-associated differences in CSF tau markers and ratios may lead to underdiagnosis of AD in African Americans. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02089555 . Retrospectively registered on 14 March 2014.

Fear and Uncertainty Do Not Influence Reported Willingness to Undergo Lumbar Punctures in a U.S. Multi-Cultural Cohort.

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease and related disorders can provide early and accurate prediction of underlying neuropathology even when the clinical symptoms are mild, but lumbar punctures (LP) to obtain CSF can be perceived as frightening and invasive. We previously demonstrated that this negative perception of the LP is strongly associated with a negative LP experience in terms of discomfort and complications, but it is not known what factors can lead to a negative perception of the LP. It has also been proposed that LP is less well-perceived by adults in the U.S. compared to Europe and elsewhere, although there is a paucity of primary data to support this. To address these knowledge gaps, we conducted a survey of 237 younger and older adults in the Atlanta area including a significant number born outside of the U.S. (n = 82, 34%) to determine demographic, medical, and experiential factors associated with the perception of LP as well as the willingness to undergo LP for medical or research purposes. Our results show that one in four respondents in this cohort with limited first-hand LP experience viewed the LP as a frightening invasive procedure, but the majority (89%) were willing to undergo LP for medical reasons. General awareness of the LP was associated with both standard and negative views of the LP, but perception did not influence willingness to undergo the procedure. Multi-variate models showed that higher annual household income, not place of birth or past experience, was associated with greater willingness to undergo LPs. We conclude that Americans (born in the U.S. or abroad) are not resistant to LPs if there is useful information to improve their health, although there is limited enthusiasm to undergo LPs solely for research purposes. At the same time, we failed to find modifiable factors to improve the perception of LP among those who already perceive it as frightening and invasive. Future recruitment effort should target adults with no preconceived notion of the LP with emphasis on data related to safety and tolerability.

Research Lumbar Punctures among African Americans and Caucasians: Perception Predicts Experience.

African Americans are under-represented in Alzheimer's disease (AD)-related biomarker studies, and it has been speculated that mistrust plays a major factor in the recruitment of African Americans for studies involving invasive procedures such as the lumbar puncture (LP). We set out to determine factors associated with non-participation in a biomarker study aiming to explore cerebrospinal fluid (CSF) AD biomarker differences between older African Americans and Caucasians. We also surveyed participants' procedure-related perception (a standard medical procedure vs. a frightening invasive procedure) and reluctance, as well as the rate and type of post-procedure discomfort and complications. Among 288 subjects approached for study participation, 145 (50.3%) refused participation with concerns over LP being the most commonly reported reason. Relatively more African Americans than Caucasians reported concerns over LP as the main reason for non-participation (46% vs. 25%, p = 0.03), but more African Americans also did not provide a specific reason for non-participation. Among those who completed study participation (including the LP), African Americans and Caucasians were similar in pre-LP perceptions and reluctance, as well as post-LP rates of discomfort or complication. Perceiving LP as a frightening invasive procedure, not race, is associated with increased likelihood of post-LP discomfort or complication (RR 6.2, 95% confidence interval 1.1-37.0). Our results indicate that LP is a well perceived procedure in a cohort of African American and Caucasian research participants, and is associated with few serious complications. The pre-procedure perception that the LP is a frightening invasive procedure significantly increases the risk of self-reported discomfort of complications, and African Americans may be more likely to turn down study participation because of the LP. Future studies will need to address factors associated with negative LP perceptions to further assure participants and reduce complication rates.

CSF complement 3 and factor H are staging biomarkers in Alzheimer's disease.

INTRODUCTION: CSF levels of established Alzheimer's disease (AD) biomarkers remain stable despite disease progression, and non-amyloid non-tau biomarkers have the potential of informing disease stage and progression. We previously identified complement 3 (C3) to be decreased in AD dementia, but this change was not found by others in earlier AD stages. We hypothesized that levels of C3 and associated factor H (FH) can potentially distinguish between mild cognitive impairment (MCI) and dementia stages of AD, but we also found their levels to be influenced by age and disease status. RESULTS: We developed a biochemical/bioinformatics pipeline to optimize the handling of complex interactions between variables in validating biochemical markers of disease. We used data from the Alzheimer's Disease Neuro-imaging Initiative (ADNI, n = 230) to build parallel machine learning models, and objectively tested the models in a test cohort (n = 73) of MCI and mild AD patients independently recruited from Emory University. Whereas models incorporating age, gender, APOE ε4 status, and CSF amyloid and tau levels failed to reliably distinguish between MCI and mild AD in ADNI, introduction of CSF C3 and FH levels reproducibly improved the distinction between the two AD stages in ADNI (p < 0.05) and the Emory cohort (p = 0.014). Within each AD stage, the final model also distinguished between fast vs. slower decliners (p < 0.001 for MCI, p = 0.007 for mild AD), with lower C3 and FH levels associated with more advanced disease and faster progression. CONCLUSIONS: We propose that CSF C3 and FH alterations may reflect stage-associated biomarker changes in AD, and can complement clinician diagnosis in diagnosing and staging AD using the publically available ADNI database as reference.

MicroRNA-152 represses VEGF and TGFß1 expressions through post-transcriptional inhibition of (Pro)renin receptor in human retinal endothelial cells.

PURPOSE: The (pro)renin receptor (PRR), a component of the renin-angiotensin system (RAS), plays an important role in the physiologic and pathophysiological regulation of blood pressure and fluid/electrolyte homeostasis. The RAS including the PRR has been identified in retinal endothelial cells and other ocular tissues. In this study, the potential involvement of miRNAs in the posttranscriptional regulation of PRR was investigated in human retinal endothelial cells (hRECs) under high glucose (HG) conditions. METHODS: miRNA-152 (miR-152) was identified in silico as a potential regulator of PRR, and this was confirmed by quantitative real-time PCR (qRT-PCR) and PRR 3'-untranslated region (UTR) reporter assays. Using RNA interference, both AT1R and PRR were implicated in the HG-mediated induction of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR-2), and transforming growth factor ß1 (TGFß1). RESULTS: The downregulation of miR-152 was observed in hRECs and rat retinal tissues under HG conditions. In parallel, PRR (target of miR-152), VEGF, VEGFR-2, and TGFß1 at mRNA levels were elevated. However, the transfection of hRECs with miR-152 mimics in HG conditions resulted in the suppression of the PRR expression, as well as reduced VEGF, VEGFR-2, and TGFß1 production. This was reversed by transfecting cells with the antisense (antagomir) of miR-152, suggesting the glucose-induced upregulation of VEGF, VEGFR-2, and TGFß1 is mediated through PRR, and this regulation is likely achieved through the HG-mediated modulation of miRNAs. CONCLUSIONS: We have demonstrated that miR-152 interacting with PRR regulates downstream VEGF, VRGFR-2, and TGFß1 expressions in hRECs in HG conditions. These studies suggest miR-152 and PRR may play a role in the pathogenesis of diabetic retinopathy (DR).

CSF beta-amyloid 1-42 - what are we measuring in Alzheimer's disease?

OBJECTIVE: To characterize biological and technical factors which influence cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarker levels, including the presence of apolipoprotein E (APOE) ε4 allele, AD diagnosis, Aß-binding proteins, sample processing, and preanalytical handling. METHODS: CSF was collected from 140 subjects with normal cognition, mild cognitive impairment, AD, and non-AD dementia. CSF levels of beta-amyloid 1-42 (Aß42), total Tau (t-Tau), and Tau phosphorylated at threonine 181 (p-Tau181) were analyzed following the standard and modified protocols. CSF levels of apoJ, apoE, albumin, and α-synuclein were measured in a subgroup (n = 69), and their effects on measured AD biomarker levels were also determined in vitro using human CSF samples. RESULTS: CSF Aß42 levels measured using the AD Neuro-imaging Initiative (ADNI) protocol (which we call suspended Aß42 or susAß) were lower than total measurable CSF Aß42 in all groups, and on average represents 57% of the latter. Logistic regression analysis showed this proportion (% susAß) to be directly correlated with CSF Aß42 and apoJ levels, but inversely correlated with CSF t-Tau levels. Finally, we showed in vitro that increasing apoE and apoJ levels directly increased % susAß. CONCLUSION: CSF susAß levels are influenced by biological and technical factors, and may represent a marker of Aß susceptible to lipoprotein-mediated clearance. Clinical trials should include total measurable Aß42 and susAß to better inform outcomes.

Global microRNA expression profiling: curcumin (diferuloylmethane) alters oxidative stress-responsive microRNAs in human ARPE-19 cells.

PURPOSE: In recent years, microRNAs (miRNAs) have been reported to play important roles in a broad range of biologic processes, including oxidative stress-mediated ocular diseases. In addition, the polyphenolic compound curcumin has been shown to possess anti-inflammatory, antioxidant, anticancer, antiproliferative, and proapoptotic activities. The aim of this study was to investigate the impact of curcumin on the expression profiles of miRNAs in ARPE-19 cells exposed to oxidative stress. METHODS: MiRNA expression profiles were measured in ARPE-19 cells treated with 20 µΜ curcumin and 200 µΜ H2O2. PCR array analysis was performed using web-based software from SABiosciences. The cytotoxicity of ARPE-19 cells was determined with the CellTiter-Blue cell viability assay. The effects of curcumin on potential miRNA targets were analyzed with quantitative real-time PCR and western blotting. RESULTS: Curcumin treatment alone for 6 h had no effect on ARPE-19 cell viability. Incubation with H2O2 (200 µM) alone for 18 h decreased cell viability by 12.5%. Curcumin alone downregulated 20 miRNAs and upregulated nine miRNAs compared with controls. H2O2 downregulated 18 miRNAs and upregulated 29 miRNAs. Furthermore, curcumin pretreatment in cells exposed to H2O2 significantly reduced the H2O2-induced expression of 17 miRNAs. As determined with quantitative real-time PCR and western blotting, curcumin increased the expression of antioxidant genes and reduced angiotensin II type 1 receptor, nuclear factor-kappa B, and vascular endothelial growth factor expression at the messenger RNA and protein levels. CONCLUSIONS: The results demonstrated that curcumin alters the expression of H2O2-modulated miRNAs that are putative regulators of antioxidant defense and renin-angiotensin systems, which have been reported to be linked to ocular diseases.

MicroRNA-30b-mediated regulation of catalase expression in human ARPE-19 cells.

BACKGROUND: Oxidative injury to retinal pigment epithelium (RPE) and retinal photoreceptors has been linked to a number of retinal diseases, including age-related macular degeneration (AMD). Reactive oxygen species (ROS)-mediated gene expression has been extensively studied at transcriptional levels. Also, the post-transcriptional control of gene expression at the level of translational regulation has been recently reported. However, the microRNA (miRNA/miR)-mediated post-transcriptional regulation in human RPE cells has not been thoroughly looked at. Increasing evidence points to a potential role of miRNAs in diverse physiological processes. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated for the first time in a human retinal pigment epithelial cell line (ARPE-19) that the post-transcriptional control of gene expression via miRNA modulation regulates human catalase, an important and potent component of cell's antioxidant defensive network, which detoxifies hydrogen peroxide (H(2)O(2)) radicals. Exposure to several stress-inducing agents including H(2)O(2) has been reported to alter miRNA expression profile. Here, we demonstrated that a sublethal dose of H(2)O(2) (200 µM) up-regulated the expression of miR-30b, a member of the miR-30 family, which inhibited the expression of endogenous catalase both at the transcript and protein levels. However, antisense (antagomirs) of miR-30b was not only found to suppress the miR-30b mimics-mediated inhibitions, but also to dramatically increase the expression of catalase even under an oxidant environment. CONCLUSIONS/SIGNIFICANCE: We propose that a microRNA antisense approach could enhance cytoprotective mechanisms against oxidative stress by increasing the antioxidant defense system.

Melatonin synthesis in retina: cAMP-dependent transcriptional regulation of chicken arylalkylamine N-acetyltransferase by a CRE-like sequence and a TTATT repeat motif in the proximal promoter.

Arylalkylamine N-acetyltransferase (AANAT) is the key regulatory enzyme controlling the daily rhythm of melatonin biosynthesis. In chicken retinal photoreceptor cells, Aanat transcription and AANAT activity are regulated in part by cAMP-dependent mechanisms. The purpose of this study was to identify regulatory elements within the chicken Aanat promoter responsible for cAMP-dependent induction. Photoreceptor-enriched retinal cell cultures were transfected with a luciferase reporter construct containing up to 4 kb of 5'-flanking region and the first exon of Aanat. Forskolin treatment stimulated luciferase activity driven by the ∼4 kb promoter construct and by all 5'-deletion constructs except the smallest, Aanat (-217 to +120)luc. Maximal basal and forskolin-stimulated expression levels were generated by the Aanat (-484 to +120)luc construct. This construct lacks a canonical cyclic AMP-response element (CRE), but contains two other potentially important elements in its sequence: an eight times TTATT repeat (TTATT8) and a CRE-like sequence. Electrophoretic mobility shift assays, luciferase reporter assays, chromatin immunoprecipitation, and siRNA experiments provide evidence that these elements bind c-Fos, JunD, and CREB to enhance basal and forskolin-stimulated Aanat transcription. We propose that the CRE-like sequence and TTATT8 elements in the 484 bp proximal promoter interact to mediate cAMP-dependent transcriptional regulation of Aanat.

Noise levels during aerobics and the potential effects on distortion product otoacoustic emissions.

UNLABELLED: The purpose of this study was to measure noise levels during aerobics classes and to examine how outer hair cell (OHC) function, using distortion product otoacoustic emissions (DPOAEs), may be affected by this exposure. Fifty individuals (48 women and 2 men, ages 19-41 years) participated in 50-min aerobics classes. Noise levels were measured using noise dosimeters placed on the collar near the test ear. The audiometric protocol consisted of a questionnaire, otoscopy, screening tympanometry, and pre- and post-aerobics DPOAEs. The minute-by-minute peak noise levels varied between 90.5 and 99.7dBA. The overall mean noise level for the aerobics classes was 87.1dBA (range=83.4-90.7dBA). Mean post-aerobic DPOAE levels were lower at most frequencies with a statistically significant 1.4dB decrease at 6000Hz. Results of this project, even with limited statistical significance, should be viewed cautiously. LEARNING OUTCOMES: The reader will be able to (1) identify the approximate dB SPL measured during aerobics classes, (2) describe what effects, if any, these levels had on DPOAEs, and (3) discuss what specific frequency had a significant decrease in DPOAE level after an aerobics class.