Elsevier

Brain Research

Volume 1678, 1 January 2018, Pages 374-383
Brain Research

Research report
MicroRNA expression patterns in human anterior cingulate and motor cortex: A study of dementia with Lewy bodies cases and controls

https://doi.org/10.1016/j.brainres.2017.11.009Get rights and content

Abstract

Overview

MicroRNAs (miRNAs) have been implicated in neurodegenerative diseases including Parkinson’s disease and Alzheimer’s disease (AD). Here, we evaluated the expression of miRNAs in anterior cingulate (AC; Brodmann area [BA] 24) and primary motor (MO; BA 4) cortical tissue from aged human brains in the University of Kentucky AD Center autopsy cohort, with a focus on dementia with Lewy bodies (DLB).

Methods

RNA was isolated from gray matter of brain samples with pathology-defined DLB, AD, AD + DLB, and low-pathology controls, with n = 52 cases initially included (n  = 23 with DLB), all with low (<4 h) postmortem intervals. RNA was profiled using Exiqon miRNA microarrays. Quantitative PCR for post hoc replication was performed on separate cases (n = 6 controls) and included RNA isolated from gray matter of MO, AC, primary somatosensory (BA 3), and dorsolateral prefrontal (BA 9) cortical regions.

Results

The miRNA expression patterns differed substantially according to anatomic location: of the relatively highly-expressed miRNAs, 150/481 (31%) showed expression that was different between AC versus MO (at p < .05 following correction for multiple comparisons), most (79%) with higher expression in MO. A subset of these results were confirmed in qPCR validation focusing on miR-7, miR-153, miR-133b, miR-137, and miR-34a. No significant variation in miRNA expression was detected in association with either neuropathology or sex after correction for multiple comparisons.

Conclusion

A subset of miRNAs (some previously associated with α-synucleinopathy and/or directly targeting α-synuclein mRNA) were differentially expressed in AC and MO, which may help explain why these brain regions show differences in vulnerability to Lewy body pathology.

Introduction

Neurodegenerative diseases (NDs) are devastating brain disorders with an enormous impact on public health. Among the prevalent NDs are Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB), and “gold-standard” diagnoses can only be made with certainty at autopsy (Beach et al., 2012, Nelson et al., 2009b, Nelson et al., 2010a). According to consensus-based recommendations, DLB pathology includes aberrant cerebral cortical deposits of insoluble α-synuclein/Lewy bodies (McKeith et al., 2004, McKeith et al., 2017). The prevalence of Lewy body pathology is 10%-30% in most series of dementia patients (Heidebrink, 2002, Neltner et al., 2016, Rahkonen et al., 2003, Sonnen et al., 2009, Zaccai et al., 2005). It is relatively challenging to study “pure” DLB cases since most persons with cortical Lewy body pathology also have comorbid AD pathology (Irwin et al., 2017, Nelson et al., 2009b, Nelson et al., 2010a, Nelson, et al., 2010b, Nelson et al., 2010a). Notably, focused duplication of the gene (SNCA) that encodes α-synuclein is enough to produce α-synuclein/Lewy body pathology (Gwinn et al., 2011, Nishioka et al., 2006, Singleton et al., 2003), so, other gene regulatory factor(s) that cause increased α-synuclein expression could potentiate the pathology.

An enigmatic characteristic of NDs is that they tend to affect specific brain areas initially, and then progress to additional brain regions in a predictable sequence. Symptoms reflect the spatiotemporal progression. In DLB, Lewy bodies are observed in the anterior cingulate gyrus (Brodmann Area [BA] 24; AC) early in the disease (Thal et al., 2004). By contrast, the primary motor cortex (BA 4; MO) and primary somatosensory (BA 3) neocortical regions are relatively resistant to pathology (Thal et al., 2004).

Brain region-specific gene expression may contribute to the initiation and/or propagation of pathology in some parts of the brain, while causing other anatomically-defined brain areas to be less vulnerable. Testing this hypothesis requires factoring in gene regulatory phenomena in addition to mRNA levels, because mRNA is an imperfect proxy for protein levels, and transcription is only one among many different nodes of gene expression regulation in the human brain (Nelson and Keller, 2007). Thus, new information about factors that affect gene regulation post-transcriptionally may be helpful to achieve better understanding of ND pathogenesis.

MicroRNAs (miRNAs) are short (∼22 nucleotides in length) noncoding RNAs that have been implicated in NDs (Liu et al., 2008, Nelson et al., 2008a). Encoded by small genes that can be located practically anywhere in the genome (Londin et al., 2015), miRNAs regulate gene expression in multiple ways – predominantly by interacting with mRNA “targets” post-transcriptionally – and each miRNA has the potential to regulate dozens or even hundreds of targeted mRNAs (Kiriakidou et al., 2004, Liu et al., 2008, Wang, et al., 2010). Relative to protein-coding genes, the human brain miRNA repertoire is smaller, with under 1000 moderately- or highly-expressed brain transcripts (Hebert and Nelson, 2012). However, individual miRNAs can be expressed at up to an order of magnitude higher transcript copies than highly expressed mRNAs (Hebert and Nelson, 2012). Furthermore, individual miRNAs have previously been implicated in AD and Lewy body pathology (Hebert and De Strooper, 2009, Nelson et al., 2008a, Pietrzak et al., 2016, Ubhi et al., 2014, Weinberg et al., 2015).

Fundamental characteristics of any miRNA include the cells it is expressed in and the factors that are associated with expression variance. For this reason, miRNA expression profiling is an important tool for understanding miRNA biology in health and in disease states. Although there have been prior expression profiling studies of miRNAs in human tissues, many brain areas have not been assessed thoroughly to date, and there are many extant unanswered questions. For example, the miRNA profiles in AC and MO regions have not been compared with each other and the relevance of miRNAs to DLB is unknown.

To address these issues, we evaluated the expression of miRNAs in well-characterized human brains. We focused specifically on AC and MO tissue from aged human brains that had been snap-frozen in liquid nitrogen at autopsy from the University of Kentucky Alzheimer’s Disease Center (UK-ADC) cohort. We were interested in testing whether the miRNA expression patterns varied according to anatomic region, sex, and/or neuropathology.

Section snippets

Results

Human brain samples were obtained at autopsies after short post-mortem interval (PMI), followed by dissection of gray matter for RNA isolation and miRNA expression profiling. The rationale for the areas of brain chosen for miRNA analyses is depicted in Fig. 1. Some aspects of RNA quality are depicted in Fig. 2. Patient characteristics, including clinical and pathological parameters, and PMI, are shown in summary form in Table 1. Provided in Supplemental material are the following additional

Discussion

The purpose of the current study was to test whether variation in miRNA levels detected in human brain tissue was associated strongly with any of the following: DLB pathology; sex; or neuroanatomical region (AC vs MO). The hypotheses that were being tested, and our interpretation of the results, are depicted in Fig. 5. Of these factors, we only found evidence of the anatomical region being associated strongly with variation in miRNA expression: many miRNAs were differentially expressed in AC

RNA isolation from a human cerebral cortex

Samples were derived from short-PMI autopsies, representing different NDs as defined according to pathology. All methods were in compliance with a University of Kentucky IRB protocol. Premortem clinical evaluations and pathological assessments were as described previously (Nelson et al., 2007, Schmitt et al., 2000, Wang et al., 2008c). Tissue used for pathologic evaluation was dissected from MO and AC (immediately adjacent to the tissues sampled for RNA studies), immersion-fixed in formalin and

Acknowledgements

The authors are deeply grateful to the study participants and clinicians who made this research possible. We thank Sonya Anderson and Ela Patel for technical support. Study funding: P30 AG028383, R21 NS085830 from the National Institutes of Health. Thanks to Ruth S. Nelson for art work and Dr. Bernard S. Wilfred for lab work. The corresponding author Dr. Peter Nelson had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the

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