James P. Bennett, Jr.1* and Paula M. Keeney1
1Neurodegeneration Therapeutics, Inc
3050A Berkmar Drive
Charlottesville, VA 22901-3450
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease of humans, is a progressive disorder that can begin outside of the central nervous system and involves multiple brain areas. We used high-density Illumina® RNA sequencing (RNA-seq) to examine the transcriptomes of ventral midbrain samples from 14 PD and 9 CTL age-, postmortem interval- and RNA quality-matched postmortem brain samples. We aligned de-multiplexed sequences against the latest UCSC-hg38 human genome using tophat2/bowtie2, then processed the resulting .bam files with either Cufflinks (FPKM normalization) or Qlucore Omics Explorer® (QOE, FPKM or TMM normalization). Gene expression data were processed using FDR (false discovery rates, Cufflinks and QOE) to compare multiple samples, PCA (principal component analysis-QOE) and GSEA (gene set expression analysis-QOE).
PD samples showed >65% loss of dopaminergic markers. At FDR<5%, 153/177 genes (86.4%) were under-expressed in PD by Cufflinks and 199/351 (56.7%) by QOE (FPKM normalization). 26 genes were co-identified by both Cufflinks and QOE (FPKM normalization) at FDR<5%, and their PD/CTL ratios (25/26 < 1.0) were linearly related (r2=0.992). GSEA analysis of PD samples showed greatest enrichment in cholesterol homeostasis (31/73 genes), mTORC1 signaling (87/198 genes) and oxidative phosphorylation (123/199 genes). PD samples were markedly heterogenous as reflected in plots of gene expression variance in PD vs CTL samples, and in PD samples against gene expression ratios (PD/CTL). The stress-response gene GADD45A/α showed small variance and was under-expressed in PD. Our systems biology approaches suggest that increased expression of GADD45A/α could benefit the majority of PD subjects.
Parkinson’s disease (PD) is a common neurodegenerative disease of adults and ranks second behind Alzheimer’s disease as an age-related affliction of societies. 7-10 million persons worldwide have PD, which is commonly (>90%) sporadic and afflicts men about 1.5 times as often as women. Older age is the major known risk factor for PD. Rare mutations of chromosomal genes are believed to be causal for autosomal dominantly or recessively inherited PD that can have variable postmortem neuropathology. [1-5]
Motor symptoms of PD are currently the basis of clinical diagnosis and are characterized by progressive bradykinesia of speech and limbs, rigidity, postural instability that can lead to increased falls and, at least once during the illness, a 4-6 Hz resting tremor of face/tongue and/or limbs. These motor deficits of tremor, rigidity and bradykinesia are believed to arise from progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. PD is commonly complicated by “secondary” symptoms, which include deficits of neuropsychiatric function, autonomic disturbances, and sleep disorders. [6-9]
Frequently, affected nigral neurons possess eosinophilic inclusions containing aggregated and post-translationally modified alpha-synuclein (A-SYN), among other proteins [5, 10-15]. Nigral DA neurons in PD show ultrastructural changes of both apoptosis and autophagy .
Neuropathological examinations of many hundreds of autopsies carried out by the Braak group have contributed significantly to our collective understanding of the correlations of PD progression and neuropathology. These studies revealed that aggregated A-SYN appears initially in gastrointestinal enteric neurons, followed temporally by rostral spread into dorsal medulla, midbrain, limbic system and frontal cortex. Braak, et al have divided PD into 6 stages, with the first 3 stages representing pre-clinical (in terms of motor deficits) progression, and the latter 3 stages representing progression of motor deficits and appearance of important secondary symptoms such as mood disorders and other neuropsychiatric symptoms and cognitive impairment. These latter 3 stages of PD correlate with spread of A-SYN aggregation pathology into rostral limbic and frontal lobe regions. [7-12, 17-40]
PD motor deficits are commonly treated by dopaminergic agents, either L-DOPA, the immediate precursor of lost dopamine (DA), or DA-ergic agonists, sometimes accompanied with agents that interfere with L-DOPA methylation or DA catabolism. These approaches are typically effective for 3-5 years, but commonly are followed by erratic responses to DA-ergic agents and troublesome side effects. Clinical responses can be improved, sometimes with reduced and/or more frequent DA-ergic drug dosing, or by continuous deep brain stimulation of basal ganglia nuclei, such as globus pallidus interna (GPi) or subthalamic nucleus (STN) [2, 3, 5, 41-44].
There are presently no known approaches that retard progression of underlying PD pathology. Should such approaches become available, then management of early clinical motor stages of PD, which generally are straightforward and successful, could be temporally extended, and later disabilities frequently derived from secondary symptoms  might be avoided.
Analyses of PD brain transcriptomes have yielded complex results.  Transcriptomes in postmortem brain represent attempts of cells to respond to stresses present at the time of death. These stresses may or may not reflect processes causal to neurodegeneration. In addition, individuals may vary in their molecular responses to similar stresses, further complicating the picture.
Because one goal of transcriptomic studies of PD postmortem brain is to attempt defining therapies that may alter deficits or overactivities of pathways that are defective in the majority of PD subjects, not a selective minority (e.g., those with a rare genetic cause), we undertook this study to try and find such a therapeutic approach. We used RNA sequencing of postmortem ventral midbrain, which consists mainly of glia and residual dopaminergic (and other phenotypic) neurons. We found significant heterogeneity of gene expression but were able to define underexpression in the majority of PD samples of a stress-activated gene (GADD45A/α) that alters many intracellular events. We propose that augmentation of expression of that gene and similar genes offers the potential to restore protective pathways in PD neurodegeneration.
Blocks of snap frozen human substantia nigra/ventral mid brain were obtained from the University of Virginia Brain Resource Facility, the Virginia Commonwealth University Brain Tissue Resource Facility or the National Disease Research Interchange (NDRI), Philadelphia, PA. Demographics of the samples are presented in Supplemental Table 1.
Isolation of total RNA
Briefly, the area of interest was dissected from fresh post mortem midbrain, immediately frozen in liquid nitrogen-chilled 2-methly butane and stored at -80 oC until sectioned. At the time of sectioning, tissue blocks were trimmed to about 1 by 1.5 cm, embedded in Neg-50 embedding compound (Richard-Allan Scientific, Kalamazoo, MI) and sectioned at -16oC. 25 sections (20 um) were collected from each sample and placed in Qiazol (Qiagen, Germantown, MD). Total RNA was isolated according to miRNeasy instructions with sonication of the tissue in Qiazol, on-column DNase digestion and an additional RPE wash. RNA quantity and quality was assessed using an Agilent Tape Station. Total RNA was sent to CoFactor Genomics for preparation and sequencing, see below.
RNA-seq and analyses
Total RNA was rRNA depleted, multiplex sequencing libraries were constructed and quantitated, and Illumina RNA paired-end 150 base sequencing carried out at CoFactor Genomics as described in [47, 48]. All bioinformatics analyses were performed “blind”, with the samples identified only as numbers and not by disease. Sequencing fastq files were examined for quality in FastQC, then Illumina sequencing adaptors removed using Trimmomatic®. Resulting sequencing files with minimum Phred scores of 33 were aligned against the UCSC-hg38 version of the human genome using Tophat2/Bowtie2®. The resulting *.bam files were processed either by Cufflinks® to yield gene expression normalized and expressed by the FPKM (Fragments Per Kilobase of exon sequenced per Million reads) method, or processed using Qlucore Genomics Explorer® with the NGS plug-in (Qlucore Genomics, Inc., www.qlucore.com) and normalized by either the TMM (Trimmed Mean of M-values) or FPKM methods. The same genome.fa and genes.gtf files were used by all programs.
Graphing and Statistics
All graphing and statistics were performed in GraphPad Prism version 7 (for Mac OSX) (www.graphpad.com). QOE (www.qlucore.com) was version 3.3(80) and included the NGS (Next Generation Sequencing) plug-in. All bioinformatic analyses were carried out on a Macintosh Pro desktop computer operating with OSX v 11 and possessing a 12-core processor and 64 GB of RAM. Sequencing data and output files were stored on a 32TB server.
PD ventral midbrain samples show loss of expression of multiple dopaminergic markers
By the time an individual dies with advanced PD, s/he has lost the majority of dopaminergic neurons in the substantia nigra [2, 13, 14]. Dopaminergic neuron loss in the more dorsal ventral tegmental area is variable in PD . Our PD ventral midbrain samples (Supplemental Table 1) showed loss of expression of tyrosine hydroxylase (TH; PD/CTL (ratios of mean PD FPKM/mean CTL FPKM) = 0.332, q (false discovery rate metric) = 0.058); dopamine transporter (DAT/SLC6A3; PD/CTL = 0.309, q=0.062); VMAT2/SLC18A2; (PD/CTL = 0.360; q= 0.063). Expression of dopa-decarboxylase, which can decarboxylate multiple L-aromatic amino acids in addition to L-DOPA, was also reduced (DDC; PD/CTL= 0.414; q=0.087).
Expression of multiple autosomal genes mutated in rare familial PD were either reduced or not statistically present in our ventral midbrain samples.
Expression of alpha-synuclein (ASYN/SNCA), the initial gene identified as mutated in familial PD [49, 50], was reduced (PD/CTL = 0.557; q=0.044), as was that of UCHL1 (PD/CTL = 0.62; q=0.056). Additional autosomal genes, mutations of which have also been associated with familial PD, were not found in our list of genes with FDR’s < 10% (q<0.1). These included LRRK2, PINK1, parkin/PARK2, DJ-1/PARK7, UPS35 and EIF4G1.
PCA plots demonstrate separable PD and CTL RNA-seq populations
PCA (principal component analysis) plots were constructed in QOE after processing the Tophat2/BowTie2-generated *.bam RNA-seq files using the NGS (Next Generation Sequencing) plug-in. Figure 1a shows the PCA plot using TMM normalization and Figure 1b shows the PCA plot obtained using FPKM normalization for the same *.bam files. Both plots showed that at a FDR (q) of <5% it was possible to separate the two populations (PD, CTL). Networks could also be constructed in QOE using TMM (Figure 1a) or FPKM (Figure 1b) normalization. Note that the same PD sample (160) overlapped in network analysis with the CTL group in both TMM and FPKM PCA plots.
Cufflinks and QOE processing showed small overlap among DEG’s (differentially expressed genes) but with nearly identical expression ratios
Processing of the *.bam sequencing files against the same hg38 genes.gtf file yielded mean FPKM values (Cufflinks) or “fold-change” values (QOE). Controlling for multiple comparisons (q<5%) yielded 177 differentially expressed genes (DEG’s) using Cufflinks and 351 DEG’s (by QOE) (Supplemental Table 2). With Cufflinks 153/177 genes (86.4%) had PD/CTL ratios < 1.0, and with QOE using FPKM normalization 199/351 genes (56.7%) had PD/CTL ratios < 1.0. There were 26 genes in common in these two datasets (Supplemental Table 3), and Figure 2 shows that the PD/CTL fold-changes in expression values derived from each analytical approach were highly correlated with each other (r2=0.992). 25/26 genes in this group were under-expressed in the PD datasets.
Heatmaps also demonstrated separability of the PD and CTL populations
Figure 3a (top, left) shows a QOE-generated heatmap of genes hierarchically clustered with q <5% and FPKM normalization, and Figure 3b shows a similar plot with q<5% and TMM normalization. In both of these heatmaps, hierarchical clustering “mis-labeled” one PD sample as belonging to the CTL cluster group. This sample (160) was the same PD sample that interacted with CTL samples in network analysis of the PCA plots (Figures 1a, 1b) Lowering of the q metric resulted in proper heirarchical cluster separation of PD and CTL samples (Figures 3c, 3d).
GSEA analyses show gene enrichment in cholesterol homeostasis, mTORC1 signaling and oxidative phosphorylation
Figure 4 shows heatmaps derived from gene set enrichment analysis (GSEA) analysis (using QOE) of cholesterol homeostasis, mTORC1 signaling and oxidative phosphorylation, the three gene sets having the highest relative enrichment in the PD samples. We used the most current “hallmark” GSEA file for these calculations. GSEA delineated that genes were represented in cholesterol homeostasis (31/73 genes), mTORC1 signaling (87/198 genes) and oxidative phosphorylation (123/199 genes).
Gene expression in PD brain samples exhibited heterogeneity.
Figure 5 shows plots of variance of gene expression (variance = standard deviation squared) in PD samples (from Cufflinks, y-axis) compared on a gene-by-gene basis to variance of expression in CTL samples (from Cufflinks, x-axis). The lower plot is an amplification of axes used in the upper plot. As can be seen, there was significant scatter in the variance values, with no clear relationships between PD and CTL.
Figure 6 shows a gene-by-gene plot of gene expression variance in the PD samples (from Cufflinks, y-axis) versus mean PD/mean CTL gene expression ratio (from Cufflinks, x-axis). The lower plot is an amplification of the axes shown in the upper plot. There is significant scatter in the values, with a suggestion of a Gaussian-type distribution. The vertical blue lines represent PD/CTL expression ratios of 1.0 (equal expression in PD and CTL).
The orange rectangle in the lower plot encompasses the genes with lowest PD variance (< 50) and expression ratios (PD/CTL) <0.5 (under-expressed in PD > 2.0-fold). These 33 genes (Supplemental Table 4), if over-expressed, should theoretically represent therapeutic targets for the majority of PD subjects.
The stress-response gene GADD45α stands out in this group for several reasons. 1.It has among the lowest variances in the PD group (5.60) and is under-expressed in PD (PD/CTL = 0.39) ; 2. It possesses pleotropic actions on multiple cell processes. 3.It may be involved in PD neurodegeneration (see Discussion for more details)
In this study we used RNA sequencing (RNA-seq) to characterize gene expression in PD and CTL ventral midbrain samples that were matched for age, postmortem intervals and RNA purity. RNA-seq, although still expensive, is rapidly becoming the state-of-the-art for gene expression studies, having replaced microarray platforms and quantitative PCR (qPCR), both 20th century technologies. There are almost 200,000 PubMed citations for this technique, and publicly available software for bioinformatic analysis of RNA-seq data has been available for ~ a decade. Commercial software is also becoming available, and we compared publicly available bioinformatic software (Tophat2/Bowtie2/Cufflinks) to that available commercially (QOE). Note that the commercial software we used still required generation of binary sequencing files (.bam files), which we did using Tophat2/Bowtie2 and the most current version (hg38) of the human genome.
Although RNA-seq is powerful and generates large datasets (which can be challenging to analyze), when applied to postmortem human brain samples from individuals that shared a common clinical phenotype, RNA-seq data must be interpreted with caution. There are multiple reasons for this caution, three of which are discussed below:
First, most individuals whose brains were donated for storage and study likely died at end-stage of disease. In the case of neurodegenerative diseases, RNA-seq may reveal molecular strategies of “survivors”, not the vulnerable neurons that have already died. Observed gene expression likely represents events mostly occurring in astroglia and represents little contribution from vulnerable neuronal populations. “Genesis events” for neurodegeneration may also not be revealed. This limitation may be addressed by use of cell lines derived from living donors, but even this approach must be used cautiously .
Second, although humans share much genetic material in common, each person is genetically/epigenetically unique, and there is no a priori reason to assume that common molecular pathogenic abnormalities will emerge from RNA-seq studies. In fact, our findings indicate great degrees of heterogeneity of gene expression across tissue samples that included quantitation of expression of thousands of genes.
Third, alterations in RNA expression may represent causal changes or may represent adaptive responses to causal events. One cannot assume that changes in RNA expression belong to either group.
These arguments suggest that our collective attempts in the past to define common sets of altered genes, even after corrections for multiple samples, is problematic in terms of defining both biological origins/changes and therapeutic relevance. In fact, there is little agreement across multiple gene expression studies as to alterations of specific genes (see  for analysis of recent Parkinson’s transcriptomic studies).
Our findings demonstrate the limited overlap among analyses that used the same *.bam sequencing files to estimate expression of genes. Even with use of common FPKM normalization, one must be cautious, as we observed very limited overlap of individual genes identified by what should be very similar approaches.
We do not believe there is a “right” or “wrong” approach. Rather, one must be aware of potential differences in estimation of gene expression. For comparative RNA-seq studies, one should use raw sequencing files and process them in the same manner.
The stress-response gene GADD45A/α (Growth Arrest and DNA Damage-inducible 45 A/α) is a rapidly-induced member of the GADD45 family that itself is non-enzymatic but serves multiple critical cell survival functions through nucleic acid and protein partner binding. Among these is the response to genotoxic DNA damage mediated by p53 induction of GADD45a expression.
GADD45α also serves an important epigenetic role by physically interacting with ten-eleven-translocation (TET) proteins such as TET1 . These dioxygenase proteins convert methyl cytosine to hydroxymethyl cytosine, which generally results in increased gene expression. GADD45α can also demethylate DNA in the course of p53 stimulation of DNA repair [53, 54] providing another theoretical mechanism to increase gene expression. GADD45 members are also involved in apoptosis, inflammation, resistance to oxidative stress and cellular senescence. [55-57]
GADD45α can be induced by vitamin D3 (1,25(OH)2D3) , by exposure to the DA-ergic neurotoxin MPP+ , or by fucoxanthin, a marine carotenoid found in brown algae , which is available as a dietary supplement. We speculate that increased expression of GADD45A/α will counter neurodegenerative mechanisms in the majority of PD subjects and slow disease progression.
Mechanistic (formerly mammalian) target of rapamycin (mTOR) is a protein kinase member of the PI3K family that combines with other protein subunits to form mTOR complexes 1 (mTORC1) and 2 (mTORC2). mTORC1 is sensitive to inhibition by rapamycin and analogues (“rapalogues”), whereas mTORC2 is relatively insensitive to inhibition by rapamycin and rapalogues. Both complexes are believed to act by sensing external nutrients and to control cell growth and differentiation by modifying downstream processes such as protein translation, cell division and growth, and autophagy. [60-64]
Our finding of decreased mTORC1 signaling in the majority of PD samples following GSEA analyses suggests that this crucial regulatory system is impaired in PD (at least in end stage disease). Reduced mTORC1 signaling would be predicted to increase autophagy, which would likely take place since we observed that all autophagy genes (ATGx) and beclin-1 (BECN1) were normally expressed in PD samples (PD/CTL ratios ~0.9-1.1). Activating autophagy has been proposed as a treatment for PD , although consideration must be given to the alternative viewpoint, that autophagy is contributing to PD pathology [16, 66]
The combination of impaired mitobiogenesis signaling [67, 68] and increased oxidative stress (which would further damage mitochondria) [46, 69, 70], sets the stage for reduced mitochondrial mass (impaired mitobiogenesis signaling, increased oxidative damage to mitochondrial components). Thus, our findings of decreased oxidative phosphorylation in GSEA analyses of the PD samples is not surprising, although one can postulate multiple origins of this energy deficit.
The above speculations do not identify a single “genesis event” for PD neurodegeneration, nor can we account for all abnormalities observed. Rather multiple interacting deficits could appear slowly over time, interacting to amplify each other’s impacts on cell survival. That concept is a major thrust of systems biology, to identify a series of interacting processes rather than individual molecules that, when combined, interact pathogenically.
In addition, it must be recalled that our samples are devoid of the majority of PD vulnerable neurons (nigral dopaminergic neurons). These neurons likely have increased oxygen consumption compared to other neurons due to mitochondrial respiration, and activities of MAO and tyrosine hydroxylase, which in turn have higher oxygen consumption compared to non-neuronal cells that likely comprise the majority of our samples. It appears likely that increased oxidative stress could be an early event in the death spiral of dopaminergic neurons in PD, but that by itself increased oxidative stress cannot likely account completely for the pathophysiology of neurodegeneration.
We propose that gene variance, or some other measure of gene variation across samples, be utilized in transcriptomic studies of human diseases, so as to define genes with both minimal variability in expression across individuals and significantly reduced expression in the disease population. Manipulation (specifically, increased expression) of these genes would be predicted to have the greatest therapeutic benefit across multiple persons, and thus they are appealing therapeutic targets. Alteration of expression of these “low variance” genes could also serve as biomarkers to predict therapeutic utility of gene expression approaches. Such approaches could seek different pathways for altering expression, including direct expression using gene vectors, administration of drugs that increase expression, or alteration of non-coding RNA’s (ie microRNA’s, etc) that indirectly alter gene expression.
Using this approach, we identified the important stress-response gene GADD45A/α as a viable candidate for therapeutic use in PD. We speculate that increased brain expression of GADD45A will slow disease progression in most PD patients.
In addition, we further speculate that sporadically occurring PD neurodegeneration is both highly heterogeneous in etiology and unlikely to be successfully managed by a traditional single protein targeting approach. Rather, we propose that low variance/low expression genes be identified in PD populations and tested for their efficacy in altering disease progression.
Figure 1. Principal component analysis (PCA) plots generated by QOE with q<5% and fold-change > 1.5. 1a. shows PCA plot generated with TMM normalization and 1b shows PCA plot generated with FPKM normalization. Note that in both 1a and 1b PD sample 160 generates an edge with one CTL sample.
Figure 2. Linear relationship among 26 genes co-identified with q < 5% using TMM normalization (QOE, y-axis ) compared to q < 5% in Cufflinks (x-axis). These genes are listed in Supplemental Table 3.
Figure 3. Heatmaps generated in QOE using q < 5%, hierarchical clustering and either FPKM (3a) or TMM (3b) normalization. Note that PD160 (black arrow) was mis-classified in both clusterings. When q is reduced to < 4.8% (3c, FPKM normalization) or < 2.9% (3d, TMM normalization), PD160 is properly clustered in the PD group.
Figure 4. Heatmaps from GSEA analyses (using the “hallmark” group h.all.v6.1.symbols.gmt, http://software.broadinstitute.org/gsea/downloads.jsp) of differentially expressed genes (DEG) using q < 5% and QOE with TMM normalization. Shown are heatmaps from the three groups with the highest “normalized enrichment”: oxidative phosphorylation (left), cholesterol homeostasis (center) and mTORC1 signaling (right). The vertical black lines define the genes which met enrichment criteria. Note that in the PD samples most genes that met enrichment criteria are under-expressed.
Figure 5. Plots of gene-by-gene variance (std dev squared) in the PD population (y-axis) compared to variance in the CTL population (x-axis). Data are from Cufflinks (FPKM) analysis. The bottom plot is from the same dataset as the top plot except the axes are enlarged.
Figure 6. Plots of gene-by-gene variance in the PD population (y-axis) vs expression ratio (mean PD FPKM/mean CTL FPKM) (x-axis). Data are from Cufflinks (FPKM) analysis. The bottom plot is the same as the top plot except the axes are enlarged. The orange rectangle represents genes whose variance is <50 and whose expression ratio is < 0.5 (>2.0-fold under-expressed in PD). These 33 genes are listed in Supplemental Table 4.
TABLE 1. Demographics of PD and CTL ventral midbrain samples used in this study.
TABLE 2. Genes identified at q<5% by Cufflinks or QOE (using FPKM normalization)
TABLE 3. Common genes identified by both Cufflinks and QOE (FPKM normalization) using q<5%
TABLE 4. 33 genes identified by Cufflinks with variance (Std.Dev.2) <50 and PD/CTL expression ratios < 0.5
Acknowledgements. This research was supported by internal funds from Neurodegeneration Therapeutics, Inc., an IRS-registered 501(c)3 non-profit medical research company. We are grateful to the many patients and families for their generous donations of postmortem brains. We particularly acknowledge the help provided by Dr. S. Churn of the VCU Brain Tissue Resource Facility that is supported by institutional funds. We also acknowledge Dr. Sara Strandberg for providing assistance in use of Qlucore software.
Author Contributions. JPB designed the study, performed all bioinformatics analyses and wrote the paper. PMK supervised the acquisition of all brain samples, sectioned all samples, extracted RNA and supervised their analyses. Both authors have reviewed and approved the final manuscript, and neither author declares any conflicts of interest.
Study data. Compressed (.gz) raw sequencing files in fastq format are the property of Neurodegeneration Therapeutics, Inc and are available from the corresponding author following execution of a Material Transfer Agreement and provision of either a FTP site URL or memory storage device sufficient for ~200GB of data.
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