James P. Bennett, Jr. M.D., Ph.D.1 and Paula M. Keeney, B.S.1
1Neurodegeneration Therapeutics, Inc.
Charlottesville, VA 22901
James P. Bennett, Jr. M.D., Ph.D.
Neurodegeneration Therapeutics, Inc.
3050A Berkmar Drive
Charlottesville, VA 22901
We used RNA sequencing (RNA-seq) to quantitate gene expression in total RNA extracts of vulnerable brain tissues from Alzheimer’s disease (AD, frontal cortical ribbon) and Parkinson’s disease (PD, ventral midbrain) subjects and phenotypically negative control subjects. Paired-end sequencing files were processed with HISAT2 aligner/Cufflinks quantitation against the hg38 human genome. We observed a significant decrease in gene expression of all mtDNA OXPHOS genes in AD and PD tissues. Gene expression of the master mitochondrial biogenesis regulator PGC-1α (PPARGC1A) was significantly reduced in AD; expression of genes for mitochondrial transcription factors A (TFAM) and B1/B2 (TFB1M/TFB2M) were not significantly changed in AD and PD tissues. 2-way ANOVAs showed significant reduction in AD brain Complex I subunits’ expressions and nearly significant reductions in PD brain. We found a significant reduction in both AD and PD brain samples of expression of genes for leucine-rich pentatricopeptide repeat containing (LRPPRC, a.k.a. LRP130), a known mtRNA-stabilizing protein. Our findings suggest that AD and PD brain tissues have a reduction in mitochondrial ATP production derived from a reduction of mitobiogenesis and mtRNA stability. If true, increased brain expression of PGC-1α and/or LRPPRC may improve bioenergetics of AD and PD and alter the course of neurodegeneration in both conditions.
The causes of neurodegeneration in sporadically occurring Alzheimer’s disease (AD) and Parkinson’s disease (PD) are not known with certainty but may derive from bioenergetic deficits arising from mitochondrial dysfunction (Onyango et al., 2006; Onyango, 2008; Onyango and Khan, 2006). If true, mitochondrial bioenergetic impairments may arise from multiple sources, including impaired energy substrate import, energy substrate processing (oxidative phosphorylation; OXPHOS), ADP (adenosine diphosphate) import/ATP (adenosine triphosphate) export, or altered mitochondrial dynamics (reduced mitobiogenesis, altered mitochondrial fission/fusion, or increased mitochondrial autophagy).
Neurons are among the most energy-intensive cell types known, and the adult human brain overall uses disproportionate energy (2-3% of body weight, 20-25% of total glucose/oxygen metabolism). Reduction of ATP production induces neuronal cell death, and AD/PD tissues have shown reductions in mitochondrial electron transport catalytic activity that is transmitted completely or in part through mitochondrial DNA (mtDNA) (Khan et al., 2000; Onyango et al., 2006; Swerdlow et al., 1996; Trimmer et al., 2004; Trimmer et al., 2000).
mtDNA is a small (16.6 kilobases in mammals), circular, independently replicating genome transmitted through maternal lines that encodes for 13 genes involved in activities of inner membrane electron transport/ATP synthesizing complexes I (NADH:ubiquinone reductase, 7 genes), III (ubiquinone:cytochrome C oxidoreductase, 1 gene), IV (cytochrome C oxidase, 3 genes), and V (ATP synthase, 2 genes) (Dasgupta, 2019). The 7 mtDNA complex I genes may be primarily involved in proton pumping compared to electron transport catalytic activity (Drose et al., 2011). mtDNA gene transcription involves the coordinated activities of several mt transcription factors and mtRNA polymerase and remains incompletely understood (Barshad et al., 2018).
Recent studies have identified PGC-1α as a master regulator of mitochondrial biogenesis (mitobiogenesis) (Barshad et al., 2018; Brohawn DG; Gleyzer and Scarpulla, 2011, 2013, 2016; Scarpulla, 2008, 2011; Shin et al., 2011; Zheng et al., 2010) that may be involved in the pathogenesis of AD (Wojtowicz et al., 2020) and PD (Pirooznia et al., 2020; Zheng et al., 2010). Other studies have shown that leucine-rich pentatricopeptide repeat containing (LRPPRC) protein is a critically important mtRNA stabilizing protein (Cui et al., 2019; Ruzzenente et al., 2012). Global loss of LRPPRC by genetic deletion is embryonic lethal (Ruzzenente et al., 2012); organ-specific knockout results in impaired mitochondrial bioenergetic function, decreased mtRNA stability and reduced polyadenylation of mitochondrial transcripts (Cui et al., 2019; Ruzzenente et al., 2012).
We used next generation RNA sequencing (RNA-seq) of AD and PD vulnerable brain tissues to quantitate expression of all 13 mtDNA genes and mitochondrial biogenesis regulators, transcription factors and LRPPRC. We found reduction of expression of all mtDNA OXPHOS genes, PGC-1α in AD, complex I subunit mtDNA genes and LRPPRC in both AD and PD brain samples.
Tissue acquisition and storage, subject demographics, isolation of total RNA, sequencing library preparation, RNA sequencing and bioinformatics analyses are all detailed in our prior publications (Bennett and Keeney, 2018; Bennett et al., 2019). Briefly, total RNA was isolated from sections of frozen (-80o C) tissue samples using Qiagen RNEasy columns. Total RNA samples were sent to Cofactor Genomics, Inc, who prepared the sequencing libraries, carried out Illumina paired-end sequencing and provided compressed, FASTQ sequencing files. Adapter-trimmed (Trimmomatic) sequencing files in FASTQ format were aligned against the hg38 human genome using HISAT2 and quantitated with Cufflinks. All graphical and statistical analyses were carried out with Prism.
|N||(9 CTL, 10 AD)||(8 CTL, 12 PD)|
|Gene||AD||p (unpaired t-test)||PD||p (unpaired t-test)|
|mtDNA OXPHOS||75.2||<0.0001 (2-way ANOVA)||83.6||0.0007 (2-way ANOVA)|
|(Complexes I, III, IV, and V)|
shows the reductions in relative levels (% Control) for the master mitobiogenesis regulator PGC-1α (PPARGC1A), for mitochondrial transcription factors A (TFAM), B1 (TFB1M) and B2 (TFB2M) and for the mtRNA stabilizing factor LRPPRC. We call attention to our finding that basal levels of LRPPRC expression in CTL tissues are identical in AD and PD samples, and that reductions of LRPPRC expression are ~ same in AD frontal cortex and PD ventral midbrain. These results are presented graphically in Figure 1.
shows the results of 2-way ANOVA analysis of expressions of all mtDNA Complex I genes in AD and PD samples. In AD, the ANOVA revealed very significant (p<0.0001) reduction of mtDNA Complex I gene expression. In PD, ANOVA revealed almost significant (p=0.0519) reduction of mtDNA Complex 1 gene expression.
Using paired-end RNA-seq, we have shown that the mitochondrial bioenergetic deficit in AD and PD brain tissues may arise from impaired mitobiogenesis signaling (in AD brain) and mtRNA stability (in both AD and PD brain) that arise from reduced expression of PGC-1α, the master mitobiogenesis regulator (in AD), and the gene for LRPPRC (a.k.a. LRP130), a mtRNA stabilizing protein (in AD and PD), respectively. While we have shown a correlation between reduced LRPPRC expression and expression of OXPHOS mtRNA’s in AD and PD vulnerable tissues, we have not demonstrated a causation.
Our study is limited by low sample numbers and cellular heterogeneity of total RNA extracted from tissue homogenates. Also, because these postmortem tissues derived from persons with advanced clinical phenotypes, we also do not know at what point in disease evolution these deficits appeared. It is conceivable that such deficits were of greater magnitude earlier in disease evolution, when neurodegeneration is more prominent.
However, we appear to have identified gene targets for neurodegenerative therapy development in AD and PD. Agents which increase mitochondrial mass and stimulate mitobiogenesis (like recombinant human TFAM, rhTFAM) (Keeney et al., 2009; Ladd et al., 2017; Thomas et al., 2012) or stimulate expression of LRPPRC may improve bioenergetic deficits of AD/PD and reduce neurodegeneration. Increased LRPPRC expression would need to be brain-specific, as peripheral neoplasms have been associated with increased LRPPRC expression (Jiang et al., 2015a; Jiang et al., 2015b).
We are grateful to the University of Virginia Brain Resource Facility (Charlottesville, VA) and the Virginia Commonwealth University Parkinson’s Center (Richmond, VA) for access to AD and PD brain samples. We thank the staff of Cofactor Genomics for assistance with the RNA-seq library preparation and Illumina sequencing. This study was supported by ALS Worldwide and private funds of Neurodegeneration Therapeutics, Inc.; both are 501c3 non-profit research corporations. All sequencing files are property of Neurodegeneration Therapeutics, Inc.
JPB designed the studies, performed all bioinformatics analyses on raw FASTQ files, analyzed the data and prepared graphs and wrote the paper draft. PMK supervised all tissue acquisition and storage, prepared RNA extracts and supervised the logistics of obtaining RNA sequencing. Both authors have seen and approved the manuscript, and neither author declares any conflicts of interest. All sequencing files with sequencing adapters removed by Trimmomatic are available upon request to the senior author (JPB).
All tissue samples were obtained either with IRB oversight (University of Virginia) or were declared “autopsy tissue” and not subject to IRB oversight (Virginia Commonwealth University). Samples were de-identified by assigning random numbers and assayed/analyzed blindly as to disease status.
Table 1. Expression (%CTL) of Relevant Mitochondrial Genes
N (9 CTL, 10 AD) (8 CTL, 12 PD)
Gene AD p (unpaired t-test) PD p (unpaired t-test)
PGC1 (PPARGC1A) 78.6 0.0072 76.1 0.092
TFAM 99.1 0.91 89.5 0.32
TFB1M 93.5 0.42 88.4 0.11
TFB2M 104 0.83 ND NA
LRPPRC 78.3 0.0010 77.1 0.0012
mtDNA OXPHOS 75.2 <0.0001 (2-way ANOVA) 83.6 0.0007 (2-way (Complexes I, III, IV, and V) ANOVA)
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