recombinant human tfam stimulates rat brain, rat cervical...
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Recombinant Human TFAM Stimulates Rat Brain, Rat Cervical Spinal Cord and
Human Neural Stem Cell Mitochondrial Bioenergetics
Ravindar R. Thomas1,*, Paula M. Keeney1,8,*, Stuart B. Berr2, Shaharyar M. Khan3,
Francisco R. Portell3, Meiram Zh. Shakenov4, Patrick F. Antkowiak2, Bijoy Kundu2,
Nicholas Tustison2, David G. Brohawn1,7, James P. Bennett, Jr.1,5,6,8
1 Parkinson’s Center, Virginia Commonwealth University, Richmond, VA
2 University of Virginia Department of Radiology, Charlottesville, VA
3 Gencia Corporation, Charlottesville, VA
4 Department of Science and Education
Republican Children's Rehabilitation Center, Astana, Kazakhstan
Departments of 5 Neurology, 6 Physiology and Biophysics, and 7Human Genetics,
Virginia Commonwealth University, Richmond, VA
8 Current Address: Neurodegeneration Therapeutics, Inc., Charlottesville, VA
* These authors contributed equally to this work.
Correspondence: James P. Bennett, Jr. M.D., Ph.D. Neurodegeneration Therapeutics, Inc. 3050A Berkmar Drive Charlottesville, VA 22901 434-529-6457 (PH) 434-529-6458 (FAX) www.NDTherapeutics.org [email protected]
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Abstract
To develop a treatment for human mitochondrial diseases, we engineered a
recombinant human mitochondrial transcription factor A (rhTFAM) that enters brain
mitochondria of animals, rapidly localizes to mitochondria of human cells and increases
mitochondrial respiration and OXPHOS capacity in human cells and animals. As part of
developing rhTFAM for use in humans, we treated rats with single or weekly i.v. injections
of highly purified rhTFAM protein and assayed brain [18F]fluorodeoxyglucose (FDG) uptake
by PET scan, forebrain mitochondrial states 3 and 4 respiration, forebrain and cervical
spinal cord mitochondrial respiratory gene expression and forebrain and cervical spinal
cord OXPHOS protein levels. rhTFAM treatments increased mitochondrial OXPHOS
function in brain and spinal cord. Total brain FDG uptake did not change, but the rate of
FDG uptake appeared to increase in the rhTFAM treated animals. Human neural stem cells
(NSC’s) exposed to 5 nM rhTFAM and analyzed by RNA-seq showed increased expression
of mitochondrial respiratory genes. 3-D gene expression visualization (Miru®) followed by
protein interaction network analysis (STRING) revealed enrichment after rhTFAM in mRNA
splicing and ribosome functions. Ingenuity Pathways Analysis showed significant over-
representation in E2F/E2F1 signaling that can modulate mitochondrial gene expression.
rhTFAM treatment could overcome mitochondrial bioenergetic deficits reported in various
human neurodegenerative conditions as well as in many “mitochondrial” diseases affecting
multiple organ systems and may use E2F signaling. (219 words).
Keywords: mitochondria, respiration, brain, spinal cord, neural stem cells, TFAM, FDG-PET
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1. Introduction
Life on Earth as we know it, in an oxygen-rich atmosphere would not be possible
without energy production by mitochondria, bacterial remnants who appeared to have
invaded early eukaryotic cells during an “endo-symbiotic” event that occurred 2-3 billion
years ago [1-5]. Mitochondria provide needed energy for differentiated tissues necessary
for complex life forms, and in addition to this critical role, control intracellular calcium
signaling [5] and initiation of cell death programs [6].
Mitochondrial density within cells varies with cellular energy demands and is
maintained by processes collectively referred to as “mitochondrial quality control”. These
include mitochondrial biogenesis (mitobiogenesis) [7, 8]) and mitochondrial removal
(mitophagy) [9, 10]), which must be balanced in order for cells to function properly.
Mitobiogenesis, in turn, depends on the coordinated expression of multiple transcription
factors that regulate respiratory gene expression (both nuclear and mitochondrial) and
replication of the mitochondrial genome needed for successful mitochondrial division [7, 8,
11, 12].
Mitochondrial transcription factor A (TFAM) is an essential downstream component
of the mitobiogenesis system, and genetic TFAM deletion is embryonic lethal [13-16].
Several years ago we described the mitobiogenesis effects of recombinant human TFAM
(rhTFAM) that we synthesized by incorporating a rapidly translocating protein transduction
domain (PTD, 11-arginines), followed by a mitochondrial matrix localization signal (MLS,
human SOD2), followed by the complete coding sequence of human TFAM [17]. We
showed that i.v. treatment with the resulting rhTFAM stimulated motor activity and brain,
skeletal muscle and cardiac mitochondrial respiration in both young and old adult mice, and
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stimulated mitobiogenesis and respiration in a variety of human cells in culture [18-21]. I.V.
rhTFAM also entered brain mitochondria and declined with a half-life of ~24-30 hrs [19].
As a result of these encouraging findings, we embarked on further pre-clinical
studies to develop rhTFAM for use in humans with mitochondrial bioenergetic deficiencies.
Essential components of these preclinical studies include demonstrations of target
engagement, signs of therapeutic activity and toxicity in experimental animals. For a
biologic therapeutic such as rhTFAM, which is a protein, the human biologic must be tested
in experimental animals, even though construction of and experimenting with animal
homologues might yield scientifically more interpretable data about TFAM actions within
mitochondria. For these reasons we tested rhTFAM in rats, even though only 146/246=59%
of amino acid residues are identical between human and rodent TFAM’s.
In the current study we used an even more highly purified rhTFAM protein than in
our earlier studies [17-19, 22-24] and extended these observations into rat forebrain and
cervical spinal cord. We initiated mechanistic studies of rhTFAM-stimulated mitobiogenesis
by exposing human neural stem cells to rhTFAM, using RNA sequencing (RNA-seq) to
define a threshold [rhTFAM] needed to stimulate mitochondrial respiratory gene expression
and develop preliminary network analyses of rhTFAM-induced gene co-expression and
protein interactions. We observed that rhTFAM treatment was free of observable toxicity,
stimulated mitochondrial functions in rat CNS and increased respiratory gene expression in
human NSC’s. These findings suggest that rhTFAM could be used to treat mitochondrial
bioenergetic deficiencies reported in adult neurodegenerative diseases and many
“mitochondrial diseases” characterized by impaired mitobiogenesis or abnormalities in
nuclear and/or mitochondrial genome-encoded proteins.
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2. Results
2.1 Acute single dose effects of rhTFAM on rat cortex and cervical spinal cord
Treatment of adult rats with a single i.v. dose of rhTFAM increased cortical
mitochondrial respiration that was greatest at 3 mg/kg dose and 24 hours after injection
(Figure 1). We did not test higher doses of rhTFAM in rats. A time course of changes in
total forebrain respiration in rats treated with 3 mg/kg i.v. rhTFAM showed highest State 3
respiration at 24 hours compared to other times after injection (Figure 2).
Because cervical spinal cord samples were limited in weight, we elected to not
assay mitochondrial respiration in spinal cord samples and instead focused on molecular
genetic studies of mtDNA-encoded gene expression. Figure 3 shows that in rat cervical
spinal cord samples, a single injection of rhTFAM increased expression of mtDNA genes
many-fold with ~equivalence at 3 compared to 7 days after injection.
2.2 Effects of multiple weekly dose of rhTFAM on rat forebrain and cervical spinal cord
mitochondrial bioenergetics
Because we are developing rhTFAM for use in human diseases characterized by
impaired CNS bioenergetics, we wished to characterize the effects of repeated rhTFAM
treatments on CNS mitochondrial function. Accordingly, we treated adult rats with 3 mg/kg
i.v. rhTFAM every week for 4 weeks (5 injections total) and assayed forebrain mitochondrial
respiration 24 hrs after the last rhTFAM injection. Because rhTFAM has a brain half-life of
~24-30 hours [19], we appreciate that this dosing schedule will not likely produce a
constant brain [rhTFAM]. However, this dosing schedule is more realistic clinically with
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humans who would receive i.v. rhTFAM. Figure 4 shows that repeated weekly rhTFAM
treatment produced a small 10-20% increase in brain mitochondrial respiration at all
respiratory complexes. The increase was significant when analyzed by matched pair 2-way
ANOVA across treatment, but no individual rat brain respiratory complexes had a
significant elevation after rhTFAM treatment compared to buffer CTL.
One month of rhTFAM injection every week also produced small elevations in
nuclear genome-encoded respiratory gene expression analyzed by SABioscience gene-
specific primer plates (Figure 5). By 2-way ANOVA rhTFAM produced a significant
elevation in Complex I and Complex IV nuclear respiratory gene expressions, but not
significant changes in nuclear gene expressions for Complexes II, III and V. We did not find
any significant increases after weekly rhTFAM treatment in forebrain OXPHOS protein
levels (not shown)
In contrast to the relatively small effects of weekly rhTFAM treatments on brain
mitochondrial bioenergetics, we found greater effects on cervical spinal cord. As shown in
Figure 6, weekly rhTFAM treatment produced greater and more significant increases in
cervical spinal cord nuclear mitochondrial gene expression (Figure 6) and increases in
representative OXPHOS proteins assayed by Western blot (Figure 7).
2.3 FDG PET Imaging
We divided the brain into 78 regions based on the publicly available Waxholm Space atlas
for Sprague Dawley rats. Using the static FDG PET images acquired 40 minutes after FDG
injection (Fig. 8), there were no statistically significant differences measured between the
rhTFAM treated versus control rats for any combination of different timepoints after rhTFAM
treatment (not shown). While there was not a difference in the overall uptake of FDG at the
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40 minute timepoint, there was a statistically significant difference in the rate at which the
FDG was taken up in the cells. Figure 9 shows the differences in the mean uptake values
for FDG at 5 minute intervals over the first 30 minutes after the injection of FDG for rhTFAM
treated rats (N=3) and control rats (N=2).
2.4 rhTFAM effects on Gene Expression in Human Neural Stem Cells
For 24 hours we exposed to 1nM or 5nM rhTFAM (or buffer) four independent
cultures of human neural stem cells we made from a 62 y.o. clinically normal male subject’s
peripheral blood mononuclear cells (PBMC). His PBMC’s were reprogrammed to iPSC’s by
electroporation of a 5-gene reprogramming plasmid [25], then neuralized as described
[26]. Following rhTFAM incubation we extracted total RNA, constructed sequencing
libraries, quantitated the libraries, then carried out multiplex paired-end RNA sequencing
using Illumina NextSeq® technology. For bioinformatic processing we used
tophat2/bowtie2 for aligning against the hg38 human genome or ENCODE human
mitochondrial genome, and quantitated gene expression using Cufflinks2.
Figure 10 shows the relationship among >20,000 gene pairs’ FPKM values in human
NSC’s in culture compared to those from averaged CTL human cervical spinal cord
sections (n=8). There was a good correlation among these gene pairs, with the greatest
scatter occurring at lower FPKM values.
Figure 11 shows that the baseline mitochondrial gene expression in the human
NSC’s was qualitatively similar to that found in CTL human cervical spinal cord sections
(n=8; Brohawn, et al, in preparation). In both groups expression levels of the two
mitochondrial rRNA’s (12S and 16S) were >20-fold higher than the highest expression level
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of any mitochondrial respiratory genes, consistent with separate transcriptional start sites in
the heavy chain of mtDNA for the 12S and 16S rRNA genes. We also found substantial
variation in expression levels of the 13 mitochondrial respiratory genes, which is puzzling,
given the current concept of single polycistronic transcription for the 13 respiratory genes
(12 from heavy chain and 1 from light chain of mtDNA). If mitochondrial protein-coding
genes are transcribed initially at ~equal levels, then our data suggest that RNA stability
varies significantly in mitochondria.
Figure 12 shows that 5 nM rhTFAM is close to a threshold [rhTFAM] for increasing
respiratory gene expression in human NSC’s. We found that 5 nM rhTFAM incubation for
24 hrs significantly increased in human NSC’s expression of ND1, ND2, ND3, CO1, CO2,
ATP6 and ATP8 determined by t-tests corrected for multiple comparisons using Holm-
Sidak method with alpha=0.05.
We then carried out 3-D visualization of gene expression in human NSC’s exposed
to 5 nM rhTFAM compared to buffer CTL. We first filtered for FPKM values >5 to reduce
noise, then calculated for each gene the expression ratio of the averaged FPKM values
after 5 nM rhTFAM divided by the averaged FPKM values for that gene from cells exposed
to buffer. We took the top 500 genes from this ratio and used Miru® (version 1.3) at high
initial correlation (Spearman r=0.9) to construct 3-D gene co-expression networks, using
the FPKM values for the rhTFAM treated cells.
Figure 13 shows the resulting 3-D gene expression network. There were two
clusters (1 and 2) that together accounted for the majority of the 500 genes. The individual
genes from each of these clusters was then analyzed for protein interactions in STRING
(www.string-db.org), yielding Figures 14 (Miru® cluster 1) and 15 (Miru® cluster 2). The
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protein clusters revealed by the STRING algorithm, when analyzed in DAVID (Database for
Analysis, Visualization and Discovery), showed very significant enrichment in processes
related to mRNA synthesis, splicing, and ribosomal function (Supplemental Table 1).
Table 1 shows the results we obtained from Ingenuity Pathways Analysis (IPA) pf
the top 500 genes used to construct Figures 13 and 14. Top canonical pathways
represented include those associated with the general transcription factor Elf2 and energy
metabolism (creatine phosphate synthesis). Top upstream regulators identified included the
mitochondrial protease LONP1 and the cell cycle regulators E2F and E2F1, both of which
have recently been shown also to control mitochondrial gene expression and mitochondrial-
mediated apoptosis [27, 28].
Thus, we found that rhTFAM, an engineered protein that localizes to mitochondria,
both appears to stimulate mitochondrial gene transcription, a process known to be
regulated by TFAM, and activates clusters of expressed genes whose proteins are involved
in critical non-mitochondrial cellular events such as mRNA splicing and protein translation
in ribosomes possibly using E2F/E2F1 signaling. How mitochondrially localized rhTFAM
brings about these non-mitochondrial cellular events is not clear and is the focus of future
studies.
3. Discussion
In the present study we treated adult rats with recombinant human TFAM (rhTFAM)
and observed increases in mitochondrial bioenergetic markers and stimulation of
mitochondrial biogenesis in forebrain (to a small extent) and cervical spinal cord (to a much
larger extent). Weekly i.v. rhTFAM treatment appeared to be non-toxic to the animals. We
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did not assay motor function as in prior studies done in adult young and old mice, where
rhTFAM treatment increased motor activity and stimulated mitobiogenesis and respiration
in both age groups of mice [19, 22].
Human TFAM is ~60% identical with rat and mouse TFAM (MacVector®, ClustaW
sequence alignment tool. Supplemental Figure 1). Given these limited identities with rodent
TFAM’s it is remarkable that any mitochondrial bioenergetic effects are observed in rodents
with the human protein that is being developed as a therapeutic for use in man.
The maximum doses used in our rat study were 3 mg/kg i.v. According to the
document “Guidance for Industry-Estimating the Maximum Safe Starting Dose in
Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” published by the US
Food and Drug Administration, 3 mg/kg in rats is equivalent to 0.48 mg/kg in humans. For
the typical 70 kg human subject, this would correspond to a dose of 33.6 mg rhTFAM.
rhTFAM has not yet been administered to humans, so we do not know about its
pharmacodynamic actions on mitochondrial function.
We observed significant bioenergetic effects of increased mitochondrial gene
expression and OXPHOS proteins in rat cervical spinal cords following weekly treatment
with rhTFAM. This was not commensurate with an increase in total brain uptake of glucose
(as measured by FDG PET), but it did seem to correlate with an increase in the rate of
glucose uptake. Because we have reported that human amyotrophic lateral sclerosis (ALS)
spinal cord sections and peripheral mononuclear cells show depressed mitochondrial gene
expression [29], our current findings support the study of rhTFAM as a mitochondrial
stimulant in human ALS. In this situation, peripheral mononuclear WBC mitochondrial gene
expression could serve as a biomarker for rhTFAM therapeutic effect.
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Our combined findings in rats (present study) and prior findings in mice [19, 22]
suggest that repeated rhTFAM treatment will at most increase slightly human cerebral
mitochondrial bioenergetics, but the limited homologies between human TFAM and TFAM’s
of rat and mice suggest caution with this prediction.
Our experiments do not provide insight into how rhTFAM, a mitochondrial matrix-
directed protein, increases expression of nuclear-encoded mitochondrial genes. We have
consistently observed this mitobiogenesis activity of rhTFAM in animals and multiple
human cell lines.
Our RNA-seq studies in human neural stem cells showed both an expected action
(increased expression of mitochondrial respiratory genes) and an unexpected action
(increased expression of gene clusters related to mRNA splicing and ribosomal functions).
Over-representation analysis (IPA) also showed nuclear-mediated effects of rhTFAM and
activation of gene programs related to E2F signaling. The E2F gene family traditionally was
associated with cell checkpoint/cell cycle regulation. More recently, this family of
transcription factors has been shown to regulate mitochondrial function and morphology,
including mitochondrial involvement in apoptosis [27, 28].
It remains unclear to us how rhTFAM brings about these nuclear genome-encoded
actions, which may represent an important component of its therapeutic effect.
There are important limitations of our studies:
First, much of our work was done using rhTFAM in rodents. This was dictated by the
regulatory need to show biological action of a human protein therapeutic in non-human
species. Clearly use of a rat TFAM would likely have provided greater insight into TFAM
molecular action, but we were not developing rat TFAM for treatment of humans.
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Second, it remains unclear why we observed such a difference between rhTFAM
mitochondrial effects in forebrain compared to spinal cord. Possible explanations include
different pharmacokinetics of rhTFAM in spinal cord compared to forebrain, greater
sensitivity of spinal cord mitochondria to rhTFAM, altered mitochondrial turnover in spinal
cord, or some combination. Other explanations are also possible.
Third, we do not yet know how our results in rodents and human cells will translate
into human persons. All indicators are that rhTFAM, being an engineered natural protein
product, will stimulate mitochondrial bioenergetics in humans, and apparently will do this
without significant toxicity. We are hopeful that soon we can begin human studies, with the
hope of improving mitochondrial function in persons with degenerative brain diseases or
peripheral mitochondrial disorders.
4. Experimental Procedures:
4.1 Animals: Normal male Sprague Dawley rats 150-200g were procured from Charles
River Laboratories and were housed two per cage in the vivarium and provided with food &
water ad libitum. The protocol was approved by the Institutional Animal Care and Use
Committees (IACUC) at Virginia Commonwealth University and the University of Virginia
and all the procedures were done in accordance with the National Institute of Health (NIH)
Guide for the Care and Use of Laboratory Animals (Eighth Edition).
4.2 rhTFAM: Recombinant human mitochondrial transcription factor A (rhTFAM) was
provided by Gencia Corporation (Charlottesville, VA) and stored at -80oC. Just before
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injection the rhTFAM was diluted in the buffer provided by Gencia to working concentration
and rats were injected intravenously (iv) by tail vein.
4.3 rhTFAM treatment Protocol:
To find out the workable concentration that will produce measurable effect rats were treated
with 1 mg/kg or 3 mg/kg rhTFAM or with control buffer (each n=1). The rats were sacrificed
after 24 hours or 3 days.
Single dose vs different time points: Based on the above experiment 3 mg/kg was
found to be effective and rats were treated at this dose and sacrificed after 0 hour, 24
hours, 3 days and 7 days (each dose n=3) along with control buffer (n=4) which were
sacrificed after 0 hour.
Chronic effect with multiple doses: To see the extended effect of rhTFAM @ 3 mg/kg
rats (n=6) were injected at 3 mg/kg per week for 5 weeks and were sacrificed 24 hours after
the last injection. Following the same protocol equal number of rats (n=6) was injected with
control buffer also.
The animals were euthanized using controlled release of CO2 in the vivarium and
the brains were removed. Half of the sagittally cut brain was snap-frozen in isopentane
chilled in dry ice and stored at -80oC until further processing. From the other half the
mitochondria were isolated and used for measuring respiration in Oxygraph.
4.4 Mitochondrial respiration: From one half of the freshly removed brain about 250 mg
was removed from the anterior forebrain and homogenized in mannitol-sucrose buffer [21].
P2 pellets were prepared by differential centrifugation method, which was rich in
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mitochondria, and dissolved in 2.4 ml MirO5 buffer
(http://www.oroboros.at/index.php?id=524#857). About 0.6 ml of this was assayed in
Oroboros Oxygraph 2 respirometer to measure mitochondrial respiration. rhTFAM or buffer
control injected brain mitochondria were assayed side by side with sequential additions of
substrates and inhibitors of Complex I to Complex IV [21]. The protein concentration of
diluted P2 pellet that was used for assay was measured (Micro BCA Protein Assay Kit,
Thermo Scientific) and was used to normalize respiration/mg protein.
4.5 RNA extraction and RT-qPCR: About 30mg brain tissue from the frontal cortex region
or spinal cord was homogenized in Qiazol and RNA was extracted using miRNeasy kit
(Qiagen). It was quantified in NanoDrop 2000C spectrometer and the quality of RNA was
analyzed by capillary electrophoresis (Experion, BioRad). 1ug of RNA was reverse
transcribed to cDNA (iScript, BioRad) using random hexamers for qPCR analysis using
CFX96 (BioRad). Primers and probes used for multiplex and SybrGreen assays were
designed by Beacon Designer®. Rat mtDNA used as external standards to quantify
mitochondrial genes, was prepared from rat liver gDNA after treating with Plasmid-Safe
ATP-Dependent DNase (Epicentre Biotechnologies) to remove all non-circular DNA.
Various genes involved in mitochondrial bioenergetics were assayed using gene specific
primers on ‘RT2 Profiler PCR Array-Rat Mitochondrial Energy Metabolism’ qPCR plates
(Qiagen). Samples were also assayed on ‘RT2 Profiler PCR Array-Rat Housekeeping
Genes’ qPCR plates (Qiagen). The qPCR data was analyzed in qbasePLUS (Biogazelle) to
select the optimal number of reference targets, which have least variability based on
GeNorm analysis in the qbasePLUS® software (Biogazelle) (Supplemental Figure 2).
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4.6 Western Blotting: Proteins extracted from brain and spinal cord samples were
electrophoresed on 4-12% Bis-Tris CriterionTM (Bio-Rad) and transferred onto nitrocellulose
membranes (iBlot, Invitrogen). MitoProfile Total OXPHOS Rodent WB Antibody Cocktail
(ab110413) was used to probe for the presence of proteins, followed by suitable secondary
antibodies (IRDye, Li-cor). The membranes were imaged in Odyssey infrared imaging
system (Li-cor) the bands were normalized by Beta-actin, which was used as loading
control.
4.7 FDG PET Imaging. We injected i.v. an average dose of 550 µCi of FDG into the rats
and imaged them on a Siemens Focus 120 PET scanner for 40 minutes. There were 5
animals in rhTFAM treatment group and 5 in the control group. Animals were injected with
rhTFAM (3 mg/kg i.v.) on Days 0, 10, 16, and 24. PET imaging was performed on Days 4,
11, 17, and 25. The Standardized Uptake Value (SUV) was calculated by normalizing the
radioactivity concentration for each voxel in the image to the animal’s body mass and the
injected dose. The PET SUV images were analyzed to see if there were significant
rhTFAM-induced alterations in glucose uptake in the 40-min static images.
To compare the 40-min static images, we combined the PET SUV data separately
for the rhTFAM treated rats and the control rats. To do this, we registered each 3D data set
to a template that was divided into 78 regions. We could then calculate the mean SUV from
each region and compared between groups to see if there was a rhTFAM-induced
significant difference in FDG uptake for any of the regions. More specifically, a region of
analysis was performed on the PET SUV images using the Advanced Normalization Tools
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(ANTs) package. Mean (shape and intensity) population-specific structural and functional
templates [30] were created from their corresponding imaging subsets. In order to create
the structural template, each PET SUV image underwent bias correction [31] and was
subsequently skull-stripped using a multi-atlas label fusion approach [32]. The structural
template was then labeled using the publicly available Waxholm Space atlas [33] providing
78 different regions over the entire brain. This atlas was registered to the population-
specific template [34] in order to transform the 78 regions to the template space. The
functional template was also normalized to the structural template in order to propagate the
Waxholm labels directly to the functional template.
After template creation, each functional image was registered to the functional
template. The labels in the template space were used to determine the mean SUV in each
ROI for each warped functional image. These mean values were used to test for
differences across regions and for each time point (Day 3, Day 16, and Day 24). False
discovery rate was used to correct for multiple comparisons [35].
In a subset of the rats (rhTFAM n=3, CTL n=2), we acquired additional data that
allowed us to see there were changes in the rate of whole-brain FDG uptake. This was
done by acquiring 5 minute scans over a period of 30 minutes, averaging the SUVs over
the whole brain, and calculating the mean and standard deviation for the SUVs for each of
the groups.
4.8 Human neural stem cell studies: We collected blood for PBMC isolation, cultured the
mononuclear cells, created iPSC’s and neuralized them to neural stem cells (NSC) as
described [26]. NSC were propagated in cell culture, and independent cultures in 6-well
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plates were incubated with 1 nM or 5 nM rhTFAM (gift of Gencia Corporation,
Charlottesville VA), or buffer, for 24 hours. All incubations were carried out in 5% oxygen.
Total RNA was isolated using Qiagen kits, depleted of ribosomal RNA, converted to cDNA
and bar-coded for multiplex RNA sequencing using Illumina Truseq® Stranded Total RNA
HT kit library construction kits, fragmented to a mean size of 150 bp, then sequenced on an
Illumina NextSeq 500 sequencer using paired-end sequencing. Raw sequencing reads
were first examined for quality (FastQC) and then trimmed of sequencing bar codes
(Trimmomatic). Trimmed reads with Phred scores >20 were aligned using tophat2/bowtie2,
and the accepted_hits.bam files were processed for FPKM values from the human hg38
transcriptome or human mitochondrial genome (ENCODE) using Cufflinks2.
4.9 Computer processing, bioinformatics algorithms and statistical analyses:
Routine graphs were constructed and t-test, ANOVA’s done using Prism® software
(GraphPad) on Macintosh computers. Tophat 2
(https://ccb.jhu.edu/software/tophat/manual.shtml) , Bowtie 2 (http://bowtie-
bio.sourceforge.net/bowtie2/index.shtml) and Cufflinks 2 (http://cole-trapnell-
lab.github.io/cufflinks/install/) algorithms were downloaded and run using native Unix on a
12-core processor, 64GB RAM MacPro with a 32TB hard drive array. Unprocessed (raw)
paired-end RNA sequences have been deposited in a public database (SRA;
www.ncbi.nlm.nih.gov/sra) as Bioproject PRJNA320380; Experiment
SRX1739676/rhTFAM_CTL42NSC; Run SRR3472844.
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5. Acknowledgements
This research was supported by the Virginia Biosciences Health Research Council
(VBHRC), the Alzheimer’s Drug Discovery Foundation (ADDF), the Parkinson’s Research
Center at Virginia Commonwealth University, the Medical College of Virginia Foundation
(MCVF) and Gencia Corporation. SMK, FRP are employees of Gencia Corporation. None
of the other authors have a financial interest in rhTFAM or Gencia Corp. None of the study
sponsors had any role in experimental design, data analysis, or manuscript preparation.
6. Figure Legends Figure1. Single i.v. rhTFAM treatments of adult rats increase brain cortex mitochondrial respiration. See Methods for details. “State 3” = respiration in the presence of ADP. “State 4” = no ADP added. “Buf” =buffer only. Figure 2. Rat forebrain mitochondrial respiration at various time points after single 3 mg/kg i.v. doses of rhTFAM. See Methods for details. “Buf” =buffer only. Figure 3. Rat cervical spinal cord mtDNA genes’ cDNA’s expression days after 3mg/kg rhTFAM treatment (outliers removed). Figure 4. Weekly rhTFAM treatment for one month increased forebrain mitochondrial respiration ~10-20%. N=6 rats each treatment. CTL=buffer only. Figure 5. Effects of weekly 3 mg/kg rhTFAM treatments on forebrain nuclear respiratory gene expression using SABiosciences gene-specific primer plates. N=6 rats each treatment. CTL=buffer only. Figure 6. Weekly 3 mg/kg rhTFAM treatments increase cervical spinal cord expression of nuclear mitochondrial genes, assayed by gene-specific primers on SABiosciences qPCR pates. N=6 rats each treatment. CTL=buffer injection. Figure 7. Effects of weekly rhTFAM (or buffer CTL) treatments on cervical spinal cord OXPHOS protein levels. N=6 rats each treatment. Figure 8. Representative FDG PET images acquired 40 minutes after injection of FDG from the three primary axes: sagittal (left), transverse (center), and coronal (right).
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Figure 9. Dynamic PET scans show an increase in the rate of FDG brain uptake in TFAM treated animals. Figure 10. X-Y plot of log2 averaged FPKM of CTL neural stem cells (1% DMSO vehicle CTL, n=2) vs. log2 of mean FPKM of CTL cervical spinal cord sections (n=8). Note the high correlation (Spearman r=0.74) for >20,000 gene pairs and that most scatter occurs at very low FPKM values. Figure 11. FPKM values for mitochondrial DNA (mtDNA)-encoded respiratory genes and 12S/16S rRNA’s in human NSC’s (top) and averaged from human CTL spinal cord sections (n=8). Although the absolute values are different, there are qualitative similarities among individuals in the two groups. Figure 12. Effects of 1 nM and 5 nM rhTFAM (incubated 24 hrs) on mtDNA gene expression in human NSC’s. I nM rhTFAM brought about minimal changes, but 5 nM rhTFAM increased expression in human NSC’s of ND1, ND2, ND3, CO1, CO2, ATP6 and ATP8 determined by t-tests corrected for multiple comparisons using Holm-Sidak method with alpha=0.05 Figure 13. 3-D output of gene co-expression from Miru® carried out with a minimum correlation of 0.9 (Spearman) for the top 500 genes in human NSC’s where ratios of FPKM’s with 5nM rhTFAM were divided by FPKM’s with buffer. Miru was asked to create co-expression networks for the FPKM’s using the four experiments with 5 nM rhTFAM, followed by Markov clustering that used the built-in “MCL” algorithm. Gene numbers in cluster 1 (top) and cluster 2 (bottom) are indicated. Note that clusters 1+ 2 accounted for 392 of the 500 genes studied. Red lines (edges) connecting spherical nodes (genes) indicate a positive association of genes’ expression. Figure 14. Protein-protein interactions determined in STRING (www.string-db.com) for the genes defined by Miru cluster 1 (see Figure 13). Note two large (circled) and 2 small clusters in the STRING output. Figure 15. Same as for Figure 14 except genes in Miru cluster 2 were used. Note two large clusters (circled) and one “hub” gene (ubiquitin). Figure 16. Results from IPA analysis of top 500 genes used to construct Figures 13-15. Supplemental Figure 1. Alignments of TFAM protein sequences from human, mouse and rat using MacVector®. Supplemental Figure 2. GeNorm analysis from qbasePLUS® (Biogazelle) of reference (“housekeeping”) gene stabilities in rat forebrain cDNA samples. geNorm analysis was carried out on 16 samples and 12 reference targets. The lower the value of geNorm M (Y-axis), the more stable is the reference gene across samples. The reference genes were provided on the SABiosciences gene-specific primer qPCR plates The optimal number of
20
reference targets selected was 2, which were Pgk1 and Hprt1 (geNorm V < 0.15) and the geometric mean of these were calculated as the optimal normalization factor. Supplemental Table 1. Representative Gene Ontology (GO) terms from STRING protein network modeling of Miru® 3-D gene clusters from RNA-seq gene expression of CTL human iPSC-derived NSC’s exposed for 24 hrs to buffer or 5nM rhTFAM. 7. References Cited [1]L.Margulis,D.Bermudes,Symbiosisasamechanismofevolution:statusofcellsymbiosistheory,Symbiosis,1(1985)101-124.[2]U.Kutschera,K.J.Niklas,Endosymbiosis,cellevolution,andspeciation,TheoryBiosci,124(2005)1-24.[3]M.Elias,J.M.Archibald,Sizingupthegenomicfootprintofendosymbiosis,Bioessays,31(2009)1273-1279.[4]P.J.Keeling,Theimpactofhistoryonourperceptionofevolutionaryevents:endosymbiosisandtheoriginofeukaryoticcomplexity,ColdSpringHarbPerspectBiol,6(2014).[5]N.W.Blackstone,Theimpactofmitochondrialendosymbiosisontheevolutionofcalciumsignaling,CellCalcium,57(2015)133-139.[6]M.X.Li,G.Dewson,Mitochondriaandapoptosis:emergingconcepts,F1000PrimeRep,7(2015)42.[7]R.C.Scarpulla,Nucleus-encodedregulatorsofmitochondrialfunction:Integrationofrespiratorychainexpression,nutrientsensingandmetabolicstress,BiochimBiophysActa,(2011).[8]R.C.Scarpulla,MetaboliccontrolofmitochondrialbiogenesisthroughthePGC-1familyregulatorynetwork,BiochimBiophysActa,1813(2011)1269-1278.[9]V.Sica,L.Galluzzi,J.M.Bravo-SanPedro,V.Izzo,M.C.Maiuri,G.Kroemer,Organelle-SpecificInitiationofAutophagy,MolCell,59(2015)522-539.[10]N.M.Held,R.H.Houtkooper,Mitochondrialqualitycontrolpathwaysasdeterminantsofmetabolichealth,Bioessays,37(2015)867-876.[11]N.D.Bonawitz,D.A.Clayton,G.S.Shadel,Initiationandbeyond:multiplefunctionsofthehumanmitochondrialtranscriptionmachinery,MolCell,24(2006)813-825.[12]R.C.Scarpulla,Transcriptionalparadigmsinmammalianmitochondrialbiogenesisandfunction,Physiologicalreviews,88(2008)611-638.[13]M.I.Ekstrand,M.Falkenberg,A.Rantanen,C.B.Park,M.Gaspari,K.Hultenby,P.Rustin,C.M.Gustafsson,N.G.Larsson,MitochondrialtranscriptionfactorAregulatesmtDNAcopynumberinmammals,HumMolGenet,13(2004)935-944.[14]K.Maniura-Weber,S.Goffart,H.L.Garstka,J.Montoya,R.J.Wiesner,TransientoverexpressionofmitochondrialtranscriptionfactorA(TFAM)issufficienttostimulatemitochondrialDNAtranscription,butnotsufficienttoincreasemtDNAcopynumberinculturedcells,NucleicAcidsRes,32(2004)6015-6027.
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[15]R.C.Scarpulla,Nuclearcontrolofrespiratorygeneexpressioninmammaliancells,Journalofcellularbiochemistry,97(2006)673-683.[16]D.Kang,S.H.Kim,N.Hamasaki,MitochondrialtranscriptionfactorA(TFAM):rolesinmaintenanceofmtDNAandcellularfunctions,Mitochondrion,7(2007)39-44.[17]S.M.Khan,J.P.Bennett,Jr.,Developmentofmitochondrialgenereplacementtherapy,Journalofbioenergeticsandbiomembranes,36(2004)387-393.[18]P.M.Keeney,C.K.Quigley,L.D.Dunham,C.M.Papageorge,S.Iyer,R.R.Thomas,K.M.Schwarz,P.A.Trimmer,S.M.Khan,F.R.Portell,K.E.Bergquist,J.P.Bennett,Jr.,MitochondrialgenetherapyaugmentsmitochondrialphysiologyinaParkinson'sdiseasecellmodel,Humangenetherapy,20(2009)897-907.[19]R.R.Thomas,S.M.Khan,F.R.Portell,R.M.Smigrodzki,J.P.Bennett,Jr.,RecombinanthumanmitochondrialtranscriptionfactorAstimulatesmitochondrialbiogenesisandATPsynthesis,improvesmotorfunctionafterMPTP,reducesoxidativestressandincreasessurvivalafterendotoxin,Mitochondrion,11(2011)108-118.[20]P.M.Keeney,C.K.Quigley,L.D.Dunham,C.M.Papageorge,S.Iyer,R.R.Thomas,K.M.Schwarz,P.A.Trimmer,S.M.Khan,F.R.Portell,K.E.Bergquist,J.P.Bennett,MitochondrialGeneTherapyAugmentsMitochondrialPhysiologyinaParkinson'sDiseaseCellModel,HumGeneTher,(2009).[21]S.Iyer,R.R.Thomas,F.R.Portell,L.D.Dunham,C.K.Quigley,J.P.Bennett,Jr.,RecombinantmitochondrialtranscriptionfactorAwithN-terminalmitochondrialtransductiondomainincreasesrespirationandmitochondrialgeneexpression,Mitochondrion,9(2009)196-203.[22]R.R.Thomas,S.M.Khan,R.M.Smigrodzki,I.G.Onyango,J.Dennis,O.M.Khan,F.R.Portelli,J.P.Bennett,Jr.,RhTFAMtreatmentstimulatesmitochondrialoxidativemetabolismandimprovesmemoryinagedmice,Aging,4(2012)620-635.[23]S.Iyer,E.Xiao,K.Alsayegh,N.Eroshenko,M.J.Riggs,J.P.Bennett,Jr.,R.R.Rao,Mitochondrialgenereplacementinhumanpluripotentstemcell-derivedneuralprogenitors,Genetherapy,(2011).[24]S.Iyer,K.Bergquist,K.Young,E.Gnaiger,R.R.Rao,J.P.Bennett,Jr.,MitochondrialGeneTherapyImprovesRespiration,Biogenesis,andTranscriptioninG11778ALeber'sHereditaryOpticNeuropathyandT8993GLeigh'sSyndromeCells,Humangenetherapy,(2012).[25]S.N.Dowey,X.Huang,B.K.Chou,Z.Ye,L.Cheng,Generationofintegration-freehumaninducedpluripotentstemcellsfrompostnatalbloodmononuclearcellsbyplasmidvectorexpression,NatProtoc,7(2012)2013-2021.[26]L.C.O'Brien,P.M.Keeney,J.P.Bennett,Jr.,DifferentiationofHumanNeuralStemCellsintoMotorNeuronsStimulatesMitochondrialBiogenesisandDecreasesGlycolyticFlux,Stemcellsanddevelopment,24(2015)1984-1994.[27]A.M.Ambrus,A.B.Islam,K.B.Holmes,N.S.Moon,N.Lopez-Bigas,E.V.Benevolenskaya,M.V.Frolov,LossofdE2Fcompromisesmitochondrialfunction,DevCell,27(2013)438-451.[28]E.V.Benevolenskaya,M.V.Frolov,EmerginglinksbetweenE2Fcontrolandmitochondrialfunction,CancerRes,75(2015)619-623.[29]A.C.Ladd,P.M.Keeney,M.M.Govind,J.P.Bennett,Jr.,MitochondrialOxidativePhosphorylationTranscriptomeAlterationsinHumanAmyotrophicLateralSclerosisSpinalCordandBlood,NeuromolecularMed,(2014).[30]B.B.Avants,P.Yushkevich,J.Pluta,D.Minkoff,M.Korczykowski,J.Detre,J.C.Gee,Theoptimaltemplateeffectinhippocampusstudiesofdiseasedpopulations,Neuroimage,49(2010)2457-2466.
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23
Figure 1
State
3 C I
State
3 C II
State
3 C IV
State
IV0
100
200
300
400%
buff
er C
TL
%Buffer Rat Cortex vs TFAM dose
Buf_24hrsTF 1mg/kg_24hrsTF 1mg/kg_3daysTF 3mg/kg_24hrsTF 3mg/kg_3days
24
Figure 2
Comp I
Comp II
Comp IV
State
40
50
100
150
200
Resp/mg Prot of Rats iv with TFAM @ 3mg/kg
% 0
hr
Mea
n of
CTL
Buf 0 hr Buf
0 hr TF24 h3 d7 d
0 hr Buf CTL to 24 hrs TFAM has significantly increased in Comp I (p <0.03,) Comp II (p<0.03) and in Comp IV (p<0.2).
**
*
*
25
Figure 3
26
Figure 4
Comp I
Comp II
Comp IV
State
40
50
100
150
Rat Forebrain Respiration/mg protein after one month of qweekly doses of rhTFAM @ 3mg/kg
p=0.011 2-way ANOVA CTL vs rhTFAM (matched pairs)%
0 h
r M
ean
of C
TL B
uf
CTL rhTFAM
27
Figure 5
Nduf
s4
Nduf
a2
Nduf
b9
Nduf
s2
Nduf
a9
Nduf
s8
Nduf
b7
Nduf
b5
Nduf
a5
Nduf
b3
Nduf
s1
Nduf
s3
Nduf
a6
Nduf
a8
Nduf
v2
Nduf
a11
Nduf
s6
Nduf
b2
Nduf
a1
Nduf
a7
Nduf
b8
Nduf
a10
Nduf
c2
Nduf
s7
Nduf
v1
Nduf
b6
Nduf
ab1
60
80
100
120
Complex I genes
% m
ean
CTL
Complex I genes CTL vs rhTFAM
p<0.0001 2-way ANOVA CTL vs rhTFAMCTLrhTFAM
Ucp1
Uqcr
c1
Uqcr
b
Ucp2
Uqcr
h
Uqcr
q
Uqcr
c2
Uqcr
fs1
Ucp3
50
100
150
Complex III genes
% m
ean
CTL
Complex III genes CTL vs rhTFAM
NS
CTLrhTFAM
Atp6
v1e2
At
p6vc
2 At
p5o
Atp5
i At
p5b
Atp5
d At
p5g2
At
p5j
Atp6
ap1
Atp5
l At
p6v0
a2
Atp5
c1
Atp5
a1
Atp5
h At
p4b
Atp1
2a
Atp5
f1
Atp5
g3
Atp4
a At
p6v1
g3At
p6v0
d2 50
100
150
200
Complex V genes
% m
ean
CTL
Complex V genes CTL vs rhTFAM
NS
CTL
rhTFAM
Slc2
5a10
Slc2
5a20
Sdhb
Slc2
5a15
Surf1
Sdhc
Sdhd
Sdha
50
100
150
Complex II genes
% m
ean
CTL
Complex II genes CTL vs rhTFAM
NS
CTL
rhTFAM
Cox6
c Co
x4i1
Co
x15
Cox7
a2
Cox8
a Co
x5b
Cox6
a1
Cox7
b Co
x17
Cox8
c Co
x7a2
l Co
x5a
Cox6
a2
Cyc1
Co
x4i2
50
100
150
Complex IV genes
% m
ean
CTL
Complex IV genes CTL vs rhTFAM
p=0.0005 2-way ANOVA CTL vs rhTFAM
CTL
rhTFAM
28
Figure 6
Ndu
fc2
Ndu
fa1
Ndu
fb3
Ndu
fb2
Ndu
fa6
Ndu
fa2
Ndu
fb7
Ndu
fs3
Ndu
fa7
Ndu
fa5
Ndu
fa8
Ndu
fab1
Ndu
fa9
Ndu
fb8
Ndu
fa10
Ndu
fb6
Ndu
fb9
Ndu
fb5
Ndu
fs6
Ndu
fs7
Ndu
fa11
Ndu
fs4
Ndu
fs8
Ndu
fs1
Ndu
fv1
Ndu
fv2
Ndu
fs20
100
200
300
gene
%m
ean
CTL
Complex Ip<0.0001 2-way ANOVA
CTL vs rhTFAMCTLrhTFAM
Cox
7bC
ox6c
Cox
7a2
Cox
6a2
Cox
17C
ox5b
Cox
6a1
Cox
8aC
ox4i
1C
ox4i
2C
ox5a
Cox
7a2l
Cox
8cC
ox15
0
100
200
300
gene
%m
ean
CTL
Complex IVp<0.0001 2-way ANOVA
CTL vs rhTFAM
CTL
rhTFAMU
cp1
Uqc
rb
Uqc
rh
Uqc
rq
Uqc
rc2
Uqc
rfs1
Uqc
rc1
Ucp
2
Ucp
30
200
400
600
gene
%m
ean
CTL
Complex IIIp=0.03 2-way ANOVA
CTL VS rhTFAM
CTLrhTFAM
Atp
5iA
tp5l
Atp
5bA
tp5j
Atp
5f1
Atp
5g2
Atp
5a1
Atp
5oA
tp5d
Atp
5c1
Atp
5hA
tp4a
Atp
5g3
Atp
6ap1
Atp
4bA
tp6v
0d2
Atp
6v1g
3A
tp6v
0a2
Atp
6v1e
2A
tp6v
c2
0
100
200
300
gene
%m
ean
CTL
ATPasep=0.0001 2-way ANOVA
CTL vs rhTFAM
CTLrhTFAM
rat cervical spinal cordrhTFAM 3 mg/kg i.v. qweek X4
29
Figure 7
CI -
ND
UFB
8
CII
- SD
HB
CIII
- U
QC
RC
2
CIV
- M
TCO
1
CV
- ATP
5A 0
100
200
300
OXPHOS protein
Nor
mal
ized
Inte
grat
ed In
tens
ity%
mea
n C
TL
%mean CTL OXPHOS proteinrat cervical spinal cord
3 mg/kg i.v. rhTFAM qWeek X4p=0.014 2-way paired ANOVA
CTL vs rhTFAM
CTL (n=6)rhTFAM (n=6)
30
Figure 8
31
Figure 9
32
Figure 10
33
Figure 11
34
Figure 12
35
Figure 13
36
Figure 14
37
Figure 15
38
Figure 16
39
Supplemental Figure 1
Formatted AlignmentsHuman TFAMMouse TFAMRat TFAM
1 401 401 40
MA F L R S MWG V L S A L G R S G A E L C T G C G S R L R S P F S F V Y L P RMA L F R GMW S V L K A L G R T G V E MC A G C G G R I P S S I S L V C I P KMA L F R GMWG V L R T L G R T G V E MC A G C G G R I P S P V S L I C I P KMA L F R GMWG V L . A L G R T G V E MC A G C G G R I P S P . S L V C I P K
Human TFAMMouse TFAMRat TFAM
41 8041 7941 79
W F S S V L A S C P K K P V S S Y L R F S K E Q L P I F K A Q N P D A K T T E LC F S S - MG S Y P K K P M S S Y L R F S T E Q L P K F K A K H P D A K L S E LC F S S - L G N Y P K K P M S S Y L R F S T E Q L P K F K A K H P D A K V S E LC F S S V L G S Y P K K P M S S Y L R F S T E Q L P K F K A K H P D A K . S E L
Human TFAMMouse TFAMRat TFAM
81 12080 11980 119
I R R I A Q RWR E L P D S K K K I Y Q D AY R A EWQ V Y K E E I S R F K E QV R K I A A LWR E L P E A E K K V Y E A D F K A EWK AY K E AV S K Y K E QI R K I A AMWR E L P E A E K K V Y E A D F K A EWK V Y K E AV S K Y K E QI R K I A A WR E L P E A E K K V Y E A D F K A EWK V Y K E AV S K Y K E Q
Human TFAMMouse TFAMRat TFAM
121 160120 159120 159
L T P S Q I M S L E K E I MD K H L K R K AM T K K K E L T L L G K P K R P R SL T P S Q L MGM E K E A R Q R R L K K K A L V K R R E L I L L G K P K R P R SL T P S Q L MG L E K E A R Q K R L K K K A Q I K R R E L I L L G K P K R P R SL T P S Q L MG L E K E A R Q K R L K K K A . K R R E L I L L G K P K R P R S
Human TFAMMouse TFAMRat TFAM
161 200160 199160 199
AY N V Y VA E R F Q E A K G D S P Q E K L K T V K E NWK N L S D S E K E L YAY N I Y V S E S F Q E A K D D S A Q G K L K L V N E AWK N L S P E E K Q AYAY N I Y V S E S F Q E A K D E S A Q G K L K L V N Q AWK N L S H D E K Q AYAY N I Y V S E S F Q E A K D D S A Q G K L K L V N E AWK N L S . E K Q AY
Human TFAMMouse TFAMRat TFAM
201 240200 238200 239
I Q H A K E D E T R Y H N E MK S WE E QM I E V G R K D L L R R T I K K Q R KI Q L A K D D R I R Y D N E MK S WE E QMA E V G R S D L I R R S V K R S - GI Q L A K D D R I R Y D N E MK S WE E QMA E V G R S D L I R R S V K R P P GI Q L A K D D R I R Y D N E MK S WE E QMA E V G R S D L I R R S V K R G
Human TFAMMouse TFAMRat TFAM
241 246239 243240 244
Y G A E E CD I S E H D I S E N D I S E C
40
Supplemental Figure 2
Average expression stability of remaining reference targets
TfrcRplp1
Hsp90
ab1
ActbNono
B2m TbpPpih Sdh
aLdha
Pgk1Hprt1
0.120
0.125
0.130
0.135
0.140
0.145
0.150
0.155
0.160
0.165
0.170
0.175
0.180
0.185
0.190
0.195
0.200
0.205
0.210
0.215
0.220
0.225
0.230
0.235
geN
orm
M
41
Supplemental Table 1. Representative Gene Ontology (GO) Terms from STRING Protein Network Modeling of Miru® 3-D Gene Clusters from RNA-seq Gene Expression of CTL Human iPSC-derived NSC’s Exposed for 24 hrs to Buffer or 5nM rhTFAM. Top 500 genes from rhTFAM/buffer gene expression. X= present; En= range of GO enrichment (fold); Bj= range of Benjamini-corrected p values
Representative Gene Ontology
(GO) terms
Cluster 1 (Fig 14,
red circle)
Cluster 1 (Fig 14,
blue circle)
Cluster 2 (Fig 15,
red circle)
Cluster 2 (Fig 15,
blue circle)
protein complex, DNA binding, RNA
polymerase, transcription
X En 28-106 fold, Bj 0.005-2.1E-8
X En 22-110
Bj 0.02-6.8E-4
ribonucleoprotein, ribonucleoprotein
complex translation,
ribosome, ncRNA
X En 4.5-132
Bj 0.004-8.7E-38
X En 9.5-49
Bj 0.03-3.5E-4
X En 7.3-339
Bj 0.008-4.2E-14
ribosome biogenesis, rRNA processing
X En 12-205
Bj 0.008-3.1E-5
RNA processing, mRNA processing
X En 25-89
Bj 1.4E-16-1.1E-22
macromolecular complex,
nucleoplasm, ribonucleoprotein complex assembly
X En 7.3-339
Bj 0.004-4.2E-14
Histone N-acetyltransferase,
chromatin organization
X En 10-73
Bj 0.035-0.003