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Mitochondrial Dysfunction: A Prelude to Neuropathogenesis of
SARS-CoV‑2
Artem Pliss, Andrey N. Kuzmin, Paras N. Prasad,*and Supriya D. Mahajan*
Cite This: ACS Chem. Neurosci. 2022, 13, 308−312
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ABSTRACT: The SARS-CoV-2 virus is notorious for its neuro-
invasive capability, causing multiple neurological conditions. The
neuropathology of SARS-CoV-2 is increasingly attributed to
mitochondrial dysfunction of brain microglia cells. However, the
changes in biochemical content of mitochondria that drive the
progression of neuro-COVID remain poorly understood. Here we
introduce a Raman microspectrometry approach that enables the
molecular profiling of single cellular organelles to characterize the
mitochondrial molecular makeup in the infected microglia cells. We
found that microglia treated with either spike protein or heat-
inactivated SARS-CoV-2 trigger a dramatic reduction in mtDNA
content and an increase in phospholipid saturation levels. At the
same time, no significant changes were detected in Golgi apparatus
and in lipid droplets, the organelles that accommodate biogenesis and storage of lipids. We hypothesize that transformations in
mitochondria are caused by increased synthesis of reactive oxygen species in these organelles. Our findings call for the development
of mitochondria-targeted therapeutic approaches to limit neuropathology associated with SARS-CoV-2.
KEYWORDS: Microglia, mitochondria, ROS, SARS-CoV-2, neuro-COVID, Raman spectrometry
■INTRODUCTION
A significant number of COVID-19 patients develop neuro-
logical symptoms, attributed to viral encephalitis, resulting in
neuroinflammation, neuronal damage, and neurocognitive
impairment. The microglia, which are the resident macro-
phages in the central nervous system, are the major players in
the brain’s immune response to SARS-CoV-2 infection.
Furthermore, it has been shown that functional mitochondria
are integral to initiation and maintenance of immune responses
by microglia, while neurological damage in COVID patients is
attributed to mitochondrial dysfunction. Mitochondria are the
primary site of ATP production and also regulate basic
metabolic functions and participate in homeostasis, cellular
proliferation, and apoptosis as well as in the synthesis of amino
acids, lipids, and nucleotides. In microglia these organelles also
mediate the antiviral immune response by releasing pro-
inflammatory cytokines, which limit viral survival and viral
replication and trigger inflammation.
1−4
Strikingly, SARS-CoV-
2 can evade the innate immune response of host cells via the
modulation of mitochondrial functions. The spike protein of
SARS-CoV-2 binds to the angiotensin-converting enzyme-2
(ACE-2) receptor on the human host cell
3
to enter the host,
and the transmembrane serine protease 2 (TMPRESS 2)
facilitates this attachment by priming the spike protein.
5
Notably, the ACE-2 receptor regulates mitochondrial func-
tion.
6
Reduced expression of ACE-2 is correlated with
decreased ATP synthesis and activation of NADPH oxidase
4, which contributes to the production of reactive oxygen
species (ROS).
4
Consistent with that, an invasion of SARS-
CoV-2 via the ACE-2 receptor compromises mitochondrial
regulation. Excessive ROS production exacerbates neuro-
inflammation, initiating apoptosis in infected cells, which
results in neurocognitive impairments. It is known that SARS-
CoV-2 infection results in a massive inflammatory response in
the brain by triggering the release of cytokines such as
interleukin (IL)-10, TNF-α, and INF-γ, which in turn further
increase mitochondrial ROS production through upregulation
of mitochondrial genes and modulation of the electron
transport chain (ETC).
7
The mitochondrial ROS stimulate
additional proinflammatory cytokine production
8
in the face of
viral persistence, leading to a “cytokine storm syndrome”,
which underlies viral encephalopathy.
7
We recently observed
an increased oxygen consumption rate (OCR) in microglial
cells treated with SARS-CoV-2 spike protein.
9
Our data
suggested that SARS-CoV-2 induced a robust inflammatory
Received: October 13, 2021
Accepted: January 18, 2022
Published: January 20, 2022
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response, significantly increasing oxidative stress and OCR, all
of which contributed to neuroinflammation and associated
neuropathology of an encephalitic coronavirus infection.
In order to evade host cell immunity and facilitate virus
replication, SARS-CoV-2 viral open reading frame (ORF) 9b
localizes in mitochondria and can directly modulate
mitochondrial function, thereby contributing to COVID-19
disease progression.
4
Thus, we hypothesize that modulating mitochondrial activity
may prevent mitochondrial dysfunction following SARS-CoV-2
infection and that mitochondria-targeted pharmacological
interventions may enhance an immune response in SARS-
CoV-2 associated neuropathogenesis.
Toward verification of this hypothesis, we analyzed the
molecular composition in the mitochondria of infected cells. It
is worth noting that characterization of the mitochondria
metabolic variations by standard biochemical approaches is
extremely challenging. Traditional molecular profiling ap-
proaches rely on cellular fractioning and extraction of the
analyte protein or lipid molecules from the studied organelles,
whichisacumbersomeprocedurethatisproneto
contamination. Additionally, the molecular extraction approach
inherently produces data averaging, thus masking hetero-
geneity between organelles obtained from different cells.
Remarkably, the capabilities of biochemical analysis have
been recently expanded with optical biosensing tools. Raman
spectrometry, one of the most valuable biosensing technolo-
gies, relies on analysis of molecular vibrational spectra and
enables the identification of diverse molecular groups in
biological samples. Because of their inherently noninvasive
properties and independence from labels, Raman-based
techniques have opened new dimensions in systemic studies
of cells and tissues.
10,11
The recently developed biomolecular
component analysis (BCA) of Raman spectra enables selective
detection and concentration measurements of the major
categories of biomolecules, including lipids, proteins, nucleic
acids, and saccharides,
12,13
in the studied samples. The high
three-dimensional resolution available in modern confocal
Raman spectrometry setups has been validated for character-
ization of microscopic subcellular structures, such as single
organelles, including the identification of abnormal biomo-
lecular signatures associated with disease progression.
14−20
In this study, we employed Raman spectrometry together
with the BCA algorithm to characterize the changes in the
molecular composition of mitochondria in response to
treatment with heat-inactivated SARS-CoV-2 or the SARS-
CoV-2 spike protein. In addition, we studied key organelles
involved in lipid metabolism: Golgi apparatus (GA) and lipid
droplets (LD). The roles lipids play in viral infection include
viral endocytosis and exocytosis, viral entry into the host cell
via membrane fusion, and viral replication, and therefore, we
were interested in potential changes of the lipid signatures in
these organelles.
Our data indicate that infection with SARS-CoV-2 causes
mitochondrial dysfunction in microglia cells, which triggers
metabolic alterations that result in a substantial increase in
glycolysis.
9
These findings suggest that a metabolic switch to
glycolysis compensates for mitochondrial dysfunction and an
energy deficit in microglia and that a consequence of this
metabolic change is an enhanced inflammatory response that
contributes to neuropathology associated with COVID-19. At
the same time, the molecular content of GA and LD was not
significantly changed, apparently because of the lack of specific
interactions between these organelles and the components of
SARS-CoV-2.
Overall, our findings support a view that viral infection of
host cells results in higher metabolic alterations to cope with
the increased anabolic demand of the cell for viral replication.
Furthermore, SARS-CoV-2-induced manipulation of the host-
cell metabolic machineries alters transcriptional regulation of
key metabolic pathways.
■METHODS
Cell Culturing and Sample Preparation. Human microglia cells
(HMC3) were obtained from ATCC (cat. no. ATCC CRL-3304) and
grown in luminescence-free 35 mm glass-bottom dishes (Fisher
Scientific Co., Hanover Park, IL). The culture medium used was
Eagle’s Minimum Essential Medium (EMEM) (cat. no. ATCC 30-
2003) supplemented with 5% fetal bovine serum (FBS), 100 units/
mL penicillin, and 100 μg/mL streptomycin, and the cells were grown
to 70% confluence at 37 °C in a humidified atmosphere containing
5% CO2. The mitochondria and GA were labeled with MitoTracker
Green FM and NBD C6 ceramide-BSA (Thermo Fisher Scientific),
respectively, as per the manufacturer-provided protocols. After
labeling, the cells were thoroughly washed in sterile phosphate-
buffered saline (PBS).
The cells were treated with the following viral constructs: 0.5 μg/
mL recombinant spike protein from SARS-related Coronavirus 2
Wuhan-Hu-1 (BEI Resources Inc., cat. no. NR-52308, lot no.
70034410) or 5 μL/mL heat-inactivated SARS-Coronavirus 2 (HI-
SARS), isolate USA-WA1/2020, (BEI Resources Inc., cat. no. NR-
52286, lot no. 70033548, pre-inactivation titer by TCID50 assay in
Vero E6 Cells = 1.6 ×105TCID50/mL), as specified.
To target acquisition of Raman spectra to specific organelles, the
mitochondria, endoplasmic reticulum (ER), and GA were labeled
using MitoTracker Green FM, ER-Tracker Green, and NBD C6
ceramide-BSA (ThermoFisher Scientific), respectively, as per the
manufacturer-provided protocols. Then the cells were thoroughly
washed in sterile PBS, and Raman spectra were acquired in the labeled
organelles.
The Raman Microscope. The spectra were measured on a DXR2
Raman microscopy setup (Thermo Fisher Scientific, Madison, WI),
equipped with a laser source unit emitting ∼60 mW at 633 nm
(ROUSB-633-PLR-70-1, Ondax), a 50 μm pinhole to shape the laser
beam to a 0.7 μm×0.7 μm×1.5 μm full width at half-maximum
(fwhm), and a Plan N 100×Olympus objective lens (NA = 1.25). In
addition, the Raman microscope was equipped with a fluorescence
illumination system (5-UR7005, Olympus), a green fluorescence cube
(488/561EX), and a fluorescence lamp (X-Cite 120 PC, Photonic
Solutions).
Acquisition of Raman Spectra. Prior to the measurements, live
cells were transferred into optically transparent Dulbecco’s Modified
Eagle’s Medium (DMEM) (Thermo Fisher Scientific) and mounted
on the microscope stage. The spectra were acquired from the labeled
organelles in live cells as recently described.
15
Fluorescence-labeled
organelles were visualized using the 488/561EX fluorescence cube.
To generate the spectra, the Raman excitation laser was overlapped
with single labeled organelles. To warrant a high-quality signal/noise
ratio, the spectra accumulation parameter was set to 6 ×20 s;
importantly, no measurable phototoxicity was observed at this
irradiation dose. During the experiments, the cells were maintained
under physiological conditions at 37 °C. We visually verified the XYZ
position of the cell before and after each measurement to ensure the
spatial precision of Raman spectra acquisition.
Biomolecular Component Analysis of Raman Spectra. The
calibration of Raman band intensities on the concentrations of
biomolecules in the sample was performed as previously
described.
15,21
Quantitative analysis of cellular spectra was performed
using BCAbox software (ACIS LLC, Buffalo, NY). The description,
interface of the BCAbox software, and schematics for the spectrum
processing algorithm are shown in Figures S1 and S2. Representative
ACS Chemical Neuroscience pubs.acs.org/chemneuro Letter
https://doi.org/10.1021/acschemneuro.1c00675
ACS Chem. Neurosci. 2022, 13, 308−312
309
examples of raw and preprocessed mitochondria spectra are shown in
Figure S3.
■RESULTS AND DISCUSSION
In our experiments, we incubated cultured microglia cells with
SARS-CoV-2 spike protein or heat-inactivated SARS-CoV-2 to
imitate viral neuroinvasion. Untreated microglia were used as
an experimental control. Mitochondria and GA were stained
with specificfluorescence probes, thus enabling acquisition of
Raman spectra in these organelles, while LD were identified by
transmitted light imaging.
The obtained Raman spectra were processed with the BCA
algorithm to quantify the concentrations of major groups of
biomolecules (Figures S6 and S7). The measurements were
performed as recently described.
18
It is worth noting that
although the Raman spectra were collected within a submicron
volume of an excitation laser focused on specific organelles, the
adjacent cytoplasm may also overlap with the laser probe and
contribute to the spectra. Nevertheless, despite this potential
contribution, there were statistically significant differences
between the molecular profiles obtained in various organelles,
which supports the sensitivity of Raman microspectrometry to
the subcellular biochemical environment. The measured values
obtained in single mitochondria of control and treated cells are
shown in Tables S1−S3.
We found that treatment with SARS-CoV-2 spike protein or
HI-SARS induced significant alterations in the concentrations
of diverse types of biomolecules in the mitochondria. First, the
concentration of mitochondrial DNA was reduced almost 2-
fold in the infected cells, from ∼2.2 mg/mL in control cells to
∼1.2 mg/mL in the cells treated with either viral agent (Figure
1), which indicates the decrease in mitochondrial DNA copy
number. At the same time, the concentration of mtRNA was
increased from ∼2.25 mg/mL in the control to 2.8 mg/mL in
HI-SARS-treated cells and ∼4.0 mg/mL in cells treated with
the spike protein; the latter difference was statistically
significant. This increase in RNA is consistent with previous
reports on mitochondrial genome upregulation in cells infected
by SARS-CoV-2.
7
We also found a significant reduction in
mitochondrial saccharides from ∼1.5 mg/mL in the control to
∼0.9 mg/mL in the HI-SARS-treated cells and ∼0.7 mg/mL in
the cells treated with the spike protein. The mitochondrial
saccharide fraction includes glucose and pyruvate, and its
reduction suggests a decrease of the respiratory function of
mitochondria.
Furthermore, we detected a significant perturbation in the
saturation of phospholipids populating the mitochondrial
lipidome. The average number of unsaturated CC bonds
per phospholipid was significantly reduced from ∼4.3 in the
control to ∼3.8 in the cells treated with the HI-SARS viral
construct and ∼3.7 in the cells treated with the spike protein.
At the same time, we did not record any significant change in
the total concentration of lipids in mitochondria (Figure 1).
We thus concluded that the shift in lipidome saturation occurs
as a result of biochemical processes inside the mitochondria
and likely is not caused by trafficking of the saturated
phospholipids to this organelle.
In parallel, we investigated the impact of HI-SARS on the
major organelles involved in the metabolism of lipids such as
GA. However, it appears that SARS-CoV-2 does not directly
influence the lipid biogenesis. We found that all of the
resolvable lipidome characteristics in the control and treated
cells for these organelles were remarkably uniform. Similarly,
the composition of LD in the treated cells remained largely
unchanged. However, we found that HI-SARS induces an
increase in the number of CC bonds in the pool of
unsaturated phospholipids stored in LD (Figures S5−S7).
In the interpretation of our data, we point to the fact that
mitochondrial lipids are predominantly synthesized in the
endoplasmic reticulum and then transported to the mitochon-
dria through the GA. While these organelles show no
differences in molecular composition between control and
treated cells, the mitochondria demonstrate substantial differ-
ences not only in phospholipid saturation but also in the
abundances of RNA, saccharides, and mtDNA (Figure 1). We
propose that these changes originate in virus-induced ROS
production, in part via oxidative damage to lipids and oxidation
of respiratory chain proteins, affecting metabolism and protein
import, which then induces DNA damage as reflected in a
sharp decrease in the mtDNA level. Furthermore, the
mechanistic link between lipid metabolism and inflammation
is well-established, wherein lipids can directly activate
inflammatory pathways.
22
Thus, significant changes in the
composition and distribution of lipids within the brain are
believed to contribute to neurocognitive decline.
23
Further-
more, SARS-CoV-2-induced oxidative stress impacts phospho-
lipid membranes, causing additional perturbations of biological
processes.
24
We propose that increased oxidative stress impacts
the fluidity of phospholipid membranes, which can affect the
interactions and activity of metabolic enzymes, resulting in
membrane remodeling. The membrane fatty acid composition
is thought to be altered in response to oxidative stress by a
Figure 1. Comparative analysis of the molecular content in
nontreated mitochondria (control) and mitochondria treated with
either heat-inactivated SARS-CoV-2 or the SARS-CoV-2 spike protein
(as indicated). The top chart shows absolute concentrations of
proteins, DNA, RNA, saccharides (Gly), and lipids in live-cell
mitochondria. The bottom chart shows a decrease in the number of
CC bonds in mitochondrial phospholipids (phospholipid unsatura-
tion parameter) in both groups of treated cells. The statistically
significant differences are indicated by horizontal brackets and p
values.
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ACS Chem. Neurosci. 2022, 13, 308−312
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decrease in the number of CC bonds, which results in higher
saturation of the organellar lipidome.
24,25
The physiological
relevance of membrane remodeling remains unclear, but it may
be an adaptive response to cellular stress. These data support
our hypothesis that mitochondrial dysfunction, oxidative stress,
and inflammation could lead to an increase in COVID-
associated neurological dysfunction. In addition, our data
support the premise that SARS-CoV-2 induces release of
pathogen-associated molecular patterns (PAMPS) and danger-
associated molecular patterns (DAMPS), ATP, oxidized lipids,
and heat shock proteins, all of which are associated with
apoptosis and autophagy.
26,27
Overall, our study clarifiestheroleofmitochondrial
dysfunction in SARS-CoV-2-induced neuropathology. Our
data suggest that mitochondrial dysfunction is among the
earliest and most prominent features of neurodegeneration. In
addition, the absence of any significant changes in the lipidome
of GA and LD indicate a targeted impact of SARS infection on
mitochondria. Therefore, examining mitochondrial function or
mitochondrial damage markers in the microglia cells in
response to interactions with SARS-CoV-2 spike protein may
help identify pathways of viral pathogenesis, unravel
mechanisms of cellular vulnerability, and aid in the discovery
of mitochondrial biomarkers relevant to SARS-CoV-2 neuro-
inflammation and progression to neuropathogenesis. Further-
more, therapeutic strategies that modulate mitochondrial
processes may be efficacious in treating patients with neuro-
COVID. Our study calls for the development of mitochondria-
targeted pharmaceutical drugs that can neutralize virus-induced
ROS production in these cellular organelles.
■ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.1c00675.
Figures S1−S7 and Tables S1−S3 (PDF)
■AUTHOR INFORMATION
Corresponding Authors
Paras N. Prasad −Institute for Lasers, Photonics and
Biophotonics and Department of Chemistry, University at
Buffalo, The State University of New York, Buffalo, New York
14260, United States; orcid.org/0000-0002-0905-7084;
Email: pnprasad@buffalo.edu
Supriya D. Mahajan −Department of Medicine, Division of
Allergy, Immunology, and Rheumatology, State University of
New York at Buffalo, Clinical Translational Research Center,
Buffalo, New York 14203, United States; Email: smahajan@
buffalo.edu
Authors
Artem Pliss −Institute for Lasers, Photonics and Biophotonics
and Department of Chemistry, University at Buffalo, The
State University of New York, Buffalo, New York 14260,
United States; orcid.org/0000-0003-4867-4074
Andrey N. Kuzmin −Institute for Lasers, Photonics and
Biophotonics and Department of Chemistry, University at
Buffalo, The State University of New York, Buffalo, New York
14260, United States; orcid.org/0000-0001-7371-4643
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.1c00675
Author Contributions
A.P., A.N.K., P.N.P., and S.D.M. conceived the project. A.P.,
A.N.K., and S.D.M. performed the experiments. All of the
authors drafted and edited the manuscript.
Funding
Funding support by the National Institute of Drug Abuse,
National Institutes of Health (Grant 5R01DA047410-02) to
S.D.M. toward experiments in this study is duly acknowledged
Notes
The authors declare no competing financial interest.
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