Pyrroloquinoline quinone drives ATP synthesis in vitro and in vivo and provides retinal ganglion cell neuroprotection

Abstract

Retinal ganglion cells are highly metabolically active requiring strictly regulated metabolism and functional mitochondria to keep ATP levels in physiological range. Imbalances in metabolism and mitochondrial mechanisms can be sufficient to induce a depletion of ATP, thus altering retinal ganglion cell viability and increasing cell susceptibility to death under stress. Altered metabolism and mitochondrial abnormalities have been demonstrated early in many optic neuropathies, including glaucoma, autosomal dominant optic atrophy, and Leber hereditary optic neuropathy. Pyrroloquinoline quinone (PQQ) is a quinone cofactor and is reported to have numerous effects on cellular and mitochondrial metabolism. However, the reported effects are highly context-dependent, indicating the need to study the mechanism of PQQ in specific systems. We investigated whether PQQ had a neuroprotective effect under different retinal ganglion cell stresses and assessed the effect of PQQ on metabolic and mitochondrial processes in cortical neuron and retinal ganglion cell specific contexts. We demonstrated that PQQ is neuroprotective in two models of retinal ganglion cell degeneration. We identified an increased ATP content in healthy retinal ganglion cell-related contexts both in in vitro and in vivo models. Although PQQ administration resulted in a moderate effect on mitochondrial biogenesis and content, a metabolic variation in non-diseased retinal ganglion cell-related tissues was identified after PQQ treatment. These results suggest the potential of PQQ as a novel neuroprotectant against retinal ganglion cell death.

Full text

Canovaietal.
Acta Neuropathologica Communications (2023) 11:146
https://doi.org/10.1186/s40478-023-01642-6
RESEARCH
Pyrroloquinoline quinone drives ATP
synthesis invitro andinvivo andprovides
retinal ganglion cell neuroprotection
Alessio Canovai1,2, James R. Tribble1, Melissa Jöe1, Daniela Y. Westerlund1, Rosario Amato2, Ian A. Trounce3,
Massimo Dal Monte2 and Pete A. Williams1*
Abstract
Retinal ganglion cells are highly metabolically active requiring strictly regulated metabolism and functional mito-
chondria to keep ATP levels in physiological range. Imbalances in metabolism and mitochondrial mechanisms can
be sufficient to induce a depletion of ATP, thus altering retinal ganglion cell viability and increasing cell susceptibility
to death under stress. Altered metabolism and mitochondrial abnormalities have been demonstrated early in many
optic neuropathies, including glaucoma, autosomal dominant optic atrophy, and Leber hereditary optic neuropathy.
Pyrroloquinoline quinone (PQQ) is a quinone cofactor and is reported to have numerous effects on cellular and mito-
chondrial metabolism. However, the reported effects are highly context-dependent, indicating the need to study
the mechanism of PQQ in specific systems. We investigated whether PQQ had a neuroprotective effect under differ-
ent retinal ganglion cell stresses and assessed the effect of PQQ on metabolic and mitochondrial processes in corti-
cal neuron and retinal ganglion cell specific contexts. We demonstrated that PQQ is neuroprotective in two models
of retinal ganglion cell degeneration. We identified an increased ATP content in healthy retinal ganglion cell-related
contexts both in in vitro and in vivo models. Although PQQ administration resulted in a moderate effect on mitochon-
drial biogenesis and content, a metabolic variation in non-diseased retinal ganglion cell-related tissues was identified
after PQQ treatment. These results suggest the potential of PQQ as a novel neuroprotectant against retinal ganglion
cell death.
Keywords Pyrroloquinoline quinone (PQQ), Mitochondria, Metabolism, Metabolomics, ATP, Retinal ganglion cell,
Retina, Optic nerve, Neuroprotection
Introduction
Retinal ganglion cells (RGCs) are the output neurons of
the retina, the axons of which form the optic nerve, con-
necting the eye to the brain. RGCs are highly metaboli-
cally and physiologically active cells, requiring a constant
supply of ATP to ensure proper function. A finely regu-
lated metabolism and perfectly balanced mitochondrial
function are fundamental to maintain ATP at a physi-
ological level. Imbalances in these mechanisms can be
detrimental to RGC viability by depleting ATP, render-
ing RGCs susceptible to damage [1]. Altered metabolism
and mitochondrial abnormalities have been reported to
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Acta Neuropathologica
Communications
*Correspondence:
Pete A. Williams
pete.williams@ki.se
1 Division of Eye and Vision, Department of Clinical Neuroscience, St. Erik
Eye Hospital, Karolinska Institutet, Stockholm, Sweden
2 Department of Biology, University of Pisa, Pisa, Italy
3 Department of Surgery, Centre for Eye Research Australia, Royal
Victorian Eye and Ear Hospital, Ophthalmology, University of Melbourne,
Melbourne, VIC, Australia
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
occur early in the pathogenesis of many optic neuropa-
thies, including glaucoma, autosomal dominant optic
atrophy (ADOA), and Leber hereditary optic neuropathy
(LHON), where progressive RGC dysfunction and degen-
eration are typical hallmarks of disease [2–8]. Reducing
bioenergetic insufficiency by buffering metabolic stress,
positively regulating mitochondrial mechanisms, and
restoring ATP levels have all been demonstrated to be
neuroprotective to RGCs [7, 9]. A treatment that can pre-
vent metabolic dysfunction and arrest RGC degeneration
is highly sought after. ere is strong potential for bioen-
ergetic compounds which could be supplemented in diet,
adjuvant to existing medication to increase RGC resil-
ience, and with better side effect profiles.
Pyrroloquinoline quinone (PQQ) is a quinone cofac-
tor first described in bacterial dehydrogenases [10]. It has
been considered as a ‘new vitamin’ given its nutritional
importance on mammalian growth, reproduction, and
development demonstrated by a wide range of abnormal-
ities when PQQ is absent in the diet [11, 12]. PQQ is not
synthetized de novo in mammals, but is instead present
in several foods, such as parsley, green pepper, spinach,
kiwi, and soybeans, thus giving the possibility to con-
sume PQQ through dietary supplementation [13]. PQQ
administration has a good safety profile, with a median
LD50 of 0.5–2.0g/kg in rats, and no signs of toxic effects
when treated long term via oral gavage [14]. is suggests
a strong potential for PQQ as a compound to support
retinal ganglion cell health.
Several studies have reported an effect of PQQ on
metabolism and mitochondrial mechanisms, acting as
an enzyme cofactor [15], regulating nicotinamide ade-
nine dinucleotide (NAD) content [16], increasing oxida-
tive phosphorylation (OXPHOS) and ATP production
[17], and altering mitochondrial dynamics and content
through the regulation of pathways particularly involved
in mitochondrial biogenesis [18, 19]. However, such
effects are highly context-dependent and heterogeneous,
dictating the necessity to study, in depth, the mechanism
of PQQ in specific systems. Although there is some evi-
dence suggesting a putative neuroprotection of PQQ in
models of acute damage in the central nervous system
(CNS) [20–23], the role of PQQ in metabolism and mito-
chondrial content in a neuronal context has not been
extensively investigated.
In this study, we explored neuroprotection exerted by
PQQ in models of RGC degeneration where bioenergetic
capacity is compromised. Given the reported ability of
PQQ to provide bioenergetic support in other systems,
we hypothesized that PQQ could provide neuroprotec-
tion by bolstering bioenergetic support. Supporting this
hypothesis, we demonstrate that PQQ is neuroprotec-
tive in two models of RGC degeneration. In addition,
we identify that PQQ administration leads to increased
neuronal ATP content in RGC-related tissues both in
invitro and invivo models. Although we identified a mild
effect exerted by PQQ on mitochondrial biogenesis and
content, we report a metabolic variation in non-diseased
RGC-related tissues. Taken together, these results sug-
gest a potential role of PQQ as a novel neuroprotective
compound to improve RGC resilience.
Materials andmethods
Animal strain andhusbandry
All breeding and experimental procedures were man-
aged in accordance with the Association for Research
for Vision and Ophthalmology Statement for the Use of
Animals in Ophthalmic and Research. Individual study
protocols were accepted by Stockholm’s Committee for
Ethical Animal Research (10389-2018). Animals were
housed in a regulated environment, (12h light/12h dark
cycle) and fed with food and water adlibitum. C57BL/6J
(B6J) and MitoV (strain information detailed below)
mouse strains were bred and used at 12–20weeks of age.
PQQ disodium salt (Mitsubishi Gas Chemical Company
Inc., Tokyo, Japan) was dissolved in DMSO and diluted
in Hank’s balanced salt solution (without CaCl2, MgCl2
and phenol red) (HBSS; Gibco). Mice were treated with
either vehicle (HBSS) or 20mg/kg PQQ i.p. either for a
single injection (short-term) or chronically every 48 h
for 2weeks (long-term) as further indicated. In a subset
of experiments, PQQ was dissolved in drinking water at
the concentration of 0.2mg/mL in order to obtain a daily
administration of 20 mg/kg considering an individual
mean drinking volume of 3mL/day. Water was protected
from light as PQQ is light sensitive.
Retina axotomy explant model
A retinal axotomy model was performed as previously
reported [7]. Briefly, B6J mice (n = 13) were euthanized
by cervical dislocation, retinas immediately dissected in
ice-cold HBSS and flat mounted on inserts for cell cul-
ture (Millicell 0.4 µm pore; Merck) with ganglion cell
layer (GCL) up. Retinas were cultured (37 °C, 5% CO2)
in media composed of Neurobasal-A supplemented with
2mM L-glutamate (GlutaMAX, Gibco), 2% B27, 1% N2,
and 1% penicillin/streptomycin (Gibco) in 6-well cul-
ture plates. After 2days, half of the media volume was
replaced. For PQQ treated retinas, PQQ was dissolved
in the culture media to a concentration of 50 or 100M.
Retinas were removed from culture to be further pro-
cessed after 3days exvivo (DEV). For controls (0 DEV),
eyes were enucleated and retinas directly fixed and pro-
cessed for immunofluorescent labelling.
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
Intravitreal rotenone model
Rotenone induced retinal degeneration model was per-
formed following established protocols [7]. B6J mice
(n = 20) underwent anesthesia by an intraperitoneal
injection of mixed ketamine (37.5 mg/kg) and medeto-
midine hydrochloride (1.25 mg/kg). Two µl of 10 mM
rotenone diluted in DMSO (all Sigma-Aldrich) or DMSO
only (control) was bilaterally injected into the vitreous
using a 33G tri-beveled needle on a 10µl glass syringe
(WPI). For PQQ treated retinas, PQQ (20 mg/kg) i.p.
was administered 24 h prior to and immediately after
rotenone injection. Mice were euthanized by cervical
dislocation 24h after rotenone injection and eyes were
immediately isolated for further analyses.
Analysis ofRGC death anddegeneration
Loss of RGCs, loss of nuclei, and nuclear shrinkage in
retinal explants (n = 5 retinas from 0 DEV, 9 from 3 DEV,
4 from 3 DEV + 50 M, 6 from 3 DEV + 100 M) and
rotenone injected retinas (n = 9 DMSO, 9 DMSO + PQQ,
10 rotenone and 9 rotenone + PQQ) were assessed by
immunofluorescence through RBPMS and DAPI label-
ling of flat mounts. Retinal explants at 3 DEV were fixed
in 3.7% PFA for 30min, detached from the cell culture
insets and transferred on slides. Eyes from the animals
injected with rotenone were fixed in PFA at the same
concentration for 1h immediately after enucleation. Reti-
nas were then dissected in ice-cold HBSS and transferred
onto slides. Tissues were isolated using a hydrophobic
barrier pen (VWR) and permeabilized with 0.5% Triton
X-100 (VWR) in 1M PBS for 1h. After blocking in 2%
bovine serum albumin (BSA, Fisher Scientific) in 1M
PBS for 1 h, retinas were immunolabelled with rabbit
polyclonal anti-RBPMS (NBP2-20112, Novusbio, 1:500)
primary antibody at 4 °C overnight. ereafter, retinas
were rinsed by 5 washes in 1M PBS for 5min each before
labelling with Alexa Fluor 568 conjugated goat anti-rabbit
secondary antibody (A11011, Invitrogen, 1:500) for 4 h
at room temperature. Tissue was then washed as before
and counterstained with DAPI (1 g/mL in 1 M PBS)
for 10min. After being rinsed once in PBS, tissue was
mounted with Fluoromount-G and glass coverslips (Inv-
itrogen). Nail-varnish was used to seal the slides which
were kept at 4°C until further imaging. e acquisition of
the images was performed on a Leica DMi8 microscope
with a CoolLED pE-300 white LED-based light source
and a Leica DFC7000 T fluorescence color camera (all
Leica). Six images per retina (40× magnification) were
acquired equidistantly at 0, 2, 4, 6, 8, and 10o’clock from
a superior to inferior line through the optic nerve head at
an eccentricity of around 1000m. Images were cropped
to 100m × 100m squares and the cell counter plugin
in Fiji was used to count RBPMS + cells and DAPI nuclei
(only round nuclei were considered, thus discarding vas-
cular endothelium). e mean of cell counts per retina
was measured across the 6 images and expressed as a
density per 0.01 mm2. For the measurement of nuclear
diameter, 30 nuclei from RBPMS-positive cells (or in as
many surviving cells in the image) per cropped image
were quantified using the line tool, providing an average
diameter. e mean diameter from each cropped image
was then averaged across the 6 images to obtain a final
average diameter per retina.
Luminometry‑based ATP andNAD assays
ATP or NAD content was analyzed invitro and invivo
following a similar protocol. In order to assess how
quickly PQQ could be used by cells invitro to increase
ATP and NAD content, ATP and NAD assays were run
on treated brain cortex first to make a dose–response
curve and then on retina, optic nerve and superior
colliculus exposed to a single concentration. A total
of 4 hemispheres were harvested from the whole cor-
tex of B6J mice. Each hemisphere was transferred to
800µl of dispase (5000 U; Corning) and incubated on
a ermomixer C heating block (Eppendorf) at 37°C,
350rpm for 30min before being dissociated by gen-
tle trituration. Cell concentration was calculated by
cell counting on a C-Chip hemocytometer (NanoEn-
tek) and each cell suspension was diluted to 2 million
cells/mL. Seven aliquots of each cell suspension for
each sample were incubated with different concentra-
tions of PQQ (0.1, 0.5, 1, 5, 10, 50M) for 2h at 37°C,
5% CO2. Cells maintained in HBSS for the same time
were used as controls. Samples were then homog-
enized for 15 s at 30,000 min−1 (VDI 12, VWR) and
ATP or NAD content was measured using a luminom-
etry-based assay (CellTiter-Glo® Luminescent Cell
Viability Assay for ATP, NAD/NADH Glo-™ for NAD;
Promega). Reagents of the kit were prepared accord-
ing to the manufacturer’s instructions. Equal volumes
(50l) of sample (reaching a concentration of 100,000
cells/well) and working reagent were combined in a
96-well plate (Nunc™ F96 MicroWell™ White Polysty-
rene plate, ermo Fisher Scientific). Luminescence
was measured using a Tecan Infinite 200 at approxi-
mately 10min for ATP and 1h for NAD from initial
mixing according to the manufacturer’s instructions.
To exclude any possible interaction between PQQ and
ATP working reagent, the assay was run on PQQ solu-
tions diluted in HBSS without cell lysates as a positive
control, demonstrating no change in luminescence with
increasing concentrations of PQQ (Additional file 2:
Fig.1). To measure the invitro ATP content on treated
retina, optic nerve and superior colliculus, 5 B6J mice
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
were euthanized by cervical dislocation, whole eyes
enucleated, and retinas dissected in HBSS. e brain
was removed, and the superior colliculus was isolated.
Optic nerves were cut at 3mm from the end proximal
to the eye, left and right segments were collected in two
different samples and stored at 4°C in HBSS until fur-
ther processing. Retinas from left and right eyes were
pooled to form a single sample. Retinas and superior
colliculi were dissociated in 500 and 800µl of dispase
respectively and processed as described above. Cells
from dissociated retinas and superior colliculi, as well
as isolated optic nerve segments, were incubated with
50M PQQ for the same time and in the same condi-
tions as above. e bioluminescent assay was then con-
ducted as previously described. e assessment of ATP
and NAD content invivo was performed on B6J mice
(n = 36) treated short-term with an injection of either
vehicle or 20mg/kg PQQ. A cohort of animals (n = 12)
was administered with either water or PQQ dissolved
in drinking water at the same dose. Animals were euth-
anized at 24, 48, 72h after the injection and 24h after
the water administration. Brains, retinas, optic nerves,
and superior colliculi were collected and processed as
described above. A direct homogenization after the cell
dilution was performed and the luminescent assay was
run with the same procedure previously indicated.
Trypan Blue assay
Cell viability was assessed by Trypan Blue staining. Cells
from brain cortex were obtained and diluted as above.
Samples were incubated with 50M PQQ for 2h, stained
with 0.4% Trypan Blue dye (ermo Fisher Scientific)
and counted on a hemocytometer (NanoEntek).
JC‑1 staining
To measure the short-term effects of PQQ on mitochon-
drial membrane potential (∆Ψ), JC-1 staining on treated
brain cortical cells (dose–response), or retina, optic nerve
and superior colliculus (single dose) was performed
according to previous protocols with some variations
[24]. Briefly, a total of 4 hemispheres from whole cor-
texes of B6J mice were harvested and brain cortical cells
were obtained as described above. Cell suspensions were
diluted at a concentration of 1 million cells/mL, then ali-
quots from each sample were incubated with solutions of
PQQ at different concentrations (0.5, 5, 50M) for 2h.
At the last 30min of incubation, JC-1 (ermo Fisher
Scientific, dissolved in DMSO) was added at a final con-
centration of 2M and cells were kept in the dye until the
end of PQQ incubation. Cells were washed from the dye,
resuspended in HBSS and 50l of sample was loaded on a
96-well plate (Nunc™ F96 MicroWell™ White Polystyrene
plate, ermo Fisher Scientific) in an alternate manner to
avoid interference between adjacent wells. Fluorescence
at 535nm (green; monomer) and 590nm (red; aggregate)
was measured using a Tecan Infinite 200 and Ψ levels
were expressed as the ratio of red to green fluorescence.
To measure ∆Ψ levels in treated retina, optic nerve and
superior colliculus, B6J mice (n = 8 for retinas, 5 for the
other tissues) were euthanized and the tissues collected
as described in the previous section. Cells from retinas
and superior colliculi were diluted at a concentration of
1 million cells/mL and incubated with 50M PQQ for
2h. JC-1 was administered at the same concentration and
times used for cortical cells. Optic nerves were incubated
with PQQ for 1h, homogenized in PQQ at 8000 min−1
speed (VDI 12, VWR) and incubated with 2M JC-1 for
30min at 37°C for a total of 2h of incubation in PQQ.
Fluorescence was measured as previously described.
Mitochondrial isolation andindividual mitochondrial
complex activity assays
A total of 6 hemispheres from whole brain cortexes of
B6J mice were isolated and dissociated as previously
detailed. Cells were then diluted at a concentration of
4 million cells/mL and incubated with 50M PQQ for
2h. ereafter, mitochondria were extracted using the
ermo Scientific™ Mitochondria Isolation Kit for Cul-
tured Cells (ermo Fisher Scientific) according to the
manufacturer’s instructions. Isolated mitochondria
were then suspended in 80 L of mitochondrial isola-
tion buffer (220mM mannitol, 70mM sucrose, 10mM
Tris–HCL, 1mM EDTA, pH = 7.2) and protein content
quantified by Pierce™ Detergent Compatible Bradford
Assay Kit (ermo Fisher Scientific). Samples were then
sonicated with 3 pulses of 3s each at ~ 4 W, aliquoted and
kept at − 80°C until further processing. Assays to evalu-
ate the activity of individual mitochondrial complexes
were performed according to the protocols described in
the supplementary materials from [25], with some adjust-
ments for plates and plate reader. All the assays were
performed in a 96 well plate (Sarstedt) and the readings
runusing a Tecan Infinite 200. Briefly, for the Complex
(C) II assay, 20–80 g/mL of mitochondrial proteins
were incubated in 20mM succinate, 2g/mL rotenone,
2g/mL antimycin A, 2mM KCN, 50 M DCPIP (all
Sigma-Aldrich) in potassium buffer for 10min and then
the reduction of DCPIP was measured after the addition
of 10mM DB (Sigma-Aldrich) at 595nm for 3min. To
assess the activity of CIII, 20 g/mL of mitochondrial
proteins were incubated in 50M decylubiquinol, 2mM
KCN, 50M cytochrome C (Cyt C) (all Sigma-Aldrich)
in sucrose/Tris buffer and the reduction of Cyt C was
monitored at 550nm for 3min. To account for CIII-inde-
pendent Cyt C reduction, samples were run both without
or with 2g/mL antimycin A. Regarding the CIV assay,
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
10 g/mL of mitochondrial proteins were mixed with
20M ferrocytochrome c (FeCyt C) in degassed potas-
sium buffer, and the oxidation of FeCyt C was measured
at 550nm for 3min. For the citrate synthase (CS) assay,
20g/mL of mitochondrial proteins were incubated with
100M DTNB, 300M acetyl-CoA and 500M oxaloac-
etate (Sigma-Aldrich) in Tris buffer, and the reduction
of DTNB was checked at 415nm for 3min. e enzyme
activity was calculated by the formula: enzyme activ-
ity (nmol min−1 mg−1) = (Absorbance/min × 1000)/
[(extinction coefficient × volume of sample used in
mL) × (sample protein concentration in mg mL−1)] [26].
e Absorbance/min was derived from the slope of the
linear phase of the reaction. e specific activity of CIII
was calculated by subtracting the activity without anti-
mycin A minus the one in presence of the inhibitor. e
activities of CII, III and IV were normalized to the CS.
Quantitative real‑time PCR
B6J treated with either vehicle or 20 mg/kg PQQ i.p.
either short-term (n = 7 mice for 24h, 8 for the other
time points) or long-term (n = 16 mice) were euthanized
by cervical dislocation. For the short-term treatment,
retinas were collected 24, 48 and 72 h after the injec-
tion, whereas for the long-term administration retinas
and optic nerves were harvested 15days after the start-
ing point. Eyes were enucleated and retinas dissected
in ice-cold HBSS. A second operator isolated the optic
nerves from the brain and cut around the optic chiasm.
A single retina comprised of a sample, whereas left and
right optic nerve segments were pooled together to make
a unique sample. All the samples were then snap frozen
in dry ice and stored at − 80°C until further processing.
A total RNA extraction was performed homogenizing
retinas and optic nerves in 350 µl buffer RLT (Qiagen)
with 1% β-mercaptoethanol (Fisher Scientific) and using
a QIAshredder kit (Qiagen) according to the manufactur-
er’s instructions. A column-based kit (RNeasy Mini Kits,
Qiagen) was used to extract RNA according to the manu-
facturer’s instructions. Isolated RNA was suspended in
nuclease-free water and RNA concentration was quanti-
fied using a NanoDrop™ One (ermo Fisher Scientific).
cDNA was generated starting from 1 g of input RNA
through an iScript™ cDNA Synthesis Kit and a MyIQ
thermocycler (both Bio-Rad) and stored at − 20 °C.
Quantitative real-time PCR was performed at the CFX96
Touch Real-Time PCR Detection System thermocycler
(Bio-Rad) using SsoAdvanced Universal SYBR Green
Supermix (Bio-Rad), cDNA (15 ng for retina; 3 ng for
optic nerve) and the appropriate DNA templates (Prime
PCR Assay, mus musculus, Bio-Rad): mt-Co2, Rsp18,
Pgc-1α, Tfam, Ndufb8, Sdhb, Uqcrc2, mt-Co1, Atp5a1.
e protocol used comprised of a 3min activation and
denaturation step at 95°C, followed by an amplification
stage composed by 50 cycles of a 15 s denaturation at
95°C and 1min annealing, and plate read at 60°C. Data
was exported and opened in the software CFX man-
ager (Bio-Rad) to visualize the amplification and melt-
ing curves. e expression levels were calculated by the
Ct method. e mtRNA/nuRNA ratio was calculated
using mt-Co2 and Rsp18 as the reference mitochondrial
and nuclear gene, respectively, as described in [27]. Rsp18
was used as housekeeping gene when the expression of
the other genes was calculated.
Western blot
B6J mice (n = 16) treated with intraperitoneal injec-
tions of either vehicle or 20mg/kg PQQ long-term were
euthanized by cervical dislocation after 15 days from
the starting point. Eyes were enucleated and retinas dis-
sected in ice-cold HBSS. A second operator isolated the
optic nerves from the brain and segments were obtained
by cutting right before the optic chiasm. Left and right
retinas or optic nerves from the same animal were pooled
to make a single sample. All the samples were then snap
frozen in dry ice and stored at − 80°C until further pro-
cessing. Samples were lysed in RIPA buffer (Santa Cruz
Biotechnology) supplemented with phosphatase and pro-
teinase inhibitor cocktails (Roche Applied Science). Pro-
tein content was measured by Micro BCA protein assay
(ermo Fisher Scientific). Equal micrograms of proteins
per sample (10µg for retinas; 20µg for optic nerves) were
separated by SDS-PAGE (4–20%; Bio-Rad) and gels were
transblotted onto nitrocellulose membranes (Bio-Rad).
Membranes were blocked with either 4% BSA (Sigma-
Aldrich) or 5% skim milk in TBS/Tween for 1h at room
temperature and incubated overnight at 4°C with mouse
monoclonal total OXPHOS rodent WB antibody cock-
tail (ab110413, Abcam, 1:250, recognizing 5 different
targets: NDUFB8, SDHB, UQCRC2, mt-CO1, ATP5a) or
mouse monoclonal anti-β-actin antibody (A2228, Sigma-
Aldrich, 1:2500). Afterward, membranes were incu-
bated for 2h at room temperature with the solution of
HRP-conjugated rabbit polyclonal anti-mouse antibody
(A9044, Sigma-Aldrich, 1:5000). Rat heart mitochondrial
extract provided by the manufacturer (ab110341, Abcam)
was diluted at 1:200 and used as a positive control. Clar-
ity western chemiluminescence substrate (Bio-Rad) was
used to develop blots, and images were acquired by the
ChemiDoc™ Imaging System (Bio-Rad). Protein levels of
the target bands were expressed by normalizing the opti-
cal density (OD) of the target calculated by Image Lab 6.0
software (Bio-Rad) to the corresponding OD of β-actin as
loading control.
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
Mitochondrial morphological analyses
To image and analyze mitochondrial morphology, reti-
nal and optic nerve cryo-sections were cut from MitoV
mice (n = 8) treated for 2weeks invivo with either vehi-
cle or 20mg/kg PQQ i.p. e MitoV mouse strain is on a
B6J background and expresses YFP under a rat neuron-
specific Eno2 promoter. YFP is localized to mitochondria
through a Cox8a gene-targeting signal fused to the YFP
N-terminus. is line was selected due to its specific-
ity in inner retinal expression (defined MitoV for visual
tissue) [7]. e strain has been further characterized in
previous reports [7]. Mice were euthanized after 15days
from the first injection and eyes enucleated. e brain
was isolated with the attached optic nerves. Tissues were
fixed in 3.7% PFA for 24h and cryo-protected by immer-
sion in 30% sucrose solution. Eyes were directly frozen
in optimal cutting temperature medium (Sakura) on dry
ice, whereas optic nerves were separated from the brain
and then included in the same medium. Blocks were
maintained at − 80 °C until use. Eyes and optic nerves
were cryo-sectioned in 20m-thick coronal and longi-
tudinal sections respectively using a cryostat (Cryostar
NX70, ermo Scientific). All the sections were stored
at − 20°C until further processing. Cryo-sections were
then air dried for 15min and rehydrated in 1M PBS for
5min before following the protocol. Tissues were isolated
using a hydrophobic barrier pen (VWR), permeabilized
with 0.1% Triton X-100 (VWR) in 1M PBS for 1h and
blocked in 2% BSA (Fisher Scientific) in 1M PBS for 1h.
Sections were then immunolabelled with chicken poly-
clonal anti-GFP (ab13970, Abcam, 1:500) and rabbit pol-
yclonal anti-TOMM20 (ab78547, Abcam, 1:500) primary
antibodies at 4°C overnight. Anti-GFP primary antibody
was used to limit loss of signal from potential bleaching
of YFP. ereafter, sections were rinsed with 5 × 5 min
washes in 1 M PBS and stained with Alexa Fluor 488
conjugated goat anti-chicken (A11039, Invitrogen, 1:500)
and Alexa Fluor 568 conjugated goat anti-rabbit (A11011,
Invitrogen, 1:500) secondary antibodies for 4h at room
temperature. Tissue was washed as before and coun-
terstained with DAPI (1g/mL in 1M PBS) for 10min.
After being rinsed once in PBS, tissue was mounted using
Fluoromount-G and glass coverslips (Invitrogen). Nail-
varnish was used to seal the slides. Images were acquired
using confocal imaging on a Zeiss LSM-980 Airy (63×,
1.5 × optical zoom, image size 89.8 × 89.8 m, 0.07 µm
pixel size, z-stacks with 0.23m optimal interval). Images
from retinal sections were acquired from central retina
at ~ 500m lateral to the optic nerve head. Images from
optic nerve sections were acquired from areas around the
optic chiasm used for reference. Alexa Fluor 488 conju-
gated and Alexa Fluor 568 conjugated secondary anti-
bodies targeting anti-GFP and anti-TOMM20 primary
antibodies respectively were imaged. DAPI channel was
also imaged for reference purposes in retinal samples.
Retinal images encompassed nerve fiber layer (NFL),
GCL and inner plexiform layer (IPL). Mitochondrial par-
ticles were reconstructed in 3D using Imaris software
(version 9.3.1). NFL/GCL and IPL were cropped for the
analyses and reconstructed separately with different set-
tings. Volume reconstructions were performed using the
surface tool and volumes under 125 voxels were filtered
and discarded from subsequent analysis to reduce noise.
Volume, surface area, sphericity (including prolate and
oblate dimensions) of each mitochondrial particle were
calculated by the software and plotted either individu-
ally or as an average per retina or optic nerve. For vol-
ume and surface area, an average of both mean and sum
of each parameter was calculated. Mean volume, volume
sum, mean surface area and surface area sum in each
retina were normalized to the number of either GFP or
TOMM20 positive cells and then to the volume crop
in NFL/GCL, or to only volume crop in IPL. e same
parameters were normalized to the volume crop in the
optic nerve images.
Metabolomics
B6J mice (n = 10 animals) treated with a single i.p. injec-
tion of either vehicle or 20mg/kg PQQ were euthanized
by cervical dislocation after 24 h from the treatment.
Eyes were enucleated and retinas were immediately dis-
sected in ice cold HBSS, wiped dry, weighed and fro-
zen on dry ice. A second investigator isolated the optic
nerves immediately after death. Optic nerves were cut at
3mm from the end proximal to the eye and frozen as for
retinas. Each sample comprised a single retina or an iso-
lated optic nerve segment. Tissue was stored at − 80 °C
and shipped kept in dry ice to the Swedish Metabolomics
Centre for sample processing. 200l of extraction buffer
(80:20 v/v MeOH:H2O) including internal standards were
added to the tubes together with 1 tungsten bead. Tissues
were shaken at 30Hz for 3min in a mixer mill and sam-
ples were then centrifuged at 4°C, 18620 g for 10min.
Afterwards, 200 l of the supernatant was transferred
to micro vials and evaporated to dryness in a speed-vac
concentrator. Samples were stored at − 80°C and small
aliquots of the remaining supernatants were pooled and
used to create quality control (QC) samples. Prior to the
analysis, samples were re-suspended in 10 + 10µl metha-
nol and elution solvent A. e samples were analyzed
in batches according to a randomized run order. Each
batch of samples was first analyzed in positive mode.
After all samples within a batch had been analyzed, the
instrument was switched to negative mode and a sec-
ond injection of each sample was performed. e chro-
matographic separation was performed on an Agilent
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
1290 Infinity UHPLC-system (Agilent Technologies,
Waldbronn, Germany). 2 l of each sample were injected
onto an Atlantis Premier BEH-Z-HILIC VanGuard FIT
(1.7µm, 2.1 × 50mm) column (Waters Corporation, Mil-
ford, MA, USA) held at 40°C. e HILIC gradient elu-
tion solvents were (A) 10mM ammonium formate, 5µM
Medronic acid in H2O, pH 9 and (B) 90:10 Acetonitrile:
[10mM ammonium formate in H2O], pH 9. Chromato-
graphic separation was achieved using a linear gradient
(flow rate of 0.4mL/ min): min 0 = 90% B, min 6 = 80% B,
min 9.5 = 20% B, min 11 = 90% B. e flow rate was then
increased to 0.7mL/min for 2min, held at this rate for
0.5min, and further reduced to 0.4mL/min for 0.5min
before the next injection. Compounds were detected with
an Agilent 6546 Q-TOF mass spectrometer equipped
with a jet stream electrospray ion source operating in
positive or negative ion mode. MSMS analysis was run on
the QC samples for identification purposes. All data pre-
processing was performed using the Agilent MassHunter
Profinder version B.10.0 SP1 (Agilent Technologies Inc.,
Santa Clara, CA, USA). Sixty-three (retina) or seventy-
three (optic nerves) low molecular weight metabolites
that could be certified with standards were detected.
e quantification of the metabolites was calculated as
area under the curve of the mass spectrometry peak and
normalized first to an internal standard for negative and
positive runs, then for the weight of the tissue for reti-
nal samples. Data were analyzed and graphs were made
using MetaboAnalyst [version 5.0; 28, 29] and R. All data
were subject to Pareto scaling [30]. Hierarchical cluster-
ing (HC) (Spearman, Average) was used to create the
dendrograms. Correlation heatmaps were created using
Spearman rank correlation. Principal component analy-
sis (PCA) was performed in R (4.1.0) using the factoextra
package. Comparisons between groups were analyzed by
two-sample t-tests with an adjusted p value (false discov-
ery rate, FDR), using a cutoff of 0.05 considered signifi-
cant. Quantitative pathway analysis was performed using
the Mus musculus KEGG library in MetaboAnalyst and a
background metabolome of all detected metabolites.
Statistical analysis
Graph Pad Prism 8.0.2 software and R were used for the
statistical analyses. A Shapiro Wilk test was used to test
the normality of the data. A Student’s t-test or one-way
ANOVA (followed by Tukey’s multiple comparison post
hoc test) were applied as appropriate to analyze normally
distributed data. A Mann–Whitney test was used to ana-
lyze non-normally distributed data. For individual mito-
chondrial particles morphology, a linear mixed effects
model through the lme4 package in R was applied when
multiple observations come from the same retina or optic
nerve, in order to reduce p value inflation and intra-class
correlation [31–33]. Differences with p < 0.05 were con-
sidered significant. For the box plots, the median is rep-
resented by the center hinge with upper and lower hinges
indicating the first and third quartiles, whereas whiskers
denote 1.5 times the interquartile range. All the graphs
were made in R or using MetaboAnalyst 5.0 for dendro-
grams and correlation heatmaps.
Results
PQQ isneuroprotective indierent models ofRGC‑related
damage
Since RGCs strictly rely on a perfectly controlled metab-
olism and PQQ has been demonstrated to regulate the
cellular bioenergetic balance [15–19], we hypothesized
that PQQ could protect RGCs under stress/injury which
compromise bioenergetic capacity. We first evaluated the
effects of PQQ in an exvivo model of RGC injury, which
reproduces an axon-specific insult by separating the ret-
ina from the optic nerve [7, 34]. Significant RGC loss was
detected after 3days exvivo (DEV), as assessed by count-
ing cells positive for RBPMS (a specific marker of RGCs).
Administration of PQQ via the culture media at both 50
and 100M provided a significant preservation of RGC
density under stress condition (Fig.1A, B). e adminis-
tration of PQQ was effective in counteracting the loss of
DAPI positive nuclei, however the nuclear shrinkage was
only partially prevented at the highest dose (Additional
file3: Fig.2A, B).
To further explore PQQ neuroprotection, we next
tested the ability of PQQ to protect RGC against Com-
plex I inhibition in a model of RGC degeneration induced
by rotenone. In this model, retinal cell death is induced
by the inhibition of mitochondrial Complex I triggered by
an intravitreal injection of rotenone, resulting in a rapid
degeneration of retinal neurons [7, 35]. A significant RGC
and retinal cell loss, as well as nuclear shrinkage, were
identified after 24h from the rotenone injection (Fig.1C,
D; Additional file3: Fig. 2C, D). While the administra-
tion of PQQ was effective in preventing RGC and retinal
cell loss, the nuclear shrinkage was persisting in retinas
of PQQ-treated mice although significantly attenuated
(Fig.1C, D; Additional file3: Fig.2C, D). ese data sup-
port a neuroprotective role of PQQ against a range of dif-
ferent stressors, such as RGC axonal damage and severe
mitochondrial dysfunction.
PQQ increases ATP content andalters mitochondrial
membrane potential incortical neurons andRGCs invitro
As PQQ has been reported to influence metabolism
and regulate the cellular bioenergetic balance in several
systems [15–19], we investigated the effect of PQQ on
neuronal ATP production. We initially assessed if PQQ
was rapidly utilized by measuring the ATP content in
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dissociated brain cortical cells. Cortical cells were
incubated with increasing concentrations of PQQ (0.1,
0.5, 1, 5, 10, 50M) for 2 h and ATP levels assessed.
A significant dose-dependent increase in ATP content
was demonstrated in cortical cells from concentra-
tions at 0.5M, reaching > 50-fold the untreated con-
trol at the highest dose tested (Fig.2A) without a loss
of viability (Additional file4: Fig.3). As NAD is a key
cofactor involved in metabolism and ATP synthesis,
we assessed if the short incubation with PQQ might
promote NAD synthesis. However, PQQ effect on
generating ATP in vitro appeared not predominantly
dependent on NAD synthesis, since NAD content was
not significantly influenced following the incubation
Fig. 1 Effects of PQQ administration on RGC survival in ex vivo and in vivo models of RGC stress. A Representative images of retinas cultured
ex vivo, immunolabeled for RNA-binding protein with multiple splicing (RBPMS, red) and counterstained with DAPI (blue). Retinal explants were
cultured in either basic or supplemented media with either 50 or 100 μM PQQ for 3 days ex vivo (DEV). Control retinas (0 DEV) were directly fixed
and processed after the dissection. B Quantification of RBPMS positive cell density per 0.01 mm2. n = 5 (0 DEV), 9 (3 DEV), 4 (3 DEV + 50 μM PQQ), 6
(3 DEV + 100 μM PQQ) retinas. C Representative images of retinas from mice injected with either DMSO or rotenone and treated with either vehicle
or 20 mg/kg PQQ. Flat mount retinas were immunolabeled for RBPMS (red) and counterstained with DAPI (blue). D Quantification of RBPMS
positive cell density per 0.01 mm2. n = 9 DMSO, 9 DMSO + PQQ, 10 rotenone and 9 rotenone + PQQ retinas. Scale bar = 20 μm. *p < 0.05, **p < 0.01
and ***p < 0.001 versus 0 DEV (explants) or DMSO (rotenone model); #p < 0.01 and ##p < 0.001 versus 3 DEV (explants) or rotenone (rotenone model)
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
with PQQ (Fig. 2B). To support these findings in an
RGC specific context wenext assessed if PQQ at 50M
(where the highest ATP increase was demonstrated)
provided a similar ATP increase in RGC-related tissues.
Dissociated retinal cells and optic nerve confirmed the
increase in ATP as seen in cortical neurons (Fig.2C).
To further investigate the mechanisms behind PQQ’s
rapid ATP increase invitro, we assessed mitochondrial
membrane potential (Ψ) in cortical cells incubated with
PQQ for 2h. e levels of Ψ dose-dependently reduced
in PQQ-incubated cells, decreasing to 47% of the control
at 50M (Fig.2D). e decline in Ψ levels was further
confirmed in dissociated retina, optic nerve, or superior
colliculus incubated at a dose of 50 M (Fig.2E), sug-
gesting that, together with a concomitant increase in
ATP levels, mitochondrial potential may be dissipated to
produce ATP [24]. Since Ψ is highly influenced by the
activity of mitochondrial complexes and enzymes from
Fig. 2 Effects of PQQ administration on ATP, NAD, mitochondrial membrane potential levels and mitochondrial function in vitro. A ATP and B
NAD content in dissociated mouse brain cortical cells incubated with different doses of PQQ (0.1, 0.5, 1, 5, 10, 50 µM) for 2 h. n = 4 different cell
suspensions from different hemispheres. C ATP content in dissociated retinal and superior colliculus cells and in isolated optic nerves incubated
with 50 µM PQQ for 2 h. The red inset shows a zoom of the graph related to the ATP content in superior colliculus. n = 5 retinal replicates made
by 2 pooled retinas, 5 optic nerve replicates made of isolated segments cut to 3 mm length, 5 superior colliculus replicates. D Mitochondrial
membrane potential (ΔΨ) levels in dissociated brain cortical cells incubated with several concentrations of PQQ (0.5, 5, 50 µM) for 2 h. n = 4 different
cell suspensions from different hemispheres. E ΔΨ levels in dissociated retinal and superior colliculus cells and in isolated optic nerves incubated
with 50 µM PQQ for 2 h. n = 8 retinal replicates made by 2 pooled retinas, 5 optic nerve replicates made of isolated segments cut to 3 mm length,
5 superior colliculus replicates. F–I Mitochondrial Complex (C) II (F), III (G), IV (H) and citrate synthase (CS; I) activity in dissociated cortical cells
incubated with 50 µM PQQ for 2 h. n = 6 different cell suspensions from different hemispheres. *p < 0.05, **p < 0.01 and ***p < 0.001 versus control
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
tricarboxylic acid (TCA) cycle (such as citrate synthase;
CS), we next assessed a direct interaction between PQQ
and each individual mitochondrial complex. Mitochon-
dria were isolated from cortical cells incubated with PQQ
and spectrophotometric assays to measure the activity
of Complexes II–IV (CII, CIII, CIV) and CS were per-
formed. No significant change in individual complexes
and citrate synthase activity was detected suggesting that
PQQ does not act directly in the mitochondrion itself
(Fig.2F–I). Taken together, these data suggest that cells
from CNS and RGC-related tissues can quickly use PQQ
and increase their ATP content.
PQQ modulates ATP andNAD content invisual system
tissues invivo
We next assessed whether the administration of PQQ
invivo leads to similar effects on metabolism. Adult B6J
mice were treated with PQQ and the levels of ATP and
NAD in visual system tissues were assessed following
a single intraperitoneal injection of 20 mg/kg PQQ. A
significant increase in ATP levels in retina, optic nerve,
and superior colliculus (a major target for RGC axons
in the brain) from PQQ-treated animals was identified
after 24 h. Higher levels of ATP in PQQ-treated mice
were also detected in retinas and superior colliculi after
48 and 72h (Fig.3A). PQQ administration significantly
increased NAD levels in the superior colliculus after 24
and 72h from the treatment, whilst having no effect on
the other tissues at all the time points analyzed (Fig.3B).
Since we confirmed an increase in ATP invivo after the
treatment with PQQ, we next assessed the efficacy of
PQQ when administered in normal drinking water. How-
ever, ATP and NAD levels were not elevated by dietary
PQQ (except for a significant increase in superior colli-
culus NAD), suggesting ineffective bioavailability of PQQ
orally (Additional file5: Fig.4).
PQQ exerts amild eect onmitochondrial content
andmorphology inRGCs
ATP production is strictly dependent on mitochon-
drial activity and content, and PQQ has been previously
reported to regulate mitochondrial biogenesis through
the activation of peroxisome proliferator-activated recep-
tor-gamma coactivator alpha (PGC-1α) and the expres-
sion of mitochondrial transcription factor A (TFAM) [18,
19]. We initially assessed this short-term transcriptional
activation of mitochondrial biogenesis in the whole ret-
ina at the same time points where ATP increases were
identified in vivo. We performed qPCR to assess the
mtRNA:nuRNA ratio, which provides an estimate of the
number of mitochondrial genome copies, an early hall-
mark of mitochondrial biogenesis and an indicator of the
number of mitochondria [27]. e expression of Pgc-1α
and Tfam were also assessed. Short-term treatment with
PQQ did not effect mtRNA:nuRNA or Pgc-1α and Tfam
mRNA levels, suggesting that a single injection of PQQ is
not adequate to induce transcriptional variations in reti-
nal mitochondrial biogenesis (Additional file6: Fig.5).
Fig. 3 Effects of PQQ administration on ATP and NAD levels in visual system tissues in vivo. A ATP and B NAD content in retina, optic nerve,
superior colliculus, and brain cortex from animals treated with either vehicle or a single injection of 20 mg/kg PQQ assessed after 24, 48 or 72 h
from the treatment. n = 6 animals per group for each time point. *p < 0.05, **p < 0.01 and ***p < 0.001 versus vehicle
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
We next questioned whether transcriptional regula-
tion of mitochondria is activated if PQQ was adminis-
tered chronically. In addition to Pgc-1α, Tfam, and the
mtRNA:nuRNA ratio, we quantified the expression of
individual mitochondrial complexes-related genes in reti-
nal samples to further assess an influence on mitochon-
drial content. mRNA levels of Ndufb8 (CI), Sdhb (CII),
Uqcrc2 (CIII), mt-Co1 (CIV), Atp5a1 (CV; ATP synthase)
were assessed. Chronic administration of PQQ had no
effect on either retinal mtRNA:nuRNA ratio or Pgc-1α
and Tfam mRNA, suggesting that PQQ is not effec-
tive in triggering mitochondrial biogenesis transcrip-
tionally short-term or long-term (Fig.4A, B). However,
PQQ-treated retinas displayed a significant increase in
Ndufb8 mRNA levels, without a change in other mito-
chondrial markers (Fig. 4C). Since whole retinas con-
tain different mixed cell populations, we next assessed if
PQQ could induce a similar response in the optic nerve,
which is an RGC enriched tissue (RGC axons). A signifi-
cant decrease in mtRNA:nuRNA ratio, as well as Ndufb8
and mt-Co1 mRNA levels, was identified in optic nerves
from PQQ-injected mice (Fig.4D–F). To further confirm
these molecular regulations, protein levels were quanti-
fied by Western blot to assess if the observed transcrip-
tional changes were strictly correlated with variations in
protein levels. A significant increase in NDUFB8 protein
levels was demonstrated in retinas from PQQ-treated
animals, suggesting that its transcriptional regulation
consequently results in its protein translation (Fig. 4G;
Additional file7: Fig.6A, C). However, no changes in the
levels of mitochondrial complexes were identified in the
optic nerve after PQQ treatment (Fig. 4H; Additional
file7: Fig.6B, D).
We next assessed whether mitochondrial morpho-
logical remodeling occurs following tissue mRNA
and metabolic changes. We first assessed the effects
of PQQ on gross mitochondrial morphology recon-
structing TOMM20-positive mitochondrial particles
in retinal GCL/NFL, IPL and in optic nerve. Mice were
administered 20mg/kg of PQQ via i.p. for 2weeks and
then euthanized and their tissue processed for high
resolution confocal microscopy. No change in total
mitochondrial morphology was observed following
PQQ administration in the retina (Fig. 5; Additional
file8: Fig.7) whereas optic nerves from PQQ-treated
mice displayed a significant decrease in individual
mitochondrial particle total surface area and changes
to sphericity (Fig.7A–I). A similar trend was identified
at an average level per optic nerve (Additional file10:
Fig.9A–E). As TOMM20 positive particles are repre-
sentative of mitochondria from multiple cell types, we
next determined whether there was an RGC-specific
response in these tissues using our recently published
mitochondrial reporter mouse which expresses YFP
under a rat neuron-specific Eno2 promoter and local-
ized to mitochondria through a Cox8a gene-targeting
signal fused to the YFP N-terminus. is strain is called
MitoV (V for visual system) and the expression of YFP is
specifically restricted to RGCs in the inner retina (with
the expression in a subset of bipolar neurons and photo-
receptors in the outer retina) [7]. A significant increase
in individual sphericity was observed in the GCL of
PQQ treated retinas, whereas no differences were
detected in all the other parameters analyzed (Fig. 6).
Taken as an average, all morphological parameters were
similar between vehicle- and PQQ- treated mice (Addi-
tional file9: Fig.8). In the optic nerve, the administra-
tion of PQQ resulted in no changed individual particles
morphology, although on average the mean volume and
the volume sum were significantly increased (Fig.7J–R;
Additional file 10: Fig. 9F–J). Taken together, all the
changes identified both at the molecular and morpho-
logical level may reflect some heterogeneity and vari-
ability across tissues and within conditions, suggesting
that the overall effect of PQQ administration results in
only a mild effect on mitochondrial content.
PQQ modies themetabolic proles innon‑diseased RGCs
Because we identified a mild effect of PQQ on mito-
chondrial content and that the ATP levels may be reg-
ulated by a crosstalk of several metabolic processes,
we set out to determine whether PQQ administration
Fig. 4 Effects of PQQ administration on molecular regulation of mitochondrial content in vivo. A mtRNA/nuRNA ratio in retinal samples
from animals treated with either vehicle or 20 mg/kg PQQ long-term, calculated using the expression of mt-Co2 and Rsp18 as mitochondrial
and nuclear reference gene, respectively. B, C Pgc-1α, Tfam (B) and Ndufb8, Sdhb, Uqcrc2, mt-Co1 and Atp5a1 (C) mRNA levels in whole retinas
from animals injected long-term with either vehicle or 20 mg/kg PQQ. Rsp18 was used as housekeeping gene. n = 15 retinas per group. D mtRNA/
nuRNA ratio in optic nerve samples from animals treated long-term with either vehicle or 20 mg/kg PQQ, calculated using the same genes
described in A as mitochondrial and nuclear reference genes. E, F Pgc-1α, Tfam (E) and Ndufb8, Sdhb, Uqcrc2, mt-Co1 and Atp5a1 (F) mRNA levels
in optic nerves from animals treated long-term with either vehicle or 20 mg/kg PQQ. Rsp18 was used as housekeeping gene. n = 8 optic nerves
per group. G, H Representative blots and densitometric analysis of NDUFB8, SDHB, UQCRC2, mt-CO1 and ATP5a protein levels in retinas (G)
and optic nerves (H) from vehicle and PQQ treated animals. Protein levels were expressed as the optical density (OD) of the target normalized
for the respective OD of β-actin used as loading control. n = 8 samples per group. *p < 0.05, **p < 0.01 and ***p < 0.001 versus vehicle
(See figure on next page.)
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
altered the metabolic profile of RGCs under basal con-
ditions. To achieve this, we performed metabolomics
across retinas and optic nerves collected from mice
treated with a single injection of PQQ after 24h from
the injection using a low molecular weight enriched
metabolomics protocol (identifying 63 low molecular
Fig. 4 (See legend on previous page.)
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
weight metabolites in the retina and 73 enriched in the
optic nerve with high accuracy and confidence; Addi-
tional file 1: Data 1). Hierarchical clustering (HC) of
both conditions and individual samples identified some
heterogeneity across and within conditions in retinal
samples, overall suggesting similarities between reti-
nas from vehicle- and PQQ- treated mice. However,
the division between samples in optic nerve was largely
distinguished (Fig.8A; Additional file11: Fig.10). Prin-
cipal component analysis (PCA) confirmed a complete
Fig. 5 Effects of PQQ administration on retinal gross mitochondrial morphology in vivo. A Representative images of retinal cross sections
from animals treated long-term with either vehicle or 20 mg/kg PQQ immunolabeled for TOMM20 (red) and counterstained with DAPI (blue). The
DAPI was used for reference to crop GCL/NFL from IPL. Dashed lines demark the boundaries between GCL/NFL, IPL and INL on retinal sections.
B–D Violin plots representing individual volume (B), surface area (C) and sphericity (D) of TOMM20-positive mitochondrial particles in GCL/NFL
of vehicle or PQQ-treated animals. n = 555 vehicle and 812 PQQ disconnected particles from 7 different retinas per group. E–G Averaged volume
sum (E), surface area sum (F) and count (G) per retina of TOMM20-positive mitochondrial particles in GCL/NFL. n = 7 retinas per group. H–J Violin
plots showing individual TOMM20-positive mitochondrial particles volume (H), surface area (I) and sphericity (J) in IPL of vehicle or PQQ-treated
animals. n = 1318 vehicle and 1361 PQQ disconnected particles from 7 different retinas per group. K–M Averaged volume sum (K), surface area
sum (L) and count (M) per retina of TOMM20-positive mitochondrial particles in IPL. n = 7 retinas per group. For individual parameters, individual
values of disconnected particles from each retina were analyzed together and a linear mixed effects model was applied to account for the multiple
observations that come from the same sample. Scale bar = 20 μm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NFL,
nerve fiber layer. The red zoom depicts the inset of data points to optimally visualize the data distribution
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
overlap between groups in retina, whilst identifying a
clear separation of samples in the optic nerve (Fig.8B).
is suggests that PQQ exerts differing effects on the
two tissues studied. Administration of PQQ resulted
in 5 changed metabolites in the retina (2 increased, 3
decreased) and 18 in the optic nerve (12 increased, 6
decreased) (Fig.8C, D; Additional file1: Data 1). Com-
parison of changed metabolites across tissues identi-
fied AMP as a commonly changed metabolite, although
demonstrating an increase in the retina and a decrease
Fig. 6 Effects of PQQ administration on RGC-specific retinal mitochondrial morphology in vivo. A Representative images of retinal cross sections
from MitoV animals treated long-term with either vehicle or 20 mg/kg PQQ immunolabeled for YFP (an antibody anti-GFP was used to limit loss
of signal from potential bleaching of YFP; the staining is indicated here as MitoV) (green) and counterstained with DAPI (blue). The DAPI was used
for reference to crop GCL/NFL from IPL. Dashed lines demark the boundaries between GCL/NFL, IPL and INL on retinal sections. (B-D) Violin
plots depicting individual volume (B), surface area (C) and sphericity (D) of MitoV-positive mitochondrial particles in GCL/NFL of animals treated
with either vehicle or PQQ. n = 176 vehicle and 268 PQQ disconnected particles in 7 different retinas per group. E–G Averaged volume sum (E),
surface area sum (F) and count (G) per retina of MitoV-positive mitochondrial particles in GCL/NFL. n = 7 retinas per group. H–J Violin plots showing
individual volume (H), surface area (I) and sphericity (J) of MitoV-positive mitochondrial particles in IPL of vehicle and PQQ-treated animals. n = 600
vehicle and 552 PQQ disconnected particles in 7 different retinas per group. K–M Averaged volume sum (K), surface area sum (L) and count (M)
per retina of MitoV-positive mitochondrial particles in IPL. n = 7 retinas per group. For individual parameters, individual values of disconnected
particles from each retina were analyzed together and a linear mixed effects model was applied to account for the multiple observations
that come from the same sample. Scale bar = 20 μm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NFL, nerve fiber layer.
The red zoom depicts the inset of data points to optimally visualize the distribution of the data. *p < 0.05 versus vehicle
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
Fig. 7 Effects of PQQ administration on general and RGC-specific mitochondrial morphology in the optic nerve in vivo. A Representative images
of optic nerve longitudinal sections from MitoV animals treated long-term with either vehicle or 20 mg/kg PQQ immunolabeled for TOMM20
(red). B–F Violin plots showing individual volume (B), surface area (C), sphericity (D), prolate (E) and oblate (F) of TOMM20-positive mitochondrial
particles in optic nerves from vehicle- or PQQ-treated animals. n = 9338 vehicle and 9293 PQQ disconnected particles in 8 different optic nerves
per group. G–I Averaged volume sum (G), surface area sum (H) and count (I) per optic nerve of TOMM20-positive particles. n = 8 optic nerves
per group. J Representative images of optic nerve longitudinal sections from MitoV animals treated long-term with either vehicle or 20 mg/kg
PQQ immunolabeled for YFP (an antibody anti-GFP was used to limit loss of signal from potential bleaching of YFP; the staining is indicated here
as MitoV) (green). K–O Violin plots representing individual volume (K), surface area (L), sphericity (M), prolate (N), oblate (O) of MitoV-positive
particles in optic nerves of vehicle and PQQ-treated animals. n = 4378 vehicle and 3947 PQQ disconnected particles in 8 different optic nerves
per group. P–R Averaged volume sum (P), surface area sum (Q) and count (R) per optic nerve of MitoV-positive particles. n = 8 optic nerves
per group. For individual parameters, individual values of disconnected particles from each optic nerve were analyzed together and a linear mixed
effects model was applied to account for the multiple observations that come from the same sample. Scale bar = 20 μm. The red zoom depicts
the inset of data points to optimally visualize the data distribution. *p < 0.05 and **p < 0.01 versus vehicle
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
in the optic nerve (Fig.8E). Pathway analysis revealed
that PQQ-induced metabolite modifications are pre-
dicted to have a minimal impact on retinal pathways,
whilst significantly affecting phenylalanine metabolism,
and arginine, phenylalanine, tyrosine, and tryptophan
biosynthesis in the optic nerve (Fig. 8F; Additional
file1: Data 1).
Taken together, our data support the potential for PQQ
as an adjuvant supplement for human retinal diseases.
As our study provides strong data for injectable routes,
but not for oral routes, further development is required
if PQQ is to be properly administered at the target doses
orally in humans.
Discussion
RGCs require strict control of ATP content due to their
high activity levels. is requires the maintenance of
functional mitochondria to provide a continuous supply
of ATP predominantly derived from OXPHOS in mito-
chondria [1, 36]. An imbalance in mitochondrial metab-
olism results in bioenergetic insufficiency, rendering
RGCs susceptible to neurodegeneration. Altered metab-
olism and mitochondrial dysfunction occur early in the
pathogenesis of many retinal diseases such as glaucoma,
ADOA, and LHON [2–8]. Improving metabolic and
mitochondrial function to increase ATP content has been
effective in counteracting RGC damage and reducing
the progression of neurodegeneration [7, 37], suggesting
the potential of ATP boosters as therapeutic compounds
against RGC-related neurodegenerative diseases.
Our data demonstrate that PQQ administration drives
an increase in ATP content invitro in cells isolated from
both cortical neurons and RGC-relevant tissues, sug-
gesting that PQQ can be rapidly utilized to enhance the
concentration of ATP. Despite similar effects in vitro,
in vivo administration of PQQ provided a differen-
tial effect on ATP generation in visual system tissues as
compared with cortex likely depending on possible dif-
ferences in drug uptake across blood-retinal barrier as
compared with blood–brain barrier [38]. In this respect,
while ATP content in the cortex remained unaltered, the
ATP-boosting effect of PQQ was consistently obtained
in visual system tissues, with a sustained elevation of
ATP over 3days after administration. e potential role
of PQQ as a potent inducer of ATP synthesis with dura-
ble effects was identified over the whole RGC-trajectory
through the visual system including retina containing
RGC soma and dendrites, optic nerve containing RGC
axons, and superior colliculus containing RGC terminals.
Noteworthy, variation in ATP content may not undoubt-
edly attributed to changes in RGC energetic balance but
rather be the result of the integration of neural, glial and
vascular cell types in each of the visual system tissues,
whose different composition might explain the variability
in response and time identified across these tissues. e
variability in ATP-boosting effect of PQQ was also dem-
onstrated by differences in its influence on NAD pool,
one of the primary factors influencing ATP generation
rate of OXPHOS. In this respect, PQQ’s effect on ATP
generation appeared unrelated with changes in NAD
pool in retina and optic nerve, while a significant incre-
ment in NAD pool was evident in superior colliculus.
Promoting mitochondrial biogenesis is a key mecha-
nism to bolster OXPHOS and ATP production by
modulating the number of functional mitochondria. In
previous studies on Hepa1-6, HepG2 and NIH/3T3 cell
lines, PQQ has been reported to bolster ATP production
by stimulating mitochondrial biogenesis through the acti-
vation of PGC-1α, a major transcriptional coactivator of
several biogenesis-related factors, among which TFAM
plays an important role in mitochondrial DNA repli-
cation and repair [18, 19, 39, 40]. However, PQQ effect
appears to be cell and system dependent, with different
outcomes depending on the cell line and the doses tested
[18, 19, 40]. In this respect, our data demonstrated that
either mitochondrial content or the expression of Pgc-1α
and Tfam were not altered in visual system tissues fol-
lowing short- or long-term administration of PQQ, thus
suggesting that the ATP-boosting effect of PQQ is likely
independent of mitochondrial biogenesis mechanisms.
On the other hand, the short-term ATP-boosting activ-
ity of PQQ was associated with a significant variation
in mitochondrial membrane potential, apparently unre-
lated to a direct effect on OXPHOS complexes activity.
Fig. 8 Effects of PQQ administration on metabolic profile of non-diseased RGC- related tissues in vivo. A, B Dendrograms (A) and principal
component analysis (PCA; B) of retinal and optic nerve samples collected from mice treated with either vehicle or a single injection of 20 mg/
kg PQQ after 24 h. C, D Bar chart (C) and volcano plots (D) indicating respectively the number and the increase/decrease of significantly changed
metabolites in retinas and optic nerves from PQQ-treated animals compared to their vehicle-injected controls (FDR < 0.05; red = increased in PQQ,
blue = decreased). E Euler plot and heatmaps showing commonly changed metabolites across tissues. The red and the blue in the heatmaps
indicates respectively the highest and the lowest value by row. F KEGG pathway analysis indicating the predicted affected pathways based
on the detected changed metabolites in retinas and optic nerves from PQQ-treated mice. Pathways were highlighted in red when FDR < 0.05
and annotated when the impact was high. The size of the points indicates the pathways hits, underlining the number of the metabolites detected
within the pathway. n = 10 retinas or optic nerves per group
(See figure on next page.)
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
Fig. 8 (See legend on previous page.)
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
e long-term administration of PQQ resulted in a mild
but significant influence on transcription and translation
of mitochondrial complex-related markers together with
a subtle variation in gross mitochondrial morphology.
Taken together, these results suggest that the ATP-boost-
ing activity of PQQ only partially consists in a direct
effect on mitochondrial content and function, at least in
healthy conditions.
To identify alternative metabolic mechanisms involved
in ATP-boosting effects of PQQ, we performed low
molecular weight metabolomics in retina and optic
nerve. We demonstrated a clear distinction between
untreated and PQQ-treated optic nerve, suggesting a
PQQ-mediated change in metabolic profile. Pathway
analysis predicted impact on phenylalanine metabo-
lism, and arginine, phenylalanine, tyrosine, and trypto-
phan biosynthesis. Changes in such pathways have also
emerged in the metabolome profile of retina and optic
nerve after the administration of nicotinamide, another
relevant ATP-boosting compound providing strong
neuroprotection in the retina via a metabolic mecha-
nism [7]. Most of the metabolites displaying a significant
increment following PQQ administration are strictly
involved in anaplerotic mechanisms providing substrates
to improve local ATP generation by fueling glycolysis or
TCA cycle and OXPHOS [41]. In particular, the PQQ-
dependent increase of L-arginine and fumarate can be
derived from the metabolization of argininosuccinate in
a reaction catalyzed by the argininosuccinate lyase [42].
Fumarate supports the activity of the TCA cycle and
could contribute to the increased PQQ-mediated ATP
content. Fumarate and its derivatives have also been
demonstrated to exert antioxidant effects by regulating
the activation of antioxidant pathways and resulting in
enhanced cytoprotective cellular resistance to free radi-
cals [43, 44]. Similarly, -arginine may act as precursor of
several endogenous polyamines reported to be neuropro-
tective in neurological and retinal diseases through the
regulation of antioxidant, anti-inflammatory and anti-
apoptotic mechanisms [45–49]. e antioxidant effects
of both metabolites may further influence ATP produc-
tion. In effect, end products of oxidative stress-induced
lipid peroxidation have been reported to alter ATP syn-
thase subunits resulting in impaired enzyme activity with
reduced conversion of ADP to ATP and energy deple-
tion [50, 51]. Reducing oxidative stress may therefore
indirectly buffer the depletion of ATP under stress, sug-
gesting an additional role of these metabolites in regu-
lating the ATP pool but may not explain an increase in
ATP seen in un-stressed systems. Low molecular weight
metabolomics also revealed a PQQ-driven increment in
-leucine, a branched-chain amino acid which has mul-
tiple functions in the brain involving the metabolism of
key neurotransmitters (glutamate), protein synthesis, and
energy production [52]. -leucine has been reported to
induce ATP synthesis alone or in combination with other
branched-chain amino acids by improving glycolysis and
protecting RGCs from degeneration in a model of glau-
coma, suggesting a potential metabolic substrate involved
in the bioenergetic support of RGCs [53]. In addition
to a, likely direct, effect in regulating ATP levels, the
metabolism of -leucine might provide carbon skeletons
to increase energy substrates fueling TCA cycle, such as
acetyl-CoA, and ketone bodies (acetoacetyl-CoA) syn-
thesis [54]. Ketone bodies in turn may be used by astro-
cytes, neurons, and oligodendrocytes to further obtain
acetyl-CoA under conditions of metabolic stress and
glucose deprivation, resulting in improved metabolism,
ATP production and RGC protection over stress [55, 56].
Supporting this, a ketogenic diet has been demonstrated
to be neuroprotective in an experimental mouse model
of glaucoma, with improved RGC survival, ameliorated
axonal transport, and reduced gliosis [56]. Glial cells have
the capacity to metabolize branched-chain amino acids,
and neurons may use these metabolites either in vitro
or in vivo under severe mitochondrial dysfunction to
replenish TCA pool intermediates, suggesting a likely
alternative way to promote metabolism and regulate the
local ATP pool [55, 57, 58].
During RGC injury, intracellular ATP seems to ini-
tially increase as an early adaptation to sustain the high
energy demand generated by the insult, declining gradu-
ally over time as the damage persists [59]. RGCs rapidly
use ATP to sustain the high cellular activity and axonal
transport of proteins required for remodeling and main-
tenance of cell cytoskeleton under stress, hence requiring
a quick ATP turnover to constantly have the necessary
metabolic substrate for their functioning [60]. us, if
ATP synthesis is inadequate in maintaining constant
optimal ATP levels under stress, RGC consume most
of the ATP pool leading to metabolic exhaustion over
time. For this reason, energy-boosting strategies provid-
ing RGCs more ATP might be neuroprotective in the
context of disease. Supplementation of exogenous ATP
in models of RGC injury gave different results under
different type of stress. Supporting this, a single supple-
mentation of ATP at either high or low dose in a model
of optic nerve crush failed to protect RGCs from death
[59]. However, multiple administration of ATP encapsu-
lated in liposomes has been effective in protecting RGC
from degeneration in a model of retinal ischemia/rep-
erfusion [61]. e discrepancy in the outcomes might
depend on the administration route, the duration of the
injury, and the poor stability of ATP which might require
multiple supplementations to become effective, raising
the question if increasing ATP directly by its exogenous
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
supplementation is an effective strategy to counteract
RGC degeneration. Given its ability to increase local
ATP content, PQQ might be a promising compound
supplying RGCs with substrates counteracting stress-
deriving bioenergetic insufficiency and the resultant
neurodegeneration.
We tested the neuroprotective efficacy of PQQ in dif-
ferent models of RGC injury where bioenergetic capacity
has been compromised. We initially assessed PQQ neu-
roprotection using an exvivo model of retinal axotomy
which results in Wallerian degeneration and loss of ATP
with significant RGC degeneration 3days post-axotomy
[7, 34, 62, 63]. Our data demonstrated that the admin-
istration of PQQ confers RGC neuroprotection in this
model, with an effective reduction of RGC cell loss and
stress-related features such as nuclear shrinkage. e
moderate PQQ neuroprotection reflects the complexity
of factors regulating RGC degeneration in this context
and might suggest that PQQ protection may act only
on some of the neurodegenerative mechanisms in this
model (e.g. supplementing ATP but not addressing neu-
roinflammation or caspase activity) [7, 34, 64]. Consider-
ing the ATP-boosting capacity of PQQ demonstrated in
healthy conditions, a possible mechanism of PQQ neu-
roprotection may be ascribed, at least in part, to a likely
counteraction of bioenergetic insufficiency through an
increased ATP reservoir. To isolate and further investi-
gate the potential contribution of PQQ in reducing RGC
stress by regulating neuronal bioenergetic balance, we
tested PQQ neuroprotection in a model where impaired
bioenergetic capacity represents the principal insult driv-
ing RGC death. To test this we used an invivo model of
bioenergetic injury initiated by the inhibition of mito-
chondrial Complex I bioenergetic injury initiated by the
inhibition of mitochondrial Complex I following intra-
vitreal injection of rotenone [35, 65]. Rotenone injec-
tion induces acute mitochondrial damage, resulting in
ATP depletion and oxidative stress which in turn cause
RGC degeneration. is models a typical feature of RGC-
specific retinal diseases such as glaucoma and genetic
optic atrophies characterized by genetic mutations in
mitochondrial-related genes as in the case of ADOA and
LHON [2, 5, 6, 8]. e preservation of RGC-density and
nuclear diameter resulting from PQQ administration
supports the strong neuroprotective efficacy of PQQ.
Since in this model RGC viability is strongly related to
altered ATP reservoirs and PQQ has an ATP-boosting
activity, the neuroprotective effects of PQQ are likely due
to an amelioration of cell bioenergetic capacity by sup-
porting ATP levels.
Conclusions
Taken in concert, our data demonstrate that PQQ is
neuroprotective in different models of RGC stress.
PQQ administration increases local ATP content and
alters metabolic profiles in non-diseased visual sys-
tem tissues. e prominent neuroprotective efficacy of
PQQ against RGC damage under a variety of stressors
is possibly related to its ATP boosting activity, although
a clear correlation would need further investigations.
e present findings support a potential role of PQQ
as a novel neuroprotective compound used as adjuvant
with other current therapies to improve RGC resilience
with a low risk of side effects. Although our data dem-
onstrated a negligible effect of short-term oral delivery,
the dietary supplementation with PQQ still remains
a valid option given the possibility to improve its bio-
availability by favoring the mobility across body bar-
riers (e.g. absorption in the gastrointestinal tract and
blood–brain barrier permeability) and by designing
slow-release formulations. Further studies are needed
to identify the optimal dose and delivery system in
humans to achieve similar effects in promoting ATP
content in health and disease before PQQ could be
considered ready for clinical use.
Abbreviations
ADOA Autosomal dominant optic atrophy
ADP Adenosine diphosphate
AMP Adenosine monophosphate
ANOVA Analysis of variance
ATP Adenosine triphosphate
ATP5a1 ATP synthase F1 subunit alpha
BSA Bovine serum albumin
CNS Central nervous system
CS Citrate synthase
Cyt C Cytochrome C
DAPI 4′,6-Diamidino-2-phenylindole
DB Decylubiquinone
DCPIP 2,6-Dichlorophenolindophenol
DEV Days ex vivo
DMSO Dimethyl sulfoxide
DTNB 5,5′-Dithiobis (2-nitrobenzoic acid)
FDR False discovery rate
GCL Ganglion cell layer
GFP Green fluorescent protein
HBSS Hank ’s balanced salt solution
HC Hierarchical clustering
IPL Inner plexiform layer
KEGG Kyoto Encyclopedia of Genes and Genomes
LD50 Lethal dose 50
LHON Leber hereditary optic neuropathy
mt-CO1 Mitochondrially Encoded Cytochrome C Oxidase I
NAD Nicotinamide adenine dinucleotide
NADH Nicotinamide adenine dinucleotide + hydrogen (reduced)
NDUFB8 NADH:Ubiquinone Oxidoreductase Subunit B8
NFL Nerve fiber layer
OD Optical density
OXPHOS Oxidative phosphorylation
PBS Phosphate-buffered saline
PCA Principal component analysis
PFA Paraformaldehyde
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Canovaietal. Acta Neuropathologica Communications (2023) 11:146
PGC-1α Proliferator-activated receptor-gamma coactivator alpha
PQQ Pyrroloquinoline quinone
RBPMS RNA-binding protein with multiple splicing
RGC Retinal ganglion cell
SDHB Succinate dehydrogenase complex iron sulfur subunit B
TBS Tris-buffered saline
TCA Tricarboxylic acid
TFAM Mitochondrial transcription factor A
TOMM20 Translocase of outer mitochondrial membrane 20
UQCRC2 Ubiquinol-cytochrome c reductase core protein 2
YFP Yellow fluorescent protein
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s40478- 023- 01642-6.
Additional le1: Dataset 1. Raw mass spectrometry data (area under
the curve), weight normalized results, comparisons, and pathway analysis.
Additional le2: Figure1. Assessment of PQQ interference on ATP assay
in vitro. Luminescence of control samples with PQQ diluted in HBSS (con-
trol vehicle) at different concentrations (0.1, 0.5, 1, 5, 10, 50 µM) without
cell lysates. n = 3 different replicates per group.
Additional le3: Figure2. Supplementary analysis of retinal cell survival
in ex vivo and in vivo models of RGC stress. (A, B) Quantification of DAPI
positive cell density per 0.01 mm2 (A) and mean DAPI nuclear diameter (B)
in GCL of retinas cultured ex vivo. Retinal explants were cultured in either
basic or supplemented media with either 50 or 100 μM PQQ for 3 days
ex vivo (DEV). Control retinas (0 DEV ) were directly fixed and processed
after the dissection. n = 5 (0 DEV), 7 (3 DEV ), 4 (3 DEV + 50 μM PQQ), 6
(3 DEV + 100 μM PQQ) retinas. (C, D) Quantification of DAPI positive cell
density per 0.01 mm2 (C) and mean DAPI nuclear diameter (D) in GCL of
retinas from animals injected either with DMSO (control) or rotenone and
treated with vehicle or 20 mg/kg i.p. PQQ. n = 9 DMSO, 9 DMSO + PQQ, 10
rotenone and 9 rotenone + PQQ retinas. GCL, ganglion cell layer. *p < 0.05,
**p < 0.01 and ***p < 0.001 versus 0 DEV (explants) or DMSO (rotenone
model); #p < 0.01 and ##p < 0.001 versus 3 DEV (explants) or rotenone
(rotenone model).
Additional le4: Figure3. Effects of PQQ administration on cell viability
in vitro. Evaluation of PQQ cell toxicity in dissociated mouse brain cortical
cells incubated with 50 μM PQQ for 2 h. Cells maintained in HBSS for
the same time were used as controls. Toxicity was assessed by Trypan
blue assay and quantified as the number of cells/mL. n = 3 different cell
suspensions from different hemispheres.
Additional le5: Figure4. Effects of PQQ administration by drinking
water on ATP and NAD levels in visual system tissues in vivo. (A) ATP and
(B) NAD content in retina, optic nerve, superior colliculus and brain cortex
measured from mice treated with either vehicle or 20 mg/kg PQQ diluted
in drinking water after 24 h. n = 6 animals per group. *p < 0.05 versus
vehicle.
Additional le6: Figure5. Effects of PQQ administration on short term
transcriptional activation of mitochondrial biogenesis in vivo. (A) mtRNA/
nuRNA ratio in whole retinal samples from animals treated with a single
i.p. injection of either vehicle or 20 mg/kg PQQ, calculated using the
expression of mt-Co2 and Rsp18 as mitochondrial and nuclear reference
gene, respectively. mtRNA/nuRNA ratio was measured 24, 48 or 72 h
after the treatment. (B) Pgc-1α and Tfam mRNA levels measured in whole
retinas from animals injected with either vehicle or 20 mg/kg PQQ after
24, 48 or 72 h. Rsp18 was used as housekeeping gene. n = 6 vehicle and 7
PQQ retinas for 24 h, 8 vehicle and 7 PQQ retinas for 48 h, 8 vehicle and 7
PQQ retinas for 72 h.
Additional le7: Figure6. Full quantified and uncropped representative
blots of Western Blot data. (A, B) Full blots of markers of mitochondrial
complexes (ATP5a, UQCRC2, mt-CO1, SDHB, NDUFB8; blots on the left) in
either retinas (A) or optic nerves (B) from vehicle-or PQQ-treated animals.
β-actin was used as loading control after membrane stripping and
reprobing (blots on the right). The optical density (OD) of each marker was
normalized for the relative OD of the β-actin to provide the quantification
reported in Fig. 3G, H. Since total OXPHOS rodent WB antibody cocktail
(ab110413, Abcam) used to detect bands recognizes 5 markers contem-
porarily, two different exposures were performed to obtain the optimal
visualization of bands (top = lower exposure; bottom = higher exposure).
Black arrows indicate which marker was quantified on each membrane
(top = ATP5a, UQCRC2 and SDHB; bottom = mt-CO1 and NDUFB8). Rat
heart mitochondrial extract provided by the manufacturer (ab110341,
Abcam) was diluted at 1:200 and run as positive control (PC). (C, D)
Uncropped membranes of the representative blots shown in Fig. 3G, H.
Additional le8: Figure7. Supplementary analysis of retinal gross mito-
chondrial morphology after PQQ administration in vivo. Individual (violin
plots) and averaged (box plots) analysis of reconstructed TOMM20-postive
particles in GCL/NFL (A-G) and IPL (H-N) in retinas of mice after long term
treatment either with vehicle or PQQ. For individual parameters, a linear
mixed effects model was applied to account for the multiple observations
that come from the same retina. Individual parameters in GCL/NFL (555
vehicle and 812 PQQ) or in IPL (1318 vehicle and 1361 PQQ) were meas-
ured on disconnected TOMM20-positive particles from 7 different retinas
per group. n = 7 retinas per group in the averaged graphs. GCL, ganglion
cell layer. IPL, inner plexiform layer. NFL, nerve fiber layer.
Additional le9: Figure8. Supplementary analysis of RGC-specific
retinal mitochondrial morphology after PQQ administration in vivo.
Individual (violin plots) and averaged (box plots) analysis of reconstructed
MitoV-positive particles in GCL/NFL (A-G) and IPL (H-N) in retinas of MitoV
mice after long term treatment either with vehicle or 20 mg/kg PQQ.
For individual parameters, a linear mixed effects model was applied to
account for the multiple observations that come from the same retina.
Individual parameters in GCL/NFL (176 vehicle and 268 PQQ) or in IPL (600
vehicle and 552 PQQ) were measured on disconnected MitoV-positive
particles from 7 different retinas per group. n = 7 retinas per group in the
averaged graphs. GCL, ganglion cell layer. IPL, inner plexiform layer. NFL,
nerve fiber layer.
Additional le10: Figure9. Supplementary analysis of general and
RGC-specific mitochondrial morphology in optic nerve in vivo. Averaged
morphological parameters per sample in TOMM20- (A-E) or MitoV-positive
(F-J) particles in optic nerves from MitoV mice treated long term with
either vehicle or 20 mg/kg PQQ. n = 8 optic nerves per group. *p < 0.05
and **p < 0.01 versus vehicle.
Additional le11: Figure10. Hierarchical clustering of retinal and optic
nerve individual samples based on the metabolic profiles derived from
the low molecular weight metabolomics in vivo. Correlation heatmaps
representing the hierarchical clustering of individual retinal and optic
nerve samples collected from animals treated with a single injection of
either vehicle or 20 mg/kg PQQ after 24 h. Heatmaps were created using
the Spearman rank correlation on Metaboanalyst 5.0 platform (red = high-
est correlation, blue = lowest correlation). n = 10 retinas or optic nerves
per group.
Acknowledgements
The authors would like to thank St. Erik Eye Hospital for financial support for
research space and facilities, the staff at the Division of Eye and Vision’s animal
facility for their assistance in animal breeding and husbandry, and the Swedish
Metabolomics Centre for metabolomics data generation.
Author contributions
AC—designed and performed experiments, analyzed data, wrote the manu-
script; JRT—designed experiments, analyzed data, wrote the manuscript;
MJ—performed experiments; DYW—performed experiments; RA—designed
experiments, wrote the manuscript; IAT—designed experiments, wrote the
manuscript; MDM—designed experiments, wrote the manuscript; PAW—con-
ceived and designed experiments, analyzed data, wrote the manuscript. All
authors read and approved the final manuscript.
Funding
Open access funding provided by Karolinska Institute. PAW is supported by
Karolinska Institutet in the form of a Board of Research Faculty Funded Career
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 21 of 23
Canovaietal. Acta Neuropathologica Communications (2023) 11:146
Position, by St. Erik Eye Hospital philanthropic donations, and Vetenskapsrådet
2018-02124 and 2022-00799. PAW is an Alcon Research Institute Young Inves-
tigator. IAT was supported by NHMRC grant GNT1159795. MDM is supported
by a grant from the Italian Ministry of University and Research to the Depart-
ment of Biology of the University of Pisa under the Department of Excellence
2023-2027 initiative, and by intramural funds of the University of Pisa.
Availability of data and materials
All data generated or analyzed during this study are included in this published
article [and its supplementary information files].
Declarations
Ethics approval and consent to participate
All breeding and experimental procedures were undertaken in accordance
with the Association for Research for Vision and Ophthalmology Statement
for the Use of Animals in Ophthalmic and Research. Individual study protocols
were approved by Stockholm’s Committee for Ethical Animal Research
(10389-2018).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Received: 7 July 2023 Accepted: 23 August 2023
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