Insulin Signaling Disruption and INF‑γ Upregulation Induce Aβ1−42
and Hyperphosphorylated-Tau Proteins Synthesis and Cell Death
after Paraquat Treatment of Primary Hippocampal Cells
Maria Luisa Abascal, Javier Sanjuan, Paula Moyano,* Emma Sola,* Andrea Flores, José Manuel Garcia,
Jimena Garcia, María Teresa Frejo, and Javier del Pino*

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ABSTRACT: Acute and long-term paraquat (PQ) exposure produces
hippocampal neurodegeneration and cognition decline. Although
some mechanisms involved in these effects were found, the rest are
unknown. PQ treatment, for 1 and 14 days, upregulated interferon-
gamma signaling, which reduced insulin levels and downregulated the
insulin pathway through phosphorylated-c-Jun N-terminal-kinase
upregulation, increasing glucose levels and the production of Aβ1−42
and phosphorylated-tau, by beta-site amyloid precursor protein
cleaving enzyme 1 (BACE1) overexpression and phosphorylated-
GSK3β (p-GSK3β; ser9) level reduction, respectively, which induced
primary hippocampal neuronal loss. This novel information on the PQ
mechanisms leading to hippocampal neurodegeneration could help
reveal the PQ actions that lead to cognition dysfunction.

Paraquat (PQ), a bipyridyl herbicide extensively used,
especially in developing countries, produces cognitive

decline following acute and repeated exposure, but some of
the processes that led to this effect remain unknown.1,2

Cognitive function is mediated by the coordinated action of
different brain regions, among which the hippocampus is a core
center for its development.3 Hippocampal neuronal loss, as
observed in dementias like Alzheimer’s disease (AD), induces
cognition dysfunction.3 PQ is redistributed and gathers in the
hippocampus,4 triggering neuronal damage and loss, following
single and long-term exposures, which are associated with the
cognitive decline induced by PQ.1,2,5 PQ induced hippocampal
neurodegeneration, in part, by beta-amyloid (Aβ) and
phosphorylated-tau (p-tau) proteins, glutamatergic neuro-
transmission dysfunction, oxidative stress (OS) generation,
BDNF/TrkB/P75NTR, and PGE2/EP1−4 signaling pathways
disruption, among other actions.5,6 However, other mechanisms
are involved.

PQ increases interferon-gamma (IFN-γ) blood levels,7 and
INF-γ knockout in mice reverts the PQ-induced expression of c-
Jun-N-terminal-kinase (JNK), activator of transcription-1
(STAT1), and proinflammatory cytokines as tumor-necrosis-
factor-α (TNF-α) and the neuronal loss induced in substantia
nigra.8 Long-term treatment with INF-γ in mice induces
hippocampal neurodegeneration and cognitive decline through
Janus kinases (JAK)/STAT1 pathway,9 which is induced by
TNF-α upregulation.10 Thus, PQ could mediate hippocampal
neurodegeneration through IFN-γ upregulation.

Additionally, PQ decreases and increases blood insulin and
glucose levels, respectively,11 and decreases insulin signaling
activation in 3T3-L1 adipocytes cells through activity repression
of its downstream targets phosphatidylinositol-3-kinase (PI3K)
and protein-kinase-beta (AKT) by reduction of their phosphor-
ylation, without any action on insulin receptors (IR) and insulin
receptor substrates (IRS). These initiate the pathway activation
after autophosphorylation of different tyrosine residues.12

Insulin signaling pathway disruption, reported in AD and
other neurodegenerative diseases, was associated with neuronal
loss and cognitive disorders.10 Besides, IFN-γ was reported to
decrease insulin levels13 and insulin pathway activation via JAK/
STAT1.14 TNF-α was described to induce insulin resistance
through increased phosphorylation of IRS1 serine residues,
mediated by JAK/STAT1 pathway upregulation.10 Therefore,
PQ could mediate the insulin signaling disruption by IFN-γ
signaling upregulation through JAK pathway upregulation,
which could induce the hippocampal neuronal loss described.

Otherwise, IFN-γ and insulin signaling pathways disruption
increase Aβ and p-tau proteins.10,15,16 Phosphorylated-tau and

Received: September 9, 2022

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Aβ proteins accumulation could be due to an increment in their
production through increased GSK3β activity that phosphory-
lated tau proteins and through amyloid precursor protein
(βAPP) processing to amyloid peptides by increasing the β-
secretase enzyme β-site amyloid precursor protein cleaving
enzyme 1 (BACE1) expression or through a reduction of their
clearance.10,15 IFN-γ and insulin signaling pathway disruption
induce GSK3β activity10,16 and βAPP and BACE1 over-
expression,10,16 leading to Aβ and p-tau proteins accumulation.
PQ increases BACE1 levels and γ-secretase and GSK3β
activities,2,17,18 suggesting it could increase Aβ and p-tau
production. Besides, PQ was shown to reduce Aβ and p-tau
clearance, in part, through HSP70 and TFEB downregulation
and proteasome P20S activity inhibition.19 Thus, PQ could
induce the Aβ and p-tau accumulation through the induction of
its production and a reduction of its clearance, mediated by IFN-
γ and insulin signaling pathways disruption.

Accordingly, we hypothesized that PQ could disrupt insulin
signaling pathways through IFN-γ signaling upregulation, which
increases Aβ and p-tau proteins production and reduces their
degradation, finally triggering hippocampal neuronal death. To
test this hypothesis, we treated unsilenced or TNF-α, βAPP,
JNK, IFN-γ, BACE1, GSK3β, and tau siRNA primary hippo-
campal neurons with 0.1 μM to 40 μM PQ alone or together
with 200 nM insulin (Sigma, Madrid, Spain), for 1 and 14 days.

The protocol by del Pino et al.5 was followed for cultures of
primary hippocampal neurons from rat fetuses, to prove our
hypothesis. In the culture, neurons and astrocytes were
identified and quantified using MAP2 and GFAP antibodies,
respectively, with neurons being predominant (91%) in the
cultures. PQ concentrations were chosen because they were
previously reported to cause Aβ and p-tau proteins aggregation
and apoptosis in primary culture hippocampal cells following 1
and 14 days treatment as well as cell death following
intrahippocampal injection in rats.5,6 The 10 μM PQ
concentration was chosen to test the role of the mechanisms
proposed because it was the lowest concentration that produces
neuronal death and increases Aβ and p-tau peptides after a single
treatment.5,6

The viability of primary hippocampal neurons was studied,
after 1 or 14 days of PQ treatment, using the MTT test.5

Induction of apoptotic death was determined with Caspase-Glo
3/7 luminescence assay kit (Promega, Madrid, Spain), following
the manufacturer’s directions. Following the producer’s
instructions, glucose levels were analyzed with a commercial
kit (Abcam, Madrid Spain). Commercial ELISA kits (Invi-
trogen, Madrid Spain) were performed to analyze insulin and tau
and Aβ proteins’ concentrations, according to the manufac-
turer’s protocol.

Validated primers (SA Biosciences) for mRNAs encoding
A C T B ( P P R 0 6 5 7 0 C ) , t a u ( P P R 4 2 7 5 7 D ) , I N F -γ
(PPR45050C), βAPP (PPR06788A), TNF-α (PPR06411F),
insulin (PPR42359A), GSK3β (PPR44848A), BACE1
(PPR50333A), and JNK (PPR43333A) were employed,
following the protocol of del Pino et al.,5 for gene expression
analysis. The cycle threshold (Ct) method20 was followed to
analyze QPCR data. INF-γ, TNF-α, p-IRS1 (panTyr), p-IRS1
(Ser307), p-AKT(Ser473), BACE1, GSK3β (Ser9), and p-
JNK(Ser63) proteins expressions were analyzed with commer-
cial ELISA kits (MBS031457, MBS697379, MBS1607535,
MBS1605652, MBS9501391, MBS1600225, MBS9511030,
and MBS2605744, respectively, MyBioSource, CA, USA),
following the producer’s guideline. siRNA (Qiagen, Barcelona,

Spain) homologous to rat βAPP (SI01488767), INF-γ
(SI01524250), TNF-α (SI02046583), tau (SI02876027), JNK
(SI03083185), GSK3β (SI01519406), and BACE1
(SI03082247) genes were used to transfect cells.5,6 siRNA
treatment efficiency was tested by analysis of gene expression
(Figure S1). PQ treatment of cells transfected with scrambled
siRNA showed no significant difference with PQ treatment of
wild-type cells (data not shown). Reported results represent all
(at least three experiments) replicates (in triplicate) performed
for each experimental condition (n = 9). Results are presented as
means ± standard error of the mean (SEM). ANOVA analyses
(one-way for concentration−response analysis, and two-way for
gene silencing/treatment), followed by Tukey posthoc test,
were performed to identify statistically significant differences
between treatments (p ≤ 0.05), using the GraphPad software
(GraphPad Software, Inc, San Diego, CA, USA).

PQ increased IFN-γ, TNF-α, p-JNK (Ser63), and p-IRS1
(Ser307) protein levels (Figure 1), but reduced insulin and p-

IRS1 (panTyr) protein levels (Figure 1) and insulin gene
expression (data not shown), following 1 (starting at 10 μM)
and 14 days of treatment (starting at 1 μM). The PQ effect on
TNF-α and p-JNK (Ser63) levels was partially abolished by
IFN-γ or TNF-α silencing (Figure 1), respectively, showing that
PQ upregulates IFN-γ, which increases TNF-α, and finally this
increases p-JNK (Ser63) levels. Additionally, the PQ effect on
insulin and p-IRS1 (Ser307) and p-IRS1 (panTyr) protein levels
was partially abolished after PQ treatment of JNK silenced cells,
pointing out that PQ mediates insulin signaling disruption, in
part, through IFN-γ signaling upregulation.

PQ was shown to increase IFN-γ blood levels,7 and revert the
expression of JNK, and TNF-α in substantia nigra through INF-
γ silencing,8 supporting our findings. Insulin was shown to be
expressed in the hippocampus,21 and, although the PQ effect on
brain insulin levels was not studied, PQ exposure decreases
insulin blood levels,12 supporting our results. However, PQ was

Figure 1. IFN-γ (A), TNF-α (B), p-JNK (C), insulin (D), p-IRS1
(panTyr) (E), and p-IRS1 (Ser307) (F) levels analysis. Each bar
represents mean ± SEM of all replicates of experiments from three
individual experimental conditions. ***p ≤ 0.001, significantly
different from controls. ###p ≤ 0.001 significantly different from PQ
treatment.

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shown not to alter IRS-1 tyrosine phosphorylation after PQ or
PQ+insulin treatment of primary rat hepatocytes.22 This
contradiction could be due to the different times of exposure,
concentrations, or model of cells used. IFN-γ decreases insulin
levels13 and insulin signaling pathway activation through JAK/
STAT1 pathway upregulation,14 which increases IRS1 phos-
phorylation of serine residues,10 supporting the results observed.
The Wnt pathway regulates the insulin pathway,23 and PQ
disrupts the Wnt signaling pathway.24 Thus, the PQ Wnt
pathway alteration could also mediate the insulin pathway
disruption.

PQ also decreased p-AKT (Ser473) (Figure 2A,B) and p-
GSK3β protein levels (Figure 3A,B) and increased glucose levels

(Figure 2C,D), following 1 (starting at 10 μM) and 14 days of
treatment (starting at 1 μM). Besides, PQ increased BACE1,
Aβ1−42, and p-tau protein levels (Figure 3). PQ decreases the
local insulin synthesis and induces slight insulin resistance,
leading to an increase in glucose levels, since insulin induced
glucose utilization by the cells. PQ decreases p-AKT (Ser473) in
SH-SY5Y cells,25 which supports our findings, but was shown
also to increase it in rat primary hepatocytes cells.22 In the cell
model used, concentration or exposure time may be responsible
for these differences. PQ also increases blood glucose levels
because of hypoinsulinemia,11 supporting our results. Besides,
PQ was shown to increase BACE1 protein levels in the mouse
cortex,2 supporting our findings, but to decrease p-GSK3β
(Tyr216) protein levels in rat hippocampus,17 which indicates
GSK3β activation and increased activity. These differences
could be due to differences in the time of exposure and doses
used. Finally, PQ increases, in a concentration dependent-way,
the Aβ1−42 and p-tau levels in the rat hippocampus and primary
hippocampal cells, confirming our results.1,2,6,19

PQ treatment of JNK silenced cells or of unsilenced cells with
insulin reversed partially the effects on glucose, p-AKT
(Ser473), p-GSK3β, BACE1, Aβ1−42, and p-tau proteins levels
observed after the PQ single treatment of wildtype cells (Figure
2 and 3). PQ cotreatment of JNK silenced cells with insulin
induced the higher reversion observed on the targets
commented, but it was incomplete (Figure 2 and 3).

Additionally, Aβ1−42 and p-tau increased levels were reversed
also after PQ treatment of the BACE1 silenced cells in the case of
Aβ1−42 or after PQ treatment of the GSK3β silenced cells in the
case of p-tau (Figure 3). PQ cotreatment of JNK and BACE
silenced cells with insulin or cotreatment of JNK and GSK3β
silenced cells with insulin induced the higher reversion observed
in the increment of Aβ1−42 and p-tau levels, but was still
incomplete (Figure 3). These results point out that the INF-γ
pathway downregulates the insulin signaling pathway, which
increases the production of Aβ1−42, through BACE1 over-
expression, and p-tau proteins, through GSK3β activation.

IFN-γ and insulin signaling pathways upregulation and
downregulation, respectively, induce GSK3β activity,10,16

BACE1 overexpression,10,16 and increase Aβ and p-tau proteins
levels,10,15,16 supporting these findings. However, other
mechanisms seem to be involved. In this sense, insulin signaling
pathway disruption decreases insulin degrading enzyme
expression,10 which participates in Aβ proteins clearance,10 so
this mechanism could also contribute. Besides, PQ was shown to
reduce Aβ and p-tau clearance, in part, through HSP70 and
TFEB downregulation and proteasome P20S activity inhib-
ition.19 Insulin regulates HSP70 expression,25 proteasome P20
activity,26 and mammalian target of rapamycin (mTOR)
activity,27 which downregulate TFEB.19 Therefore, PQ could
induce the Aβ and p-tau accumulation through its production
and a reduction of its clearance through IFN-γ and insulin
signaling pathways disruption mediated by the commented
mechanisms.

Finally, PQ (10 μM), following 1 and 14 days of treatment,
decreased cell viability (52.1% and 41.6%, respectively; Figure

Figure 2. Phosphorylated-AKT (Ser473) (A, B) and glucose (C, D)
levels analysis. Each bar represents mean ± SEM of all replicates of
experiments from three individual experimental conditions. The values
obtained were normalized by total protein concentrations. ***p ≤
0.001 correlated to control. ###p ≤ 0.001 correlated to PQ treatment.
&&&p ≤ 0.001 correlated to PQ treatment of JNK silenced cells. τττp ≤
0.001 compared to PQ+insulin treatment. γγγp ≤ 0.001 compared to
insulin treatment.

Figure 3. Phosphorylated-GSK3β (Ser9) (A, B), BACE1 (C, D), p-tau
(E), and Aβ1−42 (F) levels analysis. Each bar represents mean ± SEM of
all replicates of experiments from three individual experi-mental
conditions. The values obtained were normalized by total protein
concentrations. ***p ≤ 0.001 correlated to control. ###p ≤ 0.001
correlated to PQ treatment. &&&p ≤ 0.001 correlated to PQ treatment
of JNK silenced cells. τττp ≤ 0.001 compared to PQ+insulin treatment.
γγγp ≤ 0.001 compared to PQ treatment of BACE1 silenced cells.

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4A) and induced apoptotic cell death (172.2% and 191.9%,
respectively; Figure 4B), as previously reported.5,6,19 PQ

treatment of primary hippocampal neurons single silenced
against βAPP, JNK, or tau or cotreatment with insulin of
wildtype cells reverse partially the hippocampal neuronal loss
produced (Figure 4). PQ cotreatment of simultaneous βAPP,
JNK, and tau silenced cells with insulin produced the higher
reversion of the cell death observed after PQ treatment alone,
but was not complete (Figure 4). PQ induces primary
hippocampal neuron cell death through an increase of Aβ and
p-tau levels.6 PQ induces substantia nigra neuronal loss through
INF-γ silencing.8 INF-γ long-term treatment induces hippo-
campal neurodegeneration and cognitive decline in mice,9 and
PQ treatment of IFN-γ knockout mice reverted the substantia
nigra neuronal loss induced after PQ treatment alone.8 Insulin
signaling pathway disruption induces hippocampal neuronal loss
and produces cognitive disorders, and insulin treatment reverts
these effects.10,28,29 All these previous studies support our
results. PQ induces OS,1,2 which could induce neuroinflamma-
tion,7,8 Aβ proteins accumulation,2 and cell death,6 so PQ could
mediate the effect observed through OS generation. Finally, our
results point out that additional mechanisms may participate in
the hippocampal neuronal loss produced. The Wnt signaling
pathway maintains cognitive function and cell viability.23,30 PQ
disrupts the Wnt pathway;23 therefore, this mechanism together
with those previously described could contribute to the neuronal
loss observed and the cognitive decline described after PQ
treatment.

Accordingly, PQ (1 and 14 days of treatment) upregulates the
INF-γ signaling pathway that produces a reduction of insulin
levels and insulin signaling pathway downregulation through
JNK, which induces Aβ1−42 and tau peptides production and
hippocampal neuronal loss. Further studies are required to
determine the unknown mechanism through which PQ also
disrupts the insulin signaling pathway, mediates the Aβ1−42 and
tau peptides production, and induces hippocampal neuronal
loss. The PQ effect on insulin and INF-γ produced peripherally

could contribute to the local effect observed, and it is necessary
to perform in vivo studies to corroborate that these mechanisms
are produced and the local and peripheral contribution to the
effect observed and the induction of cognitive decline. Our
results provide new information on PQ toxic mechanisms that
may lead to hippocampal neurodegeneration, which could
clarify the PQ toxic action that triggers cognitive dysfunction
and provide additional tools to prevent and manage these
processes.

■ ASSOCIATED CONTENT
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Figure S1: Transfection efficiency for siRNA (PDF)

■ AUTHOR INFORMATION
Corresponding Authors

Paula Moyano − Department of Pharmacology and Toxicology,
Veterinary School, Complutense University of Madrid, 28040
Madrid, Spain; Phone: +34-913943836;
Email: pmoyanocires@ucm.es

Emma Sola − Department of Pharmacology and Toxicology,
Veterinary School, Complutense University of Madrid, 28040
Madrid, Spain; Phone: +34-913943836; Email: esola@
ucm.es

Javier del Pino − Department of Pharmacology and Toxicology,
Veterinary School, Complutense University of Madrid, 28040
Madrid, Spain; orcid.org/0000-0001-6937-7701;
Phone: +34-913943836; Email: jdelpino@pdi.ucm.es

Authors
Maria Luisa Abascal − Department of Pathology, Gregorio
Marañon Hospital, 28007 Madrid, Spain

Javier Sanjuan − Department of Pharmacology and Toxicology,
Veterinary School, Complutense University of Madrid, 28040
Madrid, Spain

Andrea Flores − Department of Pharmacology and Toxicology,
Veterinary School, Complutense University of Madrid, 28040
Madrid, Spain

José Manuel Garcia − Department of Pharmacology and
Toxicology, Veterinary School, Complutense University of
Madrid, 28040 Madrid, Spain

Jimena Garcia − Department of Pharmacology and Toxicology,
Veterinary School, Complutense University of Madrid, 28040
Madrid, Spain

María Teresa Frejo − Department of Pharmacology and
Toxicology, Veterinary School, Complutense University of
Madrid, 28040 Madrid, Spain

Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrestox.2c00278

Author Contributions
CRediT: Maria Luisa Abascal conceptualization, formal
analysis, investigation, writing-original draft.
Funding
This work was supported by research grants PR26/20326 from
Santander Bank/UCM.
Notes
The authors declare no competing financial interest.

Figure 4. Cell viability (A) and caspases 3/7 activity (B) analyses. Each
bar represents mean ± SEM of three separate experiments from cells of
different cultures, each one performed in triplicate. The values obtained
were normalized by total protein concentrations. ***p ≤ 0.001
correlated to control. ###p ≤ 0.001 correlated to PQ exposure. &&&p ≤
0.001 correlated to PQ+insulin treatment. γγγp ≤ 0.001 compared to tau
siRNA cells exposed to PQ.

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https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Mari%CC%81a+Teresa+Frejo"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
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https://pubs.acs.org/doi/10.1021/acs.chemrestox.2c00278?fig=fig4&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.chemrestox.2c00278?fig=fig4&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.chemrestox.2c00278?fig=fig4&ref=pdf
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https://doi.org/10.1021/acs.chemrestox.2c00278?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as


■ ACKNOWLEDGMENTS
The authors would like to thank Professor Miguel Capo for his
counseling during the preparation of the present work.

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