Effects of Celecoxib and PGE2 on SYP/PSD95 and
cAMP/PKA/CREB/BDNF signaling in N2a cells.
To identify the effects COX-2 and PGE2 in vitro, we
treated N2a cells with COX-2 selective inhibitor Celecoxib. As shown in
RT-qPCR result, the inhibition ability of Celecoxib for COX-2 was
dose-dependent, while 10μm was the most efficient among 1μm, 5μm, 10μm
and 20μm these four concentration gradients (Fig. 7A). After N2a cells
were treated with 10μm Celecoxib for 24h, evidently, we found not only
the expression of COX-2 and PSD95 protein decreased but also cAMP and
its downstream signal pathway p-PKA/PKA, p-CREB/CREB, BDNF all
downregulated. But no change was found in the SYP expression after the
treatment of Celecoxib. While all of these cytokines significantly
increased after the treatment of PGE2 (1μmol/L) for 4
hours (Fig. 7B, C). These results suggest that Celecoxib has the effect
of reducing PSD95 and cAMP/PKA/CREB/BDNF signaling by inhibiting the
releasing of PGE2 in N2a cell lines.
DISCUSSION
Evidence suggests the constitutive expression of ”inducible” COX-2 in
the brain[25], kidney[26,
27], and a few other organs[28], with basal
activity involvement in cellular functions. Previous studies have
demonstrated that neuronal COX-2 and its metabolites are essential in
memory and learning. For the first time, Yamagata established that
hippocampal neuronal COX-2 correlates with neural activity and memory
consolidation in rats [29]. COX-2 is also
implicated in various cognitive functions, such as acquiring[30], consolidating [31],
and passively avoiding [32] memories. Celecoxib is
a selective COX-2 inhibitor and could impair the spatial memory of mice
due to infusion into the hippocampal CA1 region [33,
34] without any effect on COX-1 inhibition[21]. Our data also demonstrated the importance of
COX-2 in mice’s memory, consistent with these findings.
Many studies have examined the molecular mechanisms associated with
synaptic modifications during memory development and mediated by various
proteins [35-38]. Furthermore, COX-2 is enriched
in neuronal dendritic spines with active synapses[39,40]. Synapses form the structural basis for
neuronal communication and facilitate memory functions. PSD95 and SYP
regulate the structure and function of synapses[41]. PSD95, a significantly available scaffold
protein in the postsynaptic region, helps in the signal transmission of
neurons [42]. A complete PSD95 knockout in mice
causes severe deficits in learning and memory of spatial areas,
establishing the significance of PSD95 in developing synapses[43, 44]. Therefore, COX-2 signaling plays a
crucial role in the postsynaptic region, regulating PSD95 expression and
contributing to memory formation.
Learning and memory are associated with activity-induced changes in
dendritic spine density, maturity, size, and shape[45]. New memories are directly related to the
density of dendritic spines inside the CA1 region of the hippocampal
structure [46-49]. Dendritic spines mature into
mushroom spines to achieve spatial working memory[45]. Decreased spine density or maturity is
correlated with mental retardation, memory loss, and Alzheimer’s disease[50-52]. Dendritic tree structure and arborization
provide insight into how neurons receive and transmit synaptic inputs[53]. The dendritic diameter in the cerebellum of
C57BL/6 male mice positively correlated with the total postsynaptic
density (PSD). Moreover, a greater diameter is associated with a greater
PSD area [54]. Dendrite thickness regulates
postsynaptic potential transmission by affecting electrical resistance,
with faster signal propagation among thicker dendrites[55, 56]. Evidence indicates
COX-2/PGE2 signaling alters dendrite and dendritic spine
morphology [57]. This study supported the
importance of dendritic spine density and maturity in memory formation.
The study also significantly reduced dendritic spine density in COX-2KO
mice. Thus, OX-2 was crucial in mediating memory-related neuronal
structure in the hippocampal CA1 region.
Local population activities or the assembly of neurons across multiple
brain regions cause a rhythmic fluctuation known as neural oscillation.
It is detected using electrocorticography (ECoG), local field potentials
(LFP), magnetoencephalography (MEG), and electroencephalography (EEG)
under various frequencies. These include delta (0.5–3 Hz), theta (4–12
Hz), alpha (13–15 Hz), beta (16–30 Hz), low gamma (30–60 Hz), and
high gamma(60-120 Hz) [58-60]. They regulate
neuronal spike timing on the microscale and coordinate macroscale data
dispersed in the cortical region to provide information concerning
temporospatial localizations [61]. Group
coordination is necessary for effective communication between
presynaptic and postsynaptic groups [62].
Selective COX-2 inhibitors significantly reduced LTP induction in
hippocampal dentate granule neurons in vitro . In contrast, COX-1
inhibitors were ineffective, reversed by PGE2 treatment,
but not by other PGs [63]. Additionally,
activating neuronal COX-2 was necessary to induce LTP in learningin vivo [21]. Gamma oscillations have fast
rhythms to escape inhibition through network excitation for the time
being. This elevates the effectiveness, overall precision, and selection
of communication among various network areas [64].
The changing gamma oscillation power could be a disease biomarker to
investigate novel therapies for treating cognitive impairment in
diseases such as Alzheimer’s [65, 66]. A strong
correlation was observed between the in vivo gamma oscillation
power and the cognitive functions of the test animals[67]. The present study depicts that COX-2KO in
mice effectively decreases gamma oscillation power. Thus, COX-2 could
affect learning and memory ability by modulating gamma oscillations in
the hippocampal CA1 area.
Our findings indicate that only PGE2 expression
significantly decreased in COX-2KO mice among all the five PG types. One
possible explanation for the altered PG profile would be that COX-2
predominantly metabolizes AA to PGE2. On the other hand,
COX-1 could be responsible for other PGs [68].
PGE2 is characterized as the lipid mediator, enabling
nervous system development. Meanwhile, various studies demonstrate that
abnormal PGE2 signaling leads to neurodevelopmental
disorders [69, 70]. The COX-2/PGE2signaling pathway is associated with several vital processes in
developing neurons. This includes proliferation, migration[71, 72], synaptogenesis [73,
74], and the formation and plasticity of dendritic spines[72, 75]. Some rat studies have revealed that
uncontrolled elevation or reduction of PGE2 expression
affects cerebellar [76]and hippocampal[77] dendritic arborization during critical
periods after delivery. In contrast, inhibiting PGE2production by locally injecting COX-2 inhibitors can reduce the
arborization of the dendritic tree and the density of excited synapses[78]. As depicted in the COX-2KO mouse model[79-81], many developing genes have differential
expression due to the reduced PGE2 levels. This disrupts
synaptic transmission pathways, long-term nerve potentiation, navigating
the neurites, and developing the dendritic spine [82,
83], consistent with our results.
PKA is the primary downstream kinase of cAMP and could phosphorylate
CREB. Thus, parts of the transcription factor are recruited within the
promoter regions, including the BDNFgene [84]. The
cAMP/PKA/CREB/BDNF signaling pathway promotes the survival of neurons,
modulates synaptic morphology, and strengthens synaptic transmission[85]. The pathway modulates survival, development,
cognition, and mood across epilepsy, depression, and Alzheimer’s disease
animal model neurons [86, 87]. Our study observed
lower phosphorylation expression in the COX-2KO group of cAMP, PKA, and
CREB with BDNF expression than in WT. Thus, COX-2 could participate in
the synaptic plasticity modulation of hippocampal neurons using
cAMP/PKA/CREB/BDNF signaling.
Our
findings revealed the COX-2 signaling pathway as a new mechanism for
regulating the hippocampal dendritogenic process and gamma oscillation
to affect learning and memory ability. The exogenous application of
PGE2 effectively reversed the actions caused by the
inhibition of COX-2. COX-2 could participate in hippocampal synaptic
plasticity by regulating COX-2/PGE2/cAMP/PKA/CREB/BDNF
signaling in the postsynaptic membrane PSD95 modulation. Therefore, this
study can elevate our understanding of COX-2 physiologic function and
its role in memory formation.