9. The Phosphorylation Pathways: Wip1 Expression and the Role of
p53.
Experimental data strongly suggest that the p38 MAPK pathway is central
to the development of neurodegeneration by infectious prions. The study
conducted by C Fang et al., 2018 [13] utilized a specific
neuronal culture system that distinguishes the cellular and molecular
mechanisms by which prions cause damage in neural synapses. The authors
used specific inhibitors against the three main families of MAPK, namely
a) the extracellular signal regulated kinases (ERKs), b) the Jun
amino-terminal kinases (JNKs), and c) the p38 -stress activated protein
kinases (SAPKs) in order to determine which of the distinct subfamilies
of kinases are involved in the synaptic toxicity process caused by
PrPSC. The authors concluded that the main kinases
involved in dendritic spine toxicity were those of p38 MAPK subfamily,
and, in particular, the p38α isoform. Furthermore, a p38 MAPK inhibitor,
after 24 hours of being added to the culture, was able to completely
reverse the initial synaptic toxicity effects caused by
PrPSC. Moreover, the authors also used a genetic
method of suppressing the p38 MAPK activation cascade by culturing a
hippocampal neuron cell line which is heterozygous for p38α MAPK
(T180A/Y182F), p38AF. This dominant negative mutant cell line was also
protected from PrPSC synaptic neurotoxicity in a way
comparable to the effect of the p38α inhibitor. In a relevant study, a
double mutation in the activation site of p38AF protein, at the sites of
Thr180 and Tyr182, inhibits the phosphorylation of the p38 molecule by
other kinases. Also in this study, the heterozygous mice for the p38AF
(+/-) allele show a marked reduction in a) p38-related signaling and b)
the expression of age-produced cell cycle inhibitors [80].
Additionally, the mutated p38AF animals showed increased proliferation
and regeneration of pancreatic islets, amongst other organs. Overall, in
this study, the p38AF mutated animals expressing the defective isoform
of p38α AF possessed a resistant mechanism that alleviated synaptic
toxicity caused by PrPSC (spine degeneration), thereby
bypassing the mechanism of PrPSC activation of a
localized p38-mediated signaling cascade that leads to dendritic spine
retraction [13,80]. Importantly, the p38AF, Wip1 deficient mice
showed a reduction in their cellular proliferation capacity. By
contrast, the animals that showed Wip1 overexpression retained their
cellular capacity of induced regeneration.
The Wip1 deactivation observed during the natural aging of p38AF mutated
animals, concurrent with their genetically induced loss of p38 MAPK
activation, is highly relevant to PrPSC propagation by
the SARS-CoV-2 spike protein. It shows that spike-protein-induced
neurotoxicity, as explained in more detail below, would be predicted to
be age-related. The p38 MAPK pathway, being inactivated in the p38AF
mutated animals, did not influence the Wip1 activity. Thus, these two
distinct but inter-related phosphorylation pathways are being
concurrently yet independently inactivated due to aging [80].
Under normal circumstances, the p38 MAPK pathway is activated
(phosphorylated) by the upstream induction of Toll-like receptor (TLR)
activation via Myeloid Differentiation primary response (MyD88 adapter
protein), and downstream by the TGFβ-Activated Kinase 1 (TAK1), which
becomes active through auto-phosphorylation [34]. Moreover, the
MyD88 induction involves both TLR2 and TLR4 activation (via the CD14
receptor), with the final outcome being the promotion of the NF-κB
response [81]. However, it is through the TLR4 activation and
subsequent p38 MAPK pathway follow-up of phosphorylation events that the
inflammatory response of Il-1β, Il-6 and TNF-α is being presented. The
activation of IRAK4 phosphorylation by the SARS-CoV-2 spike protein has
been shown to be induced by both TLR2 and TLR4 activation that
subsequently produces a similar interleukin-mediated inflammatory
response in human macrophages [82]. Furthermore, the same pattern of
TLR2 an TLR4 activation to produce NF-κB and the interleukin-mediated
inflammatory response is also occurring in injured or damaged microglia
and astrocytes [83].
Particularly, the TLR4 receptor serves as an upstream regulator of Wip1
phosphatase in cells in the nervous system [84,85]. In astrocytes,
Wip1 expression provides a negative feedback loop in response to the
activation of the NF-κB response. In brief, although TLR4 activation led
to an increase in Wip1 and phospho-NF-κB-p65 expressions in
LPS-stimulated primary astrocytes, the expression of p65 was further
increased when the expression of Wip1 was deactivated [84].
Similarly to the LPS-induced activation of TLR4 in human monocytes, the
SARS-CoV-2 spike protein induces a comparable interleukin (IL-1β)
response also via activating TLR4 [86]. Similar induction of IL-1β
was noticed in a differentiated neutrophil cell line that expressed TLR4
following spike protein exposure. Also, the spike protein was able to
induce an IL1β response in various murine macrophage cell lines,
specifically due to TLR4 expression.
In conditions of brain injury, the expression of Wip1 in the nervous
tissue prevents inflammation by inhibiting microglial and macrophage
accumulation [87]. In murine and human macrophages, the SARS-CoV-2
spike protein, and specifically the S1 subunit of the trimer, activates
NF-κB and c-Jun N-terminal kinase (JNK) pathways specifically via TLR4
activation [12]. Additionally, in microglial cells, that are a
specialized macrophage type of cell in the brain, the induced spike
protein neuroinflammation via TLR4 activation includes sustained NF-κB
activation, suggesting that Wip1 expression is weak and/or delayed
[10]. ROS-dependent activation of JNK causes p53 to robustly induce
apoptosis, and this is considered to be a feature in tumor cells, but it
may be worrisome when neurons are exposed to JNK activation in the
context of highly phosphorylated p53 [88].
Thus, although downregulation of Wip1 expression is positively
correlated with a better recovery from sepsis by activating neutrophil
migration and thus enhancing antimicrobial activity at the point of
infection [89], the loss of Wip1 expression in the nervous system
can be viewed as being tightly correlated with increased inflammation by
uncontrolled, p65-dependent, induction of NF-κB signaling. In that
regard, the increased p53 activity can be viewed as a normal function to
promote apoptosis in order to prevent the emergence and persistence of
cells with damaged genomes [90].