Nat Commun︱ selective inhibition of microglia activation is expected to alleviate the spread of pathological α-syn

Written by Sucre, editor in charge, Wang Sizhen Parkinson's disease (PD) is currently the second largest neurodegenerative disease in the world, and more than 6.
1 million people worldwide suffer from PD [1]
.

PD is the progressive death and loss of dopaminergic neurons in the substantia nigra projecting to the striatum, causing a neurodegenerative disease characterized by decreased dopamine levels and dyskinesia in the striatum.
The hallmark of PD is the α-process.
The nucleoprotein aggregates pathologically, forming Lewy bodies (LBs) and Lewy neurites (LNs) [2]
.

 α-synuclein (α-syn) plays an important role in the pathological process of sporadic and familial PD
.

Genetic studies have found that an increase in the copy number of the gene encoding α-syn causes an increase in the level of endogenous α-syn, which is the direct cause of early-onset PD in the family
.

The mutation of the gene encoding α-yn is another direct cause of familial PD [3]
.

In addition, α-syn is involved in the occurrence and development of many neurodegenerative diseases including PD, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) [4]
.

In view of the important role of α-syn in many diseases such as PD, targeting the transmission of α-syn is a potential intervention
.

 Previous studies have shown that neurons release intracellular pathological α-syn through exocytosis, and are taken up by surrounding glial cells and neurons, and cause chronic inflammation [5, 6]
.

As an important immune cell of the central nervous system, microglia (microglia) play an important role in mediating inflammation and removing cell debris [7]
.

At the same time, studies have shown that pathological α-syn aggregation is often accompanied by microglia activation [8]
.

However, the molecular mechanism by which microglia regulates the spread of pathological α-syn is still unclear
.

 Recently, the Kalipada Pahan team from Rush University Medical Center in the United States published a report on Nature Communications entitled "Selective targeting of the TLR2/MyD88/NF-κB pathway reduces α-synuclein spreading in vitro and in vivo "The latest research paper
.

Studies have found that prefabricated α-syn fibrils promote the interaction of TLR2 and MyD88, causing microglia activation
.

Selective inhibition of the TLR2/MyD88/NF-κB pathway can not only alleviate the activation and inflammation of microglia induced by prefabricated α-syn fibrils, but also inhibit the spread of pathological α-syn
.

In this article, the author first studied the activation of preformed α-syn fibrils (PFF) on TLR2 (Toll like receptor 2) in microglia
.

To this end, the author first prepared a PFF that can induce pathological aggregation of endogenous α-syn, and measured its structure and molecular weight with the help of electron microscopy and biochemical methods
.

Next, the author found that the interaction between TLR2 and MyD88 (Myeloid differentiation factor 88) in BV-2 microglia after PFF treatment was enhanced.
At the same time, wtTIDM (wild type TLR2-interacting domain of MyD88) polypeptide was not mTIDM.
(Mutant TLR2-interacting domain of MyD88) peptide can significantly inhibit this interaction
.

Considering that the enhanced interaction of TLR2-MyD88 can cause the activation of NF-κB, and NF-κB is a pro-inflammatory transcription factor, the authors tested the activity of NF-κB and the transcription level of inflammatory factors, and found that NF-κB The DNA binding activity and NF-κB transcription activity of NF-κB were significantly increased, and the transcription levels of iNOS and IL-1β inflammatory factors were also significantly increased, and the above effects could be significantly inhibited by wtTIDM but not mTIDM
.

These results indicate that PFF can induce the activation of TLR2 in microglia, leading to increased NF-κB activity and increased transcription levels of inflammatory factors, and this process can be effectively inhibited by wtTIDM
.

 Considering that microglia can degrade cell debris and aggregate proteins through phagocytosis in the central nervous system, the authors studied the effect of TLR2 on the phagocytosis of microglia
.

For this reason, the author knocked out TLR2 in microglia and tested its phagocytosis by fluorescently labeling monomer α-syn, and found that the phagocytic ability of TLR-/- microglia was significantly reduced
.

Interestingly, the phagocytosis of wild-type microglia treated with wtTIDM did not change significantly compared with the control group.
This phenomenon is also similar in LPS-treated microglia, although the overall phagocytosis level after LPS treatment has a certain degree of phagocytosis.
The rise
.

These results indicate that although knocking out TLR2 affects the phagocytosis of microglia, blocking the interaction of TLR2 and MyD88 specifically does not affect the phagocytosis of microglia
.

Figure 1 PFF induces TLR2 activation of microglia (picture quoted from: Dutta D, et al.
, Nat Commun.
2021) Based on the above results, under in vitro conditions, selective inhibition of TLR2 activation can be achieved without affecting microglia phagocytosis In the case of capacity, it effectively blocks the inflammatory response induced by PFF (Figure 1)
.

 Next, the authors studied the effect of wtTIDM, a selective TLR2 inhibitor, on the inflammatory response induced by PFF in vivo
.

To this end, the author first injected PFF into the intrastriatal sac of A53T (a dot at the N-terminal of α-syn) transgenic mice through a stereotaxic device [9], and started to give wtTIDM polypeptide through the nasal cavity 2 months after injection.
It lasted for 1 month, and then was tested for biochemical indicators.
The results showed that compared with control mice, the level of glial cell activation and proliferation in the SN area and the levels of inflammatory factors in the wtTIDM-treated mice were significantly inhibited
.

These results indicate that under in vivo conditions, the selective TLR2 inhibitor wtTIDM can significantly inhibit the glial cell inflammatory response induced by PFF
.

 Previous studies have shown that the activation of glial cells is often accompanied by the spread of pathological α-syn [10]
.

Next, the authors studied the effect of wtTIDM, a selective TLR2 inhibitor, on the transmission of pathological α-syn under in vivo conditions
.

Similarly, the authors injected PFF into the sac of the striatum of mice.
The experimental group was given wtTIDM intranasally, and the control group was given mTIDM intranasally.
Then the insoluble α-syn and α-syn serine in the substantia nigra and motor cortex were treated with 129 Site phosphorylation (pSyn129) levels were detected, and the results showed that compared with mTIDM, wtTIDM-treated mice, the levels of insoluble α-syn and pSyn129 in the substantia nigra and motor cortex were significantly reduced
.

These results indicate that intranasal administration of wtTIDM, a selective TLR2 inhibitor, can effectively prevent the spread of pathological α-syn in the brain (Figure 2)
.

Figure 2 TLR2 selective inhibitor wtTIDM can effectively prevent pathological α-syn transmission in the brain (picture quoted from: Dutta D, et al.
, Nat Commun.
2021) In addition, previous studies have shown that PFF can cause dopaminergic nerves in the substantia nigra Yuan died, accompanied by inflammation
.

Based on the above results, that is, wtTIDM can effectively prevent the transmission of pathological α-syn in the brain.
Then, the author conducted a study on the protective effect of wtTIDM on dopaminergic neurons
.

With the help of immunohistochemistry, western blotting, high-performance liquid chromatography, and behavioral experiments, the authors found that after 3 months of injection of PFF into the sac region of the striatum, the activity of caspas3 in the substantia nigra of A53T mice increased, and the level of TH expression decreased significantly , TH-positive neurons are significantly missing, the level of dopamine in the striatum is decreased and accompanied by dyskinesias, and intranasal administration of wtTIDM can significantly alleviate the above-mentioned pathological process
.

At the same time, in old A53T mice, wtTIDM also had a similar improvement effect on pathological changes and abnormal movements caused by PFF
.

These results indicate that wtTIDM can effectively protect neuron death caused by PFF, delay pathological changes caused by PFF, and improve movement disorders (Figure 3)
.

Figure 3 wtTIDM can effectively protect neuronal death caused by PFF, delay pathological changes and improve movement disorders (picture quoted from: Dutta D, et al.
, Nat Commun.
2021) Next, in order to further confirm the transmission of TLR2 in pathological α-syn The role of TLR2 and the above-mentioned series of protective effects of wtTIDM are TLR2-dependent.
The authors crossed A53T+/+ mice with TLR2-/- mice to obtain TLR2 knockout A53T transgenic mice, namely A53TΔTLR2
.

Similarly, pathological examination was performed 3 months after PFF was injected into the sac region of the striatum.
The results showed that compared with A53T mice, the levels of insoluble α-syn and pSyn129 in the substantia nigra and motor cortex of A53TΔTLR2 mice were significantly reduced, and accompanied by Lower levels of glial inflammation
.

Interestingly, unlike A53T mice, intranasal administration of wtTIDM could not further reduce pathological α-syn levels and inflammation in the substantia nigra of A53TΔTLR2 mice
.

These results prove that the loss of functional TLR2 significantly affects the transmission of pathological α-syn, and that the series of protective effects played by wtTIDM are TLR2-dependent (Figure 4)
.

Figure 4 Loss of functional TLR2 significantly affects the transmission of pathological α-syn (picture quoted from: Dutta D, et al.
, Nat Commun.
2021) Next, the author deeply studied the molecular mechanism of TLR2 involved in the pathological changes of α-syn
.

The expression of α-syn is a prerequisite for the formation of pathological α-syn.
However, we still do not know whether the pro-inflammatory factors derived from microglia, such as IL-1β and TNFα, can affect the expression of α-syn
.

In order to solve this problem, the author added the pro-inflammatory factors IL-1β and TNFα to MN9D nerve cells, and found that the expression level of α-syn was significantly increased in nerve cells treated with IL-1β or TNFα, and Dose-dependent
.

These results indicate that pro-inflammatory factors can increase the expression level of α-syn in nerve cells
.

 Next, the author deeply studied the molecular mechanism of pro-inflammatory factors that increase the expression level of α-syn in nerve cells
.

First, the authors used pro-inflammatory factors to treat MN9D nerve cells, and found that the NF-κB DNA binding activity and transcription activity of the cells were significantly increased after treatment.
At the same time, the specific inhibitor of NF-κB activation, wtNBD, was used to treat nerve cells.
It can significantly inhibit the up-regulation of α-syn induced by IL-1β and TNFα
.

Furthermore, the author found by searching MatInspector that NF-κB can bind to the promoter of the α-syn gene, so the author speculates that NF-κB may regulate the expression of α-syn at the transcription level
.

To this end, the authors mutated the key binding site of NF-κB on the α-syn gene promoter, and found that nerve cells treated with IL-1β and TNFα lost the ability to up-regulate the expression level of α-syn
.

At the same time, the authors found that nerve cells treated with inflammatory factors, p65, p50, CBP, p300 and RNA Pol were recruited to the promoter of the α-syn gene, thereby increasing the transcription activity of the promoter of the α-syn gene
.

Similarly, human SH-SY5Y nerve cells treated with pro-inflammatory factors also have the above-mentioned series of phenotypes
.

It is worth mentioning that the regulatory effects of the pro-inflammatory factors IL-1β and TNFα on the expression of α-syn are universal, that is, many other pro-inflammatory factors, such as MPP+, LPS, Tat, poly IC and ODN, can be passed Activate NF-κB and up-regulate the expression level of α-syn
.

These results indicate that pro-inflammatory factors increase the transcriptional activity of α-syn gene promoters by activating NF-κB, thereby achieving up-regulation of α-syn expression levels in nerve cells (Figure 5)
.

Figure 5 Pro-inflammatory factors increase the transcriptional activity of the α-syn gene promoter and up-regulate the expression level of α-syn by activating NF-κB (picture quoted from: Dutta D, et al.
, Nat Commun.
2021) Then, the author further explored The effect of pro-inflammatory factors derived from microglia on the expression level of dopaergic neuron α-syn
.

First, the author used PFF to stimulate primary microglia, and then co-cultured the stimulated microglia and primary midbrain dopaminergic neurons
.

The results showed that NF-κB activity in dopaminergic neurons was significantly increased, accompanied by an increase in α-syn mRNA and protein levels, and this result can be inhibited by wtTIDM
.

Immediately afterwards, the above results were also repeated under in vivo conditions
.

Therefore, these results prove that PFF induces pro-inflammatory factors released by microglia to up-regulate the expression of α-syn by activating dopaminergic neurons NF-κB, and this process can be effectively inhibited by wtTIDM (Figure 6)
.

Figure 6 PFF activates NF-κB in dopaminergic neurons through pro-inflammatory factors, and up-regulates the expression of α-syn (picture quoted from: Dutta D, et al.
, Nat Commun.
2021).
Based on the above results, PFF induces TLR2 activation Microglia release pro-inflammatory factors, which then up-regulate the expression of α-syn by activating NF-κB
.

Therefore, as the direct cause of the up-regulation of α-syn expression, NF-κB may be an effective target for regulating the spread of pathological α-syn
.

To this end, the author administered wtNBD, a specific inhibitor of NF-κB activation intranasally, and then tested the activation state of NF-κB and the transmission of pathological α-syn respectively.
The results showed that intranasal administration of wtNBD can effectively inhibit The activation of NF-κB and the spread of pathological α-syn in the substantia nigra and motor cortex can effectively protect dopaminergic neurons and improve motor symptoms
.

Therefore, these results indicate that in the PFF-induced A53T PD mouse model, the NF-κB specific inhibitor wtNBD can effectively inhibit the transmission of pathological α-syn and improve movement disorders (Figure 7)
.

Figure 7 NF-κB specific inhibitor wtNBD can effectively inhibit the transmission of pathological α-syn and improve dyskinesias (picture quoted from: Dutta D, et al.
, Nat Commun.
2021) Conclusion and discussion of the article, inspiration and prospects for pathology α-syn is prion-like transmission in synucleinopathies such as PD, DLB and MSA
.

However, the transmission mechanism of pathological α-syn is still unclear
.

In this article, the author found that PFF activates NF-κB by enhancing the interaction between TLR2 and MyD88, and induces microglia activation and up-regulation of pro-inflammatory factor expression
.

Using selective inhibitors of the TLR2/MyD88/NF-κB pathway, namely wtTIDM and wtNBD, to selectively inhibit the interaction of TLR2 and MyD88 and the activation of NF-κB, respectively, can significantly inhibit the inflammation caused by PFF and reduce the pathological α-syn Spread
.

It is worth mentioning that this process has significant effects under both in vitro and in vivo conditions
.

Therefore, the TLR2/MyD88/NF-κB pathway may be an important target for intervention in disease processes such as PD.
At the same time, wtTIDM and wtNBD are expected to become drugs for intervention and treatment of neurodegenerative diseases such as PD.
Moreover, both of these peptides can pass through the blood.
The brain barrier can be administered intranasally, which has natural drug administration advantages
.

 In this article, the author studied in detail the effects of PFF on microglia and discussed potential interventions
.

So, as another important type of immune cells in the central nervous system, astrocytes, can PFF cause the activation of the similar signal pathways mentioned above? It is a question worth studying
.

 In addition, the authors found that in addition to PFF, other pro-inflammatory molecules such as IL-1β, TNFα, LPS, poly IC, etc.
all activate the NF-κB pathway to up-regulate the expression of α-syn and promote the spread of pathological α-syn
.

The difference is that IFNγ does not pass through the NF-κB pathway, but can also activate α-syn agonists, up-regulate α-syn expression, and promote pathological α-syn transmission
.

This result suggests that in addition to the NF-κB pathway, there may be other inflammatory transcription factors involved in the up-regulation of α-syn expression
.

 In conclusion, this study revealed that PFF caused the activation of microglia by promoting the interaction between TLR2 and MyD88
.

Selective inhibition of the TLR2/MyD88/NF-κB pathway can not only alleviate the PFF-induced activation and inflammation of microglia, but also inhibit the spread of pathological α-syn
.

This research work has deepened our understanding of microglia's regulation of pathological α-syn transmission, and also provided potential therapeutic targets and drugs for intervention in the progression of PD and other diseases
.

Original link: https://doi.
org/10.
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Global, regional and national burden of neurological disorders, 1990–2016 :