Written by Wang Mengmeng, Ye Keqiang

Editor-in-charge - Fang Yiyi, Wang Sizhen

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Osteoporosis is a systemic bone disease characterized by the destruction of bone mass and normal bone structures resulting in increased bone fragility and risk of fracture [1

Brain-derived neurotrophic factors (BDNF) are neurotrophic factors that play an important role in the central nervous system and can also be synthesized and released by non-neuronal cells, such as fibroblasts, osteoblasts, etc.

Ye Keqiang's research group developed a prodrug R13, which can release 7,8-DHF (BDNF mimetic) to activate the TrkB receptor [7-9]; It was also found that the BDNF/TrkB signal inhibited the activation of asparagine endopeptase (AEP) through Akt phosphorylation of T322 residues[10].

In August 2022, Ye Keqiang's research group at emory University's School of Pharmacy published a study

 

To explore the role of AEP in bone remodeling, the researchers performed OVX

Figure 1 AEP knockout improves trabecular bone density in ovarian excision female mice

(Source: Xiong J, et al.

To further characterize the role of AEP in OVX-induced osteoporosis, the researchers performed H&E staining

 

Figure 2 AEP knockout inhibits bone transition in ovarian excision female mice

(Source: Xiong J, et al.
, Nat Commun, 2022)

To explore the biological role of BDNF/TrkB signaling in OVX-induced bone loss, the researchers used 12-week-old female BDNF+/- mice and WT litters
.

4 days after OVX surgery, WT and BDNF+/- mice were given oral R13 (21.
8 mg/kg) or blank vector for 8 weeks
.

OVX mice after oral R13 showed higher BV/TV, Conn.
D, lower SMI, and higher Tb.
Th and Tb.
N (Figures 3a, b); R13 after OVX increased cortical bone cortex area (Ct.
Ar) and mean cortical thickness (Ct.
Th), but the ratio of relative cortical bone area to tissue area (Ct.
Ar/Tt.
Ar) between groups was unchanged (Figure 3c).


Thus, oral R13 significantly increases bone density (Figure 3d
).

To explore whether oral R13 releases enough 7,8-DHF to trigger osteoblast differentiation, researchers conducted in vivo pharmacokinetic studies and found that 7,8-DHF distribution in the bone marrow showed a time-dependent increase, much higher than ec50
that activates TrkB in primary neurons.

Western blotting of p-TrkB signaling in the bone marrow shows that 7,8-DHF concentrations are associated with neurotrophic signal activation (supplemental Figure 2b
).

Thus, oral R13-derived 7,8-DHF activates the BDNF/TrkB signaling pathway
.

 

Figure 3 R13 treatment increased serum OPG levels and alleviated trabeculocaria-induced bone loss by WT and BDNF+/-female mice

(Source: Xiong J, et al.
, Nat Commun, 2022)

To further explore the role of BDNF signaling in bone resorption and formation after OVX, the researchers performed H&E staining analysis
of bone morphology and white fat cells in WT and BDNF mice after OVX surgery.

R13 treatment increased trabecular tissue and reduced fat cell content in post-OVX bone (Figure 4a
).

Osteoclasts were reduced (Figure 4b) and MAR and BFR (Figure 4c)
were added.

It is shown that R13 treatment relieves OVX-induced bone loss
by increasing bone formation.

After OVX, R13 treatment inhibits WT and N.
Oc/BS in BDNF+/- mice, indicating that R13 treatment induces bone formation and inhibits bone resorption
after OVX.

Notably, R13 significantly increases OPG levels without affecting RANK-L (Supplemental Figure 4
).

Thus, R13 treatment greatly prevented bone loss caused by OVX and significantly increased OPG levels
.

 

Figure 4 R13 treatment blocked bone transformation caused by oophorectomy in female mice

(Source: Xiong J, et al.
, Nat Commun, 2022)

7,8-DHF binds to the extracellular region of the TrkB receptor, where BDNF interacts with the TrkB receptor to mimic the biological role
of BDNF in a TrkB-dependent manner.

To investigate the molecular mechanisms by which 7,8-DHF stimulates elevated bone density in rodents, the researchers tested its role
in MC3T3-E1 cells in the presence of OFIM (osteogenesis induction medium).

The results showed that OIM significantly increased osterix levels, and BDNF or 7,8-DHF had a similar effect.
Both BDNF or 7,8-DHF were consistent with AEP activity (Figure 5
).

The results strongly supported the 7,8-DHF analog BDNF, and both strongly increased OPG expression and lowered the RANKL/OPG ratio, accelerating osteoblast formation
.

In addition, it inhibits the C/EBPβ/AEP pathway, thereby inhibiting the formation
of osteoclasts.

 

Fig.
5 7,8-DHF promotes MC3T3-E1 cell differentiation, mineralization and OPG secretion

(Source: Xiong J, et al.
, Nat Commun, 2022)

To further explore the mechanism by which 7,8-DHF promotes OPG expression, the researchers conducted studies
in MC3T3-E1 cells.

The results showed that 7,8-DHF 7,8-DHF mimics the action of
BDNF by activating the TrkB neurotrophic factor.

To examine which are critical for OPG expression, the corresponding gene is knocked out in MC3T3-E1 cells by specific siRNA in the presence of OIM and 7,8-DHF, and the results show that 7,8-DHF stimulates OPG expression levels by activating CREB (transcription factor downstream of the BDNF/TrkB pathway) (Figure 6).

 

Figure 6 7,8-DHF regulates OPG expression by activating the CREB forward

(Source: Xiong J, et al.
, Nat Commun, 2022)

To investigate whether 7,8-DHF promoting bone formation also involves inhibiting osteoclast production, the researchers treated RAW264.
7 cells with RANK-L, and the results showed that RANK-L promotes osteoclast production, while 7,8-DHF blocked THE RAW264.
7 osteoclast production
induced by FRANK-L associated with AEP inhibition.

Whereas R13 protects bones by increasing OPG, using RANK-L antibodies as a comparative agent, the results show that R13 shows similar efficacy to anti-RANK-L therapy in terms of bone density and various bone indices (Figure 7a-c) and that both treatment modalities significantly reduce the RANK-L/OPG ratio (Figure 7d
).

These findings suggest that R13 has a similar effect
to THAT of THE RANK-L antibody in the treatment of osteoporosis.

Given that R13 has been approved by the FDA for clinical trials for AD indications, the researchers expect that R13 may soon become a new therapeutic agent to treat osteoporosis by enhancing osteoblastic differentiation to stimulate bone formation and preventing bone resorption by blocking osteoclast production; And R13 exerts a bone protection effect through the dual mechanisms of OPG upregulation and AEP antagonism, and it is conceivable that its therapeutic effect on patients may be stronger
than that of RANK-L antibodies.

H&E staining showed that R13 treatment and anti-RANK-L increased bone marrow density and decreased white fat cell content in bone after trabecular OVX (Figure 8a
).

TRAP staining indicates that OVX-induced osteoclasts are significantly reduced in both treatments (Figure 8b
).

These results show that R13 exhibits comparable effects to anti-RANK-L in reducing bone loss induced by osteoclasts to reduce OVX (Figure 8c, d
).

 

Figure 7 R13 and Rank-L antibody therapy showed similar effects in blocking trabecular bone loss induced by oop resection in female WT mice

(Source: Xiong J, et al.
, Nat Commun, 2022)

 

Figure 8 R13 Treat osteoporosis by elevating OPG and inhibiting AEP by activating BDNF/TrkB signaling

(Source: Xiong J, et al.
, Nat Commun, 2022)

Conclusions and discussions, inspirations and prospects of the article In summary, the study found that BDNF mimic-R13 can promote bone formation by inhibiting C/EBPβ/AEP, and alleviate osteoporosis by hindering THE PRODUCTION OF RAW264.
7 OSTETHOCYTEs induced by RANK-L
.

The experimental results showed that the treatment with R13 on WT and BDNF+/- mice increased their OPG, blocked the loss of trabecular bone, and showed similar therapeutic effects
to anti-RANK-L.

In addition, knockout of AEP reduced the ratio of RANK-L/OPG in OVX mice, demonstrating that inhibition of AEP weakened bone conversion
.

The 7,8-DHF released from R13 activates TrkB and its downstream effector CREB, which is critical
for OPG enhancement.

Therefore, the study demonstrated that R13 exerts its therapeutic efficacy
against osteoporosis by inhibiting AEP and elevating OPG.

Previous studies have shown that FSH triggers bone loss, while anti-FSH increases bone density without altering estrogen concentrations, so FSH may somehow activate AEP and trigger bone loss
.

It is assumed that 7,8-DHF inhibits the production of FSH, resulting in the inhibition of AEP and an increase in bone density, which is consistent with the results of reduced bone loss and reduced RANK-L/OPG ratio after OVX in AEP KO mice in this study (Figure 8e
).

Original link: https://doi.
org/10.
1038/s41467-022-32435-5

Corresponding Author: Ye Keqiang

(Photo courtesy of Ye Keqiang Laboratory)

Selected articles from previous issues

【1】Nat Commun | Chen Zhong's team revealed the circuits and molecular mechanisms of lower support involved in temporal lobe epilepsy

[2] The Cereb Cortex-Du Yifeng/Qiu Chengxuan research group revealed a complex relationship between KIBRA gene polymorphisms and gray matter structure and olfactory function in the brain of the elderly

[3] Mol Psychiatry-Yingfei Wang's research group revealed the role of KDM6B protein in neuronal synaptic plasticity and learning and memory

[4] Cereb Cortex—The Team of Shanbao Tong and Xiangfei Hong reveals important influencing factors of EEG alpha rhythm in the visuospatial attention task

[5] Cereb Cortex: Excavation and Analysis of Imaging Subtypes for the Heterogeneity of Schizophrenia

[6] Front Cell Neurosci Review:Gamma Neural Oscillation and Central Nervous System Diseases: Mechanisms and Therapeutic Advances

[7] The NAR-He Cheng/Su Zhida team found that topoisomerase IIA can regulate adult neurogenesis in the subependymal region

[8] The Sci Adv-Liao Wenbo team has made important progress in the adaptive evolution of amphibian brain volume

[9] J Neuroinflammation—From Changchun/Jian Zhang's team found that targeted proteoglycan receptors after hemorrhagic stroke protected white matter integrity and promoted neurological recovery

[10] Front Aging Neurosci—Zeng Yanbing's team established a predictive model and revealed the effects of behavioral changes on cognitive impairment in the elderly

Recommended for high-quality scientific research training courses

【1】Special Training on Biomedical Statistics for Clinical Prediction of R Language (October 15-16, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing)

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[1] Trailer | Conference on Neuromodulation and Brain-Computer Interface (U.
S.
Pacific Time: October 12-13)

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End of this article