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Written by Zhang Haojie, Zhou Kailiang, Ni Wenfei Editor-Wang Sizhen, Fang Yiyi Editor-Wang Sizhen
Spinal cord injury (SCI) is a disabling condition that can lead to permanent loss of function and movement disorders
.
A large number of studies have shown lysosomal membrane permeability (LMP).
Occurs in spinal cord injury, causing lysosomal cathepsin extravasation into the cytoplasm, affecting lysosomal function, leading to the accumulation of neuronal autophagosomes and impaired
autophagy flow.
Wenfei Ni's research team has previously reviewed cytosolic phospholipase A2 (cPLA2).
As the main mediator of neuroinflammation, membrane phospholipids at the sn-2 position of lysosomes are broken down in pathological conditions, producing lysophospholipids and omega-3-polyunsaturated fatty acids, thereby causing LMP, producing an inflammatory environment for spinal cord injury[1].
Therefore, targeting cPLA2 to stabilize lysosomal membrane permeability may be a new strategy
for the treatment of spinal cord injury, however, due to the presence of the blood-brain barrier, the active ingredients of general drugs cannot reach the spinal cord injury area
。 Mitochondrial targeted antioxidant peptide (Elamipretide, also known as SS-31) is a novel aromatic cationic peptide due to its alternating structure of aromatic groups and cations.
It can freely cross the blood-brain barrier to reach the nerve damage site
.
Studies have confirmed that SS-31 can inhibit neuronal oxidative stress in neurodegenerative diseases, reduce the accumulation of neuroinflammatory mediators and toxic proteins, and have anti-inflammatory, anti-cell pyrosis and promote neuronal survival [2-4].
In addition, SS-31 can be widely enriched in the anionic cardiolipin inner membrane of mitochondria with cationic structure, enhance mitochondrial respiration, activate neuromitochondrial biogenesis, and enhance mitochondrial autophagy [5].
In autophagy, there is a close interaction between mitochondria and lysosomal organelles.
However, it is unknown whether mitochondrial targeted antioxidant peptide (SS-31) can also inhibit cell pyrosis and promote nerve repair of spinal cord injury by stabilizing lysosomal membrane permeability.
On January 7, 2023, the Department of Orthopedics/Zhejiang Provincial Key Laboratory of Orthopedics of the Second Affiliated Hospital of Wenzhou Medical University was held in the Journal of Neuroinflammation published a title titled "Elamipretide alleviates pyroptosis in traumatically injured spinal cord by inhibiting cPLA2-induced.
" lysosomal membrane permeabilization", using mouse spinal cord contusion models to carry out a series of studies, clarified that SS-31 improved the inflammatory environment of spinal cord injury mice and promoted the recovery of spinal cord injury function, and was found for the first time SS-31 improves lysosomal membrane permeability by inhibiting the phosphorylation level of cytoplasmic phospholipase A2 (cPLA2) by the p38-MAPK signaling pathway, thereby improving nerve cell pyrosis and impaired autophagy flow.
to promote nerve repair
in mice with spinal cord injury.
Zhang Haojie, Chen Yituo and Li Feida are the co-first authors of the paper, and Associate Professor Zhou Kailiang and Ni Wenfei The director is the co-corresponding author
of the paper.
(Further reading: Zhou Kailiang/Ni Wenfei course question group related work, see the "Logical Neuroscience" report (click to read): J.
) Neuroinflammation Review—The Ni Wenfei/Zhou Kailiang team focused on the important role of the STING pathway in neuroinflammation and cell death after CNS injury).
First, the authors determined the dose and timing of administration based on previous studies of the application of SS-31 in neurological diseases[4], and injected a daily dose of SS-31 at a dose of 5 mg/kg of SS-31 intraperitoneally in mice in the SS-31 group for three consecutive days after spinal cord injury, while Sham Mice in the group and SCI group are intraperitoneally injected with the same volume of DMSO
.
Immunofluorescence, HE/Masson staining, and gait analysis showed that SS-31 reduced glial scar area and number of neuronal deaths in mice 28 days after spinal cord injury, re-established connections between synapses, and improved motor recovery after spinal cord injury (Figure 1A-K)
。 This part of the study shows that SS-31 effectively promotes histological and functional recovery
in mice with spinal cord injury.
Fig.
1 SS-31 promotes functional recovery in mice with spinal cord injury
(Source: Zhang H, etal.
, J Neuroinflammation, 2023).
Pyroptosis eventually leads to the division of GSDMD, and its N-terminal fragment forms 10-15 nm pores in the cell membrane, causing the release of inflammatory factors, cell enlargement, and membrane rupture induces cell death
.
Previous studies have confirmed that SS-31 can reduce LPS-induced pyrosis of nucleus pulposus cells and perioperative pyrosis in elderly mice, so can SS-31 also inhibit pyroptosis in mice with spinal cord injury? In this study, Western Blot and immunofluorescence staining were used to detect the level of pyroptosis in
vivo and neurons in mice with spinal cord injury.
The results showed that SS-31 effectively inhibited pyroptosis in neurons (Figure 2A-FD).
Fig.
2 SS-31 inhibits pyroptosis levels in neurons of mice with spinal cord injury
(Source: Zhang H, etal.
, J Neuroinflammation, 2023).
To verify the effect of SS-31 on autophagy activity after spinal cord injury, the authors used PCR, WesternBlot, and immunofluorescent staining to detect autophagosome protein (Beclin-1, Beclin-1, 3 days after spinal cord injury VPS34 and LC3), lysosomal biogenesis-related biomarkers (ATP6V1B2 and LAMP1), and autophagy substrate proteins (SQSTM1/p62) as well as the level of the gene for Sqstm1
.
The results showed that spinal cord injury can lead to impaired autophagy flow, and SS-31 alleviates the blockade of autophagic flow (Figure 3A-G).
Fig.
3 SS-31 improves autophagy flow in mice with spinal cord injury (Source: Zhang H, etal.
, J Neuroinflammation, 2023)
To further clarify the interaction between SS-31 on autophagy and pyroptosis after spinal cord injury, the authors used the autophagic flow blocker chloroquine (CQ), detected by Western Blot and immunofluorescence Levels of pyroptosis and autophagy of neurons in the Sham group, Sham+CQ group, SCI group, SCI+SS-31 group and SCI+SS-31+CQ group
.
The results showed that CQ somewhat reversed the pyrotosis inhibitory effect of SS-31 after spinal cord injury, indicating that SS-31 inhibited pyroptosis by improving autophagic flow (Figure 4A-J).
。
Fig.
4 SS-31 relies on autophagy to inhibit pyroptosis in mice with spinal cord injury (Source: Zhang H, etal.
, J Neuroinflammation, 2023) Lysosomal membrane permeability (LMP)
is an important signaling pathway for inducing nerve cell pyroptosis[1].
。 In addition, lysosomes with elevated LMP can impair autophagy flow in neurological diseases and lead to abnormal accumulation
of neuronal autophagosomes.
Given this, the authors collected lysosomal enrichment components by spinal cord tissue subcellular isolation techniques
.
Determine the distribution
of lysosomal enzymes with Western Blot and immunocolocalization fluorescence.
The activity
of lysosomal enzymes in different components was tested by enzyme-linked immunosorbent assay.
A series of results showed that after spinal cord injury, lysosomal proteolytic enzyme leakage into the cytoplasm due to LMP increased, and SS-31 alleviated LMP and lysosomal proteolytic enzyme extravasation (Figures 5A-H).
。
In traumatic central nervous system injury, upregulation of cytoplasmic phospholipase (cPLA2/Pla2g4a) phosphorylation can lead to lysosomal membrane permeability[1] and cause cell pyroptosis and inhibition of autophagy flow [6]
。 Given the above effects of SS-31, the authors suspect that SS-31 can target cPLA2.
Western Blot further tested cPLA2 and p-cPLA2 in the Sham group, SCI group, and SCI+SS-31 group level
.
The results showed that the expression of cPLA2 in the three groups was comparable
.
However, spinal cord injury significantly upregulated the p-cPLA2 to p-cPLA2/cPLA2 ratio, while SS-31 attenuated this change (Figure 5I-M).
。 The results showed that SS-31 was able to inhibit the level of cPLA2 phosphorylation in neurons after spinal cord injury
.
Fig.
5 SS-31 improves lysosomal membrane permeability and inhibits the activation of neuronal p-cPLA2 (Source: Zhang H, et al.
, J Neuroinflammation, 2023)
This study further validates the inhibition of pyroptosis exerted by SS-31 in spinal cord injury, Whether the efficacy of improving lysosomal membrane permeability and restoring autophagy flow works by targeting cPLA2, using adeno-associated virus AAV-Pla2g4a transfected mice with overexpression of the Pl a2g4a gene
.
Relevant rescue experiments showed that SS-31 reduced the phosphorylation level of cPLA2 was a key target for SS-31 to improve lysosomal function, inhibit pyroptosis, and restore autophagy flow (Figures 6A-J and 7A-H)
。
In summary, a traditional spinal cord contusion model was used and treated by intraperitoneal injection of mitochondria-targeted antioxidant peptide (SS-31
).
Behaviorally, the authors used the BMS mouse scale, gait analysis, and bevel tests to evaluate the functional status
of individuals after spinal cord injury.
Histology, the authors used HE, Masson, MAP2, and SYN staining to determine spinal glial scar area, dendrites, and synapse number
.
In terms of molecular mechanism, the authors used Western Blot and immunofluorescence (IF) to detect the expression levels
of pyrozoosis and autophagy-related proteins.
In addition, the authors used lysosomal extraction kits and ultracentrifugation to separate lysosomal components and cytoplasmic components, and verified the occurrence of lysosomal membrane permeability by detecting the content of cathepsin in lysosomes and cytosolic components, and used enzyme-linked immunosorbent assay (ELISA) to detect the level of
cathepsin activity in different components.
In the above experiments, the authors found that SS-31 inhibits phosphorylation of cytoplasmic phospholipase A2 (cPLA2).
Therefore, the authors constructed AAV-Pla2g4a adeno-associated virus to transfect spinal cord-injured mice to further verify whether SS-31 was effective in SCI mice with the help of cPLA2
.
Finally, the authors examined the expression of key proteins in the MAPK signaling pathway and identified the signaling pathway
in which SS-31 targets cPLA2.
At the same time, SS-31 needs to be studied more deeply before it is clinically applied: (1) Since SS-31 is a polypeptide that targets mitochondria, it may aggregate more than
1000 times in the inner mitochondrial membrane.
Reactive oxygen species are also an important mechanism
for secondary injury to the spinal cord.
Therefore, the effect of SS-31 on reactive oxygen species in spinal cord injury needs to be further studied
.
(2) cPLA2 induces the production of many metabolites, including arachidonic acid, prostaglandin E2, lysophosphatidic acid, etc.
, which can mediate neuroinflammation
.
Whether SS-31 exerts anti-inflammatory effects by inhibiting downstream products of cPLA2 is unclear
.
(3) The authors' results suggest that SS-31 inhibits p38 rather than JNK or ERK1/2 signaling pathways
.
However, previous studies have shown that once the central nervous system is damaged, these three signaling pathways work synergistically to affect the phosphorylation
of cPLA2.
Thus, combination therapy with SS-31 and other specific JNK or ERK1/2 inhibitors may cut the positive feedback loop of cPLA2 stimulation and have additional or synergistic effects
in spinal cord injury.
(4) Oral administration, as a route of administration with high safety and good compliance, has gradually become the main route
of administration.
However, the structure and physiological function of the gastrointestinal tract make peptide drugs less bioavailable and half-life after oral administration
.
According to previous studies, SS-31 is usually administered by injection
.
In the future, SS-31 may be mixed with hydrogel and administered directly to the injured area
of the spinal cord.
This will allow the drug to be continuously released and exert its effects
over a long period of time.
Overall, the authors' study provides new preclinical evidence for SS-31 in the treatment of SCI; The SCI clinical transformation of SS-31 needs further research to promote
.
Original link: https://doi.
org/10.
1186/s12974-023-02690-4
Fund support: National Natural Science Foundation of China (8207219), Zhejiang Public Welfare Technology Project (LGF20H150003), Natural Science Foundation of Zhejiang Province (LY17H060009, Y21H060009).
), Wenzhou Science and Technology Bureau Fund Project (Y20210438).
First authors: Master Zhang Haojie (first from left), Master Yituo Chen (second from left), Master Feida Li (middle); Corresponding authors: Chief Physician Ni Wenfei (second from right), Associate Professor Zhou Kailiang (first from right).
(Photo courtesy of Ni Wenfei/Zhou Kailiang team)
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Selected articles from previous issues
[1] Redox Biol—Xinong Liu Zhigang et al.
revealed for the first time that methionine-restricted diet can improve AD cognitive dysfunction
[2] Science - heavy! A history of obesity induces epigenetic changes in the innate immune system and exacerbates neuroinflammation
[3] The SLEEP-Han Fang/Yu Hongjie team estimated the incidence of narcolepsy for the first time in China and found that it increased during the 2009 H1N1 influenza pandemic
[4] J Neurol—Brain microstructure changes in patients with essential tremor and their correlation with clinical features: a diffuse kurtosis imaging study
[5] Sci Adv—Duke University Jieyu Wu et al.
found that TMC channel proteins delay neuroaging through GABA signaling
[6] J Neurosci—Guan Xiaowei/Ge Feifei team of Nanjing University of Chinese Medicine discovered a new central mechanism of methamphetamine withdrawal anxiety
【7】 Neuroepidemiology—Zhao Guohua's research group analyzed the trend of disease burden of Parkinson's disease in China from 1990 to 2019
[8] The iScience—Wu Guangyan/Li Hongli/Sui Jianfeng team found that the anterior limbic cortex affects itching by regulating attention
[9] J Neurosci-NIH Zhengxiong Xi's team discovered a new mechanism of the non-addictive effect of cannabis
[10] Autophagy-Yingmei Lu/Han Feng team reveals new mechanisms of chemotherapy-related cognitive dysfunction
NeuroAI Book Club[1] NeuroAI Reading Club Launched—Explore the frontier intersection of neuroscience and artificial intelligence
The new issue sets sail, welcome to submit[1] Brain-X Cross-Brain Science: New Journal Officially Launched!
Selected from previous research training courses【1】High-scoring SCI Article and Tender Drawing (and AI Software Drawing) Seminar (January 14-15, 2023, Tencent Online Conference)
【2】Seminar on Multi-omics Data Integration Mining and Analysis (January 13-15, 2023, Tencent Online Conference)
Learning materials【1】Collection + Free - Brain Science Video Course & 9 Major Surgical Modeling and Cellular Molecular Research Handbook
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End of article
