Mol Neurobiol—Zhu Lixin/Guo Jiasong team revealed the important role of the basement membrane protein Perlecan in the recovery of blood-spinal cord barrier function after spinal cord injury

Editor—Summer Leaf

Spinal cord injury (SCI) is a common clinical central nervous system traumatic disease that causes loss of motor and sensory function, even paralysis and even death, with devastating consequences
for the patient's physical, mental and social health.
Spinal cord injury is pathophysiologically divided into primary injury and secondary injury
.
Primary injury destroys the blood-spinal cord barrier (BSCB), causing infiltrating lesions such as various inflammatory cytokines, neutrophils, and macrophages, resulting in secondary injury [1].

。 Subsequently, secondary injury triggers a cascade of reactions, including neuronal and glial cell death, inflammation, and glial fiber scarring, leading to axon regeneration failure [2].

Therefore, maintaining the integrity of BSCB is key
to reducing secondary injuries and facilitating spinal cord injury repair.

Perlecan is an important component of basement membrane and has a protective effect on
vascular integrity.
Spinal cord injury causes degradation and synthesis of basement membrane proteins, resulting in vascular wall structural abnormalities such as tight junctions (TJs), increasing the permeability of BSCB and secondary inflammatory response [3, 4].

。 Compared to collagen Collagen IV and laminin Laminin, Perlecan is the most sensitive basement membrane component to stroke response, shown Perlecan plays an important role in diseases of the central nervous system [5].

In addition, clinical evidence suggests that changes in Perlecan expression are associated with disease progression in patients with human brain arteriovenous malformations and cerebral amyloid angiopathy [6, 7].

However, changes and function of Perlecan after spinal cord injury have not been reported
.

On October 10, 2022, Professor Zhu Lixin of Zhujiang Hospital of Southern Medical University and Professor Guo Jiasong's research group of School of Basic Medicine jointly published a paper entitled in Molecular Neurobiology "Perlecan improves blood spinal cord barrier repair through the integrin β1/ROCK/MLC pathway after spinal cord injury
.
" The study observed that Perlecan is specifically localized to the blood-spinal barrier basement membrane
.
After spinal cord injury, Perlecan undergoes a process
of degradation/remodeling.
Overexpression of Perlecan can reduce BSCB permeability, reduce inflammatory response, and improve motor function and nerve regeneration
in mice after spinal cord injury.
In addition, the authors confirmed that Perlecan inhibits the downstream ROCK/MLC signaling pathway by interacting with integrin β1, reducing the breakdown of TJs-associated proteins , improve the integrity of BSCB after spinal cord injury, and provide new ideas
for the treatment of spinal cord injury.

This study first examined the localization of Perlecan protein expression in various components of the blood-spinal cord barrier structure of the mouse spinal cord (Figure 1A-F).

The results showed that Perlecan was specifically localized to the basement membrane (Laminin and Collagen IV) in the mouse spinal cord while being replaced by endothelial cells (CD31).
), vascular fibroblasts/pericytes (ACTA2), and astrocyte foot processes (GFAP) are surrounded, but not expressed in microglia
.
In addition, the degradation and remodeling of Perlecan can be observed after spinal cord injury (Figure 1G-J).

This suggests that Perlecan protein is associated
with blood-spinal cord barrier function after spinal cord injury.

Fig.
1 Perlecan is localized to the basement membrane and degrades/remodeling processes occur after spinal cord injury

(Source: Changnan Xie, et al.
) , Mol Neurobiol, 2022）

To further clarify the role of Perlecan in spinal cord injury, the authors used the SAM system of CRISPR/Cas9 to overexpress Perlecan and model
spinal cord injury 。 Mouse movement, neuronal survival and axon regeneration were detected by swimming experiments, footprint experiments, immunofluorescence staining, and electron microscopy
experiments.
Results showed that overexpression of Perlecan improved motor function recovery and axon regeneration after spinal cord injury (Figure 2).

It is suggested that Perlecan protein promotes motor and neurological recovery after spinal cord injury
.

Fig.
2 Overexpression of Perlecan promotes motor and neurological recovery in spinal cord injury

(Source: Changnan Xie, et al.
) , Mol Neurobiol, 2022）

Previous studies have shown that the increase in inflammatory cells and factors and the recruitment of immune cells due to spinal cord injury are closely related to the destruction of the blood-spinal cord barrier basement membrane [8].

Therefore, the authors examined the inflammatory response of overexpression of Perlecan after spinal cord injury
.
In order to verify this, the research team found that overexpression of Perlecan reduced macrophage/microglia and leukocyte infiltration after spinal cord injury and promoted the expression of anti-inflammatory factors through immunofluorescence and qPCR experiments (Figure 3).

Fig.
3 Overexpression of Perlecan reduces inflammation after spinal cord injury

(Source: Changnan Xie, et al.
, Mol Neurobiol, 2022).

To investigate whether Perlecan reduces the inflammatory response by affecting the permeability of BSCB, the authors performed Evans blue staining and immunofluorescence and found that Evans blue and fibronectin () after overexpression of Perlecan reduced spinal cord injury Fibronectin) leak (Figures 4A-C).

In addition, western blotting and electron microscopy found that TJs were destroyed after spinal cord injury, while overexpression of Perlecan reduced TJs degradation and improvement TJs structure (Figure 4D-E).

These results suggest that Perlecan improves BSCB permeability
by reducing the disassembly of TJs after spinal cord injury.

Fig.
4 Disassembly of TJs after overexpression of Perlecan reduces spinal cord injury to improve BSCB permeability

(Source: Changnan Xie, et al.
) , Mol Neurobiol, 2022）

Previous studies have shown that stress fiber formation mediated by the ROCK/MLC signaling pathway can result from centripetal tension on the binding of cell-cell/cell-extracellular matrix interactions by contracting actin TJs open and BBB permeability increase[9,10].

To investigate whether Perlecan's improvement in TJs structure and BSCB permeability is via the ROCK/MLC signaling pathway, the authors passed Western blotting and immunofluorescence experiments, observed that overexpression of Perlecan reduced ROCK1 and P-MLC expression (Fig 5）
。 In addition, endothelial cell integrin β1 interacts with the extracellular matrix to stabilize TJs and BBB/BSCB integrity [11,12].

。 The author passes qPCR, western blotting, and in vivo injection of the integrin beta1 inhibitor GRGDSP inhibit integrin beta1 Function, it was found that overexpression of Perlecan promoted integrin β1 expression, and in addition to reverse ZO-1 in spinal cord injury mice in the GRGDSP administration group , P-MLC expression and BSCB permeability (Figure 5).

These results suggest that Perlecan inhibits the ROCK/MLC signaling pathway by interacting with interginβ1, reducing TJs after spinal cord injury Disassembly of improves BSCB permeability
.

Fig.
5 Overexpression Perlecan improves BSCB after TJs disassembly after spinal cord injury is reduced by interginβ1/ROCK/MLC signaling pathway Permeability

(Source: Changnan Xie, et al.
) , Mol Neurobiol, 2022）

Fig.
6 Pattern of Perlecan BSCB repair after spinal cord injury

(Source: Changnan Xie, et al.
) , Mol Neurobiol, 2022）