Elsevier

Life Sciences

Volume 285, 15 November 2021, 119944
Life Sciences

Legumain knockout improved cognitive impairment via reducing neuroinflammation in right unilateral common carotid artery occlusion mice

https://doi.org/10.1016/j.lfs.2021.119944Get rights and content

Highlights

  • rUCCAO significantly increases the expression of legumain in the hippocampus.

  • Legumain knockout protects against behavioural dysfunctions in rUCCAO mice.

  • Legumain knockout prevents rUCCAO-induced synaptic plasticity impairments.

  • Legumain knockout suppresses the neuroinflammatory responses in rUCCAO mice.

  • Legumain knockout alleviates pyroptosis in rUCCAO mice.

Abstract

Aims

Chronic cerebral hypoperfusion (CCH) is a state of chronic cerebral blood flow reduction, and it is the main cause of cognitive impairment and neurodegenerative diseases. The abnormal upregulation of legumain, a lysosomal cysteine protease, trigger synaptic plasticity impairment and neuroinflammation, which are involved in the underlying pathophysiology of CCH. At present, few studies have reported the role of legumain in cognitive impairment caused by CCH. In our study, we aimed to investigate the involvement of legumain knockout in cognitive function and neuroinflammation in a CCH mouse model.

Main methods

In this study, right unilateral common carotid artery occlusion (rUCCAO) was used to simulate the pathological state of cerebral ischemic injury. Various behavioural tests were executed to assess cognitive performance. In vivo electrophysiological recordings were used to measure synaptic functions. Western blotting, Golgi staining, haematoxylin/eosin staining, and immunofluorescence assays were conducted to examine pathological changes and molecular mechanisms.

Key findings

The data showed that the level of legumain was significantly increased in the hippocampus of mice subjected to rUCCAO. Legumain knockout significantly improved cognitive function and synaptic plasticity induced by rUCCAO, suggesting that legumain knockout-regulation effectively protected against CCH-induced behavioural dysfunctions. Moreover, legumain knockout suppressed rUCCAO-induced microglial activation, reduced the abnormal expression of inflammatory cytokines and the inflammasome complex, and impeded the activation of P65 and pyroptosis.

Significance

These findings suggest that legumain is an effective regulator of CCH, and may be an ideal target for the development of cerebral ischemia treatments in the future.

Graphical abstract

Legumain knockout blocked microglia activation by inhibiting P65 signalling pathways, and further reduced inflammatory cytokines expression and pyroptosis. As a result, legumain knockout alleviated synaptic damage and cognitive impairment induced by right unilateral common carotid artery occlusion (rUCCAO).

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Introduction

Vascular dementia (VD) is the second most common cause of dementia worldwide, following Alzheimer's disease (AD), accounting for 20% of dementia cases [1]. Due to high morbidity and lethality, it causes serious public health problems and socio-economic burden [2]. Chronic cerebral hypoperfusion (CCH), also known as chronic cerebral ischemia, is one of the main causes of vascular dementia, and causes progressive cognitive impairment due to interference with the circulatory system. CCH has been shown to lead to neuronal damage and death, increasing the incidence of neurodegeneration [3], [4], which is involved in neurogenic inflammation and other mechanisms, such as the accumulation of amyloid, calcium influx, glutamate excitotoxicity, mitochondrial dysfunction and oxidative stress [5], [6], [7], [8].

Synaptic plasticity dysfunction and neuronal loss are important pathological features of cognitive impairment in progressive neurodegenerative diseases [9], whether in patients with ischemic brain injury or animal models of cerebral ischemia that exhibit impairments in synaptic plasticity [10]. The negative effects of CCH on the transmission and structure of synapses may induce changes in neural circuitry and affect the formation of memories. The expression of synaptic protein is decreased in patients with ischemic brain injury [11]. Additionally, the research in animal models of stroke and other ischemic injury diseases has shown that the dendritic spine density is significantly decreased [12], [13], [14]. Symptoms of cerebral ischemia were significantly alleviated by improving the function of synaptic plasticity in cortical neurons. After using melatonin to treat the transient occlusion of the right middle cerebral artery in mice, the loss of presynaptic protein and dendritic spine density in the ischemic territory was improved in the brain [15].

Legumain, also known as asparagine endopeptidase (AEP), is a cysteine protease that hydrolyses the c-terminal substrate of asparagine residues. Legumain is abnormally activated in the aging brain and participates in the aggregation of pathological proteins and neuronal damage. In pathological settings, legumain has been implicated in ischemic stroke [16], atherosclerosis [17], and other diseases [18]. Findings in the brain of AD and PD patients have shown an increased level of legumain [19]. Legumain also plays an important role in neuroinflammation during ischemic stroke and acts as an attractant for chemotherapy to invade inflammatory cells after stroke [16]. However, the molecular mechanism underlying legumain activation in cerebral ischemic pathologies remains unclear.

The inflammatory response is actively involved in mediating multiple human diseases, including ischemic reperfusion injury, Gaucher's disease, and neurodegenerative diseases [20], [21], [22]. Ischemia initiates brain inflammation, including the activation of microglia and acute inflammation, which exacerbates ischemic lesions. Under ischemic conditions, ischemia-induced mediators provoke the transcription of various genes, such as P65, and the secretion of various cytokines [23], [24]. The activation of inflammatory cytokines worsens the ischemic insult. Furthermore, inhibiting neuroinflammation can alleviate cognitive impairment [25].

Recent evidence has shown that pyroptosis contributes to inflammation-induced neuron death and neurological function impairment following ischemic stroke. Cerebral ischemic injury activates the multi-protein inflammasome complex, comprised of caspase-1, NLRP3, and ASC, triggering pyroptosis in neuron cells. Inhibition of caspase-1-mediated pyroptosis by limiting NLRP3 inflammasome oligomerisation and gasdermin-D cleavage results in the alleviation of blood-brain barrier disruption and brain injury [26], [27].

In light of previous studies, the hypothesis was raised that legumain knockout protects against cognitive deficits through inhibiting neuroinflammation in CCH. We preliminarily speculated that this may be associated with pyroptosis in neurons. Right unilateral common carotid artery occlusion (rUCCAO) was applied in order to construct the CCH model. Behavioural tests were performed, including the novel object recognition test (NOR) and Morris water maze test (MWM). In vivo electrophysiological recordings were carried out. Either an immunofluorescence assay or Western blotting was used to measure the level of synapse-related proteins and inflammation-related indicators.

Section snippets

Animals

The legumain knockout mice were generated from legumain heterozygous parents on a C57BL/6 background (obtained from Cyagen Biosciences Inc., Guangzhou, China) and compared with their wildtype littermates. A polymerase chain reaction-based method was used to genotype these mice [28]. According to the standard guidelines approved by the Animal Ethics Committee of Nankai University, animals were housed and maintained with free access to food and water at room temperature (23 ± 2 °C) in the Medical

Legumain knockout ameliorates the cognitive disabilities of rUCCAO mice

We investigated whether rUCCAO altered the expression of legumain in the hippocampus. Western blotting revealed that the legumain was markedly increased in the WT-rUCCAO group compared to the WT-Sham group (1.09 ± 0.09 vs. 3.53 ± 0.22, p < 0.001, Fig. 1b and c), and this effect was blocked in the legumain knockout mice, showing that our legumain-knockout mice model was successful.

In addition, we examined the effect of the legumain knockout on rUCCAO-induced behavioural changes. In the NOR test,

Discussion

The brain is the organ of the human body that is the most sensitive to ischemia and cerebral ischemia leads to the impairment of local brain function. Finally, the decrease in cerebral perfusion and cerebral blood flow leads to the decline of cognitive function. Legumain plays a role in the regulation of microglial activation and inflammation in the brain. In this study, we demonstrated an abnormal expression of legumain in the hippocampus of rUCCAO mice. Legumain knockout-regulation protected

Conclusion

In this study, we determined the unique role of legumain in a mouse model of chronic cerebral hypoperfusion. These data suggest that legumain knockout prevents behavioural dysfunction, avoids synaptic plasticity impairments and inhibits neuroinflammation by regulating the P65 signalling pathway. Additionally, legumain knockout may prevent pyroptosis. Our research shows that legumain is a potential novel target for chronic cerebral hypoperfusion therapy with great significance.

CRediT authorship contribution statement

Xueqing Chai: Conceptualization, Methodology, Writing – original draft. Xiaolin Li: Methodology, Formal analysis, Investigation. Wenxin Zhang: Formal analysis, Investigation. Xiaoyue Tan: Writing – review & editing. Haiyun Wang: Writing – review & editing. Zhuo Yang: Writing – review & editing, Funding acquisition, Supervision.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (81771979).

Data statement

All the data generated or analysed during this study are available from the corresponding author on reasonable request.

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