Ablation of GSDMD Improves Outcome of Ischemic Stroke Through Blocking Canonical and Non-canonical Inflammasomes Dependent Pyroptosis in Microglia

So we need human clinical trial followup. Have your stroke hospital ensure that gets done. If it doesn't have the board of directors fired. 

Ablation of GSDMD Improves Outcome of Ischemic Stroke Through Blocking Canonical and Non-canonical Inflammasomes Dependent Pyroptosis in Microglia

Kankai Wang^1,2, Zhezhe Sun^1,3, Junnan Ru^1,2, Simin Wang^1,2, Lijie Huang^1,2, Linhui Ruan^1,2, Xiao Lin^1,2, Kunlin Jin⁴, Qichuan Zhuge^1,2* and Su Yang^1,2*

• ¹Zhejiang Provincial Key Laboratory of Aging and Neurological Disorder Research, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
• ²Department of Neurosurgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
• ³Department of Cerebrovascular, The Affiliated Hospital of Medical School of Ningbo University, Ningbo, China
• ⁴Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, United States

Ischemia/reperfusion (I/R) injury is a significant cause of mortality and long-term disability worldwide. Recent evidence has proved that pyroptosis, a novel cell death form, contributes to inflammation-induced neuron death and neurological function impairment following ischemic stroke. Gasdermin D (GSDMD) is a newly discovered key molecule of cell pyroptosis, but its biological function and precise role in ischemic stroke are still unclear. The present study investigates the cleavage activity of GSDMD, localization of pyroptotic cells, and global neuroinflammation in gsdmd^−/− mice after I/R. The level of cell pyroptosis around the infarcted area was significantly increased in the acute phase of cerebral I/R injury. The ablation of GSDMD reduced the infraction volume and improved neurological function against cerebral I/R injury. Furthermore, we confirmed I/R injury induced cell pyroptosis mainly in microglia. Knockdown of GSDMD effectively inhibited the secretion of mature IL-1β and IL-18 from microglia cells but did not affect the expression of caspase-1/11 in vitro and in vivo. In summary, blocking GSDMD expression might serve as a potential therapeutic strategy for ischemic stroke.

Introduction

According to systematic analysis studies of the global burden of disease (GBD) between 1990 and 2017, acute ischemic stroke has become the leading cause of mortality and disability worldwide (1–3), causing severe economic and healthy burdens. Intravenous thrombolysis and selective mechanical thrombectomy are the optimal therapeutic measures for ischemic stroke up till now (4, 5); however, the applicable proportion of intravenous thrombolysis only accounts for 3% of total ischemic stroke patients. Therefore, further research on the pathogenesis and interventions of ischemic stroke is urgently needed.

The primary injury of ischemic stroke is mainly caused by vascular occlusion which leads to neuron death and release of damage-associated molecular patterns (DAMPs) in focal ischemic tissue. A secondary immune response is subsequently induced in the injury area, characterized by activation of resident cells (mainly microglia), recruitment of peripheral cells (neutrophils, monocytes/macrophages, and other cells) (6, 7), and rapid induction of cascaded events, including the release of pro-inflammatory mediators, blood-brain barrier (BBB) damage, brain edema, and nerve cell death (6, 8). It is reasonable to speculate that blocking the release of inflammatory mediators may be beneficial for stroke recovery.

Pyroptosis, a newly discovered proinflammatory programmed cell death, has drawn increasing attention for its unique characteristics, such as cell swelling, bulging of the plasma membrane, secretion of inflammatory cytokines, and cell lysis (9). The pyroptotic process is dependent on caspase cleavage and accompanied by the maturity and release of pro-inflammatory mediators such as IL-1β and IL-18 (10, 11). The morphological characteristics, occurrence, and regulation mechanisms of pyroptosis are different from other types of cell death such as apoptosis and necrosis. Nucleotide-binding oligomerization domain like receptor (NLR) family proteins serve as sensors that recognize DAMPs and pathogen-associated molecular patterns (PAMPs), including high cytosolic Ca²⁺ with reduced K⁺ concentrations, extracellular ATP, mitochondrial dysfunction, and lysosomal rupture (10). These stimuli initiate caspase-dependent canonical or non-canonical inflammasome assembly (12), which subsequently cleaves gasdermin, a recently discovered pyroptosis effector. The N-terminal of gasdermin forms pores on the cell membrane, causing the release of mature inflammatory mediators into the extracellular matrix, and eventually leading to a severe inflammatory cascade reaction (13). Increasing evidence indicates that pyroptosis is induced in central nervous system disease including ischemic stroke (14, 15), traumatic brain injury (TBI) (16, 17), multiple sclerosis (MS) (18), Alzheimer's Disease (AD) (19), and Parkinson's disease (PD) (20). Illuminating the pyroptotic procedure would speed up the development of a cure for those diseases.

Gasdermin D (GSDMD), a 487 amino acid cytoplasmic protein, has been discovered to form membrane pore and act as a key effector for pyroptosis. N-terminal of GSDMD (GSDMD-N) is responsible for pore-forming activity, while the C-terminal domain (GSDMD-C) exerts autoinhibition on GSDMD-induced pyroptosis by binding to the N-terminal (21–23). Studies have confirmed that GSDMD participates in a series of pathologically pyroptotic events (21, 23), including ischemia/reperfusion (I/R) injury-induced pyroptosis. Experimental findings by Lee et al. showed that increased expression of NLRP3 inflammasome components and GSDMD peaked at 48 h after penetrating ballistic-like brain injury (24). Inhibition of caspase-1 mediated pyroptosis by limiting apoptosis-associated speck-like protein containing a CRAD (ASC) oligomerization and GSDMD cleavage resulted in suppressed expression of IL-1β and IL-18, and subsequent alleviation of BBB-disruption and brain injury (16, 17). Many studies have revealed the involvement of pyroptosis in cerebral injury (25, 26). However, the main cell type for pyroptosis, as well as the detailed information regarding GSDMD cleavage and its contribution to global inflammatory profile, still needs to be clarified. This study, utilizing gsdmd^−/− mice and gsdmd^−/− microglia cells, highlights the precise function of GSDMD, main pyroptotic effector cell, and canonical or non-canonical inflammasome-dependent pyroptosis pathway in cerebral I/R injury, aiming to elucidate the mechanisms underlying GSDMD-mediated pyroptosis and to exploit new therapeutic targets for ischemic stroke.