Profiling Temporal Changes of the Pineal Transcriptomes at Single Cell Level Upon Neonatal HIBD.

Xin Ding,Li-Xiao Xu,Wenxi Huang,Xiaofeng Yang,Mei Li,Po Miao,Xing Feng,Jian Huang,Qiuyan Tian,Lauren S Hayashi,Chen-Xi Feng,Qin Liu,Fuyong Li,Bin Sun,Tao Pan

doi:10.3389/fcell.2022.794012

Abstract

Neonatal hypoxic-ischemic brain damage (HIBD) often results in various neurological deficits. Among them, a common, yet often neglected, symptom is circadian rhythm disorders. Previous studies revealed that the occurrence of cysts in the pineal gland, an organ known to regulate circadian rhythm, is associated with circadian problems in children with HIBD. However, the underlying mechanisms of pineal dependent dysfunctions post HIBD remain largely elusive. Here, by performing 10x single cell RNA sequencing, we firstly molecularly identified distinct pineal cell types and explored their transcriptome changes at single cell level at 24 and 72 h post neonatal HIBD. Bioinformatic analysis of cell prioritization showed that both subtypes of pinealocytes, the predominant component of the pineal gland, were mostly affected. We then went further to investigate how distinct pineal cell types responded to neonatal HIBD. Within pinealocytes, we revealed a molecularly defined β to α subtype conversion induced by neonatal HIBD. Within astrocytes, we discovered that all three subtypes responded to neonatal HIBD, with differential expression of reactive astrocytes markers. Two subtypes of microglia cells were both activated by HIBD, marked by up-regulation of Ccl3. Notably, microglia cells showed substantial reduction at 72 h post HIBD. Further investigation revealed that pyroptosis preferentially occurred in pineal microglia through NLRP3-Caspase-1-GSDMD signaling pathway. Taken together, our results delineated temporal changes of molecular and cellular events occurring in the pineal gland following neonatal HIBD. By revealing pyroptosis in the pineal gland, our study also provided potential therapeutic targets for preventing extravasation of pineal pathology and thus improving circadian rhythm dysfunction in neonates with HIBD.

Highlights

Neonatal hypoxic-ischemia brain damage (HIBD), which might occur during fetal development, labor or in the postnatal period, often results in permanent deficits such as cerebral palsy and developmental delay (Ferriero, 2004; Shalak and Perlman, 2004)
Dependent on the differential expression of distinct marker genes, the pinealocytes, astrocytes and microglia cells are further divided into multiple subtypes (Figures 1A–C)
When we examined changes of cellular components of the pineal gland post neonatal hypoxic-ischemic brain damage (HIBD), a prominent change is the sharp decrease of both α- and β-microglia cells (Figure 2C), which actively respond to neonatal HIBD (Figure 6)

Summary

Introduction

Neonatal hypoxic-ischemia brain damage (HIBD), which might occur during fetal development, labor or in the postnatal period, often results in permanent deficits such as cerebral palsy and developmental delay (Ferriero, 2004; Shalak and Perlman, 2004). Classic studies revealed a significant delay of the time of SWC onset by using the amplitude-integrated electroencephalography (aEEG) recording (Osredkar et al, 2005; Takenouchi et al, 2011). Such delayed onset of SWC was even observed in hypothermic-treated neonates with HIBD (Takenouchi et al, 2011). Circadian dysfunction and chronic sleep problems are not uncommon features in children with neonatal HIBD. To investigate the underlying mechanisms of HIBD caused circadian problems, we performed whole brain screening and discovered that sleep and circadian rhythm issues in children with HIBD is correlated with the occurrence of cysts in the pineal gland (Ding et al, 2016), an organ known to control circadian rhythm (Borjigin et al, 2012).

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