Study of inflammatory-related miRNAs after acute CNS injuries

Voelz, Clara Viola; Zimmer-Bensch, Geraldine Marion (Thesis advisor); Beyer, Cordian (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

Acute central nervous system (CNS) injuries lead to irreversible cell death, leaving patients with life-long disabilities or death. Acute CNS injuries can occur due to an occluded blood vessel as observed in cases of ischemic stroke (IS) or due to a traumatic impact, as is often the case in spinal cord injuries (SCI). The resulting damage is not limited to the area of injury, but occurs at a systemic level. As a result, the deregulation of a high number of genes has been described, among them: microRNAs (miRNAs). miRNAs are non-coding RNAs that bind to complementary mRNA strands and inhibit the translational process. At the injury site, an alteration of miRNA levels has been described following acute CNS injuries. This thesis seeks to investigate wheter the impact to the CNS also triggers altere miRNA levels on a systemic level. In pursuit of this, I used two different experimental set-ups of experimental middle cerebral artery occlusion (tMCAo) in rats, and spinal cord injury (SCI) in mice for in vivo studies. By insertion of a filament to the middle cerebral artery for 1 h, tMCAo was performed. To replicate a contusion of the thoracal spine, paralysis of the hindlimbs was initiated in mice. After 6, 12, 24, and 72 h, the animals were sacrified, with blood and tissue samples being collected. A third approach was hypoxic stimulation of human microglia cells (HMC-3 cell line) in vitro. Following tMCAo, an affymetrix array was conducted to assess the miRNA alterations in the cerebral cortex. Subsequently, miR-223-3p, miR-155-5p, miR-448-5p, and miR-3473 were chosen for closer examination in different brain and body parts. In cases of SCI miR-3473 was replaced with miR-124-3p, due to the former being undetectable. Expression levels of the miRNAs were measured via qRT-PCR in different brain and body parts to observe the range of miRNA alterations. Following tMCAo, an alteration of miRNAs was observed especially in the cerebral cortex at all time points and additionally in the amygdala. In cases of miR-223-3p and miR-448-5p, the alterations were also visible at the contralateral brain hemisphere. In the blood serum, an increase of miR-223-3p at 6 h was observed, whilst miR-155-5p, and miR-3473 were downregulated at all time points. Following SCI, the alterations were less pronounced. At the injury site, miR-223-3p, and miR-155-5p increased after 72 h. Also, alterations in the rostral and caudal parts of the spinal cord and the blood serum where detectable for miR-223-3p, miR-155-5p, and miR-124-3p. In peripheral organs, only a small downregulation for miR-155-5p was observed at 12 h. Not only miRNAs displayed a systemic alteration, additionally inflammation-related mRNAs such as Nlrp3, Socs1, Socs3 and Vegfa displayed upregulations in all brain and body regions following tMCAo, suggesting that the systemic alterations could be connected to immune responses. The miRNAs pass through a multistep maturation process before they can perform translational silencing. Following tMCAo and SCI, no alterations in the gene expression of involved molecules (Drosha, Dgcr8, Xpo5, Dicer, Tarbp2, Ago2) could be observed. But following in vitro hypoxia, an increase of the enzymes DROSHA, DICER, and AGO2 was found on both, transcriptional and translational levels. Additionally, a co-localization of AGO2 and G3BP, a marker for stress granules, was detected. To conclude, future studies, seeking to expand on this work should not only focus on miRNA target interaction in the cerebral cortex and the thoracal spinal cord but widen their research to the background of miRNA transportation/deregulation processes and the influence on processes like the systemic activation of the immune system.