Elsevier

Neuroscience

Volume 434, 10 May 2020, Pages 44-54
Neuroscience

Research Article
Stress Induced tRNA Halves (tiRNAs) as Biomarkers for Stroke and Stroke Therapy; Pre-clinical Study

https://doi.org/10.1016/j.neuroscience.2020.03.018Get rights and content

Highlights

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    tRNA is cleaved into 5′ and 3′tiRNA halves under stress and can be protective or toxic.

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    tRNA is cleaved during ischemia–reperfusion (I/R) injury in rats.

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    Neuroprotective therapy reduces the level of tRNA cleavage.

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    Several enzymes that regulate tRNA stability were dysregulated following I/R.

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    tiRNAs can be used as biomarkers for I/R and tRNA cleavage mechanism can be a target for stroke therapy.

Abstract

tiRNAs are small non-coding RNAs generated by angiogenin-mediated tRNA cleavage during cellular stress. Some tiRNAs were shown to be cytoprotective, while other reports indicate that the generation of tiRNAs is cytotoxic. We used rat model of focal cerebral ischemia–reperfusion (I/R) injury to study the generation and regulation of tiRNAs following in vivo I/R and the impact of neuroprotective therapy on their generation. tiRNAs were induced after I/R and Minocycline therapy reduced global tiRNA levels. Our results showed that tRNA cleavage is tRNA species specific, and neuroprotective treatment does not affect all tiRNA species. We also evaluated the temporal changes in several tRNA modifying enzymes and showed a correlation between their expression and tRNA cleavage. In conclusion, we show that tiRNAs can serve as biomarkers for stroke and stroke therapy, further adding them to the repertoire of tools that can be used to monitor and treat stroke.

Introduction

Stroke is the third leading cause of early mortality according to the global disease burden report (GBD 2017 Disease and Injury Incidence and Prevalence Collaborators, 2018). In the year 2017, the incidence of all types of stroke (ischemic and hemorrhagic) was 11931.1 thousand cases worldwide, with acute ischemic stroke making up the majority of these cases with an incidence of 7737.5 thousand cases (GBD 2017 Disease and Injury Incidence and Prevalence Collaborators, 2018). Even though previous analysis has shown a decline in stroke induced mortality and disability between the year 1990 and 2013, the global burden of stroke was rising (Feigin et al., 2017). These studies clearly demonstrate the necessity to improve the tools currently used to manage stroke. New therapies, biomarkers and strategies for stroke management, monitory and follow-up are an ever-pressing need that must be addressed.

During the past two decades, newly discovered non-coding RNA species, such as; microRNAs, circular RNAs and long non-coding RNAs (lncRNA), have been under the spotlight as biomarkers and potential therapeutic targets for different types of stroke (Dharap et al., 2009, Liu et al., 2010, Mehta et al., 2017, Tiedt et al., 2017, Wu et al., 2017, Kim et al., 2018). In recent years, tRNA was discovered to be a source of several small RNA species (Lee et al., 2009, Martinez et al., 2017, Lyons et al., 2018, Rashad et al., 2020). Of these small RNAs, two species are the most studied; the stress-induced angiogenin-cleavage mediated tRNA halves (tiRNAs) (Elkordy et al., 2018, Elkordy et al., 2019, Rashad et al., 2020) and the microRNA-like tRNA derived fragments (tRFs) (Goodarzi et al., 2015, Martinez et al., 2017).

tiRNAs were shown to be upregulated following ischemia in vitro, using neuronal cell culture (Elkordy et al., 2018, Elkordy et al., 2019), and in vivo, using rodent model of focal ischemia–reperfusion injury (I/R) (Li et al., 2016). The upregulation of 5′tiRNA fragments was shown to inhibit endothelial angiogenesis following ischemic stroke (Li et al., 2016), indicating that these small RNA fragments may play a role in modulating cerebral responses to ischemic injury. Recently, we have shown that following neuroprotective therapy using Minocycline in an in vitro model of I/R, tRNA cleavage was downregulated (Elkordy et al., 2019). This observation demonstrated a potential for tiRNAs to serve as biomarkers for stroke therapy or outcome prediction (Elkordy et al., 2019).

In this work we wanted to evaluate the potential of tiRNAs to be used as biomarkers for in vivo I/R injury, as well as their potential to be used as biomarkers for successful neuroprotective therapy.

Section snippets

Animals

Transient middle cerebral artery occlusion (tMCAO) was induced in 8 weeks old, 180–200 g male Wistar rats as previously described (Faheem et al., 2019). Animals were bought from a local vendor (Kumagai-Shoten Sendai) and housed in a controlled environment with 12 h light/dark cycle and temperature regulated at 23 °C. Animals were allowed to acclimatize for at least 24 h before any experimental procedures. All experiments were conducted according to protocols approved by the animal care facility

tRNA is cleaved after tMCAO

First, we wanted to assess tRNA cleavage after tMCAO, and the time-point of maximum cleavage. SYBR gold staining showed that tiRNAs were upregulated maximally 24 h after I/R (Fig. 1A). INB with anti-m1A antibody also confirmed that the maximal level of tiRNAs is observed after 24 h of I/R (Fig. 1B). Interestingly, we observed a low level of tiRNA fragments using SYBR gold staining and INB even in Sham animals. This was repeatedly observed and is in-line with reported physiologic role of tiRNAs

Discussion

In this work, we show for the first time, to the best of our knowledge, a translational potential of tiRNAs to serve as biomarkers for stroke. Indeed, tRNA cleavage was reported following focal ischemia in rodents (Li et al., 2016). However, it reported in the context of ischemic and post-ischemic pathophysiology. We also show how the expression of tRNA modifying enzymes, that were shown to affect tRNA stability and cleavage, correlates with tRNA cleavage in vivo, raising a potential for future

Disclosure

The authors report no conflict of interest nor there are any ethical adherences regarding this work.

Funding

This work was partially supported by JSPS KAKENHI Grant Numbers 17H01583 for Niizuma.

Author contribution

Sato: conducted experiments, collected and analyzed data. Rashad: Conception and study design, administration, conducted experiments, analyzed the data and wrote the manuscript. Niizuma: Funding acquisition and administration. Niizuma, Tominaga and Sato: critically revised the manuscript. All authors: approved the final version of the manuscript. Rashad and Niizuma: Supervised this work.

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