Cholinergic-targeted transfer RNA fragments regulate the post-stroke immune response

Katarzyna Winek ^1,2,3 Sebastian Lobentanzer ⁴ Bettina Nadorp ^1,5 Claudia Dames ⁶ Odilo Engel ³ Gilli Moshitzky ^1,2 Benjamin Hotter ⁷ Christian Meisel ⁶ Evan DeCorte ⁸ Sima Dubnova ^1,2 David Greenberg ² Sagiv Shifman ⁹ Jochen Klein ⁴ Shani Shenhar-Tsarfaty ¹⁰ Andreas Meisel ⁷ Hermona Soreq ^1,2

¹The Edmond & Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, Israel
²The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Israel
³The Department of Experimental Neurology, Charité-Universitätsmedizin Berlin, Germany
⁴Department of Pharmacology, College of Pharmacy, Goethe University, Germany
⁵The Grass Center for Bioengineering, Benin School of Computer Science and Engineering, The Hebrew University of Jerusalem, Israel
⁶The Institute for Medical Immunology, Charité-Universitätsmedizin Berlin, Germany
⁷Neurocure Clinical Research, Center for Stroke Research Berlin and the Department of Neurology, Charité-Universitätsmedizin Berlin, Germany
⁸Department of Mathematics and Statistics, McGill University, Canada
⁹The Department of Genetics, The Hebrew University of Jerusalem, Israel
¹⁰Department of Internal Medicine "C", "D" and "E", Tel Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel Aviv University, Israel

The small RNA repertoire has recently been expanded by fragments originating from transfer RNA (tRFs), which may play roles similar to microRNAs (miRs) in post-transcriptional regulation of gene expression, especially in reactions to stress and trauma. Stroke is one of leading causes of death and disability, and stroke-induced inflammation may be blocked by cholinergic signaling, which in turn increases the susceptibility to infections, leading to poor outcome. However, the molecular mechanisms of these events remain unknown. Seeking the molecular regulators involved, we performed RNA-sequencing of whole blood from a cohort of stroke patients compared to age-matched healthy controls, and discovered concomitant increases of tRFs and decreases in miRs enriched in motifs complementary to cholinergic transcripts compared to matched controls.

Segregating tRFs, miRs and coding transcripts by weighted gene co-expression network analysis (WGCNA) identified heterogenous modules linked with immunological and recovery parameters. An in-depth analysis based on the transcriptional regulatory networks of blood cells from our dataset revealed that the CD14+monocytes and CD4+ T cells are the major cells involved in cholinergic signaling, expressing molecules from the interleukin-6 family and their receptors. Furthermore, blood cells from experimental stroke mice (middle cerebral artery occlusion, MCAo) also showed altered tRFs/miRs levels compared to control animals. Our findings identify diagnostically valuable cholinergic tRF/miR co-regulators of post-stroke recovery in patients’ blood, advancing the establishment of novel personalized concepts for predicting the prospects of post-stroke recovery and advancing its therapeutics.