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The pandemic coronavirus disease 19 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is marked by thromboembolic events and an inflammatory response throughout the body, including the brain. Employing the machine learning approach BrainDead we systematically screened for SARS-CoV-2 genome-derived single-stranded (ss) RNA fragments with high potential to activate the viral RNA-sensing innate immune receptors Toll-like receptor (TLR)7 and/or TLR8. Analyzing HEK TLR7/8 reporter cells we tested such RNA fragments with respect to their potential to induce activation of human TLR7 and TLR8 and to activate human macrophages, as well as iPSC-derived human microglia, the resident immune cells in the brain. We experimentally validated several sequence-specific RNA fragment candidates out of the SARS-CoV-2 RNA fragments predicted in silico as activators of human TLR7 and TLR8. Moreover, these SARS-CoV-2 ssRNAs induced cytokine release from human macrophages and iPSC-derived human microglia in a sequence- and species-specific fashion. Our findings determine TLR7 and TLR8 as key sensors of SARS-CoV-2-derived ssRNAs and may deepen our understanding of the mechanisms how this virus triggers, but also modulates an inflammatory response through innate immune signaling.

MicroRNAs (miRNAs) canonically regulate post-transcriptional gene expression, but they can also serve as ligands for Toll-like receptors (TLRs). These receptors and their associated signalling pathways contribute to inflammatory responses involved in various central nervous system (CNS) diseases, including Alzheimer’s disease (AD). Here, we investigated the effects of extracellularly delivered miRNA in the context of neuroinflammation. We identified several miRNAs specifically dysregulated in AD and/or neuroinflammatory states, which directly activate the single-stranded RNA sensors mouse TLR7 and human TLR7/8. Among them, extracellular miR-29a-5p induced cytokine and chemokine release from murine primary microglia, altered expression of TLR signalling elements, and enhanced Aβ phagocytosis. Furthermore, this miRNA induced neuronal injury dependent on microglial TLR7 expression, but also in a cell-autonomous fashion, in vitro. Intrathecal injection of miR-29a-5p into mice led to microglial accumulation and neuronal injury in the cerebral cortex through TLR7 after 3 days. Brains of wild-type and APP/PS1 mice, an established AD mouse model, treated with multiple intrathecal miR-29a-5p injections over 120 days exhibited changes in cytokine/chemokine expression and neuronal injury. RNAseq analysis of the cerebral cortex of both miRNA-treated genotypes revealed downregulation of MAPK-associated pathways. Our study establishes AD-associated miRNAs such as miR-29a-5p as TLR7 agonists and signalling molecules for microglia, thereby altering the neuroinflammatory response.

Background Dysregulation of microRNA (miRNA) expression in the brain is a common feature of neurodegenerative diseases. Beyond their conventional role in regulating gene expression at the post-transcriptional level, certain miRNAs can act extracellularly as signaling molecules. Our study elucidates the identity of such miRNA species serving as ligands for membrane receptors expressed in central nervous system (CNS) neurons and the impact of such miRNAs on neurons in the context of neurodegenerative disease. Methods We combined a machine learning approach with the analysis of disease-associated miRNA databases to predict Alzheimer’s disease (AD)-associated miRNAs as potential signaling molecules for single-stranded RNA-sensing Toll-like receptors (TLRs) 7 and 8. TLR-expressing HEK-Blue reporter cells, primary murine microglia, and human THP-1 macrophages were used to validate the AD miRNAs as ligands for human and mouse TLR7 and/or TLR8. Interaction between mouse cortical neurons and extracellularly applied AD miRNAs was analyzed by live cell imaging and confocal microscopy. Transcriptome changes in cortical neurons exposed to AD miRNAs were assessed by RNAseq and RT-qPCR. The extracellular AD miRNAs’ effects on CNS neuron structure were investigated in cell cultures of murine primary cortical neurons and iPSC-derived human cortical neurons by immunocytochemistry. We employed a mouse model of intrathecal injection to assess effects of AD miRNAs acting as signaling molecules on neurons in vivo. Results We identified the AD-associated miRNAs miR-124-5p, miR-92a-1-5p, miR-9-5p, and miR-501-3p as novel endogenous ligands for TLR7 and/or TLR8. These miRNAs being extracellularly stable and active were taken up by murine cortical neurons via endocytosis and induced changes in neuronal inflammation-, proliferation-, and apoptosis-related gene expression. Exposure of both murine and human cortical neurons to the AD-associated miRNAs led to alterations of dendrite and axon structure, synapse protein expression, and cell viability in a sequence-dependent fashion. Extracellular introduction of the AD miRNAs into the cerebrospinal fluid of mice resulted in both changes in neuronal structure and synapses, and neuronal loss in the cerebral cortex. Most of the observed extracellular miRNA-induced effects on cortical neurons involved TLR7/8 signaling. Conclusion Neurodegenerative disease-associated miRNAs in extracellular form act as signaling molecules for CNS neurons including human cortical neurons, thereby modulating their structure and viability.

Toll-like receptor 8 (TLR8) in humans senses RNA degradation products and elicits an inflammatory immune response. In contrast, the ligand specificity and function of its murine counterpart mTLR8, long considered non-functional, remain poorly defined. Here, we established an agonist combination model of poly-deoxythymidine (poly-dT) DNA and TLR7/8 binding site 1 agonists such as uridine or the benzazepine compound TL8-506, which activates mTLR8, while suppressing mTLR7 signaling. Extensive agonist analysis based on this model revealed that 2’,3’-cyclic guanosine monophosphate (2’,3’-cGMP) serves as a natural ligand for mTLR8, suggesting functionality of its binding site 1 without engagement of site 2. In addition, 2’,3’-cyclic uridine monophosphate, bacterial single-stranded (ss) DNA, double-stranded (ds) DNA fragments, microRNAs, ssRNA derived from HIV1, SARS-CoV-2, or bacterial sources all potentiate mTLR8 sensing of site 1 agonists. All these stimuli induce distinct inflammatory responses from murine macrophages and microglia via TLR8. In vivo, intrathecal administration of TL8-506 and poly-dT led to microglial accumulation and neuronal injury in the murine cerebral cortex through TLR8, highlighting the potential neuropathological consequences of mTLR8 activation. Taken together, our study defines mTLR8 as a nucleic acid sensor detecting 2’,3’-cGMP as well as combinations of ssDNA, dsDNA, ssRNA fragments, 2’,3’-cyclic nucleotide monophosphates, and nucleosides, with implications for host defense and neuroinflammation.

Conservation programmes are always limited by available resources. Careful planning is therefore required to increase the efficiency of conservation and gap analysis can be used for this purpose. This method was used to assess the representativeness of current ex situ and in situ conservation actions of 234 priority crop wild relatives (CWR) in Indonesia. This analysis also included species distribution modelling, the creation of an ecogeographical land characterization map, and a complementarity analysis to identify priorities area for in situ conservation and for further collecting of ex situ conservation programmes. The results show that both current ex situ and in situ conservation actions are insufficient. Sixty-six percent of priority CWRs have no recorded ex situ collections. Eighty CWRs with ex situ collections are still under-represented in the national genebanks and 65 CWRs have no presence records within the existing protected area network although 60 are predicted to exist in several protected areas according to their potential distribution models. The complementarity analysis shows that a minimum of 61 complementary grid areas (complementary based on grid cells) are required to conserve all priority taxa and 40 complementary protected areas (complementary based on existing protected areas) are required to conserve those with known populations within the existing in situ protected area network. The top ten of complementary protected areas are proposed as the initial areas for the development of CWR genetic reserves network in Indonesia. It is recommended to enhanced coordination between ex situ and in situ conservation stakeholders for sustaining the long term conservation of CWR in Indonesia. Implementation of the research recommendations will provide for the first time an effective conservation planning of Indonesia’s CWR diversity and will significantly enhance the country’s food and nutritional security.