Japanese Encephalitis Virus Infected Microglial Cells Secrete Exosomes Containing let-7a/b that Facilitate Neuronal Damage via Caspase Activation

Abstract

21Extracellular microRNAs (miRNAs) are essential for the cell to cell communication in the 22 healthy and diseased brain. MicroRNAs released from the activated microglia upon 23 neurotropic virus infection may exacerbate CNS damage. Here, we identified let-7a and let-7b 24 (let-7a/b) as the overexpressed miRNAs in Japanese Encephalitis virus (JEV) infected 25 microglia and assessed their role in JEV pathogenesis. We measured the let-7a/b expressions 26 in JEV infected post-mortem human brains, mice brains and in mouse microglial N9 cells by the 27 qRT-PCR and in situ hybridization assay. The interaction between let-7a/b and NOTCH 28 signaling pathway further examined in Toll-like receptor 7 knockdown (TLR7 KD) mice to 29 assess the functions. Exosomes released from JEV infected or let-7a/b mimic transfected N9, and 30 HEK-293 cells were isolated and evaluated their function. We observed an upregulation of let-31 7a/b in the infected brains as well as in microglia. Knockdown of TLR7 or Inhibition of let-32 7a/b suppressed the JEV induced NOTCH activation possibly via NF-κB dependent manner 33 and subsequently, attenuated JEV induced TNFα production in microglial cells. Further, 34 exosomes secreted from JEV-infected microglial cells specifically contained let-7a/b. 35Exosomes overexpressed with let-7a/b were injected into BALB/c mice as well as co-incubated 36 with mouse neuronal (Neuro2a) cells, or primary cortical neuron resulted in caspase activation 37 leading to neuronal damage in the brain. Thus, our results provide evidence for the 38 multifaceted role of let-7a/b miRNAs and unravel the exosomes mediated mechanism for JEV 39 induced pathogenesis. 40 41 42 43MicroRNAs (miRNAs) are a class of regulatory non-coding RNAs that regulate many biological 44 processes within a cell by direct binding to mRNA, thus influence protein abundance (1). Recent 45 evidence demonstrates that miRNAs can also affect RNA virus replication and pathogenesis 46 through virus-mediated changes in the host transcriptome (2-9). Furthermore, microRNAs have 47 been detected extracellularly and circulating in the blood, CSF and other body fluids (10). These 48 exosomal microRNAs are thought to be released from cells as encapsulated in the vesicles, 49 called as exosomes. These miRNAs containing exosomes can be taken up by specific target cells 50 within the same or in remote tissues where they can exert their repressive function (11, 12). 51These characteristics make extracellular microRNAs ideal candidates for intercellular 52 communication over short and long distances (13). 53In the brain, the exosomes mediated neuron microglia communication recently discussed in few 54 reports (14-16). Most of the cells in the CNS, including neurons, astrocytes, oligodendrocytes, 55 and microglia shed exosomes. These extracellular vesicles are secreted by brain cells under both 56 normal and pathological conditions and transfer specific signal to the neighboring cells. Transfer 57 of miRNAs via exosome may act as a principal mediator for neuroinflamma...

Extracellular Vesicles as a Means of Viral Immune Evasion, CNS Invasion, and Glia-Induced Neurodegeneration

“…As active infection of neurons with ZIKV induces axonal degeneration and cell death, these findings suggest that EVs may contribute to the spread of neurodegeneration in ZIKV infections. Further, after infection with the Japanese encephalitis virus, expression of let-7a/b is increased in microglia, inducing an inflammatory phenotype and altering the content of EVs released from these cells to include let-7a/b (Mukherjee et al, 2019). When taken up by neurons, EVs containing let-7a/b subsequently cause neuronal damage through the activation of various caspases, indicating a specific mechanism by which viral infection may induce neurotoxicity via glial EVs (Figure 2B).…”

Exploring the role of brain-derived extracellular vesicles in viral infections: from pathological insights to biomarker potential

“…Such neuronal damage may result in neurological manifestations, such as microcephaly, in the developing embryonic brains (Zhou et al, 2019). Similar observations were done on EVs derived from Japanese Encephalitis virus (JEV) -infected microglial cells which, upon internalization by neurons, induced caspase activation and neuronal injury (Mukherjee et al, 2019).…”

Exosomes in Parkinson: Revisiting Their Pathologic Role and Potential Applications

“…In the upcoming sections, we have analyzed the results obtained from different delivery methods for miRNA-based therapy using exosomes as a core-shell to potentiate neuronal survival and differentiation. Thus, EXOs are considered a unique class of PD biomarkers, with less or no invasion [61,65,76,77]. Collectively, the previous data implies that the study of substances packaged in body fluids and released by EVs may help us comprehend the connection between systemic PD inflammation and its potential incrimination as a biomarker for PD.…”