MicroRNA 146a (miR-146a) Is Over-Expressed during Prion Disease and Modulates the Innate Immune Response and the Microglial Activation State

Increasing evidence supports the involvement of microRNAs (miRNAs) in inflammatory and immune processes in prion neuropathogenesis. MiRNAs are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. We established miR-146a over-expression in prion-infected mouse brain tissues concurrent with the onset of prion deposition and appearance of activated microglia. Expression profiling of a variety of central nervous system derived cell-lines revealed that miR-146a is preferentially expressed in cells of microglial lineage. Prominent up-regulation of miR-146a was evident in the microglial cell lines BV-2 following TLR2 or TLR4 activation and also EOC 13.31 via TLR2 that reached a maximum 24–48 hours post-stimulation, concomitant with the return to basal levels of transcription of induced cytokines. Gain- and loss-of-function studies with miR-146a revealed a substantial deregulation of inflammatory response pathways in response to TLR2 stimulation. Significant transcriptional alterations in response to miR-146a perturbation included downstream mediators of the pro-inflammatory transcription factor, nuclear factor-kappa B (NF-κB) and the JAK-STAT signaling pathway. Microarray analysis also predicts a role for miR-146a regulation of morphological changes in microglial activation states as well as phagocytic mediators of the oxidative burst such as CYBA and NOS3. Based on our results, we propose a role for miR-146a as a potent modulator of microglial function by regulating the activation state during prion induced neurodegeneration

The cell type resolved mouse transcriptome in neuron-enriched brain tissues from the hippocampus and cerebellum during prion disease

Abstract Multiple cell types and complex connection networks are an intrinsic feature of brain tissue. In this study we used expression profiling of specific microscopic regions of heterogeneous tissue sections isolated by laser capture microdissection (LCM) to determine insights into the molecular basis of brain pathology in prion disease. Temporal profiles in two mouse models of prion disease, bovine spongiform encephalopathy (BSE) and a mouse-adapted strain of scrapie (RML) were performed in microdissected regions of the CA1 hippocampus and granular layer of the cerebellum which are both enriched in neuronal cell bodies. We noted that during clinical disease the number of activated microglia and astrocytes that occur in these areas are increased, thereby likely diluting the neuronal gene expression signature. We performed a comparative analysis with gene expression profiles determined from isolated populations of neurons, microglia and astrocytes to identify transcripts that are enriched in each of these cell types. Although the incubation periods of these two models are quite different, over 300 days for BSE and ~160 days for RML scrapie, these regional microdissections revealed broadly similar profiles. Microglial and astrocyte-enriched genes contributed a profound inflammatory profile consisting of inflammatory cytokines, genes related to phagocytosis, proteolysis and genes coding for extracellular matrix proteins. CA1 pyramidal neurons displayed a net upregulation of transcription factors and stress induced genes at pre-clinical stages of disease while all tissues showed profound decrease of overlapping genes related to neuronal function, in particular transcripts related to neuronal communication including glutamate receptors, phosphatase subunits and numerous synapse-related markers. Of note, we found a small number of genes expressed in neurons that were upregulated during clinical disease including, COX6A2, FZD9, RXRG and SOX11, that may be biomarkers of neurodegeneration

Non-Productive Infection of Glial Cells with SARS-CoV-2 in Hamster Organotypic Cerebellar Slice Cultures

The numerous neurological syndromes associated with COVID-19 implicate an effect of viral pathogenesis on neuronal function, yet reports of direct SARS-CoV-2 infection in the brain are conflicting. We used a well-established organotypic brain slice culture to determine the permissivity of hamster brain tissues to SARS-CoV-2 infection. We found levels of live virus waned after inoculation and observed no evidence of cell-to-cell spread, indicating that SARS-CoV-2 infection was non-productive. Nonetheless, we identified a small number of infected cells with glial phenotypes; however, no evidence of viral infection or replication was observed in neurons. Our data corroborate several clinical studies that have assessed patients with COVID-19 and their association with neurological involvement

Early Mechanisms of Pathobiology Are Revealed by Transcriptional Temporal Dynamics in Hippocampal CA1 Neurons of Prion Infected Mice

<div><p>Prion diseases typically have long pre-clinical incubation periods during which time the infectious prion particle and infectivity steadily propagate in the brain. Abnormal neuritic sprouting and synaptic deficits are apparent during pre-clinical disease, however, gross neuronal loss is not detected until the onset of the clinical phase. The molecular events that accompany early neuronal damage and ultimately conclude with neuronal death remain obscure. In this study, we used laser capture microdissection to isolate hippocampal CA1 neurons and determined their pre-clinical transcriptional response during infection. We found that gene expression within these neurons is dynamic and characterized by distinct phases of activity. We found that a major cluster of genes is altered during pre-clinical disease after which expression either returns to basal levels, or alternatively undergoes a direct reversal during clinical disease. Strikingly, we show that this cluster contains a signature highly reminiscent of synaptic N-methyl-D-aspartic acid (NMDA) receptor signaling and the activation of neuroprotective pathways. Additionally, genes involved in neuronal projection and dendrite development were also altered throughout the disease, culminating in a general decline of gene expression for synaptic proteins. Similarly, deregulated miRNAs such as miR-132-3p, miR-124a-3p, miR-16-5p, miR-26a-5p, miR-29a-3p and miR-140-5p follow concomitant patterns of expression. This is the first in depth genomic study describing the pre-clinical response of hippocampal neurons to early prion replication. Our findings suggest that prion replication results in the persistent stimulation of a programmed response that is mediated, at least in part, by synaptic NMDA receptor activity that initially promotes cell survival and neurite remodelling. However, this response is terminated prior to the onset of clinical symptoms in the infected hippocampus, seemingly pointing to a critical juncture in the disease. Manipulation of these early neuroprotective pathways may redress the balance between degeneration and survival, providing a potential inroad for treatment.</p> </div

The endogenous expression levels in rat hippocampus of the 6 validated miRNAs that were found to be over-expressed early in prion disease.

<p>MiR-124a-3p expression levels are shown for reference purposes. NGS refers to next generation sequencing.</p>*<p>data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003002#ppat.1003002-Kye1" target="_blank">[57]</a>.</p>**<p>data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003002#ppat.1003002-Zovoilis1" target="_blank">[51]</a>.</p><p>- reflects lack of data.</p

The list of genes we further investigated using qRT-PCR.

<p>Student's t-test statistic was employed such that a deregulation in fold change between infected and control samples showed at least a p-value≤0.1. The+or−represents a 1–2 fold change; 2.1–3.0 fold change is represented by++or−−while a ≥3.1 is annotated as +++. Blank spaces represent a lack of significant signal between infected and control samples. Relative microarray fold changes are highlighted in parenthesis. Pre-clinical time period spans up to and including 110 DPI while clinical represents 130 DPI and EP.</p

Analysis of genes dysregulated by over-expression, or knock-down, in resting EOC 13.31 cells using either miR-146a mimics or anti-miRs.

<p>Genes listed in bold are those that are bioinformatically predicted to be targets of miR-146a using the TargetScan 5.1 program and IPA software.</p

A summary showing key inflammatory response-related genes whose expression is modulated upon the over-expression of miR-146a.

<p>A summary showing key inflammatory response-related genes whose expression is modulated upon the over-expression of miR-146a.</p

A dynamic temporal gene expression profile identified between prion infected and control samples.

<p>(<b>A</b>) Hierarchical clustering of 1026 genes that showed significant differential expression across time between the prion-infected and mock-infected control groups (ctrl; FDR<0.001 and at least two fold change). Red indicates increased and blue indicates decreased expression levels relative to the mean (white). The two circles highlight the “waves” of genes that are temporally deregulated during prion disease. (<b>B</b>) Gene ontology networks for up-regulated genes are grouped according to the indicated time points analyzed. Select ontologies are highlighted of which majority reflect immune-regulated genes at late stages of disease. The gene ontology involved in the stress response is highlighted by a red * that occurs at 70 DPI. (<b>C</b>) Gene ontology networks for down-regulated genes are grouped according to the indicated time points analyzed. Select ontologies are highlighted for which majority reflect neuronal-specific gene function.</p

Analysis of genes dysregulated upon over-expression, or knock-down, of miR-146a in stimulated EOC 13.31 cells using either miRNA mimics or anti-miRs.

<p><b>A.</b> Venn diagram to show the intersection between genes down-regulated in LPS stimulated EOC 13.31 cells by miR-146a over-expression, and those up-regulated upon miR-146a knock-down. Also shown are those targets bioinformatically predicted using the TargetScan 5.1 program and IPA software. <b>B.</b> Schematic showing alterations in expression in key inflammatory response-related genes in LPS stimulated EOC 13.31 cells following miR-146a over-expression. Colored green are those down-regulated genes, colored red are up-regulated genes, while those highlighted in blue are bioinformatically predicted targets of miR-146a using the TargetScan 5.1 program and IPA software.</p