Anna Majer

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

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.

DNA vaccination protects mice against Zika virus-induced damage to the testes

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract.

Molecular Evolution of the mtDNA Encoded rps3 Gene Among Filamentous Ascomycetes Fungi with an Emphasis on the Ophiostomatoid Fungi

The mitochondrial ribosomal protein S3 (rps3) gene within the fungi is extremely diverse in location and organization, some versions of this gene have been incorporated into a group I intron, others appear to have gained large insertions, microsatellite expansions, or have been invaded by homing endonucleases. Among the ascomycetes fungi the group I intron encoded version of rps3 appears to have a rather complex evolutionary history including first the acquisition of rps3 by a group I intron (mL2449), the loss of the mL2499 intron and the establishment of rps3 as a free-standing gene, and the eventual loss of the intron derived version of rps3.

MicroRNA abundance is altered in synaptoneurosomes during prion disease

Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions. MiRNAs are therefore candidate regulators of these early neurodegenerative changes and may provide clues as to the molecular pathways involved. We therefore determined changes in mature miRNA abundance within synaptoneurosomes isolated from prion-infected, as compared to mock-infected animals, at asymptomatic and symptomatic stages of disease. During preclinical disease, miRNAs that are enriched in neurons including miR-124a-3p, miR-136-5p and miR-376a-3p were elevated. At later stages of disease we found increases in miRNAs that have previously been identified as deregulated in brain tissues of prion infected mice, as well as in Alzheimers disease (AD) models. These include miR-146a-5p, miR-142-3p, miR-143-3p, miR-145a-5p, miR-451a, miR-let-7b, miR-320 and miR-150-5p. A number of miRNAs also decreased in abundance during clinical disease. These included almost all members of the related miR-200 family (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p, and miR-429-3p) and the 182 cluster (miR-182-5p and miR-183-5p).

The mtDNA rns gene landscape in the Ophiostomatales and other fungal taxa: Twintrons, introns, and intron-encoded proteins

Comparative sequence analysis of the mitochondrial small subunit ribosomal RNA (rns) gene among species of Ophiostoma, Grosmannia, Ceratocystiopsis and related taxa provides an overview of the types of introns that have invaded this gene within the ophiostomatoid fungi. The rns gene appears to be a reservoir for a number of group I and group II introns along with intron-associated open reading frames such as homing endonucleases and reverse transcriptases. This study uncovered two twintrons, one at position mS917 where a group ID intron encoding a LAGLIDADG ORF invaded another ORF-less group ID intron. Another twintron complex was detected at position mS1247 here a group IIA1 intron invaded the open reading frame embedded within a group IC2 intron. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent losses. Results of this study will make a significant contribution to the understanding of the complexity of the mitochondrial intron landscape, and offer a resource to those annotating mitochondrial genomes. It will also serve as a resource to those that bioprospect for ribozymes and homing endonucleases.

The sporadic occurrence of a group I intron-like element in the mtDNA rnl gene of Ophiostoma novo-ulmi subsp. americana

The presence of group I intron-like elements within the U7 region of the mtDNA large ribosomal subunit RNA gene (rnl) was investigated in strains of Ophiostoma novo-ulmi subsp. americana from Canada, Europe and Eurasia, and in selected strains of O. ips, O. minus, O. piceae, O. ulmi, and O. himal-ulmi. This insertion is of interest as it has been linked previously to the generation of plasmid-like mtDNA elements in diseased strains of O. novo-ulmi. Among 197 O. novo-ulmi subsp. americana strains tested, 61 contained a 1.6kb insertion within the rnl-U7 region and DNA sequence analysis suggests the presence of a group I intron (IA1 type) that encodes a potential double motif LAGLIDADG homing endonuclease-like gene (HEG). Phylogenetic analysis of rnl-U7 intron encoded HEG-like elements supports the view that double motif HEGs originated from a duplication event of a single-motif HEG followed by a fusion event that combined the two copies into one open reading frame (ORF). The data also show that rnl-U7 intron encoded ORFs belong to a clade that includes ORFs inserted into different types of group I introns, e.g. IB, ID, IC3, IA1, present within a variety of different mtDNA genes, such as the small ribosomal subunit RNA gene (rns), apo-cytochrome b gene (cob), NADH dehydrogenase subunit 5 (nad5), cytochrome oxidase subunit 1 gene (coxI), and ATPase subunit 9 gene (atp9). We also compared the occurrence of the rnl-U7 intron in our collection of 227 strains with the presence of the rnl-U11 group I intron and concluded that the U7 intron appears to be an optional element and the U11 intron is probably essential among the strains tested.

Correction: Induction of Multiple miR-200/182 Members in the Brains of Mice Are Associated with Acute Herpes Simplex Virus 1 Encephalitis

Important roles of microRNAs (miRNAs) in regulating the host response during viral infection have begun to be defined. However, little is known about the functional roles of miRNAs within an in vivo acute viral encephalitis model. We therefore identified global changes in miRNA expression during acute herpes simplex virus type 1 (HSV-1) encephalitis (HSVE) in mice. We found that many of the highly upregulated miRNAs (miR-155, miR-146a and miR-15b) detected in HSV-1 infected brain tissue are known regulators of inflammation and innate immunity. We also observed upregulation of 7 members belonging to the related group of miRNAs, the miR-200 family and miR-182 cluster (miR-200/182). Using in situ hybridization, we found that these miRNAs co-localized to regions of the brain with severe HSVE-related pathology and were upregulated in various cell types including neurons. Induction was apparent but not limited to cells in which HSV-1 was detected by immunohistochemistry, suggesting possible roles of these miRNAs in the host response to viral-induced tissue damage. Bioinformatic prediction combined with gene expression profiling revealed that the induced miR-200/182 members could regulate the biosynthesis of heparan sulfate proteoglycans. Using luciferase assays, we found that miR-96, miR-141, miR-183 and miR-200c all potentially targeted the syndecan-2 gene (Sdc2), which codes for a cell surface heparan sulfate proteoglycan involved in HSV-1 cellular attachment and entry.

Microdissection and transcriptional profiling: A window into the pathobiology of preclinical prion disease

Prion diseases share common features on a sub-cellular level with many neurodegenerative diseases including Alzheimer disease; the most prevalent neurodegenerative disease world-wide. The most obvious similarity is the accumulation of misfolded forms of the host proteins which forms aggregates in the brains of patients. Remarkably, one of the earliest pathological changes detected in degenerating brain tissue, well before clinical symptoms are observed, is synaptic dysfunction and loss. This pathology was recently shown to be reversible in early stages of mouse prion disease suggesting that synaptic regeneration and reestablishment of neuronal function is possible. Determination of the molecular events that underlie synapse degeneration and how this eventually results in neuronal loss is therefore a research priority that may contribute to the search for new therapeutic interventions for neurodegenerative disorders. Functional genomic studies using unbiased whole genome expression analyses represent one method that can provide insights into these perplexing processes. However, transcriptional profiles from brain tissues are representative of a heterogeneous mixture of cell types that effectively mask the expression of low abundance transcripts, or molecular changes that occur only in a small population of affected neurons. One method that was recently applied to address these challenges was laser capture microdissection which was used to effectively isolate the CA1 neuronal rich region of the hippocampus prior to RNA extraction. Profiling of both mRNAs and microRNAs revealed previously unidentified neuronal-specific genes and expression signatures that are relevant to understanding the pathophysiological processes involved in preclinical stages of prion disease. In this review we will highlight these molecular signatures and discuss their implications with respect to prion-induced neurodegeneration.

MicroRNA and mRNA Dysregulation in Astrocytes Infected with Zika Virus

The Zika virus (ZIKV) epidemic is an ongoing public health concern. ZIKV is a flavivirus reported to be associated with microcephaly, and recent work in animal models demonstrates the ability of the virus to cross the placenta and affect fetal brain development. Recent findings suggest that the virus preferentially infects neural stem cells and thereby deregulates gene expression, cell cycle progression, and increases cell death. However, neuronal stem cells are not the only brain cells that are susceptible to ZIKV and infection of other brain cells may contribute to disease progression. Herein, we characterized ZIKV replication in astrocytes, and profiled temporal changes in host microRNAs (miRNAs) and transcriptomes during infection. We observed the deregulation of numerous processes known to be involved in flavivirus infection, including genes involved in the unfolded protein response pathway. Moreover, a number of miRNAs were upregulated, including miR-30e-3p, miR-30e-5p, and, miR-17-5p, which have been associated with other flavivirus infections. This study highlights potential miRNAs that may be of importance in ZIKV pathogenesis.

The Role of MicroRNAs in Neurodegenerative Diseases: Implications for Early Detection and Treatment

MicroRNAs (miRNAs) are small noncoding RNAs that can posttranscriptionally regulate gene expression in development, differentiation, and in response to various stimuli. Numerous miRNAs are very specifically expressed within the central nervous system suggesting they regulate important brain functions. MiRNAs are also required for the postmitotic survival of neurons, strongly suggesting a crucial role in survival and neuroprotection. The fact that diverse arrays of miRNAs have been reported to be dysregulated in several neurodegenerative diseases implies that they can contribute to pathogenesis. As a group, the global burden of neurodegenerative disease is huge and includes conditions such as Alzheimer’s disease and other dementias, for which the numbers are steadily rising with the aging population, as well as communicable diseases caused by prions that are of public health concern. As yet, no drugs to halt or even delay the progression of these diseases are available, and this is a huge focus of global research. The best time for therapeutic intervention would be before significant memory loss and tissue destruction occurs such that interventions to boost cell repair and to promote neuroprotective mechanisms could provide significant health benefits. MicroRNA research promises to further elucidate the pathways, genes, and proteins that contribute to the neurodegenerative process that may serve as potential therapeutic targets. Furthermore, given the evidence of the neuroprotective properties of some miRNAs, these small RNA species may themselves be the focus for drug development. Here, we review recent studies that imply a link between miRNA function and neurodegeneration plus discuss how increased knowledge of miRNAs may be used in diagnosis and treatment of neurodegenerative diseases.