Engineered AAV8 capsid acquires heparin and AVB sepharose binding capacity but has altered in vivo transduction efficiency.

Naturally occurring adeno-associated virus (AAV) serotypes that bind to ligands such as AVB sepharose or heparin can be purified by affinity chromatography, which is a more efficient and scalable method than gradient ultracentrifugation. Wild-type AAV8 does not bind effectively to either of these molecules, which constitutes a barrier to using this vector when a high throughput design is required. Previously, AAV8 was engineered to contain a SPAKFA amino acid sequence to facilitate purification using AVB sepharose resin; however, in vivo studies were not conducted to examine whether these capsid mutations altered the transduction profile. To address this gap in knowledge, a mutant AAV8 capsid was engineered to bind to AVB sepharose and heparan sulfate (AAV8-AVB-HS), which efficiently bound to both affinity columns, resulting in elution yields of >80% of the total vector loaded compared to <5% for wild-type AAV8. However, in vivo comparison by intramuscular, intravenous, and intraperitoneal vector administration demonstrated a significant decrease in AAV8-AVB-HS transduction efficiency without alteration of the transduction profile. Therefore, although it is possible to engineer AAV capsids to bind various affinity ligands, the consequences associated with mutating surface exposed residues have the potential to negatively impact other vector characteristics including in vivo potency and production yield. This study demonstrates the importance of evaluating all aspects of vector performance when engineering AAV capsids.

Development of an Automated Capillary Immunoassay to Detect Prion Glycotypes in Creutzfeldt-Jakob Disease.

Creutzfeldt-Jakob disease (CJD) comprises a group of transmissible neurodegenerative diseases with vast phenotypic diversity. Sporadic CJD heterogeneity is predominantly influenced by the genotype at codon 129 of the prion-encoding gene and the molecular weight of PrPSc fragments after protease digestion, resulting in a classification of 6 subtypes of CJD (MM1, MM2, MV1, MV2, VV1, and VV2). The majority of cases with CJD can be distinguished using this classification system. However, a number of reported CJD cases are phenotypically unique from others within their same subtype, such as variably protease-sensitive prionopathies, or exist as a mixture of subtypes within the same patient. Western blotting of brain tissue, along with the genotyping of codon 129 of the prion-encoding gene, is considered the "gold standard" for the biochemical characterization of CJD. Western blotting requires a significant amount of prion protein for detection, is labor-intensive, and is also associated with high interassay variability. In addition to these limitations, a growing body of research suggests that unique subtypes of CJD are often undetected or misdiagnosed using standard diagnostic western blotting protocols. Consequently, we successfully optimized and developed a capillary-based western assay using the JESS Simple Western (ProteinSimple) to detect and characterize prion proteins from patients with CJD. We found that this novel assay consistently differentiated CJD type 1 and type 2 cases with a limit of detection 10 to 100× higher than traditional western blotting. Cases with CJD in which type 1 and type 2 coexist within the same brain region can be detected using type 1-specific and type 2-specific antibodies, and we found that there was remarkable specificity for the detection of cases with variably protease-sensitive prionopathy. The assay presented displays outstanding sensitivity, allowing for the preservation of valuable samples and enhancing current detection methods.

Intramuscular Adeno-Associated Virus-Mediated Expression of Monoclonal Antibodies Provides 100% Protection Against Ebola Virus Infection in Mice.

The 2013-2016 West Africa outbreak demonstrated the epidemic potential of Ebola virus and highlighted the need for counter strategies. Monoclonal antibody (mAb)-based therapies hold promise as treatment options for Ebola virus infections. However, production of clinical-grade mAbs is labor intensive, and immunity is short lived. Conversely, adeno-associated virus (AAV)-mediated mAb gene transfer provides the host with a genetic blueprint to manufacture mAbs in vivo, leading to steady release of antibody over many months. Here we demonstrate that AAV-mediated expression of nonneutralizing mAb 5D2 or 7C9 confers 100% protection against mouse-adapted Ebola virus infection, while neutralizing mAb 2G4 was 83% protective. A 2-component cocktail, AAV-2G4/AAV-5D2, provided complete protection when administered 7 days prior to challenge and was partially protective with a 3-day lead time. Finally, AAV-mAb therapies provided sustained protection from challenge 5 months following AAV administration. AAV-mAb may be a viable alternative strategy for vaccination against emerging infectious diseases.

MicroRNA-16 targets mRNA involved in neurite extension and branching in hippocampal neurons during presymptomatic prion disease.

The mechanisms that lead to neuronal death in neurodegenerative diseases are poorly understood. Prion diseases, like many more common disorders such as Alzheimer's and Parkinson's diseases, are characterized by the progressive accumulation of misfolded disease-specific proteins. The earliest changes observed in brain tissue include a reduction in synaptic number and retraction of dendritic spines, followed by reduced length and branching of neurites. These pathologies are observable during presymptomatic stages of disease and are accompanied by altered expression of transcripts that include miRNAs. Here we report that miR-16 localized within hippocampal CA1 neurons is increased during early prion disease. Modulating miR-16 expression in mature murine hippocampal neurons by expression from a lentivirus, thus mimicking the modest increase seen in vivo, was found to induce neurodegeneration. This was characterized by retraction of neurites and reduced branching. We performed immunoprecipitation of the miR-16 enriched RISC complex, and identified associated transcripts from the co-immunoprecipitated RNA (Ago2 RIP-Chip). These transcripts were enriched with predicted binding sites for miR-16, including the validated miR-16 targets APP and BCL2, as well as numerous novel targets. In particular, genes within the neurotrophin receptor mediated MAPK/ERK pathway were potentially regulated by miR-16; including TrkB (NTRK2), MEK1 (MAP2K1) and c-Raf (RAF). Increased miR-16 expression in neurons during presymptomatic prion disease and reduction in proteins involved in MAPK/ERK signaling represents a possible mechanism by which neurite length and branching are decreased during early stages of disease.

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 Alzheimer's 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).

A functional SNP catalog of overlapping miRNA-binding sites in genes implicated in prion disease and other neurodegenerative disorders.

The involvement of SNPs in miRNA target sites remains poorly investigated in neurodegenerative disease. In addition to associations with disease risk, such genetic variations can also provide novel insight into mechanistic pathways that may be responsible for disease etiology and/or pathobiology. To identify SNPs associated specifically with degenerating neurons, we restricted our analysis to genes that are dysregulated in CA1 hippocampal neurons of mice during early, preclinical phase of Prion disease. The 125 genes chosen are also implicated in other numerous degenerative and neurological diseases and disorders and are therefore likely to be of fundamental importance. We predicted those SNPs that could increase, decrease, or have neutral effects on miRNA binding. This group of genes was more likely to possess DNA variants than were genes chosen at random. Furthermore, many of the SNPs are common within the human population, and could contribute to the growing awareness that miRNAs and associated SNPs could account for detrimental neurological states. Interestingly, SNPs that overlapped miRNA-binding sites in the 3'-UTR of GABA-receptor subunit coding genes were particularly enriched. Moreover, we demonstrated that SNP rs9291296 would strengthen miR-26a-5p binding to a highly conserved site in the 3'-UTR of gamma-aminobutyric acid receptor subunit alpha-4.

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.

Activation of pro-survival CaMK4ß/CREB and pro-death MST1 signaling at early and late times during a mouse model of prion disease.

BACKGROUND: The signaling pathways most critical to prion disease pathogenesis are as yet incompletely characterized. We have developed a kinomics approach to identify signaling pathways that are dysregulated during prion pathogenesis. The approach is sensitive and specific enough to detect signaling pathways dysregulated in a simple in vitro model of prion pathogenesis. Here, we used this approach to identify signaling pathways dysregulated during prion pathogenesis in vivo. METHODS: Mice intraperitoneally infected with scrapie (strain RML) were euthanized at 70, 90, 110, 130 days post-infection (dpi) or at terminal stages of disease (155-190 dpi). The levels of 139 protein kinases in brainstem-cerebellum homogenates were analyzed by multiplex Western blots, followed by hierarchical clustering and analyses of activation states. RESULTS: Hierarchical and functional clustering identified CaMK4ß and MST1 signaling pathways as potentially dysregulated. Targeted analyses revealed that CaMK4ß and its downstream substrate CREB, which promotes neuronal survival, were activated at 70 and 90 dpi in cortical, subcortical and brainstem-cerebellum homogenates from scrapie-infected mice. The activation levels of CaMK4ß/CREB signaling returned to those in mock-infected mice at 110 dpi, whereas MST1, which promotes neuronal death, became activated at 130 dpi. CONCLUSION: Pro-survival CaMK4ß/CREB signaling is activated in mouse scrapie at earlier times and later inhibited, whereas pro-death MST1 signaling is activated at these later times.

Cellular prion protein regulates its own α-cleavage through ADAM8 in skeletal muscle.

The ubiquitously expressed cellular prion protein (PrP(C)) is subjected to the physiological α-cleavage at a region critical for both PrP toxicity and the conversion of PrP(C) to its pathogenic prion form (PrP(Sc)), generating the C1 and N1 fragments. The C1 fragment can activate caspase 3 while the N1 fragment is neuroprotective. Recent articles indicate that ADAM10, ADAM17, and ADAM9 may not play a prominent role in the α-cleavage of PrP(C) as previously thought, raising questions on the identity of the responsible protease(s). Here we show that, ADAM8 can directly cleave PrP to generate C1 in vitro and PrP C1/full-length ratio is greatly decreased in the skeletal muscles of ADAM8 knock-out mice; in addition, the PrP C1/full-length ratio is linearly correlated with ADAM8 protein level in myoblast cell line C2C12 and in skeletal muscle tissues of transgenic mice. These results indicate that ADAM8 is the primary protease responsible for the α-cleavage of PrP(C) in muscle cells. Moreover, we found that overexpression of PrP(C) led to up-regulation of ADAM8, suggesting that PrP(C) may regulate its own α-cleavage through modulating ADAM8 activity.

Differentiated cultures of an immortalized human neural progenitor cell line do not replicate prions despite PrPC overexpression.

Prions are misfolded proteins that accumulate within the brain in association with a rare group of fatal and infectious neurological disorders in humans and animals. A current challenge to research is a lack of in vitro model systems that are compatible with a wide range of prion strains, reproduce prion toxicity, and are amenable to genetic manipulations. In an attempt to address this need, here we produced stable cell lines that overexpress different versions of PrPC through lentiviral transduction of immortalized human neural progenitor cells (ReN VM). Differentiated cultures made from the neural progenitor cell lines overexpressed PrPC within 3D spheroid-like structures of TUBB3+ neurons and we observed evidence that PrPC modulates formation of these structures, consistent with PrPC's role in neurogenesis. However, through repeated measurements of amyloid seeding activity in 6-week time course experiments, we failed to observe any evidence of prion replication within the differentiated ReN cultures following challenge with four prion isolates (human sCJD subtypes MM1 and VV2, and rodent adapted scrapie strains RML and 263K). We attributed amyloid seeding activity detected within the cultures to residual inoculum and concluded that PrPC overexpression was insufficient to confer permissiveness of ReN cultures to prion infection. While our ReN cell prion infection model was unsuccessful, additional efforts to develop cellular models of human prion disease are highly warranted.

Neurons and Astrocytes Elicit Brain Region Specific Transcriptional Responses to Prion Disease in the Murine CA1 and Thalamus.

Progressive dysfunction and loss of neurons ultimately culminates in the symptoms and eventual fatality of prion disease, yet the pathways and mechanisms that lead to neuronal degeneration remain elusive. Here, we used RNAseq to profile transcriptional changes in microdissected CA1 and thalamus brain tissues from prion infected mice. Numerous transcripts were altered during clinical disease, whereas very few transcripts were reliably altered at pre-clinical time points. Prion altered transcripts were assigned to broadly defined brain cell types and we noted a strong transcriptional signature that was affiliated with reactive microglia and astrocytes. While very few neuronal transcripts were common between the CA1 and thalamus, we described transcriptional changes in both regions that were related to synaptic dysfunction. Using transcriptional profiling to compare how different neuronal populations respond during prion disease may help decipher mechanisms that lead to neuronal demise and should be investigated with greater detail.

Dysregulation of neuroprotective astrocytes, a spectrum of microglial activation states, and altered hippocampal neurogenesis are revealed by single-cell RNA sequencing in prion disease.

Prion diseases are neurodegenerative disorders with long asymptomatic incubation periods, followed by a rapid progression of cognitive and functional decline culminating in death. The complexity of intercellular interactions in the brain is challenging to unravel and the basis of disease pathobiology remains poorly understood. In this study, we employed single cell RNA sequencing (scRNAseq) to produce an atlas of 147,536 single cell transcriptomes from cortex and hippocampus of mice infected with prions and showing clinical signs. We identified transcriptionally distinct populations and sub-populations of all the major brain cell-types. Disease-related transcription was highly specific to not only overarching cell-types, but also to sub-populations of glia and neurons. Most striking was an apparent decrease in relative frequency of astrocytes expressing genes that are required for brain homeostasis such as lipid synthesis, glutamate clearance, synaptic modulation and regulation of blood flow. Additionally, we described a spectrum of microglial activation states that suggest delineation of phagocytic and neuroinflammatory functions in different cell subsets. Differential responses of immature and mature neuron populations were also observed, alongside abnormal hippocampal neurogenesis. Our scRNAseq library provides a new layer of knowledge on single cell gene expression in prion disease, and is a basis for a more detailed understanding of cellular interplay that leads to 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.

Generation and Characterization of a SARS-CoV-2-Susceptible Mouse Model Using Adeno-Associated Virus (AAV6.2FF)-Mediated Respiratory Delivery of the Human ACE2 Gene.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) that has caused a pandemic with millions of human infections. There continues to be a pressing need to develop potential therapies and vaccines to inhibit SARS-CoV-2 infection to mitigate the ongoing pandemic. Epidemiological data from the current pandemic indicates that there may be sex-dependent differences in disease outcomes. To investigate these differences, we proposed to use common small animal species that are frequently used to model disease with viruses. However, common laboratory strains of mice are not readily infected by SARS-CoV-2 because of differences in the angiotensin-converting enzyme 2 (ACE2), the cellular receptor for the virus. To overcome this limitation, we transduced common laboratory accessible strains of mice of different sexes and age groups with a novel a triple AAV6 mutant, termed AAV6.2FF, encoding either human ACE2 or luciferase via intranasal administration to promote expression in the lung and nasal turbinates. Infection of AAV-hACE2-transduced mice with SARS-CoV-2 resulted in high viral titers in the lungs and nasal turbinates, establishment of an IgM and IgG antibody response, and modulation of lung and nasal turbinate cytokine profiles. There were insignificant differences in infection characteristics between age groups and sex-related differences; however, there were significant strain-related differences between BALB/c vs. C57BL/6 mice. We show that AAV-hACE2-transduced mice are a useful for determining immune responses and for potential evaluation of SARS-CoV-2 vaccines and antiviral therapies, and this study serves as a model for the utility of this approach to rapidly develop small-animal models for emerging viruses.

Transcriptional modulation in a leukocyte-depleted splenic cell population during prion disease.

Prion replication in the periphery precedes neuroinvasion in many experimental rodent scrapie models, and in natural sheep scrapie and chronic wasting disease (CWD) in cervids. Prions propagate in the germinal centers of secondary lymphoid organs and are strongly associated with follicular dendritic cells (FDC) and possibly circulating dendritic cells and macrophages. Given the importance of lymphoid organs in prion disease transmission and pathogenesis, gene expression studies may reveal host factors or biological pathways related to prion replication and accumulation. A procedure was developed to enrich for FDC, dendritic cells, and macrophages prior to the investigation of transcriptional alterations in murine splenic cells during prion pathogenesis. In total, 1753 transcripts exhibited fold changes greater than three (false discovery rates less than 2%) in this population isolated from spleens of prion-infected versus uninfected mice. The gene for the small leucine-rich proteoglycan decorin (DCN) was one of the genes most overexpressed in infected mice, and the splenic protein levels mirrored this in mice infected with scrapie as well as bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD). A number of groups of functionally related genes were also significantly decreased in infected spleens. These included genes related to iron metabolism and homeostasis, pathways that have also been implicated in prion pathogenesis in the brain. These gene expression alterations provide insights into the molecular mechanisms underlying prion disease pathogenesis and may serve as a pool of potential surrogate markers for the early detection and diagnosis of some prion diseases.

Phosphorylation of prion protein at serine 43 induces prion protein conformational change.

The cause of the conformational change of normal cellular prion protein (PrP) into its disease-associated form is unknown. Posttranslational modifications, such as glycosylation, acetylation, S-nitrosylation, and phosphorylation, are known to induce protein conformational changes. Here, we investigated whether phosphorylation could induce the conformational change of PrP because PrP contains several kinase motifs and has been found recently in the cytosol, in which kinases generally reside. Neuronal cyclin-dependent kinase 5 (Cdk5) phosphorylated recombinant PrP(23-231) at serine 43 (S43) in an in vitro kinase assay. Cdk5-phosphorylated PrP became proteinase K resistant, formed Congo Red-positive fibrils, and formed aggregates that were immunostained with anti-PrP and anti-phospho-PrP(S43) (anti-pPrP(S43)). pPrP(S43) was detected in PrP/Cdk5/p25 cotransfected N2a cells. Roscovitine inhibition of Cdk5 activity or transfection of N2a cells with mutant PrP S43A eliminated the anti-pPrP(S43)-immunopositive protein. Alkaline phosphatase-sensitive and proteinase K-resistant pPrP(S43) immunoreactivity was observed in scrapie-infected but not control-injected mice brains. These results raise the possibility that phosphorylation could represent a physiological mechanism of PrP conversion in vivo.

Application of "omics" to prion biomarker discovery.

The advent of genomics and proteomics has been a catalyst for the discovery of biomarkers able to discriminate biological processes such as the pathogenesis of complex diseases. Prompt detection of prion diseases is particularly desirable given their transmissibility, which is responsible for a number of human health risks stemming from exogenous sources of prion protein. Diagnosis relies on the ability to detect the biomarker PrP(Sc), a pathological isoform of the host protein PrP(C), which is an essential component of the infectious prion. Immunochemical detection of PrP(Sc) is specific and sensitive enough for antemortem testing of brain tissue, however, this is not the case in accessible biological fluids or for the detection of recently identified novel prions with unique biochemical properties. A complementary approach to the detection of PrP(Sc) itself is to identify alternative, "surrogate" gene or protein biomarkers indicative of disease. Biomarkers are also useful to track the progress of disease, especially important in the assessment of therapies, or to identify individuals "at risk". In this review we provide perspective on current progress and pitfalls in the use of "omics" technologies to screen body fluids and tissues for biomarker discovery in prion diseases.

Exposure Risk of Chronic Wasting Disease in Humans.

The majority of human prion diseases are sporadic, but acquired disease can occur, as seen with variant Creutzfeldt-Jakob disease (vCJD) following consumption of bovine spongiform encephalopathy (BSE). With increasing rates of cervid chronic wasting disease (CWD), there is concern that a new form of human prion disease may arise. Currently, there is no evidence of transmission of CWD to humans, suggesting the presence of a strong species barrier; however, in vitro and in vivo studies on the zoonotic potential of CWD have yielded mixed results. The emergence of different CWD strains is also concerning, as different strains can have different abilities to cross species barriers. Given that venison consumption is common in areas where CWD rates are on the rise, increased rates of human exposure are inevitable. If CWD was to infect humans, it is unclear how it would present clinically; in vCJD, it was strain-typing of vCJD prions that proved the causal link to BSE. Therefore, the best way to screen for CWD in humans is to have thorough strain-typing of harvested cervids and human CJD cases so that we will be in a position to detect atypical strains or strain shifts within the human CJD population.

Downregulation of circulating miR 802-5p and miR 194-5p and upregulation of brain MEF2C along breast cancer brain metastasization.

Breast cancer brain metastases (BCBMs) have been underinvestigated despite their high incidence and poor outcome. MicroRNAs (miRNAs), and particularly circulating miRNAs, regulate multiple cellular functions, and their deregulation has been reported in different types of cancer and metastasis. However, their signature in plasma along brain metastasis development and their relevant targets remain undetermined. Here, we used a mouse model of BCBM and next-generation sequencing (NGS) to establish the alterations in circulating miRNAs during brain metastasis formation and development. We further performed bioinformatics analysis to identify their targets with relevance in the metastatic process. We additionally analyzed human resected brain metastasis samples of breast cancer patients for target expression validation. Breast cancer cells were injected in the carotid artery of mice to preferentially induce metastasis in the brain, and samples were collected at different timepoints (5 h, 3, 7, and 10 days) to follow metastasis development in the brain and in peripheral organs. Metastases were detected from 7 days onwards, mainly in the brain. NGS revealed a deregulation of circulating miRNA profile during BCBM progression, rising from 18% at 3 days to 30% at 10 days following malignant cells' injection. Work was focused on those altered prior to metastasis detection, among which were miR-802-5p and miR-194-5p, whose downregulation was validated by qPCR. Using targetscan and diana tools, the transcription factor myocyte enhancer factor 2C (MEF2C) was identified as a target for both miRNAs, and its expression was increasingly observed in malignant cells along brain metastasis development. Its upregulation was also observed in peritumoral astrocytes pointing to a role of MEF2C in the crosstalk between tumor cells and astrocytes. MEF2C expression was also observed in human BCBM, validating the observation in mouse. Collectively, downregulation of circulating miR-802-5p and miR-194-5p appears as a precocious event in BCBM and MEF2C emerges as a new player in brain metastasis development.

Activation of p53-regulated pro-apoptotic signaling pathways in PrP-mediated myopathy.

BACKGROUND: We have reported that doxycycline-induced over-expression of wild type prion protein (PrP) in skeletal muscles of Tg(HQK) mice is sufficient to cause a primary myopathy with no signs of peripheral neuropathy. The preferential accumulation of the truncated PrP C1 fragment was closely correlated with these myopathic changes. In this study we use gene expression profiling to explore the temporal program of molecular changes underlying the PrP-mediated myopathy. RESULTS: We used DNA microarrays, and confirmatory real-time PCR and Western blot analysis to demonstrate deregulation of a large number of genes in the course of the progressive myopathy in the skeletal muscles of doxycycline-treated Tg(HQK) mice. These include the down-regulation of genes coding for the myofibrillar proteins and transcription factor MEF2c, and up-regulation of genes for lysosomal proteins that is concomitant with increased lysosomal activity in the skeletal muscles. Significantly, there was prominent up-regulation of p53 and p53-regulated genes involved in cell cycle arrest and promotion of apoptosis that paralleled the initiation and progression of the muscle pathology. CONCLUSION: The data provides the first in vivo evidence that directly links p53 to a wild type PrP-mediated disease. It is evident that several mechanistic features contribute to the myopathy observed in PrP over-expressing mice and that p53-related apoptotic pathways appear to play a major role.