Prions, prionoids and protein misfolding disorders.

Prion diseases are progressive, incurable and fatal neurodegenerative conditions. The term 'prion' was first nominated to express the revolutionary concept that a protein could be infectious. We now know that prions consist of PrPSc, the pathological aggregated form of the cellular prion protein PrPC. Over the years, the term has been semantically broadened to describe aggregates irrespective of their infectivity, and the prion concept is now being applied, perhaps overenthusiastically, to all neurodegenerative diseases that involve protein aggregation. Indeed, recent studies suggest that prion diseases (PrDs) and protein misfolding disorders (PMDs) share some common disease mechanisms, which could have implications for potential treatments. Nevertheless, the transmissibility of bona fide prions is unique, and PrDs should be considered as distinct from other PMDs.

Genome-wide transcriptomics identifies an early preclinical signature of prion infection.

The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance and splicing alterations during the course of disease in prion-inoculated mice. Prion infection induced PrP-dependent transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical signs, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, could be the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed signs in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets.

Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes.

The cold shock domain is one of the most highly conserved motifs between bacteria and higher eukaryotes. Y-box-binding proteins represent a subfamily of cold shock domain proteins with pleiotropic functions, ranging from transcription in the nucleus to translation in the cytoplasm. These proteins have been investigated in all major model organisms except Caenorhabditis elegans. In this study, we set out to fill this gap and present a functional characterization of CEYs, the C. elegans Y-box-binding proteins. We find that, similar to other organisms, CEYs are essential for proper gametogenesis. However, we also report a novel function of these proteins in the formation of large polysomes in the soma. In the absence of the somatic CEYs, polysomes are dramatically reduced with a simultaneous increase in monosomes and disomes, which, unexpectedly, has no obvious impact on animal biology. Because transcripts that are enriched in polysomes in wild-type animals tend to be less abundant in the absence of CEYs, our findings suggest that large polysomes might depend on transcript stabilization mediated by CEY proteins.

Genome-wide analysis of GLD-1-mediated mRNA regulation suggests a role in mRNA storage.

Translational repression is often accompanied by mRNA degradation. In contrast, many mRNAs in germ cells and neurons are "stored" in the cytoplasm in a repressed but stable form. Unlike repression, the stabilization of these mRNAs is surprisingly little understood. A key player in Caenorhabditis elegans germ cell development is the STAR domain protein GLD-1. By genome-wide analysis of mRNA regulation in the germ line, we observed that GLD-1 has a widespread role in repressing translation but, importantly, also in stabilizing a sub-population of its mRNA targets. Additionally, these mRNAs appear to be stabilized by the DDX6-like RNA helicase CGH-1, which is a conserved component of germ granules and processing bodies. Because many GLD-1 and CGH-1 stabilized mRNAs encode factors important for the oocyte-to-embryo transition (OET), our findings suggest that the regulation by GLD-1 and CGH-1 serves two purposes. Firstly, GLD-1-dependent repression prevents precocious translation of OET-promoting mRNAs. Secondly, GLD-1- and CGH-1-dependent stabilization ensures that these mRNAs are sufficiently abundant for robust translation when activated during OET. In the absence of this protective mechanism, the accumulation of OET-promoting mRNAs, and consequently the oocyte-to-embryo transition, might be compromised.

LARP-1 promotes oogenesis by repressing fem-3 in the C. elegans germline.

LA-related protein 1 (LARP-1) belongs to an RNA-binding protein family containing a LA motif. Here, we identify LARP-1 as a regulator of sex determination. In C. elegans hermaphrodites, a complex regulatory network regulates the switch from sperm to oocyte production. We find that simultaneous depletion of larp-1 and the Nanos homologue nos-3 results in germline masculinization. This phenotype is accompanied by a strong reduction of the levels of TRA-1, a GLI-family transcription factor that promotes oogenesis. TRA-1 levels are regulated by CBC(FEM-1), a ubiquitin ligase consisting of the FEM proteins, FEM-1, FEM-2 and FEM-3 and the cullin CUL-2. We show that both the masculinization phenotype and the reduction of TRA-1 levels observed in nos-3;larp-1 mutants require fem-3 activity, suggesting that nos-3 and larp-1 regulate the sperm-oocyte switch by inhibiting the fem genes. Consistently, fem-3 mRNA levels are increased in larp-1 mutants. By contrast, levels of fem-3 mRNA are not affected in nos-3 mutants. Therefore, our data indicate that LARP-1 and NOS-3 promote oogenesis by regulating fem-3 expression through distinct mechanisms.

Ribosomal profiling during prion disease uncovers progressive translational derangement in glia but not in neurons.

Prion diseases are caused by PrPSc, a self-replicating pathologically misfolded protein that exerts toxicity predominantly in the brain. The administration of PrPSc causes a robust, reproducible and specific disease manifestation. Here, we have applied a combination of translating ribosome affinity purification and ribosome profiling to identify biologically relevant prion-induced changes during disease progression in a cell-type-specific and genome-wide manner. Terminally diseased mice with severe neurological symptoms showed extensive alterations in astrocytes and microglia. Surprisingly, we detected only minor changes in the translational profiles of neurons. Prion-induced alterations in glia overlapped with those identified in other neurodegenerative diseases, suggesting that similar events occur in a broad spectrum of pathologies. Our results suggest that aberrant translation within glia may suffice to cause severe neurological symptoms and may even be the primary driver of prion disease.

Soluble dimeric prion protein ligand activates Adgrg6 receptor but does not rescue early signs of demyelination in PrP-deficient mice.

The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as a potential therapeutic agent in treating peripheral demyelinating and dysmyelinating diseases. We designed a dimeric Fc-fusion protein comprising the myelinotrophic domain of PrP (FT2Fc), which activated Adgrg6 in vitro and exhibited favorable pharmacokinetic properties for in vivo treatment of peripheral neuropathies. While chronic FT2Fc treatment elicited specific transcriptomic changes in the sciatic nerves of PrP knockout mice, no amelioration of the early molecular signs demyelination was detected. Instead, RNA sequencing of sciatic nerves revealed downregulation of cytoskeletal and sarcomere genes, akin to the gene expression changes seen in myopathic skeletal muscle of PrP overexpressing mice. These results call for caution when devising myelinotrophic therapies based on PrP-derived Adgrg6 ligands. While our treatment approach was not successful, Adgrg6 remains an attractive therapeutic target to be addressed in other disease models or by using different biologically active Adgrg6 ligands.

Genome-wide identification of microRNAs regulating the human prion protein.

The cellular prion protein (PrPC ) is best known for its misfolded disease-causing conformer, PrPSc . Because the availability of PrPC is often limiting for prion propagation, understanding its regulation may point to possible therapeutic targets. We sought to determine to what extent the human microRNAome is involved in modulating PrPC levels through direct or indirect pathways. We probed PrPC protein levels in cells subjected to a genome-wide library encompassing 2019 miRNA mimics using a robust time-resolved fluorescence-resonance screening assay. Screening was performed in three human neuroectodermal cell lines: U-251 MG, CHP-212 and SH-SY5Y. The three screens yielded 17 overlapping high-confidence miRNA mimic hits, 13 of which were found to regulate PrPC biosynthesis directly via binding to the PRNP 3'UTR, thereby inducing transcript degradation. The four remaining hits (miR-124-3p, 192-3p, 299-5p and 376b-3p) did not bind either the 3'UTR or CDS of PRNP, and were therefore deemed indirect regulators of PrPC . Our results show that multiple miRNAs regulate PrPC levels both directly and indirectly. These findings may have profound implications for prion disease pathogenesis and potentially also for their therapy. Furthermore, the possible role of PrPC as a mediator of Aß toxicity suggests that its regulation by miRNAs may also impinge on Alzheimer's disease.

A Large Panel of Isogenic APP and PSEN1 Mutant Human iPSC Neurons Reveals Shared Endosomal Abnormalities Mediated by APP ß-CTFs, Not Aß.

Familial Alzheimer's disease (fAD) results from mutations in the amyloid precursor protein (APP) and presenilin (PSEN1 and PSEN2) genes. Here we leveraged recent advances in induced pluripotent stem cell (iPSC) and CRISPR/Cas9 genome editing technologies to generate a panel of isogenic knockin human iPSC lines carrying APP and/or PSEN1 mutations. Global transcriptomic and translatomic profiling revealed that fAD mutations have overlapping effects on the expression of AD-related and endocytosis-associated genes. Mutant neurons also increased Rab5+ early endosome size. APP and PSEN1 mutations had discordant effects on Aß production but similar effects on APP ß C-terminal fragments (ß-CTFs), which accumulate in all mutant neurons. Importantly, endosomal dysfunction correlated with accumulation of ß-CTFs, not Aß, and could be rescued by pharmacological modulation of ß-secretase (BACE). These data display the utility of our mutant iPSCs in studying AD-related phenotypes in a non-overexpression human-based system and support mounting evidence that ß-CTF may be critical in AD pathogenesis.

Whole-genome deep-learning analysis identifies contribution of noncoding mutations to autism risk.

We address the challenge of detecting the contribution of noncoding mutations to disease with a deep-learning-based framework that predicts the specific regulatory effects and the deleterious impact of genetic variants. Applying this framework to 1,790 autism spectrum disorder (ASD) simplex families reveals a role in disease for noncoding mutations-ASD probands harbor both transcriptional- and post-transcriptional-regulation-disrupting de novo mutations of significantly higher functional impact than those in unaffected siblings. Further analysis suggests involvement of noncoding mutations in synaptic transmission and neuronal development and, taken together with previous studies, reveals a convergent genetic landscape of coding and noncoding mutations in ASD. We demonstrate that sequences carrying prioritized mutations identified in probands possess allele-specific regulatory activity, and we highlight a link between noncoding mutations and heterogeneity in the IQ of ASD probands. Our predictive genomics framework illuminates the role of noncoding mutations in ASD and prioritizes mutations with high impact for further study, and is broadly applicable to complex human diseases.

Regulatory consequences of neuronal ELAV-like protein binding to coding and non-coding RNAs in human brain.

Neuronal ELAV-like (nELAVL) RNA binding proteins have been linked to numerous neurological disorders. We performed crosslinking-immunoprecipitation and RNAseq on human brain, and identified nELAVL binding sites on 8681 transcripts. Using knockout mice and RNAi in human neuroblastoma cells, we showed that nELAVL intronic and 3' UTR binding regulates human RNA splicing and abundance. We validated hundreds of nELAVL targets among which were important neuronal and disease-associated transcripts, including Alzheimer's disease (AD) transcripts. We therefore investigated RNA regulation in AD brain, and observed differential splicing of 150 transcripts, which in some cases correlated with differential nELAVL binding. Unexpectedly, the most significant change of nELAVL binding was evident on non-coding Y RNAs. nELAVL/Y RNA complexes were specifically remodeled in AD and after acute UV stress in neuroblastoma cells. We propose that the increased nELAVL/Y RNA association during stress may lead to nELAVL sequestration, redistribution of nELAVL target binding, and altered neuronal RNA splicing.

Phosphorylation of tristetraprolin by MK2 impairs AU-rich element mRNA decay by preventing deadenylase recruitment.

mRNA turnover is a critical step in the control of gene expression. In mammalian cells, a subset of mRNAs regulated at the level of mRNA turnover contain destabilizing AU-rich elements (AREs) in their 3' untranslated regions. These transcripts are bound by a suite of ARE-binding proteins (AUBPs) that receive information from cell signaling events to modulate rates of ARE mRNA decay. Here we show that a key destabilizing AUBP, tristetraprolin (TTP), is repressed by the p38 mitogen-activated protein kinase (MAPK)-activated kinase MK2 due to the inability of phospho-TTP to recruit deadenylases to target mRNAs. TTP is tightly associated with cytoplasmic deadenylases and promotes rapid deadenylation of target mRNAs both in vitro and in cells. TTP can direct the deadenylation of substrate mRNAs when tethered to a heterologous mRNA, yet its ability to do so is inhibited upon phosphorylation by MK2. Phospho-TTP is not impaired in mRNA binding but does fail to recruit the major cytoplasmic deadenylases. These observations suggest that phosphorylation of TTP by MK2 primarily affects mRNA decay downstream of RNA binding by preventing recruitment of the deadenylation machinery. Thus, TTP may remain poised to rapidly reactivate deadenylation of bound transcripts to downregulate gene expression once the p38 MAPK pathway is deactivated.