In Situ Hybridization of circRNAs in Cells and Tissues through BaseScope™ Strategy.

Imaging-based approaches are powerful strategies that nowadays have been largely used to gain insight into the function of different types of macromolecules. As for RNA, it is becoming clear how important is its intracellular localization for the control of proper cell differentiation and development and how its perturbation can be linked to several pathological states. This aspect is even more important if one thinks of highly polarized cells such as neurons.In this chapter, we describe in detail an innovative RNA-FISH approach for the detection of circular RNAs (circRNAs), a recently discovered class of noncoding RNAs, which display different subcellular localizations and whose functions still largely remain to be elucidated. The detection of these molecules represents a great challenge, above all because they share most of their sequence with the corresponding linear counterparts, from which they differ only for the back-splicing junction (BSJ) originating from the circularization reaction. This implies the use of RNA-FISH probes capable of specifically binding the BSJ and avoiding the detection of the linear counterpart. This requirement imposes the design of probes on a very small region, which implies the risk of obtaining a low and undetectable signal. The BaseScope™ Assay RNA-FISH technology overpasses this problem since it is based on branched-DNA probes. With this approach it is possible to target a specific region of the RNA, even small such as a splicing junction, and at the same time to obtain a strong and well detectable signal. All this is possible thanks to subsequent series of probes that, starting from the first hybridization to the BSJ, build a branched tree of probes that greatly amplifies the signal. Here we provide a detailed step-by-step protocol of BaseScope™ RNA-FISH on circRNAs coupled with immunofluorescence, both in cells and tissues, and we address difficulties which may arise when using this methodology that depend on cell type, specific permeabilization, image acquisition, and post-acquisition analyses.

The long noncoding RNA nHOTAIRM1 is necessary for differentiation and activity of iPSC-derived spinal motor neurons.

The mammalian nervous system is made up of an extraordinary array of diverse cells that form intricate functional connections. The programs underlying cell lineage specification, identity and function of the neuronal subtypes are managed by regulatory proteins and RNAs, which coordinate the succession of steps in a stereotyped temporal order. In the central nervous system (CNS), motor neurons (MNs) are responsible for controlling essential functions such as movement, breathing, and swallowing by integrating signal transmission from the cortex, brainstem, and spinal cord (SC) towards peripheral muscles. A prime role in guiding the progression of progenitor cells towards the MN fate has been largely attributed to protein factors. More recently, the relevance of a class of regulatory RNAs abundantly expressed in the CNS - the long noncoding RNAs (lncRNAs) - has emerged overwhelmingly. LncRNA-driven gene expression control is key to regulating any step of MN differentiation and function, and its derangement profoundly impacts neuronal pathophysiology. Here, we uncover a novel function for the neuronal isoform of HOTAIRM1 (nHOTAIRM1), a lncRNA specifically expressed in the SC. Using a model system that recapitulates spinal MN (spMN) differentiation, we show that nHOTAIRM1 intervenes in the binary cell fate decision between MNs and interneurons, acting as a pro-MN factor. Furthermore, human iPSC-derived spMNs without nHOTAIRM1 display altered neurite outgrowth, with a significant reduction of both branch and junction numbers. Finally, the expression of genes essential for synaptic connectivity and neurotransmission is also profoundly impaired when nHOTAIRM1 is absent in spMNs. Mechanistically, nHOTAIRM1 establishes both direct and indirect interactions with a number of target genes in the cytoplasm, being a novel post-transcriptional regulator of MN biology. Overall, our results indicate that the lncRNA nHOTAIRM1 is essential for the specification of MN identity and the acquisition of proper morphology and synaptic activity of post-mitotic MNs.

FUS Alters circRNA Metabolism in Human Motor Neurons Carrying the ALS-Linked P525L Mutation.

Deregulation of RNA metabolism has emerged as one of the key events leading to the degeneration of motor neurons (MNs) in Amyotrophic Lateral Sclerosis (ALS) disease. Indeed, mutations on RNA-binding proteins (RBPs) or on proteins involved in aspects of RNA metabolism account for the majority of familiar forms of ALS. In particular, the impact of the ALS-linked mutations of the RBP FUS on many aspects of RNA-related processes has been vastly investigated. FUS plays a pivotal role in splicing regulation and its mutations severely alter the exon composition of transcripts coding for proteins involved in neurogenesis, axon guidance, and synaptic activity. In this study, by using in vitro-derived human MNs, we investigate the effect of the P525L FUS mutation on non-canonical splicing events that leads to the formation of circular RNAs (circRNAs). We observed altered levels of circRNAs in FUSP525L MNs and a preferential binding of the mutant protein to introns flanking downregulated circRNAs and containing inverted Alu repeats. For a subset of circRNAs, FUSP525L also impacts their nuclear/cytoplasmic partitioning, confirming its involvement in different processes of RNA metabolism. Finally, we assess the potential of cytoplasmic circRNAs to act as miRNA sponges, with possible implications in ALS pathogenesis.

Advances in endogenous RNA pull-down: A straightforward dextran sulfate-based method enhancing RNA recovery.

Detecting RNA/RNA interactions in the context of a given cellular system is crucial to gain insights into the molecular mechanisms that stand beneath each specific RNA molecule. When it comes to non-protein coding RNA (ncRNAs), and especially to long noncoding RNAs (lncRNAs), the reliability of the RNA purification is dramatically dependent on their abundance. Exogenous methods, in which lncRNAs are in vitro transcribed and incubated with protein extracts or overexpressed by cell transfection, have been extensively used to overcome the problem of abundance. However, although useful to study the contribution of single RNA sub-modules to RNA/protein interactions, these exogenous practices might fail in revealing biologically meaningful contacts occurring in vivo and risk to generate non-physiological artifacts. Therefore, endogenous methods must be preferred, especially for the initial identification of partners specifically interacting with elected RNAs. Here, we apply an endogenous RNA pull-down to lncMN2-203, a neuron-specific lncRNA contributing to the robustness of motor neurons specification, through the interaction with miRNA-466i-5p. We show that both the yield of lncMN2-203 recovery and the specificity of its interaction with the miRNA dramatically increase in the presence of Dextran Sulfate Sodium (DSS) salt. This new set-up may represent a powerful means for improving the study of RNA-RNA interactions of biological significance, especially for those lncRNAs whose role as microRNA (miRNA) sponges or regulators of mRNA stability was demonstrated.

Circ-Hdgfrp3 shuttles along neurites and is trapped in aggregates formed by ALS-associated mutant FUS.

CircRNAs belong to a family of RNA molecules which are conserved in evolution, have tissue-specific expression, and are abundant in neuronal cells. Here, we define several features of circ-Hdgfrp3 and describe interesting alterations occurring in motor neurons (MNs) carrying ALS-associated FUS mutations. Through a highly sensitive in situ approach we describe that circ-Hdgfrp3 traffics along neurites, while upon oxidative stress it is retained in the perinuclear region. While in wild-type stressed MNs, circ-Hdgfrp3 localizes in stress granules (SGs), in MNs carrying mutant FUS, a higher proportion of circ-Hdgfrp3 was trapped into cytoplasmic aggregates. Upon stress removal, circ-Hdgfrp3 was easily freed from SGs whereas it was less efficiently released from FUS-aggregates. We found that the human circ-Hdgfrp3 counterpart was also similarly associated to mutant FUS-aggregates in stressed neuronal cells. Overall, the alteration of circ-Hdgfrp3 trafficking adds a further layer of complexity to the role of FUS-aggregates in ALS disease.

Study of Circular RNA Expression by Nonradioactive Northern Blot Procedure.

Circular RNAs (circRNAs) are covalently closed transcripts generated by back-splicing reaction. The lack of free ends endows these RNA molecules with high stability thus allowing them to accumulate in tissues and body fluids. They are widely expressed in most organisms, are modulated during development and display tissue-specific expression, resulting particularly enriched in the nervous system. Deregulation of circRNA expression has also been associated with several pathological conditions including neurological diseases and cancer.Here we present a Northern blot procedure that allows the analysis of the expression of bona fide circRNAs through the use of a digoxigenin-labeled RNA probe and the immunodetection of the signals.

Exploring the Regulatory Role of Circular RNAs in Neurodegenerative Disorders.

Circular RNAs (circRNAs) are a distinctive class of regulatory non-coding RNAs characterised by the presence of covalently closed ends. They are evolutionary conserved molecules, and although detected in different tissues, circRNAs resulted specifically enriched in the nervous system. Recent studies have shown that circRNAs are dynamically modulated during neuronal development and aging, that circRNAs are enriched at synaptic levels and resulted modulated after synaptic plasticity induction. This has suggested that circRNAs might play an important role in neuronal specification and activity. Despite the exact function of circRNAs is still poorly understood, emerging evidence indicates that circRNAs have important regulatory functions that might extensively contribute to the dynamic modulation of gene expression that supports neuronal pathways. More interestingly, deregulation of circRNAs expression has been linked with various pathological conditions. In this review, we describe current advances in the field of circRNA biogenesis and function in the nervous system both in physiological and in pathological conditions, and we specifically lay out their association with neurodegenerative diseases. Furthermore, we discuss the opportunity to exploit circRNAs for innovative therapeutic approaches and, due to their high stability, to use circRNAs as suitable biomarkers for diagnosis and disease progression.

Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset disease characterized by upper and lower motor neuron degeneration, muscle wasting and paralysis. Growing evidence suggests a link between changes in lipid metabolism and ALS. Here, we used UPLC/TOF-MS to survey the lipidome in SOD1(G86R) mice, a model of ALS. Significant changes in lipid expression were evident in spinal cord and skeletal muscle before overt neuropathology. In silico analysis also revealed appreciable changes in sphingolipids including ceramides and glucosylceramides (GlcCer). HPLC analysis showed increased amounts of GlcCer and downstream glycosphingolipids (GSLs) in SOD1(G86R) muscle compared with wild-type littermates. Glucosylceramide synthase (GCS), the enzyme responsible for GlcCer biosynthesis, was up-regulated in muscle of SOD1(G86R) mice and ALS patients, and in muscle of wild-type mice after surgically induced denervation. Conversely, inhibition of GCS in wild-type mice, following transient peripheral nerve injury, reversed the overexpression of genes in muscle involved in oxidative metabolism and delayed motor recovery. GCS inhibition in SOD1(G86R) mice also affected the expression of metabolic genes and induced a loss of muscle strength and morphological deterioration of the motor endplates. These findings suggest that GSLs may play a critical role in ALS muscle pathology and could lead to the identification of new therapeutic targets.