Neuronal activity-dependent ATP enhances the pro-growth effect of repair Schwann cell extracellular vesicles by increasing their miRNA-21 loading.

Functional recovery after peripheral nerve injuries is critically dependent on axonal regeneration. Several autonomous and non-cell autonomous processes regulate axonal regeneration, including the activation of a growth-associated transcriptional program in neurons and the reprogramming of differentiated Schwann cells (dSCs) into repair SCs (rSCs), triggering the secretion of neurotrophic factors and the activation of an inflammatory response. Repair Schwann cells also release pro-regenerative extracellular vesicles (EVs), but is still unknown whether EV secretion is regulated non-cell autonomously by the regenerating neuron. Interestingly, it has been described that nerve activity enhances axonal regeneration by increasing the secretion of neurotrophic factors by rSC, but whether this activity modulates pro-regenerative EV secretion by rSC has not yet been explored. Here, we demonstrate that neuronal activity enhances the release of rSC-derived EVs and their transfer to neurons. This effect is mediated by activation of P2Y receptors in SCs after activity-dependent ATP release from sensory neurons. Importantly, activation of P2Y in rSCs also increases the amount of miRNA-21 present in rSC-EVs. Taken together, our results demonstrate that neuron to glia communication by ATP-P2Y signaling regulates the content of SC-derived EVs and their transfer to axons, modulating axonal elongation in a non-cell autonomous manner.

Ex Vivo Analysis of Axonal Degeneration Using Sciatic and Optic Nerve Preparations.

This chapter describes techniques associated to the study of axonal degeneration in the peripheral (PNS) and central nervous system (CNS) using in vitro cultured sciatic and optic nerves from mice, a technique commonly referred to as ex vivo nerve explant analysis. Degeneration of axons in this technique is induced by axotomy (or exeresis) upon dissection of nerves from the PNS or CNS. Nerves explants can be analyzed by different techniques hours or days after in vitro culture. This model has the advantage to represent an intermediate model between in vitro and in vivo. Importantly, it allows for easy administration of drugs, electrical stimulation, and is especially suited for biochemical and morphological analysis. In addition, nerve explants can be obtained from mice of different genetic backgrounds, including knockout and transgenic animals, and allows the study of Wallerian degeneration without interference from the inflammatory reaction and macrophage infiltration that takes place after nerve injury in vivo. The protocol presented here constitutes a valuable tool to analyze in vitro the mechanisms associated to axonal degeneration and the role of Schwann cells in this process.

Schwann cell reprogramming into repair cells increases miRNA-21 expression in exosomes promoting axonal growth.

Functional recovery after peripheral nerve damage is dependent on the reprogramming of differentiated Schwann cells (dSCs) into repair Schwann cells (rSCs), which promotes axonal regeneration and tissue homeostasis. Transition into a repair phenotype requires expression of c-Jun and Sox2, which transcriptionally mediates inhibition of the dSC program of myelination and activates a non-cell-autonomous repair program, characterized by the secretion of neuronal survival and regenerative molecules, formation of a cellular scaffold to guide regenerating axons and activation of an innate immune response. Moreover, rSCs release exosomes that are internalized by peripheral neurons, promoting axonal regeneration. Here, we demonstrate that reprogramming of Schwann cells (SCs) is accompanied by a shift in the capacity of their secreted exosomes to promote neurite growth, which is dependent on the expression of c-Jun (also known as Jun) and Sox2 by rSCs. Furthermore, increased expression of miRNA-21 is responsible for the pro-regenerative capacity of rSC exosomes, which is associated with PTEN downregulation and PI3-kinase activation in neurons. We propose that modification of exosomal cargo constitutes another important feature of the repair program of SCs, contributing to axonal regeneration and functional recovery after nerve injury.

In Vitro Analysis of the Role of Schwann Cells on Axonal Degeneration and Regeneration Using Sensory Neurons from Dorsal Root Ganglia.

Sensory neurons from dorsal root ganglion efficiently regenerate after peripheral nerve injuries. These neurons are widely used as a model system to study degenerative mechanisms of the soma and axons, as well as regenerative axonal growth in the peripheral nervous system. This chapter describes techniques associated to the study of axonal degeneration and regeneration using explant cultures of dorsal root ganglion sensory neurons in vitro in the presence or absence of Schwann cells. Schwann cells are extremely important due to their involvement in tissue clearance during axonal degeneration as well as their known pro-regenerative effect during regeneration in the peripheral nervous system. We describe methods to induce and study axonal degeneration triggered by axotomy (mechanical separation of the axon from its soma) and treatment with vinblastine (which blocks axonal transport), which constitute clinically relevant mechanical and toxic models of axonal degeneration. In addition, we describe three different methods to evaluate axonal regeneration using quantitative methods. These protocols constitute a valuable tool to analyze in vitro mechanisms associated to axonal degeneration and regeneration of sensory neurons and the role of Schwann cells in these processes.

Purification of Exosomes from Primary Schwann Cells, RNA Extraction, and Next-Generation Sequencing of Exosomal RNAs.

Exosomes are small (30-150 nm) vesicles of endosomal origin secreted by most cell types. Exosomes contain proteins, lipids, and RNA species including microRNA, mRNA, rRNA, and long noncoding RNAs. The mechanisms associated with exosome synthesis and cargo loading are still poorly understood. A role for exosomes in intercellular communication has been reported in physiological and pathological conditions both in vitro and in vivo. Previous studies have suggested that Schwann cell-derived exosomes regulate neuronal functions, but the mechanisms are still unclear. Here, we describe protocols to establish rat neonatal Schwann cell cultures and to isolate exosomes from the conditioned medium of these cultures by differential ultracentrifugation. To analyze the RNA content of Schwann cell-derived exosomes, we detail protocols for RNA extraction and next-generation sequencing using miRNA and mRNA libraries. The protocol also includes RNA sequencing of Schwann cells, which allows the comparison between RNA content from cells and the secreted exosomes. Identification of RNAs present in Schwann cell-derived exosomes is a valuable tool to understand novel roles of Schwann cells in neuronal function in health and disease.

Origin of axonal proteins: Is the axon-schwann cell unit a functional syncytium?

The structural homeostasis is challenging for neurons, whose axons extend up to meters in large animals, and the axoplasmic mass reaches over a thousand times that of the cell body. Thus, the protein demand may overcome the capacity of the cell body to supply the right protein species, to the right place, in the right time. In this context, a body of evidence indicates that glial cells support the axonal maintenance and regenerative responses by diverse mechanisms of intercellular communication. We showed recently that Schwann cells (SC) transfer ribosomes to axons and also enhance regeneration by means of extracellular vesicles known as exosomes that contain mRNAs, miRNAs and proteins. These findings strongly suggest that the nucleus of the SC supports the machinery for protein synthesis of the axon and participates in the specification of the phenotype of the underlying axon. That the genetic programs of many nuclei modulate the axoplasm on a local basis is akin to a syncytium but at variance with it, the nuclei belong to satellite cells. We propose that the SC-axon unit is a functional syncytium. This intercellular organization opens a novel understanding of the nervous system and a new avenue of research into its physiology and disorders © 2016 Wiley Periodicals, Inc.

Schwann Cell Exosomes Mediate Neuron-Glia Communication and Enhance Axonal Regeneration.

The functional and structural integrity of the nervous system depends on the coordinated action of neurons and glial cells. Phenomena like synaptic activity, conduction of action potentials, and neuronal growth and regeneration, to name a few, are fine tuned by glial cells. Furthermore, the active role of glial cells in the regulation of neuronal functions is underscored by several conditions in which specific mutation affecting the glia results in axonal dysfunction. We have shown that Schwann cells (SCs), the peripheral nervous system glia, supply axons with ribosomes, and since proteins underlie cellular programs or functions, this dependence of axons from glial cells provides a new and unexplored dimension to our understanding of the nervous system. Recent evidence has now established a new modality of intercellular communication through extracellular vesicles. We have already shown that SC-derived extracellular vesicles known as exosomes enhance axonal regeneration, and increase neuronal survival after pro-degenerative stimuli. Therefore, the biology nervous system will have to be reformulated to include that the phenotype of a nerve cell results from the contribution of two nuclei, with enormous significance for the understanding of the nervous system in health and disease.

Partially redundant enhancers cooperatively maintain Mammalian pomc expression above a critical functional threshold.

Cell-specific expression of many genes is conveyed by multiple enhancers, with each individual enhancer controlling a particular expression domain. In contrast, multiple enhancers drive similar expression patterns of some genes involved in embryonic development, suggesting regulatory redundancy. Work in Drosophila has indicated that functionally overlapping enhancers canalize development by buffering gene expression against environmental and genetic disturbances. However, little is known about regulatory redundancy in vertebrates and in genes mainly expressed during adulthood. Here we study nPE1 and nPE2, two phylogenetically conserved mammalian enhancers that drive expression of the proopiomelanocortin gene (Pomc) to the same set of hypothalamic neurons. The simultaneous deletion of both enhancers abolished Pomc expression at all ages and induced a profound metabolic dysfunction including early-onset extreme obesity. Targeted inactivation of either nPE1 or nPE2 led to very low levels of Pomc expression during early embryonic development indicating that both enhancers function synergistically. In adult mice, however, Pomc expression is controlled additively by both enhancers, with nPE1 being responsible for ∼80% and nPE2 for ∼20% of Pomc transcription. Consequently, nPE1 knockout mice exhibit mild obesity whereas nPE2-deficient mice maintain a normal body weight. These results suggest that nPE2-driven Pomc expression is compensated by nPE1 at later stages of development, essentially rescuing the earlier phenotype of nPE2 deficiency. Together, these results reveal that cooperative interactions between the enhancers confer robustness of Pomc expression against gene regulatory disturbances and preclude deleterious metabolic phenotypes caused by Pomc deficiency in adulthood. Thus, our study demonstrates that enhancer redundancy can be used by genes that control adult physiology in mammals and underlines the potential significance of regulatory sequence mutations in common diseases.

A fast-evolving human NPAS3 enhancer gained reporter expression in the developing forebrain of transgenic mice.

The developmental brain gene NPAS3 stands out as a hot spot in human evolution because it contains the largest number of human-specific, fast-evolving, conserved, non-coding elements. In this paper we studied 2xHAR142, one of these elements that is located in the fifth intron of NPAS3. Using transgenic mice, we show that the mouse and chimp 2xHAR142 orthologues behave as transcriptional enhancers driving expression of the reporter gene lacZ to a similar NPAS3 expression subdomain in the mouse central nervous system. Interestingly, the human 2xHAR142 orthologue drives lacZ expression to an extended expression pattern in the nervous system. Thus, molecular evolution of 2xHAR142 provides the first documented example of human-specific heterotopy in the forebrain promoted by a transcriptional enhancer and suggests that it may have contributed to assemble the unique properties of the human brain.

Convergent evolution of two mammalian neuronal enhancers by sequential exaptation of unrelated retroposons.

The proopiomelanocortin gene (POMC) is expressed in a group of neurons present in the arcuate nucleus of the hypothalamus. Neuron-specific POMC expression in mammals is conveyed by two distal enhancers, named nPE1 and nPE2. Previous transgenic mouse studies showed that nPE1 and nPE2 independently drive reporter gene expression to POMC neurons. Here, we investigated the evolutionary mechanisms that shaped not one but two neuron-specific POMC enhancers and tested whether nPE1 and nPE2 drive identical or complementary spatiotemporal expression patterns. Sequence comparison among representative genomes of most vertebrate classes and mammalian orders showed that nPE1 is a placental novelty. Using in silico paleogenomics we found that nPE1 originated from the exaptation of a mammalian-apparent LTR retrotransposon sometime between the metatherian/eutherian split (147 Mya) and the placental mammal radiation (≈ 90 Mya). Thus, the evolutionary origin of nPE1 differs, in kind and time, from that previously demonstrated for nPE2, which was exapted from a CORE-short interspersed nucleotide element (SINE) retroposon before the origin of prototherians, 166 Mya. Transgenic mice expressing the fluorescent markers tomato and EGFP driven by nPE1 or nPE2, respectively, demonstrated coexpression of both reporter genes along the entire arcuate nucleus. The onset of reporter gene expression guided by nPE1 and nPE2 was also identical and coincidental with the onset of Pomc expression in the presumptive mouse diencephalon. Thus, the independent exaptation of two unrelated retroposons into functional analogs regulating neuronal POMC expression constitutes an authentic example of convergent molecular evolution of cell-specific enhancers.

The estrogen receptor α colocalizes with proopiomelanocortin in hypothalamic neurons and binds to a conserved motif present in the neuron-specific enhancer nPE2.

The gene encoding the prohormone proopiomelanocortin (POMC) is mainly expressed in two regions in vertebrates, namely corticotrophs and melanotrophs in the pituitary and a small population of neurons in the arcuate nucleus of the hypothalamus. In this latter region, POMC-derived peptides participate in the control of energy balance and sensitivity to pain. Neuronal expression of POMC is conferred by two enhancers, nPE1 and nPE2, which are conserved in most mammals, but no transcription factors are yet known to bind to these enhancers. In this work, by means of a one-hybrid screening, we identify that nPE2 possesses an element recognized by transcription factors of the nuclear receptor superfamily. This element, named NRBE, is conserved in all known nPE2 enhancers and is necessary to confer full enhancer strength to nPE2-driven reporter gene expression in transgenic mice assays, indicating that the phylogenetic conservation of the element is indicative of its functional importance. In a search for candidate nuclear receptors that might control POMC we observed that estrogen receptor alpha (ESR1) - a known regulator of energy balance at the hypothalamic level - can bind to the NRBE element in vitro. In addition we observed by immunofluorescence that ESR1 is coexpressed with POMC in around 25-30% of hypothalamic neurons of males and females during late embryonic stages and adulthood. Thus, our results indicate that hypothalamic expression of POMC is controlled by nuclear receptors and establish ESR1 as a candidate regulator of POMC.

Transcriptional regulation of pituitary POMC is conserved at the vertebrate extremes despite great promoter sequence divergence.

The stress response involves complex physiological mechanisms that maximize behavioral efficacy during attack or defense and is highly conserved in all vertebrates. Key mediators of the stress response are pituitary hormones encoded by the proopiomelanocortin gene (POMC). Despite conservation of physiological function and expression pattern of POMC in all vertebrates, phylogenetic footprinting analyses at the POMC locus across vertebrates failed to detect conserved noncoding sequences with potential regulatory function. To investigate whether ortholog POMC promoters from extremely distant vertebrates are functionally conserved, we used 5'-flanking sequences of the teleost fish Tetraodon nigroviridis POMCalpha gene to produce transgenic mice. Tetraodon POMCalpha promoter targeted reporter gene expression exclusively to mouse pituitary cells that normally express Pomc. Importantly, transgenic expression in mouse corticotrophs was increased after adrenalectomy. To understand how conservation of precise gene expression mechanisms coexists with great sequence divergence, we investigated whether very short elements are still conserved in all vertebrate POMC promoters. Multiple local sequence alignments that consider phylogenetic relationships of ortholog regions identified a unique 10-bp motif GTGCTAA(T/G)CC that is usually present in two copies in POMC 5'-flanking sequences of all vertebrates. Underlined nucleotides represent totally conserved sequences. Deletion of these paired motifs from Tetraodon POMCalpha promoter markedly reduced its transcriptional activity in a mouse corticotropic cell line and in pituitary POMC cells of transgenic mice. In mammals, the conserved motifs correspond to reported binding sites for pituitary-specific nuclear proteins that participate in POMC transcriptional regulation. Together, these results demonstrate that mechanisms that participate in pituitary-specific and hormonally regulated expression of POMC have been preserved since mammals and teleosts diverged from a common ancestor 450 million years ago despite great promoter sequence divergence.