Extracellular vesicles and their role in peripheral nerve regeneration.

Peripheral nerve injuries often result in sensory and motor dysfunction in respective parts of the body. Regeneration after peripheral nerve injuries is a complex process including the differentiation of Schwann cells, recruiting of macrophages, blood vessel growth and axonal regrowth. Extracellular vesicles (EVs) are considered to play a pivotal role in intercellular communication and transfer of biological information. Specifically, their bioactivity and ability to deliver cargos of various types of nucleic acids and proteins have made them a potential vehicle for neurotherapeutics. However, production, characterization, dosage and targeted delivery of EVs still pose challenges for the clinical translation of EV therapeutics. This review summarizes the current knowledge of EVs in the context of the healthy and injured peripheral nerve and addresses novel concepts for modification of EVs as therapeutic agents for peripheral nerve regeneration.

SoxD transcription factor deficiency in Schwann cells delays myelination in the developing peripheral nervous system.

The three SoxD proteins, Sox5, Sox6 and Sox13, represent closely related transcription factors with important roles during development. In the developing nervous system, SoxD proteins have so far been primarily studied in oligodendroglial cells and in interneurons of brain and spinal cord. In oligodendroglial cells, Sox5 and Sox6 jointly maintain the precursor state, interfere with terminal differentiation, and thereby ensure the proper timing of myelination in the central nervous system. Here we studied the role of SoxD proteins in Schwann cells, the functional counterpart of oligodendrocytes in the peripheral nervous system. We show that Schwann cells express Sox5 and Sox13 but not Sox6. Expression was transient and ceased with the onset of terminal differentiation. In mice with early Schwann cell-specific deletion of both Sox5 and Sox13, embryonic Schwann cell development was not substantially affected and progressed normally into the promyelinating stage. However, there was a mild and transient delay in the myelination of the peripheral nervous system of these mice. We therefore conclude that SoxD proteins-in stark contrast to their action in oligodendrocytes-promote differentiation and myelination in Schwann cells.

Autism Spectrum Disorders: Multiple Routes to, and Multiple Consequences of, Abnormal Synaptic Function and Connectivity.

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders of genetic and environmental etiologies. Some ASD cases are syndromic: associated with clinically defined patterns of somatic abnormalities and a neurobehavioral phenotype (e.g., Fragile X syndrome). Many cases, however, are idiopathic or non-syndromic. Such disorders present themselves during the early postnatal period when language, speech, and personality start to develop. ASDs manifest by deficits in social communication and interaction, restricted and repetitive patterns of behavior across multiple contexts, sensory abnormalities across multiple modalities and comorbidities, such as epilepsy among many others. ASDs are disorders of connectivity, as synaptic dysfunction is common to both syndromic and idiopathic forms. While multiple theories have been proposed, particularly in idiopathic ASDs, none address why certain brain areas (e.g., frontotemporal) appear more vulnerable than others or identify factors that may affect phenotypic specificity. In this hypothesis article, we identify possible routes leading to, and the consequences of, altered connectivity and review the evidence of central and peripheral synaptic dysfunction in ASDs. We postulate that phenotypic specificity could arise from aberrant experience-dependent plasticity mechanisms in frontal brain areas and peripheral sensory networks and propose why the vulnerability of these areas could be part of a model to unify preexisting pathophysiological theories.

FGF21 impedes peripheral myelin development by stimulating p38 MAPK/c-Jun axis.

Fibroblast growth factor 21 (FGF21) as a metabolic stress hormone, is mainly secreted by the liver. In addition to its well-defined roles in energy homeostasis, FGF21 has been shown to promote remyelination after injury in the central nervous system. In the current study, we sought to examine the potential roles of FGF21 in the peripheral nervous system (PNS) myelination. In the PNS myelin development, Fgf21 expression was reversely correlated with myelin gene expression. In cultured primary Schwann cells (SCs), the application of recombinant FGF21 greatly attenuates myelination-associated gene expression, including Oct6, Krox20, Mbp, Mpz, and Pmp22. Accordingly, the injection of FGF21 into neonatal rats markedly mitigates the myelination in sciatic nerves. On the contrary, the infusion of the anti-FGF21 antibody accelerates the myelination. Mechanistically, both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) were stimulated by FGF21 in SCs and sciatic nerves. Following experiments including pharmaceutical intervention and gene manipulation revealed that the p38 MAPK/c-Jun axis, rather than ERK, is targeted by FGF21 for mediating its repression on myelination in SCs. Taken together, our data provide a new aspect of FGF21 by acting as a negative regulator for the myelin development process in the PNS via activation of p38 MAPK/c-Jun.

A Budding Relationship: Bacterial Extracellular Vesicles in the Microbiota-Gut-Brain Axis.

The discovery of the microbiota-gut-brain axis has revolutionized our understanding of systemic influences on brain function and may lead to novel therapeutic approaches to neurodevelopmental and mood disorders. A parallel revolution has occurred in the field of intercellular communication, with the realization that endosomes, and other extracellular vesicles, rival the endocrine system as regulators of distant tissues. These two paradigms shifting developments come together in recent observations that bacterial membrane vesicles contribute to inter-kingdom signaling and may be an integral component of gut microbe communication with the brain. In this short review we address the current understanding of the biogenesis of bacterial membrane vesicles and the roles they play in the survival of microbes and in intra and inter-kingdom communication. We identify recent observations indicating that bacterial membrane vesicles, particularly those derived from probiotic organisms, regulate brain function. We discuss mechanisms by which bacterial membrane vesicles may influence the brain including interaction with the peripheral nervous system, and modulation of immune activity. We also review evidence suggesting that, unlike the parent organism, gut bacteria derived membrane vesicles are able to deliver cargo, including neurotransmitters, directly to the central nervous system and may thus constitute key components of the microbiota-gut-brain axis.

How Does Protein Zero Assemble Compact Myelin?

Myelin protein zero (P0), a type I transmembrane protein, is the most abundant protein in peripheral nervous system (PNS) myelin-the lipid-rich, periodic structure of membrane pairs that concentrically encloses long axonal segments. Schwann cells, the myelinating glia of the PNS, express P0 throughout their development until the formation of mature myelin. In the intramyelinic compartment, the immunoglobulin-like domain of P0 bridges apposing membranes via homophilic adhesion, forming, as revealed by electron microscopy, the electron-dense, double "intraperiod line" that is split by a narrow, electron-lucent space corresponding to the extracellular space between membrane pairs. The C-terminal tail of P0 adheres apposing membranes together in the narrow cytoplasmic compartment of compact myelin, much like myelin basic protein (MBP). In mouse models, the absence of P0, unlike that of MBP or P2, severely disturbs myelination. Therefore, P0 is the executive molecule of PNS myelin maturation. How and when P0 is trafficked and modified to enable myelin compaction, and how mutations that give rise to incurable peripheral neuropathies alter the function of P0, are currently open questions. The potential mechanisms of P0 function in myelination are discussed, providing a foundation for the understanding of mature myelin development and how it derails in peripheral neuropathies.

Expression of Toll-like receptors 4 and 7 in murine peripheral nervous system development.

BACKGROUND: Toll-Like Receptors (TLRs) play a critical role in the innate and adaptive immune system. They are the mammalian orthologs of Drosophila melanogaster protein Toll, which has been proved to have an early morphogenetic role in invertebrate embryogenesis that in the adult switches to an immune function. AIM: The aim of this study was to evaluate the expression of TLR4 and TLR7 during dorsal root ganglia (DRG), paravertebral ganglia (PVG), and enteric nervous system (ENS) murine development. METHODS: Mouse embryos from different stages (i.e. E12 to E18) were processed for immunolocalization analysis on formalin-fixed paraffin-embedded sections, and isolated intestine were processed for whole-mount preparations. RESULTS: We observed a differentially regulated expression of TLR4 and TLR7 during embryogenesis and an overall increased expression of both receptors during development. While TLR4 was detectable in neurons of DRG and PVG starting from E14 and only from E18 in the ENS, TLR7 was already expressed in scattered neurons of all the investigated regions at E12. CONCLUSIONS: TLR4 and TRL7 expression temporal patterns suggest a morphogenetic role for these receptors in the development of neural crest derivatives in mammals.

Role of neurotrophins in pregnancy, delivery and postpartum.

Neurotrophins (NTs) are a family of polypeptides whose functions have been extensively studied in the past two decades. In particular, Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF) play a major role in the development, nutrition and growth of the central and peripheral nervous system and in the pathogenesis of neurodegenerative, cardiometabolic and (auto)immune diseases. However, NGF and BDNF have subtle functions for follicular development, implantation, and placentation. This short narrative review summarizes the existing evidence, published between 2000 and 2019, about the role of NTs in many different conditions that might affect women during and after pregnancy such as preeclampsia, gestational diabetes, obesity, depression, anxiety, smoking and alcohol abuse. Literature suggests that the dysregulation of synthesis and release of NTs may lead to decisive effects on both maternal and fetal health. Some piece of evidences was found about a possible association between NGF/BDNF and breastfeeding. Additional studies on human models are necessary to further characterize the role of NTs in life-changing experiences like labor and delivery.

Construction and reconstruction of brain circuits: normal and pathological axon guidance.

Perception of our environment entirely depends on the close interaction between the central and peripheral nervous system. In order to communicate each other, both systems must develop in parallel and in coordination. During development, axonal projections from the CNS as well as the PNS must extend over large distances to reach their appropriate target cells. To do so, they read and follow a series of axon guidance molecules. Interestingly, while these molecules play critical roles in guiding developing axons, they have also been shown to be critical in other major neurodevelopmental processes, such as the migration of cortical progenitors. Currently, a major hurdle for brain repair after injury or neurodegeneration is the absence of axonal regeneration in the mammalian CNS. By contrasts, PNS axons can regenerate. Many hypotheses have been put forward to explain this paradox but recent studies suggest that hacking neurodevelopmental mechanisms may be the key to promote CNS regeneration. Here we provide a seminar report written by trainees attending the second Flagship school held in Alpbach, Austria in September 2018 organized by the International Society for Neurochemistry (ISN) together with the Journal of Neurochemistry (JCN). This advanced school has brought together leaders in the fields of neurodevelopment and regeneration in order to discuss major keystones and future challenges in these respective fields.

Migratory Neural Crest Cells Phagocytose Dead Cells in the Developing Nervous System.

During neural tube closure and spinal cord development, many cells die in both the central and peripheral nervous systems (CNS and PNS, respectively). However, myeloid-derived professional phagocytes have not yet colonized the trunk region during early neurogenesis. How apoptotic cells are removed from this region during these stages remains largely unknown. Using live imaging in zebrafish, we demonstrate that neural crest cells (NCCs) respond rapidly to dying cells and phagocytose cellular debris around the neural tube. Additionally, NCCs have the ability to enter the CNS through motor exit point transition zones and clear debris in the spinal cord. Surprisingly, NCCs phagocytosis mechanistically resembles macrophage phagocytosis and their recruitment toward cellular debris is mediated by interleukin-1ß. Taken together, our results reveal a role for NCCs in phagocytosis of debris in the developing nervous system before the presence of professional phagocytes.

Neuron-Glia Signaling in Synapse Elimination.

Maturation of neuronal circuits requires selective elimination of synaptic connections. Although neuron-intrinsic mechanisms are important in this process, it is increasingly recognized that glial cells also play a critical role. Without proper functioning of these cells, the number, morphology, and function of synaptic contacts are profoundly altered, resulting in abnormal connectivity and behavioral abnormalities. In addition to their role in synaptic refinement, glial cells have also been implicated in pathological synapse loss and dysfunction following injury or nervous system degeneration in adults. Although mechanisms regulating glia-mediated synaptic elimination are still being uncovered, it is clear this complex process involves many cues that promote and inhibit the removal of specific synaptic connections. Gaining a greater understanding of these signals and the contribution of different cell types will not only provide insight into this critical biological event but also be instrumental in advancing knowledge of brain development and neural disease.

Ral GTPases in Schwann cells promote radial axonal sorting in the peripheral nervous system.

Small GTPases of the Rho and Ras families are important regulators of Schwann cell biology. The Ras-like GTPases RalA and RalB act downstream of Ras in malignant peripheral nerve sheath tumors. However, the physiological role of Ral proteins in Schwann cell development is unknown. Using transgenic mice with ablation of one or both Ral genes, we report that Ral GTPases are crucial for axonal radial sorting. While lack of only one Ral GTPase was dispensable for early peripheral nerve development, ablation of both RalA and RalB resulted in persistent radial sorting defects, associated with hallmarks of deficits in Schwann cell process formation and maintenance. In agreement, ex vivo-cultured Ral-deficient Schwann cells were impaired in process extension and the formation of lamellipodia. Our data indicate further that RalA contributes to Schwann cell process extensions through the exocyst complex, a known effector of Ral GTPases, consistent with an exocyst-mediated function of Ral GTPases in Schwann cells.

Engulfing cells promote neuronal regeneration and remove neuronal debris through distinct biochemical functions of CED-1.

Two important biological events happen coincidently soon after nerve injury in the peripheral nervous system in C. elegans: removal of axon debris and initiation of axon regeneration. But, it is not known how these two events are co-regulated. Mutants of ced-1, a homolog of Draper and MEGF10, display defects in both events. One model is that those events could be related. But our data suggest that they are actually separable. CED-1 functions in the muscle-type engulfing cells in both events and is enriched in muscle protrusions in close contact with axon debris and regenerating axons. Its two functions occur through distinct biochemical mechanisms; extracellular domain-mediated adhesion for regeneration and extracellular domain binding-induced intracellular domain signaling for debris removal. These studies identify CED-1 in engulfing cells as a receptor in debris removal but as an adhesion molecule in neuronal regeneration, and have important implications for understanding neural circuit repair after injury.

Gli2 is required for the induction and migration of Xenopus laevis neural crest.

The neural crest (NC) is a multipotent migratory embryonic population that is formed during late gastrulation and gives rise to a wide array of derivatives, including cells from the peripheral nervous system (PNS), the craniofacial bones and cartilages, peripheral glial cells, and melanocyte cells, among others. In this work we analyzed the role of the Hedgehog signaling pathway effector gli2 in Xenopus NC. We provide evidence that the gli2 gene is expressed in the prospective, premigratory and migratory NC. The use of a specific morpholino against gli2 and the pharmacological specific inhibitor GANT61 in different experimental approaches allowed us to determine that gli2 is required for the induction and specification of NC cells as a transcriptional activator. Moreover, gli2 also acts by reducing apoptosis in the NC without affecting its cell proliferation status. We also demonstrated that gli2 is required cell-autonomously for NC migration, and for the formation of NC derivatives such as the craniofacial cartilages, melanocytes and the cranial ganglia. Altogether, our results showed that gli2 is a key transcriptional activator to accomplish the proper specification and development of Xenopus NC cells.

Axon Regeneration in the Central Nervous System: Facing the Challenges from the Inside.

After an injury in the adult mammalian central nervous system (CNS), lesioned axons fail to regenerate. This failure to regenerate contrasts with axons' remarkable potential to grow during embryonic development and after an injury in the peripheral nervous system (PNS). Several intracellular mechanisms-including cytoskeletal dynamics, axonal transport and trafficking, signaling and transcription of regenerative programs, and epigenetic modifications-control axon regeneration. In this review, we describe how manipulation of intrinsic mechanisms elicits a regenerative response in different organisms and how strategies are implemented to form the basis of a future regenerative treatment after CNS injury.

Essential Role of Linx/Islr2 in the Development of the Forebrain Anterior Commissure.

Linx is a member of the leucine-rich repeat and immunoglobulin family of membrane proteins which has critical roles in the development of the peripheral nervous system and forebrain connectivity. A previous study showed that Linx is expressed in projection neurons in the cortex and in cells that comprise the passage to the prethalamus that form the internal capsule, indicating the involvement of Linx in axon guidance and cell-cell communication. In this study, we found that Linx-deficient mice develop severe hydrocephalus and die perinatally by unknown mechanisms. Importantly, mice heterozygous for the linx gene exhibited defects in the development of the anterior commissure in addition to hydrocephalus, indicating haploinsufficiency of the linx gene in forebrain development. In N1E-115 neuroblastoma cells and primary cultured hippocampal neurons, Linx depletion led to impaired neurite extension and an increase in cell body size. Consistent with this, but of unknown significance, we found that Linx interacts with and upregulates the activity of Rho-kinase, a modulator of many cellular processes including cytoskeletal organization. These data suggest a role for Linx in the regulation of complex forebrain connectivity, and future identification of its extracellular ligand(s) will help clarify this function.

The peripheral nervous system of the ascidian tadpole larva: Types of neurons and their synaptic networks.

Physical and chemical cues from the environment are used to direct animal behavior through a complex network of connections originating in exteroceptors. In chordates, mechanosensory and chemosensory neurons of the peripheral nervous system (PNS) must signal to the motor circuits of the central nervous system (CNS) through a series of pathways that integrate and regulate the output to motor neurons (MN); ultimately these drive contraction of the tail and limb muscles. We used serial-section electron microscopy to reconstruct PNS neurons and their hitherto unknown synaptic networks in the tadpole larva of a sibling chordate, the ascidian, Ciona intestinalis. The larva has groups of neurons in its apical papillae, epidermal neurons in the rostral and apical trunk, caudal neurons in the dorsal and ventral epidermis, and a single tail tip neuron. The connectome reveals that the PNS input arises from scattered groups of these epidermal neurons, 54 in total, and has three main centers of integration in the CNS: in the anterior brain vesicle (which additionally receives input from photoreceptors of the ocellus), the motor ganglion (which contains five pairs of MN), and the tail, all of which in turn are themselves interconnected through important functional relay neurons. Some neurons have long collaterals that form autapses. Our study reveals interconnections with other sensory systems, and the exact inputs to the motor system required to regulate contractions in the tail that underlie larval swimming, or to the CNS to regulate substrate preference prior to the induction of larval settlement and metamorphosis.

Identifying and monitoring neurons that undergo metamorphosis-regulated cell death (metamorphoptosis) by a neuron-specific caspase sensor (Casor) in Drosophila melanogaster.

Activation of caspases is an essential step toward initiating apoptotic cell death. During metamorphosis of Drosophila melanogaster, many larval neurons are programmed for elimination to establish an adult central nervous system (CNS) as well as peripheral nervous system (PNS). However, their neuronal functions have remained mostly unknown due to the lack of proper tools to identify them. To obtain detailed information about the neurochemical phenotypes of the doomed larval neurons and their timing of death, we generated a new GFP-based caspase sensor (Casor) that is designed to change its subcellular position from the cell membrane to the nucleus following proteolytic cleavage by active caspases. Ectopic expression of Casor in vCrz and bursicon, two different peptidergic neuronal groups that had been well-characterized for their metamorphic programmed cell death, showed clear nuclear translocation of Casor in a caspase-dependent manner before their death. We found similar events in some cholinergic neurons from both CNS and PNS. Moreover, Casor also reported significant caspase activities in the ventral and dorsal common excitatory larval motoneurons shortly after puparium formation. These motoneurons were previously unknown for their apoptotic fate. Unlike the events seen in the neurons, expression of Casor in non-neuronal cell types, such as glial cells and S2 cells, resulted in the formation of cytoplasmic aggregates, preventing its use as a caspase sensor in these cell types. Nonetheless, our results support Casor as a valuable molecular tool not only for identifying novel groups of neurons that become caspase-active during metamorphosis but also for monitoring developmental timing and cytological changes within the dying neurons.

TNFa/TNFR2 signaling is required for glial ensheathment at the dorsal root entry zone.

Somatosensory information from the periphery is routed to the spinal cord through centrally-projecting sensory axons that cross into the central nervous system (CNS) via the dorsal root entry zone (DREZ). The glial cells that ensheath these axons ensure rapid propagation of this information. Despite the importance of this glial-axon arrangement, how this afferent nerve is assembled during development is unknown. Using in vivo, time-lapse imaging we show that as centrally-projecting pioneer axons from dorsal root ganglia (DRG) enter the spinal cord, they initiate expression of the cytokine TNFalpha. This induction coincides with ensheathment of these axons by associated glia via a TNF receptor 2 (TNFR2)-mediated process. This work identifies a signaling cascade that mediates peripheral glial-axon interactions and it functions to ensure that DRG afferent projections are ensheathed after pioneer axons complete their navigation, which promotes efficient somatosensory neural function.

Tridimensional Visualization and Analysis of Early Human Development.

Generating a precise cellular and molecular cartography of the human embryo is essential to our understanding of the mechanisms of organogenesis in normal and pathological conditions. Here, we have combined whole-mount immunostaining, 3DISCO clearing, and light-sheet imaging to start building a 3D cellular map of the human development during the first trimester of gestation. We provide high-resolution 3D images of the developing peripheral nervous, muscular, vascular, cardiopulmonary, and urogenital systems. We found that the adult-like pattern of skin innervation is established before the end of the first trimester, showing important intra- and inter-individual variations in nerve branches. We also present evidence for a differential vascularization of the male and female genital tracts concomitant with sex determination. This work paves the way for a cellular and molecular reference atlas of human cells, which will be of paramount importance to understanding human development in health and disease. PAPERCLIP.