Deciphering the dynamic characteristics of non-neuronal cells in dorsal root ganglion of rat at different developmental stage based on single cell transcriptome data / 生物工程学报

Dorsal root ganglia (DRG) is an essential part of the peripheral nervous system and the hub of the peripheral sensory afferent. The dynamic changes of neuronal cells and their gene expression during the development of dorsal root ganglion have been studied through single-cell RNAseq analysis, while the dynamic changes of non-neuronal cells have not been systematically studied. Using single cell RNA sequencing technology, we conducted a research on the non-neuronal cells in the dorsal root ganglia of rats at different developmental stage. In this study, primary cell suspension was obtained from using the dorsal root ganglions (DRGs, L4-L5) of ten 7-day-old rats and three 3-month-old rats. The 10×Genomics platform was used for single cell dissociation and RNA sequencing. Twenty cell subsets were acquired through cluster dimension reduction analysis, and the marker genes of different types of cells in DRG were identified according to previous researches about DRG single cell transcriptome sequencing. In order to find out the non-neuronal cell subsets with significant differences at different development stage, the cells were classified into different cell types according to markers collected from previous researches. We performed pseudotime analysis of 4 types Schwann cells. It was found that subtype Ⅱ Schwann cells emerged firstly, and then were subtype Ⅲ Schwann cells and subtype Ⅳ Schwann cells, while subtype Ⅰ Schwann cells existed during the whole development procedure. Pseudotime analysis indicated the essential genes influencing cell fate of different subtypes of Schwann cell in DRG, such as Ntrk2 and Pmp2, which affected cell fate of Schwann cells during the development period. GO analysis of differential expressed genes showed that the up-regulated genes, such as Cst3 and Spp1, were closely related to biological process of tissue homeostasis and multi-multicellular organism process. The down regulated key genes, such as Col3a1 and Col4a1, had close relationship with the progress of extracellular structure organization and negative regulation of cell adhesion. This suggested that the expression of genes enhancing cell homestasis increased, while the expression of related genes regulating ECM-receptor interaction pathway decreased during the development. The discovery provided valuable information and brand-new perspectives for the study on the physical and developmental mechanism of Schwann cell as well as the non-neuronal cell changes in DRG at different developmental stage. The differential gene expression results provided crucial references for the mechanism of somatosensory maturation during development.

Septin 7: Actin cross-organization is required for axonal association of Schwann cells

Myelin sheaths present two distinct domains: compacted myelin spirals and flanking non-compacted cytoplasmic channels, where lipid and protein segregation is established by unknown mechanisms. Septins, a conserved family of membrane and cytoskeletal interacting GTPases, form intracellular diffusion barriers during cell division and neurite extension and are expressed in myelinating cells. Septins, particularly septin 7 (Sept7), the central constituent of septin polymers, are associated with the cytoplasmic channels of myelinating cells. Here we show that Schwann cells deprived of Sept7 fail to wrap around axons from dorsal root ganglion neurons and exhibit disorganization of the actin cytoskeleton. Likewise, Sept7 distribution is dependent on microfilament but not microtubule organization.

Slow axoplasmic transport under scrutiny

The origin of axoplasmic proteins is central for the biology of axons. For over fifty years axons have been considered unable to synthesize proteins and that cell bodies supply them with proteins by a slow transport mechanism. To allow for prolonged transport times, proteins were assumed to be stable, i.e., not degraded in axons. These are now textbook notions that configure the slow transport model (STM). The aim of this article is to cast doubts on the validity of STM, as a step toward gaining more understanding about the supply of axoplasmic proteins. First, the stability of axonal proteins claimed by STM has been disproved by experimental evidence. Moreover, the evidence for protein synthesis in axons indicates that the repertoire is extensive and the amount sizeable, which disproves the notion that axons are unable to synthesize proteins and that cell bodies supply most axonal proteins. In turn, axoplasmic protein synthesis gives rise to the metabolic model (MM). We point out a few inconsistencies in STM that MM redresses. Although both models address the supply of proteins to axons, so far they have had no crosstalk. Since proteins underlie every conceivable cellular function, it is necessary to re-evaluate in-depth the origin of axonal proteins. We hope this will shape a novel understanding of the biology of axons, with impact on development and maintenance of axons, nerve repair, axonopathies and plasticity, to mention a few fields.

Schwann cell expression of an oligodendrocyte-like remyelinating pattern after ethidium bromide injection in the rat spinal cord / Expressão pelas células de Schwann de um padrão de remielinização semelhante ao oligodendroglial após injeção de brometo de etídio na medula espinhal de ratos

Schwann cells are recognized by their capacity of producing single internodes of myelin around axons of the peripheral nervous system. In the ethidium bromide (EB) model of primary demyelination in the brainstem, it is observed the entry of Schwann cells into the central nervous system in order to contribute to the myelin repair performed by the oligodendrocytes that survived to the EB gliotoxic action, being able to even remyelinate more than one axon at the same time, in a pattern of repair similar to the oligodendroglial one. The present study was developed in the spinal cord to observe if Schwann cells maintained this competence of attending simultaneously different internodes. It was noted that, on the contrary of the brainstem, Schwann cells were the most important myelinogenic cells in the demyelinated site and, although rare, also presented the capacity of producing more than one internode of myelin in distinct axons.

As células de Schwann são reconhecidas por sua capacidade de produzir internodos de mielina únicos ao redor de axônios do sistema nervoso periférico. No modelo de desmielinização primária do brometo de etídio (BE) no tronco encefálico, tem sido observada a entrada destas células no sistema nervoso central. Isso pode contribuir para o reparo mielínico desempenhado pelos oligodendrócitos que sobreviveram à ação glitóxica do BE, chegando a remielinizar mais de um axônio ao mesmo tempo, em um padrão de reparo semelhante ao oligodendroglial. O presente estudo foi realizado na medula espinhal para observar se as células de Schwann mantinham esta competência de atender simultaneamente diferentes internodos. Foi observado que, ao contrário do tronco encefálico, as células de Schwann foram as células mielinogênicas mais importantes no sítio de desmielinização induzida pelo BE e, embora raro, também apresentaram a capacidade de produzir mais de um internodo de mielina em axônios distintos.

Morphological and functional characterizations of Schwann cells stimulated with Mycobacterium leprae

Nerve damage, a characteristic of leprosy, is the cause of patient deformities and a consequence of Schwann cells (SC) infection by Mycobacterium leprae. Although function/dysfunction of SC in human diseases like leprosy is difficult to study, many in vitro models, including SC lines derived from rat and/or human Schwannomas, have been employed. ST88-14 is one of the cell lineages used by many researchers as a model for M. leprae/SC interaction. However, it is necessary to establish the values and limitations of the generated data on the effects of M. leprae in these SC. After evaluating the cell line phenotype in the present study, it is close to non-myelinating SC, making this lineage an ideal model for M. leprae/SC interaction. It was also observed that both M. leprae and PGL-1, a mycobacterial cell-wall component, induced low levels of apoptosis in ST88-14 by a mechanism independent of Bcl-2 family members.

Terminal schwann cell distribution at the neuromuscular junction of the dystrophin-deficient MDX mice

The murine model of muscular dystrophy, the mdx mice, is widely used to study the pathogenesis of muscular dystrophies. These mice suffer an X-linked dystrophin deficiency and present cycles of muscle fiber degeneration-regeneration beginning at 21 days of age. At the present, we studied neuromuscular junction organization in the sternomastoid muscle of mdx mice, focusing on the distribution of terminal Schwann cells during early development and adults. Seven and 14 days after birth (n=200 endplates for each age), before the onset of muscle degeneration-regeneration, fluorescence confocal microscopy showed that there were no detectable differences in the pattern of Schwann cell distribution in the mdx compared to controls of the same age. Schwann cells had a diffuse pattern of distribution, covering the plaques of acetylcholine receptors. In adult mdx muscles, terminal Schwann cell processes filled the center of acetylcholine receptors islands, similar to nerve terminal distribution, at the majority of the junctions (n=200; 100%). Conversely, all of the adult control junctions (n=200) showed continuous processes of Schwann cells covering the continuous branches of acetylcholine receptors. These observations indicate that remodeling of the three components of the neuromuscular junction occurs only after the onset of the cycles of muscle fiber degeneration-regeneration, in the mdx mice.

Mycobacterium leprae interactions with the host cell: recent advances.

The significance of Hansen disease, or leprosy, is underscored by fact that detection of this disease has remained stable over the past 10 yr, even though disease prevalence is reduced. Due to the long incubation time of the organism, health experts predict that leprosy will be with us for decades to come. Despite the fact that Mycobacterium leprae, the causative agent of leprosy, cannot be cultured in the laboratory, researchers are using innovative and imaginative techniques to discern the interactions of M. leprae with host cells both in vitro and in vivo to identify the host and bacterial factors integral to establishment of disease. The studies described in this review present a new and evolving picture of the many interactions between M. leprae and the host. Specific attention will be given to interactions of M. leprae bacilli with host Schwann cells, macrophages, dendritic cells and endothelial cells. The findings described also have implications for the prevention and treatment of leprosy.

Delayed Schwann cell and oligodendrocyte remyelination after ethidium bromide injection in the brainstem of Wistar rats submitted to streptozotocin diabetogenic treatment

Schwann cell disturbance followed by segmental demyelination in the peripheral nervous system occurs in diabetic patients. Since Schwann cell and oligodendrocyte remyelination in the central nervous system is a well-known event in the ethidium bromide (EB) demyelinating model, the aim of this investigation was to determine the behavior of both cell types after local EB injection into the brainstem of streptozotocin diabetic rats. Adult male Wistar rats received a single intravenous injection of streptozotocin (50 mg/kg) and were submitted 10 days later to a single injection of 10 æL 0.1 percent (w/v) EB or 0.9 percent saline solution into the cisterna pontis. Ten microliters of 0.1 percent EB was also injected into non-diabetic rats. The animals were anesthetized and perfused through the heart 7 to 31 days after EB or saline injection and brainstem sections were collected and processed for light and transmission electron microscopy. The final balance of myelin repair in diabetic and non-diabetic rats at 31 days was compared using a semi-quantitative method. Diabetic rats presented delayed macrophage activity and lesser remyelination compared to non-diabetic rats. Although oligodendrocytes were the major remyelinating cells in the brainstem, Schwann cells invaded EB-induced lesions, first appearing at 11 days in non-diabetic rats and by 15 days in diabetic rats. Results indicate that short-term streptozotocin-induced diabetes hindered both oligodendrocyte and Schwann cell remyelination (mean remyelination scores of 2.57 ± 0.77 for oligodendrocytes and 0.67 ± 0.5 for Schwann cells) compared to non-diabetic rats (3.27 ± 0.85 and 1.38 ± 0.81, respectively).

Schwann cells for spinal cord repair

The complex nature of spinal cord injury appears to demand a multifactorial repair strategy. One of the components that will likely be included is an implant that will fill the area of lost nervous tissue and provide a growth substrate for injured axons. Here we will discuss the role of Schwann cells (SCs) in cell-based, surgical repair strategies of the injured adult spinal cord. We will review key studies that showed that intraspinal SC grafts limit injury-induced tissue loss and promote axonal regeneration and myelination, and that this response can be improved by adding neurotrophic factors or anti-inflammatory agents. These results will be compared with several other approaches to the repair of the spinal cord. A general concern with repair strategies is the limited functional recovery, which is in large part due to the failure of axons to grow across the scar tissue at the distal graft-spinal cord interface. Consequently, new synaptic connections with spinal neurons involved in motor function are not formed. We will highlight repair approaches that did result in growth across the scar and discuss the necessity for more studies involving larger, clinically relevant types of injuries, addressing this specific issue. Finally, this review will reflect on the prospect of SCs for repair strategies in the clinic.

Behaviour of oligodendrocytes and Schwann cells in an experimental model of toxic demyelination of the central nervous system

Oligodendrocytes and Schwann cells are engaged in myelin production, maintenance and repairing respectively in the central nervous system (CNS) and the peripheral nervous system (PNS). Whereas oligodendrocytes act only within the CNS, Schwann cells are able to invade the CNS in order to make new myelin sheaths around demyelinated axons. Both cells have some limitations in their activities, i.e. oligodendrocytes are post-mitotic cells and Schwann cells only get into the CNS in the absence of astrocytes. Ethidium bromide (EB) is a gliotoxic chemical that when injected locally within the CNS, induce demyelination. In the EB model of demyelination, glial cells are destroyed early after intoxication and Schwann cells are free to approach the naked central axons. In normal Wistar rats, regeneration of lost myelin sheaths can be achieved as early as thirteen days after intoxication; in Wistar rats immunosuppressed with cyclophosphamide the process is delayed and in rats administered cyclosporine it may be accelerated. Aiming the enlightening of those complex processes, all events concerning the myelinating cells in an experimental model are herein presented and discussed

Cross-talk between neurons and glia: highlights on soluble factors

The development of the nervous system is guided by a balanced action between intrinsic factors represented by the genetic program and epigenetic factors characterized by cell-cell interactions which neural cells might perform throughout nervous system morphogenesis. Highly relevant among them are neuron-glia interactions. Several soluble factors secreted by either glial or neuronal cells have been implicated in the mutual influence these cells exert on each other. In this review, we will focus our attention on recent advances in the understanding of the role of glial and neuronal trophic factors in nervous system development. We will argue that the functional architecture of the brain depends on an intimate neuron-glia partnership

Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp) and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin

Non-neuronal cells are not the limiting factor for the low axonal regeneration in C57BL/6J mice

Peripheral axonal regeneration was investigated in adult male mice of the C57BL/6J (C), BALB/cJ (B) and A/J (A) strains and in their F1 descendants using a predegenerated nerve transplantation model. Four types of transplants were performed: 1) isotransplants between animals of the C, B and A strains; 2) donors of the C strain and recipients of the C x B and C x A breeding; 3) donors of the B strain and recipients of the C x B breeding, and 4) donors of the A strain and recipients of the C x A breeding. Donors had the left sciatic nerve transected and two weeks later a segment of the distal stump was transplanted into the recipient. Four weeks after transplantation the regenerated nerves were used to determine the total number of regenerated myelinated fibers (TMF), diameter of myelinated fibers (FD) and myelin thickness (MT). The highest TMF values were obtained in the groups where C57BL/6J mice were the donors (C to F1 (C x B) = 4658 + OR - 304; C to F1 (C x A) = 3899 + OR - 198). Also, A/J grafts led to a significantly higher TMF (A to F1 (C x A) = 3933 + OR - 565). Additionally, isotransplant experiments showed that when the nerve is previously degenerated, C57BL/6J mice display the largest number of myelinated fibers (C to C = 3136 + OR - 287; B to B = 2759 + OR - 170, and A to A = 2835 + OR - 239). We also observed that when C57BL/6J was the graft donor, FD was the highest and MT did not differ significantly when compared with the other groups. These morphometric results reinforce the idea that Schwann cells and the nerve environment of C57BL/6J provide enough support to the regenerative process. In this respect, the present results support the hypothesis that the non-neuronal cells, mainly Schwann cells, present in the sciatic nerve of C57BL/6J mice are not the main limiting factor responsible for low axonal regeneration

Biology of the repair of central nervous system demyelinated lesions

The integrity of myelin sheaths is maintained by oligodendrocytes and Schwann cells respectively in the central nervous system (CNS) and in the peripheral nervous system. The process of demyelination consistin of the withdrawal of myelin sheaths from their axons is a characteristic feature of multiple sclerosis, the most common human demyelinating disease. Many experimental models have been designed to study the biology of demyelination and remyelination (repair of the lost myelin) in the CNS, due to the difficulties in studying human material. In the ethidium bromide (an intercalating gliotoxic drug) model of demyelination, CNS remyelination may be carried out by surviving oligodendrocytes and/or by cells differentiated from the primitive cell lines or either by Schwann cells that invade the CNS. However, some factor such as the age of the experimental anmnals, intensity and time of exposure to the intercalating clinical and the topography of the lesions have marked influente on the repair of the tissue.

Utilización de cultivos de células de Schwann para el estudio de la infección in vitro con Mycobacterium leprae / Use of Schwann cell cultures in the study of Mycobacterium leprae in vitro infection

Mycobacterium leprae es un patógeno intracelular con marcada afinidad por células de Schwann (CS) y macrófagos del sistema reticuloendotelial, que causa una neuropatía periférica en el hombre. Es un microorganismo no cultivable en medios convencionales y numerosos intentos por cultivarlo han sido infructuosos. Los modelos animales utilizados, han sido los de elección, entre estos el del cojinete plantar del ratón que es la prueba oro utilizada en el diagnóstico y el estudio de la patogenesis de la enfermedad, la viabilidad y la resistencia a antibióticos por parte del M. leprae. Conociendo que todos los tipos de lepra (LL,LT, etc.) cursan con una lepra de tipo neural, consideramos que un modelo in vitro de CS de ratón permitiría una gran aproximación al estudio de la interacción del M. leprae y su célula blanco específica en la infección, la cual se puede obtener en cultivo, conservando propiedades morfológicas y funcionales tales como es su capacidad de asociación con neuritas. Aunque las CS comprenden el 90 por ciento de la población inicial en cultivos de ganglios de raíz dorsal (DRG), su número declina debido a la rápida rata mitótica de los fibroblastos en presencia de suero fetal bovino. Sin embargo el tratamiento de los cultivos con agentes activadores del AMP cíclico inhiben el crecimiento de fibroblastos sin causar toxicidad a la CS, permitiendo su proliferación y enriquecimiento. Utilizando un tratamiento con agentes activadores del AMPc se obtuvieron cultivos enriquecidos en células de Schwann, lo cual fue corroborado por recuentos basados en sus características morfológicas y su inmunoreactividad al anticuerpo dirigido contra la proteína S-100, una proteína reguladora de la concentración de calcio intracelular, asociada a filamentos intermedios, presente en la célula de Schwann y ausente en los fibroblastos. Obtuvimos cultivos con un 88 por ciento de células de Schwann los cuales han sido usados en los ensayos de infección. Para la infección de células de Schwann por el Mycobacterium leprae, se utilizó la técnica recomendada por la O.M.S. de obtención y cuantificación de los bacilos a partir de lepromas obtenidos de pacientes con lepra lepromatosa no tratados. Cultivos enriquecidos en células de Schwann se incubaron con este M. leprae y se realizaron estudios morfológicos por microscopía óptica, usando la coloración de Zielh Neelsen caliente cuantificando el número de bacilos por célula y el porcentaje de células infectadas, estudios de...

Desmielinizaçäo tóxica do sistema nervoso central: II. Aspectos biológicos das células de Schwann observados durante o processo de reparaçäo do tecido / Toxic demyelination of the central nervous system: II. Biological aspects of Schwann cells observed during the process of tissue repair

A droga gliotóxica brometo de etídio, quando injetada localmente na medula espinhal do rato, induziu áreas de desmielinizaçäo que se desenvolveram em tempos diferentes de acordo com a dose empregada. Doses altas induziram lesöes de desenvolvimento rápido (tipo I) e intermediárias (tipo III), enquanto doses baixas induziram lesöes lentas (tipo II). Após o processo desmielinizante, os azônios desprovidos de suas bainhas de mielina foram remielinizados por oligodendrócitos ou células de Schwann (CS) dependendo de sua localizaçäo em áreas contendo ou näo astrócitos, respectivamente. Na maioria das lesöes, a área remielinizada pelas CS era proeminente. Nas lesöes que repararam mais lentamente foi possível observar os fatores que influenciaram o comportamento dessas células dentro dos limites do sistema nervoso central. Após a invasäo inicial a partir da superfície pial e dos espaços preivasculares, a expansäo das CS dependeu da presenca de matriz extracelular estável. nas lesöes de tipo I, esta matriz estava presente devido a natureza inflamatória do processo. Nas lesöes de tipo II a matriz näo ocorreu e as CS só podiam migrar entre os axônios desmielizados usando-os tal como uma passadeira. Entre as células contíguas podiam ser observadas fibras colágenas de diâmetro pequeno. Näo foi evidenciada migraçäo das CS ao longo dos axônios

Relaciones funcionales neurona-neuroglia en la fibra nerviosa gigante del calamar / Neuron-neuroglia functional relation in the squid giant nerve fiber

El presente trabajo llevados a cabo en la fibra nerviosa gigante del calamar tropical Sepioteuthis sepioidea, oritentado a explorar la posible participación de distintos segundos mensajeros y sistemas receptores en la potencial de membrana de la célula de Schwann a la excitación del potencial de membrana de la célula de Schwann a la excitación axonal. Estos estudios proveen evidencia experimental sobre la existencia de receptores diferentes para ACh, octopamina y un péptido endógeno similar a VIP en la membrana plasmática de la célula de Schwann. Ellos indican también que el nucleótido cíclico de adenosina 3', t' monofosfato puede estar desempeñando un papel como segundo mensajero para las acciones de los receptores colinérgicos nicotínicos y los octopaminérgicos de la célula de Schwann, en tanto que el receptor de VIP puede estar actuando a través de la hidrólisis de polifosfatidilinositoles. Estos hallazgos experimentales son discutidos como evidencia para la existencia de una compleja interacción entre el axón gigante y su célula satélite neuroglial, la célula de Schwann