Transection and Crush Models of Nerve Injury to Measure Repair and Remyelination in Peripheral Nerve.

Injury to the peripheral nervous system begins a well-characterized process within both neurons and Schwann cells to allow axonal regrowth, remyelination, and functional repair. Models of peripheral nerve injury have been widely used to study the behavior of Schwann cells, neurons, and other cell types such as macrophages as the events of Wallerian degeneration and regeneration take place. The most commonly used approaches in rodent models to model nerve injury in human patients are sciatic nerve transection and nerve crush, and both have well established time courses of demyelination, immune cell influx, axonal regrowth, and remyelination. We describe the techniques of sciatic nerve surgery for transection and crush injury, together with methods for the analysis of events within peripheral nerve repair in these two models

Expression patterns of Slit and Robo family members in adult mouse spinal cord and peripheral nervous system.

The secreted glycoproteins, Slit1-3, are classic axon guidance molecules that act as repulsive cues through their well characterised receptors Robo1-2 to allow precise axon pathfinding and neuronal migration. The expression patterns of Slit1-3 and Robo1-2 have been most characterized in the rodent developing nervous system and the adult brain, but little is known about their expression patterns in the adult rodent peripheral nervous system. Here, we report a detailed expression analysis of Slit1-3 and Robo1-2 in the adult mouse sciatic nerve as well as their expression in the nerve cell bodies within the ventral spinal cord (motor neurons) and dorsal root ganglion (sensory neurons). Our results show that, in the adult mouse peripheral nervous system, Slit1-3 and Robo1-2 are expressed in the cell bodies and axons of both motor and sensory neurons. While Slit1 and Robo2 are only expressed in peripheral axons and their cell bodies, Slit2, Slit3 and Robo1 are also expressed in satellite cells of the dorsal root ganglion, Schwann cells and fibroblasts of peripheral nerves. In addition to these expression patterns, we also demonstrate the expression of Robo1 in blood vessels of the peripheral nerves. Our work gives important new data on the expression patterns of Slit and Robo family members within the peripheral nervous system that may relate both to nerve homeostasis and the reaction of the peripheral nerves to injury

Transforming growth factor beta (TGF beta) mediates schwann cell death in vitro and in vivo: Examination of c-jun activation, interactions with survival signals, and the relationship of TGF beta-mediated death to schwann cell differentiation

In some situations, cell death in the nervous system is controlled by an interplay between survival factors and negative survival signals that actively induce apoptosis. The present work indicates that the survival of Schwann cells is regulated by such a dual mechanism involving the negative survival signal transforming growth factor beta (TGF beta), a family of growth factors that is present in the Schwann cells themselves. We analyze the interactions between this putative autocrine death signal and previously defined paracrine and autocrine survival signals and show that expression of a dominant negative c-Jun inhibits TGF beta -induced apoptosis. This and other findings pinpoint activation of c-Jun as a key downstream event in TGF beta -induced Schwann cell death. The ability of TGF beta to kill Schwann cells, like normal Schwann cell death in vivo, is under a strong developmental regulation, and we show that the decreasing ability of TGF beta to kill older cells is attributable to a decreasing ability of TGF beta to phosphorylate c-Jun in more differentiated cells

Loss of SOX10 function contributes to the phenotype of human Merlin-null schwannoma cells.

Loss of the Merlin tumour suppressor causes abnormal de-differentiation and proliferation of Schwann cells and formation of schwannoma tumours in patients with neurofibromatosis type 2. Within the mature peripheral nerve the normal development, differentiation and maintenance of myelinating and non-myelinating Schwann cells is regulated by a network of transcription factors that include SOX10, OCT6 (now known as POU3F1), NFATC4 and KROX20 (also known as Egr2). We have examined for the first time how their regulation of Schwann cell development is disrupted in primary human schwannoma cells. We find that induction of both KROX20 and OCT6 is impaired, whereas enforced expression of KROX20 drives both myelin gene expression and cell cycle arrest in Merlin-null cells. Importantly, we show that human schwannoma cells have reduced expression of SOX10 protein and messenger RNA. Analysis of mouse SOX10-null Schwann cells shows they display many of the characteristics of human schwannoma cells, including increased expression of platelet derived growth factor receptor beta (PDGFRB) messenger RNA and protein, enhanced proliferation, increased focal adhesions and schwannoma-like morphology. Correspondingly, reintroduction of SOX10 into human Merlin-null cells restores the ability of these cells to induce KROX20 and myelin protein zero (MPZ), localizes NFATC4 to the nucleus, reduces cell proliferation and suppresses PDGFRB expression. Thus, we propose that loss of the SOX10 protein, which is vital for normal Schwann cell development, is also key to the pathology of Merlin-null schwannoma tumours

Single Cell Transcriptome Data Analysis Defines the Heterogeneity of Peripheral Nerve Cells in Homeostasis and Regeneration

The advances in single-cell RNA sequencing technologies and the development of bioinformatics pipelines enable us to more accurately define the heterogeneity of cell types in a selected tissue. In this report, we re-analyzed recently published single-cell RNA sequencing data sets and provide a rationale to redefine the heterogeneity of cells in both intact and injured mouse peripheral nerves. Our analysis showed that, in both intact and injured peripheral nerves, cells could be functionally classified into four categories: Schwann cells, nerve fibroblasts, immune cells, and cells associated with blood vessels. Nerve fibroblasts could be sub-clustered into epineurial, perineurial, and endoneurial fibroblasts. Identified immune cell clusters include macrophages, mast cells, natural killer cells, T and B lymphocytes as well as an unreported cluster of neutrophils. Cells associated with blood vessels include endothelial cells, vascular smooth muscle cells, and pericytes. We show that endothelial cells in the intact mouse sciatic nerve have three sub-types: epineurial, endoneurial, and lymphatic endothelial cells. Analysis of cell type-specific gene changes revealed that Schwann cells and endoneurial fibroblasts are the two most important cell types promoting peripheral nerve regeneration. Analysis of communication between these cells identified potential signals for early blood vessel regeneration, neutrophil recruitment of macrophages, and macrophages activating Schwann cells. Through this analysis, we also report appropriate marker genes for future single cell transcriptome data analysis to identify cell types in intact and injured peripheral nerves. The findings from our analysis could facilitate a better understanding of cell biology of peripheral nerves in homeostasis, regeneration, and disease.</jats:p

Inhibition of YAP/TAZ-driven TEAD activity prevents growth of NF2-null schwannoma and meningioma.

Schwannoma tumours typically arise on the 8th cranial nerve and are mostly caused by loss of the tumour suppressor Merlin (NF2). There are no approved chemotherapies for these tumours and the surgical removal of the tumour carries a high risk of damage to the 8th or other close cranial nerve tissue. New treatments for schwannoma and other NF2-null tumours such as meningioma are urgently required. Using a combination of human primary tumour cells and mouse models of schwannoma, we have examined the role of the Hippo signalling pathway in driving tumour cell growth. Using both genetic ablation of the Hippo effectors YAP and TAZ as well as novel TEAD palmitoylation inhibitors, we show that Hippo signalling may be successfully targeted in vitro and in vivo to both block and, remarkably, regress schwannoma tumour growth. In particular, successful use of TEAD palmitoylation inhibitors in a pre-clinical mouse model of schwannoma points to their potential future clinical use. We also identify the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) as a Hippo signalling target, driven by the TAZ protein in human and mouse NF2-null schwannoma cells, as well as in NF2-null meningioma cells, and examine the potential future role of this new target in halting schwannoma and meningioma tumour growth

A systematic review of biomarkers for disease progression in Parkinson's disease

Peer reviewedPublisher PD

Annotation of two large contiguous regions from the Haemonchus contortus genome using RNA-seq and comparative analysis with Caenorhabditis elegans

The genomes of numerous parasitic nematodes are currently being sequenced, but their complexity and size, together with high levels of intra-specific sequence variation and a lack of reference genomes, makes their assembly and annotation a challenging task. Haemonchus contortus is an economically significant parasite of livestock that is widely used for basic research as well as for vaccine development and drug discovery. It is one of many medically and economically important parasites within the strongylid nematode group. This group of parasites has the closest phylogenetic relationship with the model organism Caenorhabditis elegans, making comparative analysis a potentially powerful tool for genome annotation and functional studies. To investigate this hypothesis, we sequenced two contiguous fragments from the H. contortus genome and undertook detailed annotation and comparative analysis with C. elegans. The adult H. contortus transcriptome was sequenced using an Illumina platform and RNA-seq was used to annotate a 409 kb overlapping BAC tiling path relating to the X chromosome and a 181 kb BAC insert relating to chromosome I. In total, 40 genes and 12 putative transposable elements were identified. 97.5% of the annotated genes had detectable homologues in C. elegans of which 60% had putative orthologues, significantly higher than previous analyses based on EST analysis. Gene density appears to be less in H. contortus than in C. elegans, with annotated H. contortus genes being an average of two-to-three times larger than their putative C. elegans orthologues due to a greater intron number and size. Synteny appears high but gene order is generally poorly conserved, although areas of conserved microsynteny are apparent. C. elegans operons appear to be partially conserved in H. contortus. Our findings suggest that a combination of RNA-seq and comparative analysis with C. elegans is a powerful approach for the annotation and analysis of strongylid nematode genomes