Laser microdissection-based expression analysis of key genes involved in muscle regeneration in mdx mice

We have used the mdx mice strain (C57BL/10ScSn-mdx) as an experimental subject for the study of reiterative skeletal muscle necrosis-regeneration with basement membrane preservation. In young mdx muscle, by means of Hematoxylin-Eosin staining, different types of degenerative-regenerative groups (DRG) can be recognized and assigned to a defined muscle regeneration phase. To evaluate the expression of known key-regulatory genes in muscle regeneration, we have applied Laser Capture Microdissection technique to obtain tissue from different DRGs encompassing the complete skeletal muscle regenerative process. The expression of MyoD, Myf-5 and Myogenin showed a rapid increase in the first two days post-necrosis, which were followed by MRF4 expression, when newly regenerating fibers started to appear (3-5days post-necrosis). MHCd mRNA levels, undetectable in mature non-injured fibers, increased progressively from the first day post-necrosis and reached its maximum level of expression in DRGs showing basophilic regenerating fibers. TGFbeta-1 mRNA expression showed a prompt and strong increase following fiber necrosis that persisted during the inflammatory phase, and progressively decreased when new regenerating fibers began to appear. In contrast, IGF-2 mRNA expression decreased during the first days post-necrosis but was followed by a progressive rise in its expression coinciding with the appearance of the newly formed myofibers, reaching the maximum expression levels in DRGs composed of medium caliber basophilic regenerating myofibers (5-7 days post-necrosis). mdx degenerative-regenerative group typing, in conjunction with laser microdissection-based gene expression analysis, opens up a new approach to the molecular study of skeletal muscle regeneration.     

Wallerian degeneration of the inferior cerebellar peduncle depicted by diffusion weighted imaging

Wallerian degeneration of the inferior cerebellar peduncle has never been demonstrated on imaging studies. We describe a case in which it was depicted by thin slice diffusion weighted imaging. Location to the inferior cerebellar peduncle was confirmed by a fibre tracking method.     

Evidence that Wallerian degeneration and localized axon degeneration induced by local neurotrophin deprivation do not involve caspases.

The selective degeneration of an axon, without the death of the parent neuron, can occur in response to injury, in a variety of metabolic, toxic, and inflammatory disorders, and during normal development. Recent evidence suggests that some forms of axon degeneration involve an active and regulated program of self-destruction rather than a passive "wasting away" and in this respect and others resemble apoptosis. Here we investigate whether selective axon degeneration depends on some of the molecular machinery that mediates apoptosis, namely, the caspase family of cysteine proteases. We focus on two models of selective axon degeneration: Wallerian degeneration of transected axons and localized axon degeneration induced by local deprivation of neurotrophin. We show that caspase-3 is not activated in the axon during either form of degeneration, although it is activated in the dying cell body of the same neurons. Moreover, caspase inhibitors do not inhibit or retard either form of axon degeneration, although they inhibit apoptosis of the same neurons. Finally, we cannot detect cleaved substrates of caspase-3 and its close relatives immunocytochemically or caspase activity biochemically in axons undergoing Wallerian degeneration. Our results suggest that a neuron contains at least two molecularly distinct self-destruction programs, one for caspase-dependent apoptosis and another for selective axon degeneration.     

Identification of key genes involved in post-traumatic stress disorder: Evidence from bioinformatics analysis

BACKGROUNDPost-traumatic stress disorder (PTSD) is a serious stress-related disorder.AIMTo identify the key genes and pathways to uncover the potential mechanisms of PTSD using bioinformatics methods.METHODSGene expression profiles were obtained from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified by using GEO2R. Gene functional annotation and pathway enrichment were then conducted. The gene-pathway network was constructed with Cytoscape software. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was applied for validation, and text mining by Coremine Medical was used to confirm the connections among genes and pathways.RESULTSWe identified 973 DEGs including 358 upregulated genes and 615 downregulated genes in PTSD. A group of centrality hub genes and significantly enriched pathways (MAPK, Ras, and ErbB signaling pathways) were identified by using gene functional assignment and enrichment analyses. Six genes (KRAS, EGFR, NFKB1, FGF12, PRKCA, and RAF1) were selected to validate using qRT-PCR. The results of text mining further confirmed the correlation among hub genes and the enriched pathways. It indicated that these altered genes displayed functional roles in PTSD via these pathways, which might serve as key signatures in the pathogenesis of PTSD.CONCLUSIONThe current study identified a panel of candidate genes and important pathways, which might help us deepen our understanding of the underlying mechanism of PTSD at the molecular level. However, further studies are warranted to discover the critical regulatory mechanism of these genes via relevant pathways in PTSD.     

Roles of palmitoylation in axon growth,degeneration and regeneration

The protein‐lipid modification palmitoylation plays important roles in neurons, but most attention has focused on roles of this modification in the regulation of mature pre‐ and post‐synapses. However, exciting recent findings suggest that palmitoylation is also critical for both the growth and integrity of neuronal axons and plays previously unappreciated roles in conveying axonal anterograde and retrograde signals. Here we review these emerging roles for palmitoylation in the regulation of axons in health and disease. © 2017 Wiley Periodicals, Inc.     

Weighted gene co-expression network analysis identifies specific modules and hub genes related to subsyndromal symptomatic depression

Abstract

Objectives: The identification of the potential molecule targets for subsyndromal symptomatic depression (SSD) is critical for improving the effective clinical treatment on the mental illness. In the current study, we mined the genome-wide expression profiling and investigated the novel biological pathways associated with SSD.

Methods: Expression of differentially expressed genes (DEGs) were analysed with microarrays of blood tissue cohort of eight SSD patients and eight healthy subjects. The gene co-expression is calculated by WGCNA, an R package software. The function of the genes was annotated by gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.

Results: We identified 11 modules from the 9,427 DEGs. Three co-expression modules (blue, cyan and red) showed striking correlation with the phenotypic trait between SSD and healthy controls. Gene ontology and KEGG pathway analysis demonstrated that the function of these three modules was enriched with the pathway of inflammatory response and type II diabetes mellitus. Finally, three hub genes, NT5DC1, SGSM2 and MYCBP, were identified from the blue module as significant genes.

Conclusions: This first blood gene expression study in SSD observed distinct patterns between cases and controls which may provide novel insight into understanding the molecular mechanisms of SSD.     

Bioinformatic identification of key candidate genes and pathways in axon regeneration after spinal cord injury in zebrafish

Zebrafish and human genomes are highly homologous;however,despite this genomic similarity,adult zebrafish can achieve neuronal proliferation,regeneration and functional restoration within 6–8 weeks after spinal cord injury,whereas humans cannot.To analyze differentially expressed zebrafish genes between axon-regenerated neurons and axon-non-regenerated neurons after spinal cord injury,and to explore the key genes and pathways of axonal regeneration after spinal cord injury,microarray GSE56842 was analyzed using the online tool,GEO2R,in the Gene Expression Omnibus database.Gene ontology and protein-protein interaction networks were used to analyze the identified differentially expressed genes.Finally,we screened for genes and pathways that may play a role in spinal cord injury repair in zebrafish and mammals.A total of 636 differentially expressed genes were obtained,including 255 up-regulated and 381 down-regulated differentially expressed genes in axon-regenerated neurons.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results were also obtained.A protein-protein interaction network contained 480 node genes and 1976 node connections.We also obtained the 10 hub genes with the highest correlation and the two modules with the highest score.The results showed that spectrin may promote axonal regeneration after spinal cord injury in zebrafish.Transforming growth factor beta signaling may inhibit repair after spinal cord injury in zebrafish.Focal adhesion or tight junctions may play an important role in the migration and proliferation of some cells,such as Schwann cells or neural progenitor cells,after spinal cord injury in zebrafish.Bioinformatic analysis identified key candidate genes and pathways in axonal regeneration after spinal cord injury in zebrafish,providing targets for treatment of spinal cord injury in mammals.     

Role of microtubule dynamics in Wallerian degeneration and nerve regeneration after peripheral nerve injury

Wallerian degeneration,the progressive disintegration of distal axons and myelin that occurs after peripheral nerve injury,is essential for creating a permissive microenvironment for nerve regeneration,and involves cytoskeletal reconstruction.However,it is unclear whether microtubule dynamics play a role in this process.To address this,we treated cultured sciatic nerve explants,an in vitro model of Wallerian degeneration,with the microtubule-targeting agents paclitaxel and nocodazole.We found that paclitaxel-induced microtubule stabilization promoted axon and myelin degeneration and Schwann cell dedifferentiation,whereas nocodazole-induced microtubule destabilization inhibited these processes.Evaluation of an in vivo model of peripheral nerve injury showed that treatment with paclitaxel or nocodazole accelerated or attenuated axonal regeneration,as well as functional recovery of nerve conduction and target muscle and motor behavior,respectively.These results suggest that microtubule dynamics participate in peripheral nerve regeneration after injury by affecting Wallerian degeneration.This study was approved by the Animal Care and Use Committee of Southern Medical University,China(approval No.SMUL2015081) on October 15,2015.     

The cytokine network of Wallerian degeneration: IL-10 and GM-CSF

Wallerian degeneration (WD) is the inflammatory response of peripheral nerves to injury. Evidence is provided that granulocyte macrophage colony stimulating factor (GM-CSF) contributes to the initiation and progression of WD by activating macrophages and Schwann, whereas IL-10 down-regulates WD by inhibiting GM-CSF production. A significant role of activated macrophages and Schwann for future regeneration is myelin removal by phagocytosis and degradation. We studied the timing and magnitude of GM-CSF and IL-10 production, macrophage and Schwann activation, and myelin degradation in C57BL/6NHSD and C57BL/6-WLD/OLA/NHSD mice that display normal rapid-WD and abnormal slow-WD, respectively. We observed the following events in rapid-WD. The onset of GM-CSF production is within 5 h after injury. Production is steadily augmented during the first 3 days, but is attenuated thereafter. The onset of production of the macrophage and Schwann activation marker Galectin-3/MAC-2 succeeds that of GM-CSF. Galectin-3/MAC-2 production is up-regulated during the first 6 days, but is down-regulated thereafter. The onset of myelin degradation succeeds that of Galectin-3/MAC-2, and is almost complete within 1 week. IL-10 production displays two phases. An immediate low followed by a high that begins on the fourth day, reaching highest levels on the seventh. The timing and magnitude of GM-CSF production thus enable the rapid activation of macrophages and Schwann that consequently phagocytose and degrade myelin. The timing and magnitude of IL-10 production suggest a role in down-regulating WD after myelin is removed. In contrast, slow-WD nerves produce low inefficient levels of GM-CSF and IL-10 throughout. Therefore, deficient IL-10 levels cannot account for inefficient GM-CSF production, whereas deficient GM-CSF levels may account, in part, for slow-WD.     

Identification of key genes involved in recovery from spinal cord injury in adult zebrafish

Zebrafish are an effective vertebrate model to study the mechanisms underlying recovery after spinal cord injury.The subacute phase after spinal cord injury is critical to the recovery of neurological function,which involves tissue bridging and axon regeneration.In this study,we found that zebrafish spontaneously recovered 44%of their swimming ability within the subacute phase(2 weeks)after spinal cord injury.During this period,we identified 7762 differentially expressed genes in spinal cord tissue:2950 were up-regulated and 4812 were down-regulated.These differentially expressed genes were primarily concentrated in the biological processes of the respiratory chain,axon regeneration,and cell-component morphogenesis.The genes were also mostly involved in the regulation of metabolic pathways,the cell cycle,and gene-regulation pathways.We verified the gene expression of two differentially expressed genes,clasp2 up-regulation and h1m down-regulation,in zebrafish spinal cord tissue in vitro.Pathway enrichment analysis revealed that up-regulated clasp2 functions similarly to microtubule-associated protein,which is responsible for axon extension regulated by microtubules.Down-regulated h1m controls endogenous stem cell differentiation after spinal cord injury.This study provides new candidate genes,clasp2 and h1m,as potential therapeutic intervention targets for spinal cord injury repair by neuroregeneration.All experimental procedures and protocols were approved by the Animal Ethics Committee of Tianjin Institute of Medical&Pharmaceutical Sciences(approval No.IMPS-EAEP-Q-2019-02)on September 24,2019.     

Changes in excitability properties associated with axonal regeneration in human neuropathy and mouse Wallerian degeneration.

OBJECTIVE: The aim of this study was to investigate changes in excitability properties associated with axonal regeneration in human neuropathy and a mouse Wallerian degeneration model. METHODS: Threshold tracking was used to measure axonal excitability indices such as strength-duration time constant (SDTC), threshold electrotonus, supernormality in median motor axons at the wrist of 13 patients with vasculitic neuropathy in their recovery phase, and in tibial motor axons at the ankle of mice with sciatic nerve crush. In the mouse model, excitability testing was performed 4, 8, 12, and 20weeks after the nerve crush. RESULTS: In patients, there were longer SDTC, greater threshold changes at 0.2ms in latent addition, and greater threshold changes in depolarizing and hyperpolarizing threshold electrotonus, compared with controls. The pattern of changes in excitability indices was similar to those in experimental nerve crush, in which the indices remained abnormal for 20weeks after the crush. These changes suggest an increase in nodal persistent sodium currents, whereas multiple factors may also contribute to changes in excitability properties, such as axonal hyperpolarization, increased internodal resistance, and altered potassium currents. CONCLUSIONS: Excitability properties in regenerating axons are characterized by increased nodal persistent currents with variable combination of changes in passive properties, membrane potential, and potassium currents. SIGNIFICANCE: Increased persistent sodium currents are potential reasons for positive symptoms in patients with axonal neuropathy. Sodium channel blockers could be considered a treatment option.     

Expression changes and bioinformatic analysis of Wallerian degeneration after sciatic nerve injury in rat

Wallerian degeneration (WD) remains an important research topic. Many genes are differentially expressed during the process of WD, but the precise mechanisms responsible for these differentiations are not completely understood. In this study, we used microarrays to analyze the expression changes of the distal nerve stump at 0, 1, 4, 7, 14, 21 and 28 days after sciatic nerve injury in rats. The data revealed 6 076 differentially-expressed genes, with 23 types of expression, specifically enriched in genes associated with nerve development and axonogenesis, cytokine biosynthesis, cell differentiation, cytokine/chemokine production, neuron differentiation, cytokinesis, phosphorylation and axon regeneration. Kyoto Encyclopedia of Genes and Genomes pathway analysis gave findings related mainly to the MAPK signaling pathway, the Jak-STAT signaling pathway, the cell cycle, cytokine-cytokine receptor interaction, the p53 signaling pathway and the Wnt signaling pathway. Some key factors were NGF, MAG, CNTF, CTNNA2, p53, JAK2, PLCB1, STAT3, BDNF, PRKC, collagen II, FGF, THBS4, TNC and c-Src, which were further validated by real-time quantitative PCR, Western blot, and immunohistochemistry. Our findings contribute to a better understanding of the functional analysis of differentially-expressed genes in WD and may shed light on the molecular mechanisms of nerve degeneration and regeneration.     

Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP-H mice

We investigated the usefulness of YFP-H transgenic mice [Neuron 28 (2000) 41] which express yellow fluorescent protein (YFP) in a restricted subset of neurons to study Wallerian degeneration in the PNS. Quantification of YFP positive axons and myelin basic protein (MBP) immunocytochemistry revealed that YFP was randomly distributed to approximately 3% of myelinated motor and sensory fibres. Axotomy-induced Wallerian degeneration appeared as fragmentation of fluorescent signals in individual YFP positive axons with a morphology and timing similar to Wallerian degeneration observed by more traditional methods. In YFP-H transgenic mice co-expressing a high dosage of WldS, a chimeric gene that protects from Wallerian degeneration [Nat Neurosci. 4 (2001) 1199], axonal fragmentation in distal tibial nerves after sciatic nerve axotomy was approximately 10 times delayed. Considerable retardations of Wallerian degeneration using the same transgenic expression system were also observed in cultures of nerve explants, enabling in vitro real-time imaging of axonal fragmentation. Remarkably, single YFP-labelled axons could be traced in peripheral nerves for unusually long distances of up to 2.9 cm exploiting confocal fluorescence imaging. Altogether transgenic YFP-H mice prove to be a valuable tool to study mechanisms of Wallerian degeneration in vivo and in vitro.     

酒精使用障碍的潜在关键基因生物信息学分析

背景 酒精使用障碍（AUD）是一类慢性复发性脑病,遗传因素在AUD的发病过程中起重要作用。明确AUD的关键分子标志物对于进一步阐明疾病的发病机制,探索新的治疗靶点和预防复发具有重要意义。目的 应用生物信息学方法,筛选AUD的核心基因和信号通路,为AUD的防治提供新的方向。方法 从基因表达综合数据库（GEO）下载基因表达数据集GSE161986。应用R软件的limma包筛选差异表达基因（DEGs）。使用DAVID数据库进行基因富集分析（GSEA）。使用STRING数据库和Cytoscape软件构建蛋白-蛋白互作网络（PPI）并寻找网络核心基因。使用GSE44456验证筛选潜在核心基因。结果 共筛选出114个DEGs。富集分析表明,差异基因主要参与信号转导、蛋白结合、细胞膜以及MAPK信号通路等功能的调控。PPI及验证分析显示,GAD1、TIMP1和CD44可能是AUD发病的潜在关键基因。结论 GAD1和TIMP1表达异常以及MAPK信号通路可能在AUD的发病过程中起关键作用,并可能作为AUD诊断和治疗的潜在分子靶点。     

Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons

Most current studies quantify axon regeneration by immunostaining regeneration-associated proteins,representing indirect measurement of axon lengths from both sensory neurons in the dorsal root ganglia and motor neurons in the spinal cord.Our recently developed method of in vivo electroporation of plasmid DNA encoding for enhanced green fluorescent protein into adult sensory neurons in the dorsal root ganglia provides a way to directly and specifically measure regenerating sensory axon lengths in whole-mount nerves.A mouse model of sciatic nerve compression was established by squeezing the sciatic nerve with tweezers.Plasmid DNA carrying enhanced green fluorescent protein was transfected by ipsilateral dorsal root ganglion electroporation 2 or 3 days before injury.Fluorescence distribution of dorsal root or sciatic nerve was observed by confocal microscopy.At 12 and 18 hours,and 1,2,3,4,5,and 6 days of injury,lengths of regenerated axons after sciatic nerve compression were measured using green fluorescence images.Apoptosis-related protein caspase-3 expression in dorsal root ganglia was determined by western blot assay.We found that in vivo electroporation did not affect caspase-3 expression in dorsal root ganglia.Dorsal root ganglia and sciatic nerves were successfully removed and subjected to a rapid tissue clearing technique.Neuronal soma in dorsal root ganglia expressing enhanced green fluorescent protein or fluorescent dye-labeled microRNAs were imaged after tissue clearing.The results facilitate direct time course analysis of peripheral nerve axon regeneration.This study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University,China(approval No.GLMC201503010)on March 7,2014.     

An 8.5-kb segment of the PMP22 promoter responds to loss of axon signals during Wallerian degeneration, but does not respond to specific axonal signals during nerve regeneration

Altered expression of the PMP22 gene causes Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). We have examined the promoter activity of 8.5 kb upstream of the first coding exon of the rat peripheral myelin protein-22 (rPmp22) gene in transgenic mice. We found that the -8.5 kb rPmp22/chloramphenicol acetyl transferase (CAT)/beta-galactosidase (lacZ) construct directs reporter gene expression in a weakly developmental and tissue-specific pattern, consistent with the expression pattern of the endogenous Pmp22 gene. The -8.5 kb rPmp22/CAT/lacZ transgene responds to loss of axonal signals during Wallerian degeneration but unlike the endogenous Pmp22 gene, the transgene fails to respond to axonal signals during nerve regeneration after a sciatic nerve crush injury. In conclusion, the function of the -8.5 kb rPmp22/CAT/lacZ transgene suggests that there are separable regulatory elements in the rPmp22 gene that respond differently to axonal signals received by Schwann cells during nerve development, and during remyelination.     

Plasma membrane pores of the Schwann cell in Wallerian degeneration: a morphometric analysis

Using the freeze-fracture technique, myelinated fibres were examined from the rabbit sciatic nerve at 48 h after a proximal nerve crush. Employing computer-aided morphometric techniques the distribution of Schwann cell plasma membrane pores was analysed. In both normal control and crushed nerves membrane pores were restricted to the cytoplasmic circumferential bands and longitudinal columns, which characterize the surface of the myelinated nerve fibre, and were absent from the flat plaque-like areas delimited by the bands and columns. In approximately half of the myelinated fibres from the crushed nerves there was a five-fold increase in the density of plasma membrane pores. This response of the Schwann cell was interpreted in terms of an increase in pinocytosis and related to regenerative phenomena in the peripheral nerve.