Antigen dependently activated cluster of differentiation 8-positive T cells cause perforin-mediated neurotoxicity in experimental stroke.

Neuroinflammation plays a key role in secondary brain damage after stroke. Although deleterious effects of proinflammatory cytokines are well characterized, direct cytotoxic effects of invading immune cells on the ischemic brain and the importance of their antigen-dependent activation are essentially unknown. Here we examined the effects of adaptive and innate immune cells-cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells-that share the direct perforin-mediated cytotoxic pathway on outcome after cerebral ischemia in mice. Although CTLs and NK cells both invaded the ischemic brain, only brain-infiltrating CTLs but not NK cells were more activated than their splenic counterparts. Depletion of CTLs decreased infarct volumes and behavioral deficit in two ischemia models, whereas NK cell depletion had no effect. Correspondingly, adoptive CTL transfer from wild-type into Rag1 knock-out mice increased infarct size. Adoptive CTL transfer from perforin knock-out or interferon-γ knock-out mice into Rag1 knock-out mice revealed that CTL neurotoxicity was mediated by perforin. Accordingly, CTLs isolated from wild-type or interferon-γ knock-out but not from perforin knock-out mice induced neuronal cell death in vitro. CTLs derived from ovalbumin-specific T-cell receptor transgenic mice were not activated and infiltrated less into the ischemic brain compared with wild-type CTLs. Their transfer did not increase the infarct size of Rag1 knock-out mice, indicating antigen-dependent activation as an essential component of CTL neurotoxicity. Our findings underscore the importance of antigen-dependent, direct cytotoxic immune responses in stroke and suggest modulation of CTLs and their effector pathways as a potential new strategy for stroke therapy.

Constitutive activation of distinct NF-κB signals in EBV-associated nasopharyngeal carcinoma.

As a distinct type of head and neck cancer, non-keratinizing nasopharyngeal carcinoma (NPC) is closely associated with EBV infection and massive lymphoid infiltration. The unique histological features suggest that local inflammation plays an important role in NPC tumourigenesis. We comprehensively characterized NF-κB signalling, a key inflammatory pathway which might contribute to the tumourigenesis of this EBV-associated cancer. By EMSA, western blotting, and immunohistochemical staining, constitutive activation of distinct NF-κB complexes, either p50/p50/Bcl3 or p50/RelB, was found in almost all EBV-positive NPC tumours. siRNA or chemical inhibition of NF-κB signalling significantly inhibited the growth of EBV-positive NPC cells C666-1. Gene expression profiling identified a number of NF-κB target genes involved in cell proliferation, apoptosis, immune response, and transcription. We further confirmed that p50 signals modulate the expression of multiple oncogenes (MYB, BCL2), chemokines, and chemokine receptors (CXCL9, CXCL10, CX3CL1, and CCL20). The findings support a crucial role of these constitutively activated NF-κB signals in NPC tumourigenesis and local inflammation. In addition to expression of the viral oncoprotein LMP1, genetic alteration of several NF-κB regulators (eg TRAF3, TRAF2, NFKBIA, A20) also contributes to the aberrant NF-κB activation in EBV-associated NPC. Except for LMP1-expressing C15 cells, all NPC tumour lines harbour at least one of these genetic alterations. Importantly, missense mutations of TRAF3, TRAF2, and A20 were also detected in 3/33 (9.1%) primary tumours. Taken together with the reported LTBR amplification in 7.3% of primary NPCs, genetic alterations in NF-κB pathways occurred in at least 16% of cases of this cancer. The findings indicate that distinct NF-κB signals are constitutively activated in EBV-positive NPC cells by either multiple genetic changes or EBV latent genes.

Single-cell Stereo-seq reveals induced progenitor cells involved in axolotl brain regeneration.

The molecular mechanism underlying brain regeneration in vertebrates remains elusive. We performed spatial enhanced resolution omics sequencing (Stereo-seq) to capture spatially resolved single-cell transcriptomes of axolotl telencephalon sections during development and regeneration. Annotated cell types exhibited distinct spatial distribution, molecular features, and functions. We identified an injury-induced ependymoglial cell cluster at the wound site as a progenitor cell population for the potential replenishment of lost neurons, through a cell state transition process resembling neurogenesis during development. Transcriptome comparisons indicated that these induced cells may originate from local resident ependymoglial cells. We further uncovered spatially defined neurons at the lesion site that may regress to an immature neuron-like state. Our work establishes spatial transcriptome profiles of an anamniote tetrapod brain and decodes potential neurogenesis from ependymoglial cells for development and regeneration, thus providing mechanistic insights into vertebrate brain regeneration.

The engine initiating tissue regeneration: does a common mechanism exist during evolution?

A successful tissue regeneration is a very complex process that requires a precise coordination of many molecular, cellular and physiological events. One of the critical steps is to convert the injury signals into regeneration signals to initiate tissue regeneration. Although many efforts have been made to investigate the mechanisms triggering tissue regeneration, the fundamental questions remain unresolved. One of the major obstacles is that the injury and the initiation of regeneration are two highly coupled processes and hard to separate from one another. In this article, we review the major events occurring at the early injury/regeneration stage in a range of species, and discuss the possible common mechanisms during initiation of tissue regeneration.

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes.

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Publisher Correction: Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).

In the version of this protocol originally published, the recipe for CAS9 buffer was incorrectly identified as a recipe for sodium acetate solution, and vice versa. These errors have been corrected in the PDF and HTML versions of the paper.

An Immune-CNS Axis Activates Remote Hippocampal Stem Cells Following Spinal Transection Injury.

External stimuli such as injury, learning, or stress influence the production of neurons by neural stem cells (NSCs) in the adult mammalian brain. These external stimuli directly impact stem cell activity by influencing areas directly connected or in close proximity to the neurogenic niches of the adult brain. However, very little is known on how distant injuries affect NSC activation state. In this study, we demonstrate that a thoracic spinal transection injury activates the distally located hippocampal-NSCs. This activation leads to a transient increase production of neurons that functionally integrate to improve animal's performance in hippocampal-related memory tasks. We further show that interferon-CD95 signaling is required to promote injury-mediated activation of remote NSCs. Thus, we identify an immune-CNS axis responsible for injury-mediated activation of remotely located NSCs.

Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).

Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR-Cas9 system. The protocol, which takes 7-8 weeks to complete, describes generation of targeted gene knockouts and knock-ins and includes site-specific integration of large targeting constructs. The direct use of purified CAS9-NLS (CAS9 containing a C-terminal nuclear localization signal) protein allows the prompt formation of guide RNA (gRNA)-CAS9-NLS ribonucleoprotein (RNP) complexes, which accelerates the creation of double-strand breaks (DSBs) at targeted genomic loci in single-cell-stage axolotl eggs. With this protocol, a substantial number of F0 individuals harboring a homozygous-type frameshift mutation can be obtained, allowing phenotype analysis in this generation. In the presence of targeting constructs, insertions of exogenous genes into targeted axolotl genomic loci can be achieved at efficiencies of up to 15% in a non-homologous end joining (NHEJ) manner. Our protocol bypasses the long generation time of axolotls and allows direct functional analysis in F0 genetically manipulated axolotls. This protocol can be potentially applied to other animal models, especially to organisms with a well-characterized transcriptome but lacking a well-characterized genome.

Regulation of Adult CNS Axonal Regeneration by the Post-transcriptional Regulator Cpeb1.

Adult mammalian central nervous system (CNS) neurons are unable to regenerate following axonal injury, leading to permanent functional impairments. Yet, the reasons underlying this regeneration failure are not fully understood. Here, we studied the transcriptome and translatome shortly after spinal cord injury. Profiling of the total and ribosome-bound RNA in injured and naïve spinal cords identified a substantial post-transcriptional regulation of gene expression. In particular, transcripts associated with nervous system development were down-regulated in the total RNA fraction while remaining stably loaded onto ribosomes. Interestingly, motif association analysis of post-transcriptionally regulated transcripts identified the cytoplasmic polyadenylation element (CPE) as enriched in a subset of these transcripts that was more resistant to injury-induced reduction at the transcriptome level. Modulation of these transcripts by overexpression of the CPE binding protein, Cpeb1, in mouse and Drosophila CNS neurons promoted axonal regeneration following injury. Our study uncovered a global evolutionarily conserved post-transcriptional mechanism enhancing regeneration of injured CNS axons.

In vivo interrogation of central nervous system translatome by polyribosome fractionation.

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting 'translatomics' to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics. Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.