Gene expression data are accumulating exponentially in public repositories. Reanalysis and integration of themed collections from these studies may provide new insights, but requires further human ...curation. Here we report a crowdsourcing project to annotate and reanalyse a large number of gene expression profiles from Gene Expression Omnibus (GEO). Through a massive open online course on Coursera, over 70 participants from over 25 countries identify and annotate 2,460 single-gene perturbation signatures, 839 disease versus normal signatures, and 906 drug perturbation signatures. All these signatures are unique and are manually validated for quality. Global analysis of these signatures confirms known associations and identifies novel associations between genes, diseases and drugs. The manually curated signatures are used as a training set to develop classifiers for extracting similar signatures from the entire GEO repository. We develop a web portal to serve these signatures for query, download and visualization.
Mutations in the aristaless-related homeobox (ARX) gene are associated with multiple neurologic disorders in humans. Studies in mice indicate Arx plays a role in neuronal progenitor proliferation and ...development of the cerebral cortex, thalamus, hippocampus, striatum, and olfactory bulbs. Specific defects associated with Arx loss of function include abnormal interneuron migration and subtype differentiation. How disruptions in ARX result in human disease and how loss of Arx in mice results in these phenotypes remains poorly understood. To gain insight into the biological functions of Arx, we performed a genome-wide expression screen to identify transcriptional changes within the subpallium in the absence of Arx. We have identified 84 genes whose expression was dysregulated in the absence of Arx. This population was enriched in genes involved in cell migration, axonal guidance, neurogenesis, and regulation of transcription and includes genes implicated in autism, epilepsy, and mental retardation; all features recognized in patients with ARX mutations. Additionally, we found Arx directly repressed three of the identified transcription factors: Lmo1, Ebf3 and Shox2. To further understand how the identified genes are involved in neural development, we used gene set enrichment algorithms to compare the Arx gene regulatory network (GRN) to the Dlx1/2 GRN and interneuron transcriptome. These analyses identified a subset of genes in the Arx GRN that are shared with that of the Dlx1/2 GRN and that are enriched in the interneuron transcriptome. These data indicate Arx plays multiple roles in forebrain development, both dependent and independent of Dlx1/2, and thus provides further insights into the understanding of the mechanisms underlying the pathology of mental retardation and epilepsy phenotypes resulting from ARX mutations.
Human Ntera-2 (NT2) cells can be differentiated in vitro into well-characterized populations of NT2N neurons that engraft and mature when transplanted into the adult CNS of rodents and humans. They ...have shown promise as treatments for neurologic disease, trauma, and ischemic stroke. Although these features suggest that NT2N neurons would be an excellent platform for ex vivo gene therapy in the CNS, stable gene expression has been surprisingly difficult to achieve in these cells. In this report we demonstrate stable, efficient, and nontoxic gene transfer into undifferentiated NT2 cells using a pseudotyped lentiviral vector encoding the human elongation factor 1-α promoter and the reporter gene eGFP. Expression of eGFP was maintained when the NT2 cells were differentiated into NT2N neurons after treatment with retinoic acid. When transplanted into the striatum of adult nude mice, transduced NT2N neurons survived, engrafted, and continued to express the reporter gene for long-term time points in vivo. Furthermore, transplantation of NT2N neurons genetically modified to express nerve growth factor significantly attenuated cognitive dysfunction following traumatic brain injury in mice. These results demonstrate that defined populations of genetically modified human NT2N neurons are a practical and effective platform for stable ex vivo gene delivery into the CNS.
We investigated whether new neurons generated in the adult rat brain following lateral fluid percussion traumatic brain injury (TBI) are capable of projecting axons along the mossy fiber pathway to ...the CA3 region of the hippocampus. Dividing cells were labeled by intraperitoneal injection of bromodeoxyuridine (BrdU) on the day of surgery/injury, and neurons that extended axons to the CA3 region were retrogradely labeled by fluorescent tracers (FluoSpheres), stereotactically injected into the CA3 region of both the ipsi- and contralateral hippocampus at 1 or 12 days following TBI (n = 12) or sham injury (n = 12) in anaesthetized rats. Animals (n = 6 injured and n = 6 sham-injured controls per time point) were sacrificed at either 3 or 14 days post-injury. Another group of animals (n = 3) was subjected to experimental TBI and BrdU administration and sacrificed 3 days after TBI to be processed for BrdU and immunohistochemistry for polysialylated neural cell adhesion molecule (PSA-NCAM), a growth-related protein normally observed during CNS development. A fivefold bilateral increase in the number of mitotically active (BrdU+) cells was noted within the dentate gyrus when compared to uninjured controls as early as 3 days following TBI. A subgroup of dividing cells was also immunoreactive for PSA-NCAM at 3 days following TBI. By 2 weeks post-injury the number of BrdU+ cells within the dentate gyrus was increased twofold compared to the uninjured counterparts and a proportion of these newly generated cells showed cytoplasmic staining for the fluorescent tracer. These findings document rapid neurogenesis following TBI and show, for the first time, that newly generated granule neurons are capable of extending projections along the hippocampal mossy fiber pathway in the acute post-traumatic period.
Axons show a poor regenerative capacity following traumatic central nervous system (CNS) injury, partly due to the expression of inhibitors of axonal outgrowth, of which Nogo-A is considered the most ...important. We evaluated the acute expression of Nogo-A, the Nogo-66 receptor (NgR) and the novel small proline-rich repeat protein 1A (SPRR1A, previously undetected in brain), following experimental lateral fluid percussion (FP) brain injury in rats. Immunofluorescence with antibodies against Nogo-A, NgR and SPRR1A was combined with antibodies against the neuronal markers NeuN and microtubule-associated protein (MAP)-2 and the oligodendrocyte marker RIP, while Western blot analysis was performed for Nogo-A and NgR. Brain injury produced a significant increase in Nogo-A expression in injured cortex, ipsilateral external capsule and reticular thalamus from days 1–7 post-injury (
P < 0.05) compared to controls. Increased expression of Nogo-A was observed in both RIP- and NeuN positive (+) cells in the ipsilateral cortex, in NeuN (+) cells in the CA3 region of the hippocampus and reticular thalamus and in RIP (+) cells in white matter tracts. Alterations in NgR expression were not observed following traumatic brain injury (TBI). Brain injury increased the extent of SPRR1A expression in the ipsilateral cortex and the CA3 at all post-injury time-points in NeuN (+) cells. The marked increases in Nogo-A and SPRR1A in several important brain regions suggest that although inhibitors of axonal growth may be upregulated, the injured brain is also capable of expressing proteins promoting axonal outgrowth following TBI.
During the past decade, there has been accumulating evidence of the involvement of passive and active cell death mechanisms in both the clinical setting and in experimental models of traumatic brain ...injury (TBI). Traditionally, research for a treatment of TBI consists of strategies to prevent cell death using acute pharmacological therapy. However, to date, encouraging experimental work has not been translated into successful clinical trials. The development of cell replacement therapies may offer an alternative or a complementary strategy for the treatment of TBI. Recent experimental studies have identified a variety of candidate cell lines for transplantation into the injured CNS. Additionally, the characterization of the neurogenic potential of specific regions of the adult mammalian brain and the elucidation of the molecular controls underlying regeneration may allow for the development of neuronal replacement therapies that do not require transplantation of exogenous cells. These novel strategies may represent a new opportunity of great interest for delayed intervention in patients with TBI.
The regional activation (via phosphorylation) of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways was examined using immunoblotting and ...immunohistochemistry following experimental brain injury. Anesthetized rats were subjected to lateral fluid-percussion brain injury of moderate severity (2.4–2.6 atm) and euthanized at 2, 6, 24, and 72 h after injury; sham-injured animals were surgically prepared but were not injured. Immunohistochemical evidence of activation of JNK and ERK1/2 pathways was observed predominantly in regions that exhibit neural cell apoptosis and axonal damage following brain trauma. Activation of the ERK1/2 pathway was observed as early as 2 h and up to 72 h postinjury in nonneuronal cells in all layers of the cortex at the site of maximal injury, in the white matter below the site of maximal cortical damage and in the thalamus. In contrast, activation of JNK signaling was observed only at 24 and 72 h postinjury in a few neurons at the core of the cortical injury site. However, robust JNK activation was observed between 2 and 72 h postinjury in both axons and nonneuronal cells in the white matter below the site of maximal cortical damage and in the thalamus. Activation of ERK1/2, but not JNK, was observed in cells in the dentate hilus in the hippocampus in both hemispheres between 2 and 24 h postinjury. Immunoblotting analyses of extracts from various brain regions did not reveal significant alterations in intensities of either total or phosphorylated proteins underscoring the focal nature of the immunohistochemical observations. However, these results suggest that activation of MAP kinase signaling pathways may be associated with posttraumatic cell damage and are indicative of the heterogeneous nature of the mechanisms underlying regional cell death following TBI.
Experimental research during the past decade has greatly increased our understanding of the pathophysiology of traumatic brain injury (TBI) and allowed us to develop neuroprotective pharmacological ...therapies. Encouraging results of experimental pharmacological interventions, however, have not been translated into successful clinical trials, to date. Traumatic brain injury is now believed to be a progressive degenerative disease characterized by cell loss. The limited capacity for self-repair of the brain suggests that functional recovery following TBI is likely to require cellular transplantation of exogenous cells to replace those lost to trauma. Recent advances in central nervous system transplantation techniques involve technical and experimental refinements and the analysis of the feasibility and efficacy of transplantation of a range of stem cells, progenitor cells and postmitotic cells. Cellular transplantation has begun to be evaluated in several models of experimental TBI, with promising results. The following is a compendium of these new and exciting studies, including a critical discussion of the rationale and caveats associated with cellular transplantation techniques in experimental TBI research. Further refinements in future research are likely to improve results from transplantation-based treatments for TBI.
Human Ntera-2 (NT2) cells can be differentiated in vitro into well-characterized populations of NT2N neurons that engraft and mature when transplanted into the adult CNS of rodents and humans. They ...have shown promise as treatments for neurologic disease, trauma, and ischemic stroke. Although these features suggest that NT2N neurons would be an excellent platform for ex vivo gene therapy in the CNS, stable gene expression has been surprisingly difficult to achieve in these cells. In this report we demonstrate stable, efficient, and nontoxic gene transfer into undifferentiated NT2 cells using a pseudotyped lentiviral vector encoding the human elongation factor 1-alpha promoter and the reporter gene eGFP. Expression of eGFP was maintained when the NT2 cells were differentiated into NT2N neurons after treatment with retinoic acid. When transplanted into the striatum of adult nude mice, transduced NT2N neurons survived, engrafted, and continued to express the reporter gene for long-term time points in vivo. Furthermore, transplantation of NT2N neurons genetically modified to express nerve growth factor significantly attenuated cognitive dysfunction following traumatic brain injury in mice. These results demonstrate that defined populations of genetically modified human NT2N neurons are a practical and effective platform for stable ex vivo gene delivery into the CNS.
Mutations in the X-linked aristaless-related homeobox gene (ARX) have been linked to structural brain anomalies as well as multiple neurocognitive deficits. The generation of Arx-deficient mice ...revealed several morphological anomalies, resembling those observed in patients and an interneuron migration defect but perinatal lethality precluded analyses of later phenotypes. Interestingly, many of the neurological phenotypes observed in patients with various ARX mutations can be attributed, in part, to interneuron dysfunction. To directly test this possibility, mice carrying a floxed Arx allele were generated and crossed to Dlx5/6CRE-IRES-GFP(Dlx5/6CIG) mice, conditionally deleting Arx from ganglionic eminence derived neurons including cortical interneurons. We now report that Arx−/y;Dlx5/6CIG (male) mice exhibit a variety of seizure types beginning in early-life, including seizures that behaviourally and electroencephalographically resembles infantile spasms, and show evolution through development. Thus, this represents a new genetic model of a malignant form of paediatric epilepsy, with some characteristics resembling infantile spasms, caused by mutations in a known infantile spasms gene. Unexpectedly, approximately half of the female mice carrying a single mutant Arx allele (Arx−/+;Dlx5/6CIG) also developed seizures. We also found that a subset of human female carriers have seizures and neurocognitive deficits. In summary, we have identified a previously unrecognized patient population with neurological deficits attributed to ARX mutations that are recapitulated in our mouse model. Furthermore, we show that perturbation of interneuron subpopulations is an important mechanism underling the pathogenesis of developmental epilepsy in both hemizygous males and carrier females. Given the frequency of ARX mutations in patients with infantile spasms and related disorders, our data unveil a new model for further understanding the pathogenesis of these disorders.
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