The Jun-dependent axon regeneration gene program: Jun promotes regeneration over plasticity.

The regeneration-associated gene (RAG) expression program is activated in injured peripheral neurons after axotomy and enables long-distance axon re-growth. Over 1000 genes are regulated, and many transcription factors are upregulated or activated as part of this response. However, a detailed picture of how RAG expression is regulated is lacking. In particular, the transcriptional targets and specific functions of the various transcription factors are unclear. Jun was the first-regeneration-associated transcription factor identified and the first shown to be functionally important. Here we fully define the role of Jun in the RAG expression program in regenerating facial motor neurons. At 1, 4 and 14 days after axotomy, Jun upregulates 11, 23 and 44% of the RAG program, respectively. Jun functions relevant to regeneration include cytoskeleton production, metabolic functions and cell activation, and the downregulation of neurotransmission machinery. In silico analysis of promoter regions of Jun targets identifies stronger over-representation of AP1-like sites than CRE-like sites, although CRE sites were also over-represented in regions flanking AP1 sites. Strikingly, in motor neurons lacking Jun, an alternative SRF-dependent gene expression program is initiated after axotomy. The promoters of these newly expressed genes exhibit over-representation of CRE sites in regions near to SRF target sites. This alternative gene expression program includes plasticity-associated transcription factors and leads to an aberrant early increase in synapse density on motor neurons. Jun thus has the important function in the early phase after axotomy of pushing the injured neuron away from a plasticity response and towards a regenerative phenotype.

Genome-wide microRNA profiling of human temporal lobe epilepsy identifies modulators of the immune response.

Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disorder characterized by recurrent seizures. The pathogenic mechanisms underlying mTLE may involve defects in the post-transcriptional regulation of gene expression. MicroRNAs (miRNAs) are non-coding RNAs that control the expression of genes at the post-transcriptional level. Here, we performed a genome-wide miRNA profiling study to examine whether miRNA-mediated mechanisms are affected in human mTLE. miRNA profiles of the hippocampus of autopsy control patients and two mTLE patient groups were compared. This revealed segregated miRNA signatures for the three different patient groups and 165 miRNAs with up- or down-regulated expression in mTLE. miRNA in situ hybridization detected cell type-specific changes in miRNA expression and an abnormal nuclear localization of select miRNAs in neurons and glial cells of mTLE patients. Of several cellular processes implicated in mTLE, the immune response was most prominently targeted by deregulated miRNAs. Enhanced expression of inflammatory mediators was paralleled by a reduction in miRNAs that were found to target the 3'-untranslated regions of these genes in reporter assays. miR-221 and miR-222 were shown to regulate endogenous ICAM1 expression and were selectively co-expressed with ICAM1 in astrocytes in mTLE patients. Our findings suggest that miRNA changes in mTLE affect the expression of immunomodulatory proteins thereby further facilitating the immune response. This mechanism may have broad implications given the central role of astrocytes and the immune system in human neurological disease. Overall, this work extends the current concepts of human mTLE pathogenesis to the level of miRNA-mediated gene regulation.

Age-related loss of axonal regeneration is reflected by the level of local translation.

Regeneration capacity is reduced as CNS axons mature. Using laser-mediated axotomy, proteomics and puromycin-based tagging of newly-synthesized proteins in a human embryonic stem cell-derived neuron culture system that allows isolation of axons from cell bodies, we show here that efficient regeneration in younger axons (d45 in culture) is associated with local axonal protein synthesis (local translation). Enhanced regeneration, promoted by co-culture with human glial precursor cells, is associated with increased axonal synthesis of proteins, including those constituting the translation machinery itself. Reduced regeneration, as occurs with the maturation of these axons by d65 in culture, correlates with reduced levels of axonal proteins involved in translation and an inability to respond by increased translation of regeneration promoting axonal mRNAs released from stress granules. Together, our results provide evidence that, as in development and in the PNS, local translation contributes to CNS axon regeneration.

PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS.

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol (3,4,5)-trisphosphate (PIP3 ). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP3 decreases with development at the time when axon transport declines and regenerative competence is lost. Overexpressing p110α in CNS neurons had no effect; however, expression of p110δ restored axonal PIP3 and increased regenerative axon transport. p110δ expression enhanced CNS regeneration in both rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110α. Furthermore, viral delivery of p110δ promoted robust regeneration after optic nerve injury. These findings establish a deficit of axonal PIP3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion.

Selective rab11 transport and the intrinsic regenerative ability of CNS axons.

Neurons lose intrinsic axon regenerative ability with maturation, but the mechanism remains unclear. Using an in-vitro laser axotomy model, we show a progressive decline in the ability of cut CNS axons to form a new growth cone and then elongate. Failure of regeneration was associated with increased retraction after axotomy. Transportation into axons becomes selective with maturation; we hypothesized that selective exclusion of molecules needed for growth may contribute to regeneration decline. With neuronal maturity rab11 vesicles (which carry many molecules involved in axon growth) became selectively targeted to the somatodendritic compartment and excluded from axons by predominant retrograde transport However, on overexpression rab11 was mistrafficked into proximal axons, and these axons showed less retraction and enhanced regeneration after axotomy. These results suggest that the decline of intrinsic axon regenerative ability is associated with selective exclusion of key molecules, and that manipulation of transport can enhance regeneration.

Lrig2 Negatively Regulates Ectodomain Shedding of Axon Guidance Receptors by ADAM Proteases.

Many guidance receptors are proteolytically cleaved by membrane-associated metalloproteases of the ADAM family, leading to the shedding of their ectodomains. Ectodomain shedding is crucial for receptor signaling and function, but how this process is controlled in neurons remains poorly understood. Here, we show that the transmembrane protein Lrig2 negatively regulates ADAM-mediated guidance receptor proteolysis in neurons. Lrig2 binds Neogenin, a receptor for repulsive guidance molecules (RGMs), and prevents premature Neogenin shedding by ADAM17 (TACE). RGMa reduces Lrig2-Neogenin interactions, providing ADAM17 access to Neogenin and allowing this protease to induce ectodomain shedding. Regulation of ADAM17-mediated Neogenin cleavage by Lrig2 is required for neurite growth inhibition by RGMa in vitro and for cortical neuron migration in vivo. Furthermore, knockdown of Lrig2 significantly improves CNS axon regeneration. Together, our data identify a unique ligand-gated mechanism to control receptor shedding by ADAMs and reveal functions for Lrigs in neuron migration and regenerative failure.

Determinants of limitations in unpaid work after major trauma: a prospective cohort study with 15 months follow-up.

OBJECTIVE: To identify determinants of limitations in unpaid work (household work, shopping, caring for children and odd jobs around the house) in patients who had suffered major trauma (ISS≥16) and who were in full-time employment (≥80%) at the time of injury. DESIGN: Prospective cohort study. SETTING: University Medical Centre Utrecht, a level 1 trauma centre in the Netherlands. METHOD: All severely injured (ISS≥16) adult (age≥16) trauma survivors admitted from January 1999 to December 2000 who were full-time employed at time of the injury were invited for follow-up (n=214). Outcome was assessed with the 'Health and Labour Questionnaire' (HLQ) at a mean of 15 months (SD=1.5) after injury. The HLQ was completed by 211 patients. RESULTS: Response rate was 93%. Logistic regression analyses identified the percentage of permanent impairment (% PI), level of participation (RtW), co-morbidity, lower extremity injury (LEI) and female gender as determinants of limitations in unpaid work. Patients with a post-injury status of part-time or no return to work experienced more limitations in unpaid work than those who returned to full-time employment. CONCLUSIONS: Resuming paid work after major trauma is not associated with reductions in unpaid activities. To assess the long-term outcome of rehabilitation programmes, we recommend a measure that combines patient's satisfaction in their post-injury jobs with a satisfactory level of activities in their private lives.

A role for Bicaudal-D2 in radial cerebellar granule cell migration.

Bicaudal-D (BICD) belongs to an evolutionary conserved family of dynein adaptor proteins. It was first described in Drosophila as an essential factor in fly oogenesis and embryogenesis. Missense mutations in a human BICD homologue, BICD2, have been linked to a dominant mild early onset form of spinal muscular atrophy. Here we further examine the in vivo function of BICD2 in Bicd2 knockout mice. BICD2-deficient mice develop disrupted laminar organization of cerebral cortex and the cerebellum, pointing to impaired radial neuronal migration. Using astrocyte and granule cell specific inactivation of BICD2, we show that the cerebellar migration defect is entirely dependent upon BICD2 expression in Bergmann glia cells. Proteomics analysis reveals that Bicd2 mutant mice have an altered composition of extracellular matrix proteins produced by glia cells. These findings demonstrate an essential non-cell-autonomous role of BICD2 in neuronal cell migration, which might be connected to cargo trafficking pathways in glia cells.

Structure of the repulsive guidance molecule (RGM)-neogenin signaling hub.

Repulsive guidance molecule family members (RGMs) control fundamental and diverse cellular processes, including motility and adhesion, immune cell regulation, and systemic iron metabolism. However, it is not known how RGMs initiate signaling through their common cell-surface receptor, neogenin (NEO1). Here, we present crystal structures of the NEO1 RGM-binding region and its complex with human RGMB (also called dragon). The RGMB structure reveals a previously unknown protein fold and a functionally important autocatalytic cleavage mechanism and provides a framework to explain numerous disease-linked mutations in RGMs. In the complex, two RGMB ectodomains conformationally stabilize the juxtamembrane regions of two NEO1 receptors in a pH-dependent manner. We demonstrate that all RGM-NEO1 complexes share this architecture, which therefore represents the core of multiple signaling pathways.

Semaphorin signaling: molecular switches at the midline.

To establish axonal connections growth cones must navigate multiple intermediate targets before reaching their final target. During this journey growth cones are guided by extracellular repulsive and attractive signals. Although initially identified as repulsive molecules, members of the semaphorin family include both attractants and repellents. How a navigating growth cone responds to a specific semaphorin is not absolute but instead depends on the biological context in which this cue is encountered. Here we review recent breakthroughs in our understanding of the extrinsic signals and molecular processes that control growth cone responses to class 3 semaphorins (Sema3s) at a well-characterized intermediate target, the spinal cord midline.

Pitx3 potentiates Nurr1 in dopamine neuron terminal differentiation through release of SMRT-mediated repression.

In recent years, the meso-diencephalic dopaminergic (mdDA) neurons have been extensively studied for their association with Parkinson's disease. Thus far, specification of the dopaminergic phenotype of mdDA neurons is largely attributed to the orphan nuclear receptor Nurr1. In this study, we provide evidence for extensive interplay between Nurr1 and the homeobox transcription factor Pitx3 in vivo. Both Nurr1 and Pitx3 interact with the co-repressor PSF and occupy the promoters of Nurr1 target genes in concert. Moreover, in vivo expression analysis reveals that Nurr1 alone is not sufficient to drive the dopaminergic phenotype in mdDA neurons but requires Pitx3 for full activation of target gene expression. In the absence of Pitx3, Nurr1 is kept in a repressed state through interaction with the co-repressor SMRT. Highly resembling the effect of ligand activation of nuclear receptors, recruitment of Pitx3 modulates the Nurr1 transcriptional complex by decreasing the interaction with SMRT, which acts through HDACs to keep promoters in a repressed deacetylated state. Indeed, interference with HDAC-mediated repression in Pitx3(-/-) embryos efficiently reactivates the expression of Nurr1 target genes, bypassing the necessity for Pitx3. These data position Pitx3 as an essential potentiator of Nurr1 in specifying the dopaminergic phenotype, providing novel insights into mechanisms underlying development of mdDA neurons in vivo, and the programming of stem cells as a future cell replacement therapy for Parkinson's disease.