Search

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., Highlights • Laser-mediated axotomy of hESC-derived neurons is used as a model for axonal regeneration and local protein translation. • Pull down of newly synthesized axonal proteins is used to identify local protein synthesis following axonal injury. • Reduced axonal regeneration is associated with reduced levels of local translation. • Enhancing regeneration by co-culturing with human glial precursor cells is associated with enhanced levels of translation. • Proteomics profiling of axonal proteins during aging reveals reduced protein translation machinery as axons age.

In the originally published version of this manuscript, there were errors in the details and enumeration for authors' affiliations.

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 (PIP 3). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP 3 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 PIP 3 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 PIP 3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion., CNS axons lose the ability to regenerate with maturity, whilst PNS axons do not. This study shows that PIP3 levels decline in CNS neurons at the time when regenerative ability is lost. CNS overexpression of one isoform of PI3K, p110δ, enhances axonal PIP3, axon transport, and regenerative ability.

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 found that neuronal PIP3 decreases with maturity in line with regenerative competence, firstly in the cell body and subsequently in the axon. We show that adult PNS neurons utilise two catalytic subunits of PI3K for efficient regeneration: p110 and p110{delta}. Overexpressing p110 in CNS neurons had no effect, however expression of p110{delta} restored axonal PIP3 and enhanced CNS regeneration in rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110. Furthermore, viral delivery of p110{delta} promoted robust regeneration after optic nerve injury. These findings demonstrate a deficit of axonal PIP3 as a reason for intrinsic regeneration failure and show that native p110{delta} facilitates axon regeneration by functioning in a hyperactive fashion.

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., eLife digest The nerves in the brain and spinal cord can be damaged by trauma, stroke and other conditions. Damage to these nerve fibres can destroy the connections they form with each other, which may lead to paralysis, loss of sensation and loss of body control. If we could stimulate the regeneration and reconnection of the damaged nerve fibres then neurological function could be restored. However, although embryonic nerve fibres can regenerate when they are transplanted into the adult central nervous system, this regenerative ability appears to be lost as the nerve fibres mature. To investigate when and why nerve fibres lose the ability to regenerate, Koseki et al. first developed a tissue culture assay in which individual nerve fibres were cut with a laser and imaged for several hours to track their regeneration (or failure to regenerate). The results demonstrate that nerve fibres from the central nervous system progressively lose the ability to grow and regenerate as they mature. To investigate why mature nerve fibres cannot regenerate, Koseki et al. measured whether nerve fibres can transport some of the molecules needed for growth and regeneration to sites of damage. This showed that the compartments in which some key growth molecules are transported become excluded from mature nerve fibres. These compartments are marked by a protein called rab11, and Koseki et al. found that forcing rab11 back into mature nerve fibres restored their ability to regenerate. There is still a lot of work needed before these findings can lead to a new regeneration treatment for patients, but it is a crucial step forwards. Furthermore, the assay developed by Koseki et al. could be used to develop and test such treatments.

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. (C) 2013 Elsevier Ltd. All rights reserved.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00018-012-0992-7) contains supplementary material, which is available to authorized users.

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.

Nature Communications 5: Article number: 3411 (2014); Published: 11 March 2014; Updated: 7 July 2014. The affiliation details for Susan van Erp are incorrect in this Article. The correct affiliation details for this author are given below: Department of Translational Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands.

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.

RGM Proteins Members of the repulsive guidance molecule (RGM) family of proteins can be secreted or reside on the surface of cells where they bind to the cell surface receptor, neogenin. The RGM proteins are named for their role in axon guidance for developing neurons, but their function is also linked to a range of human diseases, including inflammation, multiple sclerosis, and cancer. Bell et al. (p. 77 ) solved the crystal structures of the external portions of the RGMB protein with portions of neogenin. The structures revealed interactions of dimers of RGMB with neogenin in which ligand binding induced conformational changes that may initiate intracellular signaling from the receptor. RGM proteins contain a site of autocatalytic cleavage that affects secretion of the proteins, and some disease-associated mutations in RGM proteins were clustered at this site.

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.