Properties of intact and injured sensory neurons with nerve growth factor receptors

The role of nerve growth factor (NGF), a neurotrophic molecule, and its high-affinity receptor in intact and injured adult rat lumbar sensory neurons was examined at a cellular level using quantitative receptor radioautography to localize the NGF high-affinity receptor-positive subpopulation, in conjunction with histochemistry on adjacent sections. The 40-50% of sensory neurons displaying NGF receptors were characterized. Virtually all neurons containing substance P or CGRP were NGF receptor-positive, but not those with somatostatin or thiamine monophosphatase activity. The ability of a neuron to bind NGF with high-affinity correlates positively with growth-associated protein (GAP43) expression but not with neurofilament (NFM) expression. Following injury, sensory neurons atrophy, lose NGF receptors, decrease NFM expression, while GAP43 expression is elevated in all neurons irrespective of their ability to bind NGF. Infusion of NGF for 1 week, at the time of injury or 3 weeks following injury counteracts NGF receptor loss, cell atrophy, and decreased NFM expression, but only in those neurons bearing NGF receptors. GAP43 expression remained high in all neurons despite infusion. NGF's function in normal sensory neurons appears to be modulatory, permitting regulation of intrinsic properties. Injury disrupts this permissive state, which can be restored with exogenous NGF

Efficient gene targeting and foreign DNA removal by homologous recombination in the picoeukaryote Ostreococcus

With fewer than 8000 genes and a minimalist cellular organization, the green picoalga Ostreococcus tauri is one of the simplest photosynthetic eukaryotes. Ostreococcus tauri contains many plant-specific genes but exhibits a very low gene redundancy. The haploid genome is extremely dense with few repeated sequences and rare transposons. Thanks to the implementation of genetic transformation and vectors for inducible overexpression/knockdown this picoeukaryotic alga has emerged in recent years as a model organism for functional genomics analyses and systems biology. Here we report the development of an efficient gene targeting technique which we use to knock out the nitrate reductase and ferritin genes and to knock in a luciferase reporter in frame to the ferritin native protein. Furthermore, we show that the frequency of insertion by homologous recombination is greatly enhanced when the transgene is designed to replace an existing genomic insertion. We propose that a natural mechanism based on homologous recombination may operate to remove inserted DNA sequences from the genome

Delayed nerve stimulation promotes axon-protective neurofilament phosphorylation, accelerates immune cell clearance and enhances remyelination in vivo in focally demyelinated nerves.

Rapid and efficient axon remyelination aids in restoring strong electrochemical communication with end organs and in preventing axonal degeneration often observed in demyelinating neuropathies. The signals from axons that can trigger more effective remyelination in vivo are still being elucidated. Here we report the remarkable effect of delayed brief electrical nerve stimulation (ES; 1 hour @ 20 Hz 5 days post-demyelination) on ensuing reparative events in a focally demyelinated adult rat peripheral nerve. ES impacted many parameters underlying successful remyelination. It effected increased neurofilament expression and phosphorylation, both implicated in axon protection. ES increased expression of myelin basic protein (MBP) and promoted node of Ranvier re-organization, both of which coincided with the early reappearance of remyelinated axons, effects not observed at the same time points in non-stimulated demyelinated nerves. The improved ES-associated remyelination was accompanied by enhanced clearance of ED-1 positive macrophages and attenuation of glial fibrillary acidic protein expression in accompanying Schwann cells, suggesting a more rapid clearance of myelin debris and return of Schwann cells to a nonreactive myelinating state. These benefits of ES correlated with increased levels of brain derived neurotrophic factor (BDNF) in the acute demyelination zone, a key molecule in the initiation of the myelination program. In conclusion, the tremendous impact of delayed brief nerve stimulation on enhancement of the innate capacity of a focally demyelinated nerve to successfully remyelinate identifies manipulation of this axis as a novel therapeutic target for demyelinating pathologies

Endogenous BDNF regulates induction of intrinsic neuronal growth programs in injured sensory neurons

Identification of the molecule(s) that globally induce a robust regenerative state in sensory neurons following peripheral nerve injury remains elusive. A potential candidate is brain-derived neurotrophic factor (BDNF), the sole neurotrophin upregulated in sensory neurons after peripheral nerve injury. Here we tested the hypothesis that BDNF plays a critical role in the regenerative response of mature rat sensory neurons following peripheral nerve lesion. Neutralization of endogenous BDNF was performed by infusing BDNF antibodies intrathecally via a mini-osmotic pump for 3 days at the level of the fifth lumbar dorsal root ganglion, immediately following unilateral spinal nerve injury. This resulted in decreased expression of the injury/regeneration-associated genes growth-associated protein-43 and T alpha 1 tubulin in the injured sensory neurons as compared to injury plus control IgG infused or injury alone animals. Similar results were observed following inhibition of BDNF expression by intrathecal delivery of small interfering RNAs (siRNA) targeting BDNF starting 3 days prior to injury. The reduced injury/regeneration-associated gene expression correlated with a significantly reduced intrinsic capacity of these neurons to extend neurites when assayed in vitro. In contrast, delayed infusion of BDNF antibody for 3 days beginning 1 week post-lesion had no discernible influence on the elevated expression of these regeneration-associated markers. These results support an important role for endogenous BDNF in induction of the cell body response in injured sensory neurons and their intrinsic ability to extend neurites, but BDNF does not appear to be necessary for maintaining the response once it is induced. (C) 2009 Elsevier Inc. All rights reserved

Incidence, level and distribution of PACAP mRNA expression in DRG neurons after delayed antibody infusion.

<p>Representative scatterplots depicting relative changes in PACAP mRNA hybridization signal over individual neurons in relation to neuronal size after delayed control IgG (A) or anti-BDNF (B) infusions. Each dot represents hybridization signal intensity over a quantified neuron. Solid lines divide the plots into unlabeled (shaded) and labeled (≥6X background labeling) populations, and dashed lines separate moderately from highly labeled populations (≥32 X background). Numbers and percentages of DRG neurons expressing detectable PACAP mRNA after delayed intrathecal control (C) or anti-BDNF (D) antibody treatment for 3 days following 7 days of injury (n = 6, 3 animals analysed per treatment group). Note, delayed intrathecal anti-BDNF infusion did not have any significant effect on the injury-induced increase in PACAP mRNA expression after axotomy.</p