Fred De Winter

Semaphorin 3A displays a punctate distribution on the surface of neuronal cells and interacts with proteoglycans in the extracellular matrix

Secreted semaphorins are essential for neural development and continue to be expressed in subpopulations of adult neurons, where they subserve as yet unknown functions. We employed functional myc- and GFP-tagged Sema3A proteins to obtain insight in the localization of Sema3A in neuronal cells. Sema3A localized to both axons and dendrites of cortical neurons. GFP-Sema3A exhibited a characteristic punctate distribution on the surface of Neuro-2a cells, localized to migratory pathways of cultured cells, and co-localized with and induced clustering of its receptor component neuropilin-1. Treatment with excess glycosaminoglycans and chondroitinase ABC resulted in the removal of cell surface Sema3A. Heparin enhanced Sema3As binding to neuropilin-1-expressing cells and potentiated its growth cone collapsing activity. Together, these results indicate that association with proteoglycans in the extracellular matrix of neuronal cells plays an important role in the localization of the chemorepulsive guidance cue Sema3A, and that this interaction may enhance its biological activity.

The expression of the chemorepellent Semaphorin 3A is selectively induced in terminal Schwann cells of a subset of neuromuscular synapses that display limited anatomical plasticity and enhanced vulnerability in motor neuron disease

Neuromuscular synapses differ markedly in their plasticity. Motor nerve terminals innervating slow muscle fibers sprout vigorously following synaptic blockage, while those innervating fast-fatigable muscle fibers fail to exhibit any sprouting. Here, we show that the axon repellent Semaphorin 3A is differentially expressed in terminal Schwann cells (TSCs) on different populations of muscle fibers: postnatal, regenerative and paralysis induced remodeling of neuromuscular connections is accompanied by increased expression of Sema3A selectively in TSCs on fast-fatigable muscle fibers. To our knowledge, this is the first demonstration of a molecular difference between TSCs on neuromuscular junctions of different subtypes of muscle fibers. Interestingly, also in a mouse model for amyotrophic lateral sclerosis (ALS), Sema3A is expressed at NMJs of fast-fatigable muscle fibers. We propose that expression of Sema3A by TSCs not only suppresses nerve terminal plasticity at specific neuromuscular synapses, but may also contribute to their early and selective loss in the motor neuron disease ALS.

Rescue and sprouting of motoneurons following ventral root avulsion and reimplantation combined with intraspinal adeno-associated viral vector-mediated expression of glial cell line-derived neurotrophic factor or brain-derived neurotrophic factor

Following avulsion of a spinal ventral root, motoneurons that project through the avulsed root are axotomized. Avulsion between, for example, L2 and L6 leads to denervation of hind limb muscles. Reimplantation of an avulsed root directed to the motoneuron pool resulted in re-ingrowth of some motor axons. However, most motoneurons display retrograde atrophy and subsequently die. Two neurotrophic factors, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), promote the survival of motoneurons after injury. The long-term delivery of these neurotrophic factors to the motoneurons in the ventral horn of the spinal cord is problematic. One strategy to improve the outcome of the neurosurgical reinsertion of the ventral root following avulsion would involve gene transfer with adeno-associated viral (AAV) vectors encoding these neurotrophic factors near the denervated motoneuron pool. Here, we show that AAV-mediated overexpression of GDNF and BDNF in the spinal cord persisted for at least 16 weeks. At both 1 and 4 months post-lesion AAV-BDNF- and -GDNF-treated animals showed an increased survival of motoneurons, the effect being more prominent at 1 month. AAV vector-mediated overexpression of neurotrophins also promoted the formation of a network of motoneuron fibers in the ventral horn at the avulsed side, but motoneurons failed to extent axons into the reinserted L4 root towards the sciatic nerve nor to improve functional recovery of the hind limbs. This suggests that high levels of neurotrophic factors in the ventral horn promote sprouting, but prevent directional growth of axons of a higher number of surviving motoneurons into the implanted root.

ALS as a distal axonopathy: molecular mechanisms affecting neuromuscular junction stability in the presymptomatic stages of the disease

Amyotrophic Lateral Sclerosis (ALS) is being redefined as a distal axonopathy, in that many molecular changes influencing motor neuron degeneration occur at the neuromuscular junction (NMJ) at very early stages of the disease prior to symptom onset. A huge variety of genetic and environmental causes have been associated with ALS, and interestingly, although the cause of the disease can differ, both sporadic and familial forms of ALS show a remarkable similarity in terms of disease progression and clinical manifestation. The NMJ is a highly specialized synapse, allowing for controlled signaling between muscle and nerve necessary for skeletal muscle function. In this review we will evaluate the clinical, animal experimental and cellular/molecular evidence that supports the idea of ALS as a distal axonopathy. We will discuss the early molecular mechanisms that occur at the NMJ, which alter the functional abilities of the NMJ. Specifically, we focus on the role of axon guidance molecules on the stability of the cytoskeleton and how these molecules may directly influence the cells of the NMJ in a way that may initiate or facilitate the dismantling of the neuromuscular synapse in the presymptomatic stages of ALS.

Neuropilin and class 3 semaphorins in nervous system regeneration.

Injury to the mature mammalian central nervous system (CNS) is often accompanied by permanent loss of function of the damaged neural circuits. The failure of injured CNS axons to regenerate is thought to be caused, in part, by neurite outgrowth inhibitory factors expressed in and around the lesion. These include several myelin associated inhibitors, proteoglycans, and tenascin-R. Recent studies have documented the presence of class 3 semaphorins in fibroblast-like meningeal cells present in the core of the neural scar formed following CNS injury. Class 3 semaphorins display neurite growth-inhibitory effects on growing axons during embryonic development. The induction of the expression of class 3 semaphorins in the neural scar and the persistent expression of their receptors, the neuropilins and plexins, by injured CNS neurons suggest that they contribute to the regenerative failure of CNS neurons. Neuropilins are also expressed in the neural scar in a subpopulation of meningeal fibroblast and in neurons in the vicinity of the scar. Semaphorin/neuropilin signaling might therefore also be important for cell migration, angiogenis and neuronal cell death in or around neural scars. In contrast to neurons in the CNS, neuropilin/plexin positive neurons in the PNS do display long distance regeneration following injury. Injured PNS neurons do not encounter a semaphorin positive neural scar. Furthermore, Semaphorin 3A is downregulated in the regenerating spinal motor neurons themselves. This was accompanied by a transient upregulation of Semaphorin 3A in the target muscle. These observations suggest that the injury induced regulation of Semaphorin 3A in the PNS contributes to successful regeneration and target reinnervation. Future studies in genetically modified mice should provide more insight into the mechanisms by which neuropilins and semaphorins influence nervous system regeneration and degeneration.

The chemorepulsive axon guidance protein semaphorin3A is a constituent of perineuronal nets in the adult rodent brain

In the adult rodent brain, subsets of neurons are surrounded by densely organised extracellular matrix called perineuronal nets (PNNs). PNNs consist of hyaluronan, tenascin-R, chondroitin sulphate proteoglycans (CSPGs), and the link proteins Crtl1 and Bral2. PNNs restrict plasticity at the end of critical periods and can be visualised with Wisteria floribunda agglutinin (WFA). Using a number of antibodies raised against the different regions of semaphorin3A (Sema3A) we demonstrate that this secreted chemorepulsive axon guidance protein is localised to WFA-positive PNNs around inhibitory interneurons in the cortex and several other PNN-bearing neurons throughout the brain and co-localises with aggrecan, versican, phosphacan and tenascin-R. Chondroitinase ABC (ChABC) was injected in the cortex to degrade glycosaminoglycans (GAGs) from the CSPGs, abolishing WFA staining of PNNs around the injection site. Sema3A-positive nets were no longer observed in the area devoid of WFA staining. In mice lacking the link protein Crtl1 in the CNS only vestigial PNNs are present, and in these mice there were no Sema3A-positive PNN structures. A biochemical analysis shows that Sema3A protein binds with high-affinity to CS-GAGs and aggrecan and versican extracted from PNNs in the adult rat brain, and a significant proportion of Sema3A is retrieved in brain extracts that are enriched in PNN-associated GAGs. The Sema3A receptor components PlexinA1 and A4 are selectively expressed by inhibitory interneurons in the cortex that are surrounded by Sema3A positive PNNs. We conclude that the chemorepulsive axon guidance molecule Sema3A is present in PNNs of the adult rodent brain, bound to the GAGs of the CSPGs. These observations suggest a novel concept namely that chemorepulsive axon guidance molecules like Sema3A may be important functional attributes of PNNs in the adult brain.

Proteomics of the Injured Rat Sciatic Nerve Reveals Protein Expression Dynamics During Regeneration

Using proteomics, we investigated the temporal expression profiles of proteins in rat sciatic nerve after experimental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. Of the ∼1,500 protein spots resolved on each gel, 121 showed significant regulation during at least one time point. Using cluster analysis, these proteins were grouped into two expression profiles of down-regulation and four of up-regulation. These profiles mainly reflected differences in cellular origins in addition to different functional roles. Mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute-phase proteins, antioxidant proteins, and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization, and in lipid metabolism. Several proteins not previously implicated in nerve regeneration were identified, e.g. translationally controlled tumor protein, annexin A9/31, vitamin D-binding protein, α-crystallin B, α-synuclein, dimethylargininases, and reticulocalbin. Real-time PCR analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. In conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants.

Role for semaphorin III and its receptor neuropilin-1 in neuronal regeneration and scar formation?

Publisher Summary The vigorous regrowth of injured axons in the peripheral nervous system (PNS) contrasts with the observed failure of axonal regeneration in the mammalian central nervous system (CNS). This dichotomy is due to a different microenvironment at the site of the lesion and a differential intrinsic capacity of CNS and PNS neurons to activate a program of gene expression that supports regeneration. This chapter focuses on recent observations that suggest a possible role for semaphorin III and its recently identified receptor neuropilin- 1 in axonal regeneration and scar formation. First, a general introduction on inhibitory mechanisms that appear to prevent successful axonal regeneration in the CNS is provided. Subsequently, recent evidence for a role of semaphorin/neuropilin signaling in axonal regeneration in the PNS and CNS is reviewed. The recently observed downregulation of semaphorin III in motor neurons following a peripheral nerve crush and the induction of semaphorin III in CNS scar tissue argues that the regulation of the expression of some members of the semaphorin gene family may contribute significantly to the failure or success of the neuroregeneration process.

Expression of a Mutant SEMA3A Protein with Diminished Signalling Capacity Does Not Alter ALS-Related Motor Decline, or Confer Changes in NMJ Plasticity after BotoxA-Induced Paralysis of Male Gastrocnemic Muscle

Terminal Schwann cells (TSCs) are specialized cells that envelop the motor nerve terminal, and play a role in the maintenance and regeneration of neuromuscular junctions (NMJs). The chemorepulsive protein semaphorin 3A (SEMA3A) is selectively up-regulated in TSCs on fast-fatigable muscle fibers following experimental denervation of the muscle (BotoxA-induced paralysis or crush injury to the sciatic nerve) or in the motor neuron disease amyotrophic lateral sclerosis (ALS). Re-expression of SEMA3A in this subset of TSCs is thought to play a role in the selective plasticity of nerve terminals as observed in ALS and following BotoxA-induced paralysis. Using a mouse model expressing a mutant SEMA3A with diminished signaling capacity, we studied the influence of SEMA3A signaling at the NMJ with two denervation paradigms; a motor neuron disease model (the G93A-hSOD1 ALS mouse line) and an injury model (BotoxA-induced paralysis). ALS mice that either expressed 1 or 2 mutant SEMA3A alleles demonstrated no difference in ALS-induced decline in motor behavior. We also investigated the effects of BotoxA-induced paralysis on the sprouting capacity of NMJs in the K108N-SEMA3A mutant mouse, and observed no change in the differential neuronal plasticity found at NMJs on fast-fatigable or slow muscle fibers due to the presence of the SEMA3A mutant protein. Our data may be explained by the residual repulsive activity of the mutant SEMA3A, or it may imply that SEMA3A alone is not a key component of the molecular signature affecting NMJ plasticity in ALS or BotoxA-induced paralysis. Interestingly, we did observe a sex difference in motor neuron sprouting behavior after BotoxA-induced paralysis in WT mice which we speculate may be an important factor in the sex dimorphic differences seen in ALS.