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Sensory axons cut within the spinal cord do not regenerate spontaneously. This failure is due in part to absence of an appropriate adhesion molecule on sensory axons, able to interact with the CNS environment. Previous work has shown that transduction of sensory neurons with α9 integrin, which combines with endogenous β1 as α9β1 together with the integrin activator kindlin-1 enables regeneration of sensory axons from a dorsal root crush to the brainstem. The integrin provides an adhesion molecule that recognizes Tenascin-C (which is upregulated in damaged spinal cord) and signals to upregulate the regeneration-associated gene programme. Regeneration from sensory neurons transduced with α9 integrin and kindlin-1 was examined after C4 and after T10 dorsal column lesions with C6,7 and L4,5 sensory ganglia injected with AAV1 vectors. In α9 integrin and kindlin-1 treated animals sensory axons regenerated through tenascin-C-expressing connective tissue strands and bridges across the lesion then re-entered CNS tissue. Many axons regenerated rostrally to the level of the medulla but were excluded from the sensory nuclei. Regenerated axons were seen particularly at the white matter/grey matter boundary in the dorsal cord. Many branches into the dorsal horn were seen tipped with synaptic swellings. Stimulation of the sciatic nerve led to many neurons rostral to the injury being activated to express cFos. Behavioural recovery was seen in heat and pressure sensation and tape removal. Treatment of sensory neurons with α9 integrin and kindlin-1 therefore enables partial reconstruction of the spinal sensory pathway.Significance StatementFull recovery from spinal cord injury can only occur if the axon pathways that connect the brain and spinal cord regenerate to restore motor and sensory connections. Neither sensory nor motor axons in mammals can regenerate spontaneously. A critical factor missing in adult neurons is an adhesion molecule that can enable them to interact with the environment of the damaged spinal cord. In this study in rats an integrin adhesion molecule together with its activator was expressed in sensory neurons using a viral vector. Possession of this adhesion molecule enabled sensory axons to regenerate through the spinal cord injury and all the way back to the brainstem, restoring the sensory pathway. The animal regained touch sensation, enabling sensory behaviours.

Funder: This research was funded by a Nathalie Rose Barr award (NRB110) from the International Spinal Research Trust, and support from Medical Research Council (MR/R004544/1 & MR/R004463/1), NWO (013-16-002), Czech Ministry of Education (CZ.02.1.01/0.0./0.0/15_003/0000419), ERA-NET NEURON AxonRepair, Christopher and Dana Reeve Foundation, International Foundation for Research in Paraplegia, Hersenstichting Nederland., Adeno-associated viral vectors are widely used as vehicles for gene transfer to the nervous system. The promoter and viral vector serotype are two key factors that determine the expression dynamics of the transgene. A previous comparative study has demonstrated that AAV1 displays efficient transduction of layer V corticospinal neurons, but the optimal promoter for transgene expression in corticospinal neurons has not been determined yet. In this paper, we report a side-by-side comparison between four commonly used promoters: the short CMV early enhancer/chicken β actin (sCAG), human cytomegalovirus (hCMV), mouse phosphoglycerate kinase (mPGK) and human synapsin (hSYN) promoter. Reporter constructs with each of these promoters were packaged in AAV1, and were injected in the sensorimotor cortex of rats and mice in order to transduce the corticospinal tract. Transgene expression levels and the cellular transduction profile were examined after 6 weeks. The AAV1 vectors harbouring the hCMV and sCAG promoters resulted in transgene expression in neurons, astrocytes and oligodendrocytes. The mPGK and hSYN promoters directed the strongest transgene expression. The mPGK promoter did drive expression in cortical neurons and oligodendrocytes, while transduction with AAV harbouring the hSYN promoter resulted in neuron-specific expression, including perineuronal net expressing interneurons and layer V corticospinal neurons. This promoter comparison study contributes to improve transgene delivery into the brain and spinal cord. The optimized transduction of the corticospinal tract will be beneficial for spinal cord injury research.

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.

BackgroundDysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer’s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain.MethodsA plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTLwith amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTLprotein was employed to study interaction of CERTLwith amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTLwas overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay.ResultsHere, we report that CERTLbinds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTLin vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype.ConclusionOur results demonstrate a crucial role of CERTLin regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.

Perineuronal nets (PNNs) are condensed structures in the extracellular matrix that mainly surround GABA-ergic parvalbumin-positive interneurons in the adult brain. Previous studies revealed a parallel between PNN formation and the closure of the critical period. Moreover, ocular dominance plasticity is enhanced in response to PNN manipulations in adult animals. However, the mechanisms through which perineuronal nets modulate plasticity are still poorly understood. Recent work indicated that perineuronal nets may convey molecular signals by binding and storing proteins with important roles in cellular communication. Here we report that semaphorin3A (Sema3A), a chemorepulsive axon guidance cue known to bind to important perineuronal net components, is necessary to dampen ocular dominance plasticity in adult rats. First, we showed that the accumulation of Sema3A in PNNs in the visual cortex correlates with critical period closure, following the same time course of perineuronal nets maturation. Second, the accumulation of Sema3A in perineuronal nets was significantly reduced by rearing animals in the dark in the absence of any visual experience. Finally, we developed and characterized a tool to interfere with Sema3A signaling by means of AAV-mediated expression of receptor bodies, soluble proteins formed by the extracellular domain of the endogenous Sema3A receptor (neuropilin1) fused to a human IgG Fc fragment. By using this tool to antagonize Sema3A signaling in the adult rat visual cortex, we found that the specific inhibition of Sema3A promoted ocular dominance plasticity. Thus, Sema3A accumulates in perineuronal nets in an experience-dependent manner and its presence in the mature visual cortex inhibits plasticity.

Mesenchymal stromal cells (MSC) are used for cell therapy for spinal cord injury (SCI) because of their ability to support tissue repair by paracrine signaling. Preclinical and clinical research testing MSC transplants for SCI have revealed limited success, which warrants the exploration of strategies to improve their therapeutic efficacy. MSC are sensitive to the microenvironment and their secretome can be altered in vitro by exposure to different culture media. Priming MSC with inflammatory stimuli increases the expression and secretion of reparative molecules. We studied the effect of macrophage-derived inflammation priming on MSC transplants and of primed MSC (pMSC) acute transplants (3 days) on spinal cord repair using an adult rat model of moderate–severe contusive SCI. We found a decrease in long-term survival of pMSC transplants compared with unprimed MSC transplants. With a pMSC transplant, we found significantly more anti-inflammatory macrophages in the contusion at 4 weeks post transplantation (wpt). Blood vessel presence and maturation in the contusion at 1 wpt was similar in rats that received pMSC or untreated MSC. Nervous tissue sparing and functional recovery were similar across groups. Our results indicate that macrophage-derived inflammation priming does not increase the overall therapeutic potential of an MSC transplant in the adult rat contused spinal cord.

A human neuroma-in continuity (NIC), formed following a peripheral nerve lesion, impedes functional recovery. The molecular mechanisms that underlie the formation of a NIC are poorly understood. Here we show that the expression of multiple genes of the Wnt family, including Wnt5a, is changed in NIC tissue from patients that underwent reconstructive surgery. The role of Wnt ligands in NIC pathology and nerve regeneration is of interest because Wnt ligands are implicated in tissue regeneration, fibrosis, axon repulsion and guidance. The observations in NIC prompted us to investigate the expression of Wnt ligands in the injured rat sciatic nerve and in the dorsal root ganglia (DRG). In the injured nerve, four gene clusters were identified with temporal expression profiles corresponding to particular phases of the regeneration process. In the DRG up- and down regulation of certain Wnt receptors suggests that nerve injury has an impact on the responsiveness of injured sensory neurons to Wnt ligands in the nerve. Immunohistochemistry showed that Schwann cells in the NIC and in the injured nerve are the source of Wnt5a, whereas the Wnt5a receptor Ryk is expressed by axons traversing the NIC. Taken together, these observations suggest a central role for Wnt signalling in peripheral nerve regeneration.

During restricted time windows of postnatal life, called critical periods, neural circuits are highly plastic and are shaped by environmental stimuli. In several mammalian brain areas, from the cerebral cortex to the hippocampus and amygdala, the closure of the critical period is dependent on the formation of perineuronal nets. Perineuronal nets are a condensed form of an extracellular matrix, which surrounds the soma and proximal dendrites of subsets of neurons, enwrapping synaptic terminals. Experimentally disrupting perineuronal nets in adult animals induces the reactivation of critical period plasticity, pointing to a role of the perineuronal net as a molecular brake on plasticity as the critical period closes. Interestingly, in the adult brain, the expression of perineuronal nets is remarkably dynamic, changing its plasticity-associated conditions, including memory processes. In this review, we aimed to address how perineuronal nets contribute to the maturation of brain circuits and the regulation of adult brain plasticity and memory processes in physiological and pathological conditions.

Pre-clinical and clinical studies revealed that mesenchymal stromal cell (MSC) transplants elicit tissue repair. Conditioning MSC prior to transplantation may boost their ability to support repair. We investigated macrophage-derived inflammation as a means to condition MSC by comprehensively analyzing their transcriptome and secretome. Conditioning MSC with macrophage-derived inflammation resulted in 3208 differentially expressed genes, which were annotated with significantly enriched GO terms for 1085 biological processes, 85 cellular components, and 79 molecular functions. Inflammation-mediated conditioning increased the secretion of growth factors that are key for tissue repair, including vascular endothelial growth factor, hepatocyte growth factor, nerve growth factor and glial-derived neurotrophic factor. Furthermore, we found that inflammation-mediated conditioning induces transcriptomic changes that challenge the viability and mobility of MSC. Our data support the notion that macrophage-derived inflammation stimulates MSC to augment their paracrine repair-supporting activity. The results suggest that inflammatory pre-conditioning enhances the therapeutic potential of MSC transplants.

Funder: Alzheimers research UK, Perineuronal nets (PNNs) are chondroitin sulphate proteoglycan-containing structures on the neuronal surface that have been implicated in the control of neuroplasticity and memory. Age-related reduction of chondroitin 6-sulphates (C6S) leads to PNNs becoming more inhibitory. Here, we investigated whether manipulation of the chondroitin sulphate (CS) composition of the PNNs could restore neuroplasticity and alleviate memory deficits in aged mice. We first confirmed that aged mice (20-months) showed memory and plasticity deficits. They were able to retain or regain their cognitive ability when CSs were digested or PNNs were attenuated. We then explored the role of C6S in memory and neuroplasticity. Transgenic deletion of chondroitin 6-sulfotransferase (chst3) led to a reduction of permissive C6S, simulating aged brains. These animals showed very early memory loss at 11 weeks old. Importantly, restoring C6S levels in aged animals rescued the memory deficits and restored cortical long-term potentiation, suggesting a strategy to improve age-related memory impairment.

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

Background Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer’s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. Methods A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. Results Here, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype. Conclusion Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s13195-021-00780-0.

Background: Deregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer’s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers, crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain.Methods: The plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno associated virus (AAV) in a familial mouse model of familial AD (5xFAD). Ten weeks after transduction animal were challenged with behavior tests for memory, anxiety and locomotion. At week twelve brains were investigated for sphingolipid levels by mass spectrometry, plaques and neuroinflammation by immunohistochemistry, gene expression and/or immunoassay.Results: Here, we report that CERTL, binds to APP, modifies Aβ aggregation and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male transgenic mice, modelling familial AD (5xFAD). CERTL in vivo over-expression has a mild effect on animal locomotion and decreases Aβ formation and modulates microglia by decreasing their pro-inflammatory phenotype.Conclusion: Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.

Significance Understanding mechanisms underlying learning and memory is crucial in view of tackling cognitive decline occurring during aging or following neurological disorders. The cerebellum offers an ideal system to achieve this goal because of the well-characterized forms of motor learning that it controls. It is so far unclear whether cerebellar memory processes depend on changes in perineuronal nets (PNNs). PNNs are assemblies of extracellular matrix molecules around neurons, which regulate neural plasticity. Here we demonstrate that during eyeblink conditioning (EBC), which is a form of cerebellar motor learning, PNNs in the mouse deep cerebellar nuclei are dynamically modulated, and PNN changes are essential for the formation and storage of EBC memories. Together, these results unveil an important mechanism controlling motor associative memories., Perineuronal nets (PNNs) are assemblies of extracellular matrix molecules, which surround the cell body and dendrites of many types of neuron and regulate neural plasticity. PNNs are prominently expressed around neurons of the deep cerebellar nuclei (DCN), but their role in adult cerebellar plasticity and behavior is far from clear. Here we show that PNNs in the mouse DCN are diminished during eyeblink conditioning (EBC), a form of associative motor learning that depends on DCN plasticity. When memories are fully acquired, PNNs are restored. Enzymatic digestion of PNNs in the DCN improves EBC learning, but intact PNNs are necessary for memory retention. At the structural level, PNN removal induces significant synaptic rearrangements in vivo, resulting in increased inhibition of DCN baseline activity in awake behaving mice. Together, these results demonstrate that PNNs are critical players in the regulation of cerebellar circuitry and function.

Ventral root avulsion leads to severe motoneuron degeneration and prolonged distal nerve denervation. After a critical period, a state of chronic denervation develops as repair Schwann cells lose their pro-regenerative properties and inhibitory factors such as CSPGs accumulate in the denervated nerve. In rats with ventral root avulsion injuries, we combined timed GDNF gene therapy delivered to the proximal nerve roots with the digestion of inhibitory CSPGs in the distal denervated nerve using sustained lentiviral-mediated chondroitinase ABC (ChABC) enzyme expression. Following reimplantation of lumbar ventral roots, timed GDNF-gene therapy enhanced motoneuron survival up to 45 weeks and improved axonal outgrowth, electrophysiological recovery, and muscle reinnervation. Despite a timed GDNF expression period, a subset of animals displayed axonal coils. Lentiviral delivery of ChABC enabled digestion of inhibitory CSPGs for up to 45 weeks in the chronically denervated nerve. ChABC gene therapy alone did not enhance motoneuron survival, but led to improved muscle reinnervation and modest electrophysiological recovery during later stages of the regeneration process. Combining GDNF treatment with digestion of inhibitory CSPGs did not have a significant synergistic effect. This study suggests a delicate balance exists between treatment duration and concentration in order to achieve therapeutic effects.

Schwann cell grafts support axonal growth following spinal cord injury, but a boundary forms between the implanted cells and host astrocytes. Axons are reluctant to exit the graft tissue in large part due to the surrounding inhibitory environment containing chondroitin sulphate proteoglycans (CSPGs). We use a lentiviral chondroitinase ABC, capable of being secreted from mammalian cells (mChABC), to examine the repercussions of CSPG digestion upon Schwann cell behaviour in vitro. We show that mChABC transduced Schwann cells robustly secrete substantial quantities of the enzyme causing large-scale CSPG digestion, facilitating the migration and adhesion of Schwann cells on inhibitory aggrecan and astrocytic substrates. Importantly, we show that secretion of the engineered enzyme can aid the intermingling of cells at the Schwann cell-astrocyte boundary, enabling growth of neurites over the putative graft/host interface. These data were echoed in vivo. This study demonstrates the profound effect of the enzyme on cellular motility, growth and migration. This provides a cellular mechanism for mChABC induced functional and behavioural recovery shown in in vivo studies. Importantly, we provide in vitro evidence that mChABC gene therapy is equally or more effective at producing these effects as a one-time application of commercially available ChABC.

Memory loss is a usual consequence of ageing and aged mice show progressive deficits in memory tasks. In aged brains, perineuronal nets (PNNs), which are implicated in plasticity and memory, become inhibitory due to decreased 6-sulphation of their glycan chains (C6S). Removal of PNNs or digestion of their glycosaminoglycans rescued age-related memory loss. Premature reduction of permissive C6S by transgenic deletion of chondroitin 6-sulfotransferase led to very early memory loss. However, restoring C6S levels in aged animals by AAV delivery or transgenic expression of 6-sulfotransferase restored memory. Low C6S levels caused loss of cortical long-term potentiation, which was restored by AAV-mediated 6-sulfotransferase delivery. The study shows that loss of C6S in the aged brain leads to declining memory and cognition. Age-related memory impairment was restored by C6S replacement or other interventions targeting perineuronal nets

A spinal root avulsion is the most severe proximal peripheral nerve lesion possible. Avulsion of ventral root filaments disconnects spinal motoneurons from their target muscles, resulting in complete paralysis. In patients that undergo brachial plexus nerve repair, axonal regeneration is a slow process. It takes months or even years to bridge the distance from the lesion site to the distal targets located in the forearm. Following ventral root avulsion, without additional pharmacological or surgical treatments, progressive death of motoneurons occurs within 2 weeks (Koliatsos et al., 1994). Reimplantation of the avulsed ventral root or peripheral nerve graft can act as a conduit for regenerating axons and increases motoneuron survival (Chai et al., 2000). However, this beneficial effect is transient. Combined with protracted and poor long-distance axonal regeneration, this results in permanent function loss. To overcome motoneuron death and improve functional recovery, several promising intervention strategies are being developed. Here, we focus on GDNF gene-therapy. We first introduce the experimental ventral root avulsion model and discuss its value as a proxy to study clinical neurotmetic nerve lesions. Second, we discuss our recent studies showing that GDNF gene-therapy is a powerful strategy to promote long-term motoneuron survival and improve function when target muscle reinnervation occurs within a critical post-lesion period. Based upon these observations, we discuss the influence of timing of the intervention, and of the duration, concentration and location of GDNF delivery on functional outcome. Finally, we provide a perspective on future research directions to realize functional recovery using gene therapy.

Large peripheral nerve (PN) defects require bridging substrates to restore tissue continuity and permit the regrowth of sensory and motor axons. We previously showed that cell-free PN segments repopulated ex vivo with Schwann cells (SCs) transduced with lentiviral vectors (LV) to express different growth factors (BDNF, CNTF or NT-3) supported the regeneration of axons across a 1 cm peroneal nerve defect (Godinho et al., 2013). Graft morphology, the number of regrown axons, the ratio of myelinated to unmyelinated axons, and hindlimb locomotor function differed depending on the growth factor engineered into SCs. Here we extend these observations, adding more LVs (expressing GDNF or NGF) and characterising regenerating sensory and motor neurons after injection of the retrograde tracer Fluorogold (FG) into peroneal nerve distal to grafts, 10 weeks after surgery. Counts were also made in rats with intact nerves and in animals receiving autografts, acellular grafts, or grafts containing LV-GFP transduced SCs. Counts and analysis of FG positive (+) DRG neurons were made from lumbar (L5) ganglia. Graft groups contained fewer labeled sensory neurons than non-operated controls, but this decrease was only significant in the LV-GDNF group. These grafts had a complex fascicular morphology that may have resulted in axon trapping. The proportion of FG+ sensory neurons immunopositive for calcitonin-gene related peptide (CGRP) varied between groups, there being a significantly higher percentage in autografts and most neurotrophic factor groups compared to the LV-CNTF, LV-GFP and acellular groups. Furthermore, the proportion of regenerating isolectin B4+ neurons was significantly greater in the LV-NT-3 group compared to other groups, including autografts and non-lesion controls. Immunohistochemical analysis of longitudinal graft sections revealed that all grafts contained a reduced number of choline acetyltransferase (ChAT) positive axons, but this decrease was significant only in the GDNF and NT-3 graft groups. We also assessed the number and phenotype of regrowing lumbar FG+ motor neurons in non-lesioned animals, and in rats with autografts, acellular grafts, or in grafts containing SCs expressing GFP, CNTF, NGF or NT-3. The overall number of FG+ motor neurons per section was similar in all groups; however in tissue immunostained for NeuN (expressed in α- but not γ-motor neurons) the proportion of NeuN negative FG+ neurons ranged from about 40–50% in all groups except the NT-3 group, where the percentage was 82%, significantly more than the SC-GFP group. Immunostaining for the vesicular glutamate transporter VGLUT-1 revealed occasional proprioceptive terminals in ‘contact’ with regenerating FG+ α-motor neurons in PN grafted animals, the acellular group having the lowest counts. In sum, while all graft types supported sensory and motor axon regrowth, there appeared to be axon trapping in SC-GDNF grafts, and data from the SC-NT-3 group revealed greater regeneration of sensory CGRP+ and IB4+ neurons, preferential regeneration of γ-motor neurons and perhaps partial restoration of monosynaptic sensorimotor relays.

The failure of axons to regenerate in the damaged mammalian CNS is the main impediment to functional recovery. There are many molecules and structures in the environment of the injured nervous system that can inhibit regeneration, but even when these are removed or replaced with a permissive environment, most CNS neurons exhibit little regeneration of their axons. This contrasts with the extensive and vigorous axon growth that may occur when embryonic neurons are transplanted into the adult CNS. In the peripheral nervous system, the axons usually respond to axotomy with a vigorous regenerative response accompanied by a regenerative program of gene expression, usually referred to as the regeneration-associated gene (RAG) program. These different responses to axotomy in the mature and immature CNS and the PNS lead to the concept of the intrinsic regenerative response of axons. Analysis of the many mechanisms and issues that affect the intrinsic regenerative response is the topic of this special issue of Developmental Neurobiology. The review articles highlight the control of expression of growth and regeneration-associated genes, emphasizing the role of epigenetic mechanisms. The reviews also discuss changes within axons that lead to the developmental loss of regenerative ability. This is caused by changes in axonal transport and trafficking, in the cytoskeleton and in signaling pathways.

Spinal cord injury results in destructive events that lead to tissue loss and functional impairments. A hallmark of spinal cord injury is the robust and persistent presence of inflammatory macrophages. Mesenchymal stem cells (MSCs) are known to benefit repair of the damaged spinal cord often associated with improved functional recovery. Transplanted MSCs immediately encounter the abundance of inflammatory macrophages in the injury site. It is known that MSCs interact closely and reciprocally with macrophages during tissue healing. Here, we will review the roles of (transplanted) MSCs and macrophages in spinal cord injury and repair. Molecular interactions between MSCs and macrophages and the deficiencies in our knowledge about the underlying mechanisms will be reviewed. We will discuss possible ways to benefit from the MSC-macrophage choreography for developing repair strategies for the spinal cord. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-018-0629-0) contains supplementary material, which is available to authorized users.

The neuron‐intrinsic response to axonal injury differs markedly between neurons of the peripheral and central nervous system. Following a peripheral lesion, a robust axonal growth program is initiated, whereas neurons of the central nervous system do not mount an effective regenerative response. Increasing the neuron‐intrinsic regenerative response would therefore be one way to promote axonal regeneration in the injured central nervous system. The large‐diameter sensory neurons located in the dorsal root ganglia are pseudo‐unipolar neurons that project one axon branch into the spinal cord, and, via the dorsal column to the brain stem, and a peripheral process to the muscles and skin. Dorsal root ganglion neurons are ideally suited to study the neuron‐intrinsic injury response because they exhibit a successful growth response following peripheral axotomy, while they fail to do so after a lesion of the central branch in the dorsal column. The dorsal column injury model allows the neuron‐intrinsic regeneration response to be studied in the context of a spinal cord injury. Here we will discuss the advantages and disadvantages of this model. We describe the surgical methods used to implement a lesion of the ascending fibers, the anatomy of the sensory afferent pathways and anatomical, electrophysiological, and behavioral techniques to quantify regeneration and functional recovery. Subsequently we review the results of experimental interventions in the dorsal column lesion model, with an emphasis on the molecular mechanisms that govern the neuron‐intrinsic injury response and manipulations of these after central axotomy. Finally, we highlight a number of recent advances that will have an impact on the design of future studies in this spinal cord injury model, including the continued development of adeno‐associated viral vectors likely to improve the genetic manipulation of dorsal root ganglion neurons and the use of tissue clearing techniques enabling 3D reconstruction of regenerating axon tracts. © 2018 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 00: 000–000, 2018

Repulsive guidance molecule member a (RGMa) is a membrane-associated or released guidance molecule that is involved in axon guidance, cell patterning, and cell survival. In our previous work, we showed that RGMa is significantly upregulated in the substantia nigra of patients with Parkinson's disease. Here we demonstrate the expression of RGMa in midbrain human dopaminergic (DA) neurons. To investigate whether RGMa might model aspects of the neuropathology of Parkinson's disease in mouse, we targeted RGMa to adult midbrain dopaminergic neurons using adeno-associated viral vectors. Overexpression of RGMa resulted in a progressive movement disorder, including motor coordination and imbalance, which is typical for a loss of DA release in the striatum. In line with this, RGMa induced selective degeneration of dopaminergic neurons in the substantia nigra (SN) and affected the integrity of the nigrostriatal system. The degeneration of dopaminergic neurons was accompanied by a strong microglia and astrocyte activation. The behavioral, molecular, and anatomical changes induced by RGMa in mice are remarkably similar to the clinical and neuropathological hallmarks of Parkinson's disease. Our data indicate that dysregulation of RGMa plays an important role in the pathology of Parkinson's disease, and antibody-mediated functional interference with RGMa may be a disease modifying treatment option.SIGNIFICANCE STATEMENTParkinson's disease (PD) is a neurodegenerative disease characterized by severe motor dysfunction due to progressive degeneration of mesencephalic dopaminergic (DA) neurons in the substantia nigra. To date, there is no regenerative treatment available. We previously showed that repulsive guidance molecule member a (RGMa) is upregulated in the substantia nigra of PD patients. Adeno-associated virus-mediated targeting of RGMa to mouse DA neurons showed that overexpression of this repulsive axon guidance and cell patterning cue models the behavioral and neuropathological characteristics of PD in a remarkable way. These findings have implications for therapy development as interfering with the function of this specific axon guidance cue may be beneficial to the survival of DA neurons.

Avulsion of spinal nerve roots is a severe proximal peripheral nerve lesion. Despite neurosurgical repair, recovery of function in human patients is disappointing, because spinal motor neurons degenerate progressively, axons grow slowly and the distal Schwann cells which are instrumental to supporting axon extension lose their pro-regenerative properties. We have recently shown that timed GDNF gene therapy (dox-i-GDNF) in a lumbar plexus injury model promotes axon regeneration and improves electrophysiological recovery but fails to stimulate voluntary hind paw function. Here we report that dox-i-GDNF treatment following avulsion and re-implantation of cervical ventral roots leads to sustained motoneuron survival and recovery of voluntary function. These improvements were associated with a twofold increase in motor axon regeneration and enhanced reinnervation of the hand musculature. In this cervical model the distal hand muscles are located 6,5 cm from the reimplantation site, whereas following a lumber lesion this distance is twice as long. Since the first signs of muscle reinnervation are observed 6 weeks after the lesion, this suggests that regenerating axons reached the hand musculature before a critical state of chronic denervation has developed. These results demonstrate that the beneficial effects of timed GDNF-gene therapy are more robust following spinal nerve avulsion lesions that allow reinnervation of target muscles within a relatively short time window after the lesion. This study is an important step in demonstrating the potential of timed GDNF-gene therapy to enhance axon regeneration after neurosurgical repair of a severe proximal nerve lesion.