Marc J. Ruitenberg

Expression of the gene encoding the chemorepellent semaphorin III is induced in the fibroblast component of neural scar tissue formed following injuries of adult but not neonatal CNS

This study evaluates the expression of the chemorepellent semaphorin III (D)/collapsin-1 (sema III) following lesions to the rat CNS. Scar tissue, formed after penetrating injuries to the lateral olfactory tract (LOT), cortex, perforant pathway, and spinal cord, contained numerous spindle-shaped cells expressing high levels of sema III mRNA. The properties of these cells were investigated in detail in the lesioned LOT. Most sema III mRNA-positive cells were located in the core of the scar and expressed proteins characteristic for fibroblast-like cells. Neuropilin-1, a sema III receptor, was expressed in injured neurons with projections to the lesion site, in a subpopulation of scar-associated cells and in blood vessels around the scar. In contrast to lesions made in the mature CNS, LOT transection in neonates did not induce sema III mRNA expression within cells in the lesion and was followed by vigorous axonal regeneration. The concomitant expression of sema III and its receptor neuropilin-1 in the scar suggests that sema III/neuropilin-1-mediated mechanisms are involved in CNS scar formation. The expression of the secreted chemorepellent sema III following CNS injury provides the first evidence that chemorepulsive semaphorins may contribute to the inhibitory effects exerted by scars on the outgrowth of injured CNS neurites. The vigorous regrowth of injured axons in the absence of sema III following early neonatal lesions is consistent with this notion. The inactivation of sema III in scar tissue by either antibody perturbation or by genetic or pharmacological intervention could be a powerful means to promote long-distance regeneration in the adult CNS.

Injury-induced class 3 semaphorin expression in the rat spinal cord

In this study we evaluate the expression of all members of the class 3 semaphorins and their receptor components following complete transection and contusion lesions of the adult rat spinal cord. Following both types of lesions the expression of all class 3 semaphorins is induced in fibroblast in the neural scar. The distribution of semaphorin-positive fibroblasts differs markedly in scars formed after transection or contusion lesion. In contusion lesions semaphorin expression is restricted to fibroblasts of the meningeal sheet surrounding the lesion, while after transection semaphorin-positive fibroblast penetrate deep into the center of the lesion. Two major descending spinal cord motor pathways, the cortico- and rubrospinal tract, continue to express receptor components for class 3 semaphorins following injury, rendering them potentially sensitive to scar-derived semaphorins. In line with this we observed that most descending spinal cord fibers were not able to penetrate the semaphorin positive portion of the neural scar formed at the lesion site. These results suggest that the full range of secreted semaphorins contributes to the inhibitory nature of the neural scar and thereby may inhibit successful regeneration in the injured spinal cord. Future studies will focus on the neutralization of class 3 semaphorins, in order to reveal whether this creates a more permissive environment for regeneration of injured spinal cord axons.

Viral vector-mediated gene expression in olfactory ensheathing glia implants in the lesioned rat spinal cord

Implantation of olfactory ensheathing glia (OEG) is a promising strategy to augment long-distance regeneration in the injured spinal cord. In this study, implantation of OEG following unilateral hemisection of the dorsal cervical spinal cord was combined with ex vivo gene transfer techniques. We report, to our knowledge for the first time, that purified cultures of primary OEG are capable of expressing a foreign gene following adenoviral (AdV) and lentiviral (LV) vector-mediated gene transfer. OEG implants subjected to AdV vector-mediated gene transfer expressed high levels of transgenic protein in both intact and lesioned spinal cord at 7 days after implantation. However, the levels of transgene expression gradually declined between 7 and 30 days after implantation in lesioned spinal cord. Infection with LV vectors resulted in stable transduction of primary OEG cultures and transgene expression persisted for at least 4 months after implantation. Genetic engineering of OEG opens the possibility of expressing additional neurotrophic genes and create optimal ‘bridging’ substrates to support spinal axon regeneration. Furthermore, stable transduction of OEG allows us to reliably study the behaviour of implanted cells and to obtain better understanding of their regeneration supporting properties.

Microglia Modulate Hippocampal Neural Precursor Activity in Response to Exercise and Aging

Exercise has been shown to positively augment adult hippocampal neurogenesis; however, the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here, we used transgenic Csf1r-GFP mice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely, microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX3CL1, a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX3CL1 receptor, CX3CR1, but not control IgG, dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX3CL1 was observed following running, reduced levels of this chemokine were found in the aged brain. Lower levels of CX3CL1 with advancing age correlated with the natural decline in neural precursor cell activity, a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus, and that signaling through the CX3CL1–CX3CR1 axis critically contributes toward this process.

Amelioration of chronic neuropathic pain after partial nerve injury by adeno-associated viral (AAV) vector-mediated over-expression of BDNF in the rat spinal cord

Changing the levels of neurotrophins in the spinal cord micro-environment after nervous system injury has been proposed to recover normal function, such that behavioral response to peripheral stimuli does not lead to chronic pain. We have investigated the effects of recombinant adeno-associated viral (rAAV)-mediated over-expression of brain-derived neurotrophic factor (BDNF) in the spinal cord on chronic neuropathic pain after unilateral chronic constriction injury (CCI) of the sciatic nerve. The rAAV-BDNF vector was injected into the dorsal horn at the thirteenth thoracic spinal cord vertebra (L1 level) 1 week after CCI. Allodynia and hyperalgesia induced by CCI in the hindpaws were permanently reversed, beginning 1 week after vector injection, compared with a similar injection of a control rAAV-GFP vector (green fluorescent protein) or saline. In situ hybridization for BDNF demonstrated that both dorsal and ventral lumbar spinal neurons contained an intense signal for BDNF mRNA, at 1 to 8 weeks after vector injection. There was no similar BDNF mRNA over-expression associated with either injections of saline or rAAV-GFP. These data suggest that chronic neuropathic pain is sensitive to early spinal BDNF levels after partial nerve injury and that rAAV-mediated gene transfer could potentially be used to reverse chronic pain after nervous system injuries in humans.

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.

Adeno-associated viral vector-mediated gene transfer of brain-derived neurotrophic factor reverses atrophy of rubrospinal neurons following both acute and chronic spinal cord injury.

Rubrospinal neurons (RSNs) undergo marked atrophy after cervical axotomy. This progressive atrophy may impair the regenerative capacity of RSNs in response to repair strategies that are targeted to promote rubrospinal tract regeneration. Here, we investigated whether we could achieve long-term rescue of RSNs from lesion-induced atrophy by adeno-associated viral (AAV) vector-mediated gene transfer of brain-derived neurotrophic factor (BDNF). We show for the first time that AAV vectors can be used for the persistent transduction of highly atrophic neurons in the red nucleus (RN) for up to 18 months after injury. Furthermore, BDNF gene transfer into the RN following spinal axotomy resulted in counteraction of atrophy in both the acute and chronic stage after injury. These novel findings demonstrate that a gene therapeutic approach can be used to reverse atrophy of lesioned CNS neurons for an extended period of time.

Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used as tools for gene therapy, and clinical trials have begun in patients with genetically linked retinal disorders. Intravitreal injection is optimal for the transduction of retinal ganglion cells (RGCs), although complete selectivity has not been achieved. There may also be advantages in using intravitreal approaches for the transduction of photoreceptors. Here we compared the cellular tropism and transduction efficiency of rAAV2/1, -2/2, -2/3, -2/4, -2/5, -2/6 and -2/8 in adult rat retina after intravitreal injection. Each vector encoded green fluorescent protein (GFP), and the number, laminar distribution and morphology of transduced GFP+ cells were determined using fluorescent microscopy. Assessment of transduced cell phenotype was based on cell morphology and immunohistochemistry. rAAV2/2 and rAAV2/6 transduced the greatest number of cells, whereas rAAV2/5 and rAAV2/8 were least efficient. Most vectors primarily transduced RGCs; however, rAAV2/6 had a more diverse tropism profile, with 46% identified as amacrine or bipolar cells, 23% as RGCs and 22% as Müller cells. Müller cells were also frequently transduced by rAAV2/4. The highest photoreceptor transduction was seen after intravitreal rAAV2/3 injection. These data facilitate the design and selection of rAAV vectors to target specific retinal cells, potentially leading to an improved gene therapy for various human retinal pathologies.

Complement activation in the injured central nervous system: another dual-edged sword?

The complement system, a major component of the innate immune system, is becoming increasingly recognised as a key participant in physiology and disease. The awareness that immunological mediators support various aspects of both normal central nervous system (CNS) function and pathology has led to a renaissance of complement research in neuroscience. Various studies have revealed particularly novel findings on the wide-ranging involvement of complement in neural development, synapse elimination and maturation of neural networks, as well as the progression of pathology in a range of chronic neurodegenerative disorders, and more recently, neurotraumatic events, where rapid disruption of neuronal homeostasis potently triggers complement activation. The purpose of this review is to summarise recent findings on complement activation and acquired brain or spinal cord injury, i.e. ischaemic-reperfusion injury or stroke, traumatic brain injury (TBI) and spinal cord injury (SCI), highlighting the potential for complement-targeted therapeutics to alleviate the devastating consequences of these neurological conditions.

Immature Doublecortin-Positive Hippocampal Neurons Are Important for Learning But Not for Remembering

It is now widely accepted that hippocampal neurogenesis underpins critical cognitive functions, such as learning and memory. To assess the behavioral importance of adult-born neurons, we developed a novel knock-in mouse model that allowed us to specifically and reversibly ablate hippocampal neurons at an immature stage. In these mice, the diphtheria toxin receptor (DTR) is expressed under control of the doublecortin (DCX) promoter, which allows for specific ablation of immature DCX-expressing neurons after administration of diphtheria toxin while leaving the neural precursor pool intact. Using a spatially challenging behavioral test (a modified version of the active place avoidance test), we present direct evidence that immature DCX-expressing neurons are required for successful acquisition of spatial learning, as well as reversal learning, but are not necessary for the retrieval of stored long-term memories. Importantly, the observed learning deficits were rescued as newly generated immature neurons repopulated the granule cell layer upon termination of the toxin treatment. Repeat (or cyclic) depletion of immature neurons reinstated behavioral deficits if the mice were challenged with a novel task. Together, these findings highlight the potential of stimulating neurogenesis as a means to enhance learning.