Claude Gravel

BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain

Neuropathic pain that occurs after peripheral nerve injury depends on the hyperexcitability of neurons in the dorsal horn of the spinal cord. Spinal microglia stimulated by ATP contribute to tactile allodynia, a highly debilitating symptom of pain induced by nerve injury. Signalling between microglia and neurons is therefore an essential link in neuropathic pain transmission, but how this signalling occurs is unknown. Here we show that ATP-stimulated microglia cause a depolarizing shift in the anion reversal potential (Eanion) in spinal lamina I neurons. This shift inverts the polarity of currents activated by GABA (γ-amino butyric acid), as has been shown to occur after peripheral nerve injury. Applying brain-derived neurotrophic factor (BDNF) mimics the alteration in Eanion. Blocking signalling between BDNF and the receptor TrkB reverses the allodynia and the Eanion shift that follows both nerve injury and administration of ATP-stimulated microglia. ATP stimulation evokes the release of BDNF from microglia. Preventing BDNF release from microglia by pretreating them with interfering RNA directed against BDNF before ATP stimulation also inhibits the effects of these cells on the withdrawal threshold and Eanion. Our results show that ATP-stimulated microglia signal to lamina I neurons, causing a collapse of their transmembrane anion gradient, and that BDNF is a crucial signalling molecule between microglia and neurons. Blocking this microglia–neuron signalling pathway may represent a therapeutic strategy for treating neuropathic pain.

Vasculature Guides Migrating Neuronal Precursors in the Adult Mammalian Forebrain via Brain-Derived Neurotrophic Factor Signaling

Adult neuronal precursors retain the remarkable capacity to migrate long distances from the posterior (subventricular zone) to the most anterior [olfactory bulb (OB)] parts of the brain. The knowledge about the mechanisms that keep neuronal precursors in the migratory stream and organize this long-distance migration is incomplete. Here we show that blood vessels precisely outline the migratory stream for new neurons in the adult mammalian forebrain. Real-time video imaging of cell migration in the acute slices demonstrate that neuronal precursors are retained in the migratory stream and guided into the OB by blood vessels that serve as a physical substrate for migrating neuroblasts. Our data suggest that endothelial cells of blood vessels synthesize brain-derived neurotrophic factor (BDNF) that fosters neuronal migration via p75NTR expressed on neuroblasts. Interestingly, GABA released from neuroblasts induces Ca2+-dependent insertion of high-affinity TrkB receptors on the plasma membrane of astrocytes that trap extracellular BDNF. We hypothesize that this renders BDNF unavailable for p75NTR-expressing migrating cells and leads to their entrance into the stationary period. Our findings provide new insights into the functional organization of substrates that facilitate the long-distance journey of adult neuronal precursors.

THE MODULAR CEREBELLUM

The putative modular organization of the cerebellum can be revealed in three ways: functionally through receptive field mapping, anatomically through tract tracing studies, and biochemically by using a range of molecular probes. The present review concentrates on the last of these, the evidence for cerebellar modules that comes from molecular studies, in particular our own

Protection of axotomized retinal ganglion cells by adenovirally delivered BDNF in vivo

Following intraorbital transection of the optic nerve (ON) in rats, more than 80% of the retinal ganglion cell (RGC) population die by apoptosis within 14 days. Repeated intraocular injection of brain‐derived neurotrophic factor (BDNF) has been efficient in enhancing RGC survival following ON axotomy. The present study was designed to define a potential survival‐promoting effect of adenovirally administered BDNF on axotomized RGCs. A single injection of an adenoviral vector expressing the human BDNF gene from a CMV promoter/enhancer (Ad‐BDNF) enhanced RGC survival 14 days after axotomy by 40.3%. Moreover, a combinatory treatment regimen consisting of intraocular Ad‐BDNF administration and systemic application of the free radical scavenger, N‐tert‐butyl‐(2‐sulphophenyl)‐nitrone (S‐PBN), enhanced RGC survival by 63.0%. Our data demonstrate that adenoviral delivery of neurotrophic factors to the vitreous body is a feasible approach for the prevention of axotomy‐induced RGC death. Further, as shown for S‐PBN, therapeutic regimens that combine local virus‐mediated gene delivery with systemic administration of protective compounds, may offer promising strategies for future treatment also in human neurodegenerative conditions.

Myoblast transplantation in monkeys: control of immune response by FK506.

Myoblasts were grown from monkey muscle biopsies and infected in vitro with a defective retroviral vector containing a cytoplasmic β-galactosidase (β-gal) gene. These myoblasts were then transplanted to 14 different monkeys, 6 of which were immunosuppressed with FK506. Without immunosuppression, only a few myoblasts and myotubes expressing β-gal were observed 1 week after the transplantation, but no cells expressing β-gal were observed after 4 weeks. This result was attributed to immune responses since infiltration by CD4+ or CD8+ lymphocytes was abundant 1 week after transplantation but not after 4 weeks. The expression of interleukin 6 (IL-6), interleukin 2 (IL-2), granulocyte/macrophage colony stimulating factor (GM-CSF), transforming growth factor-beta (TGF-β) and granzyme B mRNAs was increased in the myoblast-injected muscle indicating that the infiltrating lymphocytes were activated. Moreover, antibodies against the donor myoblasts were detected in 3 out of 6 cases. When the monkeys were immunosuppressed with FK506, muscle fibers expressing beta-galactosidase (β-gal) were present 1, 4 and 12 weeks after the transplantation. There was neither significant infiltration by CD4 or CD8 lymphocytes, nor antibodies detected. The mRNA expression of most cytokines was significantly reduced as compared to the nonimmunosuppressed monkeys. These results indicate that FK506 is effective in controlling short-term immune reactions following myoblast transplantation in monkeys and suggest that it may prove useful for myoblast transplantation in Duchenne Muscular Dystrophy patients.

Extra neurofilament NF-L subunits rescue motor neuron disease caused by overexpression of the human NF-H gene in mice.

Previous studies demonstrated that transgenic mice overexpressing human neurofilament heavy (hNF-H) protein develop a progressive motor neuron disease characterized by the perikaryal accumulations of neurofilaments resembling those found in amyotrophic lateral sclerosis (ALS). To further investigate this neurofilament-induced pathology, we generated transgenic mice expressing, solely or concomitantly, the hNF-H and the human neurofilament light (hNF-L) proteins. We report here that the motor neuron disease caused by excess hNF-H proteins can be rescued by overexpression of hNF-L in a dosage-dependent fashion. In hNF-H transgenic mice, the additional hNF-L led to reduction of perikaryal swellings, relief of axonal transport defect and restoration of axonal radial growth. A gene delivery approach based on recombinant adenoviruses bearing the hNF-L gene also demonstrated the possibility to reduce perikaryal swellings after their formation in adult mice. The finding that extra NF-L can protect against NF-H-mediated pathogenesis is of potential importance for ALS, particularly for cases with NF-H abnormalities.

Zebrins: Molecular Markers of Compartmentation in the Cerebellum

Ordered projections in the brain are established in several stages. Initially, the formation of an afferent pathway depends on white matter interactions, such as contact guidance along genetically determined pathways and selective axon fasciculation, to guide neurites to the correct target fields. Subsequently, target cell recognition by afferent growth cones and competition between growth cones for targets (and between targets for inputs) serve to eliminate superfluous or incorrect projections and may refine the topography. Many regions, including the neocortex, the superior colliculus, the striatum, and the dorsal column nuclei, are functionally organized in the form of patches or stripes that correspond to the discrete segregation of the afferent or efferent axons. The same appears to be true in the cerebellum. Studies using the retrograde-anterograde axonal transport of tracers, electrophysiological recording, somatotopic mapping, and molecular mapping have all revealed a parasagittal bandlike topographical organization of the cerebellar cortex and its afferent and efferent connections. We are using pattern formation in the cerebellar cortex as a model to explore the rules that give rise to topographically ordered projections.

Dose-dependent rescue of axotomized rat retinal ganglion cells by adenovirus-mediated expression of glial cell-line derived neurotrophic factor in vivo.

Adult rat retinal ganglion cells undergo degeneration after optic nerve transection. Repeated intraocular injection of glial cell‐line derived neurotrophic factor (GDNF) has been shown to be efficient in enhancing retinal ganglion cell survival following optic nerve axotomy. In the present study we evaluated the potential survival‐promoting effect of adenovirally administered GDNF on axotomized retinal ganglion cells. A single intravitreal injection [7 × 107 plaque‐forming units (pfu) or 7 × 108 pfu] of an adenoviral vector expressing the rat GDNF gene from a cytomegalovirus promoter enhanced retinal ganglion cell survival 14 days after axotomy by 67 and 125%, respectively, when compared to control animals. Intraocular administration of the vector rescued 12.6 and 23%, respectively, of the retinal ganglion cells which would otherwise have died after axotomy. An increase in retinal GDNF protein and specific virally transduced GDNF mRNA expression was detected following intraocular vector application. Our data support previous findings showing that adenoviral delivery of neurotrophic factors to the vitreous body is a feasible approach for the prevention of axotomy‐induced retinal ganglion cell death in vivo and may constitute a relevant strategy for future treatment in traumatic brain injury and ensuing neurodegeneration.

Adeno-associated Virus-mediated Delivery of a Recombinant Single-chain Antibody Against Misfolded Superoxide Dismutase for Treatment of Amyotrophic Lateral Sclerosis

There is emerging evidence that the misfolding of superoxide dismutase 1 (SOD1) may represent a common pathogenic event in both familial and sporadic amyotrophic lateral sclerosis (ALS). To reduce the burden of misfolded SOD1 species in the nervous system, we have tested a novel therapeutic approach based on adeno-associated virus (AAV)–mediated tonic expression of a DNA construct encoding a secretable single-chain fragment variable (scFv) antibody composed of the variable heavy and light chain regions of a monoclonal antibody (D3H5) binding specifically to misfolded SOD1. A single intrathecal injection of the AAV encoding the single-chain antibody in SOD1G93A mice at 45 days of age resulted in sustained expression of single-chain antibodies in the spinal cord, and it delayed disease onset and extension of life span by up to 28%, in direct correlation with scFv titers in the spinal cord. The treatment caused attenuation of neuronal stress signals and reduction in levels of misfolded SOD1 in the spinal cord of SOD1G93A mice. From these results, we propose that an immunotherapy based on intrathecal inoculation of AAV encoding a secretable scFv against misfolded SOD1 should be considered as potential treatment for ALS, especially for individuals carrying SOD1 mutations.

Excess target-derived brain-derived neurotrophic factor preserves the transient uncrossed retinal projection to the superior colliculus.

During early postnatal development, a widespread ipsilateral projection to the superior colliculus is secondarily restricted to a small topographically defined region by elimination of ipsilaterally projecting retinal ganglion cells. Brain-derived neurotrophic factor (BDNF) has been proposed as the target-derived neurotrophic factor for retinal ganglion cells in several studies. Here we investigated the long-term effects of excess BDNF in the retinal ganglion cell target on naturally occurring retinal ganglion cell (RGC) elimination and on the restriction of the ipsilateral projection. To this end, sustained overexpression of BDNF was achieved in the postnatal superior colliculus using an adenoviral vector. While the total number of retinal ganglion cells in the adenovirus-BDNF treated animals was unchanged, a much higher proportion of RGCs retained a projection to the ipsilateral superior colliculus. We conclude that an excess of target-derived BDNF does not reduce the net amount of naturally occurring cell death in the retino-collicular system, but prevents the negative selection of retinal ganglion cells making inappropriate topographic connections.