The central hypothesis of excitotoxicity is that excessive stimulation of neuronal NMDA-sensitive glutamate receptors is harmful to neurons and contributes to a variety of neurological disorders. Glial cells have been proposed to participate in excitotoxic neuronal loss, but their precise role is defined poorly. In this in vivo study, we show that NMDA induces profound nuclear factor B (NF-B) activation in Müller glia but not in retinal neurons. Intriguingly, NMDA-induced death of retinal neurons is effectively blocked by inhibitors of NF-B activity. We demonstrate that tumor necrosis factor ␣ (TNF␣) protein produced in Müller glial cells via an NMDA-induced NF-B-dependent pathway plays a crucial role in excitotoxic loss of retinal neurons. This cell loss occurs mainly through a TNF␣-dependent increase in Ca 2ϩ -permeable AMPA receptors on susceptible neurons. Thus, our data reveal a novel non-cell-autonomous mechanism by which glial cells can profoundly exacerbate neuronal death following excitotoxic injury.

This study identifies a GPCR, S1PR2, as a receptor for the Nogo-A-Δ20 domain of the membrane protein Nogo-A, which inhibits neuronal growth and synaptic plasticity.

Nogo-A is an important axonal growth inhibitor in the adult and developing CNS. In vitro, Nogo-A has been shown to inhibit migration and cell spreading of neuronal and nonneuronal cell types. Here, we studied in vivo and in vitro effects of Nogo-A on vascular endothelial cells during angiogenesis of the early postnatal brain and retina in which Nogo-A is expressed by many types of neurons. Genetic ablation or virus-mediated knock down of Nogo-A or neutralization of Nogo-A with an antibody caused a marked increase in the blood vessel density in vivo. In culture, Nogo-A inhibited spreading, migration, and sprouting of primary brain microvascular endothelial cells (MVECs) in a dose-dependent manner and induced the retraction of MVEC lamellipodia and filopodia. Mechanistically, we show that only the Nogo-A–specific Delta 20 domain exerts inhibitory effects on MVECs, but the Nogo-66 fragment, an inhibitory domain common to Nogo-A, -B, and -C, does not. Furthermore, the action of Nogo-A Delta 20 on MVECs required the intracellular activation of the Ras homolog gene family, member A (Rho-A)-associated, coiled-coil containing protein kinase (ROCK)-Myosin II pathway. The inhibitory effects of early postnatal brain membranes or cultured neurons on MVECs were relieved significantly by anti–Nogo-A antibodies. These findings identify Nogo-A as an important negative regulator of developmental angiogenesis in the CNS. They may have important implications in CNS pathologies involving angiogenesis such as stroke, brain tumors, and retinopathies.

No abstract

Axonal damage leads to permanent deficits in the adult central nervous system (CNS) not only because of the weak intrinsic ability of adult neurons to activate their growth program but importantly also because of the presence of specific growth inhibitors in the CNS tissue and the environment of the damaged axons. The well-studied myelin-derived protein Nogo-A is involved in various cellular and molecular events contributing to the failure of CNS axons to regrow and reconnect after transection. Recent studies have shown that, by acting in a negative way on the cytoskeleton and on the growth program of axotomized neurons, Nogo-A exerts fast and chronic inhibitory effects on neurite outgrowth. On the other hand, the blockade of Nogo-A results in a marked enhancement of compensatory and regenerative axonal extension in vivo; this enhancement is often paralleled by significant functional recovery, for example, of locomotion or skilled forelimb reaching after spinal cord or stroke lesions in rats and monkeys. Surprisingly, the blockade of Nogo-A or its receptor NgR in the hippocampus has recently been demonstrated to enhance long-term potentiation. A role of Nogo-A in synaptic plasticity/stability might therefore represent an additional, new and important aspect of CNS circuit remodeling. Function-blocking anti-Nogo-A antibodies are currently being tested in a clinical trial for improved outcome after spinal cord injury.

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.

No abstract

Trauma or disease in the CNS often leads to neuronal death and consequent loss of functional connections. The idea has been put forward that strategies aimed at repairing the injured CNS involve stimulation of both neuronal survival and axon regeneration. We tested this hypothesis in the adult rat retinocollicular system by combining two strategies: (i) exogenous administration of brain-derived neurotrophic factor (BDNF), a potent survival factor for damaged retinal ganglion cells (RGCs) and (ii) lens injury, which promotes robust growth of transected RGC axons. Our results demonstrate that BDNF and lens injury interact synergistically to promote neuronal survival: 71% of RGCs were alive at 2 weeks after optic nerve injury, a time when only approximately 10% of these neurons remain without treatment. Intravitreal injection of BDNF, however, led to regeneration failure following lens injury. The effect of BDNF could not be generalized to other growth factors, as ciliary neurotrophic factor did not cause a significant reduction of lens injury-induced regeneration. Growth arrest in optic nerves treated with BDNF and lens injury correlated with the formation of hypertrophic axonal swellings in the proximal optic nerve. These swellings were filled with numerous vesicular bodies, disorganized neurofilaments and degenerating organelles. Our results demonstrate that: (i) increased neuronal survival does not necessarily lead to enhanced axon regeneration and (ii) activation of survival and growth pathways may produce axonal dystrophy similar to that found in neurodegenerative disorders including glaucoma, Alzheimer's disease and multiple sclerosis. We propose that loss of axonal integrity may limit neuronal recovery in the injured, adult CNS.