Vincent Pernet

Excitotoxic Death of Retinal Neurons In Vivo Occurs via a Non-Cell-Autonomous Mechanism

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 κB (NF-κB) activation in Müller glia but not in retinal neurons. Intriguingly, NMDA-induced death of retinal neurons is effectively blocked by inhibitors of NF-κB activity. We demonstrate that tumor necrosis factor α (TNFα) protein produced in Müller glial cells via an NMDA-induced NF-κB-dependent pathway plays a crucial role in excitotoxic loss of retinal neurons. This cell loss occurs mainly through a TNFα-dependent increase in Ca2+-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.

Nogo-A and Myelin-Associated Glycoprotein Differently Regulate Oligodendrocyte Maturation and Myelin Formation

Nogo-A is one of the most potent oligodendrocyte-derived inhibitors for axonal regrowth in the injured adult CNS. However, the physiological function of Nogo-A in development and in healthy oligodendrocytes is still unknown. In the present study, we investigated the role of Nogo-A for myelin formation in the developing optic nerve. By quantitative real-time PCR, we found that the expression of Nogo-A increased faster in differentiating oligodendrocytes than that of the major myelin proteins MBP (myelin basic protein), PLP (proteolipid protein)/DM20, and CNP (2′,3′-cyclic nucleotide 3′-phosphodiesterase). The analysis of optic nerves and cerebella of mice deficient for Nogo-A (Nogo-A−/−) revealed a marked delay of oligodendrocyte differentiation, myelin sheath formation, and axonal caliber growth within the first postnatal month. The combined deletion of Nogo-A and MAG caused a more severe transient hypomyelination. In contrast to MAG−/− mice, Nogo-A−/− mutants did not present abnormalities in the structure of myelin sheaths and Ranvier nodes. The common binding protein for Nogo-A and MAG, NgR1, was exclusively upregulated in MAG−/− animals, whereas the level of Lingo-1, a coreceptor, remained unchanged. Together, our results demonstrate that Nogo-A and MAG are differently involved in oligodendrocyte maturation in vivo, and suggest that Nogo-A may influence also remyelination in pathological conditions such as multiple sclerosis.

The Sphingolipid Receptor S1PR2 Is a Receptor for Nogo-A Repressing Synaptic Plasticity

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 a negative regulator of CNS angiogenesis

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.

PAX6-positive Müller glia cells express cell cycle markers but do not proliferate after photoreceptor injury in the mouse retina

In lower vertebrates, such as fish, Müller glia plays an essential role in the restoration of visual function after retinal degeneration by transdifferentiating into photoreceptors and other retinal neurons. During this process, Müller cells re‐enter the cell cycle, proliferate, and migrate from the inner nuclear layer (INL) to the photoreceptor layer where they express photoreceptor‐specific markers. This process of Müller cell transdifferentiation is absent in mammals, and the loss of photoreceptors leads to permanent vision deficits. The mechanisms underlying the failure of mammalian Müller cells to behave as stem cells after photoreceptor degeneration are poorly understood. In the present study, we show that photoreceptor injury induces migration of PAX6‐positive Müller cell nuclei toward the outer part of the INL and into the inner part of the outer nuclear layer. These cells express markers of the cell cycle, suggesting an attempt to re‐enter the cell cycle similarly to lower vertebrates. However, mouse Müller cells do not proliferate in response to photoreceptor injury implying a blockade of the S‐phase transition. Our results suggest that a release of the S‐phase blockade may be crucial for Müller cells to successfully transdifferentiate and replace injured photoreceptors in mammals.

Calsyntenin-1 Regulates Targeting of Dendritic NMDA Receptors and Dendritic Spine Maturation in CA1 Hippocampal Pyramidal Cells during Postnatal Development

Calsyntenin-1 is a transmembrane cargo-docking protein important for kinesin-1-mediated fast transport of membrane-bound organelles that exhibits peak expression levels at postnatal day 7. However, its neuronal function during postnatal development remains unknown. We generated a knock-out mouse to characterize calsyntenin-1 function in juvenile mice. In the absence of calsyntenin-1, synaptic transmission was depressed. To address the mechanism, evoked EPSPs were analyzed revealing a greater proportion of synaptic GluN2B subunit-containing receptors typical for less mature synapses. This imbalance was due to a disruption in calsyntenin-1-mediated dendritic transport of NMDA receptor subunits. As a consequence of increased expression of GluN2B subunits, NMDA receptor-dependent LTP was enhanced at Schaffer collateral–CA1 pyramidal cell synapses. Interestingly, these defects were accompanied by a decrease in dendritic arborization and increased proportions of immature filopodia-like dendritic protrusions at the expense of thin-type dendritic spines in CA1 pyramidal cells. Thus, these results highlight a key role for calsyntenin-1 in the transport of NMDA receptors to synaptic targets, which is necessary for the maturation of neuronal circuits during early development.

Nogo-A deletion increases the plasticity of the optokinetic response and changes retinal projection organization in the adult mouse visual system

The inhibitory action of Nogo-A on axonal growth has been well described. However, much less is known about the effects that Nogo-A could exert on the plasticity of neuronal circuits under physiological conditions. We investigated the effects of Nogo-A knock-out (KO) on visual function of adult mice using the optokinetic response (OKR) and the monocular deprivation (MD)-induced OKR plasticity and analyzed the anatomical organization of the eye-specific retinal projections. The spatial frequency sensitivity was higher in intact Nogo-A KO than in wild-type (WT) mice. After MD, Nogo-A KO mice reached a significantly higher spatial frequency and contrast sensitivity. Bilateral ablation of the visual cortex did not affect the OKR sensitivity before MD but reduced the MD-induced enhancement of OKR by approximately 50xa0% in Nogo-A KO and WT mice. These results suggest that cortical and subcortical brain structures contribute to the OKR plasticity. The tracing of retinal projections to the dorsal lateral geniculate nucleus (dLGN) revealed that the segregation of eye-specific terminals was decreased in the adult Nogo-A KO dLGN compared with WT mice. Strikingly, MD of the right eye led to additional desegregation of retinal projections in the left dLGN of Nogo-A KO but not in WT mice. In particular, MD promoted ectopic varicosity formation in Nogo-A KO dLGN axons. The present data show that Nogo-A restricts visual experience-driven plasticity of the OKR and plays a role in the segregation and maintenance of retinal projections to the brain.

The Ephrin receptor EphA4 restricts axonal sprouting and enhances branching in the injured mouse optic nerve.

The lack of axonal regeneration in the adult central nervous system is in part attributable to the presence of inhibitory molecules present in the environment of injured axons such as the myelin‐associated proteins Nogo‐A and MAG and the repulsive guidance molecules Ephrins, Netrins and Semaphorins. In the present study, we hypothesized that EphA4 and one of its potential binding partners EphrinA3 may participate in the inhibition of adult axon regeneration in the model of adult mouse optic nerve injury. Axonal regeneration was analysed in three dimensions after tissue clearing of EphA4 knockout (KO), EphrinA3 KO and wild‐type (WT) optic nerves. By immunohistochemistry, EphA4 was highly expressed in Müller glia endfeet in the retina and in astrocytes in the retina and the optic nerve, while EphrinA3 was present in retinal ganglion cells and oligodendrocytes. Optic nerve crush did not cause expression changes. Significantly more axons grew in the crushed optic nerve of EphA4 KO mice than in WT or EphrinA3 KO animals. Single axon analysis revealed that EphA4 KO axons were less prone to form aberrant branching than axons in the other mouse groups. The expression of growth‐associated proteins Sprr1a and Gap‐43 did not vary between EphA4 KO and WT retinae. However, glial fibrillary acidic protein‐expressing astrocytes were withdrawn from the perilesional area in EphA4 KO, suggesting that gliosis down‐regulation may locally contribute to improve axonal growth at the injury site. In summary, our three‐dimensional analysis of injured mouse optic nerves reveals beneficial effects of EphA4 ablation on the intensity and the pattern of optic nerve axon regeneration.

Nonamyloidogenic processing of amyloid beta precursor protein is associated with retinal function improvement in aging male APPswe/PS1ΔE9 mice

Vision declines during normal aging and in Alzheimers disease (AD). Although the toxic role of amyloid beta (Aβ) has been established in AD pathogenesis, its influence on the aging retina is unclear. Using APPswe/PS1ΔE9 transgenic (TG) mice, a classical AD model, the retinal cell function and survival was assessed by electroretinogram (ERG) recordings and immunofluorescent stainings. Strikingly, photopic ERG measurements revealed that the retinal response mediated by cones was preserved in aging TG mice relative to WT controls. In contrast to the cortex, the expression of mutated APPswe and PS1ΔE9 did not allow to detect Aβ or amyloid plaques in 13-month-old male TG retinae. In addition, the CTFβ/CTFα ratio was significantly lower in retinal samples than that in cortical extracts, suggesting that the nonamyloidogenic pathway may endogenously limit Aβ formation in the retina of male mice. Collectively, our data suggest that retinal-specific processing of amyloid may confer protection against AD and selectively preserve cone-dependent vision during aging.

Sphingosine 1‐phosphate receptor 1 is required for retinal ganglion cell survival after optic nerve trauma

In this study, we used a classical optic nerve injury model to address the function of the sphingosine 1‐phosphate (S1P)–S1P receptor (S1PR) axis in retinal ganglion cell (RGC) death and axonal growth. After lesion, the expression of S1PR1 was generally reduced in axotomized RGCs but persisted in αRGCs, a subpopulation of injury‐resistant RGCs. Silencing S1PR1 with an adeno‐associated virus serotype 2 (AAV2) containing a shRNA specific to S1PR1 (AAV2.shRNA‐S1PR1) exacerbated the loss of RGCs induced by optic nerve crush; the rate of RGC survival was decreased by more than 24% in retinae infected with AAV2.shRNA‐S1PR1 compared with AAV2.shRNA‐scrambled or AAV2.GFP control treatments. In the superior and temporal regions of the retina, cell death rose by more than ~ 35% and ~ 50%, respectively, in comparison with control groups. In the optic nerve, S1PR1 silencing markedly reduced axonal sprouting after the lesion relative to control animals. Early after optic nerve crush, 67% of αRGCs stained for osteopontin were lost in retinae infected with AAV2.shRNA‐S1PR1, whereas the number of intrinsically photosensitive RGCs expressing melanopsin, another injury‐resistant RGC type, was not affected. Moreover, retinal infection with AAV2.shRNA‐S1PR1 down‐regulated mammalian target of rapamycin pathway activation in αRGCs. Together, our results reveal that S1PR1 contributes to survival and growth mechanisms in injured RGCs by regulating the mammalian target of rapamycin pathway.