Sandrine Joly

Cooperative phagocytes: resident microglia and bone marrow immigrants remove dead photoreceptors in retinal lesions.

Phagocytosis is essential for the removal of photoreceptor debris following retinal injury. We used two mouse models, mice injected with green fluorescent protein-labeled bone marrow cells or green fluorescent protein-labeled microglia, to study the origin and activation patterns of phagocytic cells after acute blue light-induced retinal lesions. We show that following injury, blood-borne macrophages enter the eye via the optic nerve and ciliary body and soon migrate into the injured retinal area. Resident microglia are also activated rapidly throughout the entire retina and adopt macrophage characteristics only in the injured region. Both blood-borne- and microglia-derived macrophages were involved in the phagocytosis of dead photoreceptors. No obvious breakdown of the blood-retinal barrier was observed. Ccl4, Ccl12, Tgfb1, Csf1, and Tnf were differentially expressed in both the isolated retina and the eyecup of wild-type mice. Debris-laden macrophages appeared to leave the retina into the general circulation, suggesting their potential to become antigen-presenting cells. These experiments provide evidence that both local and immigrant macrophages remove apoptotic photoreceptors and cell debris in the injured retina.

Leukemia Inhibitory Factor Extends the Lifespan of Injured Photoreceptors In Vivo

Survival and death of photoreceptors in degenerative diseases of the retina is controlled by a multitude of genes and endogenous factors. Some genes may be involved in the degenerative process itself whereas others may be part of an endogenous defense system. We show in two models of retinal degeneration that photoreceptor death strongly induces expression of leukemia inhibitory factor (LIF) in a subset of Muller glia cells in the inner nuclear layer of the retina. LIF expression is essential to induce an extensive intraretinal signaling system which includes Muller cells and photoreceptors and is characterized by an upregulation of Edn2, STAT3, FGF2 and GFAP. In the absence of LIF, Muller cells remain quiescent, the signaling system is not activated and retinal degeneration is strongly accelerated. Intravitreal application of recombinant LIF induces the full molecular pathway including the activation of Muller cells in wild-type and Lif –/– mice. Interruption of the signaling cascade by an Edn2 receptor antagonist increases whereas activation of the receptor decreases photoreceptor cell death. Thus, LIF is essential and sufficient to activate an extensive molecular defense response to photoreceptor injury. Our data establish LIF as a Muller cell derived neuronal survival factor which controls an intrinsic protective mechanism that includes Edn2 signaling to support photoreceptor cell survival and to preserve vision in the injured retina.

Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve

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.

Long-distance axonal regeneration induced by CNTF gene transfer is impaired by axonal misguidance in the injured adult optic nerve

The optic nerve crush injury is a well-accepted model to study the mechanisms of axonal regeneration after trauma in the CNS. The infection of retinal ganglion cells (RGCs) with an adeno-associated virus serotype 2 - ciliary neurotrophic factor (AAV2.CNTF) was previously shown to stimulate axonal regeneration. However, the transfection of axotomized neurons themselves may not be optimal to promote full axonal regeneration in the visual system. Here, we show that the release of CNTF by glial cells is a very powerful stimulus for optic fiber regeneration and RGC survival after optic nerve crush. After 8 weeks, long-distance regeneration of severed optic axons was induced by CNTF until and beyond the optic chiasm. Regenerated axons stayed for at least 6 months in the damaged optic nerve. Strikingly, however, many regenerated axons showed one or several sharp U-turns along their course, suggesting that guidance cues are missing and that long-distance axonal regeneration is limited by the return of the growing axons toward the retina. Even more surprisingly, massive axonal sprouting was observed within the eye, forming a dense plexus of neurites at the inner surface of the retina. These results indicate that massive stimulation of the neuronal growth program can lead to aberrant growth; the absence of local regulatory and guidance factors in the adult, injured optic nerve may therefore represent a major, so far underestimated obstacle to successful axon regeneration.

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.

Neuronal Nogo-A upregulation does not contribute to ER stress-associated apoptosis but participates in the regenerative response in the axotomized adult retina

Nogo-A, an axonal growth inhibitory protein known to be mostly present in CNS myelin, was upregulated in retinal ganglion cells (RGCs) after optic nerve injury in adult mice. Nogo-A increased concomitantly with the endoplasmic reticulum stress (ER stress) marker C/EBP homologous protein (CHOP), but CHOP immunostaining and the apoptosis marker annexin V did not co-localize with Nogo-A in individual RGC cell bodies, suggesting that injury-induced Nogo-A upregulation is not involved in axotomy-induced cell death. Silencing Nogo-A with an adeno-associated virus serotype 2 containing a short hairpin RNA (AAV2.shRNA-Nogo-A) or Nogo-A gene ablation in knock-out (KO) animals had little effect on the lesion-induced cell stress or death. On the other hand, Nogo-A overexpression mediated by AAV2.Nogo-A exacerbated RGC cell death after injury. Strikingly, however, injury-induced sprouting of the cut axons and the expression of growth-associated molecules were markedly reduced by AAV2.shRNA-Nogo-A. The axonal growth in the optic nerve activated by the intraocular injection of the inflammatory molecule Pam3Cys tended to be lower in Nogo-A KO mice than in WT mice. Nogo-A overexpression in RGCs in vivo or in the neuronal cell line F11 in vitro promoted regeneration, demonstrating a positive, cell-autonomous role for neuronal Nogo-A in the modulation of axonal regeneration.

Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve.

Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre+/−xRtn4/Nogo-Aflox/flox) and neuron-specific (Thy1-Cretg+xRtn4flox/flox) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4flox/flox animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS.

Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia‐inducible factor‐1α expression in photoreceptors

Hypoxic preconditioning stabilizes hypoxia‐inducible factor (HIF) 1α in the retina and protects photoreceptors against light‐induced cell death. HIF‐1α is one of the major transcription factors responding to low oxygen tension and can differentially regulate a large number of target genes. To analyse whether photoreceptor‐specific expression of HIF‐1α is essential to protect photoreceptors by hypoxic preconditioning, we knocked down expression of HIF‐1α specifically in photoreceptor cells, using the cyclization recombinase (Cre)–lox system. The Cre‐mediated knockdown caused a 20‐fold reduced expression of Hif‐1α in the photoreceptor cell layer. In the total retina, RNA expression was reduced by 65%, and hypoxic preconditioning led to only a small increase in HIF‐1α protein levels. Accordingly, HIF‐1 target gene expression after hypoxia was significantly diminished. Retinas of Hif‐1α knockdown animals did not show any pathological alterations, and tolerated hypoxic exposure in a comparable way to wild‐type retinas. Importantly, the strong neuroprotective effect of hypoxic preconditioning against light‐induced photoreceptor degeneration persisted in knockdown mice, suggesting that hypoxia‐mediated survival of light exposure does not depend on an autocrine action of HIF‐1α in photoreceptor cells. Hypoxia‐mediated stabilization of HIF‐2α and phosphorylation of signal transducer and activator of transcription 3 (STAT 3) were not affected in the retinas of Hif‐1α knockdown mice. Thus, these factors are candidates for regulating the resistance of photoreceptors to light damage after hypoxic preconditioning, along with several potentially neuroprotective genes that were similarly induced in hypoxic knockdown and control mice.

Animal models for retinal degeneration

Retinal degeneration is often used to describe a category of human eye diseases, which are characterized by photoreceptor loss leading to severe visual impairment and blindness. An important, yet heterogeneous group of such diseases is called Retinitis Pigmentosa (RP). To understand the molecular mechanisms of disease induction and progression and to develop therapeutical strategies for the preservation of vision in RP patients, appropriate animal models are used in many research laboratories worldwide. The largest category of models consists of mutant (spontaneous and genetically engineered) mice. However, in recent years, zebrafish has been established as a highly valuable tool for the study of various biological problems, including retinal degeneration. In this review, we summarize the currently available mouse and zebrafish models to study retinal degeneration and give a short overview about recent developments in the field.

LIF-dependent JAK3 activation is not essential for retinal degeneration.

J. Neurochem. (2010) 113, 1210–1220.