Activation of BMP-Smad1/5/8 Signaling Promotes Survival of Retinal Ganglion Cells after Damage In Vivo

While the essential role of bone morphogenetic protein (BMP) signaling in nervous system development is well established, its function in the adult CNS is poorly understood. We investigated the role of BMP signaling in the adult mouse retina following damage in vivo. Intravitreal injection of N-Methyl-D-aspartic acid (NMDA) induced extensive retinal ganglion cell death by 2 days. During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells. Expression of Inhibitor of differentiation 1 (Id1; a known BMP-Smad1/5/8 target) was also upregulated in the retina. This activation of BMP-Smad1/5/8 signaling was also observed following light damage, suggesting that it is a general response to retinal injuries. Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone. Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells. These data demonstrate that BMP-Smad1/5/8 signaling is neuroprotective for retinal ganglion cells after damage, and suggest that stimulation of this pathway can serve as a potential target for neuroprotective therapies in retinal ganglion cell diseases, such as glaucoma

TGF-β Is Required for Vascular Barrier Function, Endothelial Survival and Homeostasis of the Adult Microvasculature

Pericyte-endothelial cell (EC) interactions are critical to both vascular development and vessel stability. We have previously shown that TGF-β signaling between EC and mural cells participates in vessel stabilization in vitro. We therefore investigated the role of TGF-β signaling in maintaining microvessel structure and function in the adult mouse retinal microvasculature. TGF-β signaling was inhibited by systemic expression of soluble endoglin (sEng) and inhibition was demonstrated by reduced phospho-smad2 in the adult retina. Blockade of TGF-β signaling led to increased vascular and neural cell apoptosis in the retina, which was associated with decreased retinal function, as measured by electroretinogram (ERG). Perfusion of the inner retinal vasculature was impaired and was accompanied by defective autoregulation and loss of capillary integrity. Fundus angiography and Evans blue permeability assay revealed a breakdown of the blood-retinal-barrier that was characterized by decreased association between the tight junction proteins zo-1 and occludin. Inhibition of TGF-β signaling in cocultures of EC and 10T1/2 cells corroborated the in vivo findings, with impaired EC barrier function, dissociation of EC from 10T1/2 cells, and endothelial cell death, supporting the role of EC-mesenchymal interactions in TGF-β signaling. These results implicate constitutive TGF-β signaling in maintaining the integrity and function of the adult microvasculature and shed light on the potential role of TGF-β signaling in vasoproliferative and vascular degenerative retinal diseases

Autophagy in the eye:from physiology to pathophysology

Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye

Development of astrocytes in the vertebrate eye

Astrocytes represent the earliest glial population in the embryonic optic nerve, contributing critically to retinal angiogenesis and formation of brain-retinal-barrier. Despite of many developmental and clinical implications of astrocytes, answers to some of the most fundamental questions of this unique type of glial cells remain elusive. This review provides an overview of the current knowledge about the origination, proliferation, and differentiation of astrocytes, their journey from the optic nerve toward the neuroretina, and their involvement in physiological and pathological development of the visual system

Regulation of peripheral nerve regeneration by the mTOR pathway

While neurons in the central nervous system: CNS) have limited capacity for regrowth after damage, neurons in the peripheral nervous system: PNS) have a robust ability to regenerate their axons following injury. Successful regeneration depends upon both extrinsic cues in the environment and the activation of intrinsic mechanisms to promote regrowth. A number of inhibitory molecules in the CNS environment that prevent axonal regrowth have been identified, but less is known regarding the signaling mechanisms that regulate regenerative ability in PNS neurons. Here, we explored multiple components of injury signaling in the PNS, including the retrograde transport of local axonal injury signals, enhancement of axonal growth capacity in the cell body, and the response of Schwann cells that myelinate the damaged axon. We first addressed how axonal injury triggers enhancement of axonal growth capacity in PNS neurons. The lack of regenerative ability of CNS neurons has been linked to downregulation of the mammalian target of rapamycin: mTOR) pathway. We find that PNS dorsal root ganglia neurons: DRGs) activate mTOR following damage, and that this activity contributes to enhance axonal growth capacity following injury. Furthermore, upregulation of mTOR activity by deletion of tuberous sclerosis complex 2: TSC2) in DRGs is sufficient to enhance axonal growth capacity in vitro and in vivo. We identified GAP-43 as a downstream target of this pathway, which may contribute to enhance regenerative ability. However, while genetic upregulation of mTOR activity in sensory neurons facilitates axonal regrowth, it also leads to a number of developmental and functional defects, including aberrant target innervation. Thus, while manipulation of the mTOR activity could stimulate nerve regeneration in the PNS, fine control of mTOR activity may be required for proper target innervation and functional recovery. mTOR activation in the damaged neuron is likely to represent one of several signaling events that mediate nerve regeneration. We thus also explored other aspects of peripheral nerve injury signaling, including the retrograde transport of local injury signals by axonal vesicles, and the response of myelinating Schwann cells to axonal damage. Our results indicate that several classes of signaling pathways occurring both in axons and Schwann cells cooperate to generate a robust regenerative response. A better understanding of the signaling pathways leading to increased regenerative growth ability of PNS neurons may guide new strategies to enhance nerve regeneration in the CNS

Exploiting the impact of the secretome of MSCs isolated from different tissue sources on neuronal differentiation and axonal growth

Cell transplantation using Mesenchymal stem cell (MSC) secretome have recently been presented as a possible free-based therapy for CNS related disorders. MSC secretome is rich in several bio-factors that act synergically towards the repair of damaged tissues, thus making it an ideal candidate for regenerative applications. Great effort is currently being made to map the molecules that compose the MSC secretome. Previous proteomic characterization of the secretome (in the form of conditioned media - CM) of MSCs derived from adipose tissue (ASC), bone-marrow (BMSC) and umbilical cord (HUCPVC) was performed by our group, where proteins relevant for neuroprotection, neurogenic, neurodifferentiation, axon guidance and growth functions were identified. Moreover, we have found significant differences among the expression of several molecules, which may indicate that their therapeutic outcome might be distinct. Having this in mind, in the present study, the neuroregulatory potential of ASC, BMSC and HUCPVC CM in promoting neurodifferentiation and axonal outgrowth was tested in vitro, using human telencephalon neuroprogenitor cells and dorsal root ganglion explants, respectively. The CM from the three MSC populations induced neuronal differentiation from human neural progenitor cells, as well as neurite outgrowth from dorsal root ganglion explants. Moreover, all the MSC populations promoted the same extent of neurodifferentiation, while ASC CM demonstrated higher potential in promoting axonal growth.The authors acknowledge the financial support by Premios Santa Casa Neurociencias - Prize Melo e Castro for Spinal Cord ^ Injury Research (MC-17-2013 and MC-04-2017); Portuguese Foundation for Science and Technology (Doctoral fellowships PDE/ BDE/113596/2015 and SFRH/BD/120124/2016 to R.C Assunçao Silva ~ and B. Mendes-Pinheiro, respectively; Post-doctoral fellowhip to F.G. Teixeira and Patrícia Patrício - SFRH/BPD/118408/2016 and SFRH/BPD/116249/2016; IF Starting Grant to L. Pinto and IF Development Grant to A. J. Salgado); Canada Research Chair in Biomedical Engineering (LAB). This work is funded by national funds through FCT under the scope of grante reference TUBITAK/0007/ 2014. This article has been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology, under the scope of the project POCI-01-0145-FEDER-007038. HUCPVCs and ASCs were kindly provided by Prof. John E. Davies (University of Toronto, Canada) and Prof. Jeff Gimble (LaCell Inc, USA).info:eu-repo/semantics/publishedVersio

The Controversial Role of TGF-β in Neovascular Age-Related Macular Degeneration Pathogenesis.

The multifunctional transforming growth factors-beta (TGF-βs) have been extensively studied regarding their role in the pathogenesis of neovascular age-related macular degeneration (nAMD), a major cause of severe visual loss in the elderly in developed countries. Despite this, their effect remains somewhat controversial. Indeed, both pro- and antiangiogenic activities have been suggested for TGF-β signaling in the development and progression of nAMD, and opposite therapies have been proposed targeting the inhibition or activation of the TGF-β pathway. The present article summarizes the current literature linking TGF-β and nAMD, and reviews experimental data supporting both pro- and antiangiogenic hypotheses, taking into account the limitations of the experimental approaches

Role of Wnt and Notch signalling pathways on the neural differentiation of human Müller stem cells and their modulation by growth factors

Müller glia mediates retina regeneration in zebrafish. Despite exhibiting Müller glial with stem cell (hMSC) characteristics in vitro by the human retina, retinal regeneration mediated by Müller glia following disease or injury has not been demonstrated. Notch, Wnt, TGFβ and HBEGF signalling is well known to regulate retinal neurogenesis and inflammation, but the roles of these molecules in the neural differentiation of hMSC are not known. This thesis aimed to establish whether there is an interaction between these signalling pathways and the role that these factors play during retinal ganglion cell (RGC) and photoreceptor differentiation of hMSC. The research showed that inhibition of Notch signalling caused downregulation of components of the canonical Wnt signalling pathway in these cells, as demonstrated by a decrease in mRNA expression of the Wnt ligand WNT2B and its target genes WISP-1 and AXIN2. Addition of TGFβ1 did not significantly change the expression of the Notch signalling target HES1 or the RGC marker BRN3A/B. Culture of hMSC with a combination of factors that induce their photoreceptor differentiation (FGF2, taurine, retinoic acid and Insulin growth factor; collectively called FTRI), markedly upregulated the expression of components of the canonical Wnt signalling pathway, including WNT2B, DKK1 and active β-catenin. Although FTRI did not modify mRNA expression of WNT5B, a component of the non-canonical/planar cell polarity Wnt pathway, it upregulated its secretion. Furthermore, TGFβ1 not only decreased WNT2B expression, but inhibited FTRI-induced photoreceptor differentiation of hMSC, as determined by expression of the photoreceptor markers NR2E3, rhodopsin and recoverin. Inhibition of TGFβ1 signalling by an ALK5 inhibitor prevented TGFβ1 induced changes in the expression of the two Wnt ligands examined. More importantly, inhibition of the canonical Wnt signalling by XAV-939 prevented FTRI-induced photoreceptor differentiation. Similarly, HBEGF, a factor shown to be upregulated by FTRI also decreased Wnt signalling components such as WNT2B, WISP-1, DKK1 and AXIN2. Inhibition of HBEGF by its specific inhibitor CRM197 prevented photoreceptor differentiation. These observations suggest that both Notch and Wnt signalling pathways can regulate the neurogenicity of hMSC in vitro and that TGFβ as well as HBEGF play important roles in mediating key pathways leading to either RGC or photoreceptor differentiation of hMSC. Targeting components of both Notch and Wnt pathways may constitute targets for potential induction of endogenous regeneration of the human retina and this merits further studies

Downregulation of the Canonical WNT Signaling Pathway by TGF beta 1 Inhibits Photoreceptor Differentiation of Adult Human Muller Glia with Stem Cell Characteristics

Muller glia are responsible for the retina regeneration observed in zebrafish. Although the human retina harbors Muller glia with stem cell characteristics, there is no evidence that they regenerate the retina after disease or injury. Transforming growth factor-b (TGFb) and Wnt signaling regulate retinal neurogenesis and inflammation, but their roles in the neural differentiation of human Mu¨ller stem cells (hMSC) are not known. We examined hMSC lines in vitro for the expression of various Wnt signaling components and for their modulation by TGFb1, as well as the effect of this cytokine on the photoreceptor differentiation of these cells. Culture of hMSC with a combination of factors that induce photoreceptor differentiation of hMSC (FGF2, taurine, retinoic acid, and insulin-like growth factor type1; FTRI), markedly upregulated the expression of components of the canonical Wnt signaling pathway, including WNT2B, DKK1, and active b-CATENIN. Although FTRI did not modify mRNA expression of WNT5B, a component of the noncanonical/planar cell polarity Wnt pathway, it upregulated its secretion. Furthermore, TGFb1 not only decreased WNT2B expression, but also inhibited FTRI-induced photoreceptor differentiation of hMSC, as determined by expression of the photoreceptor markers NR2E3, RHODOPSIN, and RECOVERIN. Inhibition of TGFb1 signaling by an ALK5 inhibitor prevented TGFb1-induced changes in the expression of the two Wnt ligands examined. More importantly, inhibition of the canonical WNT signaling by XAV-939 prevented FTRI-induced photoreceptor differentiation. These observations suggest that TGFb may play a key role in preventing neural differentiation of hMSC and may constitute a potential target for induction of endogenous regeneration of the human retina