PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS.

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol (3,4,5)-trisphosphate (PIP3 ). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP3 decreases with development at the time when axon transport declines and regenerative competence is lost. Overexpressing p110α in CNS neurons had no effect; however, expression of p110δ restored axonal PIP3 and increased regenerative axon transport. p110δ expression enhanced CNS regeneration in both rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110α. Furthermore, viral delivery of p110δ promoted robust regeneration after optic nerve injury. These findings establish a deficit of axonal PIP3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion.

Expression of Developmentally Important Axon Guidance Cues in the Adult Optic Chiasm.

Purpose: Regeneration of optic nerve axons after injury can be facilitated by several approaches, but misguidance at the optic chiasm is often observed. We characterized guidance cues in the embryonic visual system and adult optic chiasm before and after optic nerve crush (ONC) injury to better understand barriers to optic nerve regeneration in adults. Methods: Radial glial (RC2/BLBP/Slit1), developmental (Pax2) and extracellular markers (CSPG: H2B/CS-56) were assessed in C57BL/6J mice by immunohistochemistry. RC2, BLBP, Slit1, and CSPG are known inhibitory guidance cues while Pax2 is a permissive guidance cue. Results: At embryonic day 15.5 (E.15.5), RC2 and BLBP were identified superior to, and extending through, the optic chiasm. The optic chiasm was BLBP-ve in adult uninjured mice but BLBP+ve in adult mice 10 days after ONC injury. The reverse was true for RC2. Both BLBP and RC2 were absent in adult mice 6 weeks post-ONC. Slit1 was present in the optic chiasm midline and optic tracts in embryonic samples but was absent in uninjured adult tissue. Slit1 was observed superior to and at the midline of the optic chiasm 10 days post-ONC but absent 6 weeks after injury. Pax2 was expressed at the junction between the optic nerve and optic chiasm in embryonic brain tissue. In embryonic sections, CS-56 was observed at the junction between the optic chiasm and optic tract, and immediately superior to the optic chiasm. Both 2H6 and CS-56 staining was absent in uninjured and ONC-injured adult brains. Conclusion: Differences in guidance cue expression during development, in adulthood and after injury may contribute to misguidance of regenerating RGC axons in the adult optic chiasm.

Neuroprotection of retinal ganglion cells by a novel gene therapy construct that achieves sustained enhancement of brain-derived neurotrophic factor/tropomyosin-related kinase receptor-B signaling.

Previous studies have demonstrated that intravitreal delivery of brain-derived neurotrophic factor (BDNF) by injection of recombinant protein or by gene therapy can alleviate retinal ganglion cell (RGC) loss after optic nerve injury. BDNF gene therapy can improve RGC survival in experimental models of glaucoma, the leading cause of irreversible blindness worldwide. However, the therapeutic efficacy of BDNF supplementation alone is time limited at least in part due to BDNF receptor downregulation. Tropomyosin-related receptor kinase-B (TrkB) downregulation has been reported in many neurological diseases including glaucoma, potentially limiting the effect of sustained or repeated BDNF delivery.Here, we characterize a novel adeno-associated virus (AAV) gene therapy (AAV2 TrkB-2A-mBDNF) that not only increases BDNF production but also improves long-term neuroprotective signaling by increasing expression of the BDNF receptor (TrkB) within the inner retina. This approach leads to significant and sustained elevation of survival signaling pathways ERK and AKT within RGCs over 6 months and avoids the receptor downregulation which we observe with treatment with AAV2 BDNF alone. We validate the neuroprotective efficacy of AAV2 TrkB-2A-mBDNF in a mouse model of optic nerve injury, where it outperforms conventional AAV2 BDNF or AAV2 TrkB therapy, before showing powerful proof of concept neuroprotection of RGCs and axons in a rat model of chronic intraocular pressure (IOP) elevation. We also show that there are no adverse effects of the vector on retinal structure or function as assessed by histology and electroretinography in young or aged animals. Further studies are underway to explore the potential of this vector as a candidate for progression into clinical studies to protect RGCs in patients with glaucoma and progressive visual loss despite conventional IOP-lowering treatment.

Identification of a critical sulfation in chondroitin that inhibits axonal regeneration.

The failure of mammalian CNS neurons to regenerate their axons derives from a combination of intrinsic deficits and extrinsic factors. Following injury, chondroitin sulfate proteoglycans (CSPGs) within the glial scar inhibit axonal regeneration, an action mediated by the sulfated glycosaminoglycan (GAG) chains of CSPGs, especially those with 4-sulfated (4S) sugars. Arylsulfatase B (ARSB) selectively cleaves 4S groups from the non-reducing ends of GAG chains without disrupting other, growth-permissive motifs. We demonstrate that ARSB is effective in reducing the inhibitory actions of CSPGs both in in vitro models of the glial scar and after optic nerve crush (ONC) in adult mice. ARSB is clinically approved for replacement therapy in patients with mucopolysaccharidosis VI and therefore represents an attractive candidate for translation to the human CNS.

Transplanted Donor- or Stem Cell-Derived Cone Photoreceptors Can Both Integrate and Undergo Material Transfer in an Environment-Dependent Manner.

Human vision relies heavily upon cone photoreceptors, and their loss results in permanent visual impairment. Transplantation of healthy photoreceptors can restore visual function in models of inherited blindness, a process previously understood to arise by donor cell integration within the host retina. However, we and others recently demonstrated that donor rod photoreceptors engage in material transfer with host photoreceptors, leading to the host cells acquiring proteins otherwise expressed only by donor cells. We sought to determine whether stem cell- and donor-derived cones undergo integration and/or material transfer. We find that material transfer accounts for a significant proportion of rescued cells following cone transplantation into non-degenerative hosts. Strikingly, however, substantial numbers of cones integrated into the Nrl-/- and Prph2rd2/rd2, but not Nrl-/-;RPE65R91W/R91W, murine models of retinal degeneration. This confirms the occurrence of photoreceptor integration in certain models of retinal degeneration and demonstrates the importance of the host environment in determining transplantation outcome.

Molecular Mechanisms Mediating Retinal Reactive Gliosis Following Bone Marrow Mesenchymal Stem Cell Transplantation.

A variety of diseases lead to degeneration of retinal ganglion cells (RGCs) and their axons within the optic nerve resulting in loss of visual function. Although current therapies may delay RGC loss, they do not restore visual function or completely halt disease progression. Regenerative medicine has recently focused on stem cell therapy for both neuroprotective and regenerative purposes. However, significant problems remain to be addressed, such as the long-term impact of reactive gliosis occurring in the host retina in response to transplanted stem cells. The aim of this work was to investigate retinal glial responses to intravitreally transplanted bone marrow mesenchymal stem cells (BM-MSCs) to help identify factors able to modulate graft-induced reactive gliosis. We found in vivo that intravitreal BM-MSC transplantation is associated with gliosis-mediated retinal folding, upregulation of intermediate filaments, and recruitment of macrophages. These responses were accompanied by significant JAK/STAT3 and MAPK (ERK1/2 and JNK) cascade activation in retinal Muller glia. Lipocalin-2 (Lcn-2) was identified as a potential new indicator of graft-induced reactive gliosis. Pharmacological inhibition of STAT3 in BM-MSC cocultured retinal explants successfully reduced glial fibrillary acidic protein expression in retinal Muller glia and increased BM-MSC retinal engraftment. Inhibition of stem cell-induced reactive gliosis is critical for successful transplantation-based strategies for neuroprotection, replacement, and regeneration of the optic nerve.

Müller glia activation in response to inherited retinal degeneration is highly varied and disease-specific.

Despite different aetiologies, most inherited retinal disorders culminate in photoreceptor loss, which induces concomitant changes in the neural retina, one of the most striking being reactive gliosis by Müller cells. It is typically assumed that photoreceptor loss leads to an upregulation of glial fibrilliary acidic protein (Gfap) and other intermediate filament proteins, together with other gliosis-related changes, including loss of integrity of the outer limiting membrane (OLM) and deposition of proteoglycans. However, this is based on a mix of both injury-induced and genetic causes of photoreceptor loss. There are very few longitudinal studies of gliosis in the retina and none comparing these changes across models over time. Here, we present a comprehensive spatiotemporal assessment of features of gliosis in the degenerating murine retina that involves Müller glia. Specifically, we assessed Gfap, vimentin and chondroitin sulphate proteoglycan (CSPG) levels and outer limiting membrane (OLM) integrity over time in four murine models of inherited photoreceptor degeneration that encompass a range of disease severities (Crb1rd8/rd8, Prph2+/Δ307, Rho-/-, Pde6brd1/rd1). These features underwent very different changes, depending upon the disease-causing mutation, and that these changes are not correlated with disease severity. Intermediate filament expression did indeed increase with disease progression in Crb1rd8/rd8 and Prph2+/Δ307, but decreased in the Prph2+/Δ307 and Pde6brd1/rd1 models. CSPG deposition usually, but not always, followed the trends in intermediate filament expression. The OLM adherens junctions underwent significant remodelling in all models, but with differences in the composition of the resulting junctions; in Rho-/- mice, the adherens junctions maintained the typical rod-Müller glia interactions, while in the Pde6brd1/rd1 model they formed predominantly between Müller cells in late stage of degeneration. Together, these results show that gliosis and its associated processes are variable and disease-dependent.

Photoreceptor replacement therapy: challenges presented by the diseased recipient retinal environment.

Vision loss caused by the death of photoreceptors is the leading cause of irreversible blindness in the developed world. Rapid advances in stem cell biology and techniques in cell transplantation have made photoreceptor replacement by transplantation a very plausible therapeutic strategy. These advances include the demonstration of restoration of vision following photoreceptor transplantation and the generation of transplantable populations of donor cells from stem cells. In this review, we present a brief overview of the recent progress in photoreceptor transplantation. We then consider in more detail some of the challenges presented by the degenerating retinal environment that must play host to these transplanted cells, how these may influence transplanted photoreceptor cell integration and survival, and some of the progress in developing strategies to circumnavigate these issues.

Migration, integration and maturation of photoreceptor precursors following transplantation in the mouse retina.

Retinal degeneration leading to loss of photoreceptors is a major cause of untreatable blindness. Recent research has yielded definitive evidence for restoration of vision following the transplantation of rod photoreceptors in murine models of blindness, while advances in stem cell biology have enabled the generation of transplantable photoreceptors from embryonic stem cells. Importantly, the amount of visual function restored is dependent upon the number of photoreceptors that migrate correctly into the recipient retina. The developmental stage of the donor cells is important for their ability to migrate; they must be immature photoreceptor precursors. Little is known about how and when donor cell migration, integration, and maturation occurs. Here, we have performed a comprehensive histological analysis of the 6-week period following rod transplantation in mice. Donor cells migrate predominately as single entities during the first week undergoing a stereotyped sequence of morphological changes in their translocation from the site of transplantation, through the interphotoreceptor matrix and into the recipient retina. This includes initial polarization toward the outer nuclear layer (ONL), followed by formation of an apical attachment and rudimentary segment during migration into the ONL. Strikingly, acquisition of a nuclear architecture typical of mature rods was accelerated compared with normal development and a feature of migrating cells. Once within the ONL, precursors formed synaptic-like structures and outer segments in accordance with normal maturation. The restoration of visual function mediated by transplanted photoreceptors correlated with the later expression of rod α-transducin, achieving maximal function by 5 weeks.

Repair of the degenerate retina by photoreceptor transplantation.

Despite different aetiologies, age-related macular degeneration and most inherited retinal disorders culminate in the same final common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into a mouse model of stationary night-blindness, raising the critical question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase (Prph2(+/Δ307)), decrease (Crb1(rd8/rd8), Gnat1(-/-), Rho(-/-)), or remain constant (PDE6ß(rd1/rd1), Prph2(rd2/rd2)) with disease progression, depending upon the gene defect, with no correlation with severity. Robust integration is possible even in late-stage disease. Glial scarring and outer limiting membrane integrity, features that change with degeneration, significantly affect transplanted photoreceptor integration. Combined breakdown of these barriers markedly increases integration in a model with an intact outer limiting membrane, strong gliotic response, and otherwise poor transplantation outcome (Rho(-/-)), leading to an eightfold increase in integration and restoration of visual function. Thus, it is possible to achieve robust integration across a broad range of inherited retinopathies. Moreover, transplantation outcome can be improved by administering appropriate, tailored manipulations of the recipient environment.

Long-term survival of photoreceptors transplanted into the adult murine neural retina requires immune modulation.

Stem cell therapy presents an opportunity to replace photoreceptors that are lost as a result of inherited and age-related degenerative disease. We have previously shown that murine postmitotic rod photoreceptor precursor cells, identified by expression of the rod-specific transcription factor Nrl, are able to migrate into and integrate within the adult murine neural retina. However, their long-term survival has yet to be determined. Here, we found that integrated Nrl.gfp(+ve) photoreceptors were present up to 12 months post-transplantation, albeit in significantly reduced numbers. Surviving cells had rod-like morphology, including inner/outer segments and spherule synapses. In a minority of eyes, we observed an early, marked reduction in integrated photoreceptors within 1 month post-transplantation, which correlated with increased numbers of amoeboid macrophages, indicating acute loss of transplanted cells due to an inflammatory response. In the majority of transplants, similar numbers of integrated cells were observed between 1 and 2 months post-transplantation. By 4 months, however, we observed a significant decrease in integrated cell survival. Macrophages and T cells were present around the transplantation site, indicating a chronic immune response. Immune suppression of recipients significantly increased transplanted photoreceptor survival, indicating that the loss observed in unsuppressed recipients resulted from T cell-mediated host immune responses. Thus, if immune responses are modulated, correctly integrated transplanted photoreceptors can survive for extended periods of time in hosts with partially mismatched H-2 haplotypes. These findings suggest that autologous donor cells are optimal for therapeutic approaches to repair the neural retina, though with immune suppression nonautologous donors may be effective.