Ariel Wilson

The molecular basis of retinal ganglion cell death in glaucoma.

Glaucoma is a group of diseases characterized by progressive optic nerve degeneration that results in visual field loss and irreversible blindness. A crucial element in the pathophysiology of all forms of glaucoma is the death of retinal ganglion cells (RGCs), a population of CNS neurons with their soma in the inner retina and axons in the optic nerve. Strategies that delay or halt RGC loss have been recognized as potentially beneficial to preserve vision in glaucoma; however, the success of these approaches depends on an in-depth understanding of the mechanisms that lead to RGC dysfunction and death. In recent years, there has been an exponential increase in valuable information regarding the molecular basis of RGC death stemming from animal models of acute and chronic optic nerve injury as well as experimental glaucoma. The emerging landscape is complex and points at a variety of molecular signals - acting alone or in cooperation - to promote RGC death. These include: axonal transport failure, neurotrophic factor deprivation, toxic pro-neurotrophins, activation of intrinsic and extrinsic apoptotic signals, mitochondrial dysfunction, excitotoxic damage, oxidative stress, misbehaving reactive glia and loss of synaptic connectivity. Collectively, this body of work has considerably updated and expanded our view of how RGCs might die in glaucoma and has revealed novel, potential targets for neuroprotection.

Gene therapy for retinal ganglion cell neuroprotection in glaucoma

Glaucoma is the leading cause of irreversible blindness worldwide. The primary cause of glaucoma is not known, but several risk factors have been identified, including elevated intraocular pressure and age. Loss of vision in glaucoma is caused by the death of retinal ganglion cells (RGCs), the neurons that convey visual information from the retina to the brain. Therapeutic strategies aimed at delaying or halting RGC loss, known as neuroprotection, would be valuable to save vision in glaucoma. In this review, we discuss the significant progress that has been made in the use of gene therapy to understand mechanisms underlying RGC degeneration and to promote the survival of these neurons in experimental models of optic nerve injury.

ASPP1/2 Regulate p53-Dependent Death of Retinal Ganglion Cells through PUMA and Fas/CD95 Activation In Vivo

The transcription factor p53 mediates neuronal death in a variety of stress-related and neurodegenerative conditions. The proapoptotic activity of p53 is tightly regulated by the apoptosis-stimulating proteins of p53 (ASPP) family members: ASPP1 and ASPP2. However, whether ASPP1/2 play a role in the regulation of p53-dependent neuronal death in the CNS is currently unknown. To address this, we asked whether ASPP1/2 contribute to the death of retinal ganglion cells (RGCs) using in vivo models of acute optic nerve damage in mice and rats. Here, we show that p53 is activated in RGCs soon after injury and that axotomy-induced RGC death is attenuated in p53 heterozygote and null mice. We demonstrate that ASPP1/2 proteins are abundantly expressed by injured RGCs, and that short interfering (si)RNA-based ASPP1 or ASPP2 knockdown promotes robust RGC survival. Comparative gene expression analysis revealed that siASPP-mediated downregulation of p53-upregulated-modulator-of-apoptosis (PUMA), Fas/CD95, and Noxa depends on p53 transcriptional activity. Furthermore, siRNA against PUMA or Fas/CD95 confers neuroprotection, demonstrating a functional role for these p53 targets in RGC death. Our study demonstrates a novel role for ASPP1 and ASPP2 in the death of RGCs and provides evidence that blockade of the ASPP–p53 pathway is beneficial for central neuron survival after axonal injury.

MicroRNA signatures in vitreous humour and plasma of patients with exudative AMD.

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide affecting individuals over the age of 50. The neovascular form (NV AMD) is characterized by choroidal neovascularization (CNV) and responsible for the majority of central vision impairment. Using non-biased microRNA arrays and individual TaqMan qPCRs, we profiled miRNAs in the vitreous humour and plasma of patients with NV AMD. We identified a disease-associated increase in miR-146a and a decrease in miR-106b and miR-152 in the vitreous humour which was reproducible in plasma. Moreover, miR-146a/miR-106b ratios discriminated patients with NV AMD with an area under the Receiver Operating Characteristic curve (ROC AUC) of 0,977 in vitreous humour and 0,915 in plasma suggesting potential for a blood-based diagnostic. Furthermore, using the AMD Gene Consortium (AGC) we mapped a NV AMD-associated SNP (rs1063320) in a binding site for miR-152-3p in the HLA-G gene. The relationship between our detected miRNAs and NV AMD related genes was also investigated using gene sets derived from the Ingenuity Pathway Analysis (IPA). To our knowledge, our study is the first to correlate vitreal and plasma miRNA signatures with NV AMD, highlighting potential future worth as biomarkers and providing insight on NV AMD pathogenesis.

Inhibitor of Apoptosis-Stimulating Protein of p53 (iASPP) Is Required for Neuronal Survival after Axonal Injury

The transcription factor p53 mediates the apoptosis of post-mitotic neurons exposed to a wide range of stress stimuli. The apoptotic activity of p53 is tightly regulated by the apoptosis-stimulating proteins of p53 (ASPP) family members: ASPP1, ASPP2 and iASPP. We previously showed that the pro-apoptotic members ASPP1 and ASPP2 contribute to p53-dependent death of retinal ganglion cells (RGCs). However, the role of the p53 inhibitor iASPP in the central nervous system (CNS) remains to be elucidated. To address this, we asked whether iASPP contributes to the survival of RGCs in an in vivo model of acute optic nerve damage. We demonstrate that iASPP is expressed by injured RGCs and that iASPP phosphorylation at serine residues, which increase iASPP affinity towards p53, is significantly reduced following axotomy. We show that short interference RNA (siRNA)-induced iASPP knockdown exacerbates RGC death, whereas adeno-associated virus (AAV)-mediated iASPP expression promotes RGC survival. Importantly, our data also demonstrate that increasing iASPP expression in RGCs downregulates p53 activity and blocks the expression of pro-apoptotic targets PUMA and Fas/CD95. This study demonstrates a novel role for iASPP in the survival of RGCs, and provides further evidence of the importance of the ASPP family in the regulation of neuronal loss after axonal injury.

Gut microbiota influences pathological angiogenesis in obesity-driven choroidal neovascularization.

Age‐related macular degeneration in its neovascular form (NV AMD) is the leading cause of vision loss among adults above the age of 60. Epidemiological data suggest that in men, overall abdominal obesity is the second most important environmental risk factor after smoking for progression to late‐stage NV AMD. To date, the mechanisms that underscore this observation remain ill‐defined. Given the impact of high‐fat diets on gut microbiota, we investigated whether commensal microbes influence the evolution of AMD. Using mouse models of NV AMD, microbiotal transplants, and other paradigms that modify the gut microbiome, we uncoupled weight gain from confounding factors and demonstrate that high‐fat diets exacerbate choroidal neovascularization (CNV) by altering gut microbiota. Gut dysbiosis leads to heightened intestinal permeability and chronic low‐grade inflammation characteristic of inflammaging with elevated production of IL‐6, IL‐1β, TNF‐α, and VEGF‐A that ultimately aggravate pathological angiogenesis.

Truncated netrin-1 contributes to pathological vascular permeability in diabetic retinopathy

Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in the working-age population. Impaired blood-retinal barrier function leads to macular edema that is closely associated with the deterioration of central vision. We previously demonstrated that the neuronal guidance cue netrin-1 activates a program of reparative angiogenesis in microglia within the ischemic retina. Here, we provide evidence in both vitreous humor of diabetic patients and in retina of a murine model of diabetes that netrin-1 is metabolized into a bioactive fragment corresponding to domains VI and V of the full-length molecule. In contrast to the protective effects of full-length netrin-1 on retinal microvasculature, the VI-V fragment promoted vascular permeability through the uncoordinated 5B (UNC5B) receptor. The collagenase matrix metalloprotease 9 (MMP-9), which is increased in patients with diabetic macular edema, was capable of cleaving netrin-1 into the VI-V fragment. Thus, MMP-9 may release netrin-1 fragments from the extracellular matrix and facilitate diffusion. Nonspecific inhibition of collagenases or selective inhibition of MMP-9 decreased pathological vascular permeability in a murine model of diabetic retinal edema. This study reveals that netrin-1 degradation products are capable of modulating vascular permeability, suggesting that these fragments are of potential therapeutic interest for the treatment of DR.

Neuropilin-1 expression in adipose tissue macrophages protects against obesity and metabolic syndrome

Neuropilin-1–expressing adipose tissue macrophages are critical mediators of protective inflammation during weight gain. Keeping the calm in the adipose tissue Obesity is associated with chronic inflammation and accumulation of immune cells in adipose tissue. Here, Wilson et al. report that macrophages that express neuropilin-1 (NRP1) play an essential role in limiting obesity-associated inflammation. In mice placed on a high-fat diet, deletion of NRP1 in myeloid cells accelerated both weight gain and development of insulin resistance. The authors found that NRP1 regulated fatty acid uptake in macrophages and that deficiency of NRP1 skewed their metabolism toward glycolysis, which is associated with a more aggressive inflammation. The studies add to the growing recognition of the importance of immune cells in obesity and other metabolic syndromes. Obesity gives rise to metabolic complications by mechanisms that are poorly understood. Although chronic inflammatory signaling in adipose tissue is typically associated with metabolic deficiencies linked to excessive weight gain, we identified a subset of neuropilin-1 (NRP1)–expressing myeloid cells that accumulate in adipose tissue and protect against obesity and metabolic syndrome. Ablation of NRP1 in macrophages compromised lipid uptake in these cells, which reduced substrates for fatty acid β-oxidation and shifted energy metabolism of these macrophages toward a more inflammatory glycolytic metabolism. Conditional deletion of NRP1 in LysM Cre-expressing cells leads to inadequate adipose vascularization, accelerated weight gain, and reduced insulin sensitivity even independent of weight gain. Transfer of NRP1+ hematopoietic cells improved glucose homeostasis, resulting in the reversal of a prediabetic phenotype. Our findings suggest a pivotal role for adipose tissue–resident NRP1+-expressing macrophages in driving healthy weight gain and maintaining glucose tolerance.

Neurons and guidance cues in retinal vascular diseases.

Retinal vasculopathies such as diabetic retinopathy (DR), retinopathy of prematurity (ROP), and age-related macular degeneration (AMD) are the most common causes of vision loss in the industrialized world. According to the National Eye Institute, 7.7 million Americans are affected by DR, an additional 10 million have AMD, and of the 3.9 million infants born annually in the U.S., ∼15 000 are affected to some degree by ROP. In wet AMD, unchecked proliferation of choroidal vessels leads to photoreceptor compromise and vision loss. In ischemic retinopathies such as ROP and DR, degeneration of retinal vessels is followed by misguided pathological neovascularization. Recent studies on DR, ROP and AMD have revealed the paramount role for neurons and neuronal guidance cues in disease progression [1-6]. During retinal embryogenesis, coordinated interplay between neurons, blood vessels and immune cells is critical for proper retinal development. Although neurovascular and neuroimmune crosstalk shapes vascular development in the retina, it has received limited attention in disease etiology. A better understanding of this crosstalk may provide novel druggable targets to counter vasodegenerative and vasoproliferative eye disease. Here we briefly highlight new evidence suggesting that CNS neurons and guidance cues secreted by neurons such as retinal ganglion cells (RGCs) and photoreceptors have an inherent ability to influence vascular and immune responses in retinopathies. Photoreceptors were likely the first neurons suggested to influence the progression of pathological retinal neovascularization. For instance, a negative correlation was drawn between incidences of retinitis pigmentosa, which causes photoreceptor degeneration, and proliferative DR [7]. Further investigation demonstrated that retinal neovascularization associated with long-standing diabetes mellitus spontaneously regressed with the onset of retinitis pigmentosa [8]. This observation held true in animal models where mice with genetically ablated photoreceptors failed to mount reactive retinal neovascularization in a model of oxygen-induced retinopathy (OIR), which mimics the vasodegenerative phase of ROP and loosely represents the vasoproliferative phases of ROP and DR [8]. These studies draw a link between neuronal energy demand and retinal neovascularization. A mechanism via which central neurons communicate with their environment is through production of classical neuronal guidance cues such as semaphorins, netrins, and ephrins. These proteins are now widely regarded to have angiogenic and inflammatory functions, underscoring their pleiotropic nature. While these signaling proteins were initially thought to exclusively pattern the nervous system, it is now clear they also play a critical role in blood vessel development and influence the immune response during embryogenesis, and tissue homeostasis in adulthood. If discovered by an immunologist, they would have been classified as cytokines, by a vascular biologist, they would have been labeled angiomodulatory factors. In addition to photoreceptors, RGCs have the propensity to significantly influence their retinal angiogenesis (either positively [3] or negatively [4]), and are directly apposed to the retinal vasculature that degenerates in DR and ROP. For example, in the mouse model of OIR, the guidance cue SEMA3A is secreted by hypoxic neurons, repelling regenerating vessels from the most ischemic regions of the retina [6]. Silencing Sema3A enhances physiological vascular regeneration of the hypoxic retina [6]. In non-proliferative DR, SEMA3A is produced by RGCs in hyperglycemic conditions and contributes to inner blood-retinal barrier disruption and consequent macular edema [1]. Therapeutic neutralization of SEMA3A reduces pathological retinal vascular permeability [1]. In addition to its influence on blood vessels, SEMA3A also affects immune cells such as mononuclear phagocytes (microglia/macrophages) that are central to the progression of proliferative retinopathies. While cytokine signatures responsible for retinal inflammation in DR and ROP are well characterized (notably IL-4, IL-6 and TNFα), the pro-inflammatory properties of neuronally expressed SEMA3A (and VEGF) in retinopathies highlight their worth as therapeutic targets. Hypoxic retinal neurons secrete SEMA3A and VEGF, attracting pro-angiogenic mononuclear phagocytes to sites of pathological neovascularization [2]. These mononuclear phagocytes infiltrate the retina, take on a microglial phenotype and actively partake in vascular degeneration and later pathological angiogenesis [2]. Therapeutic inhibition of SEMA3A (and VEGF) consequently reduces ischemia-driven retinal inflammation [2]. In counterpart, certain guidance cues are critical for maintenance of retinal homeostasis. Under physiological conditions, Netrin-1 polarizes retinal microglia towards a reparative phenotype [3]. In retinopathy, ischemia activates pathways of ER-Stress that degrade Netrin-1 and hence short circuit reparative neuroimmune communication. Degradation ofNetrin-1 by the IRE1α arm of ER stress impedes physiological vascular regeneration [3]. Furthermore, vasorepellent SEMA3F is expressed in healthy, avascular outer layers of mouse and human retinas, at the photoreceptor/retinal pigment epithelium (RPE) interface [5]. SEMA3F is significantly downregulated in the RPE layer of patients with AMD showing pathological choroidal neovascularization [5]. Reduction in levels of SEMA3F in the outer retina may therefore compromise a chemical barrier that maintains the photoreceptor layer avascular. In sum, there is growing evidence that neuronal guidance cues actively partake in retinal vasculopathies such as ROP, DR and wet AMD. While current therapeutic strategies for these blinding diseases focus largely on blocking pathological angiogenesis through inhibition of VEGF, deleterious off-target effects are emerging due to the vaso- and neurotrophic properties of this factor. Pharmacological modulation of guidance cues such as semaphorins and netrins may offer alternative strategies to restore healthy functional retinal vasculature to ischemic zones of the retina and consequently reduce the destructive inflammation associated with these diseases.

in vivo laser-mediated retinal ganglion cell optoporation using KV1.1 conjugated gold nanoparticles

Vision loss caused by retinal diseases affects hundreds of millions of individuals worldwide. The retina is a delicate central nervous system tissue stratified into layers of cells with distinct roles. Currently, there is a void in treatments that selectively target diseased retinal cells, and current therapeutic paradigms present complications associated with off-target effects. Herein, as a proof of concept, we introduce an in vivo method using a femtosecond laser to locally optoporate retinal ganglion cells (RGCs) targeted with functionalized gold nanoparticles (AuNPs). We provide evidence that AuNPs functionalized with an antibody toward the cell-surface voltage-gated K+ channel subunit KV1.1 can selectively deliver fluorescently tagged siRNAs or fluorescein isothiocyanate-dextran dye into retinal cells when irradiated with an 800 nm 100 fs laser. Importantly, neither AuNP administration nor irradiation resulted in RGC death. This system provides a novel, non-viral-based approach that has the potential to selectively target retinal cells in diseased regions while sparing healthy areas and may be harnessed in future cell-specific therapies for retinal degenerative diseases.