Elevated intraocular pressure (IOP) is a significant risk factor for glaucoma, causing structural and functional damage to the eye. Increased IOP compromises the metabolic and structural integrity of retinal ganglion cell (RGC) axons, leading to progressive degeneration and influencing the ocular immune response. This study investigated early cellular and molecular changes in the retina and optic nerve (ON) following ocular hypertension (OHT). A pigmented rat model was used, with OHT induced through low-temperature cauterization of the limbal vascular plexus. To assess the effects at early time points after OHT, transmission electron microscopy (TEM) was employed to analyze ultrastructural changes in the retina and ON, while immunofluorescence was used to evaluate cellular responses. Flow cytometry was used to examine alterations in immune-cell populations. Within 24 h post-OHT, ultrastructural changes were detected in the cytoplasm of RGCs, indicating early cellular alterations undetectable by conventional microscopy. These ultrastructural modifications progressed further at 48 and 72 h, despite the absence of overt RGC loss or disruptions in retinal layer integrity. Changes in the axons and nodes of Ranvier were evident within the first 24 h after ocular hypertension, becoming more pronounced by 72 h. These findings offer novel insights into the early pathogenesis of glaucoma, highlighting critical early impacts that could guide the development of new therapeutic strategies to prevent irreversible RGC loss.

Retinal ganglion cells (RGCs) are the projection neurons of the retina, and their death promotes an irreversible blindness. Several factors were described to control their genesis during retinal development. These include Atoh7, a major orchestrator of the RGC program, and downstream targets of this transcription factor, including Pou4f factors, that in turn regulate key aspects of terminal differentiation. The absence of POU4F family genes results in defects in RGC differentiation, aberrant axonal elaboration and, ultimately, RGC death. This confirms the requirement of POU4F factors for RGC development and survival, with a crucial role in regulating RGC axon outgrowth and pathfinding. Here, we have investigated in vivo whether ectopic Pou4f2 expression in late retinal progenitor cells (late RPCs) is sufficient to induce the generation of cells with RGC properties, including long-range axon projections. We show that Pou4f2 overexpression generates RGC-like cells that share morphological and transcriptional features with RGCs that are normally generated during early development and extend axonal projections up to the brain. In conclusion, these results show that POU4F2 alone is sufficient to promote the crucial properties of projection neurons that arise from retinal progenitors outside their developmental window.

Glaucoma, the leading cause of irreversible blindness worldwide, is a neurodegenerative disease characterized by chronic axonal damages and progressive loss of retinal ganglion cells, with increased intraocular pressure (IOP) as the primary risk factor. While current treatments focus solely on reducing IOP, understanding glaucoma through experimental models is essential for developing new therapeutic strategies and biomarkers for early diagnosis. Our research group developed an ocular hypertension rat model based on limbal plexus cautery, which provides significant glaucomatous neurodegeneration up to four weeks after injury. We evaluated long-term morphological, functional, and vascular alterations in this model. Our results showed that transient ocular hypertension, lasting approximately one week, can lead to progressive increase in optic nerve cupping and retinal ganglion cells loss. Remarkably, the pressure insult caused several vascular changes, such as arteriolar and venular thinning, and permanent choroidal vascular swelling. This study provides evidence of the longitudinal effects of a pressure insult on retinal structure and function using clinical modalities and techniques. The multifactorial changes reported in this model resemble the complex retinal ganglion cell degeneration found in glaucoma patients, and therefore may also provide a unique tool for the development of novel interventions to either halt or slow down disease progression.

Glaucoma is a progressive neurodegenerative disease characterized by the gradual loss of retinal ganglion cells (RGCs), leading to visual impairment and potential blindness.1 The degeneration of RGCs is often associated with increased intraocular pressure (IOP), resulting from an imbalance in fluid dynamics within the eye. Elevated IOP leads to mechanical stress and compromises blood flow to the optic nerve, thereby triggering a cascade of neurodegenerative events. The various types of glaucoma are the major cause of irreversible vision loss worldwide.

Glaucoma is a chronic and multifactorial neurodegenerative disease marked by structural damage to the optic nerve with axonal loss, progressive retinal ganglion cell degeneration, and optic disc excavation. Both high intraocular pressure and aging are important risk factors, but not essential to the progression of glaucomatous neurodegeneration. Current treatments are based on controlling intraocular pressure, which is not always effective in avoiding the progression of visual loss. In this sense, novel therapeutic strategies to glaucoma should aim to promote the neuroprotection of both the cell soma of retinal ganglion cells and the axons of the optic nerve. Gene therapy is a new therapeutical approach to glaucoma with a great capacity to overcome neurodegeneration. It consists of the transfer of exogenous genetic material to target cells with a therapeutic purpose. Gene therapy strategies for glaucoma include both the neuroprotection aiming to prevent cell soma and axonal loss and the regeneration of optic nerve axons. In this chapter, we review the most promising current gene therapies for glaucoma that address the various aspects of glaucoma pathology. We also discuss the potential of combining neuroprotective and regenerative strategies to reach a synergic effect for the treatment of glaucoma.

IntroductionInfection with human T cell lymphotropic virus type 1 (HTLV-1) is endemic in Brazil and is linked with pro-inflammatory conditions including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic neuroinflammatory incapacitating disease that culminates in loss of motor functions. The mechanisms underlying the onset and progression of HAM/TSP are incompletely understood. Previous studies have demonstrated that inflammation and infectious agents can affect the expression of cellular prion protein (PrPC) in immune cells.MethodsHere, we investigated whether HTLV-1 infection affected PrPC content in cell lines and primary CD4+cells in vitro using flow cytometry and western blot assays.ResultsWe found that HTLV-1 infection decreased the expression levels of PrPC and HTLV-1 Orf I encoded p12, an endoplasmic reticulum resident protein also known to affect post-transcriptionally cellular proteins such as MHC-class I and the IL-2 receptor. In addition, we observed a reduced percentage of CD4+ T cells from infected individuals expressing PrPC, which was reflected by IFN type II but not IL-17 expression.DiscussionThese results suggested that PrPC downregulation, linked to both HTLV-1 p12 and IFN-γ expression in CD4+ cells, may play a role in the neuropathogenesis of HTLV-1 infection.

Recombinant adeno-associated viral vectors (rAAV) are the safest and most effective gene delivery platform to drive the treatment of many inherited eye disorders in well-characterized animal models. The use in rAAV of ubiquitous promoters derived from viral sequences such as CMV/CBA (chicken β-actin promoter with cytomegalovirus enhancer) can lead to unwanted side effects such as pro-inflammatory immune responses and retinal cytotoxicity, thus reducing therapy efficacy. Thus, an advance in gene therapy is the availability of small promoters, that potentiate and direct gene expression to the cell type of interest, with higher safety and efficacy. In this study, we used six human mini-promoters packaged in rAAV2 quadruple mutant (Y–F) to test for transduction of the rat retina after intravitreal injection. After four weeks, immunohistochemical analysis detected GFP-labeled cells in the ganglion cell layer (GCL) for all constructs tested. Among them, Ple25sh1, Ple25sh2 and Ple53 promoted a widespread reporter-transgene expression in the GCL, with an increased number of GFP-expressing retinal ganglion cells when compared with the CMV/CBA vector. Moreover, Ple53 provided the strongest levels of GFP fluorescence in both cell soma and axons of retinal ganglion cells (RGCs) without any detectable adverse effects in retina function. Remarkably, a nearly 50-fold reduction in the number of intravitreally injected vector particles containing Ple53 promoter, still attained levels of transgene expression similar to CMV/CBA. Thus, the tested MiniPs show great potential for protocols of retinal gene therapy in therapeutic applications for retinal degenerations, especially those involving RGC-related disorders such as glaucoma.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

Purpose Based on our preview evidence that reduced nuclear content of the transcription factor Myc-associated protein X (MAX) is an early event associated with degeneration of retinal ganglion cells (RGCs), in the present study, our purpose was to test whether the overexpression of human MAX had a neuroprotective effect against RGC injury. Methods Overexpression of either MAX or green fluorescent protein (GFP) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs). Lister Hooded rats were used in three models of RGC degeneration: (1) cultures of retinal explants for 30 hours ex vivo from the eyes of 14-day-old rats that had received intravitreal injections of rAAV2-MAX or the control vector rAAV2-GFP at birth; (2) an optic nerve crush model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were operated on; and (3) an ocular hypertension (OHT) glaucoma model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were subject to cauterization of the limbal plexus. Cell death was estimated by detection of pyknotic nuclei and TUNEL technique and correlated with MAX immunocontent in an ex vivo model of retinal explants. MAX expression was detected by quantitative RT-PCR. In the OHT model, survival of RGCs was quantified by retrograde labeling with DiI or immunostaining for BRN3a at 14 days after in vivo injury. Functional integrity of RGCs was analyzed through pattern electroretinography, and damage to the optic nerve was examined in semithin sections. Results In all three models of RGC insult, gene therapy by overexpression of MAX prevented RGC death. Also, ON degeneration and electrophysiologic deficits were prevented in the OHT model. Conclusions Our experiments offer proof of concept for a novel neuroprotective gene therapy for glaucomatous neurodegeneration based on overexpression of MAX.

EDITORIAL article Front. Neurosci., 22 November 2022Sec. Neurodegeneration https://doi.org/10.3389/fnins.2022.1076486

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

Mitochondria are key players of aerobic respiration and the production of adenosine triphosphate and constitute the energetic core of eukaryotic cells. Furthermore, cells rely upon mitochondria homeostasis, the disruption of which is reported in pathological processes such as liver hepatotoxicity, cancer, muscular dystrophy, chronic inflammation, as well as in neurological conditions including Alzheimer's disease, schizophrenia, depression, ischemia and glaucoma. In addition to the well-known spontaneous cell-to-cell transfer of mitochondria, a therapeutic potential of the transplant of isolated, metabolically active mitochondria has been demonstrated in several in vitro and in vivo experimental models of disease. This review explores the striking outcomes achieved by mitotherapy thus far, and the most relevant underlying data regarding isolated mitochondria transplantation, including mechanisms of mitochondria intake, the balance between administration and therapy effectiveness, the relevance of mitochondrial source and purity and the mechanisms by which mitotherapy is gaining ground as a promising therapeutic approach.

Glaucoma leads to irreversible vision loss and current therapeutic strategies are often insufficient to prevent the progression of the disease and consequent blindness. Elevated intraocular pressure is an important risk factor, but not required for the progression of glaucomatous neurodegeneration. The demise of retinal ganglion cells represents the final common pathway of glaucomatous vision loss. Still, lifelong control of intraocular pressure is the only current treatment to prevent severe vision loss, although it frequently fails despite best practices. This scenario calls for the development of neuroprotective and pro-regenerative therapies targeting the retinal ganglion cells as well as the optic nerve. Several experimental studies have shown the potential of gene modulation as a tool for neuroprotection and regeneration. In this context, gene therapy represents an attractive approach as a persistent treatment for glaucoma. Viral vectors engineered to promote overexpression of a broad range of cellular factors have been shown to protect retinal ganglion cells and/or promote axonal regeneration in experimental models. Here, we review the mechanisms involved in glaucomatous neurodegeneration and regeneration in the central nervous system. Then, we point out the current limitations of gene therapy platforms and review a myriad of studies that use viral vectors to manipulate genes in retinal ganglion cells, as a strategy to promote neuroprotection and regeneration. Finally, we address the potential of combining neuroprotective and regenerative gene therapies as an approach to glaucomatous neurodegeneration.

Systemic multimorbidity is highly prevalent in the elderly and, remarkably, coexisting neuropathological markers of Alzheimer's (AD) and cerebrovascular (CVD) diseases are found at autopsy in most brains of patients clinically diagnosed as AD. Little is known on neurodegeneration peculiar to comorbidities, especially at early stages when pathogenesis may propagate at subclinical levels. We developed a novel in vitro model of comorbid CVD/AD in organotypic hippocampal cultures, by combining oxygen-glucose deprivation (OGD) and exposure to amyloid-A beta oligomers (A beta Os), both applied at levels subtoxic to neurons when used in isolation. We focused on synaptic proteins and the roles of glutamate receptors, which have been implicated in many basic and clinical approaches to either CVD or AD. Subtoxic insults by OGD and A beta Os synergized to reduce levels of synaptophysin (SYP) and PSD-95 without cell death, while effects of antagonists of either metabotropic or ionotropic glutamate receptors were distinct from reports in models of isolated CVD or AD. In particular, modulation of glutamate receptors differentially impacted SYP and PSD-95, and antagonists of a single receptor subtype had distinct effects when either isolated or combined. Our findings highlight the complexity of CVD/AD comorbidity, help understand variable responses to glutamate receptor antagonists in patients diagnosed with AD and may contribute to future development of therapeutics based on investigation of the pattern of progressive comorbidity.

Alcohol dependence is one of the main reasons for inpatient admission to psychiatric hospitals. The abuse of this chemical substance can cause modifications in our organism and among them, variations in the oxidative stress parameters. Therefore, the aim of this study is to evaluate patients admitted to a psychiatric hospital unit to treat alcohol dependence, comparing oxidative stress, renal and hepatic function parameters from the moment of admission to those obtained at discharge. Hepatic function was verified through gamma-glutamyl-transferase (GGT), alkaline-phosphatase (ALP), aspartate-aminotransferase (AST) and alanine-aminotransferase (ALT) activity measurements. Urea and creatinine serum levels were measured for kidney function evaluation. Oxidative stress was evaluated by superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), ferric reducing antioxidant power (FRAP) and malondialdehyde (MDA). Medications used during hospital stay were record and their influence over the measured parameters analyzed. Twenty-eight patients (82% male, 44 +/- 13 years old) were included in this study. A significant increase in BMI of patients after the period of hospitalization could be observed. There were reductions in creatinine, AST, ALT, GGT and ALP serum levels. SOD levels were lower at discharge, while GPx and FRAP presented higher levels. Chlorpromazine use showed influence over some hepatic function markers (ALT, GGT and ALP) and oxidative stress parameters (CAT and GPx); while carbamazepine use influenced GGT and FRAP. Patients on alcohol dependence treatment had significant improvements of renal and hepatic function parameters and higher GPx and FRAP values after the hospitalization period, which indicates reversion of alcohol effects over oxidative stress parameters.