Assessment of adaptive behavior in people with intellectual disabilities: Design and development of a new test battery.

Background: Adaptive behavior is an important characteristic of people with intellectual disabilities, and it has been associated with a person's performance in social and work contexts. Indeed, adaptive behavior denotes what a person does independently, without help, support, reminders, or prompts. In Peru, available measures of adaptive behavior are commercial; thus, there is a need for an open-access tool to assess the adaptive behavior of people with intellectual disabilities. For this reason, the aim of the study was to design and develop a new Adaptive Behavior Test Battery for people from 13 to 60 years old with intellectual disabilities who have an interest in being part of the economically active population. Methods: A cross-sectional design was defined, starting with a qualitative approach to designing and constructing the item pool for the test battery. Then, quantitative indexes Aiken's V for content validity and Krippendorff's alpha for inter-observer reliability were estimated, resulting in a first version of the three subscales that comprised the test battery. The initial versions were tested on a sample of 566 persons with intellectual disabilities from two regions of Peru: Lima (Coast) and San Martín (Jungle). The internal structure was analyzed under a factor analysis approach, along with internal consistency measures of reliability. Further analyses of invariance regarding gender, region, and age were carried out. Results: Three observer subscales were proposed: Daily living activities (11 items), Instrumental skills (4 items), and Communication (9 items). All subscales showed excellent psychometric properties denoted by the Aiken's V coefficient, Krippendorff's alpha, factor analysis, internal consistency analysis, and invariance analyses. Conclusion: The developed a new Adaptive Behavior Test Battery is a useful tool for the measurement of adaptive behavior and the monitoring of social and labor inclusion programs for people with intellectual disabilities.

Neurovascular dysfunction in glaucoma.

Retinal ganglion cells, the neurons that die in glaucoma, are endowed with a high metabolism requiring optimal provision of oxygen and nutrients to sustain their activity. The timely regulation of blood flow is, therefore, essential to supply firing neurons in active areas with the oxygen and glucose they need for energy. Many glaucoma patients suffer from vascular deficits including reduced blood flow, impaired autoregulation, neurovascular coupling dysfunction, and blood-retina/brain-barrier breakdown. These processes are tightly regulated by a community of cells known as the neurovascular unit comprising neurons, endothelial cells, pericytes, Müller cells, astrocytes, and microglia. In this review, the neurovascular unit takes center stage as we examine the ability of its members to regulate neurovascular interactions and how their function might be altered during glaucomatous stress. Pericytes receive special attention based on recent data demonstrating their key role in the regulation of neurovascular coupling in physiological and pathological conditions. Of particular interest is the discovery and characterization of tunneling nanotubes, thin actin-based conduits that connect distal pericytes, which play essential roles in the complex spatial and temporal distribution of blood within the retinal capillary network. We discuss cellular and molecular mechanisms of neurovascular interactions and their pathophysiological implications, while highlighting opportunities to develop strategies for vascular protection and regeneration to improve functional outcomes in glaucoma.

Pericyte dysfunction and loss of interpericyte tunneling nanotubes promote neurovascular deficits in glaucoma.

Reduced blood flow and impaired neurovascular coupling are recognized features of glaucoma, the leading cause of irreversible blindness worldwide, but the mechanisms underlying these defects are unknown. Retinal pericytes regulate microcirculatory blood flow and coordinate neurovascular coupling through interpericyte tunneling nanotubes (IP-TNTs). Using two-photon microscope live imaging of the mouse retina, we found reduced capillary diameter and impaired blood flow at pericyte locations in eyes with high intraocular pressure, the most important risk factor to develop glaucoma. We show that IP-TNTs are structurally and functionally damaged by ocular hypertension, a response that disrupted light-evoked neurovascular coupling. Pericyte-specific inhibition of excessive Ca2+ influx rescued hemodynamic responses, protected IP-TNTs and neurovascular coupling, and enhanced retinal neuronal function as well as survival in glaucomatous retinas. Our study identifies pericytes and IP-TNTs as potential therapeutic targets to counter ocular pressure-related microvascular deficits, and provides preclinical proof of concept that strategies aimed to restore intrapericyte calcium homeostasis rescue autoregulatory blood flow and prevent neuronal dysfunction.

AMPK hyperactivation promotes dendrite retraction, synaptic loss, and neuronal dysfunction in glaucoma.

BACKGROUND: The maintenance of complex dendritic arbors and synaptic transmission are processes that require a substantial amount of energy. Bioenergetic decline is a prominent feature of chronic neurodegenerative diseases, yet the signaling mechanisms that link energy stress with neuronal dysfunction are poorly understood. Recent work has implicated energy deficits in glaucoma, and retinal ganglion cell (RGC) dendritic pathology and synapse disassembly are key features of ocular hypertension damage. RESULTS: We show that adenosine monophosphate-activated protein kinase (AMPK), a conserved energy biosensor, is strongly activated in RGC from mice with ocular hypertension and patients with primary open angle glaucoma. Our data demonstrate that AMPK triggers RGC dendrite retraction and synapse elimination. We show that the harmful effect of AMPK is exerted through inhibition of the mammalian target of rapamycin complex 1 (mTORC1). Attenuation of AMPK activity restores mTORC1 function and rescues dendrites and synaptic contacts. Strikingly, AMPK depletion promotes recovery of light-evoked retinal responses, improves axonal transport, and extends RGC survival. CONCLUSIONS: This study identifies AMPK as a critical nexus between bioenergetic decline and RGC dysfunction during pressure-induced stress, and highlights the importance of targeting energy homeostasis in glaucoma and other neurodegenerative diseases.

Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells.

Small and cell-type restricted promoters are important tools for basic and preclinical research, and clinical delivery of gene therapies. In clinical gene therapy, ophthalmic trials have been leading the field, with over 50% of ocular clinical trials using promoters that restrict expression based on cell type. Here, 19 human DNA MiniPromoters were bioinformatically designed for rAAV, tested by neonatal intravenous delivery in mouse, and successful MiniPromoters went on to be tested by intravitreal, subretinal, intrastromal, and/or intravenous delivery in adult mouse. We present promoter development as an overview for each cell type, but only show results in detail for the recommended MiniPromoters: Ple265 and Ple341 (PCP2) ON bipolar, Ple349 (PDE6H) cone, Ple253 (PITX3) corneal stroma, Ple32 (CLDN5) endothelial cells of the blood-retina barrier, Ple316 (NR2E1) Müller glia, and Ple331 (PAX6) PAX6 positive. Overall, we present a resource of new, redesigned, and improved MiniPromoters for ocular gene therapy that range in size from 784 to 2484 bp, and from weaker, equal, or stronger in strength relative to the ubiquitous control promoter smCBA. All MiniPromoters will be useful for therapies involving small regulatory RNA and DNA, and proteins ranging from 517 to 1084 amino acids, representing 62.9-90.2% of human proteins.

New MiniPromoter Ple345 (NEFL) Drives Strong and Specific Expression in Retinal Ganglion Cells of Mouse and Primate Retina.

Retinal gene therapy is leading the neurological gene therapy field, with 32 ongoing clinical trials of recombinant adeno-associated virus (rAAV)-based therapies. Importantly, over 50% of those trials are using restricted promoters from human genes. Promoters that restrict expression have demonstrated increased efficacy and can limit the therapeutic to the target cells thereby reducing unwanted off-target effects. Retinal ganglion cells are a critical target in ocular gene therapy; they are involved in common diseases such as glaucoma, rare diseases such as Leber's hereditary optic neuropathy, and in revolutionary optogenetic treatments. Here, we used computational biology and mined the human genome for the best genes from which to develop a novel minimal promoter element(s) designed for expression in restricted cell types (MiniPromoter) to improve the safety and efficacy of retinal ganglion cell gene therapy. Gene selection included the use of the first available droplet-based single-cell RNA sequencing (Drop-seq) dataset, and promoter design was bioinformatically driven and informed by a wide range of genomics datasets. We tested seven promoter designs from four genes in rAAV for specificity and quantified expression strength in retinal ganglion cells in mouse, and then the single best in nonhuman primate retina. Thus, we developed a new human-DNA MiniPromoter, Ple345 (NEFL), which in combination with intravitreal delivery in rAAV9 showed specific and robust expression in the retinal ganglion cells of the nonhuman-primate rhesus macaque retina. In mouse, we also developed MiniPromoters expressing in retinal ganglion cells, the hippocampus of the brain, a pan neuronal pattern in the brain, and peripheral nerves. As single-cell transcriptomics such as Drop-seq become available for other cell types, many new opportunities for additional novel restricted MiniPromoters will present.

The glial cell modulator ibudilast attenuates neuroinflammation and enhances retinal ganglion cell viability in glaucoma through protein kinase A signaling.

Glaucoma is a neurodegenerative disease and the leading cause of irreversible blindness worldwide. Vision deficits in glaucoma result from the selective loss of retinal ganglion cells (RGC). Glial cell-mediated neuroinflammation has been proposed to contribute to disease pathophysiology, but whether this response is harmful or beneficial for RGC survival is not well understood. To test this, we characterized the role of ibudilast, a clinically approved cAMP phosphodiesterase (PDE) inhibitor with preferential affinity for PDE type 4 (PDE4). Here, we demonstrate that intraocular administration of ibudilast dampened macroglia and microglia reactivity in the retina and optic nerve hence decreasing production of proinflammatory cytokines in a rat model of ocular hypertension. Importantly, ibudilast promoted robust RGC soma survival, prevented axonal degeneration, and improved anterograde axonal transport in glaucomatous eyes without altering intraocular pressure. Intriguingly, ocular hypertension triggered upregulation of PDE4 subtype A in Müller glia, and ibudilast stimulated cAMP accumulation in these cells. Co-administration of ibudilast with Rp-cAMPS, a cell-permeable and non-hydrolysable cAMP analog that inhibits protein kinase A (PKA), completely blocked ibudilast-induced neuroprotection. Collectively, these data demonstrate that ibudilast, a safe and well-tolerated glial cell modulator, attenuates gliosis, decreases levels of proinflammatory mediators, and enhances neuronal viability in glaucoma through activation of the cAMP/PKA pathway. This study provides insight into PDE4 signaling as a potential target to counter the harmful effects associated with chronic gliosis and neuroinflammation in glaucoma.

Tau Accumulation, Altered Phosphorylation, and Missorting Promote Neurodegeneration in Glaucoma.

UNLABELLED: Glaucoma, the leading cause of irreversible blindness worldwide, is characterized by the selective death of retinal ganglion cells (RGCs). Ocular hypertension is the most significant known risk factor for developing the disease, but the mechanism by which elevated pressure damages RGCs is currently unknown. The axonal-enriched microtubule-associated protein tau is a key mediator of neurotoxicity in Alzheimer's disease and other tauopathies. Using a well characterized in vivo rat glaucoma model, we show an age-related increase in endogenous retinal tau that was markedly exacerbated by ocular hypertension. Early alterations in tau phosphorylation, characterized by epitope-dependent hyperphosphorylation and hypophosphorylation, correlated with the appearance of tau oligomers in glaucomatous retinas. Our data demonstrate the mislocalization of tau in the somatodendritic compartment of RGCs subjected to high intraocular pressure. In contrast, tau was depleted from RGC axons in the optic nerve of glaucomatous eyes. Importantly, intraocular administration of short interfering RNA against tau effectively reduced retinal tau accumulation and promoted robust survival of RGC somas and axons, supporting a critical role for tau alterations in ocular hypertension-induced neuronal damage. Our study reveals that glaucoma displays signature pathological features of tauopathies, including tau accumulation, altered phosphorylation, and missorting; and identifies tau as a novel target to counter RGC neurodegeneration in glaucoma and prevalent optic neuropathies. SIGNIFICANCE STATEMENT: In this study, we investigated the role of tau in retinal ganglion cell (RGC) damage in glaucoma. We demonstrate that high intraocular pressure leads to a rapid increase in endogenous retinal tau with altered phosphorylation profile and the formation of tau oligomers. Tau accumulation was primarily observed in RGC dendrites, while tau in RGC axons within the optic nerve was depleted. Attenuation of endogenous retinal tau using a targeted siRNA led to striking protection of RGC somas and axons from hypertension-induced damage. Our study identifies novel and substantial alterations of endogenous tau protein in glaucoma, including abnormal subcellular distribution, an altered phosphorylation profile, and neurotoxicity.

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice.

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Soluble Tumor Necrosis Factor Alpha Promotes Retinal Ganglion Cell Death in Glaucoma via Calcium-Permeable AMPA Receptor Activation.

Loss of vision in glaucoma results from the selective death of retinal ganglion cells (RGCs). Tumor necrosis factor α (TNFα) signaling has been linked to RGC damage, however, the mechanism by which TNFα promotes neuronal death remains poorly defined. Using an in vivo rat glaucoma model, we show that TNFα is upregulated by Müller cells and microglia/macrophages soon after induction of ocular hypertension. Administration of XPro1595, a selective inhibitor of soluble TNFα, effectively protects RGC soma and axons. Using cobalt permeability assays, we further demonstrate that endogenous soluble TNFα triggers the upregulation of Ca(2+)-permeable AMPA receptor (CP-AMPAR) expression in RGCs of glaucomatous eyes. CP-AMPAR activation is not caused by defects in GluA2 subunit mRNA editing, but rather reflects selective downregulation of GluA2 in neurons exposed to elevated eye pressure. Intraocular administration of selective CP-AMPAR blockers promotes robust RGC survival supporting a critical role for non-NMDA glutamate receptors in neuronal death. Our study identifies glia-derived soluble TNFα as a major inducer of RGC death through activation of CP-AMPARs, thereby establishing a novel link between neuroinflammation and cell loss in glaucoma. SIGNIFICANCE STATEMENT: Tumor necrosis factor α (TNFα) has been implicated in retinal ganglion cell (RGC) death, but how TNFα exerts this effect is poorly understood. We report that ocular hypertension, a major risk factor in glaucoma, upregulates TNFα production by Müller cells and microglia. Inhibition of soluble TNFα using a dominant-negative strategy effectively promotes RGC survival. We find that TNFα stimulates the expression of calcium-permeable AMPA receptors (CP-AMPAR) in RGCs, a response that does not depend on abnormal GluA2 mRNA editing but on selective downregulation of the GluA2 subunit by these neurons. Consistent with this, CP-AMPAR blockers promote robust RGC survival supporting a critical role for non-NMDA glutamate receptors in glaucomatous damage. This study identifies a novel mechanism by which glia-derived soluble TNFα modulates neuronal death in glaucoma.

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.