Evaluating the performance of OCT in assessing static and potential dynamic properties of the retinal ganglion cells and nerve fiber bundles in the living mouse eye.

Glaucoma is a group of eye diseases characterized by the thinning of the retinal nerve fiber layer (RNFL), which is primarily caused by the progressive death of retinal ganglion cells (RGCs). Precise monitoring of these changes at a cellular resolution in living eyes is significant for glaucoma research. In this study, we aimed to assess the effectiveness of temporal speckle averaging optical coherence tomography (TSA-OCT) and dynamic OCT (dOCT) in examining the static and potential dynamic properties of RGCs and RNFL in living mouse eyes.

Retrolaminar Demyelination of Structurally Intact Axons in Nonhuman Primate Experimental Glaucoma.

Purpose: To determine if structurally intact, retrolaminar optic nerve (RON) axons are demyelinated in nonhuman primate (NHP) experimental glaucoma (EG).

Methods: Unilateral EG NHPs (n = 3) were perfusion fixed, EG and control eyes were enucleated, and foveal Bruch's membrane opening (FoBMO) 30° sectoral axon counts were estimated. Optic nerve heads were trephined; serial vibratome sections (VSs) were imaged and colocalized to a fundus photograph establishing their FoBMO location.

Glaucoma-associated Optineurin mutations increase transmitophagy in a vertebrate optic nerve.

We previously described a process referred to as transmitophagy where mitochondria shed by retinal ganglion cell (RGC) axons are transferred to and degraded by surrounding astrocytes in the optic nerve head of mice. Since the mitophagy receptor Optineurin (OPTN) is one of few large-effect glaucoma genes and axonal damage occurs at the optic nerve head in glaucoma, here we explored whether OPTN mutations perturb transmitophagy. Live-imaging of optic nerves revealed that diverse human mutant but not wildtype OPTN increase stationary mitochondria and mitophagy machinery and their colocalization within, and in the case of the glaucoma-associated OPTN mutations also outside of, RGC axons.

Dual leucine zipper kinase is necessary for retinal ganglion cell axonal regeneration in .

Retinal ganglion cell (RGC) axons of the African clawed frog, , unlike those of mammals, are capable of regeneration and functional reinnervation of central brain targets following injury. Here, we describe a tadpole optic nerve crush (ONC) procedure and assessments of brain reinnervation based on live imaging of RGC-specific transgenes which, when paired with CRISPR/Cas9 injections at the one-cell stage, can be used to assess the function of regeneration-associated genes in vivo in F0 animals. Using this assay, we find that map3k12, also known as dual leucine zipper kinase (Dlk), is necessary for RGC axonal regeneration and acts in a dose-dependent manner.

Optic Nerve Head Myelin-Related Protein, GFAP, and Iba1 Alterations in Non-Human Primates With Early to Moderate Experimental Glaucoma.

Purpose: The purpose of this study was to test if optic nerve head (ONH) myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), glial fibrillary acidic protein (GFAP), and ionized calcium binding adaptor molecule 1 (Iba1) proteins are altered in non-human primate (NHP) early/moderate experimental glaucoma (EG).

Methods: Following paraformaldehyde perfusion, control and EG eye ONH tissues from four NHPs were paraffin embedded and serially (5 µm) vertically sectioned. Anti-MBP, CNPase, GFAP, Iba1, and nuclear dye-stained sections were imaged using sub-saturating light intensities.

Neuron-astrocyte transmitophagy is altered in Alzheimer's disease.

Under physiological conditions in vivo astrocytes internalize and degrade neuronal mitochondria in a process called transmitophagy. Mitophagy is widely reported to be impaired in neurodegeneration but it is unknown whether and how transmitophagy is altered in Alzheimer's disease (AD). Here we report that the internalization of neuronal mitochondria is significantly increased in astrocytes isolated from AD mouse brains.

The basic science of optic nerve regeneration.

Diverse insults to the optic nerve result in partial to total vision loss as the axons of retinal ganglion cells are destroyed. In glaucoma, axons are injured at the optic nerve head; in other optic neuropathies, axons can be damaged along the entire visual pathway. In all cases, as mammals cannot regenerate injured central nervous system cells, once the axons are lost, vision loss is irreversible.

Analysis of the retinal capillary plexus layers in a murine model with diabetic retinopathy: effect of intravitreal injection of human CD34 bone marrow stem cells.

Background: Diabetic retinopathy is a retinal vasculopathy involving all three retinal capillary plexus layers. Since human CD34 bone marrow stem cells (BMSCs) have the potential to promote revascularization of ischemic tissue, this study tests the hypothesis that intravitreal injection of human CD34 BMSCs can have protective effects on all layers of the retinal vasculature in eyes with diabetic retinopathy.

Methods: Streptozotocin (STZ)-induced diabetic mice were injected intravitreally with 50,000 human CD34 BMSCs or phosphate-buffered saline (PBS) into the right eye.

The remote enhancer provides transcriptional robustness during retinal ganglion cell development.

The retinal ganglion cell (RGC) competence factor ATOH7 is dynamically expressed during retinal histogenesis. transcription is controlled by a promoter-adjacent primary enhancer and a remote shadow enhancer (SE). Deletion of the human SE causes nonsyndromic congenital retinal nonattachment (NCRNA) disease, characterized by optic nerve aplasia and total blindness.

Programmed switch in the mitochondrial degradation pathways during human retinal ganglion cell differentiation from stem cells is critical for RGC survival.

Retinal ganglion cell (RGC) degeneration is the root cause for vision loss in glaucoma as well as in other forms of optic neuropathy. A variety of studies have implicated abnormal mitochondrial quality control (MQC) as contributing to RGC damage and degeneration in optic neuropathies. The ability to differentiate human pluripotent stem cells (hPSCs) into RGCs provides an opportunity to study RGC MQC in great detail.

Effects of intravitreal injection of human CD34 bone marrow stem cells in a murine model of diabetic retinopathy.

Human CD34  stem cells are mobilized from bone marrow to sites of tissue ischemia and play an important role in tissue revascularization. This study used a murine model to test the hypothesis that intravitreal injection of human CD34  stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy.

Autocrine Mfge8 Signaling Prevents Developmental Exhaustion of the Adult Neural Stem Cell Pool.

Adult neurogenesis, arising from quiescent radial-glia-like neural stem cells (RGLs), occurs throughout life in the dentate gyrus. How neural stem cells are maintained throughout development to sustain adult mammalian neurogenesis is not well understood. Here, we show that milk fat globule-epidermal growth factor (EGF) 8 (Mfge8), a known phagocytosis factor, is highly enriched in quiescent RGLs in the dentate gyrus.

Neuroinflammation in glaucoma: A new opportunity.

Mounting evidence suggests neuroinflammation is a key process in glaucoma, yet the precise roles are not known. Understanding these complex processes, which may also be a key in other common neurodegenerations such as Alzheimer's disease, will lead to targeted therapeutics for a disease that affects as many as 80 million people worldwide. Here, we define neuroinflammation as any immune-relevant response by a variety of cell types including astrocytes, microglia, and peripherally derived cells occurring in the optic nerve head and/or retina.

Endocannabinoid signaling enhances visual responses through modulation of intracellular chloride levels in retinal ganglion cells.

Type 1 cannabinoid receptors (CB1Rs) are widely expressed in the vertebrate retina, but the role of endocannabinoids in vision is not fully understood. Here, we identified a novel mechanism underlying a CB1R-mediated increase in retinal ganglion cell (RGC) intrinsic excitability acting through AMPK-dependent inhibition of NKCC1 activity. Clomeleon imaging and patch clamp recordings revealed that inhibition of NKCC1 downstream of CB1R activation reduces intracellular Cl(-) levels in RGCs, hyperpolarizing the resting membrane potential.

Translational profiling of retinal ganglion cell optic nerve regeneration in Xenopus laevis.

Unlike adult mammals, adult frogs regrow their optic nerve following a crush injury, making Xenopus laevis a compelling model for studying the molecular mechanisms that underlie neuronal regeneration. Using Translational Ribosome Affinity Purification (TRAP), a method to isolate ribosome-associated mRNAs from a target cell population, we have generated a transcriptional profile by RNA-Seq for retinal ganglion cells (RGC) during the period of recovery following an optic nerve injury. Based on bioinformatic analysis using the Xenopus laevis 9.

Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.

Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology.

Evidence for accelerated tauopathy in the retina of transgenic P301S tau mice exposed to repetitive mild traumatic brain injury.

Chronic traumatic encephalopathy (CTE) is associated with repetitive mild traumatic brain injury (mTBI) in the context of contact and collision sports, but not all exposed individuals develop this condition. In addition, experiments in animal models in several laboratories have shown that non-transgenic mice do not develop tauopathy after exposure to repetitive mTBI schedules. It is thus reasonable to assume that genetic factors may play an etiological role in the development of CTE.

Astrocytes phagocytose focal dystrophies from shortening myelin segments in the optic nerve of Xenopus laevis at metamorphosis.

Oligodendrocytes can adapt to increases in axon diameter through the addition of membrane wraps to myelin segments. Here, we report that myelin segments can also decrease their length in response to optic nerve (ON) shortening during Xenopus laevis metamorphic remodeling. EM-based analyses revealed that myelin segment shortening is accomplished by focal myelin-axon detachments and protrusions from otherwise intact myelin segments.

Discovery and implications of transcellular mitophagy.

The mitochondrial quality control system regulating mitochondria biogenesis, dynamics, and degradation has been extensively studied because of its roles in normal cell homeostasis and dysfunction due to aging or disease. Mitochondria degradation is generally thought to occur by autophagy and has therefore been viewed as a cell-autonomous process. In a recent study, we demonstrated that a large fraction of retinal ganglion cell mitochondria undergo lysosomal degradation within the astrocytes of the optic nerve head.

Repetitive mild traumatic brain injury with impact acceleration in the mouse: Multifocal axonopathy, neuroinflammation, and neurodegeneration in the visual system.

Repetitive mild traumatic brain injury (mTBI) is implicated in chronic neurological illness. The development of animal models of repetitive mTBI in mice is essential for exploring mechanisms of these chronic diseases, including genetic vulnerability by using transgenic backgrounds. In this study, the rat model of impact acceleration (IA) was redesigned for the mouse cranium and used in two clinically relevant repetitive mTBI paradigms.

Transcellular degradation of axonal mitochondria.

It is generally accepted that healthy cells degrade their own mitochondria. Here, we report that retinal ganglion cell axons of WT mice shed mitochondria at the optic nerve head (ONH), and that these mitochondria are internalized and degraded by adjacent astrocytes. EM demonstrates that mitochondria are shed through formation of large protrusions that originate from otherwise healthy axons.

Synucleins antagonize endoplasmic reticulum function to modulate dopamine transporter trafficking.

Synaptic re-uptake of dopamine is dependent on the dopamine transporter (DAT), which is regulated by its distribution to the cell surface. DAT trafficking is modulated by the Parkinson's disease-linked protein alpha-synuclein, but the contribution of synuclein family members beta-synuclein and gamma-synuclein to DAT trafficking is not known. Here we use SH-SY5Y cells as a model of DAT trafficking to demonstrate that all three synucleins negatively regulate cell surface distribution of DAT.

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs.

Cell type-specific translational profiling in the Xenopus laevis retina.

Background: Translating Ribosome Affinity Purification (TRAP), a method recently developed to generate cell type-specific translational profiles, relies on creating transgenic lines of animals in which a tagged ribosomal protein is placed under regulatory control of a cell type-specific promoter. An antibody is then used to affinity purify the tagged ribosomes so that cell type-specific mRNAs can be isolated from whole tissue lysates.

Results: Here, cell type-specific transgenic lines were generated to enable TRAP studies for retinal ganglion cells and rod photoreceptors in the Xenopus laevis retina.

Selective pattern of motor system damage in gamma-synuclein transgenic mice mirrors the respective pathology in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterised by substantial loss of both upper and lower motor neuron function, with sensory and cognitive systems less affected. Though heritable forms of the disease have been described, the vast majority of cases are sporadic with poorly defined underlying pathogenic mechanisms. Here we demonstrate that the neurological pathology induced in transgenic mice by overexpression of γ-synuclein, a protein not previously associated with ALS, recapitulates key features of the disease, namely selective damage and loss of discrete populations of upper and lower motor neurons and their axons, contrasted by limited effects upon the sensory system.