Adult Mouse Retina Research Articles

Stimulating the regenerative capacity of the human retina with proneural transcription factors in 3D cultures

Retinal diseases often lead to degeneration of specific retinal cell types with currently limited therapeutic options to replace the lost neurons. Previous studies have reported that overexpression of ASCL1 or combinations of proneural factors in Müller glia (MG) induce regeneration of functional neurons in the adult mouse retina. Recently, we applied the same strategy in dissociated cultures of fetal human MG and although we stimulated neurogenesis from MG, our effect in 2D cultures was modest and our analysis of newborn neurons was limited. In this study, we aimed to improve our MG reprogramming strategy in a more intact retinal environment. For this purpose, we used an in vitro culture system of human fetal retinal tissue and adult human postmortem retina. To stimulate reprogramming, we used lentiviral vectors to deliver constructs with a glial-specific promoter (HES1) driving ASCL1 alone or in combination with additional developmental transcription factors (TFs) such as ATOH1 and NEUROD1. Combining IHC, scRNA-seq, and electrophysiology, we show that human MG can generate new neurons even in adults. This work constitutes a key step toward a future clinical application of this regenerative medicine approach for retinal degenerative disorders.

Dopaminergic amacrine cells express HCN channels in the developing and adult mouse retina.

To determine the molecular and functional expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in developing and mature dopaminergic amacrine cells (DACs), the sole source of ocular dopamine that plays a vital role in visual function and eye development. HCN channels are encoded by isoforms 1-4. HCN1, HCN2, and HCN4 were immunostained in retinal slices obtained from mice at postnatal day 4 (P4), P8, and P12 as well as in adults. Each HCN channel isoform was also immunostained with tyrosine hydroxylase, a marker for DACs, at P12 and adult retinas. Genetically-marked DACs were recorded in flat-mount retina preparation using a whole-cell current-clamp technique. HCN1 was expressed in rods/cones, amacrine cells, and retinal ganglion cells (RGCs) at P4, along with bipolar cells by P12. Different from HCN1, HCN2 and HCN4 were each expressed in amacrine cells and RGCs at P4, along with bipolar cells by P8, and in rods/cones by P12. Double immunostaining shows that each of the three isoforms was expressed in approximately half of DACs at P12 but in almost all DACs in adults. Electrophysiology results demonstrate that HCN channel isoforms form functional HCN channels, and the pharmacological blockade of HCN channels reduced the spontaneous firing frequency in most DACs. Each class of retinal neurons may use different isoforms of HCN channels to function during development. HCN1, HCN2, and HCN4 form functional HCN channels in DACs, which appears to modulate their spontaneous firing activity.

Predation without direction selectivity

Across the animal kingdom, visual predation relies on motion-sensing neurons in the superior colliculus (SC) and its orthologs. These neurons exhibit complex stimulus preferences, including direction selectivity, which is thought to be critical for tracking the unpredictable escape routes of prey. The source of direction selectivity in the SC is contested, and its contributions to predation have not been tested experimentally. Here, we use type-specific cell removal to show that narrow-field (NF) neurons in the mouse SC guide predation. In vivo recordings demonstrate that direction-selective responses of NF cells are independent of recently reported stimulus-edge effects. Monosynaptic retrograde tracing reveals that NF cells receive synaptic input from direction-selective ganglion cells. When we eliminate direction selectivity in the retina of adult mice, direction-selective responses in the SC, including in NF cells, are lost. However, eliminating retinal direction selectivity does not affect the hunting success or strategies of mice, even when direction selectivity is removed after mice have learned to hunt, and despite abolishing the gaze-stabilizing optokinetic reflex. Thus, our results identify the retinal source of direction selectivity in the SC. They show that NF cells in the SC guide predation, an essential spatial orienting task, independent of their direction selectivity, revealing behavioral multiplexing of complex neural feature preferences and highlighting the importance of feature-selective manipulations for neuroethology.

Non-haematopoietic Sca-1+ Cells in the Retina of Adult Mice Express Functional TLR2.

The mammal retina does not have the capacity to regenerate throughout life, although some stem and progenitor cells persist in the adult retina and might retain multipotentiality, as previously described in many tissues. In this work we demonstrate the presence of a small lineage- Sca-1+ cell population in the adult mouse retina which expresses functional TLR2 receptors as in vitro challenge with the pure TLR2 agonist Pam3CSK4 increases cell number and upregulates TLR2. Therefore, this population could be of interest in neuroregeneration studies to elucidate its role in these processes.

Does retinal microglia respond to peripheral nervous system injury?

The nervous system is the principal integration and coordination centre of the organism receiving, processing and transmitting information to produce a response that regulates and maintains homeostasis. In the central nervous system (CNS), homeostasis is regulated by haemato‐neuronal barriers that prevent free entry to immune system cells, although there is increasing evidence that neuro‐immunological interactions during injury, inflammation and chronic peripheral disease may contribute to the establishment and development of chronic neurodegenerative pathologies at the CNS level.Inflammation plays a significant role in the resolution and sequelae of CNS damage. In recent years, special attention has been dedicated to the role and phenotype of phagocytic cells acting in the CNS and, in particular, to the study of which macrophage subpopulations contribute to excessive or detrimental inflammation or, on the contrary, to beneficial repair or resolution of the lesion.All this raises the question of how local peripheral neuroimmunological interaction induces an inflammatory response in the CNS and how this can be better understood. The study of this phenomenon in the retina is of particular interest, as the retina does not receive neural afferents from other areas of the CNS.The use of experimental models of peripheral lesions of the peripheral nervous system, such as sciatic nerve trauma, or inflammatory lesions, such as the induction of a peripheral arthritic joint process, aims to investigate the time course of the response in the CNS to local peripheral inflammation in the retina of adult mice. The results reveal qualitative and quantitative cellular changes compatible with microglial activation processes in the retina, but no significant changes occur in its neuronal population. The presence and activation of haematic mononuclear‐phagocytic cells of myelopoietic origin are also observed in the retina, where they differentiate into microglia.In the absence of local retinal injury, we postulate that the detected neuroinflammatory reaction may be due to a preventive CNS defence reaction favouring neuroprotection since microglial cells derived from the haematic monocyte/macrophage cell population may consider within the M2 macrophage subtype, characterized by their ability to suppress proinflammatory immune responses and promote the expression of neuroprotective and repair genes.On the other hand, we must consider that activation of the mononuclear phagocytic system in the retina caused by a peripheral inflammatory process could potentially be detrimental to patients with neurodegenerative retinal diseases or contribute to their development.

Characterization of the whole Advillin expressing retinal ganglion cell population in the rodent retina

Aims/Purpose: We have quantified and examined the topography of the total population of retinal ganglion cells (RGC) expressing Advillin (Advillin+RGCs), an actin‐binding protein expressed in somatic sensory neurons, in the retinas, as well as their projections to the brain, in control adult Advillin‐Cretg/+ mice (Zurborg et al. Mol Pain 2011, 7:66).Methods: Retinas from adult Advillin‐Cretg/+ Ai14fl/fl C57BL/6J mice (Frutos‐Rincón et al., Int J Mol Sci 2022, 23:2997) expressing tdTomato were prepared as whole‐mounts, labelled with antibodies recognizing Brn3a and melanopsin. The entire Advillin+RGC population was quantified and its retinal distribution analysed by nearest neighbour maps. The brains were serial coronal crysectioned and the axonal Advillin+RGC projections in the brain were anatomically analysed.Results: Advillin protein was mainly located in the somas and axons of a subset of RGCs. Total number of Advillin+RGCs was 1947 ± 157 (mean ± SD; n = 7), which constitutes 5% of the total RGC population. Approximately 17.5% (340 ± 76) and 0% (3 ± 1) of the Advillin+RGC population co‐expressed Brn3a or melanopsin proteins, respectively. The Advillin+RGC population was distributed across the entire retina, with a higher concentration in the temporal region. The axons of this RGC subpopulation project to the main retinal recipient regions in the brain, such as the lateral geniculate nucleus (LGN) and superior colliculus (SC). However, none of these axons project to the suprachiasmatic nucleus. These data indicates that Advillin+RGCs is not a subpopulation of ipRGCs.Conclusions: The Advillin protein, which is characteristic of sensory neurons, is also expressed in a specific RGC subpopulation of the adult mouse retina. A small percentage of Advillin+RGCs expressed the Brn3a transcription factor, while none expressed melanopsin. The axons of Advillin+RGCs project to the typical retino‐recipient areas in the brain, excluding the suprachiasmatic nucleus.

Consequences of early life stress on the structure and function of the adult mouse retina.

Consequences of early life stress on the structure and function of the adult mouse retina.

Invited Session II: Retinal remodeling and regeneration: Stimulation of functional neural regeneration in adult mouse retina.

Neurons are not regenerated in the adult mammalian retina. However, in non-mammalian vertebrates, specialized glial cells (Muller glia, MG) spontaneously generate neural progenitors, which go on to replace neurons after injury. We have recently developed strategies to stimulate regeneration of functional neurons in the adult mouse retina by over-expressing developmental transcription factors, including Ascl1, Atoh1, Islet1 and Pou4f2. We have found that over-expressing Ascl1 in MG in vivo, followed by injury and TSA, induces regeneration of functional retinal neurons in adult mice. Adding additional TFs, such as Atoh1, substantially increases the efficiency of neurogenesis from the MG, while combining other TFs with Ascl1 can alter the types of neurons that are regenerated after injury. Together our data show that we can considerably recapitulate in mice much of the regeneration that occurs naturally in fish.

ASCL1 induces neurogenesis in human Müller glia

In mammals, loss of retinal cells due to disease or trauma is an irreversible process that can lead to blindness. Interestingly, regeneration of retinal neurons is a well established process in some non-mammalian vertebrates and is driven by the Müller glia (MG), which are able to re-enter the cell cycle and reprogram into neurogenic progenitors upon retinal injury or disease. Progress has been made to restore this mechanism in mammals to promote retinal regeneration: MG can be stimulated to generate new neurons invivo in the adult mouse retina after the over-expression of the pro-neural transcription factor Ascl1. In this study, we applied the same strategy to reprogram human MG derived from fetal retina and retinal organoids into neurons. Combining single cell RNA sequencing, single cell ATAC sequencing, immunofluorescence, and electrophysiology we demonstrate that human MG can be reprogrammed into neurogenic cells invitro.

Distribution of intracellular Ca2+-ATPases in the mouse retina and their involvement in light-induced cone degeneration

Calcium signalling is involved in many processes in mammalian retina, from development to mature functions and neurodegeneration. Although proteins involved in Ca2+ entry in retinal cells have been well studied, less is known about Ca2+-clearance. Among the Ca2+ pumps, plasma membrane Ca2+-ATPases (PMCAs) have been identified as key proteins extruding Ca2+ across the plasma membrane with specific distribution in developing and adult retina. However, the two main isoforms of intracellular Ca2+-ATPases in the central nervous system, the sarco(endo)plasmic reticulum (ER) Ca2+-ATPase 2b (SERCA2b) and the secretory pathway Ca2+-ATPase 1 (SPCA1), which remove cytosolic Ca2+ into intracellular stores, have been less or not at all analysed, respectively. In this study, we described for the first time the SPCA1 localisation in adult mouse retina and we report differential distributions of SERCA2b and SPCA1 transporters within various classes of retinal neurons and distinct subcellular localisations. In addition, we studied the expression and localisation of both Ca2+ pumps in 661W cells, a cone photoreceptor-derived cell line. Since continuous exposure to high light intensity induces photodegeneration, we analysed the effect of LED light exposure on these cells and SERCA2b and SPCA1 distribution. We found that continuous mild LED-light exposure compromised cell survival and produced stress in the ER and Golgi, the Ca2+ stores where the two pumps are localised. These effects were reversed after halting light exposure and washing. This study demonstrates that Ca2+ signalling may be involved in light-induced photoreceptor cell damage and points to previously unrecognised functions of intracellular Ca2+-ATPases in retina physiology.

Long-term Effects of the pituitary-adenylate cyclase-activating Polypeptide (PACAP38) in the Adult Mouse Retina: Microglial Activation and Induction of Neural Proliferation

The degenerative retinal disorders characterized by progressive cell death and exacerbating inflammation lead ultimately to blindness. The ubiquitous neuropeptide, PACAP38 is a promising therapeutic agent as its proliferative potential and suppressive effect on microglia might enable cell replacement and attenuate inflammation, respectively. Our previous finding that PACAP38 caused a marked increase of the amacrine cells in the adult (1-year-old) mouse retina, served as a rationale of the current study. We aimed to determine the proliferating elements and the inflammatory status of the PACAP38-treated retina. Three months old mice were intravitreally injected with 100 pmol PACAP38 at 3 months intervals (3X). Retinas of 1-year-old animals were dissected and effects on cell proliferation, and expression of inflammatory regulators were analyzed. Interestingly, both mitogenic and anti-mitogenic actions were detected after PACAP38-treatment. Further analysis of the mitogenic effect revealed that proliferating cells include microglia, endothelial cells, and neurons of the ganglion cell layer but not amacrine cells. Furthermore, PACAP38 stimulated retinal microglia to polarize dominantly into M2-phenotype but also might cause subsequent angiogenesis. According to our results, PACAP38 might dampen pro-inflammatory responses and help tissue repair by reprogramming microglia into an M2 phenotype, nonetheless, with angiogenesis as a warning side effect.

Erratum for the Research Article: “Reprogramming Müller glia to regenerate ganglion-like cells in adult mouse retina with developmental transcription factors”

Erratum for the Research Article: “Reprogramming Müller glia to regenerate ganglion-like cells in adult mouse retina with developmental transcription factors”

Developmental Expression of the Cell Cycle Regulator p16INK4a in Retinal Glial Cells: A Novel Marker for Immature Ocular Astrocytes?

Retinal astrocytes are vital for neuronal homeostasis in the retina. Together with Müller glia, they provide retinal cells with neurotrophic factors, antioxidative support, and defense mechanisms such as the formation of the blood-retinal barrier. Substantial heterogeneity of astrocyte morphology and function represents a challenge for identification of distinct subtypes which may be potential targets for therapeutic purposes. Hence, identification of novel markers of astrocyte subpopulations is highly relevant to better understand the molecular mechanisms involved in retinal development, homeostasis, and pathology. In this study, we observed that the cell cycle regulator, p16INK4a, is expressed in immature astrocytes in the mouse retina. Immunohistochemical analysis showed p16INK4a expression in the optic nerve of wild-type mice from 3 days to 3 months of age and in the nerve fiber layer of the adult mouse retina. Colocalization of p16INK4a expression and glial fibrillary acidic protein (immature/mature astrocyte marker) tends to decrease with age. However, colocalization of p16INK4a expression and vimentin (immature astrocyte marker) remains high in the optic nerve from the early postnatal period to adulthood. The observations from this study provide a valuable tool for further investigations of ocular astrocytes in the developing retina as well as in degenerative retinopathies.

New AAV tools fail to detect Neurod1-mediated neuronal conversion of Müller glia and astrocytes invivo.

Reprogramming resident glial cells to convert them into neurons invivo represents a potential therapeutic strategy that could replenish lost neurons, repair damaged neural circuits, and restore function. AAV (adeno-associated virus)-based expression systems are powerful tools for invivo gene delivery in glia-to-neuron reprogramming, however, recent studies show that AAV-based gene delivery of Neurod1 into the mouse brain can cause severe leaky expression into endogenous neurons leading to misinterpretation of glia-to-neuron conversion. AAV-based delivery systems were modified for improved invivo delivery of Neurod1, Math5, Ascl1, and Neurog2 in the adult mouse retina and brain. To examine whether bona fide glia-to-neuron conversion occurs, stringent fate mapping experiments were performed to trace the lineage of glial cells. The neuronal leakage is prevalent after AAV-GFAP-mediated delivery of Neurod1, Math5, Ascl1, and Neurog2. The transgene-dependent leakage cannot be corrected after lowering the AAV doses, using alterative AAV serotypes or injection routes. Importantly, we report the development of two new AAV-based tools that can significantly reduce neuronal leakage. Using the new AAV-based tools, we provide evidence that Neurod1 gene transfer fails to convert lineage traced glial cells into neurons. Stringent fate mapping techniques independently of an AAV-based expression system are the golden standard for tracing the fate of glia cells during neuronal reprogramming. The newly developed AAV-based systems are invaluable tools for glia-to-neuron reprogramming invivo. The work in Chen lab was supported by National Institutes of Health (NIH) grants R01 EY024986 and R01 EY028921, an unrestricted challenge grant from Research to Prevent Blindness, the New York Eye and Ear Infirmary Foundation, and The Harold W. McGraw, Jr. Family Foundation for Vision Research. The work in Zhang lab was supported by NIH (R01 NS127375 and R01 NS117065) and The Decherd Foundation.

Temporal single-cell atlas of non-neuronal retinal cells reveals dynamic, coordinated multicellular responses to central nervous system injury.

Non-neuronal cells are key to the complex cellular interplay that follows central nervous system insult. To understand this interplay, we generated a single-cell atlas of immune, glial and retinal pigment epithelial cells from adult mouse retina before and at multiple time points after axonal transection. We identified rare subsets in naive retina, including interferon (IFN)-response glia and border-associated macrophages, and delineated injury-induced changes in cell composition, expression programs and interactions. Computational analysis charted a three-phase multicellular inflammatory cascade after injury. In the early phase, retinal macroglia and microglia were reactivated, providing chemotactic signals concurrent with infiltration of CCR2+ monocytes from the circulation. These cells differentiated into macrophages in the intermediate phase, while an IFN-response program, likely driven by microglia-derived type I IFN, was activated across resident glia. The late phase indicated inflammatory resolution. Our findings provide a framework to decipher cellular circuitry, spatial relationships and molecular interactions following tissue injury.

Functional Changes in the Adult Mouse Retina using an Alpha7 Nicotinic Acetylcholine Receptor Agonist after Blast Exposure

Currently, there is a lack of treatments for retinal neurotrauma. To address this issue, this study uses an alpha7 nAChR agonist, PNU-282987, to determine it effects on functional activity in the retina shortly after a traumatic blast exposure. The objectives of this research include: (1) examination of the cellular and functional damage associated with ocular blast exposure, and (2) evaluation of structural and functional changes that occur post PNU-282987 treatment. Significant ocular blast damage was induced in adult mice after exposure to a single blast of 35 psi to the left eye. Blast-exposed transgenic mice expressing tdTomato Müller glia were treated daily with eyedrops containing PNU-282987 for 4 weeks following the blast exposure. Antibody staining studies in these transgenic mice was conducted to examine lineage tracing and electroretinograms (ERGs) were obtained to examine functional changes. Blast exposure caused a significant loss of cells in all retinal layers after 4 weeks. Immunohistochemical analysis demonstrated tdTomato-positive labeled photoreceptors and retinal ganglion cells in blast-exposed mice treated with PNU-282987. ERG recordings were taken from control animals, from blast-damaged animals and from animals exposed to blast followed by 4 weeks of PNU-282987 treatment. Scotopic ERG recordings from blast-exposed mice had significantly decreased amplitudes of a-wave, b-wave, oscillatory potentials and flicker frequencies, which were prevented after PNU-282987 treatment. In photopic experiments, the PhNR response was reduced significantly after blast exposure but the decrease was prevented after treatment with PNU-282987. These are the first experiments that demonstrate preservation of retinal function after blast exposure using an alpha7 nAChR agonist.

Retinal Explant of the Adult Mouse Retina as an <em>Ex Vivo</em> Model for Studying Retinal Neurovascular Diseases

Retinal Explant of the Adult Mouse Retina as an <em>Ex Vivo</em> Model for Studying Retinal Neurovascular Diseases

Retinal Explant of the Adult Mouse Retina as an <em>Ex Vivo</em> Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Reprogramming Müller glia to regenerate ganglion-like cells in adult mouse retina with developmental transcription factors.

Many neurodegenerative diseases cause degeneration of specific types of neurons. For example, glaucoma leads to death of retinal ganglion cells, leaving other neurons intact. Neurons are not regenerated in the adult mammalian central nervous system. However, in nonmammalian vertebrates, glial cells spontaneously reprogram into neural progenitors and replace neurons after injury. We have recently developed strategies to stimulate regeneration of functional neurons in the adult mouse retina by overexpressing the proneural factor Ascl1 in Müller glia. Here, we test additional transcription factors (TFs) for their ability to direct regeneration to particular types of retinal neurons. We engineered mice to express different combinations of TFs in Müller glia, including Ascl1, Pou4f2, Islet1, and Atoh1. Using immunohistochemistry, single-cell RNA sequencing, single-cell assay for transposase-accessible chromatin sequencing, and electrophysiology, we find that retinal ganglion-like cells can be regenerated in the damaged adult mouse retina in vivo with targeted overexpression of developmental retinal ganglion cell TFs.

Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina.

Direct reprogramming of retinal Müller glia is a promising avenue for replacing photoreceptors and retinal ganglion cells lost to retinal dystrophies. However, questions have recently been raised about the accuracy of studies claiming efficient glia-to-neuron reprogramming in retina that were conducted using GFAP mini promoter-driven adeno-associated virus (AAV) vectors. In this study, we have addressed these questions using GFAP mini promoter-driven AAV constructs to simultaneously overexpress the mCherry reporter and candidate transcription factors predicted to induce glia-to-neuron conversion, in combination with prospective genetic labeling of retinal Müller glia using inducible Cre-dependent GFP reporters. We find that, while control GFAP-mCherry constructs express faithfully in Müller glia, 5 out of 7 transcription factor overexpression constructs tested are predominantly expressed in amacrine and retinal ganglion cells. These findings demonstrate strong insert-dependent effects on AAV-based GFAP mini promoter specificity that preclude its use in inferring cell lineage relationships when studying glia-to-neuron conversion in retina.