examination-anatomical-features-retinal-ganglion-cells-under-n-methyl

Examination of Anatomical Features of Retinal Ganglion Cells Under N-methyl-D-aspartic Acid (NMDA)-induced Excitotoxicity

Retinal ganglion cells (RGCs) are the output neurons of the retina, responsible for transmitting visual information to the brain. In the mature mouse ...

University of Texas Health Science Center at Houston (UTHealth)

Mitochondrial dysfunction has been implicated in the pathologies of a number of retinal degenerative diseases in both the outer and inner retina. In the outer retina, photoreceptors are particularly vulnerable to mutations affecting mitochondrial function due to their high energy demand and sensitivity to oxidative stress. However, it is unclear how defective mitochondrial biogenesis affects neural development and contributes to neural degeneration. In this report, we investigated the in vivo function of nuclear respiratory factor 1 (Nrf1), a major transcriptional regulator of mitochondrial biogenesis in both proliferating retinal progenitor cells (RPCs) and postmitotic rod photoreceptor cells (PRs).

Essential roles of mitochondrial biogenesis regulator Nrf1 in retinal development and homeostasis.

In the mouse retina, more than 30 retinal ganglion cell (RGC) subtypes have been classified based on a combined metric of morphological and functional characteristics. RGCs arise from a common pool of retinal progenitor cells during embryonic stages and differentiate into mature subtypes in adult retinas. However, the cellular and molecular mechanisms controlling formation and maturation of such remarkable cellular diversity remain unknown. Here, we demonstrate that T-box transcription factor T-brain 1 (Tbr1) is expressed in two groups of morphologically and functionally distinct RGCs: the orientation-selective J-RGCs and a group of OFF-sustained RGCs with symmetrical dendritic arbors. When Tbr1 is genetically ablated during retinal development, these two RGC groups cannot develop. Ectopically expressing Tbr1 in M4 ipRGCs during development alters dendritic branching and density but not the inner plexiform layer stratification level. Our data indicate that Tbr1 plays critical roles in regulating the formation and dendritic morphogenesis of specific RGC types.

Essential Roles of Tbr1 in the Formation and Maintenance of the Orientation-Selective J-RGCs and a Group of OFF-Sustained RGCs in Mouse.

In situ hybridization (ISH) techniques provide important information regarding gene expression in cells and tissues. Especially, ISH details complex spatial RNA expression in highly heterogeneous tissues, such as developing and mature central nervous systems, where rare genes involved in many fundamental developmental or biological events are expressed. Although several techniques have been developed to detect low levels of RNA expression, there are still problematic issues caused by a low signal-to-noise ratio after signal amplification. RNAscope is a recently developed ISH technique with high sensitivity and low background. RNAscope utilizes a unique probe system (double Z probe) to amplify signal from rare RNAs. Additionally, the double Z probe enables a significant reduction in nonspecific signal amplification. Here we report detailed procedures of the brown-color RNAscope ISH on embryonic and adult mouse retinas.

Ultrasensitive RNAscope In Situ Hybridization System on Embryonic and Adult Mouse Retinas.

The stereotypic dendritic morphology is one of the landmark characteristics for classifying retinal ganglion cell (RGC) subtypes. These unique dendritic morphologies and their corresponding stratification level in the inner plexiform layer are indicators of their physiological function and presynaptic connection with other neurons. Mis-patterned dendritic morphologies underlie many neurological disease conditions. To streamline the morphological analysis of RGCs, here, we describe a simple protocol using Cre-/lox-dependent genetically directed sparse labeling strategy on flat-mounted retinas to inspect dendritic morphology of specific RGC subtypes.

Genetically Directed Sparse Labeling System for Anatomical Studies of Retinal Ganglion Cells.

Mouse photoreceptors are electrically coupled via gap junctions, but the relative importance of rod/rod, cone/cone, or rod/cone coupling is unknown. Furthermore, while connexin36 (Cx36) is expressed by cones, the identity of the rod connexin has been controversial. We report that FACS-sorted rods and cones both express  but no other connexins. We created rod- and cone-specific  knockout mice to dissect the photoreceptor network. In the wild type, Cx36 plaques at rod/cone contacts accounted for more than 95% of photoreceptor labeling and paired recordings showed the transjunctional conductance between rods and cones was ~300 pS. When  was eliminated on one side of the gap junction, in either conditional knockout, Cx36 labeling and rod/cone coupling were almost abolished. We could not detect direct rod/rod coupling, and cone/cone coupling was minor. Rod/cone coupling is so prevalent that indirect rod/cone/rod coupling via the network may account for previous reports of rod coupling.

Molecular and functional architecture of the mouse photoreceptor network.

The mammalian retina contains more than 40 retinal ganglion cell (RGC) subtypes based on their unique morphologies, functions, and molecular profiles. Among them, intrinsically photosensitive RGCs (ipRGCs) are the first specified RGC type emerging from a common retinal progenitor pool during development. Previous work has shown that T-box transcription factor T-brain 2 (Tbr2) is essential for the formation and maintenance of ipRGCs, and that Tbr2-expressing RGCs activate Opn4 expression upon native ipRGC ablation, suggesting that Tbr2 RGCs contain a reservoir for ipRGCs. However, the identity of Tbr2 RGCs has not been fully vetted. Here, using genetic sparse labeling and single cell recording, we showed that Tbr2-expressing retinal neurons include RGCs and a subset of GABAergic displaced amacrine cells (dACs). Most Tbr2 RGCs are intrinsically photosensitive and morphologically resemble native ipRGCs with identical retinofugal projections. Tbr2 RGCs also include a unique and rare Pou4f1-expressing OFF RGC subtype. Using a loss-of-function strategy, we have further demonstrated that Tbr2 is essential for the survival of these RGCs and dACs, as well as maintaining the expression of Opn4. These data set a strong foundation to study how Tbr2 regulates ipRGC development and survival, as well as the expression of molecular machinery regulating intrinsic photosensitivity.

Characterization of Tbr2-expressing retinal ganglion cells.

The heterogeneous family of complexes comprising Polycomb repressive complex 1 (PRC1) is instrumental for establishing facultative heterochromatin that is repressive to transcription. However, two PRC1 species, ncPRC1.3 and ncPRC1.5, are known to comprise novel components, AUTS2, P300, and CK2, that convert this repressive function to that of transcription activation. Here, we report that individuals harboring mutations in the HX repeat domain of AUTS2 exhibit defects in AUTS2 and P300 interaction as well as a developmental disorder reflective of Rubinstein-Taybi syndrome, which is mainly associated with a heterozygous pathogenic variant in CREBBP/EP300. Moreover, the absence of AUTS2 or mutation in its HX repeat domain gives rise to misregulation of a subset of developmental genes and curtails motor neuron differentiation of mouse embryonic stem cells. The transcription factor nuclear respiratory factor 1 (NRF1) has a novel and integral role in this neurodevelopmental process, being required for ncPRC1.3 recruitment to chromatin.

NRF1 association with AUTS2-Polycomb mediates specific gene activation in the brain.

The retina, the accessible part of the central nervous system, has served as a model system to study the relationship between energy utilization and metabolite supply. When the metabolite supply cannot match the energy demand, retinal neurons are at risk of death. As the powerhouse of eukaryotic cells, mitochondria play a pivotal role in generating ATP, produce precursors for macromolecules, maintain the redox homeostasis, and function as waste management centers for various types of metabolic intermediates. Mitochondrial dysfunction has been implicated in the pathologies of a number of degenerative retinal diseases. It is well known that photoreceptors are particularly vulnerable to mutations affecting mitochondrial function due to their high energy demand and susceptibility to oxidative stress. However, it is unclear how defective mitochondria affect other retinal neurons. Nuclear respiratory factor 1 (Nrf1) is the major transcriptional regulator of mitochondrial biogenesis, and loss of  leads to defective mitochondria biogenesis and eventually cell death. Here, we investigated how different retinal neurons respond to the loss of . We provide in vivo evidence that the disruption of -mediated mitochondrial biogenesis results in a slow, progressive degeneration of all retinal cell types examined, although they present different sensitivity to the deletion of , which implicates differential energy demand and utilization, as well as tolerance to mitochondria defects in different neuronal cells. Furthermore, transcriptome analysis on rod-specific  deletion uncovered a previously unknown role of Nrf1 in maintaining genome stability.

Differential Susceptibility of Retinal Neurons to the Loss of Mitochondrial Biogenesis Factor Nrf1.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) pathway is a powerful tool to combat tumor growth and progression; however, drug resistance frequently emerges. We identify CD5L (CD5 antigen-like precursor) as an important gene upregulated in response to antiangiogenic therapy leading to the emergence of adaptive resistance. By using both an RNA-aptamer and a monoclonal antibody targeting CD5L, we are able to abate the pro-angiogenic effects of CD5L overexpression in both in vitro and in vivo settings. In addition, we find that increased expression of vascular CD5L in cancer patients is associated with bevacizumab resistance and worse overall survival. These findings implicate CD5L as an important factor in adaptive resistance to antiangiogenic therapy and suggest that modalities to target CD5L have potentially important clinical utility.

Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L.

More than 40 retinal ganglion cell (RGC) subtypes have been categorized in mouse based on their morphologies, functions, and molecular features. Among these diverse subtypes, orientation-selective Jam2-expressing RGCs (J-RGCs) has two unique morphologic characteristics: the ventral-facing dendritic arbor and the OFF-sublaminae stratified terminal dendrites in the inner plexiform layer. Previously, we have discovered that T-box transcription factor  () is expressed in J-RGCs. We further found that  is essential for the expression of , and Tbr1 regulates the formation and the dendritic morphogenesis of J-RGCs. However, Tbr1 begins to express in terminally differentiated RGCs around perinatal stage, suggesting that it is unlikely involved in the initial fate determination for J-RGC and other upstream transcription factors must control  expression and J-RGC formation. Using the Cleavage Under Targets and Tagmentation technique, we discovered that Pou4f1 binds to  on the evolutionary conserved exon 6 and an intergenic region downstream of the 3'UTR, and on a region flanking the promoter and the first exon of . We showed that Pou4f1 is required for the expression of  and , indicating Pou4f1 as a direct upstream regulator of  and . Most interestingly, the Pou4f1-bound element in exon 6 of  possesses high-level enhancer activity, capable of directing reporter gene expression in J-RGCs. Together, these data revealed a  genetic hierarchy as a critical pathway in the formation of J-RGC subtype.

Article	Year
Essential roles of mitochondrial biogenesis regulator Nrf1 in retinal development and homeostasis. Molecular neurodegeneration\| PubMed ID: 30333037	2018
Essential Roles of Tbr1 in the Formation and Maintenance of the Orientation-Selective J-RGCs and a Group of OFF-Sustained RGCs in Mouse. Cell reports\| PubMed ID: 30995485	2019
Ultrasensitive RNAscope In Situ Hybridization System on Embryonic and Adult Mouse Retinas. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 31786787	2020
Genetically Directed Sparse Labeling System for Anatomical Studies of Retinal Ganglion Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 31786789	2020
Molecular and functional architecture of the mouse photoreceptor network. Science advances\| PubMed ID: 32832605	2020
Characterization of Tbr2-expressing retinal ganglion cells. The Journal of comparative neurology\| PubMed ID: 34245014	2021
NRF1 association with AUTS2-Polycomb mediates specific gene activation in the brain. Molecular cell\| PubMed ID: 34637754	2021
Differential Susceptibility of Retinal Neurons to the Loss of Mitochondrial Biogenesis Factor Nrf1. Cells\| PubMed ID: 35883647	2022
Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L. Nature communications\| PubMed ID: 37100807	2023
Pou4f1-Tbr1 transcriptional cascade controls the formation of Jam2-expressing retinal ganglion cells. Frontiers in ophthalmology\| PubMed ID: 38469155	2023

Chai-An Mao

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