Biology subjects

Palazzo, I.

Publications and source records attributed to Palazzo, I..

10 recordsLinked to original sources

bioRxiv2025

Comparative single-cell multiomic analysis reveals evolutionarily conserved and species-specific cellular mechanisms mediating natural retinal aging.

Biological age is a major risk factor in the development of common degenerative retinal diseases such as age-related macular degeneration and glaucoma. To systematically characterize molecular mechanisms underlying retinal aging, we performed integrated single- cell RNA- and ATAC-Seq analyses of the retina and retinal pigment epithelium (RPE) across the natural lifespan in zebrafish, mice, and humans. By profiling gene expression and chromatin accessibility, we identified extensive cell type- and species-specific aging-dependent changes, with a much smaller number of broadly expressed and conserved genes that include regulators of inflammation and autophagy. We constructed predictive aging clocks for retinal cell types and observed dynamic, reversible shifts in cellular age following acute injury. Spatial transcriptomic analysis revealed region-specific aging signatures and proximity effects, with Muller glia exhibiting pro-rejuvenating influences on neighboring neurons. Targeted Muller glia-specific induction of Yamanaka factors reduced molecular age in rod photoreceptors and bipolar cells without altering glial age. Our findings define conserved and divergent regulatory and signaling pathways mediating retinal aging, highlighting Muller glia as potential therapeutic targets for combating age-associated retinal dystrophies.

neuroscience↗

bioRxiv2024

Viral-mediated Oct4 overexpression and inhibition of Notch signaling synergistically induce neurogenic competence in mammalian Muller glia.

Retinal Muller glia in cold-blooded vertebrates can reprogram into neurogenic progenitors to replace neurons lost to injury, but mammals lack this ability. While recent studies have shown that transgenic overexpression of neurogenic bHLH factors and glial-specific disruption of NFI family transcription factors and Notch signaling induce neurogenic competence in mammalian Muller glia, induction of neurogenesis in wild-type glia has thus far proven elusive. Here, we report that viral-mediated overexpression of the pluripotency factor Oct4 (Pou5f1) induces transdifferentiation of mouse Muller glia into bipolar neurons, and synergistically stimulates glial-derived neurogenesis in parallel with Notch loss of function. Single cell multiomic analysis shows that Oct4 overexpression leads to widespread changes in gene expression and chromatin accessibility, inducing activity of both the neurogenic transcription factor Rfx4 and the Yamanaka factors Sox2 and Klf4. This study demonstrates that viral-mediated overexpression of Oct4 induces neurogenic competence in retinal Muller glia, identifying mechanisms that could be used in cell-based therapies for treating retinal dystrophies.

neuroscience↗

bioRxiv2023

Molecular regulation of retinal regeneration is context specific

Many genes are known to regulate retinal regeneration following widespread tissue damage. Conversely, genes controlling regeneration following limited retinal cell loss, akin to disease conditions, are undefined. Combining a novel retinal ganglion cell (RGC) ablation-based glaucoma model, single cell omics, and rapid CRISPR/Cas9-based knockout methods to screen 100 genes, we identified 18 effectors of RGC regeneration kinetics. Surprisingly, 32 of 33 previously known/implicated regulators of retinal tissue regeneration were not required for RGC replacement; 7 knockouts accelerated regeneration, including sox2, olig2, and ascl1a. Mechanistic analyses revealed loss of ascl1a increased "fate bias", the propensity of progenitors to produce RGCs. These data demonstrate plasticity and context-specificity in how genes function to control regeneration, insights that could help to advance disease-tailored therapeutics for replacing lost retinal cells. One sentence summaryWe discovered eighteen genes that regulate the regeneration of retinal ganglion cells in zebrafish.

neuroscience↗

bioRxiv2023

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes. This regeneration requires Muller glia (MG) to reprogram and divide asymmetrically to produce a multipotent Muller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, does loss of different retinal cell subtypes induce unique MG regeneration responses? Second, do MG reprogram to a developmental retinal progenitor cell state? And finally, to what extent does regeneration recapitulate retinal development? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. While MG reprogram to a state similar to late-stage retinal progenitors in developing retinas, there are transcriptional differences between reprogrammed MG/MGPCs and late progenitors, as well as reprogrammed MG in outer and inner retinal damage models. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. This work identifies major differences between gene regulatory networks activated following the selective loss of different subtypes of retina neurons, as well as between retinal regeneration and development.

neuroscience↗

bioRxiv2023

Formation of Müller glia-derived progenitor cells in retinas depleted of microglia

Recent studies have demonstrated the complex coordination of pro-inflammatory signaling and reactive microglia/macrophage on the formation Muller glial-derived progenitor cells (MGPCs) in the retinas of fish, birds and mice. We generated scRNA-seq libraries to identify transcriptional changes in Muller glia (MG) that result from the depletion of microglia from the chick retina. We found significant changes in different networks of genes in MG in normal and damaged retinas when the microglia are ablated. We identified a failure of MG to upregulate Wnt-ligands, Heparin binding epidermal growth factor (HBEGF), Fibroblast growth factor (FGF), retinoic acid receptors and genes related to Notch-signaling. Inhibition of GSK3{beta}, to simulate Wnt-signaling, failed to rescue the deficit in formation of proliferating MGPCs in damaged retinas missing microglia. By comparison, application of HBEGF or FGF2 completely rescued the formation of proliferating MGPCs in microglia-depleted retinas. Similarly, injection of a small molecule inhibitor to Smad3 or agonist to retinoic acid receptors partially rescued the formation of proliferating MGPCs in microglia-depleted damaged retinas. According to scRNA-seq libraries, patterns of expression of ligands, receptors, signal transducers and/or processing enzymes to cell-signaling via HBEGF, FGF, retinoic acid and TGF{beta} are rapidly and transiently upregulated by MG after neuronal damage, consistent with important roles for these cell-signaling pathways in regulating the formation of MGPCs. We conclude that quiescent and activated microglia have a significant impact upon the transcriptomic profile of MG. We conclude that signals produced by reactive microglia in damaged retinas stimulate MG to upregulate cell signaling through HBEGF, FGF and retinoic acid, and downregulate signaling through TGF{beta}/Smad3 to promote the reprogramming on MG into proliferating MGPCs.

neuroscience↗

bioRxiv2022

Patterns of NFkB activation resulting from damage, reactive microglia, cytokines, and growth factors in the mouse retina

Muller glia are a cellular source for neuronal regeneration in vertebrate retinas. However, the capacity for retinal regeneration varies widely across species. Understanding the mechanisms that regulate the reprogramming of Muller glia into progenitor cells is key to reversing the loss of vision that occurs with retinal diseases. In the mammalian retina, NF{kappa}B signaling promotes glial reactivity and represses the reprogramming of Muller glia into progenitor cells. Here we investigate different cytokines, growth factors, cell signaling pathways, and damage paradigms that influence NF{kappa}B-signaling in the mouse retina. We find that exogenous TNF and IL1{beta} potently activate NF{kappa}B-signaling in Muller glia in undamaged retinas, and this activation is independent of microglia. By comparison, TLR1/2 agonist indirectly activates NF{kappa}B-signaling in Muller glia, and this activation depends on the presence of microglia as Tlr2 is predominantly expressed by microglia, but not other types of retinal cells. Exogenous FGF2 did not activate NF{kappa}B-signaling, whereas CNTF, Osteopontin, WNT4, or inhibition of GSK3{beta} activated NF{kappa}B in Muller glia in the absence of neuronal damage. By comparison, dexamethasone, a glucocorticoid agonist, suppressed NF{kappa}B-signaling in Muller glia in damaged retinas, in addition to reducing numbers of dying cells and the accumulation of reactive microglia. Although NMDA-induced retinal damage activated NF{kappa}B in Muller glia, optic nerve crush had no effect on NF{kappa}B activation within the retina, whereas glial cells within the optic nerve were responsive. We conclude that the NF{kappa}B pathway is activated in retinal Muller glia in response to many different cell signaling pathways, and activation often depends on signals produced by reactive microglia.

neuroscience↗

bioRxiv2021

NFkB-signaling suppresses Müller glia-mediated neuron regeneration in the mammalian retina

Muller glia (MG) in mammalian retinas are incapable of regenerating neurons after damage, whereas the MG in lower vertebrates regenerate functional neurons. Identification networks that regulate MG-mediated regeneration is key to harnessing the regenerative potential of MG. Here we study how NFkB-signaling influences glial responses to damage and reprogramming of MG into neurons in the rodent retina. We find activation of NFkB and dynamic expression of NFkB-associated genes in MG after damage, however NFkB activity is inhibited by microglia ablation. Knockout of NFkB in MG suppressed the accumulation of immune cells after damage. Inhibition of NFkB following NMDA-damage significantly enhanced the reprogramming of Ascl1-overexpressing MG into neuron-like cells. scRNA-seq of retinal glia following inhibition of NFkB reveals coordination with signaling via TGF{beta}2 and suppression of NFI and Id transcription factors. Inhibition of Smad3 or Id transcription factors increased numbers of neuron-like cells produced by Ascl1-overexpressing MG. We conclude that NFkB is a key signaling hub that is activated in MG after damage, mediates the accumulation of immune cells, and suppresses the neurogenic potential of MG.

neuroscience↗

bioRxiv2020

Midkine in chick and mouse retinas: neuroprotection, glial reactivity and the formation of Müller glia-derived progenitor cells

Recent studies have shown that midkine (MDK), a basic heparin-binding growth factor, is involved in the development and regeneration of the zebrafish retina. However, very little is known about MDK in the retinas of warm-blooded vertebrates. We investigate the expression patterns of MDK and related factors, roles in neuronal survival, and influence upon the formation of Muller glia-derived progenitor cells (MGPCs) in chick and mouse model systems. By using single-cell RNA-sequencing, we find that MDK is upregulated during Muller glia (MG) maturation in chick development and when stimulated to reprogram into MGPCs after NMDA damage or FGF2/Insulin treatment. Interestingly, MDK is significantly up-regulated by MG in damaged chick retinas, but down-regulated by MG in damaged mouse retinas. In both chick and mouse retinas, exogenous MDK selectively up-regulates cFOS and pS6 (a readout of mTOR-signaling) in MG. In the chick, intraocular injections of MDK before injury is neuroprotective with an observed decrease in dying neurons and microglial reactivity, inducing fewer proliferating MGPCs. Blocking MDK signaling with Na3VO4 following blocks neuroprotective effects with an increase the number of dying cells and negates the pro-proliferative effects on MGPCs. Inhibitors of PP2A and Pak1 associated with MDK integrin {beta}1 signaling had MG specific inhibitory effects on MGPC formation. In mice, MDK administration with NMDA damage drives a small but significant increase in MGPCs. We conclude that MDK expression is dynamically regulated in reactive Muller glia and during reprogramming into MGPCs. MDK acts to coordinate glial activity, neuronal survival, and may act in an autocrine manner to influence the re-programming of Muller glia into proliferating MGPCs.

neuroscience↗

bioRxiv2019

NF-κB signaling regulates the formation of proliferating Muller glia-derived progenitor cells in the avian retina.

Neuronal regeneration in the retina is a robust, effective process in some cold-blooded vertebrates, but this process is ineffective in warm-blooded vertebrates. Understanding the mechanisms and cell-signaling pathways that restrict the reprogramming of Muller glia into proliferating neurogenic progenitors is key to harnessing the regenerative potential of the retina. Inflammation and reactive microglia are known to influence the formation of Muller glia-derived progenitor cells (MGPCs), but the mechanisms underlying this response are unknown. Using the chick retina in vivo as a model system, we investigate the role of the Nuclear Factor kappa B (NF-{kappa}B) signaling, a critical regulator of inflammation. We find that components of the NF-{kappa}B pathway are expressed by Muller glia and are dynamically regulated after neuronal damage or treatment with growth factors. Inhibition of NF-{kappa}B enhances, whereas activation suppresses the formation of proliferating MGPCs. Additionally, activation of NF-{kappa}B promotes glial differentiation from MGPCs in damaged retinas. With microglia ablated, the effects of NF-{kappa}B-agonists/antagonists on MGPC formation are reversed, suggesting that the context and timing of signals provided by reactive microglia influence how NF-{kappa}B-signaling impacts the reprogramming of Muller glia. We propose that NF-{kappa}B-signaling is an important signaling \"hub\" that suppresses the reprogramming of Muller glia into proliferating MGPCs and this \"hub\" coordinates signals provided by reactive microglia.

neuroscience↗

bioRxiv2019

Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia

Injury induces retinal Muller glia of cold-blooded, but not mammalian, vertebrates to regenerate neurons. To identify gene regulatory networks that control neuronal reprogramming in retinal glia, we comprehensively profiled injury-dependent changes in gene expression and chromatin accessibility in Muller glia from zebrafish, chick and mice using bulk RNA-Seq and ATAC-Seq, as well as single-cell RNA-Seq. Cross-species integrative analysis of these data, together with functional validation, identified evolutionarily conserved and species-specific gene networks controlling glial quiescence, gliosis and neurogenesis. In zebrafish and chick, transition from the resting state to gliosis is essential for initiation of retinal regeneration, while in mice a dedicated network suppresses neurogenic competence and restores quiescence. Selective disruption of NFI family transcription factors, which maintain and restore quiescence, enables Muller glia to proliferate and generate neurons in adult mice following retinal injury. These findings may aid in the design of cell-based therapies aimed at restoring retinal neurons lost to degenerative disease. Summary sentenceThis study identifies gene regulatory networks controlling proliferative and neurogenic competence in retinal Muller glia.

neuroscience↗