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Background Microglia and inflammation have context-specific impacts upon neuronal survival in different models of central nervous system (CNS) disease. Herein, we investigate how inflammatory mediators, including microglia, interleukin 1 beta (IL1β), and signaling through interleukin 1 receptor type 1 (IL-1R1), influence the survival of retinal neurons in response to excitotoxic damage. Methods Excitotoxic retinal damage was induced via intraocular injections of NMDA. Microglial phenotype and neuronal survival were assessed by immunohistochemistry. Single-cell RNA sequencing was performed to obtain transcriptomic profiles. Microglia were ablated by using clodronate liposome or PLX5622. Retinas were treated with IL1β prior to NMDA damage and cell death was assessed in wild type, IL-1R1 null mice, and mice expressing IL-1R1 only in astrocytes. Results NMDA-induced damage included neuronal cell death, microglial reactivity, upregulation of pro-inflammatory cytokines, and genes associated with IL1β-signaling in different types of retinal neurons and glia. Expression of the IL1β receptor, IL-1R1, was evident in astrocytes, endothelial cells, some Müller glia, and OFF bipolar cells. Ablation of microglia with clodronate liposomes or Csf1r antagonist (PLX5622) resulted in elevated cell death and diminished neuronal survival in excitotoxin-damaged retinas. Exogenous IL1β stimulated the proliferation and reactivity of microglia in the absence of damage, reduced numbers of dying cells in damaged retinas, and increased neuronal survival following an insult. IL1β failed to provide neuroprotection in the IL-1R1-null retina, but IL1β-mediated neuroprotection was rescued when expression of IL-1R1 was restored in astrocytes. Conclusions We conclude that reactive microglia provide protection to retinal neurons, since the absence of microglia is detrimental to survival. We propose that, at least in part, the survival-influencing effects of microglia may be mediated by IL1β, IL-1R1, and interactions of microglia and other macroglia.

Retinal Müller 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 Müller glia, induction of neurogenesis in wildtype glia has thus far proven elusive. Here, we report that viral-mediated overexpression of the pluripotency factor Pou5f1 (Oct4) induces transdifferentiation of mouse Müller glia into bipolar neurons, and synergistically stimulates glial-derived neurogenesis in parallel with Notch loss of function. Single-cell multiomic analysis shows that Pou5f1 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 Pou5f1 induces neurogenic competence in adult mouse Müller glia, identifying mechanisms that could be used in cell-based therapies for treating retinal dystrophies.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes through Müller glia (MG) reprogramming and asymmetric cell division that produces a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, do MG reprogram to a developmental retinal progenitor cell (RPC) state? Second, to what extent does regeneration recapitulate retinal development? And finally, does loss of different retinal cell subtypes induce unique MG regeneration responses? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. Here we show that injury induces MG to reprogram to a state similar to late-stage RPCs. However, there are major transcriptional differences between MGPCs and RPCs, as well as major transcriptional differences between activated MG and MGPCs when different retinal cell subtypes are damaged. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. The molecular mechanisms controlling injury-dependent neuronal regeneration are largely unknown. Here, the authors use integrated multiomic analysis to characterize gene regulatory networks controlling injury-induced neurogenesis in zebrafish retina

Evolutionary adaptation to diurnal vision in ground squirrels has led to the development of a cone-dominant retina, in stark contrast to the rod-dominant retinas of most mammals. The molecular mechanisms driving this shift remain largely unexplored. Here, we perform single-cell RNA sequencing and chromatin accessibility profiling (scATAC-Seq) across developmental retinal neurogenesis in the 13-lined ground squirrel (13LGS) to uncover the regulatory basis of this adaptation. We find that 13LGS cone photoreceptors arise not only from early-stage neurogenic progenitors, as seen in rod-dominant species like mice, but also from late-stage neurogenic progenitors. This extended period of cone generation is driven by a heterochronic shift in transcription factor expression, with cone-promoting factors such as Onecut2 , Pou2f1 , and Zic3 remaining active in late-stage progenitors, and factors that promote cone differentiation such as Thrb , Rxrg , and Mef2c expressed precociously in late-stage neurogenic progenitors. Functional analyses reveal that Zic3 and Mef2c are sufficient to promote cone and repress rod photoreceptor-specific gene expression and act through species-specific regulatory elements that drive their expression in late-stage progenitors. These results demonstrate that modifications to gene regulatory networks underlie the development of cone-dominant retinas and provide insight into mechanisms of sensory adaptation and potential strategies for cone photoreceptor regeneration in vision disorders.

Retinal Müller 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 Müller glia, induction of neurogenesis in wildtype glia has thus far proven elusive. Here, we report that viral-mediated overexpression of the pluripotency factor Pou5f1 (Oct4) induces transdifferentiation of mouse Müller glia into bipolar neurons, and synergistically stimulates glial-derived neurogenesis in parallel with Notch loss of function. Single-cell multiomic analysis shows that Pou5f1 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 Pou5f1 induces neurogenic competence in adult mouse Müller glia, identifying mechanisms that could be used in cell-based therapies for treating retinal dystrophies.

Evolutionary adaptation to diurnal vision in ground squirrels has led to the development of a cone-dominant retina, in stark contrast to the rod-dominant retinas of most mammals. The molecular mechanisms driving this shift remain largely unexplored. Here, we perform single-cell RNA sequencing and chromatin accessibility profiling (scATAC-Seq) across developmental retinal neurogenesis in the 13-lined ground squirrel (13LGS) to uncover the regulatory basis of this adaptation. We find that 13LGS cone photoreceptors arise not only from early-stage neurogenic progenitors, as seen in rod-dominant species like mice, but also from late-stage neurogenic progenitors. This extended period of cone generation is driven by a heterochronic shift in transcription factor expression, with cone-promoting factors such as Onecut2 , Pou2f1 , and Zic3 remaining active in late-stage progenitors, and factors that promote cone differentiation such as Thrb , Rxrg , and Mef2c expressed precociously in late-stage neurogenic progenitors. Functional analyses reveal that Zic3 and Mef2c are sufficient to promote cone and repress rod photoreceptor-specific gene expression and act through species-specific regulatory elements that drive their expression in late-stage progenitors. These results demonstrate that modifications to gene regulatory networks underlie the development of cone-dominant retinas and provide insight into mechanisms of sensory adaptation and potential strategies for cone photoreceptor regeneration in vision disorders.

Aging is a major risk factor for the progression of neurodegenerative diseases, including Huntington disease (HD). Reduced neuronal IGF1 or Irs2 signaling have been shown to extend life span in mice. To determine whether Irs2 signaling modulates neurodegeneration in HD, we genetically modulated Irs2 concentrations in the R6/2 mouse model of HD. Increasing Irs2 levels in the brains of R6/2 mice significantly reduced life span and increased neuronal oxidative stress and mitochondrial dysfunction. In contrast, reducing Irs2 levels throughout the body (except in β cells, where Irs2 expression is needed to prevent diabetes onset; R6/2•Irs2+/-•Irs2βtg mice) improved motor performance and extended life span. The slower progression of HD-like symptoms was associated with increased nuclear localization of the transcription factor FoxO1 and increased expression of FoxO1-dependent genes that promote autophagy, mitochondrial function, and resistance to oxidative stress. Mitochondrial function improved and the number of autophagosomes increased in R6/2•Irs2+/-•Irs2βtg mice, whereas aggregate formation and oxidative stress decreased. Thus, our study suggests that Irs2 signaling can modulate HD progression. Since we found the expression of Irs2 to be normal in grade II HD patients, our results suggest that decreasing IRS2 signaling could be part of a therapeutic approach to slow the progression of HD.

Retinal degeneration is a leading cause of blindness. Progressive death of retinal neurons is irreversible and results in loss of vision. Currently, there are no therapeutic treatments for loss of vision caused by retinal degeneration. However, the ability of various vertebrate species to utilize endogenous cellular sources for neuronal replacement provides promise for retinal regeneration. Müller glia are the primary support cell of the retina, and they possess the capacity to de-differentiate from mature support cells into progenitor cells that give rise to various types of neurons throughout the retina. However, the capacity for Müller glia-mediated neuronal regeneration varies widely across phylogeny. Cold-blooded vertebrates, such as zebrafish, exhibit a robust regenerative capacity where Müller glia reprogram into proliferating progenitor cells that subsequently differentiate into all the various subtypes of retinal neurons to restore a fully functional retina after damage. This capacity is diminished in avian models where only a small proportion of neurons regenerate from Müller glia-derived progenitor cells. This capacity for regeneration is refractory in the mammalian system, with Müller glia failing to reprogram after damage. Understanding the molecular mechanisms that control Müller glia reprogramming and identifying mechanisms that permit reprogramming in the fish and prevent reprogramming in the mammalian system is essential to harnessing the capacity of these cells for potential therapeutic interventions to regenerate neurons and prevent blindness.The primary focus of this dissertation is how neuroinflammation regulates glial response to damage in the retina. The first data chapter focuses on how neuroinflammation impacts neuronal death after excitotoxin-induced retinal degeneration. We describe how microglia, the resident immune cell of the retina, become activated after damage and communicate with astrocytes, via cytokine signaling, to convey neuroprotection. The next two chapters focus on how neuroinflammatory signaling impacts neurogenic reprogramming of Müller glia after damage. We take a cross-species approach to investigate the role of the master pro-inflammatory regulator, Nuclear Factor kappa-light-chain-enhancer of activated B cells (NFkB), in Müller glia reprogramming. We find that NFkB is rapidly activated in Müller glia in both chick and mouse models after NMDA-induced excitotoxic retinal injury. We find that inhibition of NFkB signaling in the damaged chick retina promotes the formation and proliferation of Müller glia-derived progenitor cells, however, this is dependent on the presence of microglia. Additionally, we find that inhibition of NFkB signaling in the damaged mouse retina is neuroprotective and diminishes the recruitment of peripheral immune cells to the retina. Since mammalian Müller glia do not reprogram in response to retinal damage, we utilized transgenic overexpression of the pro-neural transcription factor Ascl1, which results in moderate neurogenesis from adult Müller glia. In these mice, we find inhibition of NFkB signaling promotes Müller glia-derived neuron formation. Single cell RNA-sequencing (scRNA-seq) analysis indicates that this increased neurogenesis mediated by NFkB inhibition is a result of diminished glial reactivity and decreased activation of pro-glial/anti-neural gene regulatory networks in damaged retina.The final chapter of this dissertation focuses on how NFkB signaling communicates with the key signaling pathways identified to regulate Müller glia reprogramming in the zebrafish, chick, and mouse retina. Collectively, these data describe a mechanism by which pro-inflammatory signaling interferes with neuron regeneration in the retina across species, thus proposing a promising therapeutic target to reverse loss of vision.

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.