Excitatory neurotransmission activates compartmentalized calcium transients in Müller glia without affecting lateral process motility

Neural activity has been implicated in the motility and outgrowth of glial cell processes throughout the central nervous system. Here we explore this phenomenon in Müller glia, which are specialized radial astroglia that are the predominant glial type of the vertebrate retina. Müller glia extend fine filopodia-like processes into retinal synaptic layers, in similar fashion to brain astrocytes and radial glia which exhibit perisynaptic processes. Using two-photon volumetric imaging, we found that during the second postnatal week, Müller glial processes were highly dynamic, with rapid extensions and retractions that were mediated by cytoskeletal rearrangements. During this same stage of development, retinal waves led to increases in cytosolic calcium within Müller glial lateral processes and stalks. These comprised distinct calcium compartments, distinguished by variable participation in waves, timing, and sensitivity to an M1 muscarinic acetylcholine receptor antagonist. However, we found that motility of lateral processes was unaffected by the presence of pharmacological agents that enhanced or blocked wave-associated calcium transients. Finally, we found that mice lacking normal cholinergic waves in the first postnatal week also exhibited normal Müller glial process morphology. Hence, outgrowth of Müller glial lateral processes into synaptic layers is determined by factors that are independent of neuronal activity.

Develop an efficient and specific AAV-based labeling system for Muller glia in mice

Cell degeneration in the retina leads to several ocular diseases and vision loss. Considerable research efforts focus on reprogramming Muller glia (MG) into functional cells to rescue vision as a promising therapeutic strategy, although whether MG can convert into functional amacrine cells, bipolar cells, retinal ganglia cells (RGCs), rods or cones in mammals remains controversial. The broad applicability of tracking MG differentiation thus presents a need for improved labeling efficiency and specificity. Here, we compared AAV-based labeling strategies with conventional lineage-tracking by crossing transgenic mouse lines. We found that reporter expression was weak and not MG-specific in mGFAP-Cre transgenic mice. Different AAV serotypes showed a range of efficiency and specificity in labeling MG, leading us to optimize a human GFAP-Cre reporter system packaged in the AAV9 serotype with the WPRE (WPRE, woodchuck hepatitis virus post-transcriptional regulatory element) removed. The hGFAP-Cre-ΔWPRE reporter could label 20-73.8% MGs, with non-specific RGC labeling rates ranging from 0-0.08% at doses of 1 × 108 to 1010 vector genomes (vg) per eye, an approximate 40-fold reduction compared with the AAV9-hGFAP-Cre-WPRE labeling system. The AAV9-hGFAP-Cre-ΔWPRE system thus represents a highly efficient and specific labeling system for Muller glia, providing a valuable tool for tracking cell fate in vivo.

A Novel and Atypical Nf-Kb Proinflammatory Program Regulated by A Camkii-Proteasome Axis Is Involved in the Early Activation of Muller Glia by High Glucose

BackgroundDiabetic retinopathy (DR) is a microvascular complication of diabetes with a heavy impact on the life-quality of subjects and with a dramatic burden for health and economic systems on a global scale.Although the pathogenesis of DR is largely unknown, several preclinical data have pointed out to a main role of Muller glia, a cell type which spans across the retina layers providing nourishment and support for Retina Ganglion Cells (RGCs), in sensing glycemia and in acquiring a proinflammatory polarization in response to this insult.ResultsBy using a validated experimental model of DR in vitro, the rMC1 cells challenged with high glucose, we uncovered the induction of an early (within minutes) and atypical NF-kB signalling pathway regulated by a CamKII-proteasome axis. Phosphorylation of proteasome subunit Rpt6 (at serine 120) by CamKII stimulated the accelerated turnover of IkBα (i.e., the natural inhibitor of p65-50 transcription factor), regardless of the phosphorylation at serine 32 which labels canonical NF-kB signalling. This event allowed the p65-p50 heterodimer to migrate into the nucleus and to induce the selective transcription of IL-8, Il-1β and MCP-1. Pharmacological inhibition of CamKII or proteasome stopped this proinflammatory program, whereas introduction of a Rpt6 phospho-dead mutant (Rpt6-S120A) stimulated a paradoxical effect on NF-kB probably through the activation of a compensatory mechanism which may involve phosphorylation of 20S α4 subunit.ConclusionsThis study introduces a novel pathway of MG activation by high glucose and casts some light on the biological relevance of proteasome post-translational modifications in modulating pathways regulated through targeted proteolysis.

Ikaros family proteins regulate developmental windows in the mouse retina through convergent and divergent transcriptional programs

ABSTRACTTemporal identity factors regulate the competence of neural progenitors to generate specific cell types in a time-dependent manner, but how they operate remains poorly defined. In the developing mouse retina, the Ikaros zinc finger transcription factor Ikzf1 regulates the production of early-born cell types, except cone photoreceptors. In this study we show that Ikzf4, another Ikaros family protein, cooperates with Ikzf1 to control cone photoreceptor production during early stages of retinal development, whereas at late stages, when Ikzf1 is no longer expressed in progenitors, Ikzf4 is instead required for Müller glia production. Using CUT&RUN sequencing, we find that both Ikzf1 and Ikzf4 generally bind to the same genes involved in cone development and other early-born fates, but at different cis-regulatory elements. In late-stage progenitors, Ikzf4 re-localizes to bind target genes involved in Müller glia development and regulate their expression. Specifically, we show that Ikzf4 maintains Hes1 expression in differentiating cells using two Ikzf GGAA binding sites at the Hes1 promoter, thereby favouring Müller glia fate commitment. These results uncover a combinatorial role for Ikaros family members in nervous system development and provide mechanistic insights on how they temporally regulate cell fate output.

Müller glial responses compensate for degenerating photoreceptors in retinitis pigmentosa

Photoreceptor degeneration caused by genetic defects leads to retinitis pigmentosa, a rare disease typically diagnosed in adolescents and young adults. In most cases, rod loss occurs first, followed by cone loss as well as altered function in cells connected to photoreceptors directly or indirectly. There remains a gap in our understanding of retinal cellular responses to photoreceptor abnormalities. Here, we utilized single-cell transcriptomics to investigate cellular responses in each major retinal cell type in retinitis pigmentosa model (P23H) mice vs. wild-type littermate mice. We found a significant decrease in the expression of genes associated with phototransduction, the inner/outer segment, photoreceptor cell cilium, and photoreceptor development in both rod and cone clusters, in line with the structural changes seen with immunohistochemistry. Accompanying this loss was a significant decrease in the expression of genes involved in metabolic pathways and energy production in both rods and cones. We found that in the Müller glia/astrocyte cluster, there was a significant increase in gene expression in pathways involving photoreceptor maintenance, while concomitant decreases were observed in rods and cones. Additionally, the expression of genes involved in mitochondrial localization and transport was increased in the Müller glia/astrocyte cluster. The Müller glial compensatory increase in the expression of genes downregulated in photoreceptors suggests that Müller glia adapt their transcriptome to support photoreceptors and could be thought of as general therapeutic targets to protect against retinal degeneration.

mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia

Retinal progenitor cells (RPCs) divide in limited numbers to generate the cells comprising vertebrate retina. The molecular mechanism that leads RPC to the division limit, however, remains elusive. Here, we find that the hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) in an RPC subset by deletion of tuberous sclerosis complex 1 (Tsc1) makes the RPCs arrive at the division limit precociously and produce Müller glia (MG) that degenerate from senescence-associated cell death. We further show the hyperproliferation of Tsc1-deficient RPCs and the degeneration of MG in the mouse retina disappear by concomitant deletion of hypoxia-induced factor 1-a (Hif1a), which induces glycolytic gene expression to support mTORC1-induced RPC proliferation. Collectively, our results suggest that, by having mTORC1 constitutively active, an RPC divides and exhausts mitotic capacity faster than neighboring RPCs, and thus produces retinal cells that degenerate with aging-related changes.