Biology subjects

Fischer, A. J.

Publications and source records attributed to Fischer, A. J..

18 recordsLinked to original sources

bioRxiv2026

Particle size determines mucociliary transport mechanisms in normal and cystic fibrosis airways

A wide spectrum of microparticles is inhaled with each breath, deposited on airway surfaces, entrapped in the mucus, and removed by mucociliary transport (MCT). However, the influence of particle size on MCT remains largely unknown. Here, we investigated the MCT of microparticles with a trachea-on-a-chip method that integrates a micro-machined device with a trachea explant from newborn pigs. This method preserves airway structures for mucus secretion and cilia beating (e.g., airway surface epithelia and submucosal glands), maintains physiological air-liquid-interface on the airway surface, and allows tracks motion of microparticles with high resolution. Using this method, we found that, in normal airways, 6 {micro}m polystyrene particles clear rapidly, whereas 102 {micro}m particles clear slower and require mucus strands for motion. In cystic fibrosis (CF) airways, MCT of microparticles reduces, but particle size-dependence persists. Methacholine increases particle motion in normal airways, but not in CF airways. These findings suggest two distinct MCT processes, in which large particles rely on mucus strands for clearance, small particles can be cleared independent of mucus strands, and CF disrupts both. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/735890v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@3e3026org.highwire.dtl.DTLVardef@cb11fdorg.highwire.dtl.DTLVardef@a28a3org.highwire.dtl.DTLVardef@f1f1a9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

bioengineering↗

bioRxiv2026

In Utero CFTR Modulation Alleviates Disease in G551D Cystic Fibrosis Pigs

Previous studies indicate that pigs with CFTR-null and CFTR-{Delta}F508 mutations develop multiorgan disease similar to that in people with cystic fibrosis (CF). At birth, their airways exhibit host defense defects that predispose to airway infection, inflammation, and mucus accumulation. The CFTR-G551D mutation causes CF by producing CFTR channels that localize correctly but have reduced channel activity. Ivacaftor (VX-770) is a small molecule drug developed to potentiate CFTR activity. To test the phenotype of the CFTR-G551D mutation in pigs and determine whether ivacaftor can rescue CF abnormalities, we developed CFTRG551D/G551D (CF-G551D) pigs through homologous recombination in fetal fibroblasts and somatic cell nuclear transfer. Newborn CF-G551D piglets exhibited phenotypes similar to CF-null piglets, including meconium ileus, exocrine pancreatic destruction, micro-gallbladder, vas deferens destruction, and airway structural abnormalities. Compared to wild-type pigs, CF-G551D pigs had reduced forskolin-stimulated short-circuit current in airway and intestinal tissues. Ivacaftor increased the single-channel open state probability of CFTR-G551D and increased short-circuit current to near wild-type levels. Similar to our other CF pig models, we found that 100% of CF-G551D pigs were born with meconium ileus. To test whether in utero ivacaftor treatment could prevent or alleviate meconium ileus, pregnant sows were treated with ivacaftor beginning at day 35 of gestation and continuing until delivery. This treatment rescued the pancreas, gallbladder, and vas deferens phenotype in the majority of CF-G551D pigs. Animals that were spared from meconium ileus were able to survive without ivacaftor treatment. Airway disease developed similar to other CF pig models. These findings indicate that this model may be useful for studies in which CFTR function can be reversed, for investigating in utero CFTR correction strategies, and for longitudinal studies in CF pigs.

physiology↗

bioRxiv2025

Sphingosine-1-phosphate signaling through Müller glia regulates neuroprotection and the accumulation of immune cells in the rodent retina

The purpose of this study was to investigate how Sphingosine-1-phosphate (S1P) signaling regulates glial phenotype, neuroprotection, and reprogramming of Muller glia (MG) into neurogenic MG-derived progenitor cells (MGPCs) in the adult mouse retina. We found that S1P-related genes were dynamically regulated following retinal damage. S1pr1 (encoding S1P receptor 1) and Sphk1 (encoding sphingosine kinase 1) are expressed at low levels by resting MG and are rapidly upregulated following acute damage. Overexpression of the neurogenic bHLH transcription factor Ascl1 in MG downregulates S1pr1, and inhibition of Sphk1 and S1pr1/3 enhances Ascl1-driven differentiation of bipolar-like cells and suppresses glial differentiation. Treatments that activate S1pr1 or increase retinal levels of S1P initiate pro-inflammatory NF{kappa}B-signaling in MG, whereas treatments that inhibit S1pr1 or decreased levels of S1P suppress NF{kappa}B-signaling in MG in damaged retinas. Conditional knock-out of NF{kappa}B-signaling in MG increases glial expression of S1pr1 but decreases levels of S1pr3 and Sphk1. Conditional knock-out (cKO) of S1pr1 in MG, but not Sphk1, enhances the accumulation of immune cells in acutely damaged retinas. cKO of S1pr1 is neuroprotective to ganglion cells, whereas cKO of Sphk1 is neuroprotective to amacrine cells in NMDA-damaged retinas. Consistent with these findings, pharmacological treatments that inhibit S1P receptors or inhibit Sphk1 had protective effects upon inner retinal neurons. We conclude that the S1P-signaling pathway is activated in MG after damage and this pathway acts secondarily to restrict the accumulation of immune cells, impairs neuron survival and suppresses the reprogramming of MG into neurogenic progenitors in the adult mouse retina.

neuroscience↗

bioRxiv2024

Sphingosine-1-phosphate signaling regulates the ability of Müller glia to become neurogenic, proliferating progenitor-like cells

The purpose of these studies is to investigate how Sphingosine-1-phosphate (S1P) signaling regulates glial phenotype, dedifferentiation of Muller glia (MG), reprogramming into proliferating MG-derived progenitor cells (MGPCs), and neuronal differentiation of the progeny of MGPCs in the chick retina. We found that S1P-related genes are highly expressed by retinal neurons and glia, and levels of expression were dynamically regulated following retinal damage. Drug treatments that activate S1P receptor 1 (S1PR1) or increase levels of S1P suppressed the formation of MGPCs. Conversely, treatments that inhibit S1PR1 or decrease levels of S1P stimulated the formation of MGPCs. Inhibition of S1P receptors or S1P synthesis significantly enhanced the neuronal differentiation of the progeny of MGPCs. We report that S1P-related gene expression in MG is modulated by microglia and inhibition of S1P receptors or S1P synthesis partially rescues the loss of MGPC formation in damaged retinas missing microglia. Finally, we show that TGF{beta}/Smad3 signaling in the resting retina maintains S1PR1 expression in MG. We conclude that the S1P signaling is dynamically regulated in MG and MGPCs in the chick retina, and activation of S1P signaling depends, in part, on signals produced by reactive microglia.

neuroscience↗

bioRxiv2024

Development and Initial Characterization of Pigs with DNAI1 Mutations and Primary Ciliary Dyskinesia

Mutations in more than 50 different genes cause primary ciliary dyskinesia (PCD) by disrupting the activity of motile cilia that facilitate mucociliary transport (MCT). Knowledge of PCD has come from studies identifying disease-causing mutations, characterizing structural cilia abnormalities, finding genotype-phenotype relationships, and studying the cell biology of cilia. Despite these important findings, we still lack effective treatments and people with PCD have significant pulmonary impairment. As with many other diseases, a better understanding of pathogenic mechanisms may lead to effective treatments. To pursue disease mechanisms, we used CRISPR-Cas9 to develop a PCD pig with a disrupted DNAI1 gene. PCD pig airway cilia lacked the outer dynein arm and had impaired beating. MCT was impaired under both baseline conditions and after cholinergic stimulation in PCD pigs. Neonatal PCD pigs developed neonatal respiratory distress with evidence of atelectasis, air trapping, and airway mucus obstruction. Despite airway mucus accumulation, lung bacterial counts were similar between neonatal wild-type and PCD pigs. Sinonasal disease was present in all neonatal PCD pigs. Older PCD pigs developed worsening airway mucus obstruction, inflammation, and bacterial infection. This pig model closely mimics the disease phenotype seen in people with PCD and can be used to better understand the pathophysiology of PCD airway disease.

physiology↗

bioRxiv2024

Ibudilast Protects Retinal Bipolar Cells from Excitotoxic Retinal Damage and Activates the mTOR Pathway

Ibudilast, an inhibitor of macrophage migration inhibitory factor (MIF) and phosphodiesterase (PDE), has been recently shown to have neuroprotective effects in a variety of neurologic diseases. We utilize a chick excitotoxic retinal damage model to investigate ibudilasts potential to protect retinal neurons. Using single cell RNA-sequencing (scRNA-seq), we find that MIF, putative MIF receptors CD74 and CD44, and several PDEs are upregulated in different retinal cells during damage. Intravitreal ibudilast is well tolerated in the eye and causes no evidence of toxicity. Ibudilast effectively protects neurons in the inner nuclear layer from NMDA-induced cell death, restores retinal layer thickness on spectral domain optical coherence tomography, and preserves retinal neuron function, particularly for the ON bipolar cells, as assessed by electroretinography. PDE inhibition seems essential for ibudilasts neuroprotection, as AV1013, the analogue that lacks PDE inhibitor activity, is ineffective. scRNA-seq analysis reveals upregulation of multiple signaling pathways, including mTOR, in damaged Muller glia (MG) with ibudilast treatment compared to AV1013. Components of mTORC1 and mTORC2 are upregulated in both bipolar cells and MG with ibudilast. The mTOR inhibitor rapamycin blocked accumulation of pS6 but did not reduce TUNEL positive dying cells. Additionally, through ligand-receptor interaction analysis, crosstalk between bipolar cells and MG may be important for neuroprotection. We have identified several paracrine signaling pathways that are known to contribute to cell survival and neuroprotection and might play essential roles in ibudilast function. These findings highlight ibudilasts potential to protect inner retinal neurons during damage and show promise for future clinical translation. Graphical AbstractO_ST_ABSMain PointsC_ST_ABS- Ibudilast, a MIF and PDE inhibitor, preserves the form and function of the retina, especially bipolar cells, during excitotoxic damage - Ibudilast upregulates multiple signaling pathways, including mTOR, in damaged Muller glia and bipolar cells O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/585556v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@18d9916org.highwire.dtl.DTLVardef@13db1b2org.highwire.dtl.DTLVardef@15d3d8dorg.highwire.dtl.DTLVardef@742b0c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Protein phosphatases regulate the formation of Müller glia-derived progenitor cells in the chick retina

Different kinase-dependent cell signaling pathways are known to play important roles in glia-mediated neuroprotection and reprogramming of Muller glia (MG) into Muller glia-derived progenitor cells (MGPCs) in the retina. However, very little is known about the phosphatases that regulate kinase-dependent signaling in MG. Using single-cell RNA-sequencing (scRNA-seq) databases, we investigated patterns of expression of Dual Specificity Phosphatases (DUSP1/6) and other protein phosphatases in normal and damaged chick retinas. We found that DUSP1, DUSP6, PPP3CB, PPP3R1 and PPPM1A/B/D/E/G are dynamically expressed by MG and MGPCs in retinas during the process of reprogramming. We find that inhibition of DUSP1/6 and PP2C phosphatases enhances the formation of proliferating MGPCs in damaged retinas and in retinas treated with insulin in FGF2 in the absence of damage. By contrast, inhibition of PP2B phosphatases suppressed the formation of proliferating MGPCs, but increased numbers of proliferating MGPCs in undamaged retinas treated with insulin and FGF2. In damaged retinas, inhibition of DUSP1/6 increased levels of pERK1/2 and cFos in MG whereas inhibition of PP2Bs decreased levels of pStat3 and pS6 in MG. Analyses of scRNA-seq libraries identified numerous differentially activated gene modules in MG in damaged retinas versus MG in retinas treated with insulin+FGF2 suggesting significant differences in kinase-dependent signaling pathways that converge on the formation of MGPCs. Inhibition of phosphatases had no significant effects upon numbers of dying cells in damaged retinas. We conclude that the activity of different protein phosphatases "fine-tune" the cell signaling responses of MG in damaged retinas and during the reprogramming of MG into MGPCs.

neuroscience↗

bioRxiv2023

ID transcription factors regulate the ability of Muller glia to become proliferating neurogenic progenitor-like cells

The purpose of this study was to investigate how ID transcription factors (TFs) regulate the ability of Muller glia (MG) to reprogram into proliferating MG-derived progenitor cells (MGPCs) in the chick retina. We found that ID1 is transiently expressed by maturing MG, whereas ID4 is upregulated and maintained in maturing MG in embryonic retinas. In mature retinas, ID4 was prominently expressed by resting MG, but in response to retinal damage ID4 was rapidly upregulated and then downregulated in MGPCs. By contrast, ID1, ID2 and ID3 were low in resting MG and then upregulated by MGPCs. Inhibition of ID TFs following retinal damage decreased numbers of proliferating MGPCs. Inhibition of IDs after the proliferation of MGPCs significantly increased numbers of progeny that differentiate as neurons. In damaged or undamaged retinas inhibition of IDs increased levels of p21Cip1 in MG. In response to damage or insulin+FGF2 levels of CDKN1A message and p21Cip1 protein were decreased, absent in proliferating MGPCs, and elevated in MG returning to a resting phenotype. Inhibition of Notch- or gp130/Jak/Stat-signaling in damaged retinas increased levels of ID4 but not p21Cip1 in MG. Although ID4 is the predominant isoform expressed by MG in the chick retina, id1 and id2a are predominantly expressed by resting MG and downregulated in activated MG and MGPCs in zebrafish retinas. We conclude that ID TFs have a significant impact on regulating the responses of MG to retinal damage, controlling the ability of MG to proliferate by regulating levels of p21Cip1, and suppressing the neurogenic potential of MGPCs.

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↗

bioRxiv2023

Parallel Evolution of Linezolid Resistant Staphylococcus aureus in Patients with Cystic Fibrosis

BackgroundLinezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). ObjectivesThe goals of this study were to determine the incidence of linezolid resistance in CF and identify molecular mechanisms for linezolid resistance. MethodsWe identified patients with S. aureus resistant to linezolid (MIC > 4) at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Main ResultsBetween 2008 and 2018, 111 patients received linezolid and 4 of these patients cultured linezolid resistant S. aureus. We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS- mutL- hypermutating S. aureus that produced 5 resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. ConclusionsLinezolid resistance evolved in 4 of 111 patients in this study. Linezolid resistance occurred by multiple genetic mechanisms. All resistant strains developed in ST5 or ST105 MRSA backgrounds. Key PointLinezolid resistance arises through multiple genetic mechanisms and could be facilitated by mutator phenotypes. Linezolid resistance was transient, possibly due to growth disadvantage.

microbiology↗

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↗

bioRxiv2022

The polycomb repressor complex 2 and Enhancer of Zeste 2 regulate glial reactivity and are required for the formation of Muller glia-derived progenitor cells in the retina

Chromatin access and epigenetic control over gene expression play important roles in regulating developmental processes. However, little is known about how chromatin access and epigenetic gene silencing influence mature glial cells and retinal regeneration. Herein we investigate the expression and functions of S-Adenosylhomocysteine Hydrolase (SAHH; AHCY) and Histone Methyltransferases (HMTs) during the formation of Muller glia-derived progenitor cells (MGPCs) in the chick and mouse retinas. In chick, AHCY, AHCYL1, AHCYL2 and many different HMTs are dynamically expressed by MG and MGPCs in damaged retinas. Inhibition of SAHH reduced levels of H3K27me3 and potently blocks the formation of proliferating MGPCs. By using a combination of single cell RNA-seq and single cell ATAC-seq, we find significant changes in gene expression and chromatin access in MG with SAHH inhibition and NMDA-treatment; many of these genes are associated with glial and neuronal differentiation. A strong correlation across gene expression, chromatin access, and transcription factor motif access in MG was observed for transcription factors known to covey glial identity and promote retinal development. By comparison, in the mouse retina, inhibition of SAHH has no influence on the formation of neuron-like cells from Ascl1-overexpressing MG. We conclude that in the chick, but not the mouse, the activity of SAHH and HMTs are required for the reprogramming of MG into MGPCs by regulating chromatin access to transcription factors associated with glial differentiation and retinal development.

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↗

bioRxiv2021

Fatty acid-binding proteins and fatty acid synthase influence glial reactivity and promote the formation of Müller glia-derived progenitor cells in the avian retina

The capacity for retinal regeneration varies greatly across vertebrates species. A recent comparative epigenetic and transcriptomic investigation of Muller glial (MG) in the retinas of fish, birds and mice revealed that Fatty Acid Binding Proteins (FABPs) are among the most highly up-regulated genes in activated chick MG (Hoang et al., 2020). Herein we provide an in-depth follow-up investigation to describe patterns of expression and how FABPs and fatty acid synthase (FASN) influence glial cells in the chick retina. During development, FABP7 is highly expressed by embryonic retinal progenitor cells (eRPCs) and maturing MG, whereas FABP5 is gradually up-regulated in maturing MG and remains elevated in mature glial cells. PMP2 (FABP8) is expressed by oligodendrocytes and FABP5 is expressed by non-astrocytic inner retinal glial cells, and both of these FABPs are significantly up-regulated in activated MG in damaged or growth factor-treated retinas. In addition to suppressing the formation of MGPCs, we find that FABP-inhibition suppressed the accumulation of proliferating microglia, although the microglia appeared highly reactive. scRNA-seq analyses of cells treated with FABP-inhibitor revealed distinct changes in patterns of expression suggesting that FABPs are involved in the transitions of MG from a resting state to a reactive state and conversion from reactive MG to MGPCs. Inhibition of FABPs in undamaged retinas had a significant impact upon the transcriptomic profiles of MG, with up-regulation of genes associated with gliogenesis, decreases in genes associated with neurogenesis, and suppression of the ability of MG to become MGPCs. scRNA-seq analyses of microglia indicated that FABP inhibition enhances gene modules related to reactivity, proliferation and cytokine signaling. We find that the proliferation of retinal progenitors in the circumferential marginal zone (CMZ) is unaffected by FABP-inhibitor. Upstream of FABP activity, we inhibited FASN in damaged retinas, which reduced numbers of dying cells, increased the proliferation of microglia, and potently suppressed the formation MGPCs in damaged retinas. We conclude that the activity of FASN and FABPs are required early during the formation of proliferating MGPCs. Fatty acid metabolism and cell signaling involving fatty acids are important in regulating glial homeostasis in the retina, and the dedifferentiation and proliferation of microglia and MGPCs.

developmental biology↗

bioRxiv2021

Nuclear Factor I in neurons, glia and during the formation of Muller glia-derived progenitor cells in avian, porcine and primate retinas

The regenerative potential of Muller glia (MG) is extraordinary in fish, poor in chick and terrible in mammals. In the chick model, MG readily reprogram into proliferating Muller glia-derived progenitor cells (MGPCs), but neuronal differentiation is very limited. The factors that suppress the neurogenic potential of MGPCs in the chick are slowly being revealed. Isoforms of Nuclear Factor I (NFI) are cell-intrinsic factors that limit neurogenic potential; these factors are required for the formation of MG in the developing mouse retina (Clark et al., 2019) and deletion of these factors reprograms MG into neuron-like cells in mature mouse retina (Hoang et al., 2020). Accordingly, we sought to characterize the patterns of expression NFIs in the developing, mature and damaged chick retina. In addition, we characterized patterns of expression of NFIs in the retinas of large mammals, pigs and monkeys. Using a combination of single cell RNA-sequencing (scRNA-seq) and immunolabeling we probed for patterns of expression. In embryonic chick, levels of NFIs are very low in early E5 (embryonic day 5) retinal progenitor cells (RPCs), up-regulated in E8 RPCs, further up-regulated in differentiating MG at E12 and E15. NFIs are maintained in mature resting MG, microglia and neurons. Levels of NFIs are reduced in activated MG in retinas treated with NMDA and/or insulin+FGF2, and further down-regulated in proliferating MGPCs. However, levels of NFIs in MGPCs were significantly higher than those seen in RPCs. Immunolabeling for NFIA and NFIB closely matched patterns of expression revealed in different types of retinal neurons and glia, consistent with findings from scRNA-seq. In addition, we find expression of NFIA and NFIB through progenitors in the circumferential marginal zone at the far periphery of the retina. We find similar patterns of expression for NFIs in scRNA-seq databases for pig and monkey retinas. Patterns of expression of NFIA and NFIB were validated with immunofluorescence in pig and monkey retinas wherein these factors were predominantly detected in MG and a few types of inner retinal neurons. In summary, NFIA and NFIB are prominently expressed in developing chick retina and by mature neurons and glia in the retinas of chicks, pigs and monkeys. Although levels of NFIs are decreased in chick, in MGPCs these levels remain higher than those seen in neurogenic RPCs. We propose that the neurogenic potential of MGPCs in the chick retina is suppressed by NFIs.

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↗

bioRxiv2020

MgrA regulates interaction of Staphylococcus aureus with mucin

BackgroundTo defend the lungs, mucus adheres to bacterial cells and facilitates their removal by ciliary transport. Our goals were to measure the affinity of mucus for the respiratory pathogen Staphylococcus aureus and identify bacterial genes that regulate this interaction. MethodsS. aureus was added to pig tracheas to determine whether it binds mucus or epithelial cells. To quantify its affinity for mucus, we developed a competition assay in microtiter plates. Mucin was added over a dose range as an inhibitor of bacterial attachment. We then examined how transcriptional regulator MgrA and cell wall transpeptidase sortase (SrtA) affect bacterial interaction with mucin. ResultsIn pig tracheas, S. aureus bound mucus strands from submucosal glands more than epithelial cells. In microtiter plate assays, {Delta}srtA failed to attach even in the absence of mucin. Mucin blocked wild type S. aureus attachment in a dose-dependent manner. Higher concentrations were needed to inhibit binding of {Delta}mgrA. Co-deletion of ebh and sraP, which encode surface proteins repressed by MgrA, suppressed the {Delta}mgrA binding phenotype. No differences between {Delta}mgrA and wild type were observed when methylcellulose or heparin sulfate were substituted for mucin, indicating specificity. ConclusionsMucin decreases attachment of S. aureus to plastic, consistent with its physiologic role in host defense. S. aureus deficient in MgrA has decreased affinity for mucin. Ebh and SraP, which are normally repressed by MgrA, may function as inhibitors of attachment to mucin. These data show that specific bacterial factors may regulate the interaction of S. aureus with mucus.

microbiology↗

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↗