KLF9 and JNK3 Interact to Suppress Axon Regeneration in the Adult CNS

Neurons in the adult mammalian CNS decrease in intrinsic axon growth capacity during development in concert with changes in Krüppel-like transcription factors (KLFs). KLFs regulate axon growth in CNS neurons including retinal ganglion cells (RGCs). Here, we found that knock-down of KLF9, an axon growth suppressor that is normally upregulated 250-fold in RGC development, promotes long-distance optic nerve regeneration in adult rats of both sexes. We identified a novel binding partner, MAPK10/JNK3 kinase, and found that JNK3 (c-Jun N-terminal kinase 3) is critical for KLF9\u27s axon-growth-suppressive activity. Interfering with a JNK3-binding domain or mutating two newly discovered serine phosphorylation acceptor sites, Ser106 and Ser110, effectively abolished KLF9\u27s neurite growth suppression in vitro and promoted axon regeneration in vivo. These findings demonstrate a novel, physiologic role for the interaction of KLF9 and JNK3 in regenerative failure in the optic nerve and suggest new therapeutic strategies to promote axon regeneration in the adult CNS

Biology of Amacrine Cells and Retinal Ganglion Cells in the Developing Retina

How do neighboring neurons differ? How are they similar? How do they relate to each other? Retinal ganglion cells (RGCs) and amacrine cells are born during the same developmental window, from the same population of progenitor cells, and some amacrine cells even migrate to the same retinal layer where RGCs reside, the ganglion cell layer. Amacrine cells are presynaptic to RGCs, whose axons subsequently project through the optic nerve and carry visual information to the brain. Although the cell biology of RGCs has been studied in some detail, relatively little is known about the cell and molecular biology of amacrine cells. What distinguishes these neighboring cell types and in what ways are they similar? Here I present a series of studies examining amacrine cells and RGCs, with a view towards better understanding the development and cell biology of these neurons. The retina has been long used as a model system to study central nervous system (CNS) development and regeneration: RGCs fail to regrow their axons after injury, and RGC survival is compromised in optic neuropathies such as glaucoma. In contrast, amacrine cells survive even after the loss of their targets, RGCs, in glaucoma and other optic neuropathies. While the signaling for RGC survival in vitro has been widely studied, little is known about the molecular mechanisms that may underlie amacrine cellsâ?? resistance to neurodegeneration. Taking advantage of our unique method to highly purify amacrine cells away from other retinal neighbors and glial cells, I found that amacrine cells can survive at very low densities in culture and that they do not require addition of exogenous trophic factors, unlike RGCs. Interestingly, blocking of MEK1/2 or PI3K signaling pathways significantly impaired survival, suggesting that these intracellular signaling pathways are necessary for amacrine cell survival. Thus, while amacrine cell and RGC survival seem to be regulated through similar signaling pathways, these two cell types have different requirements for exogenous peptide trophic factors Because amacrine cells were able to survive in our low density cultures in serum and peptide trophic-free media, it is possible that amacrine cell survival is regulated by autocrine signaling, or by hormones and/or antioxidants. Thus these retinal interneurons may not depend on target RGCs for peptide trophic support. Amacrine cells are a heterogeneous group of interneurons that modulate retinal signaling of visual information onto RGCs. There are more than 30 subtypes described in the mammalian retina, characterized by myriad morphologies and the secretion of different neurotransmitters. Despite their apparent inability to differentiate axons and dendrites, purified amacrine cells in vitro extended neurites with varied lengths and morphologies, raising the hypothesis that the regulation of these processes has an intrinsic component. Specifically, I asked whether purified amacrine cell subpopulations would extend neurites similarly in vivo and in vitro. Surprisingly, three purified amacrine cell subpopulations recapitulated aspects of their in vivo morphology in vitro, consistent with the existence of intrinsic mechanisms of neurite growth and patterning in the developing retina. Thus, I have demonstrated that there is an intrinsic regulatory component that contributes to the varied morphology of amacrine cell neurites found in vivo. To further characterize differences between amacrine cells and RGCs, I generated a database of amacrine cell gene expression during development and compared it to the transcriptome of RGCs at the same developmental ages. I found ~75% similarity among the genes expressed in RGCs and amacrine cells during development. However, I focused my interest in genes that were differentially regulated because they might underlie amacrine cellsâ?? resistance to neurodegeneration and could help understand the differences in polarity between amacrine cells and RGCs. Comparing the gene expression profiles of these two cell types, I found that RGCs expressed higher levels of the pro-apoptotic molecules Bax and Bad. This raises the interesting hypothesis that amacrine cells may be more resistant to degeneration than RGCs because they do not express as many pro-apoptotic molecules as RGCs do. In addition, I generated a list of polarity-associated candidate genes that are differentially expressed in amacrine cells and RGCs. Together, these data could be combined for therapeutical purposes. Switching dying RGCs to an amacrine cell-like state may help preserve these cells in neurodegenerative diseases like glaucoma. Conversely, regulating polarity genes in amacrine cells might induce changes in their neurite outgrowth ability that could help understand the mechanisms of cell polarization and axon growth, two critical components to achieve CNS regeneration. How might these presynaptic amacrine cells influence their neighboring RGCsâ?? cell biological phenotypes? Previous findings in the laboratory demonstrate that purified RGCs undergo an irreversible loss of their intrinsic axon growth ability during development, and that the process can be signaled by amacrine cells. Thus, amacrine cells are sufficient to signal RGCs to decrease their intrinsic axon growth ability during development. It is not known, however, whether amacrine cells are necessary for this process. I hypothesized that in the absence of amacrine cells, RGCsâ?? axon growth might be dysregulated in vivo. The creation of the Foxn4-/- mouse by the Xiang laboratory allowed me to address this question. Foxn4 is a forkhead transcription factor that is required for amacrine cell genesis during retinal development, and as a result the Foxn4 knockout mice have fewer amacrine cells. I found that in the context of a reduced number of amacrine cells, RGCs projected fewer dendrites to the inner plexiform layer in the retina. In addition, RGCsâ?? axon projection to their target (the superior colliculus) was developmentally delayed, and they failed to penetrate into the retinorecipient layers of the superior colliculus. Finally, Foxn4-/- mice showed disrupted optic nerve architecture, albeit the fluorescence intensity of labeled RGC axons in optic nerve cross- sections was similar among animals. Taken together, these data demonstrate a role for a pre-synaptic partner, amacrine cells, in regulating a neuronâ??s intrinsic axon growth ability and intrinsic patterning. In conclusion, together these data paint a portrait of how two neighboring retinal cell types differ: amacrine cells are resistant to neurodegeneration whereas RGCs express genes that are associated with apoptosis and glaucoma; amacrine cells do not require the presence of exogenous trophic factors to survive in vitro whereas RGCs do require trophic support. Finally, while amacrine cells and RGCs may differ in their mechanisms for establishing axon and dendrite specification, they both exhibit an intrinsic capacity to grow neurites in vitro that recapitulates their phenotype in vivo. These differences in developmental cell biology may point to new approaches to understanding retinal and retino-collicular patterning, neuronal survival and perhaps even optic nerve regeneration

Amacrine Cell Subtypes Differ in Their Intrinsic Neurite Growth Capacity

PURPOSE. Amacrine cell neurite patterning has been extensively studied in vivo, and more than 30 subpopulations with varied morphologies have been identified in the mammalian retina. It is not known, however, whether the complex amacrine cell morphology is determined intrinsically, is signaled by extrinsic cues, or both. METHODS. Here we purified rat amacrine cell subpopulations away from their retinal neighbors and glial-derived factors to ask questions about their intrinsic neurite growth ability. In defined medium strongly trophic for amacrine cells in vitro, we characterized survival and neurite growth of amacrine cell subpopulations defined by expression of specific markers. RESULTS. We found that a series of amacrine cell subtype markers are developmentally regulated, turning on through early postnatal development. Subtype marker expression was observed in similar fractions of cultured amacrine cells as was observed in vivo, and was maintained with time in culture. Overall, amacrine cell neurite growth followed principles very similar to those in postnatal retinal ganglion cells, but embryonic retinal ganglion cells demonstrated different features, relating to their rapid axon growth. Surprisingly, the three subpopulations of amacrine cells studied in vitro recapitulated quantitatively and qualitatively the varied morphologies they have in vivo. CONCLUSIONS. Our data suggest that cultured amacrine cells maintain intrinsic fidelity to their identified in vivo subtypes, and furthermore, that cell-autonomous, intrinsic factors contribute to the regulation of neurite patterning

Novel Identity and Functional Markers for Human Corneal Endothelial Cells

PURPOSE: Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy. METHODS: Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays. RESULTS: Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs. CONCLUSIONS: In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction

Microarray analysis indicated that hESC-CEC and HCEC have the most similar genetic profile compared to HUVEC, HEK 293, and pancreatic Islet cells.

<p>Publicly available datasets for HUVEC, HEK293, and human pancreatic Islet cells were compared to ESC-CEC and pHCEC. A. Correlation analysis indicates that samples within groups have little variation. B. Principal component analysis indicates that ESC-CEC were mostly closely grouped to pHCEC, with HUVEC showing the next closest similarity. HEK 293 (HEK) and pancreatic Islet cells (ISL) were the least similar. C. Venn diagram of total probes. Of note, 76 genes appear to be uniquely expressed by ESC-CECs and HCECs which could be potential novel identifiers for corneal endothelial cells.</p

Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation

<div><p>Aim</p><p>To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies.</p><p>Materials and Methods</p><p>Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.</p><p>Results</p><p>hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na⁺/K⁺ATPase<b>α</b>1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet’s membrane, Collagen VIII<b>α</b>1 and VIII<b>α</b>2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.</p><p>Conclusion</p><p>hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.</p></div

Microarray analysis indicates hESC-CEC were highly similar to primary cultured adult HCECs.

<p>A. Correlation analysis. Red color indicated little global variability between samples within group of human corneal endothelial cells (pHCECs) and human embryonic stem cell derived corneal endothelial cells (ESC-CEC). B. Volcano plot indicated most genes expressed by pHCECs and ESC-CEC were within 2 fold.</p

Genes expressed by corneal endothelial cells were present in hESC-CEC.

<p>A. COL8A1, a major component of the Descemet’s membrane was expressed is upregulated after 1 week of induction compared to hESC by QPCR utilizing the <b>ΔΔ</b>Ct method of analysis and the endogenous control PGK1. mRNA levels peaked around the second week which may have indicated that the cornea endothelial cells no longer need to produce as much COL8A1 to form Descemet’s membrane in vitro. B, C. hESC-CEC secreted COL8A1 and COL8A2 in vitro. The subcellular extracellular matrix secreted by hESC-CEC was analyzed by standard Western blot analysis for the presence of COL8A1 and COL8A2. D. Aquaporin 1 (AQP1) is a water pump that functions as part of the endothelial pump that keeps the cornea dehydrates. The increase in AQP1 expression may indicate increased ability of hESC-CEC to function as a pump. E. Many components of the corneal endothelial pump function were enriched in hESC-CEC. Carbonic Anhydrase 2 (CA2), Carbonic anhydrase 4 (CA4), Cystic Fibrosis Transmembrane conductance receptor (CFTR), Solute Carrier Family 16, member 3(SLC16A3)/Monocarboxylic acid transporter 4 (MCT4), Solute Carrier Family 16, member 7 (SLC16A7)/ Monocarboxylic acid transporter 2 (MCT2), and Solute carrier family 4, sodium bicarbonate cotransporter, member 4 (SLC4A4)/Sodium Bicarbonate cotransporter 1 (MBC1) were enriched in hESC-CECs at 1–4 weeks after induction with the exception of CFTR at 4 weeks, where it was expressed at levels similar to undifferentiated hESCs. F. hESC-CEC weeks 1–4 express about 5 fold less COL8A1 than 2 separate samples of human cultured CEC (HCEC). Normalized to hESC and the endogenous controls of 18S and GAPDH. G. hESC-CEC weeks 1–3 express similar levels of AQP1 as HCEC with the exception of 4 weeks. Normalized to hESC and the endogenous controls of 18S and GAPDH. Wk = week.</p

hESC-CEC expressed ZO-1 and Na⁺K⁺ATPaseα1 at the boundaries of cells, but not vascular endothelial markers vWF and CD31.

<p>A. Zona Occuldins-1, ZO-1 (red), an adherens tight junction marker, was expressed at the cell borders in hESC derived CEC indicating that the cells were tightly adhered and were hexagonal/polygonal in shape. B. Nuclear marker DAPI (blue) was on a different plane than ZO-1. C. Merge of ZO-1 and DAPI. D. Representative phase contrast picture from same experiment. E. Na⁺K⁺ATPase<b>α</b>1 (red) was localized to the cell borders in hESC-CEC indicating that cells had properly localized a component of the endothelial pump function. E. Nuclear marker DAPI (blue) was on a different plane than Na⁺K⁺ATPase<b>α</b>1. F. Merge of Na⁺K⁺ATPase<b>α</b>1 and DAPI. G. Human umbilical cord vein endothelial cells (HUVEC) expressed von Willebond factor (vWF, red) a marker of vascular endothelial cells, but not ZO-1 (green). H. hESC-CEC expressed ZO-1 (green), but not vWF (red). I. HUVEC expressed Platelet endothelial cell adhesion 1 (PECAM1 or CD31) but not ZO-1 (green). J. hESC-CEC expressed ZO-1 (green), but not CD31 (red). DAPI (blue) stained nucleus. Scale bars = 10 <b>μ</b>m.</p