MTP18 is a Novel Regulator of Mitochondrial Fission in CNS Neuron Development, Axonal Growth, and Injury Responses.

The process of mitochondrial fission-fusion has been implicated in diverse neuronal roles including neuronal survival, axon degeneration, and axon regeneration. However, whether increased fission or fusion is beneficial for neuronal health and/or axonal growth is not entirely clear, and is likely situational and cell type-dependent. In searching for mitochondrial fission-fusion regulating proteins for improving axonal growth within the visual system, we uncover that mitochondrial fission process 1,18 kDa (MTP18/MTFP1), a pro-fission protein within the CNS, is critical to maintaining mitochondrial size and volume under normal and injury conditions, in retinal ganglion cells (RGCs). We demonstrate that MTP18's expression is regulated by transcription factors involved in axonal growth, Kruppel-like factor (KLF) transcription factors-7 and -9, and that knockdown of MTP18 promotes axon growth. This investigation exposes MTP18's previously unexplored role in regulating mitochondrial fission, implicates MTP18 as a downstream component of axon regenerative signaling, and ultimately lays the groundwork for investigations on the therapeutic efficacy of MTP18 expression suppression during CNS axon degenerative events.

Magnetic Human Corneal Endothelial Cell Transplant: Delivery, Retention, and Short-Term Efficacy.

Purpose: Corneal endothelial dysfunction leads to corneal edema, pain, and vision loss. Adequate animal models are needed to study the safety and efficacy of novel cell therapies as an alternative to corneal transplantation. Methods: Primary human corneal endothelial cells (HCECs) were isolated from cadaveric donor corneas, expanded in vitro, transduced to express green fluorescent protein (GFP), loaded with superparamagnetic nanoparticles, and injected into the anterior chamber of adult rabbits immediately after endothelial cell or Descemet's membrane stripping. The same volume of balanced salt solution plus (BSS+) was injected in control eyes. We compared different models for inducing corneal edema in rabbits, and examined the ability of transplanted HCECs to reduce corneal edema over time by measuring central corneal thickness and tracking corneal clarity. GFP-positive donor cells were tracked in vivo using optical coherence tomography (OCT) fluorescence angiography module, and the transplanted cells were confirmed by human nuclei immunostaining. Results: Magnetic HCECs integrated onto the recipient corneas with intact Descemet's membrane, and donor identity was confirmed by GFP expression and immunostaining for human nuclei marker. Donor HCECs formed a monolayer on the posterior corneal surface and expressed HCEC functional markers of tight junction formation. No GFP-positive cells were observed in the trabecular meshwork or on the iris, and intraocular pressure remained stable through the length of the study. Conclusions: Our results demonstrate magnetic cell-based therapy efficiently delivers HCECs to restore corneal transparency without detectable toxicity or adverse effect on intraocular pressure. Magnetic delivery of HCECs may enhance corneal function and should be explored further for human therapies.

The Krüppel-Like Factor Gene Target Dusp14 Regulates Axon Growth and Regeneration.

Purpose: Adult central nervous system (CNS) neurons are unable to regenerate their axons after injury. Krüppel-like transcription factor (KLF) family members regulate intrinsic axon growth ability in vitro and in vivo, but mechanisms downstream of these transcription factors are not known. Methods: Purified retinal ganglion cells (RGCs) were transduced to express exogenous KLF9, KLF16, KLF7, or KLF11; microarray analysis was used to identify downstream genes, which were screened for effects on axon growth. Dual-specificity phosphatase 14 (Dusp14) was further studied using genetic (siRNA, shRNA) and pharmacologic (PTP inhibitor IV) manipulation to assess effects on neurite length in vitro and survival and regeneration in vivo after optic nerve crush in rats and mice. Results: By screening genes regulated by KLFs in RGCs, we identified Dusp14 as a critical gene target limiting axon growth and regeneration downstream of KLF9's ability to suppress axon growth in RGCs. The KLF9-Dusp14 pathway inhibited activation of mitogen-activated protein kinases normally critical to neurotrophic signaling of RGC survival and axon elongation. Decreasing Dusp14 expression or disrupting its function in RGCs increased axon growth in vitro and promoted survival and optic nerve regeneration after optic nerve injury in vivo. Conclusions: These results link intrinsic and extrinsic regulators of axon growth and suggest modulation of the KLF9-Dusp14 pathway as a potential approach to improve regeneration in the adult CNS after injury.

A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function.

Purpose: Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods: HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors ("proliferative media") to media without those factors ("stabilizing media"). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results: Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions: HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies.

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's 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's 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.SIGNIFICANCE STATEMENT Injured CNS nerves fail to regenerate spontaneously. Promoting intrinsic axon growth capacity has been a major challenge in the field. Here, we demonstrate that knocking down Krüppel-like transcription factor 9 (KLF9) via shRNA promotes long-distance axon regeneration after optic nerve injury and uncover a novel and important KLF9-JNK3 interaction that contributes to axon growth suppression in vitro and regenerative failure in vivo These studies suggest potential therapeutic approaches to promote axon regeneration in injury and other degenerative diseases in the adult CNS.

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.

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

AIM: To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. MATERIALS AND METHODS: 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. RESULTS: hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPaseα1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (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. CONCLUSION: 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.

Magnetic field-guided cell delivery with nanoparticle-loaded human corneal endothelial cells.

To improve the delivery and integration of cell therapy using magnetic cell guidance for replacement of corneal endothelium, here we assess magnetic nanoparticles' (MNPs') effects on human corneal endothelial cells (HCECs) in vitro. Biocompatible, 50 nm superparamagnetic nanoparticles endocytosed by cultured HCECs induced no short- or long-term change in viability or identity. Assessment of guidance of the magnetic HCECs in the presence of different magnet shapes and field strengths showed a 2.4-fold increase in delivered cell density compared to gravity alone. After cell delivery, HCECs formed a functional monolayer, with no difference in tight junction formation between MNP-loaded and control HCECs. These data suggest that nanoparticle-mediated magnetic cell delivery may increase the efficiency of cell delivery without compromising HCEC survival, identity or function. Future studies may assess the safety and efficacy of this therapeutic modality in vivo. From the clinical editor: The authors show in this article that magnetic force facilitates the delivery of human corneal endothelial cells loaded by superparamagnetic nanoparticles to cornea, without changing their morphology, identity or functional properties. This novel idea can potentially have vast impact in the treatment of corneal endothelial dystrophies by providing self-endothelial cells after ex-vivo expansion.

Regenerative Cell Therapy for Corneal Endothelium.

Endothelial cell dysfunction as in Fuchs dystrophy or pseudophakic bullous keratopathy, and the limited regenerative capacity of human corneal endothelial cells (HCECs), drive the need for corneal transplant. In response to limited donor corneal availability, significant effort has been directed towards cell therapy as an alternative to surgery. Stimulation of endogenous progenitors, or transplant of stem cell-derived HCECs or in vitro-expanded, donor-derived HCECs could replace traditional surgery with regenerative therapy. Ex vivo expansion of HCECs is technically challenging, and the basis for molecular identification of functional HCECs is not established. Delivery of cells to the inner layer of the human cornea is another challenge: different techniques, from simple injection to artificial corneal scaffolds, are being investigated. Despite remaining questions, corneal endothelial cell therapies, translated to the clinic, represent the future for the treatment of corneal endotheliopathies.

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.

Foxn4 is required for retinal ganglion cell distal axon patterning.

The regulation of retinal ganglion cell (RGC) axon growth and patterning in vivo is thought to be largely dependent on interactions with visual pathway and target cells. Here we address the hypothesis that amacrine cells, RGCs' presynaptic partners, regulate RGC axon growth or targeting. We asked whether amacrine cells play a role in RGC axon growth in vivo using Foxn4(-/-) mice, which have fewer amacrine cells, but a normal complement of RGCs. We found that Foxn4(-/-) mice have a similar reduction in most subtypes of amacrine cells examined. Remarkably, spontaneous retinal waves were not affected by the reduction of amacrine cells in the Foxn4(-/-) mice. There was, however, a developmental delay in the distribution of RGC projections to the superior colliculus. Furthermore, RGC axons failed to penetrate into the retinorecipient layers in the Foxn4(-/-) mice. Foxn4 is not expressed by RGCs and was not detectable in the superior colliculus itself. These findings suggest that amacrine cells are critical for proper RGC axon growth in vivo, and support the hypothesis that the amacrine cell-RGC interaction may contribute to the regulation of distal projections and axon patterning.

Amacrine cell gene expression and survival signaling: differences from neighboring retinal ganglion cells.

PURPOSE. To describe how developing amacrine cells and retinal ganglion cells (RGCs) differ in survival signaling and global gene expression. METHODS. Amacrine cells were immunopurified and processed for gene microarray analysis. For survival studies, purified amacrine cells were cultured at low density in serum-free medium, with and without peptide trophic factors and survival pathway inhibitors. The differences in gene expression between amacrine cells and RGCs were analyzed by comparing the transcriptomes of these two cell types at the same developmental ages. RESULTS. The amacrine cell transcriptome was very dynamic during development. Amacrine cell gene expression was remarkably similar to that of RGCs, but differed in several gene ontologies, including polarity- and neurotransmission-associated genes. Unlike RGCs, amacrine cell survival in vitro was independent of cell density and the presence of exogenous trophic factors, but necessitated Erk activation via MEK1/2 and AKT signaling. Finally, comparison of the gene expression profile of amacrine cells and RGCs provided a list of polarity-associated candidate genes that may explain the inability of amacrine cells to differentiate axons and dendrites as RGCs do. CONCLUSIONS. Comparison of the gene expression profile between amacrine cells and RGCs may improve our understanding of why amacrine cells fail to differentiate axons and dendrites during retinal development and of what makes amacrine cells differ in their resistance to neurodegeneration. Switching RGCs to an amacrine cell-like state could help preserve their survival in neurodegenerative diseases like glaucoma, and amacrine cells could provide a ready source of replacement RGCs in such optic neuropathies.

Disease gene candidates revealed by expression profiling of retinal ganglion cell development.

To what extent do postmitotic neurons regulate gene expression during development or after injury? We took advantage of our ability to highly purify retinal ganglion cells (RGCs) to profile their pattern of gene expression at 13 ages from embryonic day 17 through postnatal day 21. We found that a large proportion of RGC genes are regulated dramatically throughout their postmitotic development, although the genes regulated through development in vivo generally are not regulated similarly by RGCs allowed to age in vitro. Interestingly, we found that genes regulated by developing RGCs are not generally correlated with genes regulated in RGCs stimulated to regenerate their axons. We unexpectedly found three genes associated with glaucoma, optineurin, cochlin, and CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), previously thought to be primarily expressed in the trabecular meshwork, which are highly expressed by RGCs and regulated through their development. We also identified several other RGC genes that are encoded by loci linked to glaucoma. The expression of glaucoma-linked genes by RGCs suggests that, at least in some cases, RGCs may be directly involved in glaucoma pathogenesis rather than indirectly involved in response to increased intraocular pressure. Consistent with this hypothesis, we found that CYP1B1 overexpression potentiates RGC survival.