Budd A. Tucker

Endogenous VEGF Is Required for Visual Function: Evidence for a Survival Role on Müller Cells and Photoreceptors

Background Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical. Methods and Findings Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective. Conclusions These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.

Transplantation of Adult Mouse iPS Cell-Derived Photoreceptor Precursors Restores Retinal Structure and Function in Degenerative Mice

This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs), could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.

Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa

Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. We used exome sequencing to identify a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) as the cause of disease in an isolated individual with RP. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands are of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. Induced pluripotent stem cells were derived from the skin of the proband and a patient with non-MAK–associated RP (RP control). In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies.

The synergistic effects of NGF and IGF‐1 on neurite growth in adult sensory neurons: convergence on the PI 3‐kinase signaling pathway

Nerve growth factor (NGF) and insulin‐like growth factor‐1 (IGF‐1) play an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons. Adult DRG neurons exhibit neurotrophin‐independent survival, providing an excellent system with which to study trophic factor effects on neurite growth in the absence of significant survival effects. Using young adult rat DRG neurons we have demonstrated a synergistic effect of NGF plus IGF (N + I), compared with either factor alone, in promoting neurite growth. Not only does the presence of NGF and IGF‐1 enhance neurite initiation, it also significantly augments the extent of neurite branching and elongation. We have also examined potential mechanism(s) underlying this synergistic effect. Immunoblotting experiments of classical growth factor intermediary signalling pathways (PI 3‐K‐Akt‐GSK‐3 and Ras‐Raf‐MAPK) were performed using phospho‐specific antibodies to assess activation state. We found that activation of Akt and MAPK correlated with neurite elongation and branching. However, using pharmacological inhibitors, we observed that a PI 3‐K pathway involving both Akt and GSK‐3 appeared to be more important for neurite extension and branching than MAPK‐dependent signalling. In fact, inhibition of activation of MAPK with U0126 resulted in increased neuritic branching, possibly as a result of the concomitant increase observed in phospho‐Akt. Furthermore, inhibition of GSK3 (which is negatively regulated by phosphorylation on S9/S21) also resulted in increased growth. Our data point to signalling convergence upon the PI 3‐K‐Akt‐GSK‐3 pathway that underlies the NGF plus IGF synergism. In addition, to our knowledge, this is the first report in primary neurons that inhibition of GSK3 results in an enhanced neurite growth.

Endurance exercise regimens induce differential effects on brain-derived neurotrophic factor, synapsin-I and insulin-like growth factor I after focal ischemia

The optimal amount of endurance exercise required to elevate proteins involved in neuroplasticity during stroke rehabilitation is not known. This study compared the effects of varying intensities and durations of endurance exercise using both motorized and voluntary running wheels after endothelin-I-induced focal ischemia in rats. Hippocampal levels of brain-derived neurotrophic factor, insulin-like growth factor I and synapsin-I were elevated in the ischemic hemisphere even in sedentary animals suggesting an intrinsic restorative response 2 weeks after ischemia. In the sensorimotor cortex and the hippocampus of the intact hemisphere, one episode of moderate walking exercise, but not more intense running, resulted in the greatest increases in levels of brain-derived neurotrophic factor and synapsin-I. Exercise did not increase brain-derived neurotrophic factor, insulin-like growth factor I or synapsin-I in the ischemic hemisphere. In voluntary running animals, both brain and serum insulin-like growth factor I appeared to be intensity dependent and were associated with decreasing serum levels of insulin-like growth factor I and increasing hippocampal levels of insulin-like growth factor I in the ischemic hemisphere. This supports the notion that exercise facilitates the movement of insulin-like growth factor I across the blood-brain barrier. Serum corticosterone levels were elevated by all exercise regimens and were highest in rats exposed to motorized running of greater speed or duration. The elevation of corticosterone did not seem to alter the expression of the proteins measured, however, graduated exercise protocols may be indicated early after stroke. These findings suggest that relatively modest exercise intervention can increase proteins involved in synaptic plasticity in areas of the brain that likely subserve motor relearning after stroke.

Exercise intensity influences the temporal profile of growth factors involved in neuronal plasticity following focal ischemia.

Exercise increases brain-derived neurotrophic factor (BDNF), phosphorylated cAMP response-element binding protein (pCREB), insulin-like growth factor (IGF-I) and synapsin-I, each of which has been implicated in neuroplastic processes underlying recovery from ischemia. In this study we examined the temporal profile (0, 30, 60 and 120 min following exercise) of these proteins in the hippocampus and sensorimotor cortex following both motorized (60 min) and voluntary (12 h) running, 2 weeks after focal ischemia. Our goal was to identify the optimal training paradigms (intensity, duration and frequency) needed to integrate endurance exercise in stroke rehabilitation. Therefore we utilized telemetry to measure changes in heart rate with both exercise methods. Our findings show that although the more intense, motorized running exercise induced a rapid increase in BDNF, the elevation was more short-lived than with voluntary running. Motorized running was also associated with higher levels of synapsin-I in several brain regions but simultaneously, a more pronounced increase in the stress hormone, corticosterone. Furthermore, both forms of exercise resulted in decreased phosphorylation of CREB and downregulation of synapsin-I in hippocampus beginning 30 to 60 min after the exercise bout. This phenomenon was more robust after motorized running, the method that generated higher heart rate and serum corticosterone levels. This immediate stress response is likely specific to acute exercise and may diminish with repeated exercise exposure. The present data illustrate a complex interaction between different forms of exercise and proteins implicated in neuroplasticity. For clinical application, frequent lower intensity exercise episodes (as in voluntary running wheels), which may be safer to provide to patients with stroke, has a delayed but sustained effect on BDNF that may support brain remodeling after stroke.

ABCB5 is a limbal stem cell gene required for corneal development and repair

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

CNS Progenitor Cells Promote a Permissive Environment for Neurite Outgrowth via a Matrix Metalloproteinase-2-Dependent Mechanism

Transplantation of progenitor cells to the CNS has shown promise in neuronal and glial replacement and as a means of rescuing host neurons from apoptosis. Here we examined the effect of progenitor grafts on neurite extension in the degenerating retina of rd1 (retinal degeneration 1) mice. Transplantation of retinal progenitor cells induced increased matrix metalloproteinase-2 (MMP2) secretion, partly from activated glial cells, which was then activated by neuronally expressed MMP14. Active MMP2 resulted in proteolysis of the neurite outgrowth inhibitors CD44 and neurocan in the degenerative retina, allowing significantly increased neurite outgrowth across the border between abutting nondystrophic and rd1 retinas. Progenitor-induced enhancement of outgrowth was abrogated by an MMP inhibitor or by coculture with retinal explants from MMP2−/− mice. This study provides the first identification of an MMP2-dependent mechanism by which exogenous progenitor cells alter the host environment to promote neural regeneration. This suggests a novel therapeutic role for progenitor cells in the treatment of CNS degenerative diseases.

Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa

Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient’s keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient’s mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient’s other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1−/− mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration. DOI: http://dx.doi.org/10.7554/eLife.00824.001

Use of a Synthetic Xeno‐Free Culture Substrate for Induced Pluripotent Stem Cell Induction and Retinal Differentiation

The purpose of this study was to determine whether a proprietary xeno‐free synthetic culture surface could be used to aid in the production and subsequent retinal‐specific differentiation of clinical‐grade induced pluripotent stem cells (iPSCs). iPSCs were generated using adult somatic cells via infection with either a single cre‐excisable lentiviral vector or four separate nonintegrating Sendai viruses driving expression of the transcription factors OCT4, SOX2, KLF4, and c‐MYC. Retinal precursor cells were derived via targeted differentiation of iPSCs with exogenous delivery of dkk‐1, noggin, insulin‐like growth factor‐1, basic fibroblast growth factor, acidic fibroblast growth factor, and DAPT. Phase contrast microscopy, immunocytochemistry, hematoxylin and eosin staining, and reverse transcription‐polymerase chain reaction were used to determine reprogramming efficiency, pluripotency, and fate of undifferentiated and differentiated iPSCs. Following viral transduction, cells underwent prototypical morphological changes resulting in the formation of iPSC colonies large enough for manual isolation/passage at 3–4 weeks postinfection. Both normal and disease‐specific iPSCs expressed markers of pluripotency and, following transplantation into immune‐compromised mice, formed teratomas containing tissue comprising all three germ layers. When subjected to our established retinal differentiation protocol, a significant proportion of the xeno‐free substrate‐derived cells expressed retinal cell markers, the number of which did not significantly differ from that derived on traditional extracellular matrix‐coated dishes. Synthetic cell culture substrates provide a useful surface for the xeno‐free production, culture, and differentiation of adult somatic cell‐derived iPSCs. These findings demonstrate the potential utility of these surfaces for the production of clinical‐grade retinal neurons for transplantation and induction of retinal regeneration.