Iqbal Ahmad - US grants

The acquisition of neuronal diversity is central to the development and organization of the mammalian central nervous system (CNS). Our understanding of some of the mechanisms by which this diversity is achieved has been greatly facilitated by studies of neuronal differentiation in retina, a well characterized model of the CNS. Lineage analyses of neuronal precursors in vivo using retroviral infections and progressive lineage restriction during cell-type specification. However, our understanding of the molecular basis of these processes remains unclear for the lack of information regarding genes involved in the mammalian neurogenesis. One of the approaches is to identify and study the retina-specific expression of the mammalian homologs of Drosophila genes whose role in neurogenesis has been well established by mutation analyses. Depending upon their spatio-temporal expression and functions during early neurogenesis these genes belong to two different classes, the proneural genes of the achaete-scute (AS-C) complex that encode transcription factors of basic Helix-Loop-Helix (bHLH) class and the neurogenic genes of which Notch is a member, that encodes a membrane protein. Members of both proneural and neurogenic genes have been shown to play important roles in the development of Drosophila eye. The function of these genes in early neurogenesis in general and in the development of the eye in particular make their mammalian homologues ideal candidates for the investigation of the spatio-temporal expression of the mammalian homologs of AS-C and Notch genes in developing retina in order to know their cell-specific expression and to formulate a hypothesis regarding their function. Both genes may be involved in mediating intercellular interactions during retinal neurogenesis and cell-type specification. While AS-C homologs can activate the differentiation program by influencing the genome, Notch on the other had can link the activation f the genome with microenvironment in which the differentiation is taking place. In order to evaluate such possibilities, the expression of these genes will be studied in vitro in response to various growth factors that have been shown to modulate cell proliferation and differentiation in retina. The AS-C homologs, analogous to myogenic gene, MyoD may function as master regulatory genes involved in the activation of downstream neuron-specific genes in precursors during the early stages of neurogenesis. This hypothesis will lbs initially tested by analyzing the ability of AS-C homologs expressed in developing retina to interact with putative cis- acting elements (E-box) and evaluating their transcriptional activity by transactivation experiments. This information will be critical for the identification of downstream, neuron-specific genes and evaluation of AS- C homologs expression as the nodal point in neurogenesis. Studies of the homologs of Notch and AS-C genes during the retinal development will help us in our long term goal of obtaining a comprehensive picture of the molecular events underlying neurogenesis and cell-type specification. The information obtained from these studies will help us in understanding some of the processes involved in retinal degeneration and formulated hypotheses regarding interventions at the molecular and cellular levels to prolong and promote the survival of specific neurons by recapitulation of developmental mechanisms during retinal degeneration.

Establishing cellular diversity is central to the development, structure and function of the brain. Underlying this diversity is a population of neural progenitors with stem cell properties that generates region-specific neurons and glia. Therefore understanding the molecular and cellular biology of neural progenitors holds the key to the mechanism(s) of development of specific regions of the brain including retina. Recapitulation of these developmental mechanisms is likely to open new avenues for treating the impairments of functions that arise due to death of specific neuronal populations as in the case of retinitis pigmentosa and macular degeneration. With these objectives in mind we have proposed to characterize neural progenitors isolated from derivatives of ocular neuroepithelium, the retina and the ciliary body, for their proliferative capacity, self-renewal, maintenance, and developmental potentials in vivo and in vitro under the following specific aims. First, proliferative and differentiation potentials of ocular progenitors will be characterized in the context of their responsiveness to the mitogens, FGF2 and EGF. We will use flow cytometry to examine their proliferation and survival, limiting dilution analysis to estimate their frequency to form clones, immunocytochemical and RT-PCR analyses of cell-type specific markers to test their multipotentiality, and clonal density culture to determine their self-renewal capacity. In addition, we will determine the distribution of mitogen receptors in different sub-populations of ocular progenitors in order to understand the basis of their responsiveness and relationships. Second, the role of Notch signaling in the maintenance of ocular progenitors will be evaluated. We will analyze proliferation and differentiation of these cells in response to gain-of-function and loss-of-function perturbations of Notch signaling. Third, the differentiation potentials of ocular progenitors will be determined in vitro in conditions that promote differentiation. We will use immunocytochemical and electrophysiological analyses to evaluate their ability to acquire specific phenotypes in response to growth factors and neurotrophins, and in co-culture conditions. Fourth, the potential of ocular progenitors to generate site-specific cells in vivo will be evaluated. We will use homotopic and heterotopic transplantation and in vivo activation of progenitors in response to injuries to achieve the aim. Accomplishing these aims will provide valuable information about the underlying mechanism(s) of retinal development and will allow the use of ocular progenitors in stem cell therapy to address degenerative changes in the retina whether inherited, age-related or due to injuries.

DESCRIPTION (provided by applicant): Glaucoma is the most prevalent optic neuropathy where a progressive degeneration of retinal ganglion cells (RGCs) leads to vision loss. Our long-term goal is to help prevent the degeneration of glaucomatous RGCs by characterizing induced-pluripotent stem cells (iPSCs) as a renewable source of retinal progenitors for autologous ex vivo cell therapy. The objective of this application is to optimize the use of limbal iPSCs to generate RGCs that are functional, safe, and practical for clinical use. The central hypothesis of the proposed study is that the molecular mechanism underlying RGC differentiation is active in iPSC-derived retinal progenitors and recruited in response to specific extrinsic cues to generate RGCs with target specificity. Our reasoning is based on the following observations:(1) retinal progenitors can be derived from limbal iPSCs, generated through safe non-nucleic acid method (2) iPSC-derived retinal progenitors respond to cues conducive for RGC differentiation, and (3) iPSC-derived RGCs demonstrate target specificity. The rationale for the proposed research is that once conditions are identified, we can efficiently generate RGC precursors to treat RGC degeneration through transplantation, and develop a robust model system for testing drugs and genetic approaches for optic neuropathy. Based on our preliminary data the following specific aims are proposed to test the hypothesis: Specific Aim 1: To determine the conditions for generating retinal progenitors from iPSCs, Specific Aim 2: To determine conditions for the generation of RGCs from iPSC-derived retinal progenitors, and Specific Aim 3: To determine the target specificity and in vivo differentiation of iPSC-derived RGCs. The retinal potential will be examined in limbal iPSCs generated by non-nucleic acid means, pioneered in our lab. This approach of reprogramming by recruiting endogenous pluripotency genes instead of introducing exogenous genes, which can lead to insertional mutagenesis, addresses a significant barrier to iPSC-based therapy. Controls will include limbal iPSC derived by a conventional nucleic acid method to compare the effects of two different approaches of reprogramming on the acquisition of retinal and RGC potential. The induction of iPSCs along a neural lineage, their subsequent specification into retinal progenitors, and their final differentiation into RGCs will be accomplished non-cell autonomously by perturbing specific signaling pathways to recapitulate developmental mechanism. Therefore, our research proposed is innovative because it presents an entirely different and a safe approach for reprogramming somatic cells to a pluripotent state and generating RGCs without using nucleic acids or forced expression of exogenous factors. The emerging information will be significant because it will not only address each of the barriers that currently make the ex-vivo stem cell therapy approach impractical but also lead to the development of a robust model system for testing normal mechanisms of RGC development and for screening drugs and genes for additional new approaches for addressing glaucomatous retinal degeneration.

ABSTRACT Glaucoma is the most prevalent optic neuropathy where a progressive degeneration of retinal ganglion cells (RGCs) leads to vision loss. Our long-term goal is to help prevent the degeneration of glaucomatous RGCs by characterizing pluripotent stem cells as a renewable source of RGCs for autologous ex vivo cell therapy. The objective of this renewal application is to address the next question relevant to the potential clinical application of human pluripotent cell-derived RGCs: whether or not these cells can elaborate guidable axons that can navigate out of the host retina and seek bonafide targets, essential for reversing vision loss. To our knowledge this question, essential for practical ex-vivo stem cell approach to glaucomatous degeneration, remains unanswered. The central hypothesis of the proposed study is that human induced pluripotent stem cells derived RGCs (hiPSC-RGCs) elaborate guidable axons, regulated by the mTOR pathway, an intrinsic regulator axonogenesis and regeneration. Our reasoning is based on our observations that hiPSC-RGCs are (1) stable, functional, and safe (2) express guidance receptors and respond to both proximal (intra- retinal) and distal (extra-retinal) guidance cues, and (3) have active mTOR pathway, regulating development and neuritogenesis. Our rationale is that the ability of hiPSC-RGCs to recapitulate the mechanism of axon growth and guidance will posit them as a viable reagent to functionally replace degenerated RGCs in glaucoma. The following specific aims are proposed to test the hypothesis: Aim 1: To determine the competence of hiPSC-RGCs for axon guidance and target specificity, Aim 2: To determine the competence of hiPSC-RGCs for mTOR-dependent axonogenesis and regeneration in vitro, and Aim 3: To determine mTOR-dependent hiPSC-RGC axonogenesis in neonatal and adult retina. The potential of hiPSC-RGCs for axonogenesis and axon guidance will be examined in co-culture paradigm using the microfluidic system in controlled conditions. Immunocytochemical analysis of known pathways and transcriptional profiling would identify candidate regulatory factors. The regenerative ability of hiPSC-RGCs in the context of mTOR pathway will be examined in a microfluidic model of the axotomy model, established in our lab. Transcription profile at pre-axotomy, axotomy, and post-axotomy stages would identify regenerative gene regulatory network. Finally, regenerative capacity of hiPSC-RGCs and the influence of the mTOR pathway will be examined in vivo in neonatal retina, where environment is conducive for axon growth and in a degenerative adult environment in animal model of glaucoma. Our research proposal is innovative because it will determine whether the de novo generated neurons can functionally replace those that make long distance connections such as RGCs and bridge a gap in our knowledge about human RGC development and axon path finding, a barrier to optic nerve regeneration. The emerging information will be significant because it will not only address each of the most significant barriers that currently make the ex-vivo stem cell therapy approach impractical but also lead to the development of a robust model system for testing normal/pathological mechanisms of RGC development and for screening drugs and genes for additional new therapeutic approaches for glaucomatous retinal degeneration.

PROJECT SUMMARY Glaucoma represents a group of diseases, which are associated with multiple risk factors and genetic variants. The unifying theme among these diseases is the progressive degeneration of optic nerve and retinal ganglion cells (RGCs) degenerate, leading to irreversible blindness. Based on this observation we propose a hypothesis that RGCs are intrinsically vulnerable to glaucoma risk factors and genetic variants. Since RGCs are born embryonically and glaucoma is an adult onset disease our knowledge about the emergence of and mechanism underlying RGC susceptibility, important for early diagnosis and formulating therapeutic approaches, remain rudimentary. Our objective is to examine the impact of genetic variations associated with glaucoma on the development, phenotype, and regeneration of RGCs, using the induced pluripotent stem cell (iPSC) model of primary open angle glaucoma (POAG), the most prevalent type of the disease. Here, we will test the hypothesis that RGCs in the SIX6 risk allele (Asn141His, rs33912345) in POAG are developmentally compromised in normal phenotype and function, and that this affects their survival and regeneration. The SIX6 variant is a suitable target for the analysis because SIX6 is one of the eye-field genes involved in retinal development and the missense mutation (Asn141His) in the DNA binding region of SIX6 is accompanied by degenerative changes that include reduction in the retinal nerve fiber layer (RNFL) thickness in POAG. Thus, the generation of SIX6 risk allele-POAG RGCs in a dish model of the disease would allow the characterization of developmental and phenotype abnormalities, shedding light on glaucomatous RGC susceptibility. We have proposed three aims to test the central hypothesis. First, we will determine the impact of the SIX6 risk allele-POAG on the development and phenotype of patient-specific RGCs by systematic characterization of sequential steps of retinal inductions and RGC differentiation of POAG patient-specific and age- and sex-matched control iPSCs, under the influence of a stage-specific and chemically defined protocol. Second, we will examine the impact of the SIX6 risk allele-POAG on patient-specific RGC axon growth and regeneration in the context of mTOR signaling, a regulator of retinal development and regeneration, which is inhibited in POAG patient- specific RGCs. Lastly, we will identify the molecular pathway(s) underlying abnormal RGC development and phenotype. We will carry out hypothesis-driven (e.g., HMGA2 and KLF-4 as candidate hub genes) genome wide transcriptional analysis of POAG patient-specific RGCs during development and regeneration to understand the molecular mechanism underlying SIX6 risk allele associated RGC abnormalities. Information emerging from our study will bridge a gap in our knowledge of the intrinsic vulnerability of RGCs in glaucoma. The information on dysregulated processes and pathways will reveal biomarkers for early diagnosis, and will allow strategies for therapeutic regeneration of glaucomatous RGCs.