Significance: Nearly 20% of individuals with neurofibromatosis type 1 (NF1) develop tumors along the anterior visual pathway, involving optic nerves, optic chiasm and/or optic tracts, which are collectively referred to as NF1- associated optic pathway gliomas (NF1-OPGs). Despite no risk for malignant transformation, one-third of NF1- OPG patients will develop progressive diseases, exhibiting vision loss, often to the point of legal blindness. Most NF1-OPGs are diagnosed in children younger than 7 years of age with a peak incidence between ages 4-5. Thus, blindness will lead to a lifelong disability in nearly 7% of children with NF1. Since NF1-OPGs often grow extensively along the optic pathway, surgery is a high-risk treatment option and, consequently, human tumor tissues are rarely available for research. Radiation therapy is avoided in NF1 patients due to its potential to induce secondary malignant tumors (that can cause death of patients) and vascular abnormalities. Although chemotherapy, including a recently developed molecularly targeted therapy with MEK inhibitor (MEKi), can be effective in shrinking these tumors, frequently there is a mismatch between the amount of tumor shrinkage and the patient’s vision after treatment. Recent studies suggest that loss of retinal ganglion cells (RGCs) – the only nerve cells that connect the eye to the brain, is at least one of the mechanisms causing NF1-OPG associated vision loss. Because chemotherapeutic agents are not expected to regenerate RGCs or re-establish neuronal circuits, the most effective therapy will be prevention or early intervention – the treatment that is delivered before irreversible neurological deficits such as death of nerve cells occur. The main objective of this proposal is to investigate the mechanism by which NF1-OPG causes death of RGCs and vision loss as well as develop novel therapeutic strategies that can preserve and/or restore vision.

Research Questions/Concepts: Clinical studies have provided the evidence that loss of RGCs is not simply caused by the compressive effect of NF1-OPGs on the optic nerve. For example, some patients can lose vision despite having relatively small tumors, while some with larger growing tumors may have well maintained vision. Moreover, some patients with NF1-OPGs lost vision despite stable radiographic responses to chemotherapy. The lack of association between tumor size and visual functions in both untreated and treated patients suggests the existence of a “chemical” signal(s) in the tumor microenvironment, independent of physical compression of tumors, which triggers an inflammatory response, nerve damage, death of RGCs, and consequently vision loss.

Specific Aims and Study Design: In our preliminary studies, we developed a series of genetically engineered mouse (GEM) models of NF1-OPG in which tumor initiation occurs at birth or postnatal day 0.5 (P0.5) with a peak expansion of tumor cells during the first 3 weeks (P21), thereby establishing the first NF1-OPG model arising from the developing optic nerve. More importantly, we observed an inflammatory response with abnormal accumulation of microglia during tumor initiation before degeneration of the optic nerve or death of RGCs occurs. Thus, we hypothesize that abnormal infiltration of microglia induced by Nf1-/- OPG cells triggers a long-sought “chemical” signal(s), inducing OPG-associated axonal/myelin degeneration and RGC death. To test this hypothesis, we propose three specific aims. In Aim 1, we will use a green fluorescent reporter to isolate and characterize this abnormally infiltrating inflammatory cells and perform multi-omics experiments, including sophisticated genomic, epigenomic and transcriptomic assays, to study them during OPG initiation and progression. In Aim 2, we will either transiently eliminate or metabolically modulate this inflammatory cell population(s) as a means to prevent or alleviate OPG-associated nerve damage, RGC death and vision loss. In Aim 3, we will develop a novel model using the newly established genetic system called Mosaic Analysis with Double Markers (MADM) model, which tags sibling cells with or without Nf1 by green and red fluorescent protein, respectively. Multi-omics approaches will be performed on cells with or without Nf1 from the same optic nerve isolated by green and red fluorescence, allowing us to identify signals that induce inflammatory responses.

Impact: Due a lack of human surgical specimen or patient-derived cell lines, the development of the new series of NF1-OPG GEM models, including the MADM-Nf1 model, will provide important research tools to investigate disease mechanisms and perform preclinical testing to develop novel therapies. We propose to study and treat NF1-OPG as a nerve injury and neuronal degenerative diseases by targeting both inflammatory cells (e.g., microglia) in the tumor microenvironment and OPG cells. If successful, we will provide a strategy to treat patients with NF1-OPGs before visual impairment becomes irreversible, which will significantly enhance the quality of life of the children afflicted with NF1-OPG as well as many blinding diseases caused by degeneration of RGCs.