Research Overview
Our research focuses on restoration of cellular homeostasis to improve neuronal differentiation, maturation and protection, ultimately for developing neuroprotection therapy such as for glaucoma. Disrupted mitochondrial homeostasis with metabolic abnormalities mark the onset of certain central nervous system disorders including glaucoma and ALS. Current glaucoma therapies focus on managing intraocular pressure (IOP), but many patients continue to experience vision impairments despite effective IOP management. Retinal ganglion cells (RGCs) are the projection neurons of retina that deliver visual signal from eye to the brain and progressively degenerate in glaucoma.
There is a significant gap in research on restoring metabolic homeostasis as a means of developing neuroprotection for affected neurons in glaucoma. Our lab differentiates human embryonic stem cells (ESCs) and patient-derived induced pluripotent stem cells (iPSCs) with glaucoma causing mutations to study disease mechanisms related to mitochondrial biology. Our goal is to restore mitochondrial and metabolic homeostasis for developing neuroprotection therapy.
We examine not only the differentiated neurons but also the cellular homeostasis mechanisms in the origin stem cells. This approach allows us to explore the developmental aspects of disease mechanisms, genotype-to-phenotype specificity by comparing different neurons derived from the same mutant stem cells, and the potential for perturbing cellular quality control pathways to enhance differentiation. These insights could contribute to both regenerative medicine and neuroprotection.
Our work is highly interdisciplinary and encompasses a range of studies, from stem cells to animal models, driven by highly interdisciplinary and collaborative efforts.
Metabolic Homeostasis Restoration for Glaucoma Neuroprotection Therapy
The defining pathophysiological event in glaucoma is the degeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve. Intraocular pressure (IOP) is the only treatable risk factor, though many glaucoma patients continue to lose RGCs and vision even when IOP is managed effectively. Early progression involves metabolic dysfunctions with depleted ATP supply in RGCs as manifest by degradation of anterograde axonal transport from eye to brain, requiring more ATP than retrograde transport, results in buildup of active mitochondria in the unmyelinated axons of optic nerve head. This is partly due to the inefficiency of the long unmyelinated RGC axon segment spanning the retina through the lamina cribrosa, which requires high ATP consumption for slow transport predisposing to oxidative stress. Even though these defects mark early onset, it provides a window of intervention to protect RGCs from irreversible loss.
Our objective here is to test the efficacy and mechanisms of a cell intrinsic intervention that may restore metabolic homeostasis and RGC axon function. Prior interventions that target mitochondrial metabolism include supplementation with metabolic intermediates of mitochondrial oxidative phosphorylation (OXPHOS), such as pyruvate and nicotinamide, and constitutive changes in mitochondrial fusion and transport. However, these approaches, in long-term possess considerable risk owing to the constitutive changes in mitochondrial morphology, distribution, and OXPHOS which would simultaneously increase mitochondrial toxic byproducts, such as reactive oxygen species (ROS) and cytochrome c. Our approach avoids these risks. Recently, we discovered that a cell intrinsic transient activation of mitochondrial biogenesis restores mitochondrial health leading to neuroprotection against mitochondrial stress in human stem cell differentiated RGCs (hRGCs) containing the glaucoma causing E50K mutation in the Optineurin (OPTN) gene, which is critical for maintaining mitochondrial homeostasis. Using a high-throughput screening for improved mitochondrial health as outcome, we discovered several compounds that both shows increased mitochondrial mass as well as reduced mass in the hRGCs (Fig. 1). We then tested manually these compounds if they reproduce the screening data, and next tested if they reduce caspase activity in the hRGCs as potential neuroprotective compounds. The compounds indicated by “neuroprotective (NP)” in Fig. 1 are the ones that showed reduced caspase activity. Among them, we discovered a compound NP1 which is an antagonist of the Gi/o protein-coupled receptor. Our studies show NP1 transiently activates multiple neuroprotective pathways in hRGCs with robust in-vivo effect for acute optic nerve crush (ONC) injury and chronic high IOP glaucoma mice. Shown here examples of neuroprotective and/or regenerative effect of the treatment for ONC injury where animals are treated intraperitoneally with the NP1 compound prior to ONC until the tissue collection. The treatment shows remarkable RGC body protection in the retina (Fig. 2A, B) and axon regeneration in the optic nerve (Fig. 2C, D).
Based on these results, we are investigating the central hypothesis that cell intrinsic transient activation of neuroprotective mechanisms by NP1 protects against RGC axonopathy by preventing early metabolic impairment in glaucoma. Our aims here illustrated in Fig. 3 and include: (i) Investigate the neuroprotective mechanisms by NP1 involving mitochondria dependent and/or independent mechanisms in hRGCs using pharmacological, genetic and biochemical approaches. (ii) Determine if the treatment restores metabolic homeostasis in hRGCs and RGCs isolated from glaucoma mice by performing ATP, ADP level measurements; and by performing integrated omic analysis on the metabolomic and RNAseq data. (iii) Test if NP1 treatment prevents glaucoma axonopathy by measuring compound action potential on RGC axons in the mouse optic nerve, and by active uptake mediated anterograde transport of cholera toxin B (CTB) subunit from retina to visual cortex.
In addition to above compound, we are testing the other NP compounds for their in-vivo efficacy for potential in-depth mechanistic analysis as outlined above.
These studies, based on technical and conceptual innovative approaches will pave the path for glaucoma neuroprotection therapy that can also be adopted for other neurodegenerative conditions with metabolic disruption as a major risk factor.
Collaborators: David Calkins (Vanderbilt University), Harry Quigley (Johns Hopkins University), Thomas Johnson (Johns Hopkins), Larry Benowitz (HMS), Thomas O’Connell (IUSM), Jason Meyer (IUSM), Jingwei Meng (IUSM)
Figure 1. Small molecule screen on H7-hRGC (differentiated from H7-hESC reporter line) identified drugs showing >40% change (outside of blue box) to mitochondria mass by live cell Mito Tracker Deep Red (MTDR) dye.
Figure 2. (A - D) C57BL/6J (C57) mice were treated daily with NP1 (5 mg/kg) by intraperitoneal injection (IP) 5 days before ONC until tissue collection on indicated “day post crush (dpc)”. (A) Confocal IHC images showing RBPMS positive RGCs on retinal flat-mount. (B) Quantification of RGC numbers/mm2 from central and peripheral retina shows significant protection by on 6 and 14 dpc. Two-way ANOVA with Tukey’s, ****, p < 0.0001, ***, p < 0.001, n = 24-48 images from 3-6 mice for each group. (C) Regenerated or protected axons are detected and (D) quantified at 0.5 mm from crush site on serial cryosections by confocal IHC imaging against GAP43 following published methods by Larry Benowitz lab. GAP43 antibody is shared by Larry Benowitz. Student’s t-test, **, p < 0.007, n = 3 mice per group. Error bars are SEM
Figure 3. Overview of the project where we will resolve the molecular mechanisms of RGC protection by NP1 (Aim1), mechanisms for in-vivo efficacy (Aim2) and if the treatment prevents glaucoma axonopathy (Aim3).
