Mishal Rao

All Publications

• Generating ESC-Derived RGCs for Cell Replacement Therapy. Methods in molecular biology (Clifton, N.J.) Rao, M., Liu, C. C., Wang, S., Chang, K. C. 2025; 2848: 187-196

  Abstract

  In several ocular diseases, degeneration of retinal neurons can lead to permanent blindness. Transplantation of stem cell (SC)-derived RGCs has been proposed as a potential therapy for RGC loss. Although there are reports of successful cases of SC-derived RGC transplantation, achieving long-distance regeneration and functional connectivity remains a challenge. To address these hurdles, retinal organoids are being used to study the regulatory mechanism of stem cell transplantation. Here we present a modified protocol for differentiating human embryonic stem cells (ESCs) into retinal organoids and transplanting organoid-derived RGCs into the murine eyes.

  View details for DOI 10.1007/978-1-0716-4087-6_12

  View details for PubMedID 39240524

  View details for PubMedCentralID PMC12164832

• Report 3D-printed plugs enhance cell usage efficiency for single-cell migration and neuron axon guidance assays CELL REPORTS METHODS Cheng, J., Rock, E. C., Rao, M., Chen, H., Ma, Y., Chang, K., Chen, Y. 2025; 5 (8): 101117

  Abstract

  This paper reports a 3D-printed plug as a meso-scale interface solution that minimizes sample loss and enhances cell usage efficiency, seamlessly connecting microfluidic systems to conventional well plates. The plug concentrates cells near the region of interest for chemotaxis, reducing cell number requirements and featuring tapered structures for efficient manual or robotic liquid handling. Comprehensive testing showed that the plug increased cell usage efficiency in single-cell migration assays by 8-fold, maintaining accuracy and sensitivity. We also extended our approach to neuron axon guidance assays, where limited cell availability is a constraint, and observed substantial improvements in assay outcomes. This integration of 3D printing with microfluidics establishes low-loss interfaces for precious samples, advancing the capabilities of microfluidic technology.

  View details for DOI 10.1016/j.crmeth.2025.101117

  View details for Web of Science ID 001554472400001

  View details for PubMedID 40730156

• Insights from TPPP3 and its family member proteins in neuronal diseases. Neural regeneration research Rao, M., Chang, K. 2025

  Abstract

  Tubulin polymerization-promoting protein family member 3 (TPPP3) is a neuronal-specific protein involved in cytoskeletal stability, axonal maintenance, and neuronal survival. Dysregulation of TPPP3 is implicated in neurodegenerative diseases such as Parkinson's disease and diabetic retinopathy. Unlike TPPP1, which is oligodendrocyte-specific, TPPP3 was reported to primarily promote neuronal regeneration and serve as a therapeutic target for neurodegenerative diseases such as Parkinson's disease and glaucoma. Beyond the nervous system, TPPP3 has been linked to oncogenesis and tissue regeneration, suggesting potential roles in tumor suppression and wound healing. This review summarizes neuronal functions of TPPP3, therapeutic opportunities, and future research directions. Understanding the molecular mechanisms underlying function of TPPP3 could provide valuable insights into its therapeutic applications in neuroprotection.

  View details for DOI 10.4103/NRR.NRR-D-25-00345

  View details for PubMedID 40817731

• Tppp3 is a novel molecule for retinal ganglion cell identification and optic nerve regeneration. Acta neuropathologica communications Rao, M., Luo, Z., Liu, C. C., Chen, C. Y., Wang, S., Nahmou, M., Tanasa, B., Virmani, A., Byrne, L., Goldberg, J. L., Sahel, J. A., Chang, K. C. 2024; 12 (1): 204

  Abstract

  Mammalian central nervous system (CNS) axons cannot spontaneously regenerate after injury, creating an unmet need to identify molecular regulators to promote axon regeneration and reduce the lasting impact of CNS injuries. While tubulin polymerization promoting protein family member 3 (Tppp3) is known to promote axon outgrowth in amphibians, its role in mammalian axon regeneration remains unknown. Here we investigated Tppp3 in retinal ganglion cells (RGCs) neuroprotection and axonal regeneration using an optic nerve crush (ONC) model in the rodent. Single-cell RNA sequencing identified the expression of Tppp3 in RGCs of mice, macaques, and humans. Tppp3 overexpression enhanced neurite outgrowth in mouse primary RGCs in vitro, promoted axon regeneration, and improved RGC survival after ONC. Bulk RNA sequencing indicated that Tppp3 overexpression upregulates axon regeneration genes such as Bmp4 and neuroinflammatory pathways. Our findings advance regenerative medicine by developing a new therapeutic strategy for RGC neuroprotection and axon regeneration.

  View details for DOI 10.1186/s40478-024-01917-6

  View details for PubMedID 39734233

  View details for PubMedCentralID 8931466

• GDF-15 Attenuates the Epithelium-Mesenchymal Transition and Alleviates TGFβ2-Induced Lens Opacity. Translational vision science & technology Wang, S., Chen, C. Y., Liu, C. C., Stavropoulos, D., Rao, M., Petrash, J. M., Chang, K. C. 2024; 13 (7): 2

  Abstract

  We sought to evaluate the efficacy of growth differentiation factor (GDF)-15 treatment for suppressing epithelial-mesenchymal transition (EMT) and alleviating transforming growth factor β2 (TGFβ2)-induced lens opacity.To test whether GDF-15 is a molecule that prevents EMT, we pretreated the culture with GDF-15 in neural progenitor cells, retinal pigment epithelial cells, and lens epithelial cells and then treated with factors that promote EMT, GDF-11, and TGFβ2, respectively. To further investigate the efficacy of GDF-15 on alleviating lens opacity, we used mouse lens explant culture to mimic secondary cataracts. We pretreated the lens culture with GDF-15 and then added TGFβ2 to develop lens opacity (n = 3 for each group). Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to measure EMT protein and gene expression, respectively.In cell culture, GDF-15 pretreatment significantly attenuated EMT marker expression in cultured cells induced by treatment with GDF-11 or TGFβ2. In the lens explant culture, GDF-15 pretreatment also reduced mouse lens opacity induced by exposure to TGFβ2.Our results indicate that GDF-15 could alleviate TGFβ2-induced EMT and is a potential therapeutic agent to slow or prevent posterior capsular opacification (PCO) progression after cataract surgery.Cataracts are the leading cause of blindness worldwide, with the only current treatment involving surgical removal of the lens and replacement with an artificial lens. However, PCO, also known as secondary cataract, is a common complication after cataract surgery. The development of an adjuvant that slows the progression of PCO will be beneficial to the field of anterior complications.

  View details for DOI 10.1167/tvst.13.7.2

  View details for PubMedID 38949633

  View details for PubMedCentralID PMC11221611

• A potential therapeutic target for optic nerve regeneration Rao, M., Luo, Z., Liu, C., Nahmou, M., Tanasa, B., Virmani, A., Byrne, L., Goldberg, J., Sahel, J., Chang, K. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2024

  View details for Web of Science ID 001312227707102

• Aldose reductase is a potential therapeutic target for neurodegeneration. Chemico-biological interactions Rao, M., Chang, K. C. 2024; 389: 110856

  Abstract

  Neurodegeneration is a complex process involving various inflammatory mediators and cellular responses. Aldose reductase (AR) is a key enzyme in the polyol pathway, which converts glucose to sorbitol. Beyond its metabolic role, AR has also been found to play a significant role in modulating neuroinflammation. This review aims to provide an overview of the current knowledge regarding the involvement of AR inhibition in attenuating neuroinflammation and complications from diabetic neuropathies. Here, we review the literature regarding AR and neuropathy/neurodegeneration. We discuss the mechanisms underlying the influence of AR inhibitors on ocular inflammation, beta-amyloid-induced neurodegeneration, and optic nerve degeneration. Furthermore, potential therapeutic strategies targeting AR in neurodegeneration are explored. The understanding of AR's role in neurodegeneration may lead to the development of novel therapeutic interventions for other neuroinflammatory disorders.

  View details for DOI 10.1016/j.cbi.2024.110856

  View details for PubMedID 38185272

  View details for PubMedCentralID PMC10842418

• Aldose reductase inhibition decelerates optic nerve degeneration by alleviating retinal microglia activation. Scientific reports Rao, M., Huang, Y. K., Liu, C. C., Meadows, C., Cheng, H. C., Zhou, M., Chen, Y. C., Xia, X., Goldberg, J. L., Williams, A. M., Kuwajima, T., Chang, K. C. 2023; 13 (1): 5592

  Abstract

  As part of the central nervous system (CNS), retinal ganglion cells (RGCs) and their axons are the only neurons in the retina that transmit visual signals from the eye to the brain via the optic nerve (ON). Unfortunately, they do not regenerate upon injury in mammals. In ON trauma, retinal microglia (RMG) become activated, inducing inflammatory responses and resulting in axon degeneration and RGC loss. Since aldose reductase (AR) is an inflammatory response mediator highly expressed in RMG, we investigated if pharmacological inhibition of AR can attenuate ocular inflammation and thereby promote RGC survival and axon regeneration after ON crush (ONC). In vitro, we discovered that Sorbinil, an AR inhibitor, attenuates BV2 microglia activation and migration in the lipopolysaccharide (LPS) and monocyte chemoattractant protein-1 (MCP-1) treatments. In vivo, Sorbinil suppressed ONC-induced Iba1 + microglia/macrophage infiltration in the retina and ON and promoted RGC survival. Moreover, Sorbinil restored RGC function and delayed axon degeneration one week after ONC. RNA sequencing data revealed that Sorbinil protects the retina from ONC-induced degeneration by suppressing inflammatory signaling. In summary, we report the first study demonstrating that AR inhibition transiently protects RGC and axon from degeneration, providing a potential therapeutic strategy for optic neuropathies.

  View details for DOI 10.1038/s41598-023-32702-5

  View details for PubMedID 37019993

  View details for PubMedCentralID PMC10076364

• Aldose reductase inhibition promotes retinal ganglion cell survival after optic nerve injury Rao, M., Huang, Y., Meadows, C., Liu, C., Cheng, H., Zhou, M., Cheng, Y., Xia, X., Goldberg, J. L., Kuwajima, T., Chang, K. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2022

  View details for Web of Science ID 000844401304279

• The retinal pigment epithelium: Development, injury responses, and regenerative potential in mammalian and non-mammalian systems. Progress in retinal and eye research George, S. M., Lu, F., Rao, M., Leach, L. L., Gross, J. M. 2021; 85: 100969

  Abstract

  Diseases that result in retinal pigment epithelium (RPE) degeneration, such as age-related macular degeneration (AMD), are among the leading causes of blindness worldwide. Atrophic (dry) AMD is the most prevalent form of AMD and there are currently no effective therapies to prevent RPE cell death or restore RPE cells lost from AMD. An intriguing approach to treat AMD and other RPE degenerative diseases is to develop therapies focused on stimulating endogenous RPE regeneration. For this to become feasible, a deeper understanding of the mechanisms underlying RPE development, injury responses and regenerative potential is needed. In mammals, RPE regeneration is extremely limited; small lesions can be repaired by the expansion of adjacent RPE cells, but large lesions cannot be repaired as remaining RPE cells are unable to functionally replace lost RPE tissue. In some injury paradigms, RPE cells proliferate but do not regenerate a morphologically normal monolayer, while in others, proliferation is pathogenic and results in further disruption to the retina. This is in contrast to non-mammalian vertebrates, which possess tremendous RPE regenerative potential. Here, we discuss what is known about RPE formation during development in mammalian and non-mammalian vertebrates, we detail the processes by which RPE cells respond to injury, and we describe examples of RPE-to-retina and RPE-to-RPE regeneration in non-mammalian vertebrates. Finally, we outline barriers to RPE-dependent regeneration in mammals that could potentially be overcome to stimulate a regenerative response from the RPE.

  View details for DOI 10.1016/j.preteyeres.2021.100969

  View details for PubMedID 33901682

  View details for PubMedCentralID PMC8536801

• Dynamics of replication origin over-activation. Nature communications Fu, H., Redon, C. E., Thakur, B. L., Utani, K., Sebastian, R., Jang, S. M., Gross, J. M., Mosavarpour, S., Marks, A. B., Zhuang, S. Z., Lazar, S. B., Rao, M., Mencer, S. T., Baris, A. M., Pongor, L. S., Aladjem, M. I. 2021; 12 (1): 3448

  Abstract

  Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions (re-replicating cells). These cells exhibited slow replication, increased frequency of replication initiation events, and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (dormant) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins.

  View details for DOI 10.1038/s41467-021-23835-0

  View details for PubMedID 34103496

  View details for PubMedCentralID PMC8187443

• Unique patterns of organization and migration of FGF-expressing cells during Drosophila morphogenesis. Developmental biology Du, L., Zhou, A., Patel, A., Rao, M., Anderson, K., Roy, S. 2017; 427 (1): 35-48

  Abstract

  Fibroblast growth factors (FGF) are essential signaling proteins that regulate diverse cellular functions in developmental and metabolic processes. In Drosophila, the FGF homolog, branchless (bnl) is expressed in a dynamic and spatiotemporally restricted pattern to induce branching morphogenesis of the trachea, which expresses the Bnl-receptor, breathless (btl). Here we have developed a new strategy to determine bnl- expressing cells and study their interactions with the btl-expressing cells in the range of tissue patterning during Drosophila development. To enable targeted gene expression specifically in the bnl expressing cells, a new LexA based bnl enhancer trap line was generated using CRISPR/Cas9 based genome editing. Analyses of the spatiotemporal expression of the reporter in various embryonic stages, larval or adult tissues and in metabolic hypoxia, confirmed its target specificity and versatility. With this tool, new bnl expressing cells, their unique organization and functional interactions with the btl-expressing cells were uncovered in a larval tracheoblast niche in the leg imaginal discs, in larval photoreceptors of the developing retina, and in the embryonic central nervous system. The targeted expression system also facilitated live imaging of simultaneously labeled Bnl sources and tracheal cells, which revealed a unique morphogenetic movement of the embryonic bnl- source. Migration of bnl- expressing cells may create a dynamic spatiotemporal pattern of the signal source necessary for the directional growth of the tracheal branch. The genetic tool and the comprehensive profile of expression, organization, and activity of various types of bnl-expressing cells described in this study provided us with an important foundation for future research investigating the mechanisms underlying Bnl signaling in tissue morphogenesis.

  View details for DOI 10.1016/j.ydbio.2017.05.009

  View details for PubMedID 28502613

  View details for PubMedCentralID PMC5731658

• A replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells. Nature communications Zhang, Y., Huang, L., Fu, H., Smith, O. K., Lin, C. M., Utani, K., Rao, M., Reinhold, W. C., Redon, C. E., Ryan, M., Kim, R., You, Y., Hanna, H., Boisclair, Y., Long, Q., Aladjem, M. I. 2016; 7: 11748

  Abstract

  Mammalian chromosome replication starts from distinct sites; however, the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. Here we identify a replication-initiation determinant (RepID) protein that binds a subset of replication-initiation sites. A large fraction of RepID-binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from RepID-bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID-depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication-initiation events. These observations are consistent with a model, suggesting that RepID facilitates replication initiation at a distinct group of human replication origins.

  View details for DOI 10.1038/ncomms11748

  View details for PubMedID 27272143

  View details for PubMedCentralID PMC4899857