Browsing by Author "Harkin, Jade"

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    Acquisition of neurodegenerative features in isogenic OPTN(E50K) human stem cell-derived retinal ganglion cells associated with autophagy disruption and mTORC1 signaling reduction
    (Springer Nature, 2024-10-18) Huang, Kang‑Chieh; Gomes, Cátia; Shiga, Yukihiro; Belforte, Nicolas; VanderWall, Kirstin B.; Lavekar, Sailee S.; Fligor, Clarisse M.; Harkin, Jade; Hetzer, Shelby M.; Patil, Shruti V.; Di Polo, Adriana; Meyer, Jason S.; Biology, School of Science
    The ability to derive retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) has led to numerous advances in the field of retinal research, with great potential for the use of hPSC-derived RGCs for studies of human retinal development, in vitro disease modeling, drug discovery, as well as their potential use for cell replacement therapeutics. Of all these possibilities, the use of hPSC-derived RGCs as a human-relevant platform for in vitro disease modeling has received the greatest attention, due to the translational relevance as well as the immediacy with which results may be obtained compared to more complex applications like cell replacement. While several studies to date have focused upon the use of hPSC-derived RGCs with genetic variants associated with glaucoma or other optic neuropathies, many of these have largely described cellular phenotypes with only limited advancement into exploring dysfunctional cellular pathways as a consequence of the disease-associated gene variants. Thus, to further advance this field of research, in the current study we leveraged an isogenic hPSC model with a glaucoma-associated mutation in the Optineurin (OPTN) protein, which plays a prominent role in autophagy. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor AMPK, along with subsequent neurodegeneration in OPTN(E50K) RGCs differentiated from hPSCs, and have further validated some of these findings in a mouse model of ocular hypertension. Pharmacological inhibition of mTORC1 in hPSC-derived RGCs recapitulated disease-related neurodegenerative phenotypes in otherwise healthy RGCs, while the mTOR-independent induction of autophagy reduced protein accumulation and restored neurite outgrowth in diseased OPTN(E50K) RGCs. Taken together, these results highlighted that autophagy disruption resulted in increased autophagic demand which was associated with downregulated signaling through mTORC1, contributing to the degeneration of RGCs.
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    Astrocytes modulate neurodegenerative phenotypes associated with glaucoma in OPTN(E50K) human stem cell-derived retinal ganglion cells
    (Elsevier, 2022) Gomes, Cátia; VanderWall, Kirstin B.; Pan, Yanling; Lu, Xiaoyu; Lavekar, Sailee S.; Huang, Kang-Chieh; Fligor, Clarisse M.; Harkin, Jade; Zhang, Chi; Cummins, Theodore R.; Meyer, Jason S.; Medical and Molecular Genetics, School of Medicine
    Although the degeneration of retinal ganglion cells (RGCs) is a primary characteristic of glaucoma, astrocytes also contribute to their neurodegeneration in disease states. Although studies often explore cell-autonomous aspects of RGC neurodegeneration, a more comprehensive model of glaucoma should take into consideration interactions between astrocytes and RGCs. To explore this concept, RGCs and astrocytes were differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated OPTN(E50K) mutation along with corresponding isogenic controls. Initial results indicated significant changes in OPTN(E50K) astrocytes, including evidence of autophagy dysfunction. Subsequently, co-culture experiments demonstrated that OPTN(E50K) astrocytes led to neurodegenerative properties in otherwise healthy RGCs, while healthy astrocytes rescued some neurodegenerative features in OPTN(E50K) RGCs. These results are the first to identify disease phenotypes in OPTN(E50K) astrocytes, including how their modulation of RGCs is affected. Moreover, these results support the concept that astrocytes could offer a promising target for therapeutic intervention in glaucoma.
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    Autophagy disruption reduces mTORC1 activation leading to retinal ganglion cell neurodegeneration associated with glaucoma
    (Cold Spring Harbor Laboratory, 2023-01-04) Huang, Kang-Chieh; Gomes, Cátia; Shiga, Yukihiro; Belforte, Nicolas; VanderWall, Kirstin B.; Lavekar, Sailee S.; Fligor, Clarisse M.; Harkin, Jade; Di Polo, Adriana; Meyer, Jason S.; Biology, School of Science
    Autophagy dysfunction has been associated with several neurodegenerative diseases including glaucoma, characterized by the degeneration of retinal ganglion cells (RGCs). However, the mechanisms by which autophagy dysfunction promotes RGC damage remain unclear. Here, we hypothesized that perturbation of the autophagy pathway results in increased autophagic demand, thereby downregulating signaling through mammalian target of rapamycin complex 1 (mTORC1), a negative regulator of autophagy, contributing to the degeneration of RGCs. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor adenosine monophosphate-activated protein kinase (AMPK), along with subsequent neurodegeneration in RGCs differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated variant of Optineurin (OPTN-E50K). Similarly, the microbead occlusion model of glaucoma resulting in ocular hypertension also exhibited autophagy disruption and mTORC1 downregulation. Pharmacological inhibition of mTORC1 in hPSC-derived RGCs recapitulated disease-related neurodegenerative phenotypes in otherwise healthy RGCs, while the mTOR-independent induction of autophagy reduced protein accumulation and restored neurite outgrowth in diseased OPTN-E50K RGCs. Taken together, these results highlight an important balance between autophagy and mTORC1 signaling essential for RGC homeostasis, while disruption to these pathways contributes to neurodegenerative features in glaucoma, providing a potential therapeutic target to prevent neurodegeneration.
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    Defective Hand1 phosphoregulation uncovers essential roles for Hand1 in limb morphogenesis
    (The Company of Biologists Ltd, 2017-07-01) Firulli, Beth A.; Milliar, Hannah; Toolan, Kevin P.; Harkin, Jade; Fuchs, Robyn K.; Robling, Alex G.; Firulli, Anthony B.; Anatomy and Cell Biology, School of Medicine
    The morphogenesis of the vertebrate limbs is a complex process in which cell signaling and transcriptional regulation coordinate diverse structural adaptations in diverse species. In this study, we examine the consequences of altering Hand1 dimer choice regulation within developing vertebrate limbs. Although Hand1 deletion via the limb-specific Prrx1-Cre reveals a non-essential role for Hand1 in mouse limb morphogenesis, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, results in a severe truncation of proximal-anterior limb elements. Molecular analysis reveals a non-cell-autonomous mechanism that causes widespread cell death within the embryonic limb bud. In addition, we observe changes in proximal-anterior gene regulation, including a reduction in the expression of Irx3, Irx5, Gli3 and Alx4, all of which are upregulated in Hand2 limb conditional knockouts. A reduction of Hand2 and Shh gene dosage improves the integrity of anterior limb structures, validating the importance of the Twist-family bHLH dimer pool in limb morphogenesis., Summary: Altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, results in a severe truncation of anterior-proximal limb elements in mice.
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    Development of a three-dimensional organoid model to explore early retinal phenotypes associated with Alzheimer’s disease
    (Springer Nature, 2023-08-24) Lavekar, Sailee S.; Harkin, Jade; Hernandez, Melody; Gomes, Cátia; Patil, Shruti; Huang, Kang‑Chieh; Puntambekar, Shweta S.; Lamb, Bruce T.; Meyer, Jason S.; Biology, School of Science
    Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of Aβ plaques and neurofibrillary tangles, resulting in synaptic loss and neurodegeneration. The retina is an extension of the central nervous system within the eye, sharing many structural similarities with the brain, and previous studies have observed AD-related phenotypes within the retina. Three-dimensional retinal organoids differentiated from human pluripotent stem cells (hPSCs) can effectively model some of the earliest manifestations of disease states, yet early AD-associated phenotypes have not yet been examined. Thus, the current study focused upon the differentiation of hPSCs into retinal organoids for the analysis of early AD-associated alterations. Results demonstrated the robust differentiation of retinal organoids from both familial AD and unaffected control cell lines, with familial AD retinal organoids exhibiting a significant increase in the Aβ42:Aβ40 ratio as well as phosphorylated Tau protein, characteristic of AD pathology. Further, transcriptional analyses demonstrated the differential expression of many genes and cellular pathways, including those associated with synaptic dysfunction. Taken together, the current study demonstrates the ability of retinal organoids to serve as a powerful model for the identification of some of the earliest retinal alterations associated with AD.
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    Elucidating the Influence of Microglia on Retinal Ganglion Cells in a Human Pluripotent Stem Cell Model
    (2024-06) Harkin, Jade; Meyer, Jason; Sheets, Patrick; Landreth, Gary; Block, Michelle; Sharma, Tasneem; Gomes, Catia
    Glaucoma is a complex disease that leads to irreversible blindness, characterized by the loss of retinal ganglion cells (RGCs), which are the cells that transmit visual information from your eye into your brain. Evidence suggests that microglia, the resident immune cells in the central nervous system, may have a detrimental role in the onset and the progression of glaucoma. Microglia become activated in response to damage, pathogens and toxins and are initially thought to be beneficial to RGCs. However, when these cells are activated for excessive periods of time, they are thought to be harmful to RGCs. Thus, we sought to develop novel human pluripotent stem cell (hPSC)-derived microglia, astrocyte and RGC co-cultures to determine how microglia activation modulates RGC phenotypes in a human cellular model. Healthy and LPS-activated microglia were first co-cultured with RGCs for up to 3 weeks and the effects of microglia upon RGCs were assessed. Additionally, healthy and LPS-activated microglia were also co-cultured with astrocytes and RGCs for up to three weeks to assess if LPS-treated microglia can activate astrocytes and the effects this would have on RGCs. Results showed that when co-cultured with RGCs alone for 1 week, microglia activation is initially beneficial to RGCs. However, when co-cultured with RGCs for 3 weeks, microglia activation leads to RGC damage. Consequently, when astrocytes are present, microglia activation is harmful to RGCs in both short-term and long-term co-cultures, suggesting an additional role for microglia modulation of astrocytes, further leading to neurodegeneration. Taken together, our results have allowed for the precise study of how individual cell types are adversely affected in disease-relevant states, how microglia can directly influence RGCs, and how multiple co-cultures of human microglia, astrocytes and RGCs allows for a more sophisticated investigation of cellular interactions in disease states relevant to glaucoma.
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    Exploring dysfunctional barrier phenotypes associated with glaucoma using a human pluripotent stem cell-based model of the neurovascular unit
    (Springer Nature, 2024-11-14) Lavekar, Sailee S.; Hughes, Jason M.; Gomes, Cátia; Huang, Kang-Chieh; Harkin, Jade; Canfield, Scott G.; Meyer, Jason S.; Biology, School of Science
    Glaucoma is a neurodegenerative disease that results in the degeneration of retinal ganglion cells (RGCs) and subsequent loss of vision. While RGCs are the primary cell type affected in glaucoma, neighboring cell types selectively modulate RGCs to maintain overall homeostasis. Among these neighboring cell types, astrocytes, microvascular endothelial cells (MVECs), and pericytes coordinate with neurons to form the neurovascular unit that provides a physical barrier to limit the passage of toxic materials from the blood into neural tissue. Previous studies have demonstrated that these barrier properties may be compromised in the progression of glaucoma, yet mechanisms by which this happens have remained incompletely understood. Thus, the goals of this study were to adapt a human pluripotent stem cell (hPSC)-based model of the neurovascular unit to the study of barrier integrity relevant to glaucoma. To achieve this, hPSCs were differentiated into the cell types that contribute to this barrier, including RGCs, astrocytes, and MVECs, then assembled into an established Transwell®-insert model. The ability of these cell types to contribute to an in vitro barrier model was tested for their ability to recapitulate characteristic barrier properties. Results revealed that barrier properties of MVECs were enhanced when cultured in the presence of RGCs and astrocytes compared to MVECs cultured alone. Conversely, the versatility of this system to model aspects of barrier dysfunction relevant to glaucoma was tested using an hPSC line with a glaucoma-specific Optineurin (E50K) mutation as well as a paired isogenic control, where MVECs then exhibited reduced barrier integrity. To identify factors that could result in barrier dysfunction, results revealed an increased expression of TGFβ2 in glaucoma-associated OPTN(E50K) astrocytes, indicating a potential role for TGFβ2 in disease manifestation. To test this hypothesis, we explored the ability to modulate exogenous TGFβ2 in both isogenic control and OPTN(E50K) experimental conditions. Collectively, the results of this study indicated that the repurposing of this in vitro barrier model for glaucoma reliably mimicked some aspects of barrier dysfunction, and may serve as a platform for drug discovery, as well as a powerful in vitro model to test the consequences of barrier dysfunction upon RGCs in glaucoma.
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    Extension of retinofugal projections in an assembled model of human pluripotent stem cell-derived organoids
    (Cell Press, 2021-09-14) Fligor, Clarisse M.; Lavekar, Sailee S.; Harkin, Jade; Shields, Priya K.; VanderWall, Kirstin B.; Huang, Kang-Chieh; Gomes, Cátia; Meyer, Jason S.; Biology, School of Science
    The development of the visual system involves the coordination of spatial and temporal events to specify the organization of varied cell types, including the elongation of axons from retinal ganglion cells (RGCs) to post-synaptic targets in the brain. Retinal organoids recapitulate many features of retinal development, yet have lacked downstream targets into which RGC axons extend, limiting the ability to model projections of the human visual system. To address these issues, retinal organoids were generated and organized into an in vitro assembloid model of the visual system with cortical and thalamic organoids. RGCs responded to environmental cues and extended axons deep into assembloids, modeling the projections of the visual system. In addition, RGC survival was enhanced in long-term assembloids, overcoming prior limitations of retinal organoids in which RGCs are lost. Overall, these approaches will facilitate studies of human visual system development, as well as diseases or injuries to this critical pathway.
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    The HAND1 frameshift A126FS mutation does not cause hypoplastic left heart syndrome in mice
    (Oxford University Press, 2017-12-01) Firulli, Beth A.; Toolan, Kevin P.; Harkin, Jade; Millar, Hannah; Pineda, Santiago; Firulli, Anthony B.; Pediatrics, School of Medicine
    Aims: To test if a human Hand1 frame shift mutation identified in human samples is causative of hypoplastic left heart syndrome (HLHS). Methods and results: HLHS is a poorly understood single ventricle congenital heart defect that affects two to three infants in every 10 000 live births. The aetiologies of HLHS are largely unknown. The basic helix-loop-helix transcription factor HAND1 is required for normal heart development. Interrogation of HAND1 sequence from fixed HLHS tissues identified a somatic frame-shift mutation at Alanine 126 (NP_004812.1 p.Ala126Profs13X defined as Hand1A126fs). Hand1A126fs creates a truncated HAND1 protein that predictively functions as dominant negative. To determine if this mutation is causative of HLHS, we engineered a conditional Hand1A126fs mouse allele. Activation of this allele with Nkx2.5Cre results in E14.5 lethality accompanied by cardiac outflow tract and intraventricular septum abnormalities. Using αMHC-Cre or Mef2CAHF-Cre to activate Hand1A126fs results in reduced phenotype and limited viability. Left ventricles of Hand1A126FS mutant mice are not hypoplastic. Conclusions: Somatically acquired Hand1A126FS mutation is not causative of HLHS. Hand1A126FS mutation does exhibit embryonic lethal cardiac defects that reflect a dominant negative function supporting the critical role of Hand1 in cardiogenesis.
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    HAND1 loss-of-function within the embryonic myocardium reveals survivable congenital cardiac defects and adult heart failure
    (Oxford University Press, 2020-03) Firulli, Beth A.; George, Rajani M.; Harkin, Jade; Toolan, Kevin P.; Gao, Hongyu; Liu, Yunlong; Zhang, Wenjun; Field, Loren J.; Liu, Ying; Shou, Weinian; Payne, Ronald Mark; Rubart-von der Lohe, Michael; Firulli, Anthony B.; Pediatrics, School of Medicine
    Aims: To examine the role of the basic Helix-loop-Helix (bHLH) transcription factor HAND1 in embryonic and adult myocardium. Methods and results: Hand1 is expressed within the cardiomyocytes of the left ventricle (LV) and myocardial cuff between embryonic days (E) 9.5-13.5. Hand gene dosage plays an important role in ventricular morphology and the contribution of Hand1 to congenital heart defects requires further interrogation. Conditional ablation of Hand1 was carried out using either Nkx2.5 knockin Cre (Nkx2.5Cre) or α-myosin heavy chain Cre (αMhc-Cre) driver. Interrogation of transcriptome data via ingenuity pathway analysis reveals several gene regulatory pathways disrupted including translation and cardiac hypertrophy-related pathways. Embryo and adult hearts were subjected to histological, functional, and molecular analyses. Myocardial deletion of Hand1 results in morphological defects that include cardiac conduction system defects, survivable interventricular septal defects, and abnormal LV papillary muscles (PMs). Resulting Hand1 conditional mutants are born at Mendelian frequencies; but the morphological alterations acquired during cardiac development result in, the mice developing diastolic heart failure. Conclusion: Collectively, these data reveal that HAND1 contributes to the morphogenic patterning and maturation of cardiomyocytes during embryogenesis and although survivable, indicates a role for Hand1 within the developing conduction system and PM development.