Browsing by Author "Lasagna Reeves, Cristian A."

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    Elucidating Cellular Mechanisms Underlying Retinal Ganglion Cell Neurodegeneration in a Human Pluripotent Stem Cell-Derived Model
    (2022-12) Huang, Kang-Chieh; Cummins, Theodore R.; Meyer, Jason S.; Marrs, James A.; Perrin, Benjamin J.; Lasagna Reeves, Cristian A.
    Glaucoma is a leading cause of blindness characterized by the progressive loss of retinal ganglion cells (RGCs), essentially severing the connection between the eye and the brain. Among many underlying causes of the disease, mutations in the Optineurin (OPTN) gene result in severe RGC neurodegeneration in the absence of elevated intraocular pressure, providing a novel opportunity to study molecular mechanisms that lead to RGC neurodegeneration associated with glaucoma. Efforts of this study establishing a human pluripotent stem cell (hPSC)-derived in vitro disease model by inserting OPTN(E50K) mutation via CRISPR/Cas9 genome editing and investigate the cellular mechanisms of RGC neurodegeneration associated with glaucoma. OPTN(E50K) RGCs revealed neurodegeneration phenotypes, including downregulation of RGCs transcription factors, neurite retraction, and hyperexcitability, suggesting that OPTN(E50K) RGCs can serve as an appropriate disease model to study glaucoma-associated neurodegeneration. Since OPTN serves a primary role as an autophagy receptor, we further hypothesized that the OPTN(E50K) mutation disrupts autophagy in RGCs, and modulation of autophagy by mammalian target of rapamycin (mTOR)-independent pathways can preserve RGC phenotypes by maintaining mTOR signaling. OPTN(E50K) RGCs exhibited a higher number of OPTN puncta along with an overall reduced expression of OPTN protein, indicating a gain of toxic protein accumulation or loss of protein function. Furthermore, OPTN(E50K) RGCs revealed an accumulation of the autophagosome protein LC3 in a punctal manner as well as increased expression of lysosomal proteins, suggesting a disruption of degradation pathway in autophagosome and lysosome fusion. As mTOR complex 1 (mTORC1) signaling serves as a negative regulator of autophagy, a downregulation of mTORC1 signaling via activation of stress sensor adenosine monophosphate-activated protein kinase (AMPK) was observed as a possible compensatory mechanism for autophagy deficits in OPTN(E50K) RGCs. Pharmacological inhibition of mTOR in wild-type hRGCs resulted in similar disease-related phenotypes, while preservation of the mTOR pathway in OPTN(E50K) RGCs by treatment with the mTOR-independent autophagy modulator trehalose cleared OPTN accumulated puncta, preserving mTORC1 signaling, as well as rescuing neurodegenerative phenotypes. To further validate these associations in an animal model, the microbead occlusion mouse model was established by injection of magnetic microbeads in the anterior chamber to block aqueous outflow resulting ocular hypertension. In agreement with our findings in hRGCs, a decrease in mTOR signaling associated with an increase in the expression of autophagy-associated proteins was observed in RGCs in the microbead occlusion model. Additionally, these disease-related phenotypes were observed specifically within RGCs but not cortical neurons with an underlying OPTN(E50K) mutation, demonstrating that autophagy represents an essential pathway in RGCs to maintain homeostasis, and selective disrupt of autophagy in RGCs leads to neurodegeneration. Taken together, the results of this study highlight an essential balance between autophagy and mTORC1 signaling that is essential for the homeostasis of RGCs, while disruption to these signaling pathways contributes to neurodegenerative features in glaucoma. These results also demonstrated the ability to pharmacologically intervene to experimentally manipulate these pathways and rescue neurodegenerative phenotypes, providing a potential therapeutic target to prevent glaucoma-associated neurodegeneration.
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    The Role of Microglial-Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) in Neuronal Homeostasis and Tau Pathogenesis
    (2021-06) Jadhav, Vaishnavi Sunil; Landreth, Gary E.; Lamb, Bruce T.; Oblak, Adrian L.; Apostolova, Liana G.; Lasagna Reeves, Cristian A.
    The importance of microglia in neurodegeneration has been highlighted by recent identification of microglial genes associated with increased risk for dementia. Among these, several variants of ‘Triggering Receptor expressed on myeloid cells 2’ (TREM2), confer higher risk for different types of dementia including Alzheimer’s disease and frontotemporal-like dementia with early onset. The mechanism by which alterations in TREM2 predisposes individuals to early dementia and how TREM2 influences proteinopathies, especially tauopathy remains unclear. The first part of this thesis focused on the role of TREM2 in neuronal homeostasis using a novel Trem2 p.Y38C mouse model (Trem2Y38C/Y38C) and mice lacking Trem2 (Trem2-/-). Young adult Trem2Y38C/Y38C and Trem2-/- mice exhibited synaptic impairments with reduced long-term potentiation accompanied by oligodendrocyte/myelin impairments. These pathologies are reminiscent of the clinical manifestation in patients with TREM2 p.Y38C mutation and functional loss of TREM2. Since these alterations were detected in wildtype Trem2Y38C/Y38C and Trem2-/- mice in the absence of any pathological insults, these results demonstrate that TREM2 directly impacts neuronal functions and homeostasis independent of the triggers such as pathological tau. In the second part of the thesis, we addressed the role of TREM2 in tau pathogenesis using aforementioned Trem2Y38C/Y38C and Trem2-/- mouse models crossed to human wildtype tau expressing ‘htau’ mice. Loss of functional TREM2 does not alter the overall phosphorylated tau burden but shifts localization of tau to the interstitial fluid in a tau species and sex-dependent manner. Female htau mice lacking functional TREM2 showed lower insoluble tau (largely intracellular) but higher tau levels in the interstitial fluid (extracellular). Transcriptomic analysis reveal alterations in genes associated with neuroinflammation and microglial phagocytic pathways in htau;Trem2Y38C/Y38C and htau;Trem2-/- mice . These alterations likely suggest compromised uptake and/or clearance of extracellular tau leading to the accumulation of tau in the ISF, which has been shown to be detrimental to the synapses. These results demonstrate that TREM2 is important for microglial, neuronal, and white matter functions and provides unique insights on the aspects of tau pathogenesis impacted by TREM2.