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Principal Investigator: David Wang
Organization: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
Fiscal Year: 2024
Award: $827,865
Funding agency: National Institute of Neurological Disorders and Stroke
Summary:
Specific aim 1:
To develop in vitro 2D neuronal and 3D brain organoid models derived from human adult peripheral CD34+ cells to study neural development and degeneration and infectious diseases involving human brain. Using the brain-on-the-chip microfluidic culture, we worked on 3D brain cultures with mixed neurons, microglia, astroglia in one channel and endothelial cells in another channel, to reconstitute human blood brain barrier (BBB). We confirmed the integrity of the BBB with leakage tests on different sizes of fluorescent labeled molecules and used it to study the possible toxicity of SARS-CoV2 on BBB. By comparing recombinant spike protein of SARS-CoV2, spike protein plus spike protein targeting antibody, we found that the combination of spoke protein and antibody caused moderate disruption of BBB. We are working to delineate whether addition of the complements in human serum may enhance the toxicity. In another approach, we standardized our protocol to make brain organoids containing microglia by derive the embryoid bodies from mixed iPSC and iPSC-differentiated hematopoietic progenitor cells. By invitation, we submitted the protocol for publication to JoVE, a scientific video Journal. We also presented our variety of 3D models and findings in a special 3D brain organoid workshop on the 11th IBRO World Congress of Neuroscience, Granada, Spain and drew warm discussions and interests from the international researchers as well as potential collaborations. As a contribution to the neural stem cell research community, Dr. Wang guest edited a special Experimental Neurology issue on Neural Stem Cells.
Specific aim 2: To study the roles of HERV-K on brain development. We adopted a published protocol to differentiate microglia from iPSCs. Use this method, we generated microglia with confirmed microglial marker IBA1 expression. We found that HERV-K Env expression changed during different stages of the microglial differentiation and maturation and phorbol myristate acetate (PMA) treatment on the microglia induced HERV-K env activation, which was inhibited by siRNA targeting KU80, a protein responsible for initiating DNA repairing processes. The inhibition effect was also observed in prevented PMA-activated cGAS-Sting pathway, which are responsible for sensing intracellular DNA. Our observation indicates that HERV-K activation in microglia is regulated partly by KU80. KU80 may play an important role in regulating microglial response to virus infection and retrovirus may interfere this process. Targeting KU80 could be used as an important approach for modulating microglial activity, which plays important roles in neuroinflammation and neuroinfectious disorders.
Specific aim 3: To study the association of HERV-K and motor neuron degeneration like ALS. We continued our study of retro virus activation and motor neuron degeneration. We found that C9orf72 iPSC-differentiated motor neurons produced less mitochondria than healthy controls, in a collaboration with Dr. InHong Yang from UNC to develop high-throughput apparatus monitoring mitochondrial trafficking through axons. We are in collaboration with Dr. Kory Johnson to explore the possibility of using Artificial intelligence to facilitate the image processing and develop criteria for mitochondrial normality in iPSC-differentiated human motor neurons for possible novel ALS diagnostic development. Furthermore, using Western-blot assay, we found that C9orf72 iPSC derived motor neurons produced phosphorylated-TDP43, a protein observed in degenerated motor neurons. More promisingly, by treating the motor neurons with antisense Oligonucleotides (ASOs) targeting HERV-K Env or C9orf72 repeats, we decreased the phos-TDP43 products significantly. Our results indicate that the iPSC-differentiated motor neurons preserved some of the C9orf72 ALS properties and could be used for study the pathogenesis of the disease. By doing that, we confirmed the roles of C9orf72 mutation and HERV-K activation in the degeneration of the motor neurons. Further study for more efficient ASOs could provide treatment for the disease.
Specific aim 4: To facilitate research and therapeutic developments for neurological disorders using our models and methods. We have trained additional staff from Dr. Farinaz Safavi’s lab on the 3D brain organoids. We have collaborated with Dr. Safavi and cultured more brain organoids with PI3KR mutations at variety sites. These affected brain organoids showed consistent morphological differences compared with the healthy control and gene corrected controls. The differences between them were also confirmed by scRNA-Seq analysis. We are working on delineating the functions of specific mutations using more patient derived iPSC lines. We also helped Dr.Christopher Batley to train his postdoc on differentiating microglial from patient iPSCs and provided iPSC cell lines as healthy controls. In another collaborative project, we provided trainings to Dr. Pankaj Seth from India National Brain Research Center on 3D brain organoids and explored the possibility of using ASOs to treat Dengue virus infection in human brain. Our preliminary results found that the Dengue virus can infect the brain organoid and specifically designed ASOs can penetrate the brain organoid and inhibit the virus production. Further studies will be done with the collaboration with Dr. Lisa Henderson from SINS on the project, aiming to a potential treatment using ASOs for the virus infection.
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