Study the pathogenesis of neurological disorders using human neural cultures derived from patient peripheral blood CD34 cells

National Institute Of Neurological Disorders And Stroke

Investigators

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Abstract

Specific aim 1: Develop in vitro 3D brain organoid models derived from human adult peripheral CD34+ cells to study neural development and degeneration and infectious diseases involving human brain. We collaborated with Dr. Mark Cooksons lab in NIA to compare the endothelial cells incorporated 3D organoids with human brain datasets from the Allen Brain Atlas. The result showed the 3D organoids consist of a variety of neurons including both inhibitory and excitatory neurons, similar to the human brain. Furthermore, endothelial and microglial cells as well as oligodendrocytes were also confirmed in the new analysis. We further derived both endothelial incorporated neuronal 3D organoids and cortical brain organoids from six different human iPSC lines in order to see if endothelial cell incorporation could affect the health of organoids. We observed that while the cortical brain organoids showed significant cell stress, including growth pause and cell number loss, after 4 weeks in cultures, the 3D organoids with endothelial cells were free of stress markers beyond three months of in vitro cultures. RNA-Seq was performed on all the organoids to further compare the cell stress markers between the groups. We also purchased a new 10x Genomic controller for scRNA-Seq studies. Specific aim 2: Study the role of HERV-K on human neural development and tumorigenicity. While we continue to study the role of HERV-K on human neural development, we also helped our collaborators with professional advice and materials obtained from the study. As a result, we published a review on the topic and co-authored two publications with Dr. Iordanski and Dr. Doucet-OHare. We will continue the study on the lipid metabolism during neuronal differentiation and neuropathogenesis, collaborating with Dr. Haughey. Specific aim 3: Study the association of HERV-K and ALS. Data suggest that retro elements including HERV-K in human genome may be associated with motor neuron disorder ALS, especially in a subset of ALS with C9orf72 mutations. We have been generating motor neuron lines from PBMC samples from ALS patients with C9orf72 mutations to study the effect of DNA damage and repair processes on HERV-K activation. We have determined the best sequence for antisense oligos (ASO) and with the help of Dr. Lisa Henderson from SINS lab, designed and synthesized an ASO with a lipid tail which can easily go across plasma membranes such as an endosome, thus increasing efficacy and eliminating the need for a transfection reagent. We used this improved tool and found that C9orf72 expansions caused HERV-K Env expression in motor neurons, which was at least partially mediated by activated KU80, a critical element for DNA repair and other transcriptional functions. The inhibition of C9or72 expression or KU80 decreased HERV-K Env activation and provided protection specifically to motor neurons. We are currently using 3D motor neuronal cultures to confirm these observations. Specific aim 4: facilitate research and therapeutic developments for neurological disorders using our models and methods. We are in collaboration with other investigators by providing material support and technique trainings of the iNSC/iPSC generation and 3D modeling. We are continuously helping Dr. Henry Levin's lab with technique support, cell lines and expertise on developing a project studying the retroelements in neurological disorders using neural stem cells. We are also collaborating with Dr. In-Hong Yang in UNC and on developing a high-throughput in vitro model to study mitochondrial trafficking in motor neuron processes and a 3D model to study the magnetic stimulation effect on oligodendrocyte maturation. We collaborated with Dr. Kousa from National Childrens Hospital to study the pathogenesis of ZIKA virus on human brain development. Using the endothelial incorporated 3D brain organoids, we have successfully established a ZIKA infection model which showed the ZIKA infection caused significant toxicity in the organoids, mediated by mTOR pathways. We are now determining the genetic factors that influence ZIKA viral replication in the brain.