Specific aim 1: Develop in vitro 3D brain organoid models derived from human adult peripheral CD34+ cells to study neural development and degeneration. Interactions with non-neuronal cells plays an important role in neural development and neurological disorders. We have established protocols to produce brain organoids with endothelial cells. We used RNA-seq data to study the genes expressed in the organoids and their relevance in studying neural developmental disorders. In collaboration with Dr. Kousa from CNHS, who have generated gene sets containing 7972 genes that associated with 52 neural development and degenerative disorders. we found 7673 (96%) genes were expressed in the 3D organoids, suggesting they can be used as model for studying these disorders. A proposal has been submitted to use this model to study the genes associated with ZIKA infection-induced defect in brain development. We also took advantage of recently published protocols that enabled microglial differentiation to make microglia incorporated brain organoids. By incorporating iPSC-derived CD34+ blood progenitor cells into the 3D organoids, following neuronal differentiation, we observed that microglia were generated within the organoids, which numbers were further increased by microglial-inducing cytokines. This model when optimized, will be useful to study microglial associated neurodegenerative disorders such as Parkinsons disease.
Specific aim 2 : Study the role of HERV-K on human neural development. We have found that human endogenous retroviruses K (HERV-K) is expressed on human iPSC. Inhibition of HERV-K Env protein enhanced neuronal differentiation through inhibiting mTOR pathway and LPCAT1 activation. It was reported that mTOR activation may be a main factor differentiating human and other primate brain developments. To see if HERV-K play a role in human brain evolution, we generated neural progenitor cells from fibroblasts of Rhesus monkey, which contains no HERV-K. By transfecting the rhesus neural cells with plasmid containing HERV-K envelope, we observed that incorporation of HERV-K envelope in the rhesus neuronal cells dramatically increased expression of rps6, a key effector of mTOR pathway, and LPCAT1, while impaired the spontaneous neuronal differentiation. Our results indicate that HERV-K incorporation may have played a role in the human brain evolution.
Specific aim 3 : Study the role of association of HERV-K and ALS. It has been implied 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. To confirm such relations and study the possible mechanism between HERV-K expression and ALS, we generated motor neuron lines from five PBMC samples from ALS patients with C9orf72 mutations. Preliminary result showed an increased expression of HERV-K in ALS motor neurons compared to cells generated from health donors. We will use this in vitro model to delineate the mechanism.
Specific aim 4 : facilitate the research and therapeutic developments for neurological disorders using our models and methods. We are in collaboration with other investigators by providing material support and training of the iNSC/iPSC generation. We are helping Dr. Henry Levis lab with technique support, cell lines and expertise on developing a project studying the retroelements in neurological disorders using neural stem cells. The project has resulted in decent result that showed certain retroelement may regulate adjacent gene expression and participate in neurological disorders. A manuscript based on the collaboration is in preparation. The collaboration with Dr. Katherine Roche and Dr. Sergey Iordanskiy also resulted in additional manuscripts.
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