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Principal Investigator: Avindra Nath
Organization: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
Fiscal Year: 2020
Award: $1,054,273
Funding agency: National Institute of Neurological Disorders and Stroke
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 continued to characterize the 3D brain organoids with multiple cell types including neurons, glial cells and endothelial cells. Use single-cell RNA-Seq analysis and optimized 3D clearance techniques accompanying immunostaining, we were able to determine cell types within the 3D organoids in a single cell level. We confirmed the 3D organoids generated using our in-house protocol consist of our targeting cells including neurons, astroglia, microglia and endothelial cells, besides stem cells. Furthermore, the long-term culture (15 weeks) produced organoids with endothelial cells building blood vessel-like structures. These results suggest that we successfully generated a human brain organoid model similar to human developing brains. We then used this model to study the expression of genes important for COVID-19. COVID-19 is the ongoing pandemic caused by a novel corona virus SARS-CoV-2. SARS-CoV-2 infects human cells through receptor ACE2 with the help of an enzyme TMPRESS2, which processes the spike protein of SARS-CoV-2 to make it suitable to bind ACE2 on the human cell surfaces. Using our 3D brain organoids, we found that there were few (< 1%) neurons and astroglia cells express ACE2 and TMPRESS2. The result is in agreement with the published data (Allen brain Atlas) which shows very low levels of ACE2 and TMPRESS2 expressions in human brains. These results suggest that human brain is not a suitable target for SARS-CoV-2 direct infection. The brain symptoms of COVID-19 could be either a reaction to SARS-CoV-2 induced inflammation or by brain infection mediated by other pathways. The 3D brain organoids could be a suitable model to study COVID-19 related brain dysfunction. A manuscript is in preparation on this topic.
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 iPSCs. Inhibition of HERV-K Env protein enhanced neuronal differentiation through inhibiting mTOR pathway and LPCAT1 activation. We further confirmed the importance of HERV-K Env on human brain evolution by study forced expression of HERV-K Env on Rhesus neural progenitor cells, which showed increased activation of mTOR pathway, and LPCAT1. The results have been published in PNAS. As phospholipid metabolism has been associated to energy metabolism and disease progression. We further studied the effect of LPCAT1 on regulations of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), two most abundant phospholipids in mammalian cell membranes. We studied the neuronal induction on the changes of profiles of lipids in iPSCs and whether inhibition of LPCAT1 using inhibitory siRNA could change the profiles in a similar way. Our preliminary results indicate a trend change of lipid profiling (PE/PC) similar to the effect of LPCAT1 inhibition during neuronal differentiation. We are recruiting more accurate techniques to help determine the precise mechanism of the regulation. As lipid metabolism is understudied in neurodegenerative disorders, we are working to fill this gap and provide possible novel targets for disease intervention.
Specific aim 3: Study the 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. 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 antisense oligos (ASO) that efficiently decrease C9orf72 expression and is using this as a tool to study the effect of C9orf72 on HERV-K activation and subsequent neurotoxicity.
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 technique trainings of the iNSC/iPSC generation. We are continuously 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 a couple of meeting abstracts and posters and a manuscript is in preparation. We also provided ALS cell lines to Dr. In-Hong Yang in UNC and in close collaboration on developing a high-throughput in vitro model to study mitochondrial trafficking in motor neuron processes. Other collaborations including the study on the mechanism of African trypanosomes induced neuronal toxicity with Dr. Dennis Grab. We continually support Dr. Katherine Roche to study the function of NLGN4 in autism which have resulted in a new publication in journal Neuron.
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