Herpes simplex virus 1 (HSV-1) encephalitis (HSE) is the most common form of sporadic viral encephalitis in Western countries. We discovered that forebrain HSE (via olfactory neurons) in otherwise healthy children can result from inborn errors of the TLR3 pathway (TLR3, UNC93B1, TRIF, TRAF3, TBK1, and IRF3), whereas brainstem HSE (via trigeminal (TG) neurons) can result from inborn errors of RNA lariat metabolism due to bi-allelic mutations in DBR1. We also found that children with a broader defect of impaired production of (mutations in NEMO) or response to IFN-α/β and IFN-λ (STAT1) are prone to HSE and other infections. Thanks to NIH R01NS072381, we analyzed the cellular basis of HSE using induced pluripotent stem cell (iPSC)-based peripheral and central nervous system (CNS) cell differentiation technology. We first showed that forebrain HSE in TLR3 pathway-deficient patients results from impaired anti-HSV1 intrinsic (i.e. non-hematopoietic) immunity in forebrain cortical neurons and pro-oligodendrocytes, whereas astrocytes and neural stem cells were not affected by TLR3 deficiency (microglial cells were not tested). More recently, we showed that iPSC-derived trigeminal (TG) neurons do not rely on TLR3 to control HSV1 (olfactory neurons were not tested). These data indicated that childhood HSE results from inborn errors of non-hematopoietic, CNS-specific, cell-intrinsic immunity, affecting cortical neurons and oligodendrocytes in particular. We have since identified new forebrain HSE-causing genes that are connected to the canonical TLR3-IFN circuit (MEX3B, IFNAR1), to the non-canonical TLR3-necroptosis pathway (RIPK3), or that underlie HSE via other mechanisms (SNORA31, TMEFF1). The goal of this renewal application is to dissect in greater breadth and depth the CNS-specific and -intrinsic pathogenesis of HSE. We will test the hypotheses that (1) the new genetic etiologies of forebrain HSE impair intrinsic immunity in CNS but not olfactory neurons, (2) DBR1 deficiency impairs intrinsic immunity in brainstem and/or TG neurons, (3) the interaction between different CNS cell types during HSV-1 infection also contributes to the HSE pathogenesis. We will first devise novel protocols to efficiently differentiate iPSCs into olfactory and brainstem neurons and microglial cells, and study the TLR3 responses and HSV-1 infection; second, we will assess the responses of iPSC-derived cortical and olfactory neurons, and microglia cells, from patients carrying mutations in new forebrain HSE-causing genes, to stimulation via TLR3, MAVS, STING, IFN-α/β, or IFN-λ, and by HSV-1; we will also study pre-mRNA processing in DBR1-mutated brainstem and TG neurons, with and without HSV-1 infection; third, we will analyze HSV-1 infection in iPSC-derived cortical, olfactory, brainstem or TG neurons. To demonstrate the disease-causing role of these genetic defects, we will also test isogenic iPSC lines in which the mutation has been corrected or introduced by CRISPR-Cas9 gene editing. All technologies needed have been successfully set up in a synergistic manner across our four laboratories. Exciting preliminary data have been obtained, including (1) novel genetic etiologies of childhood HSE (mutations in MEX3B, IFNAR1, RIPK3, SNORA31, TMEFF1), (2) novel protocols to differentiate olfactory, brainstem, and microglial cells. The pursuit of this human iPSC-based study will enable us to dissect in greater breadth and depth the cellular basis of HSE in children with inborn errors of CNS-intrinsic immunity to HSV-1. This path-breaking collaborative study has far-reac
|Effective start/end date||6/1/20 → 3/31/21|
- Rockefeller University (SUB00000183 AMD 2 // 5R01NS072381-11)
- National Institute of Neurological Disorders and Stroke (SUB00000183 AMD 2 // 5R01NS072381-11)
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