The role of non-coding RNAs in epilepsy of tuberous sclerosis complex and focal cortical dysplasia type 2B

Wayne State University, Detroit MI

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Abstract

Cortical tubers in tuberous sclerosis complex (TSC) and focal cortical dysplasia type IIB (FCD2B) are malformations of cortical development (MCD) that share similar histopathology and are associated with a high incidence of intractable epilepsy. The molecular events that lead to seizures originating from TSC/FCD2B lesions are poorly understood, and multiple mechanisms may be involved. Mechanistic variability requires biomarkers that can stratify epileptogenic lesions by mechanistic features to enable precision pharmacological targeting. Our long-term objective is to address this need and develop improved diagnostic methods and therapeutic approaches for the treatment of intractable epilepsy in children. Mounting evidence indicates that immune and inflammatory signaling may contribute to the perpetuation of seizures arising from some lesions. Positron emission tomography (PET) imaging with the tracer alpha[C-11]methyl-L-tryptophan (AMT) has nearly 100% specificity for identifying epileptogenic lesions. Uptake of AMT is increased in lesions having neuroinflammatory activation of the kynurenine pathway (KP). Our prior results demonstrate that ?hot? lesions with elevated AMT uptake have a distinct molecular signature and innate immune activation and suggest a key event is activation of toll-like receptors 7/8 (TLR7/8). These receptors reside in endosomes of cells and sense single-stranded RNAs (ssRNA) from pathogens. However, intercellular signaling of endogenous ssRNA delivered by exosomes can activate TLR7/8. We hypothesize that non-coding RNAs in extracellular exosomes secreted from ?hot? tubers activate endosomal TLR7/8 and key downstream pathways that are known to contribute to seizure susceptibility. Importantly, drugs are currently available to inhibit the TLR7/8 pathway, so AMT-PET imaging offers a precise diagnostic marker to identify epileptogenic lesions where neuroinflammatory signaling and key downstream pathways can be disrupted. Confirmation of our hypothesis would link a clinical biomarker to a pathway for which inhibitors already exist, thus enabling a ?theranostic? approach to treating intractable epilepsy in TSC/FCD2B. We propose to test our hypothesis by integrative analyses of epilepsy surgery brain tissue specimens using quantitative proteomics, RNA-seq, in vitro functional assays, and in vivo molecular imaging. We will characterize the RNA cargo of exosomes extracted from TSC/FCD2B lesions and link it to TLR7/8 activation and AMT uptake in the source tissues. In vitro assays will measure the ability of extracted exosomes to activate TLR7/8 signaling. We will use expansion microscopy with nanoscale resolution to determine if TLR7/8 and activating RNAs are co-localized in source tissue. We expect to link AMT-PET to TLR7/8 activation by exosomal RNAs in AMT hot lesions. The proposed research is innovative because the role of TLR7/8 in epilepsy of TSC/FCD2B has not been investigated, and the outcomes are expected to propel the field in a new direction, including a new approach for precision pharmacological targeting to disrupt the ?vicious cycle? of inflammatory signaling in TSC/FCD2B epilepsy. !