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CODING AND SMALL NON-CODING TRANSCRIPTIONAL LANDSCAPE OF TSC
64, suggesting that drugs targeting the TREM-1 signaling may have potential therapeutic benefit for seizures in TSC. Evaluation of fetal TSC brain revealed an activation of the innate and adaptive immune response early during brain development65 suggesting that the induction of this pathway could be intrinsic to the TSC developmental pathology and linked to the deregulation of the mTOR pathway66-68. Accordingly, a recent study shows over-activation of pro-inflammatory signaling pathways in astrocytes before epi- lepsy onset in a mouse model of TSC, pointing to the role of mTOR-mediated inflamma- tory mechanisms in TSC69. Amongst the significantly over-expressed genes in TSC sub- jects there was enrichment for microglial and astrocyte specific genes. Both microglia and astrocytes are involved in inflammation driven processes throughout the brain70, 71, suggesting that the TSC inflammatory processes may be driven by activation of microg- lia and astrocytes. Understanding whether changes in innate and/or adaptive immune gene expression represent the cause or the consequence of chronic pharmacoresistant seizure activity is extremely difficult since epilepsy surgery targeting cortical tubers or autopsy is performed several months or years after the start of seizures. Moreover, a comparison with non-epileptogenic tubers could not be performed, since this tissue is not resected during epilepsy surgery and infrequently available at autopsy. Nevertheless these findings provide further evidence for the activation of both the innate and adap- tive immune response cascades in the pathophysiology of TSC.
Besides the protein coding transcriptome we also mapped the small non-cod- ing RNA in TSC brain tissue relative to controls. We found that not only miRNA, exten- sively investigated as epigenetic effector molecules, were significantly altered in the TSC brain transcriptome but also other species of small RNA, notably snoRNAs, snRNAs and scaRNAs. These small RNA species represent an ancient group of non-coding RNA mol- ecules, which in particular to snoRNA (including the H/ACA box and C/D box categories) may spread throughout the genome by retrotransposition, representing a new family of mobile genetic elements 72, 73. These recently discovered small RNA have been shown to modify the chemical properties of ribosomal RNAs and transfer RNAs, impacting on mRNA translation, RNA silencing, alternative splicing74-76, as well as methylation and pseudouridylation of spliceosomal RNAs in the Cajal bodies of the nucleus77, 78. Notably, the disruption of snoRNA expression and/or function has been implicated in neurodevel- opmental disorders such as Prader-Willi syndrome and autism spectrum disorders79, 80. These as yet under-explored classes of small non-coding RNA certainly warrant further investigation in TSC pathogenesis.
Through the use of an unsupervised systems-based computational technique, that is gene co-expression network construction, we identified multiple modules of tightly inter-correlating protein coding genes. This approach allowed for the construc- tion of a holistic transcriptome model, thus not only those functions biased towards the response in TSC relative to control subjects (supervised analysis). This aspect is supported by the finding of modules enriched for genes involved in neuronal attuned biological functions, such as neurogenesis and glutamate receptor signaling, which would otherwise have been missed. Moreover, WGCNA has emerged as an important tool in integrative bioinformatics/genomics81. We reasoned that since miRNA are known to target and influence the expression of multiple genes involved in shared and dis- tinct cellular processes, a modular approach may uncover functional units of distinct
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