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Figure 5 Neurite outgrowth modulated by transfection with miR34b-5p in mouse hippocampal neurons (a,b) Representative images of mouse hippocampal dissociated neurons co-transfected with GFP vector and mimics for miR34b-5p (miR34b) or NC-1 (negative control; Scr); Scale bar, 100 μm. (c) Graphical representation of neurite outgrowth analysis using the NeuroMath soft- ware showing total length of neurites, total number of neurites, cell area, longest neurite and to- tal number of branches. Student’s t-test: *p < 0.05; n = 3 experiments, 3 independent transfections each experiment.
Heightened expression of genes involved in innate and adaptive immune path- ways, such as complement system, TREM1 signaling and CD28 signaling was an overarch- ing feature of the TSC cortical tuber protein coding transcriptome. These data extend on our previous study, a genome wide microarray analysis of a small cohort of TSC cortical tubers and four controls demonstrating up-regulation of genes related to the inflammatory response, including complement factors, chemokines and several cyto- kines14. Others have also shown differential inflammatory gene expression in TSC corti- cal tubers; albeit by targeted RT-qPCR and immunocytochemistry14, 52, 53, indicating that our data on mRNA expression changes in TSC cortical tubers were consistent across different sample cohorts and studies. The presence of biologically active fragments of C3 (C3c and C3d); and C4 (C4b) in TSC cortical tubers indicates that the activation of the complement cascade may be a key driver of the inflammatory response consistently observed in the TSC brain14, 52, 53. The complement cascade has been implicated in sei- zure generation and progression in numerous experimental epilepsy models54-58, human temporal lobe epilepsy59, and in epileptogenic glioneuronal tumors60. Also, our data sug- gest a role for TREM-1 signaling in TSC. TREM-1 can amplify Toll like receptor signaling, thereby increasing the production of a wide range of cytokines and chemokines61, 62. Interestingly, experimental studies support the role of these pathways in seizures 57, 63,