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observed. This suggests that an overexpression of miR34b-5p in astrocytes could acti- vate an inflammatory response in astrocytes.
miR34b modulates neurite outgrowth in mouse hippocampal neuronal cultures
To further explore the capacity of miR34 family members to modulate neuronal char- acteristics, as predicted by our miRNA-to-module interaction framework, we selected miR34b for further functional studies in vitro. In particular we investigated the impact of miR34b overexpression on neurite outgrowth in a mouse hippocampal neuron model. Primary mouse neuronal cultures prepared from the hippocampi of postnatal day 0 (P0) C57Bl/6 mice were transfected at 1 day in vitro with either the miR34b-5p mimic or the miRNA mimic negative control (Scr) and analyzed for neurite outgrowth at 4 days in vitro. Neurons transfected with miR34b-5p mimic showed an increased number of lon- ger neurites as compared to Scr transfected cultures (Fig. 5a,b). Quantification using the NeuroMath software showed a significant increase in the total length, number of neur- ites and an increase in soma size (Fig. 5c) of the miR34b-5p mimic transfected neurons compared to Scr transfected cultures.
Discussion
In this study we report a comprehensive analysis of the brain transcriptome in TSC patients relative to control subjects. Evaluation of both the protein coding and small non-coding RNA by RNA-seq revealed substantial alterations in TSC brain tissue. In par- ticular, TSC cortical tubers had elevated expression of genes involved in innate immune pathways, such as complement system, concomitant with decreased expression of genes predominantly associated with neuronal cellular pathways, including neurogenesis and glutamate receptor signaling. A variety of small non-coding RNA transcripts, includ- ing miRNAs, were also significantly altered in TSC cortical tubers relative to controls. Application of a gene co-expression network approach and miRNA-to-target predictions allowed for the identification of functional modules of the brain transcriptome poten- tially modulated by miRNAs. Based on these predictions miR34 family members, notably miR34b, were assessed by functional in vitro studies and shown to possess a capacity to modulate neurite outgrowth and to activate an inflammatory response in astrocytes.
Figure 4 In situ hybridization of miR34a-5p and miR34b-5p in Tuberous Sclerosis Complex (TSC) cortical tubers. Panels a-d: miR34a-5p. Control cortex (a) shows moderate expression of mi- R34a-5p in few neuronal cells (arrows); not detectable expression is observed in control white matter (c). Panels b and d (TSC) show strong expression of miR34a-5p within the dysplastic re- gion with several positive dysmorphic neurons (arrows in b) and glial cells (arrowheads in b,d); insert in b: miR34b-5p in a NeuN positive cell; insert in d shows colocalization with GFAP. Expres- sion of miR34a-5p is also detected in giant cells within the tuber white matter (arrows in d). Panels e-h: miR34b–5p. Control cortex (e) shows moderate expression of miR34b-5p in neuronal cells (arrows); very low expression is observed in control white matter (g). Panels f and h (TSC) show expression of miR34b-5p within the dysplastic region with several positive dysmorphic neurons [arrows; insert in f: miR34b-5p in a NeuN positive cell] and glial cells [arrowheads in f and insert (a) in h; insert (b) in h shows colocalization with GFAP]; arrow in h shows a positive giant cell within the tuber white matter. Scale bar in a: (a–g), 80 μm; (h), 40 μm. [arrows in h and insert (a) in h, white matter]. Scale bar in a: (a,e,f): 160 μm; (b–d,g,h) 80 μm.