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biological functions in the brain transcriptome potentially modulated by miRNA, rather than considering single transcripts. In so doing, we uncovered the miR34 family (miR34a, miR34b and miR34c) as predicted modifiers of neurogenesis and glutamate receptor signaling transcriptional output. These predictions were further tested experimentally using a miR34b-5p overexpression system in mouse hippocampal neurons, demonstrat- ing that miR34b-5p can modulate neurite outgrowth. Reduced neurogenesis during chronic epilepsy could result in loss-of-function deficits and contribute to common TSC comorbidities such cognitive impairment or depression 82, 83. Also, previous findings have demonstrated abnormal accumulation of extracellular glutamate occurring in human epileptogenic tissue, which is hypothesized as a key factor in recurrent seizures and neu- ronal death84. Mouse models have also shown that controlling seizures by antiepileptic drugs (AEDs) may act via alterations in brain glutamate dehydrogenase activity as well as the brain transcriptome including miRNA85, 86. Both miR34a and miR34b have been pre- viously identified as overexpressed miRNAs in tubers15, as well as key tumor suppressors downstream of the p53 pathway and have been suggested as potential targets of therapy in several cancers.87, 88 Members of the miR34 family have also been shown to regulate key pathways in neurodevelopment and cortical neurogenesis, such as the Notch89-91 and the Wnt signaling pathway92, 93. More specifically, it appears that miR34a plays a role in neuronal differentiation94-99, where overexpression of miR34a in mouse neural stem cells impairs both neuronal differentiation and synapse function96-100. Moreover, a recent study has demonstrated targeting of the TSC1 3′ UTR by miR34a, supporting the role of this miRNA in tuber pathology 15. In this particular study epileptogenic tubers were compared to adjacent non-tuber tissue indicating that elevated expression of miR34 members may indeed represent an important feature of tuber physiology rather than an effect of AED treatment.
Our study has limitations. TSC patients received individually tailored AED regi- mens contrary to autopsy control subjects. Moreover, AEDs were given in multiple com- binations, which precludes proper evaluation of the effects of AEDs on gene expression alterations in TSC tubers.
In conclusion, our study provides a comprehensive analysis of the coding and small non-coding transcriptional landscape of TSC cortical tubers. The TSC transcrip- tomic network reflects the prominent activation of both innate and adaptive immune response characteristic of cortical tubers, with identification of key pathways such as complement system, TREM1 and CD28 signaling. Notably, the under-expressed genes were linked to neurogenesis and glutamate receptor signaling. We identified a variety of small RNA molecules, including miRNA, snoRNA, snRNA and scaRNA, differentially expressed in TSC. Moreover, our study predicts an important role for the miR34 family (miR34a, miR34b and miR34c) as modifiers of neurogenesis and glutamate receptor sig- naling in TSC, which may potentially provide an epigenetic-driven therapeutic tool for epilepsy and cognitive disabilities in TSC.
Acknowledgements
This work was supported by European Union’s Seventh Framework Programme (FP7/2007-2013) under the grant agreement no. 602391 (project acronym EPISTOP; AI, JvS, AM, EA, FI, KK, SK, AJ, LL, PC, DK); Epilepsiefonds (WAR 15-05; RJP); European Union’s