Hypoxia-regulated MicroRNA-210 in neuronal plasticity

Abstract

In recent years increasing evidence has established microRNAs as critical regulators of neuronal development and plasticity that can act locally, in an activity-dependent manner. The hypoxia-regulated microRNA-210 (miR-210) is a highly conserved miRNA, widely studied for its role in regulating cellular response to low oxygen conditions. Previously, our lab identified miR-210 to be involved in honeybee learning and memory using olfactory conditioning indicating it may have a role in neuronal function as well. Within the nervous system, miR-210 dysregulation has also been associated with epilepsy, being significantly upregulated in rodent temporal lobe epilepsy models and with Alzheimer’s disease (AD), being significantly downregulated in brain tissue and cerebrospinal fluid of AD patients. This project aimed to further investigate the role of miR-210 in mammalian neuronal plasticity both in vitro and in vivo using various molecular, cellular and behavioural techniques to identify neuronal regulatory targets of miR-210, determine neuronal expression and induction of miR-210 and functionally characterise the effects of miR-210 dysregulation. Using a biotin pull-down approach in the human-derived SH-SY5Y neuroblastoma cell line, 620 unique target genes of miR-210 were experimentally identified by RNAseq. Among these targets there was a significant enrichment of neurodegenerative KEGG pathways including Huntington’s, Alzheimer’s and Parkinson’s disease as well as a number of specific genes known to be regulated by neuronal activity and with neural-plasticity function. Using dual-luciferase assay validation, miR-210 was shown to directly interact with and down-regulate a number of oxidative phosphorylation genes associated with neurodegeneration (ATP5G2, ATP5D, COX8A, COX6A1, NDUFS7, NDUFS8, NDUFA4L2, CYC1), as well as MAPK/VEGF signalling genes (EIF4EBP1, MAP2K2, VEGFB) and genes with a known role in synaptic function or plasticity (GRINA, AP2S1, TMUB1, ATP6V0C, ACTB). Differentiated SH-SY5Y cells were also used as a human neuronal-like model to examine effects of miR-210 dysregulation and measures related to cellular metabolism, including generation of reactive oxygen species and mitochondrial membrane potential, were found to be altered. To further elucidate the regulation and role of miR-210 in endogenous neuronal systems, an in vitro neuronal model was established from primary mouse hippocampal cultures and a conditional neuronal knockout mouse line (miR-210-/-;Nestin-Cre) was generated. In wild-type hippocampal cultures quantification of miR-210 following (K+)-induced activity found that miR-210 expression was induced in response to neuronal activation. Quantification in vivo also found miR-210 displayed expression relevant to learning and memory, being significantly increased within the mouse hippocampus compared to other brain regions. Analysis of hippocampal neurons cultured from miR-210-/-;Nestin-Cre mice and littermate controls (miR-210loxP/loxP) found miR-210 knockout also disrupted neuronal metabolism and altered dendritic morphology in vitro. To assess cognitive performance of mice, visual discrimination and reversal learning tasks were conducted using touchscreen operant conditioning and miR-210-/-;Nestin-Cre mice found to display reduced perseverative behaviour and increased motivation during reversal learning. This data provides the first characterisation of mammalian miR-210 neuronal function both in vitro and in vivo and has highlighted novel targeting pathways of miR-210 as well as potential functional roles of miR-210 in neuronal plasticity. Identifying the molecular links between neuronal activation, altered metabolism and plasticity is critical to understanding how dysregulated metabolism may lead to cognitive deficits in neurodegenerative conditions and characterising genes such as miR-210, and other sensors of metabolism, provides insight into mechanisms translating environmental signal into neural response.