The mammalian brain expresses an extraordinary diversity of long non-coding RNAs (lncRNAs), many of which exhibit region- and cell-type-specific expression patterns and are enriched in subnuclear or cytoplasmic domains. These lncRNAs are increasingly recognised as key regulators of brain development and cognition, yet the functions of most remain unexplored. We examined the subcellular localisation of hippocampal and cerebellum enriched lncRNAs in mouse brain using RNAscope Multiplex Fluorescent in situ hybridisation and super-resolution microscopy. LncRNAs displayed unique expression patterns across CNS regions, with the majority found in both nuclear and cytoplasmic compartments. Two lncRNAs, Gomafu and Oip5os1, are localised to dendrites and axons, suggesting synaptic functions, while the expression profiles of two others, Rodin and Dory, were highly enriched in the hippocampus, a key brain structure involved in learning and memory. Rodin was almost exclusively expressed in excitatory and inhibitory neurons in the dentate gyrus, while Dory is enriched in CA2 and dentate gyrus subregions and expressed in excitatory, inhibitory and immature neurons.
We generated CRISPR-Cas9-mediated knockout (KO) mice for 6 lncRNAs and performed a battery of cognitive and motor behavioural assays. Five of the 6 lncRNAs displayed cognitive and/or motor phenotypes. Rodin KO mice exhibited deficits in hippocampus-dependent memory consolidation and motor learning deficits. Molecular profiling of KO hippocampus revealed dysregulation of genes and proteins implicated in neurodevelopmental and behavioural disorders. Dory KO mice displayed impaired spatial memory and motor coordination in females but not males. RNA-seq and mass spectrometry revealed divergent molecular responses between sexes, suggesting a role for Dory in sex-specific neural circuitry.
In confirmatory studies, we are using gapmer antisense oligonucleotides to knock down Rodin and Dory in multiple experimental models. In vitro experiments using mouse hippocampal neuron cultures, human neuronal cultures, and human cerebral organoids, will examine knockdown effects on neuronal differentiation, conductance patterns, and dendritic and synapse density. In vivo knockdown in rat hippocampus will be followed by behavioural assessments similar to KO mice but including contextual fear conditioning. Our findings highlight brain-expressed lncRNAs as key regulators of cognitive and motor behaviours, with their spatial and subcellular localisation offering important clues to their function. By integrating localisation, knockout and knockdown studies with behavioural and molecular profiling, we establish a framework to uncover how lncRNAs contribute to neural circuit function and complex behaviours, and potential roles in brain disorders.