Role of Densin-180 in neuronal development

Abstract

Neurodevelopmental disorders (NDDs) are heritable diseases with complex symptoms. Current treatments rely on supportive drugs which lack accuracy in targeting the underlying pathophysiology. Genome-wide association studies have been performed and implicated postsynaptic proteins as one of the major contributors of NDDs. Densin-180, which is encoded by LRRC7 (Leucine-rich-repeat-containing protein 7), is a scaffold protein in the postsynaptic density. LRRC7 is associated with several NDDs including childhood emotional dysregulation and autism spectrum disorders (ASD). Our group has generated a mouse line with an insertional mutation of Lrrc7. Pilot study of mutant mice revealed anxiety-like behaviour. At the cellular level, neurons had reduced neurite length and reduced mGluR5 expression in the synaptosomal fraction. However, the role of Densin-180 in childhood emotional dysregulation and ASD remains unclear. In this study, I examined mutant mice on core symptoms of these disorders, including anxiety, impaired social function, working memory deficits and repetitive behaviour. The underlying cellular and molecular mechanisms were investigated. Based on these molecular findings, pharmacological treatment was adopted to rescue the cellular and behavioural defects. Behavioural characterization of Lrrc7 mutant mice showed that homozygous mutants followed their littermates and initiated juvenile fight before weaning stage. Their behaviour was further evaluated when they reached 7 weeks old. Investigation of aggression by the one-chamber and tube tests confirmed that homozygous mutants were not aggressive. They exhibited significant anxiety-like behaviour in open field test and light/dark box. Increased social interaction with stranger mice was evident in one-chamber and three-chamber tests. Mutant mice also had impaired working memory based on the Y-maze but not repetitive behaviour based on the marble burying test. Analysis of neuronal morphology of CA1 hippocampal neurons indicated reduced neurite branching and elongated spines in homozygous mutants. From primary hippocampal neurons isolated at embryonic day 16.5, homozygous mutant neurons exhibited delayed neuritogenesis on first 3 days and reduced neurite length and branching at 7 days in culture. Next, the role of mGluR5 in Densin-mediated neurite growth was examined using primary neuronal cultures. Addition of mGluR5 antagonist MPEP reduced neurite length and branching in wildtype but not in mutant neurons. Furthermore, enhancement of mGluR5 signalling by the positive allosteric modulator CDPPB rescued neurite defects. These data demonstrated the importance of mGluR5 in regulating neurite growth. Disruption of Densin reduced surface expression of ZmGluR5 using surface protein biotinylation assay. Also, mGluR5-mediated ERK, JNK and AKT pathways were impaired in mutant neurons. Rescue experiments of anxiety-like behaviour and hypersociability were performed by acute administration of CDPPB 30 minutes before behavioural tests. CDPPB alleviated the anxiety-like behaviour of mutant mice in open field test and hypersociability in three-chamber test. Chronic treatment of CDPPB from postnatal 16 to 22 improved growth and alleviated fighting problems of homozygous mutant mice before weaning. In conclusion, I demonstrated that disruption of Densin in mice resulted in behavioural phenotypes analogous to childhood emotional dysregulation in patients. A novel role of mGluR5 in promoting Densin-mediated neurite growth and in rescuing anxiety-like behaviour and hypersociability in Lrrc7 mutant mice was discovered.