Cerebral Semaphorin3D is a novel risk factor for age-associated cognitive impairment.

BACKGROUND: We previously reported that miR-195 exerts neuroprotection by inhibiting Sema3A and cerebral miR-195 levels decreased with age, both of which urged us to explore the role of miR-195 and miR-195-regulated Sema3 family members in age-associated dementia. METHODS: miR-195a KO mice were used to assess the effect of miR-195 on aging and cognitive functions. Sema3D was predicted as a miR-195 target by TargetScan and then verified by luciferase reporter assay, while effects of Sema3D and miR-195 on neural senescence were assessed by beta-galactosidase and dendritic spine density. Cerebral Sema3D was over-expressed by lentivirus and suppressed by si-RNA, and effects of over-expression of Sema3D and knockdown of miR-195 on cognitive functions were assessed by Morris Water Maze, Y-maze, and open field test. The effect of Sema3D on lifespan was assessed in Drosophila. Sema3D inhibitor was developed using homology modeling and virtual screening. One-way and two-way repeated measures ANOVA were applied to assess longitudinal data on mouse cognitive tests. RESULTS: Cognitive impairment and reduced density of dendritic spine were observed in miR-195a knockout mice. Sema3D was identified to be a direct target of miR-195 and a possible contributor to age-associated neurodegeneration as Sema3D levels showed age-dependent increase in rodent brains. Injection of Sema3D-expressing lentivirus caused significant memory deficits while silencing hippocampal Sema3D improved cognition. Repeated injections of Sema3D-expressing lentivirus to elevate cerebral Sema3D for 10 weeks revealed a time-dependent decline of working memory. More importantly, analysis of the data on the Gene Expression Omnibus database showed that Sema3D levels were significantly higher in dementia patients than normal controls (p < 0.001). Over-expression of homolog Sema3D gene in the nervous system of Drosophila reduced locomotor activity and lifespan by 25%. Mechanistically, Sema3D might reduce stemness and number of neural stem cells and potentially disrupt neuronal autophagy. Rapamycin restored density of dendritic spines in the hippocampus from mice injected with Sema3D lentivirus. Our novel small molecule increased viability of Sema3D-treated neurons and might improve autophagy efficiency, which suggested Sema3D could be a potential drug target. Video Abstract CONCLUSION: Our results highlight the importance of Sema3D in age-associated dementia. Sema3D could be a novel drug target for dementia treatment.

MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development.

Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins.

Transgenic microRNA inhibition with spatiotemporal specificity in intact organisms.

MicroRNAs are important regulators of gene expression, yet the functional outputs of most microRNA-target interactions remain elusive. Here we introduce the Drosophila melanogaster microRNA sponge (miR-SP) as a powerful transgenic technology to dissect the function of every microRNA with precise spatiotemporal resolution. miR-SPs can be used to characterize tissue-specific microRNA loss-of-function phenotypes, define the spatial regulation of their effectors and uncover interactions between microRNAs and other genes. Using themiR-SP system, we identified an essential role of the conserved microRNA miR-8, in neuromuscular junction formation. Tissue-specific silencing revealed that postsynaptic activity of miR-8 is important for normal neuromuscular junction morphogenesis. Given that miR-SPs rely on a bipartite modular expression system, they could be used to elucidate the endogenous function of microRNAs in any species in which conditional expression can be achieved.

Synapse specificity: Wnts keep motor axons on target.

New studies on the molecular logic of synapse specificity in the fly and worm have brought neurobiologists back to an ancient family of morphogens best known for establishing pattern in the early embryonic nervous system.

Regulation of the Ca2+/CaM-responsive pool of CaMKII by scaffold-dependent autophosphorylation.

CaMKII is critical for structural and functional plasticity. Here we show that Camguk (Cmg), the Drosophila homolog of CASK/Lin-2, associates in an ATP-regulated manner with CaMKII to catalyze formation of a pool of calcium-insensitive CaMKII. In the presence of Ca(2+)/CaM, CaMKII complexed to Cmg can autophosphorylate at T287 and become constitutively active. In the absence of Ca(2+)/CaM, ATP hydrolysis results in phosphorylation of T306 and inactivation of CaMKII. Cmg coexpression suppresses CaMKII activity in transfected cells, and the level of Cmg expression in Drosophila modulates postsynaptic T306 phosphorylation. These results suggest that Cmg, in the presence of Ca(2+)/CaM, can provide a localized source of active kinase. When Ca(2+)/CaM or synaptic activity is low, Cmg promotes inactivating autophosphorylation, producing CaMKII that requires phosphatase to reactivate. This interaction provides a mechanism by which the active postsynaptic pool of CaMKII can be controlled locally to differentiate active and inactive synapses.

CaMKII, an enzyme on the move: regulation of temporospatial localization.

Calcium-calmodulin-dependent protein kinase II (CaMKII) is an important regulator of neuronal and behavioral plasticity. Studies in which the subcellular distribution of CaMKII has been altered argue that targeting of this enzyme to specific subcellular compartments is crucial to many of its roles. Understanding how a very abundant enzyme can achieve specificity of action over time and space requires an understanding of the functional diversity of the enzyme and its distribution. In this review we will discuss how structurally distinct isozymes, splice isoforms, and autophosphorylation states of CaMKII can affect kinase activity and localization. We will focus on the fast activity-dependent synaptic localization of the kinase and its association with postsynaptic proteins. The ability of enzyme activation to regulate protein-protein interactions with these binding partners and the potential for such binding interactions to regulate CaMKII activity in novel ways may represent new paradigm for CaMKII regulation.