Rab and Arl guanine nucleotide‐binding (G) proteins regulate trafficking pathways essential for the formation, function and composition of primary cilia, which are sensory devices associated with Sonic hedgehog (Shh) signalling and ciliopathies. Here, using mammalian cells and zebrafish, we uncover ciliary functions for Rab35, a multitasking G protein with endocytic recycling, actin remodelling and cytokinesis roles. Rab35 loss via siRNAs, morpholinos or knockout reduces cilium length in mammalian cells and the zebrafish left‐right organiser (Kupffer's vesicle) and causes motile cilia‐associated left‐right asymmetry defects. Consistent with these observations, GFP‐Rab35 localises to cilia, as do GEF (DENND1B) and GAP (TBC1D10A) Rab35 regulators, which also regulate ciliary length and Rab35 ciliary localisation. Mammalian Rab35 also controls the ciliary membrane levels of Shh signalling regulators, promoting ciliary targeting of Smoothened, limiting ciliary accumulation of Arl13b and the inositol polyphosphate 5‐phosphatase (INPP5E). Rab35 additionally regulates ciliary PI(4,5)P2 levels and interacts with Arl13b. Together, our findings demonstrate roles for Rab35 in regulating cilium length, function and membrane composition and implicate Rab35 in pathways controlling the ciliary levels of Shh signal regulators.
Donnai-Barrow syndrome, a genetic disorder associated to LRP2 (low density lipoprotein receptor 2/megalin) mutations, is characterized by unexplained neurological symptoms and intellectual deficits. Megalin is a multifunctional endocytic clearance cell-surface receptor, mostly described in epithelial cells. This receptor is also expressed in the CNS, mainly in neurons, being involved in neurite outgrowth and neuroprotective mechanisms. Yet, the mechanisms involved in the regulation of megalin in the CNS are poorly understood. Using transthyretin knockout mice, a megalin ligand, we found that transthyretin positively regulates neuronal megalin levels in different CNS areas, particularly in the hippocampus. Transthyretin is even able to rescue megalin downregulation in transthyretin knockout hippocampal neuronal cultures, in a positive feedback mechanism via megalin. Importantly, transthyretin activates a regulated intracellular proteolysis mechanism of neuronal megalin, producing an intracellular domain, which is translocated to the nucleus, unveiling megalin C-terminal as a potential transcription factor, able to regulate gene expression. We unveil that neuronal megalin reduction affects physiological neuronal activity, leading to decreased neurite number, length and branching, and increasing neuronal susceptibility to a toxic insult. Finally, we unravel a new unexpected role of megalin in synaptic plasticity, by promoting the formation and maturation of dendritic spines, and contributing for the establishment of active synapses, both in in vitro and in vivo hippocampal neurons. Moreover, these structural and synaptic roles of megalin impact on learning and memory mechanisms, since megalin heterozygous mice show hippocampal-related memory and learning deficits in several behavior tests. Altogether, we unveil a complete novel role of megalin in the physiological neuronal activity, mainly in synaptic plasticity with impact in learning and memory. Importantly, we contribute to disclose the molecular mechanisms underlying the cognitive and intellectual disabilities related to megalin gene pathologies.
Transthyretin (TTR) has intrinsic neurotrophic physiological activities independent from its thyroxine ligands, which involve activation of signaling pathways through interaction with megalin. Still, the megalin binding motif on TTR is unknown. Nanobodies (Nb) have the ability to bind “hard to reach” epitopes being useful tools for protein/structure function. In this work, we characterize two anti-TTR Nanobodies, with similar mouse TTR binding affinities, although only one is able to block its neuritogenic activity (169F7_Nb). Through epitope mapping, we identified amino acids 14–18, at the entrance of the TTR central channel, to be important for interaction with megalin, and a stable TTR K15N mutant in that region was constructed. The TTR K15N mutant lacks neuritogenic activity, indicating that K15 is critical for TTR neuritogenic activity. Thus, we identify the putative binding site for megalin and describe two Nanobodies that will allow research and clarification of TTR physiological properties, regarding its neurotrophic effects.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.