Рождественские встречи нейробиологов – 2018

Learning and memory formation in the brain involve adaptive, multi-level nerve cell morphogenesis pertinent to changes in neural network connectivity. Although serotonin receptors provide an important signaling pathways for such morphogenesis, the underlying molecular mechanisms remain poorly understood.
Recently we deciphered the role of two serotonin receptors, 5-HT4 and 5-HT7, in this process. Among other, we linked morphological changes in dendritic spines and extracellular matrix (ECM) remodeling by uncovering the signaling pathway involving the serotonin 5-HT7 receptors the matrix metalloproteinase-9 (MMP-9), the hyaluronan receptor CD44, and the small GTPase Cdc42. We highlight a physical interaction between 5-HT7R and CD44 (identified as a novel MMP 9 substrate in neurons) on the nanoscale, and find that 5-HT7R stimulation increases local MMP 9 activity triggering dendritic spines remodeling, synaptic pruning and impairment of long-term potentiation (LTP).
In addition, we found that activation of serotonin receptor 5-HT4 (5-HT4R) stimulated small GTPase RhoA, resulting in actin reorganization. In hippocampal neurons, stimulation of 5-HT4R triggered a prominent increase in the head width of dendritic spines. Time-lapse FRET monitoring of individual spines revealed 5-HT4R-activated dynamic patterns of RhoA, which lead to the accumulation of filamentous actin followed by spine enlargement. The 5-HT4R-mediated boost in dendritic spine maturation enhanced spontaneous neuronal activity and increased the LTP expression level while reducing the amplitude of evoked multi-synaptic currents which might reflect homeostatic changes in the circuitry.
The dynamic reorganization of neuronal morphology mediated by the 5-HT4 and 5-HT7 receptors emerges therefore as a novel molecular pathway underpinning memory trace formation in the brain.

Numerous genetic mutations in ion channels are associated with severe diseases, including heart arrhythmias and epilepsies. Exploring mechanisms by which such mutations cause ion channel dysfunctions is important for medicine and general physiology. We used cryo-EM and X-ray structures of sodium and calcium channels and computations with Monte Carlo energy minimizations to model channel Cav1.2 in the inactivated, open and closed states. The models suggest mechanisms by which voltage-dependent inactivation its deceleration by several mutations associated with Timothy syndrome, a rare heart disease.
Various cationic peptide toxins block the outer pore of cationic channels. Paradoxically, Cav1.2 is also blocked by glacontryphan-M, an anionic peptide. Computations predict that the toxin's γ-carboxyglutamates and the selectivity-filter carboxylates are bridged by two calcium ions, which repel incoming calcium ions. Thus, the pore of cationic channels can be blocked by permeant ions bound to either small molecule blockers or to anionic toxins.
Batrachotoxin (BTX) is known to bind in the inner of pore eukaryotic sodium channels, but allow permeant ions to move through the pore. Recently, BTX was shown to activate prokaryotic sodium channels. Common structural features of pro- and eukaryotic sodium channels allow BTX to work like a surgical stent in a blood vessel.

Трех-петельные α-нейротоксины змей, к которым принадлежит классический блокатор никотиновых холинорецепторов (нХР) α-бунгаротоксин, широко используются для исследования нХР. Однако, существует потребность в холинергических лигандах, обладающих иными фармакологическими свойствами. В результате поиска таких соединений в ядах змей нами были обнаружены новые пептидные и белковые нейротоксины. Некоторые из этих соединений являются структурными аналогами трехпетельных токсинов, иные принадлежат к другим семействам змеиных токсинов, или же представляют собой совершенно новые соединения. В частности, нами были обнаружены димеры трехпетельных токсинов, отличающиеся по специфичности взаимодействия от исходного кобратоксина. Из ядов гадюк выделены пептидные нейротоксины, взаимодействующие с ортостеричекими и аллостерическими сайтами нХР. Показано, что фосфолипазы А2 способны блокировать нХР. Обнаруженные соединения существенно расширяют набор лигандов нХР.

К настоящему времени открыто большое количество соединений пептидной природы, эффективно взаимодействующих с никотиновыми холинорецепторами (нХР), и содержащих множественные положительно заряженные аминокислотные остатки. Среди них – компоненты ядов змей (ваглерины, аземиопсин) и морских моллюсков (различные конотоксины). Более того наш опыт работы с последними показал, что введение в структуру природных конотоксинов дополнительного положительного заряда во многих случаях приводит к увеличению сродства такого мутанта к определенным подтипам нХР. Для лучшего понимания природы этого явления мы осуществили конструирование и синтез серии олигоаргининов и изучили их способность взаимодействовать с рядом различных холинорецепторов. При этом была выявлена корреляция между длиной олигоаргининов и их холинергической активностью, и при этом максимальное измеренное сродство составляло десятки наномолей/литр, что позволяет говорить о создании нового класса антагонистов нХР.

Migraine is the most common neurological disorder, but the mechanisms of migraine are remaining essential enigmatic and migraine pain is often intractable. Migraine pain is generated in meninges, which are heavily innervated by trigeminal nerve fibers and occupied by multiple mast cells. In our project, we studied the action of the migraine mediator serotonin (5-HT) on meningeal trigeminal afferents in the hemiskull preparation. This approach was complemented with the live imaging and patch-clamp recordings from isolated trigeminal neurons. We found that 5-HT evoked a long-lasting firing in meningeal afferents, the origin site of migraine pain. Cluster analysis revealed a prevailing delta- and theta-rhythm firing in active nociceptive fibers. The antagonist of Cys-loop 5-HT3-receptors MDL-72222 largely reduced the excitatory nociceptive action of 5-HT. Consistent with this, the 5-HT3 agonist mCPBG activated spikes in MDL-72222-dependent manner. In meninges, 5-HT also triggered the release of CGRP which was blocked by MDL-72222. 5-HT evoked fast membrane currents and Ca2+ transients in a fraction of trigeminal neurons. Exposure to migraine mediator CGRP increased the fraction of 5-HT responding neurons. Extracellular ATP degranulated meningeal mast cells and largely increased nociceptive firing in the WT mice. However, ATP was dramatically less effective in mice lacking mast cells in meninges. Consistent with the nociceptive action of the mast cell-derived 5-HT, the action of ATP in the WT mice was reduced by MDL-72222. In conclusion, 5-HT evokes a powerful direct pro-nociceptive peripheral action in trigeminal system transmitting migraine pain via Cys-loop ionotropic 5-HT3 receptor and indirectly supports neuroinflammation via release of CGRP.