Annotation of the results obtained in 2018
Liquid-liquid phase separation is an increasingly recognized mechanism for compartmental organization in cells. Phosphoprotein synapsin I plays critical role in organising synaptic vesicles (SVs) in the reserve pool in many central synapses. Recent in vitro studies suggest that synaptic vesicle clusters may utilize liquid-liquid phase organizational principle (Milovanovic and De Camilli, Science 2017). We tested this possibility by performing microinjections at the living giant reticulospinal synapse at rest. We found that compounds that perturb the intrinsically disordered region of synapsin, which is critical for liquid phase organization in vitro, cause dispersion of synaptic vesicles from resting clusters. Reagents that perturb SH3 domain interactions of synaptic proteins with synapsin were found ineffective at rest. Our results indicate that synaptic vesicles in a living central synapse are organized as a distinct liquid phase maintained by interactions via the intrinsically disordered region of synapsin. Using the giant reticulospinal model synapse in lamprey we show that the scaffolding protein intersectin 1 (ITSN1) regulates the synapsin 1 function during synaptic activity by forming a dynamic complex with synapsin. Like in mammalian synapses ITSN1 is a component of an extravesicular matrix in giant lamprey synapses. The complex formation with synapsin 1 is mediated by SH3A (Src-homology 3 A) domain of ITSN1, which binds to the D domain of synapsin I. An intramolecular switch within ITSN1 regulates the interaction between the proteins. Microinjection of antibodies against SH3A domain into giant synapses at rest does not perturb SV organization, while during stimulation it disrupts the vesicle clustering in the reserve pool thus supporting that ITSN1 and synapsin 1 come into interaction during synaptic activity. Our data indicate that the SH3A domain of ITSN1 serves to sequester synapsin 1 within the reserve pool when it dissociates from SV during stimulation and promotes efficient reclustering when stimulation ceased by releasing dephosphorylated synapsin within the reserve pool. Thus, our experiments uncover the molecular mechanism that regulates vesicle reclustering within the reserve pool of SVs and thus SV controls availability of vesicles during release. (Shupliakov et al., 2018; Shupliakov 2019; paper submitted, under revision) We describe a novel organization of synaptic vesicles in vertebrate axons. We detected clusters of vesicles interspersed by amyloid protein aggregates derive from synapses and are formed through a reorganization of the intravesicular protein matrix clustering synaptic vesicles in the reserve pool under normal conditions. SV's are linked to these amyloid structures as reveled by electron microscopy. This evolution of the intervesicular matrix of the reserve pool represents a novel form of synaptic plasticity and could be linked to early presynaptic pathology observed following acute perturbation of synapsin interactions. (Sopova, Shupliakov, 2018; Manuscript). It has been suggested that retraction of synapses, structural changes, and formation of protein aggregates in nerve terminals following conditional ablation of lmx1b in dopaminergic neurons in substantia nigra are primarily cased by mitophagy and mitochondrial dysfunctions (e.g., Doucet-Beaupré et al., Proc Natl Acad Sci U S A. 2016). We show that conditional ablation of mytofusin 2 in postmitotic dopaminergic neurons results in destruction of mitochondria leading to degeneration of the neurons. These perturbations do not lead to the structural changes in synaptic projections observed following ablation of lmx1b. Thus the early structural changes in nerve terminals are induced by perturbations of other singling pathways and protein-protein interactions (submitted; Li et al., 2018). Our experiments strongly suggest that synaptic vesicle clusters under certain conditions may represent the source of pathological protein aggregates in synapses. We propose that malfunctions in presynaptic mechanisms and signaling pathways linking synaptic machinery to nucleus may underlie early pathologies in Parkinson´s disease. These ideas and recent findings are summarised in our review articles published in 2018 (Brodin, Shupliakov, 2018; Sopova et al., 2018).

 

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Annotation of the results obtained in 2016
1. Our experiments suggest that mitochondria dysfunctions and disruptions of the actin cytoskeleton dynamics that occurs in neurodegenerative diseases, such as Parkinson´s disease, may affect vesicle fusion. It has been widely accepted earlier that membrane vesicles collapse into the plasma membrane once a fusion pore is formed and that this step does not require any energy consumption. Our paper published in Nature Communications shows that the fusion event may occur via pushing the vesicle membrane through the fusion pore into the plasma membrane, which is controlled by the cellular membrane tension and involves ATP hydrolysis. Actin cytoskeleton and ATP regulate such fusion event in neuroendocrine cells and neuronal synapses. Read the article at: http://www.nature.com/articles/ncomms12604. The paper has been recommended in F1000Prime as being of special significance in its field. 2. In search for potential molecular mechanisms controlling neuronal architecture effects of altered metabolism of membrane lipids, sphingolipids, were investigated. Brain-specific knockout mice, in which S1P-lyase (SPL), the enzyme responsible for irreversible of the bioactive lipid sphingosine 1-phosphate (S1P) cleavage was generated. Constitutive ablation of SPL in the brain (SPLfl/fl/Nes) caused marked accumulation of S1P. Altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles and decreased expression of several presynaptic proteins were observed in neurons. It was found that the structural and functional alterations of the presynaptic morphology were associated with an activation of the ubiquitin proteasome system (UPS) and a decreased expression of the deubiquitinating enzyme USP14. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as one of the mechanism controlling presynaptic morphology, thus suggesting that it can be a target mechanism in abnormal dopaminergic synapses in LMXa/b knockout mice. Read the article at: http://www.nature.com/articles/srep37064.

 

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Annotation of the results obtained in 2017
Several key findings were published in 2017: 1. Synaptic proteins, synucleins, are found in pathologic aggregates in human brains during neurodegenerative diseases, including Parkinson´s disease. The normal functions of these proteins in synapses are still unclear. We used cDNA cloning to determine amino acid sequences of synucleins in the central nervous system of river lamprey (Lampetra fluviatilis), which is used as a model organism to study molecular mechanisms of synaptic transmission in our project. Three genes are identified. High similarity in amino acid sequences as compared to other vertebrate species is reveled. Bioinformatic analysis predicts that the river lamprey synucleins relate to the group of gamma-synucleins. High homology with human alpha-synuclein was found. The hydrophobic region required for the formation of alpha-synuclein amyloid fibers is also present in the river lamprey synucleins. The latter suggests that this region appeared at early stages of evolution. The obtained amino acid sequences of synucleins in the river lamprey brain will allow generating novel molecular tools for dissecting physiological functions of these proteins. Cloning data are submitter to the NCBI database. 2. Our preliminary data show that protein aggregates can be induced in synapses, which contain synuclein and a scaffolding protein complex, which includes intersectin. Our studies of the functions of this protein reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery. Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with synapsins in this process was unknown. We identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. 3. Another function of the scaffolding complex that includes Intersection was investigated. Using Drosophila as a model system we found that in the presynaptic nerve terminal, the membrane-remodeling F-BAR domain protein Nervous Wreck (Nwk) relocates from the SV pool to the periactive zone during synaptic activity. It is targeted to sites of endocytosis by SH3 domain interactions with the scaffolding protein Dap160/Intersectin. These interactions also directly promote Nwk membrane binding in vitro. Genetic perturbations of Dap160/Intersectin -Nwk interactions do not block neurotransmitter release, but mutants lacking either Dap160/Intersectin -Nwk interactions or the Nwk F-bar domain exhibit altered NMJ architecture and accumulation of synaptic vesicles of different diameters at active zones. In addition an accumulation of large endocytic cisternae and appearance of enlarged clathrin-coated vesicles occur in response to strong stimulation. The cellular mechanisms for generating uniform vesicles during the synaptic vesicle cycle are not fully understood. Thus the recruitment of the F-bar domain of Nwk to the endocytic site is an important step in the synaptic vesicle recycling mechanism and it is required for the formation of uniform size vesicles. 4. The chromaffin cells of the adrenal medulla secrete catecholamines, adrenaline (epinephrine), noradrenaline (norepinephrine) and dopamine. These cells of the adrenal medulla (AM) represent the main neuroendocrine adrenergic component and are believed to differentiate from neural crest cells. It was demonstrated that large numbers of chromaffin cells arise from peripheral glial stem cells, termed Schwann cell precursors (SCPs). SCPs migrate along the visceral motor nerve to the vicinity of the forming adrenal gland where they detach from the nerve and form post-synaptic neuroendocrine chromaffin cells. An intricate molecular logic drives this transition and encompasses two separate gene programs, one unique for the distinct transient state and another for the cell type specification. Subsequently, these programs down-regulate SCP- and upregulate chromaffin-cell-gene networks. Adrenal medulla forms through limited cell expansion and requires the recruitment of numerous SCPs. These results highlight the importance of the peripheral nerve as a stem cell niche for neuroendocrine system development.

 

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