α-Synuclein in the Synaptic Vesicle Liquid Phase: Active Player or Passive Bystander?

The protein α-synuclein, which is well-known for its links to Parkinson's Disease, is associated with synaptic vesicles (SVs) in nerve terminals. Despite intensive studies, its precise physiological function remains elusive. Accumulating evidence indicates that liquid-liquid phase separation takes part in the assembly and/or maintenance of different synaptic compartments. The current review discusses recent data suggesting α-synuclein as a component of the SV liquid phase. We also consider possible implications of these data for disease.

Clathrin-mediated endocytosis cooperates with bulk endocytosis to generate vesicles.

Clathrin-mediated endocytosis, the most prominent endocytic mode, is thought to be generated primarily from relatively flat patches of the plasma membrane. By employing conventional and platinum replica electron microscopy and super-resolution STED microscopy in neuroendocrine chromaffin cells, we found that large Ω-shaped or dome-shaped plasma membrane invaginations, previously thought of as the precursor of bulk endocytosis, are primary sites for clathrin-coated pit generation after depolarization. Clathrin-coated pits are more densely packed at invaginations rather than flat membranes, suggesting that invaginations are preferred sites for clathrin-coated pit formation, likely because their positive curvature facilitates coated-pit formation. Thus, clathrin-mediated endocytosis closely collaborates with bulk endocytosis to enhance endocytic capacity in active secretory cells. This direct collaboration between two classically independent endocytic pathways is of broad importance given the central role of both clathrin-mediated endocytosis and bulk endocytosis in neurons, endocrine cells, immune cells, and many other cell types throughout the body.

Mitochondrial dysfunction in adult midbrain dopamine neurons triggers an early immune response.

Dopamine (DA) neurons of the midbrain are at risk to become affected by mitochondrial damage over time and mitochondrial defects have been frequently reported in Parkinson's disease (PD) patients. However, the causal contribution of adult-onset mitochondrial dysfunction to PD remains uncertain. Here, we developed a mouse model lacking Mitofusin 2 (MFN2), a key regulator of mitochondrial network homeostasis, in adult midbrain DA neurons. The knockout mice develop severe and progressive DA neuron-specific mitochondrial dysfunction resulting in neurodegeneration and parkinsonism. To gain further insights into pathophysiological events, we performed transcriptomic analyses of isolated DA neurons and found that mitochondrial dysfunction triggers an early onset immune response, which precedes mitochondrial swelling, mtDNA depletion, respiratory chain deficiency and cell death. Our experiments show that the immune response is an early pathological event when mitochondrial dysfunction is induced in adult midbrain DA neurons and that neuronal death may be promoted non-cell autonomously by the cross-talk and activation of surrounding glial cells.

Preformed Ω-profile closure and kiss-and-run mediate endocytosis and diverse endocytic modes in neuroendocrine chromaffin cells.

Transformation of flat membrane into round vesicles is generally thought to underlie endocytosis and produce speed-, amount-, and vesicle-size-specific endocytic modes. Visualizing depolarization-induced exocytic and endocytic membrane transformation in live neuroendocrine chromaffin cells, we found that flat membrane is transformed into Λ-shaped, Ω-shaped, and O-shaped vesicles via invagination, Λ-base constriction, and Ω-pore constriction, respectively. Surprisingly, endocytic vesicle formation is predominantly from not flat-membrane-to-round-vesicle transformation but calcium-triggered and dynamin-mediated closure of (1) Ω profiles formed before depolarization and (2) fusion pores (called kiss-and-run). Varying calcium influxes control the speed, number, and vesicle size of these pore closures, resulting in speed-specific slow (more than ∼6 s), fast (less than ∼6 s), or ultrafast (<0.6 s) endocytosis, amount-specific compensatory endocytosis (endocytosis = exocytosis) or overshoot endocytosis (endocytosis > exocytosis), and size-specific bulk endocytosis. These findings reveal major membrane transformation mechanisms underlying endocytosis, diverse endocytic modes, and exocytosis-endocytosis coupling, calling for correction of the half-a-century concept that the flat-to-round transformation predominantly mediates endocytosis after physiological stimulation.

A latent lineage potential in resident neural stem cells enables spinal cord repair.

Injuries to the central nervous system (CNS) are inefficiently repaired. Resident neural stem cells manifest a limited contribution to cell replacement. We have uncovered a latent potential in neural stem cells to replace large numbers of lost oligodendrocytes in the injured mouse spinal cord. Integrating multimodal single-cell analysis, we found that neural stem cells are in a permissive chromatin state that enables the unfolding of a normally latent gene expression program for oligodendrogenesis after injury. Ectopic expression of the transcription factor OLIG2 unveiled abundant stem cell-derived oligodendrogenesis, which followed the natural progression of oligodendrocyte differentiation, contributed to axon remyelination, and stimulated functional recovery of axon conduction. Recruitment of resident stem cells may thus serve as an alternative to cell transplantation after CNS injury.

Vesicle Clustering in a Living Synapse Depends on a Synapsin Region that Mediates Phase Separation.

Liquid-liquid phase separation is an increasingly recognized mechanism for compartmentalization in cells. Recent in vitro studies suggest that this organizational principle may apply to synaptic vesicle clusters. Here we test this possibility by performing microinjections at the living lamprey giant reticulospinal synapse. Axons are maintained at rest to examine whether reagents introduced into the cytosol enter a putative liquid phase to disrupt critical protein-protein interactions. 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 with synapsin are ineffective at rest. Our results indicate that synaptic vesicles at a living central synapse are organized as a distinct liquid phase maintained by interactions via the intrinsically disordered region of synapsin.

Vesicle Shrinking and Enlargement Play Opposing Roles in the Release of Exocytotic Contents.

For decades, two fusion modes were thought to control hormone and transmitter release essential to life; one facilitates release via fusion pore dilation and flattening (full collapse), and the other limits release by closing a narrow fusion pore (kiss-and-run). Using super-resolution stimulated emission depletion (STED) microscopy to visualize fusion modes of dense-core vesicles in neuroendocrine cells, we find that facilitation of release is mediated not by full collapse but by shrink fusion, in which the Ω-profile generated by vesicle fusion shrinks but maintains a large non-dilating pore. We discover that the physiological osmotic pressure of a cell squeezes, but does not dilate, the Ω-profile, which explains why shrink fusion prevails over full collapse. Instead of kiss-and-run, enlarge fusion, in which Ω-profiles grow while maintaining a narrow pore, slows down release. Shrink and enlarge fusion may thus account for diverse hormone and transmitter release kinetics observed in secretory cells, previously interpreted within the full-collapse/kiss-and-run framework.

Retromer in Synaptic Function and Pathology.

The retromer complex mediates export of select transmembrane proteins from endosomes to the trans-Golgi network (TGN) or to the plasma membrane. Dysfunction of retromer has been linked with slowly progressing neurodegenerative disorders, including Alzheimer's and Parkinson's disease (AD and PD). As these disorders affect synapses it is of key importance to clarify the function of retromer-dependent protein trafficking pathways in pre- and postsynaptic compartments. Here we discuss recent insights into the roles of retromer in the trafficking of synaptic vesicle proteins, neurotransmitter receptors and other synaptic proteins. We also consider evidence that implies synapses as sites of early pathology in neurodegenerative disorders, pointing to a possible role of synaptic retromer dysfunction in the initiation of disease.

Intersectin associates with synapsin and regulates its nanoscale localization and function.

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 is unknown. Here 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. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery.

Sphingosine 1-phosphate lyase ablation disrupts presynaptic architecture and function via an ubiquitin- proteasome mediated mechanism.

The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPLfl/fl/Nes) but not postnatal neuronal forebrain-restricted SPL deletion (SPLfl/fl/CaMK) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPLfl/fl/Nes mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.

Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.

Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30-300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging.

Membrane Charge Directs the Outcome of F-BAR Domain Lipid Binding and Autoregulation.

F-BAR domain proteins regulate and sense membrane curvature by interacting with negatively charged phospholipids and assembling into higher-order scaffolds. However, regulatory mechanisms controlling these interactions are poorly understood. Here, we show that Drosophila Nervous Wreck (Nwk) is autoregulated by a C-terminal SH3 domain module that interacts directly with its F-BAR domain. Surprisingly, this autoregulation does not mediate a simple "on-off" switch for membrane remodeling. Instead, the isolated Nwk F-BAR domain efficiently assembles into higher-order structures and deforms membranes only within a limited range of negative membrane charge, and autoregulation elevates this range. Thus, autoregulation could either reduce membrane binding or promote higher-order assembly, depending on local cellular membrane composition. Our findings uncover an unexpected mechanism by which lipid composition directs membrane remodeling.

An Endocytic Scaffolding Protein together with Synapsin Regulates Synaptic Vesicle Clustering in the Drosophila Neuromuscular Junction.

Many endocytic proteins accumulate in the reserve pool of synaptic vesicles (SVs) in synapses and relocalize to the endocytic periactive zone during neurotransmitter release. Currently little is known about their functions outside the periactive zone. Here we show that in the Drosophila neuromuscular junction (NMJ), the endocytic scaffolding protein Dap160 colocalizes during the SV cycle and forms a functional complex with the SV-associated phosphoprotein synapsin, previously implicated in SV clustering. This direct interaction is strongly enhanced under phosphorylation-promoting conditions and is essential for proper localization of synapsin at NMJs. In a dap160 rescue mutant lacking the interaction between Dap160 and synapsin, perturbed reclustering of SVs during synaptic activity is observed. Our data indicate that in addition to the function in endocytosis, Dap160 is a component of a network of protein-protein interactions that serves for clustering of SVs in conjunction with synapsin. During the SV cycle, Dap160 interacts with synapsin dispersed from SVs and helps direct synapsin back to vesicles. The proteins function in synergy to achieve efficient clustering of SVs in the reserve pool. SIGNIFICANCE STATEMENT: We provide the first evidence for the function of the SH3 domain interaction in synaptic vesicle (SV) organization at the synaptic active zone. Using Drosophila neuromuscular junction as a model synapse, we describe the molecular mechanism that enables the protein implicated in SV clustering, synapsin, to return to the pool of vesicles during neurotransmitter release. We also identify the endocytic scaffolding complex that includes Dap160 as a regulator of the events linking exocytosis and endocytosis in synapses.

Dopaminergic control of autophagic-lysosomal function implicates Lmx1b in Parkinson's disease.

The role of developmental transcription factors in maintenance of neuronal properties and in disease remains poorly understood. Lmx1a and Lmx1b are key transcription factors required for the early specification of ventral midbrain dopamine (mDA) neurons. Here we show that conditional ablation of Lmx1a and Lmx1b after mDA neuron specification resulted in abnormalities that show striking resemblance to early cellular abnormalities seen in Parkinson's disease. We found that Lmx1b was required for the normal execution of the autophagic-lysosomal pathway and for the integrity of dopaminergic nerve terminals and long-term mDA neuronal survival. Notably, human LMX1B expression was decreased in mDA neurons in brain tissue affected by Parkinson's disease. Thus, these results reveal a sustained and essential requirement of Lmx1b for the function of midbrain mDA neurons and suggest that its dysfunction is associated with Parkinson's disease pathogenesis.

Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses.

Neurotransmission involves the exo-endocytic cycling of synaptic vesicle (SV) membranes. Endocytic membrane retrieval and clathrin-mediated SV reformation require curvature-sensing and membrane-bending BAR domain proteins such as endophilin A. While their ability to sense and stabilize curved membranes facilitates membrane recruitment of BAR domain proteins, the precise mechanisms by which they are targeted to specific sites of SV recycling has remained unclear. Here, we demonstrate that the multi-domain scaffold intersectin 1 directly associates with endophilin A to facilitate vesicle uncoating at synapses. Knockout mice deficient in intersectin 1 accumulate clathrin-coated vesicles at synapses, a phenotype akin to loss of endophilin function. Intersectin 1/endophilin A1 complex formation is mediated by direct binding of the SH3B domain of intersectin to a non-canonical site on the SH3 domain of endophilin A1. Consistent with this, intersectin-binding defective mutant endophilin A1 fails to rescue clathrin accumulation at neuronal synapses derived from endophilin A1-3 triple knockout (TKO) mice. Our data support a model in which intersectin aids endophilin A recruitment to sites of clathrin-mediated SV recycling, thereby facilitating vesicle uncoating.

Fast neurotransmitter release regulated by the endocytic scaffold intersectin.

Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndrome-associated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs. Genetic deletion of intersectin 1 expression or acute interference with intersectin function inhibited the replenishment of release-ready vesicles, resulting in short-term depression, without significantly affecting the rate of endocytic membrane retrieval. Acute perturbation experiments suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin and with the actin regulatory GTPase CDC42. Our data indicate a role for the endocytic scaffold intersectin in fast neurotransmitter release, which may be of prime importance for information processing in the brain.

The dynamin-binding domains of Dap160/intersectin affect bulk membrane retrieval in synapses.

Dynamin-associated protein 160 kDa (Dap160)/intersectin interacts with several synaptic proteins and affects endocytosis and synapse development. The functional role of the different protein interaction domains is not well understood. Here we show that Drosophila Dap160 lacking the dynamin-binding SH3 domains does not affect the development of the neuromuscular junction but plays a key role in synaptic vesicle recycling. dap160 mutants lacking dynamin-interacting domains no longer accumulate dynamin properly at the periactive zone, and it becomes dispersed in the bouton during stimulation. This is accompanied by a reduction in uptake of the dye FM1-43 and an accumulation of large vesicles and membrane invaginations. However, we do not observe an increase in the number of clathrin-coated intermediates. We also note a depression in evoked excitatory junction potentials (EJPs) during high-rate stimulation, accompanied by aberrantly large miniature EJPs. The data reveal the important role of Dap160 in the targeting of dynamin to the periactive zone, where it is required to suppress bulk synaptic vesicle membrane retrieval during high-frequency activity.

Structural organization of the presynaptic density at identified synapses in the locust central nervous system.

In a synaptic active zone, vesicles aggregate around a densely staining structure called the presynaptic density. We focus on its three-dimensional architecture and a major molecular component in the locust. We used electron tomography to study the presynaptic density in synapses made in the brain by identified second-order neuron of the ocelli. Here, vesicles close to the active zone are organized in two rows on either side of the presynaptic density, a level of organization not previously reported in insect central synapses. The row of vesicles that is closest to the density's base includes vesicles docked with the presynaptic membrane and thus presumably ready for release, whereas the outer row of vesicles does not include any that are docked. We show that a locust ortholog of the Drosophila protein Bruchpilot is localized to the presynaptic density, both in the ocellar pathway and compound eye visual neurons. An antibody recognizing the C-terminus of the Bruchpilot ortholog selectively labels filamentous extensions of the presynaptic density that reach out toward vesicles. Previous studies on Bruchpilot have focused on its role in neuromuscular junctions in Drosophila, and our study shows it is also a major functional component of presynaptic densities in the central nervous system of an evolutionarily distant insect. Our study thus reveals Bruchpilot executes similar functions in synapses that can sustain transmission of small graded potentials as well as those relaying large, spike-evoked signals.

Synapsin I senses membrane curvature by an amphipathic lipid packing sensor motif.

Sustained neurotransmitter release at synapses during high-frequency synaptic activity involves the mobilization of synaptic vesicles (SVs) from the tightly clustered reserve pool (RP). Synapsin I (Syn I), a brain-specific peripheral membrane protein that undergoes activity-dependent cycles of SV association and dissociation, is implicated in RP organization via its ability to cluster SVs. Although Syn I has affinity for phospholipids, the mechanism for the reversible association of synapsin with SV membranes remains enigmatic. Here, we show that rat Syn I is able to sense membrane curvature via an evolutionary conserved amphipathic lipid packing sensor motif (ALPS). Deletion or mutational inactivation of the ALPS impairs the ability of Syn I to associate with highly curved membranes and with SVs. Furthermore, a Syn I mutant lacking ALPS displays defects in its ability to undergo activity-induced cycles of dispersion and reclustering in neurons and fails to induce vesicle clustering in vitro. Our data suggest a crucial role for ALPS-mediated sensing of membrane curvature in regulating synapsin function.

Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition.

Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.