Excess phosphoserine-129 α-synuclein induces synaptic vesicle trafficking and declustering defects at a vertebrate synapse.

α-Synuclein is a presynaptic protein that regulates synaptic vesicle (SV) trafficking. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), α-synuclein aberrantly accumulates throughout neurons, including at synapses. During neuronal activity, α-synuclein is reversibly phosphorylated at serine 129 (pS129). While pS129 comprises ∼4% of total α-synuclein under physiological conditions, it dramatically increases in PD and DLB brains. The impacts of excess pS129 on synaptic function are currently unknown. We show here that compared with wild-type (WT) α-synuclein, pS129 exhibits increased binding and oligomerization on synaptic membranes and enhanced vesicle "microclustering" in vitro. Moreover, when acutely injected into lamprey reticulospinal axons, excess pS129 α-synuclein robustly localized to synapses and disrupted SV trafficking in an activity-dependent manner, as assessed by ultrastructural analysis. Specifically, pS129 caused a declustering and dispersion of SVs away from the synaptic vicinity, leading to a significant loss of total synaptic membrane. Live imaging further revealed altered SV cycling, as well as microclusters of recently endocytosed SVs moving away from synapses. Thus, excess pS129 caused an activity-dependent inhibition of SV trafficking via altered vesicle clustering/reclustering. This work suggests that accumulation of pS129 at synapses in diseases like PD and DLB could have profound effects on SV dynamics.

α-Synuclein colocalizes with AP180 and affects the size of clathrin lattices.

α-Synuclein and family members ß- and γ-synuclein are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. αßγ-synuclein triple knockout neurons exhibit SV endocytosis deficits. Here, we investigated if α-synuclein affects clathrin assembly in vitro. Visualizing clathrin assembly on membranes using a lipid monolayer system revealed that α-synuclein increases clathrin lattices size and curvature. On cell membranes, we observe that α-synuclein is colocalized with clathrin and its adapter AP180 in a concentric ring pattern. Clathrin puncta that contain both α-synuclein and AP180 were significantly larger than clathrin puncta containing either protein alone. We determined that this effect occurs in part through colocalization of α-synuclein with the phospholipid PI(4,5)P2 in the membrane. Immuno-electron microscopy (EM) of synaptosomes uncovered that α-synuclein relocalizes from SVs to the presynaptic membrane upon stimulation, positioning α-synuclein to function on presynaptic membranes during or after stimulation. Additionally, we show that deletion of synucleins impacts brain-derived clathrin-coated vesicle size. Thus, α-synuclein affects the size and curvature of clathrin structures on membranes and functions as an endocytic accessory protein.

α-Synuclein-112 Impairs Synaptic Vesicle Recycling Consistent With Its Enhanced Membrane Binding Properties.

Synucleinopathies are neurological disorders associated with α-synuclein overexpression and aggregation. While it is well-established that overexpression of wild type α-synuclein (α-syn-140) leads to cellular toxicity and neurodegeneration, much less is known about other naturally occurring α-synuclein splice isoforms. In this study we provide the first detailed examination of the synaptic effects caused by one of these splice isoforms, α-synuclein-112 (α-syn-112). α-Syn-112 is produced by an in-frame excision of exon 5, resulting in deletion of amino acids 103-130 in the C-terminal region. α-Syn-112 is upregulated in the substantia nigra, frontal cortex, and cerebellum of parkinsonian brains and higher expression levels are correlated with susceptibility to Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). We report here that α-syn-112 binds strongly to anionic phospholipids when presented in highly curved liposomes, similar to α-syn-140. However, α-syn-112 bound significantly stronger to all phospholipids tested, including the phosphoinositides. α-Syn-112 also dimerized and trimerized on isolated synaptic membranes, while α-syn-140 remained largely monomeric. When introduced acutely to lamprey synapses, α-syn-112 robustly inhibited synaptic vesicle recycling. Interestingly, α-syn-112 produced effects on the plasma membrane and clathrin-mediated synaptic vesicle endocytosis that were phenotypically intermediate between those caused by monomeric and dimeric α-syn-140. These findings indicate that α-syn-112 exhibits enhanced phospholipid binding and oligomerization in vitro and consequently interferes with synaptic vesicle recycling in vivo in ways that are consistent with its biochemical properties. This study provides additional evidence suggesting that impaired vesicle endocytosis is a cellular target of excess α-synuclein and advances our understanding of potential mechanisms underlying disease pathogenesis in the synucleinopathies.

Kinetic Measurements of Endocytosis and Exocytosis in Cultured Neurons.

The measurement of synaptic vesicle recycling in live neurons transfected with vesicular glutamate transporter fused to pHluorin (vGLUT-pHluorin) allows us to study exocytosis and endocytosis in neurons. When neurons are transfected with this protein we can measure the rate of vesicles fusing and internalizing from the membrane using live total internal reflection fluorescence (TIRF) imaging. Here, we describe transfection, culturing, and imaging of wild-type and αßγ-synuclein knockout hippocampal neurons. This technique can be used to evaluate the effect of different phenotypes and treatments in the physiology of synaptic vesicle recycling in cultured neurons.

Synucleins Have Multiple Effects on Presynaptic Architecture.

Synucleins (α, ß, γ-synuclein) are a family of abundant presynaptic proteins. α-Synuclein is causally linked to the pathogenesis of Parkinson's disease (PD). In an effort to define their physiological and pathological function or functions, we investigated the effects of deleting synucleins and overexpressing α-synuclein PD mutations, in mice, on synapse architecture using electron microscopy (EM) and cryoelectron tomography (cryo-ET). We show that synucleins are regulators of presynapse size and synaptic vesicle (SV) pool organization. Using cryo-ET, we observed that deletion of synucleins increases SV tethering to the active zone but decreases the inter-linking of SVs by short connectors. These ultrastructural changes were correlated with discrete protein phosphorylation changes in αßγ-synuclein-/- neurons. We also determined that α-synuclein PD mutants (PARK1/hA30P and PARK4/hα-syn) primarily affected presynaptic cytomatrix proximal to the active zone, congruent with previous findings that these PD mutations decrease neurotransmission. Collectively, our results suggest that synucleins are important orchestrators of presynaptic terminal topography.

Synucleins regulate the kinetics of synaptic vesicle endocytosis.

Genetic and pathological studies link α-synuclein to the etiology of Parkinson's disease (PD), but the normal function of this presynaptic protein remains unknown. α-Synuclein, an acidic lipid binding protein, shares high sequence identity with ß- and γ-synuclein. Previous studies have implicated synucleins in synaptic vesicle (SV) trafficking, although the precise site of synuclein action continues to be unclear. Here we show, using optical imaging, electron microscopy, and slice electrophysiology, that synucleins are required for the fast kinetics of SV endocytosis. Slowed endocytosis observed in synuclein null cultures can be rescued by individually expressing mouse α-, ß-, or γ-synuclein, indicating they are functionally redundant. Through comparisons to dynamin knock-out synapses and biochemical experiments, we suggest that synucleins act at early steps of SV endocytosis. Our results categorize α-synuclein with other familial PD genes known to regulate SV endocytosis, implicating this pathway in PD.

Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors.

Slow and persistent synaptic inhibition is mediated by metabotropic GABAB receptors (GABABRs). GABABRs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABABRs are predominantly found on dendritic spines, adjacent to excitatory synapses, but the control of their plasma membrane availability is still controversial. Here, we explore the role of glutamate receptor activation in regulating the function and surface availability of GABABRs in central neurons. We demonstrate that prolonged activation of NMDA receptors (NMDA-Rs) leads to endocytosis, a diversion from a recycling route, and subsequent lysosomal degradation of GABABRs. These sorting events are paralleled by a reduction in GABABR-dependent activation of inwardly rectifying K+ channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABABR2 subunit, an established substrate of AMP-dependent protein kinase (AMPK). NMDA-R activation leads to a rapid increase in phosphorylation of S783, followed by a slower dephosphorylation, which results from the activity of AMPK and protein phosphatase 2A, respectively. Agonist activation of GABABRs counters the effects of NMDA. Thus, NMDA-R activation alters the phosphorylation state of S783 and acts as a molecular switch to decrease the abundance of GABABRs at the neuronal plasma membrane. Such a mechanism may be of significance during synaptic plasticity or pathological conditions, such as ischemia or epilepsy, which lead to prolonged activation of glutamate receptors.

Dendritic assembly of heteromeric gamma-aminobutyric acid type B receptor subunits in hippocampal neurons.

Understanding the mechanisms that control synaptic efficacy through the availability of neurotransmitter receptors depends on uncovering their specific intracellular trafficking routes. gamma-Aminobutyric acid type B (GABA(B)) receptors (GABA(B)Rs) are obligatory heteromers present at dendritic excitatory and inhibitory postsynaptic sites. It is unknown whether synthesis and assembly of GABA(B)Rs occur in the somatic endoplasmic reticulum (ER) followed by vesicular transport to dendrites or whether somatic synthesis is followed by independent transport of the subunits for assembly and ER export throughout the somatodendritic compartment. To discriminate between these possibilities we studied the association of GABA(B)R subunits in dendrites of hippocampal neurons combining live fluorescence microscopy, biochemistry, quantitative colocalization, and bimolecular fluorescent complementation. We demonstrate that GABA(B)R subunits are segregated and differentially mobile in dendritic intracellular compartments and that a high proportion of non-associated intracellular subunits exist in the brain. Assembled heteromers are preferentially located at the plasma membrane, but blockade of ER exit results in their intracellular accumulation in the cell body and dendrites. We propose that GABA(B)R subunits assemble in the ER and are exported from the ER throughout the neuron prior to insertion at the plasma membrane. Our results are consistent with a bulk flow of segregated subunits through the ER and rule out a post-Golgi vesicular transport of preassembled GABA(B)Rs.

Postnatal expression of V2 vasopressin receptor splice variants in the rat cerebellum.

The V(2) vasopressin receptor gene contains an alternative splice site in exon-3, which leads to the generation of two splice variants (V(2a) and V(2b)) first identified in the kidney. The open reading frame of the alternatively spliced V(2b) transcript encodes a truncated receptor, showing the same amino acid sequence as the canonical V(2a) receptor up to the sixth transmembrane segment, but displaying a distinct sequence to the corresponding seventh transmembrane segment and C-terminal domain relative to the V(2a) receptor. Here, we demonstrate the postnatal expression of V(2a) and V(2b) variants in the rat cerebellum. Most importantly, we showed by in situ hybridization and immunocytochemistry that both V(2) splice variants were preferentially expressed in Purkinje cells, from early to late postnatal development. In addition, both variants were transiently expressed in the neuroblastic external granule cells and Bergmann fibers. These results indicate that the cellular distributions of both splice variants are developmentally regulated, and suggest that the transient expression of the V(2) receptor is involved in the mechanisms of cerebellar cytodifferentiation by AVP. Finally, transfected CHO-K1 expressing similar amounts of both V(2) splice variants, as that found in the cerebellum, showed a significant reduction in the surface expression of V(2a) receptors, suggesting that the differential expression of the V(2) splice variants regulates the vasopressin signaling in the cerebellum.

The availability of surface GABA B receptors is independent of gamma-aminobutyric acid but controlled by glutamate in central neurons.

The efficacy of synaptic transmission depends on the availability of ionotropic and metabotropic neurotransmitter receptors at the plasma membrane, but the contribution of the endocytic and recycling pathways in the regulation of gamma-aminobutyric acid type B (GABA(B)) receptors remains controversial. To understand the mechanisms that regulate the abundance of GABA(B) receptors, we have studied their turnover combining surface biotin labeling and a microscopic immunoendocytosis assay in hippocampal and cortical neurons. We report that internalization of GABA(B) receptors is agonist-independent. We also demonstrate that receptors endocytose in the cell body and dendrites but not in axons. Additionally, we show that GABA(B) receptors endocytose as heterodimers via clathrin- and dynamin-1-dependent mechanisms and that they recycle to the plasma membrane after endocytosis. More importantly, we show that glutamate decreases the levels of cell surface receptors in a manner dependent on an intact proteasome pathway. These observations indicate that glutamate and not GABA controls the abundance of surface GABA(B) receptors in central neurons, consistent with their enrichment at glutamatergic synapses.