Rafael Fernández-Chacón

Synaptotagmin I functions as a calcium regulator of release probability

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors—which are the gatekeepers to neurotransmission—remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.

α-Synuclein Cooperates with CSPα in Preventing Neurodegeneration

Summary α-synuclein and cysteine-string protein-α (CSPα) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in α-synuclein cause Parkinsons disease, but the physiological role of α-synuclein remains unknown. Deletion of CSPα produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPα is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of α-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPα. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPα inhibits SNARE complex assembly; transgenic α-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPα-deficient mice, α-synuclein does not simply substitute for CSPα but acts by a downstream mechanism that requires phospholipid binding by α-synuclein. These observations reveal a powerful in vivo activity of α-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPα and SNARE proteins on the presynaptic membrane interface.

The synaptic vesicle protein CSPα prevents presynaptic degeneration

Abstract Cysteine string protein α (CSPα)—an abundant synaptic vesicle protein that contains a DNA-J domain characteristic of Hsp40 chaperones—is thought to regulate Ca 2+ channels and/or synaptic vesicle exocytosis. We now show that, in young mice, deletion of CSPα does not impair survival and causes no significant changes in presynaptic Ca 2+ currents or synaptic vesicle exocytosis as measured in the Calyx of Held synapse. At 2–4 weeks of age, however, CSPα-deficient mice develop a progressive, fatal sensorimotor disorder. The neuromuscular junctions and Calyx synapses of CSPα-deficient mice exhibit increasing neurodegenerative changes, synaptic transmission becomes severely impaired, and the mutant mice die at ∼2 months of age. Our data suggest that CSPα is not essential for the normal operation of Ca 2+ channels or exocytosis but acts as a presynaptic chaperone that maintains continued synaptic function, raising the possibility that enhanced CSPα function could attenuate neurodegenerative diseases.

A Trimeric Protein Complex Functions as a Synaptic Chaperone Machine

We identify a chaperone complex composed of (1) the synaptic vesicle cysteine string protein (CSP), thought to function in neurotransmitter release, (2) the ubiquitous heat-shock protein cognate Hsc70, and (3) the SGT protein containing three tandem tetratricopeptide repeats. These three proteins interact with each other to form a stable trimeric complex that is located on the synaptic vesicle surface, and is disrupted in CSP knockout mice. The CSP/SGT/Hsc70 complex functions as an ATP-dependent chaperone that reactivates a denatured substrate. SGT overexpression in cultured neurons inhibits neurotransmitter release, suggesting that the CSP/SGT/Hsc70 complex is important for maintenance of a normal synapse. Taken together, our results identify a novel trimeric complex that functions as a synapse-specific chaperone machine.

Synaptotagmin VII as a Plasma Membrane Ca2+ Sensor in Exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).

The Making of Neurexins

Abstract: Neurexins are neuronal cell‐surface proteins with up to thousands of isoforms. These isoforms are generated by alternative splicing of transcripts from six promoters in three genes. The structure of neurexins resembles cell‐surface receptors with a modular architecture suggestive of a sequential assembly during evolution. Neurexins probably perform multiple functions in the brain. They participate in intercellular junctions in which β‐neurexins tightly bind to a second class of neuronal cell‐surface receptors called neuroligins. Intracellularly, the neurexin/neuroligin junction is bound by CASK on the neurexin side and PSD95 on the neuroligin side. CASK and PSD95 are homologous membrane‐associated guanylate kinases that bind to the neurexin/neuroligin junction via PDZ domains, creating an asymmetric junction (neurexin/neuroligin) with similar intracellular binding partners. In addition to a function as cell‐adhesion molecules, neurexins may also serve as a signalling receptor, because a class of ligands for α‐neurexins called neurexophilins is similar to peptide hormones. Finally, at least one neurexin isoform, neurexin Iα, represents a high‐affinity receptor for α‐latrotoxin, which is a potent excitatory neurotoxin. Thus, neurexins constitute a large family of neuronal receptors that may be involved in multiple interactive functions between neurons.

Deletion of CASK in mice is lethal and impairs synaptic function

CASK is an evolutionarily conserved multidomain protein composed of an N-terminal Ca2+/calmodulin-kinase domain, central PDZ and SH3 domains, and a C-terminal guanylate kinase domain. Many potential activities for CASK have been suggested, including functions in scaffolding the synapse, in organizing ion channels, and in regulating neuronal gene transcription. To better define the physiological importance of CASK, we have now analyzed CASK “knockdown” mice in which CASK expression was suppressed by ≈70%, and CASK knockout (KO) mice, in which CASK expression was abolished. CASK knockdown mice are viable but smaller than WT mice, whereas CASK KO mice die at first day after birth. CASK KO mice exhibit no major developmental abnormalities apart from a partially penetrant cleft palate syndrome. In CASK-deficient neurons, the levels of the CASK-interacting proteins Mints, Veli/Mals, and neurexins are decreased, whereas the level of neuroligin 1 (which binds to neurexins that in turn bind to CASK) is increased. Neurons lacking CASK display overall normal electrical properties and form ultrastructurally normal synapses. However, glutamatergic spontaneous synaptic release events are increased, and GABAergic synaptic release events are decreased in CASK-deficient neurons. In contrast to spontaneous neurotransmitter release, evoked release exhibited no major changes. Our data suggest that CASK, the only member of the membrane-associated guanylate kinase protein family that contains a Ca2+/calmodulin-dependent kinase domain, is required for mouse survival and performs a selectively essential function without being in itself required for core activities of neurons, such as membrane excitability, Ca2+-triggered presynaptic release, or postsynaptic receptor functions.

Rab3D Is Not Required for Exocrine Exocytosis but for Maintenance of Normally Sized Secretory Granules

ABSTRACT Rab3D, a member of the Rab3 subfamily of the Rab/ypt GTPases, is expressed on zymogen granules in the pancreas as well as on secretory vesicles in mast cells and in the parotid gland. To shed light on the function of Rab3D, we have generated Rab3D-deficient mice. These mice are viable and have no obvious phenotypic changes. Secretion of mast cells is normal as revealed by capacitance patch clamping. Furthermore, enzyme content and overall morphology are unchanged in pancreatic and parotid acinar cells of knockout mice. Both the exocrine pancreas and the parotid gland show normal release kinetics in response to secretagogue stimulation, suggesting that Rab3D is not involved in exocytosis. However, the size of secretory granules in both the exocrine pancreas and the parotid gland is significantly increased, with the volume being doubled. We conclude that Rab3D exerts its function during granule maturation, possibly by preventing homotypic fusion of secretory granules.

Examining Synaptotagmin 1 Function in Dense Core Vesicle Exocytosis under Direct Control of Ca2

We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2+ sensor for fast neurosecretion by analyzing the intracellular Ca2+ dependence of large dense-core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin C2A domain. The mutation (R233Q) causes a twofold increase in the KD of Ca2+-dependent phospholipid binding to the double C2A-C2B domain of synaptotagmin. Using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates, longer secretory delays, and a higher intracellular Ca2+-threshold for secretion due to a twofold increase in the apparent KD of the Ca2+ sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca2+-dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca2+ dependence of exocytosis.

Multiple internalization pathways of polyelectrolyte multilayer capsules into mammalian cells.

Polyelectrolyte multilayer (PEM) capsules are carrier vehicles with great potential for biomedical applications. With the future aim of designing biocompatible, effective therapeutic delivery systems (e.g., for cancer), the pathway of internalization (uptake and fate) of PEM capsules was investigated. In particular the following experiments were performed: (i) the study of capsule co-localization with established endocytic markers, (ii) switching-off endocytotic pathways with pharmaceutical/chemical inhibitors, and (iii) characterization and quantification of capsule uptake with confocal and electron microscopy. As result, capsules co-localized with lipid rafts and with phagolysosomes, but not with other endocytic vesicles. Chemical interference of endocytosis with chemical blockers indicated that PEM capsules enter the investigated cell lines through a mechanism slightly sensitive to electrostatic interactions, independent of clathrin and caveolae, and strongly dependent on cholesterol-rich domains and organelle acidification. Microscopic characterization of cells during capsule uptake showed the formation of phagocytic cups (vesicles) to engulf the capsules, an increased number of mitochondria, and a final localization in the perinuclear cytoplasma. Combining all these indicators we conclude that PEM capsule internalization in general occurs as a combination of different sequential mechanisms. Initially, an adsorptive mechanism due to strong electrostatic interactions governs the stabilization of the capsules at the cell surface. Membrane ruffling and filopodia extensions are responsible for capsule engulfing through the formation of a phagocytic cup. Co-localization with lipid raft domains activates the cell to initiate a lipid-raft-mediated macropinocytosis. Internalization vesicles are very acidic and co-localize only with phagolysosome markers, excluding caveolin-mediated pathways and indicating that upon phagocytosis the capsules are sorted to heterophagolysosomes.