Stefan H. Gerber

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.

Three-dimensional structure of the synaptotagmin 1 C2B-domain: Synaptotagmin 1 as a phospholipid binding machine

Synaptotagmin 1 probably functions as a Ca2+ sensor in neurotransmitter release via its two C2-domains, but no common Ca2+-dependent activity that could underlie a cooperative action between them has been described. The NMR structure of the C2B-domain now reveals a beta sandwich that exhibits striking similarities and differences with the C2A-domain. Whereas the bottom face of the C2B-domain has two additional alpha helices that may be involved in specialized Ca2+-independent functions, the top face binds two Ca2+ ions and is remarkably similar to the C2A-domain. Consistent with these results, but in contrast to previous studies, we find that the C2B-domain binds phospholipids in a Ca2+-dependent manner similarly to the C2A-domain. These results suggest a novel view of synaptotagmin function whereby the two C2-domains cooperate in a common activity, Ca2+-dependent phospholipid binding, to trigger neurotransmitter release.

Conformational switch of syntaxin-1 controls synaptic vesicle fusion.

During synaptic vesicle fusion, the soluble N-ethylmaleimide-sensitive factor–attachment protein receptor (SNARE) protein syntaxin-1 exhibits two conformations that both bind to Munc18-1: a “closed” conformation outside the SNARE complex and an “open” conformation in the SNARE complex. Although SNARE complexes containing open syntaxin-1 and Munc18-1 are essential for exocytosis, the function of closed syntaxin-1 is unknown. We generated knockin/knockout mice that expressed only open syntaxin-1B. Syntaxin-1BOpen mice were viable but succumbed to generalized seizures at 2 to 3 months of age. Binding of Munc18-1 to syntaxin-1 was impaired in syntaxin-1BOpen synapses, and the size of the readily releasable vesicle pool was decreased; however, the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies.

A novel family of phosphatidylinositol 4-kinases conserved from yeast to humans.

Phosphatidylinositolpolyphosphates (PIPs) are centrally involved in many biological processes, ranging from cell growth and organization of the actin cytoskeleton to endo- and exocytosis. Phosphorylation of phosphatidylinositol at the D-4 position, an essential step in the biosynthesis of PIPs, appears to be catalyzed by two biochemically distinct enzymes. However, only one of these two enzymes has been molecularly characterized. We now describe a novel class of phosphatidylinositol 4-kinases that probably corresponds to the missing element in phosphatidylinositol metabolism. These kinases are highly conserved evolutionarily, but unrelated to previously characterized phosphatidylinositol kinases, and thus represent the founding members of a new family. The novel phosphatidylinositol 4-kinases, which are widely expressed in cells, only phosphorylate phosphatidylinositol, are potently inhibited by adenosine, but are insensitive to wortmannin or phenylarsine oxide. Although they lack an obvious transmembrane domain, they are strongly attached to membranes by palmitoylation. Our data suggest that independent pathways for phosphatidylinositol 4-phosphate synthesis emerged during evolution, possibly to allow tight temporal and spatial control over the production of this key signaling molecule.

Piccolo and bassoon maintain synaptic vesicle clustering without directly participating in vesicle exocytosis

Piccolo and bassoon are highly homologous multidomain proteins of the presynaptic cytomatrix whose function is unclear. Here, we generated piccolo knockin/knockout mice that either contain wild-type levels of mutant piccolo unable to bind Ca2+ (knockin), ∼60% decreased levels of piccolo that is C-terminally truncated (partial knockout), or <5% levels of piccolo (knockout). All piccolo mutant mice were viable and fertile, but piccolo knockout mice exhibited increased postnatal mortality. Unexpectedly, electrophysiology and electron microscopy of piccolo-deficient synapses failed to uncover a major phenotype either in acute hippocampal slices or in cultured cortical neurons. To unmask potentially redundant functions of piccolo and bassoon, we thus acutely knocked down expression of bassoon in wild-type and piccolo knockout neurons. Despite a nearly complete loss of piccolo and bassoon, however, we still did not detect an electrophysiological phenotype in cultured piccolo- and bassoon-deficient neurons in either GABAergic or glutamatergic synaptic transmission. In contrast, electron microscopy revealed a significant reduction in synaptic vesicle clustering in double bassoon/piccolo-deficient synapses. Thus, we propose that piccolo and bassoon play a redundant role in synaptic vesicle clustering in nerve terminals without directly participating in neurotransmitter release.

A conformational switch in the Piccolo C2A domain regulated by alternative splicing

C2 domains are widespread Ca2+-binding modules. The active zone protein Piccolo (also known as Aczonin) contains an unusual C2A domain that exhibits a low affinity for Ca2+, a Ca2+-induced conformational change and Ca2+-dependent dimerization. We show here that removal of a nine-residue sequence by alternative splicing increases the Ca2+ affinity, abolishes the conformational change and abrogates dimerization of the Piccolo C2A domain. The NMR structure of the Ca2+-free long variant provides a structural basis for these different properties of the two splice forms, showing that the nine-residue sequence forms a β-strand otherwise occupied by a nonspliced sequence. Consequently, Ca2+-binding to the long Piccolo C2A domain requires a marked rearrangement of secondary structure that cannot occur for the short variant. These results reveal a novel mechanism of action of C2 domains and uncover a structural principle that may underlie the alteration of protein function by short alternatively spliced sequences.

Expression of monocyte chemoattractant protein-1 cDNA in vascular smooth muscle cells: induction of the synthetic phenotype: a possible clue to SMC differentiation in the process of atherogenesis.

In the arterial wall, smooth muscle cells (SMC) normally exist in a quiescent, differentiated state, representing the contractile phenotype. During the development of atherosclerosis SMC change towards the synthetic phenotype going along with proliferation, chemotactic response and increased monocyte binding. Expression of monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant for monocytes, has been shown to be among the earliest events in atherogenesis. We investigated the effect of MCP-1 on differentiated and dedifferentiated SMC. Differentiation of SMC was induced using Matrigel as a matrix for cultivation. MCP-1 was expressed in SMC by means of a recombinant adenovirus. Expression of MCP-1 led to dedifferentiation of SMC as demonstrated by induction of cytokeratin 18, a marker for the synthetic phenotype. Concurrently, migration was only detectable in MCP-1 expressing cells, whereas SMC infected with a control virus, coding for the nuclear-targeted lacZ gene showed no migration. The expression of intercellular adhesion molecule-1 (ICAM-1) could be demonstrated in synthetic SMC and was induced after infection of differentiated cells with recombinant adenovirus, coding for MCP-1 (AdMCP-1). Expression of ICAM-1 was associated with a tenfold higher monocyte binding compared to lacZ infected cells. Our data suggest that MCP-1 plays an important role for SMC in the functional switch from the contractile to the synthetic phenotype in the course of atherogenesis.

An unusual C2-domain in the active-zone protein piccolo: implications for Ca2+ regulation of neurotransmitter release

Ca2+ regulation of neurotransmitter release is thought to require multiple Ca2+ sensors with distinct affinities. However, no low‐affinity Ca2+ sensor has been identified at the synapse. We now show that piccolo/aczonin, a recently described active‐zone protein with C‐terminal C2A‐ and C2B‐domains, constitutes a presynaptic low‐affinity Ca2+ sensor. Ca2+ binds to piccolo by virtue of its C2A‐domain via an unusual mechanism that involves a large conformational change. The distinct Ca2+‐binding properties of the piccolo C2A‐ domain are mediated by an evolutionarily conserved sequence at the bottom of the C2A‐domain, which may fold back towards the Ca2+‐binding sites on the top. Point mutations in this bottom sequence inactivate it, transforming low‐affinity Ca2+ binding (100–200 μM in the presence of phospholipids) into high‐affinity Ca2+ binding (12–14 μM). The unusual Ca2+‐binding mode of the piccolo C2A‐domain reveals that C2‐domains are mechanistically more versatile than previously envisaged. The low Ca2+ affinity of the piccolo C2A‐domain suggests that piccolo could function in short‐term synaptic plasticity when Ca2+ concentrations accumulate during repetitive stimulation.

Injection of Nerve Growth Factor Into Stellate Ganglia Improves Norepinephrine Reuptake Into Failing Hearts

An impairment of cardiac norepinephrine reuptake through the neuronal norepinephrine transporter promotes depletion of cardiac norepinephrine stores and local cardiac sympathetic activation in heart failure. Nerve growth factor regulates differentiation and survival of adult sympathetic cells and is decreased in failing hearts. We hypothesized that injection of nerve growth factor into stellate ganglia normalizes cardiac norepinephrine homeostasis in experimental heart failure. Rats with transverse aortic constriction characterized by heart failure, depleted cardiac norepinephrine stores, and impaired cardiac norepinephrine reuptake were used as an experimental model. Nerve growth factor (20 &mgr;g) or saline was directly injected into left stellate ganglia 4 weeks after transverse aortic constriction. Thirty-two hours after injection, determinants of cardiac norepinephrine homeostasis were measured. As compared with saline, nerve growth factor refilled depleted cardiac norepinephrine stores and improved cardiac [3H]-norepinephrine uptake into isolated perfused hearts of transverse aortic constricted rats. In addition, pharmacological blockade of the norepinephrine transporter led to a higher increase in the overflow of endogenous norepinephrine from hearts of nerve growth factor-injected than saline-injected transverse aortic constricted rats. Norepinephrine transporter mRNA levels and the density of cardiac sympathetic nerves were not changed. Thirty-two hours after nerve growth factor injection, echocardiography revealed an increase in fractional shortening as compared with 2 days before injection. In conclusion, nerve growth factor attenuates local cardiac sympathetic overdrive of hypertrophic hearts by improving cardiac norepinephrine reuptake and might represent a novel therapeutic principle in the treatment of heart failure.

Nicotine-induced exocytotic norepinephrine release in guinea-pig heart, human atrium and bovine adrenal chromaffin cells: Modulation by single components of ischaemia

The influence of single components of myocardial ischaemia, such as anoxia, substrate withdrawal, hyperkalemia and extracellular acidosis, on nicotine-induced norepinephrine (NE) release was investigated in the isolated perfused guinea-pig heart, in incubated human atrial tissue and in cultured bovine adrenal chromaffin cells (BCC). In normoxia, nicotine (1-1000 mumol/l) evoked a concentration-dependent release of NE (determined by high pressure liquid chromatography and electrochemical detection) from guinea-pig heart and human atrium. In contrast to selective anoxia (Po2 < 5 mmHg) or glucose withdrawal, respectively, anoxia in combination with glucose withdrawal (5-40 min) markedly potentiated nicotine-induced NE release both in guinea-pig heart and human atrium. The sensitization of cardiac sympathetic nerve endings to nicotine was characterized by a lower threshold concentration and an approximate two-fold increase of maximum NE release, peaking after 10 min of anoxia and glucose withdrawal. Cyanide intoxication (1 mmol/l) combined with glucose withdrawal resulted in a similar increase of nicotine-induced sympathetic transmitter release both in guinea-pig heart and human atrium. In contrast, the nicotine-induced (10 mumol/l) NE overflow was only slightly potentiated by 10 min of global ischaemia in guinea-pig heart. Both hyperkalemia ([K+] 16 mmol/l) and acidosis (pH 6.8-6.0) distinctly attenuated the stimulatory effect of nicotine in guinea-pig heart and human atrium under normoxic conditions. Consistent with an exocytotic release mechanism, NE release was dependent on the presence of extracellular calcium under all conditions tested. Furthermore, NE overflow from guinea-pig heart was accompanied by a release of the exocytosis marker neuropeptide Y (NPY; determined by radioimmunoassay). In BCC, nicotine (1-10 mumol/l) evoked a release of NE and NPY and a transient rise of [Ca2+]i (determined with fura-2) during normoxia which were both dependent on the presence of extracellular calcium. Both hyperkalemia and acidosis markedly reduced the exocytotic release of sympathetic transmitters and the corresponding [Ca2+]i-transients. These data demonstrate that nicotine-induced cardiac exocytotic NE release is markedly potentiated during short-term anoxia in combination with glucose withdrawal. In contrast, a brief period of ischaemia causes only a slight sensitization of cardiac sympathetic nerve endings to nicotine. This discrepancy may be due to an attentuation of nicotine-evoked NE release by hyperkalemia and by acidosis. The protective effect of these factors against anoxia-induced sensitization to nicotine appears to be related to the inhibition of nicotine-evoked [Ca2+]i-transients.