Comparative structural analysis of neuromuscular junctions in mice at different ages

The authors studied the histochemical and ultrastructural modifications that occur in the neuromuscular junctions (NMJ) of fibularis longus muscles of mice with an age range of 3 to 21 months. Twenty-four male and female animals were killed at 3, 5, 14 and 21 months of age: 7 of them at 3 months, 4 of them at 5 month, 9 at 14 months and 4 at 21 months. The fibularis longus muscles were processed and their NMJ examined with the transmission electron microscope. The most relevant changes were associated with the degeneration and retraction of terminal axons, i.e., axons poor in synaptic vesicles with degenerated mitochondria, and exhibiting multivesicular bodies and vacuoles; exposed and widened junctional folds and cytoplasmic processes of Schwann cells located in the synaptic gutter. The presence of lysosomes or lipofuchsin in the juxtajunctional sarcoplasm was also noted. These observations suggest that the phenomena of retraction and budding occur in the NMJ with advancing age, with a predominance of events associated with degeneration, leading to profound changes in NMJ shape.

Genetische und funktionelle Analyse von Vesikelmembranprotein-Mutanten in Maus und Caenorhabditis elegans

Tetraspan vesicle membrane proteins (TVPs) sind konservierte, ubiquitär vorkommende Membranproteine synaptischer Vesikel und zytoplasmatischer Transportvesikel. Bei Säugetieren lassen sie sich in die Physine, Gyrine und SCAMPs (secretory carrier-associated membrane proteins) unterteilen, die im Nematoden C. elegans jeweils nur durch ein einzelnes Polypeptid vertreten sind (Synaptophysin-1 [SPH-1], Synaptogyrin-1 [SNG-1] und SCAMP-1 [SCM-1]). Obwohl den TVPs eine Beteiligung bei der Regulation des Vesikelzyklus zugesprochen wurde, sind Synaptophysin-1-Knockout-Mäuse und vollständig TVP-defiziente Würmer gesund und weisen nur geringgradige Veränderungen auf. In dieser Arbeit sollten daher zum einen genomweite komparative Transkriptomanalysen durchgeführt werden, um mögliche Kompensationsmechanismen in der Maus und C. elegans zu finden, zum anderen sollten mit Hilfe pharmakologischer Stressassays und genetischer Verfahren Schwachstellen und Redundanzen identifiziert werden. Erstaunlicherweise konnten durch Affymetrix GeneChip-Analysen der RNA in der Retina von Synaptophysin-1-/--Mäusen keine differenziell exprimierten Gene gefunden werden. Bei der Untersuchung der C. elegans-TVP-Dreifachmutante wurden hingegen 17 Gene mit erhöhter und 3 mit erniedrigter Transkription identifiziert. Die Befunde für 12 hochregulierte Gene wurden durch quantitative Real-Time RT-PCR bestätigt. Das am stärksten hochregulierte Gen arf-1.1 kodiert für eine GTPase, die vermutlich an der Regulation der Vesikelbildung beteiligt ist. Von den ebenso identifizierten Genen cdr-2, cdr-4 und pgp-9 ist bekannt, dass sie in Stresssituationen, z. B. in Gegenwart von Cadmium, verstärkt transkribiert werden. ugt-62 und ugt-19 kodieren für Glucuronosyltransferasen. Für arf-1.1, cdr-2, ugt-62 sowie für das Gen T16G1.6, das für eine coiled-coil-Domäne kodiert, wurden im Folgenden fluoreszierende Promoterkonstrukte hergestellt, um Koexpressionsmuster mit TVPs zu bestimmen. Es stellte sich heraus, dass alle vier Promoterkonstrukte im Darm zusammen mit SPH-1 und SCM-1 im Darm transkribiert werden. Mit fluoreszierenden Translationschimären konnte weiterhin gezeigt werden, dass ARF-1.1 und CDR-2 mit den Darm-spezifischen TVPs im apikalen Bereich der Darmzellen kolokalisieren. Um mehr über die Funktion von TVPs im Vesikelzyklus zu erfahren, wurden pharmakologische und genetische Analysen von Würmern durchgeführt, in denen die Expression des Neuronen-spezifischen SNG-1 verändert ist. Deletion oder Überexpression führte zu einer Resistenz gegenüber dem Acetylcholinesterase-Inhibitor Aldicarb und zu erhöhter Empfindlichkeit gegenüber dem GABA-Rezeptor-Antagonisten Pentylentetrazol. Auf genetischer Ebene zeigte sich, dass sng-1 synthetisch mit den Genen für Synaptotagmin-1, Endophilin A sowie Synaptojanin wirkt. Die beobachteten Effekte weisen auf alternative Funktionen in der synaptischen Übertragung hin und unterstützen zugleich die Hypothese, dass SNG-1 im synaptischen Vesikelzyklus eine wichtige Funktion erfüllt, die möglicherweise einem noch unbekannten redundanten Kompartiment-spezifischen Signalweg der synaptischen Transmission zuzuordnen ist.

Quantitative analysis of the native presynaptic cytomatrix by cryoelectron tomography

The presynaptic terminal contains a complex network of filaments whose precise organization and functions are not yet understood. The cryoelectron tomography experiments reported in this study indicate that these structures play a prominent role in synaptic vesicle release. Docked synaptic vesicles did not make membrane to membrane contact with the active zone but were instead linked to it by tethers of different length. Our observations are consistent with an exocytosis model in which vesicles are first anchored by long (>5 nm) tethers that give way to multiple short tethers once vesicles enter the readily releasable pool. The formation of short tethers was inhibited by tetanus toxin, indicating that it depends on soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor complex assembly. Vesicles were extensively interlinked via a set of connectors that underwent profound rearrangements upon synaptic stimulation and okadaic acid treatment, suggesting a role of these connectors in synaptic vesicle mobilization and neurotransmitter release.

Structural plasticity of spines at giant mossy fiber synapses

The granule cells of the dentate gyrus give rise to thin unmyelinated axons, the mossy fibers. They form giant presynaptic boutons impinging on large complex spines on the proximal dendritic portions of hilar mossy cells and CA3 pyramidal neurons. While these anatomical characteristics have been known for some time, it remained unclear whether functional changes at mossy fiber synapses such as long-term potentiation (LTP) are associated with structural changes. Since subtle structural changes may escape a fine-structural analysis when the tissue is fixed by using aldehydes and is dehydrated in ethanol, rapid high-pressure freezing (HPF) of the tissue was applied. Slice cultures of hippocampus were prepared and incubated in vitro for 2 weeks. Then, chemical LTP (cLTP) was induced by the application of 25 mM tetraethylammonium (TEA) for 10 min. Whole-cell patch-clamp recordings from CA3 pyramidal neurons revealed a highly significant potentiation of mossy fiber synapses when compared to control conditions before the application of TEA. Next, the slice cultures were subjected to HPF, cryosubstitution, and embedding in Epon for a fine-structural analysis. When compared to control tissue, we noticed a significant decrease of synaptic vesicles in mossy fiber boutons and a concomitant increase in the length of the presynaptic membrane. On the postsynaptic side, we observed the formation of small, finger-like protrusions, emanating from the large complex spines. These short protrusions gave rise to active zones that were shorter than those normally found on the thorny excrescences. However, the total number of active zones was significantly increased. Of note, none of these cLTP-induced structural changes was observed in slice cultures from Munc13-1 deficient mouse mutants showing severely impaired vesicle priming and docking. In conclusion, application of HPF allowed us to monitor cLTP-induced structural reorganization of mossy fiber synapses.

Clathrin heavy chain plays multiple roles in polarizing the Drosophila oocyte downstream of Bic-D

Bicaudal-D (Bic-D), Egalitarian (Egl), microtubules and their motors form a transport machinery that localizes a remarkable diversity of mRNAs to specific cellular regions during oogenesis and embryogenesis. Bic-D family proteins also promote dynein-dependent transport of Golgi vesicles, lipid droplets, synaptic vesicles and nuclei. However, the transport of these different cargoes is still poorly understood. We searched for novel proteins that either mediate Bic-Ddependent transport processes or are transported by them. Clathrin heavy chain (Chc) co-immunopurifies with Bic-D in embryos and ovaries, and a fraction of Chc colocalizes with Bic-D. Both proteins control posterior patterning of the Drosophila oocyte and endocytosis. Although the role of Chc in endocytosis is well established, our results show that Bic-D is also needed for the elevated endocytic activity at the posterior of the oocyte. Apart fromaffecting endocytosis indirectly by its role in osk mRNA localization, Bic-D is also required to transport Chc mRNA into the oocyte and for transport and proper localization of Chc protein to the oocyte cortex, pointing to an additional,more direct role of Bic-D in the endocytic pathway. Furthermore, similar to Bic-D, Chc also contributes to proper localization of osk mRNA and to oocyte growth. However, in contrast to other endocytic components and factors of the endocytic recycling pathway, such as Rabenosyn-5 (Rbsn-5) and Rab11, Chc is needed during early stages of oogenesis (from stage 6 onwards) to localize oskmRNA correctly.Moreover,we also uncovered a novel, presumably endocytosis-independent, role of Chc in the establishment of microtubule polarity in stage 6 oocytes.

Central and peripheral defects in motor units of the diaphragm of spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) is characterized by motoneuron loss and muscle weakness. However, the structural and functional deficits that lead to the impairment of the neuromuscular system remain poorly defined. By electron microscopy, we previously found that neuromuscular junctions (NMJs) and muscle fibres of the diaphragm are among the earliest affected structures in the severe mouse SMA model. Because of certain anatomical features, i.e. its thinness and its innervation from the cervical segments of the spinal cord, the diaphragm is particularly suitable to characterize both central and peripheral events. Here we show by immunohistochemistry that, at postnatal day 3, the cervical motoneurons of SMA mice receive less stimulatory synaptic inputs. Moreover, their mitochondria become less elongated which might represent an early stage of degeneration. The NMJs of the diaphragm of SMA mice show a loss of synaptic vesicles and active zones. Moreover, the partly innervated endplates lack S100 positive perisynaptic Schwann cells (PSCs). We also demonstrate the feasibility of comparing the proteomic composition between diaphragm regions enriched and poor in NMJs. By this approach we have identified two proteins that are significantly upregulated only in the NMJ-specific regions of SMA mice. These are apoptosis inducing factor 1 (AIFM1), a mitochondrial flavoprotein that initiates apoptosis in a caspase-independent pathway, and four and a half Lim domain protein 1 (FHL1), a regulator of skeletal muscle mass that has been implicated in several myopathies.

Mechanisms of heterosynaptic modulation of transmission in Aplysia

Heterosynaptic plasticity has received considerable attention as a means to induce and maintain cell-wide, as opposed to synapse-specific, learning-related modifications. Modulatory neurotransmitters are thought to provide the attentional and motivational state for memory formation. However, the cellular and molecular mechanisms mediating the effects of most of these modulators on synaptic plasticity and learning remain unclear. A well established system for the study of heterosynaptic plasticity is the Aplysia sensorimotor synapse, which is subject regulation by at least two neuromodulators, serotonin (5-HT) and FMRFa. ^ 5-HT engages multiple second messenger cascades to induce short- and long-term facilitation (STF and LTF, respectively) of synaptic transmission. One mechanism proposed to be involved in STF is mobilization of synaptic vesicles from a storage pool to a releasable pool. To investigate this hypothesis, we examined the involvement of the protein synapsin, a central element in the regulation of the storage pool of vesicles in nerve terminals, in STF. 5-HT induced phosphorylation of synapsin and modified its subcellular distribution via PKA and p42/44 MAPK. Electrophysiological experiments and computer simulations suggested that synapsin can support heterosynaptic plasticity by regulating vesicle mobilization. ^ FMRFa induce short- and long-term synaptic depression in Aplysia . Long-term depression (LTD) correlates with morphological changes, the mechanisms of which remain elusive. LTD is also transcription- and translation-dependent, but little is known about the genes expressed and their regulation. We investigated the role of protein degradation via the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by the transcription factor CREB2, which is generally regarded as a transcription repressor. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions. ^ These and additional studies on the interaction of the 5-HT and FMRFa-activated pathways suggest that different neuromodulators, by activating several and sometimes overlapping signaling cascades, can exercise bidirectional control on synaptic gain and information processing.^

Quantitative fine-structural analysis of olfactory cortical synapses

To determine the extent to which hippocampal synapses are typical of those found in other cortical regions, we have carried out a quantitative analysis of olfactory cortical excitatory synapses, reconstructed from serial electron micrograph sections of mouse brain, and have compared these new observations with previously obtained data from hippocampus. Both superficial and deep layer I olfactory cortical synapses were studied. Although individual synapses in each of the areas—CA1 hippocampus, olfactory cortical layer Ia, olfactory cortical area Ib—might plausibly have been found in any of the other areas, the average characteristics of the three synapse populations are distinct. Olfactory cortical synapses in both layers are, on average, about 2.5 times larger than their hippocampal counterparts. The layer Ia olfactory cortical synapses have fewer synaptic vesicles than do the layer Ib synapses, but the absolute number of vesicles docked to the active zone in the layer Ia olfactory cortical synapses is about equal to the docked vesicle number in the smaller hippocampal synapses. As would be predicted from studies on hippocampus that relate paired-pulse facilitation to the number of docked vesicles, the synapses in layer 1a exhibit facilitation, whereas the ones in layer 1b do not. Although hippocampal synapses provide as a good model system for central synapses in general, we conclude that significant differences in the average structure of synapses from one cortical region to another exist, and this means that generalizations based on a single synapse type must be made with caution.

A third member of the synapsin gene family

Synapsins are a family of neuron-specific synaptic vesicle-associated phosphoproteins that have been implicated in synaptogenesis and in the modulation of neurotransmitter release. In mammals, distinct genes for synapsins I and II have been identified, each of which gives rise to two alternatively spliced isoforms. We have now cloned and characterized a third member of the synapsin gene family, synapsin III, from human DNA. Synapsin III gives rise to at least one protein isoform, designated synapsin IIIa, in several mammalian species. Synapsin IIIa is associated with synaptic vesicles, and its expression appears to be neuron-specific. The primary structure of synapsin IIIa conforms to the domain model previously described for the synapsin family, with domains A, C, and E exhibiting the highest degree of conservation. Synapsin IIIa contains a novel domain, termed domain J, located between domains C and E. The similarities among synapsins I, II, and III in domain organization, neuron-specific expression, and subcellular localization suggest a possible role for synapsin III in the regulation of neurotransmitter release and synaptogenesis. The human synapsin III gene is located on chromosome 22q12–13, which has been identified as a possible schizophrenia susceptibility locus. On the basis of this localization and the well established neurobiological roles of the synapsins, synapsin III represents a candidate gene for schizophrenia.

A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA

The topology of signal transduction is particularly important for neurons. Neurotrophic factors such as nerve growth factor (NGF) interact with receptors at distal axons and a signal is transduced by retrograde transport to the cell body to ensure survival of the neuron. We have discovered an organelle that may account for the retrograde transport of the neurotrophin signal. This organelle is derived from endocytosis of the receptor tyrosine kinase for NGF, TrkA. In vitro reactions containing semi-intact PC12 cells and ATP were used to enhance recovery of a novel organelle: small vesicles containing internalized NGF bound to activated TrkA. These vesicles were distinct from clathrin coated vesicles, uncoated primary endocytic vesicles, and synaptic vesicles, and resembled transport vesicles in their sedimentation velocity. They contained 10% of the total bound NGF and almost one-third of the total tyrosine phosphorylated TrkA. These small vesicles are compelling candidates for the organelles through which the neurotrophin signal is conveyed down the axon.

VMAT2 knockout mice: Heterozygotes display reduced amphetamine-conditioned reward, enhanced amphetamine locomotion, and enhanced MPTP toxicity

The brain vesicular monoamine transporter (VMAT2) pumps monoamine neurotransmitters and Parkinsonism-inducing dopamine neurotoxins such as 1-methyl-4-phenyl-phenypyridinium (MPP+) from neuronal cytoplasm into synaptic vesicles, from which amphetamines cause their release. Amphetamines and MPP+ each also act at nonvesicular sites, providing current uncertainties about the contributions of vesicular actions to their in vivo effects. To assess vesicular contributions to amphetamine-induced locomotion, amphetamine-induced reward, and sequestration and resistance to dopaminergic neurotoxins, we have constructed transgenic VMAT2 knockout mice. Heterozygous VMAT2 knockouts are viable into adult life and display VMAT2 levels one-half that of wild-type values, accompanied by smaller changes in monoaminergic markers, heart rate, and blood pressure. Weight gain, fertility, habituation, passive avoidance, and locomotor activities are similar to wild-type littermates. In these heterozygotes, amphetamine produces enhanced locomotion but diminished behavioral reward, as measured by conditioned place preference. Administration of the MPP+ precursor N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to heterozygotes produces more than twice the dopamine cell losses found in wild-type mice. These mice provide novel information about the contributions of synaptic vesicular actions of monoaminergic drugs and neurotoxins and suggest that intact synaptic vesicle function may contribute more to amphetamine-conditioned reward than to amphetamine-induced locomotion.

Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity

Synapsin I is a synaptic vesicle-associated phosphoprotein that has been implicated in the formation of presynaptic specializations and in the regulation of neurotransmitter release. The nonreceptor tyrosine kinase c-Src is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity. Using overlay, affinity chromatography, and coprecipitation assays, we have now shown that synapsin I is the major binding protein for the Src homology 3 (SH3) domain of c-Src in highly purified synaptic vesicle preparations. The interaction was mediated by the proline-rich domain D of synapsin I and was not significantly affected by stoichiometric phosphorylation of synapsin I at any of the known regulatory sites. The interaction of purified c-Src and synapsin I resulted in a severalfold stimulation of tyrosine kinase activity and was antagonized by the purified c-Src-SH3 domain. Depletion of synapsin I from purified synaptic vesicles resulted in a decrease of endogenous tyrosine kinase activity. Portions of the total cellular pools of synapsin I and Src were coprecipitated from detergent extracts of rat brain synaptosomal fractions using antibodies to either protein species. The interaction between synapsin I and c-Src, as well as the synapsin I-induced stimulation of tyrosine kinase activity, may be physiologically important in signal transduction and in the modulation of the function of axon terminals, both during synaptogenesis and at mature synapses.

Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion

The membrane protein syntaxin participates in several protein–protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, we microinjected into the squid giant presynaptic terminal botulinum toxin C1, which cleaves syntaxin, and the H3 domain of syntaxin, which mediates binding to other proteins. Both reagents inhibited synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. Recombinant H3 domain inhibited the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse.

Ca2+-dependent and -independent interactions of the isoforms of the α1A subunit of brain Ca2+ channels with presynaptic SNARE proteins

Fast neurotransmission requires that docked synaptic vesicles be located near the presynaptic N-type or P/Q-type calcium channels. Specific protein–protein interactions between a synaptic protein interaction (synprint) site on N-type and P/Q-type channels and the presynaptic SNARE proteins syntaxin, SNAP-25, and synaptotagmin are required for efficient, synchronous neurotransmitter release. Interaction of the synprint site of N-type calcium channels with syntaxin and SNAP-25 has a biphasic calcium dependence with maximal binding at 10–20 μM. We report here that the synprint sites of the BI and rbA isoforms of the α1A subunit of P/Q-type Ca2+ channels have different patterns of interactions with synaptic proteins. The BI isoform of α1A specifically interacts with syntaxin, SNAP-25, and synaptotagmin independent of Ca2+ concentration and binds with high affinity to the C2B domain of synaptotagmin but not the C2A domain. The rbA isoform of α1A interacts specifically with synaptotagmin and SNAP-25 but not with syntaxin. Binding of synaptotagmin to the rbA isoform of α1A is Ca2+-dependent, with maximum affinity at 10–20 μM Ca2+. Although the rbA isoform of α1A binds well to both the C2A and C2B domains of synaptotagmin, only the interaction with the C2A domain is Ca2+-dependent. These differential, Ca2+-dependent interactions of Ca2+ channel synprint sites with SNARE proteins may modulate the efficiency of transmitter release triggered by Ca2+ influx through these channels.

GS32, a Novel Golgi SNARE of 32 kDa, Interacts Preferentially with Syntaxin 6

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST–syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.