Elevated synaptic vesicle release probability in synaptophysin/gyrin family quadruple knockouts.

Synaptophysins 1 and 2 and synaptogyrins 1 and 3 constitute a major family of synaptic vesicle membrane proteins. Unlike other widely expressed synaptic vesicle proteins such as vSNAREs and synaptotagmins, the primary function has not been resolved. Here, we report robust elevation in the probability of release of readily releasable vesicles with both high and low release probabilities at a variety of synapse types from knockout mice missing all four family members. Neither the number of readily releasable vesicles, nor the timing of recruitment to the readily releasable pool was affected. The results suggest that family members serve as negative regulators of neurotransmission, acting directly at the level of exocytosis to dampen connection strength selectively when presynaptic action potentials fire at low frequency. The widespread expression suggests that chemical synapses may play a frequency filtering role in biological computation that is more elemental than presently envisioned. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Altered synaptobrevin-II trafficking in neurons expressing a synaptophysin mutation associated with a severe neurodevelopmental disorder.

Following exocytosis, synaptic vesicles (SVs) have to be reformed with the correct complement of proteins in the correct stoichiometry to ensure continued neurotransmission. Synaptophysin is a highly abundant, integral SV protein necessary for the efficient retrieval of the SV SNARE protein, synaptobrevin II (sybII). However the molecular mechanism underpinning synaptophysin-dependent sybII retrieval is still unclear. We recently identified a male patient with severe intellectual disability, hypotonia, epilepsy and callosal agenesis who has a point mutation in the juxtamembrane region of the fourth transmembrane domain of synaptophysin (T198I). This mutation had no effect on the activity-dependent retrieval of synaptophysin that was tagged with the genetically-encoded pH-sensitive reporter (pHluorin) in synaptophysin knockout hippocampal cultures. This suggested the mutant has no global effect on SV endocytosis, which was confirmed when retrieval of a different SV cargo (the glutamate transporter vGLUT1) was examined. However neurons expressing this T198I mutant did display impaired activity-dependent sybII retrieval, similar to that observed in synaptophysin knockout neurons. Interestingly this impairment did not result in an increased stranding of sybII at the plasma membrane. Screening of known human synaptophysin mutations revealed a similar presynaptic phenotype between T198I and a mutation found in X-linked intellectual disability. Thus this novel human synaptophysin mutation has revealed that aberrant retrieval and increased plasma membrane localisation of SV cargo can be decoupled in human disease.

X-linked intellectual disability-associated mutations in synaptophysin disrupt synaptobrevin II retrieval.

Synaptophysin is an integral synaptic vesicle (SV) protein that accounts for ∼10% of total SV protein cargo. Deletion of synaptophysin results in the defective retrieval of synaptobrevin II (sybII) from the plasma membrane during endocytosis, coupled with a slowing in the speed of endocytosis. Synaptophysin has been implicated in X-linked intellectual disability, with a recent study identifying four separate synaptophysin gene mutations in families affected by the disorder. To determine how these mutations may affect synaptophysin function, we expressed them in cultured neurons derived from synaptophysin knock-out mice. Two distinct truncating mutants were mislocalized throughout the axon and phenocopied the arrest of sybII retrieval in synaptophysin knock-out cultures. The remaining two mutants displayed a nerve terminal localization but did not support efficient sybII retrieval. Interestingly, one mutant fully rescued SV endocytosis kinetics, suggesting that sybII retrieval and endocytosis speed are independent from each other. These studies suggest that the efficient retrieval of sybII by synaptophysin may be key to maintaining synaptic health and perturbation of this event may contribute to the pathogenesis underlying neurodevelopmental disorders such as X-linked intellectual disability.

Synaptophysin regulates the kinetics of synaptic vesicle endocytosis in central neurons.

Despite being the most abundant synaptic vesicle membrane protein, the function of synaptophysin remains enigmatic. For example, synaptic transmission was reported to be completely normal in synaptophysin knockout mice; however, direct experiments to monitor the synaptic vesicle cycle have not been carried out. Here, using optical imaging and electrophysiological experiments, we demonstrate that synaptophysin is required for kinetically efficient endocytosis of synaptic vesicles in cultured hippocampal neurons. Truncation analysis revealed that distinct structural elements of synaptophysin differentially regulate vesicle retrieval during and after stimulation. Thus, synaptophysin regulates at least two phases of endocytosis to ensure vesicle availability during and after sustained neuronal activity.

Interaction assays in yeast and cultured cells confirm known and identify novel partners of the synaptic vesicle protein synaptophysin.

Synaptophysin (SYP) is a major protein of neurotransmitter-containing vesicles spanning the membrane four times and contributing to various aspects of the synaptic vesicle cycle. The split-ubiquitin yeast two-hybrid system was used to characterize molecular interactions of membrane-bound, full-length murine SYP. In this way, the known homophilic SYP-SYP association could be confirmed and heterophilic binding of SYP to other tetraspan vesicle membrane proteins of the secretory carrier-associated membrane- and synaptogyrin-type could be detected for the first time. SYP-binding was also observed for the vSNARE synaptobrevin2 and various membrane and membrane-associated proteins. Double labeling immunofluorescence microscopy of murine retina, co-immunoprecipitation experiments and fluorescence energy resonance transfer (FRET) analyses between fluorescent protein-tagged polypeptides were carried out to validate and further characterize the association of SYP with the tetraspan vesicle membrane proteins secretory carrier-associated membrane protein 1 and synaptogyrin3, with synaptobrevin2, and the newly identified binding partners phospholipase D4, stathmin-like3, Rho family GTPase2 and ADP-ribosylation factor interacting protein2. It was observed that the carboxyterminus of SYP is dispensable for association with integral membrane proteins while it is needed for binding to membrane-associated polypeptides. The latter appears to be regulated by phosphorylation, since src homology 2-domains were shown to attach to the multiple carboxyterminal phosphotyrosine residues of SYP. In conclusion, the association of SYP with different tetraspan vesicle membrane proteins suggests shared functions and the multiple other interactions identify SYP as part of a membrane platform acting as a facilitator of various steps of the synaptic vesicle cycle.

Compromised store-operated Ca2+ entry in aged skeletal muscle.

In aged skeletal muscle, changes to the composition and function of the contractile machinery cannot fully explain the observed decrease in the specific force produced by the contractile machinery that characterizes muscle weakness during aging. Since modification in extracellular Ca(2+) entry in aged nonexcitable and excitable cells has been recently identified, we evaluated the functional status of store-operated Ca(2+) entry (SOCE) in aged mouse skeletal muscle. Using Mn(2+) quenching of Fura-2 fluorescence and confocal-microscopic imaging of Ca(2+) movement from the transverse tubules, we determined that SOCE was severely compromised in muscle fibers isolated from aged mice (26-27 months) as compared with those from young (2-5 months) mice. While reduced SOCE in aged skeletal muscle does not appear to result from altered expression levels of STIM1 or reduced expression of mRNA for Orai, this reduction in SOCE is mirrored in fibers isolated from young mice null for mitsugumin-29, a synaptophysin-related protein that displays decreased expression in aged skeletal muscle. Our data suggest that decreased mitsugumin-29 expression and reduced SOCE may contribute to the diminished intracellular Ca(2+) homeostatic capacity generally associated with muscle aging.

When sparks get old.

Sparks are transient local elevations of Ca ion concentration observed in different types of muscle cells. Such local Ca2+ signals can be provoked in skeletal muscle cells by altering the osmotic pressure of the extracellular solution. In this issue, Weisleder et al. (see p. 639) demonstrate that the Ca2+ response to osmotic stress is substantially altered in aged muscle. The study presents evidence for a link between this finding and a reduced expression of mitsugumin 29 (MG29), a small membrane protein of the sarcoplasmic reticulum (SR).

Transcriptome comparison of murine wild-type and synaptophysin-deficient retina reveals complete identity.

Loss of synaptophysin, one of the major synaptic vesicle membrane proteins, is surprisingly well tolerated in knockout mice. To test whether compensatory gene transcription accounts for the apparent lack of functional deficiencies, comparative transcriptome analyses were carried out. The retina was selected as the most suitable tissue since morphological alterations were observed in mutant photoreceptors, most notably a reduction of synaptic vesicles and concomitant increase in clathrin-coated vesicles. Labeled cRNA was prepared in triplicate from retinae of age- and sex-matched wild-type and mutant litter mates and hybridized to high-density microarray chips. Only three differentially expressed RNAs were identified in this way, one of which was synaptophysin. Further validation by quantitative RT-PCR could only corroborate the results for the latter. We therefore conclude that, despite the distinct morphological phenotype, no significant changes in gene expression are detectable in synaptophysin-deficient animals and that therefore compensatory mechanisms are either pre-existent and/or act at the posttranscriptional level.

Loss of synaptophysin-positive boutons on lumbar motor neurons innervating the medial gastrocnemius muscle of the SOD1G93A G1H transgenic mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a common form of motor neuron disease (MND) that involves both upper and lower nervous systems. In the SOD1G93A G1H transgenic mouse, a widely used animal model of human ALS, a significant pathology is linked to the degeneration of lower motor neurons in the lumbar spinal cord and brainstem. In the current study, the number of presynaptic boutons immunoreactive for synaptophysin was estimated on retrogradely labeled soma and proximal dendrites of alpha and gamma motor neurons innervating the medial gastrocnemius muscle. No changes were detected on both soma and proximal dendrites at postnatal day 60 (P60) of alpha and gamma motor neurons. By P90 and P120, however, alpha motor neuron soma had a reduction of 14 and 33% and a dendritic reduction of 19 and 36%, respectively. By P90 and P120, gamma motor neuron soma had a reduction of 17 and 41% and a dendritic reduction of 19 and 35%, respectively. This study shows that levels of afferent innervation significantly decreased on surviving alpha and gamma motor neurons that innervate the medial gastrocnemius muscle. This finding suggests that the loss of motor neurons and the decrease of synaptophysin in the remaining motor neurons could lead to functional motor deficits, which may contribute significantly to the progression of ALS/MND.

Activation of c-Jun N-terminal kinase and p38 in an Alzheimer's disease model is associated with amyloid deposition.

The mechanisms by which neurons and synapses are lost in Alzheimer's disease (AD) are not completely understood. To characterize potential signaling events linked to AD pathogenesis, activation-specific antibodies were used to examine mitogen-activated protein kinase (MAPK) kinase pathways at various ages in mice transgenic for human amyloid precursor protein-695 with the Swedish familial AD mutations (Tg2576) and homozygous for a P264L familial AD mutation introduced by targeting of the presenilin-1 gene (PS1(P264L)). Although the c-Jun N-terminal kinase (JNK) and p38 pathways were significantly activated in the cortex at both 7 and 12 months of age, there was no significant activation of the extracellular signal-regulated kinase pathway. MAPK kinase-4, an upstream activator of JNK, was also significantly activated at 7 and 12 months, whereas c-Jun, a downstream effector of JNK-associated apoptotic signaling, was not induced. The lack of c-Jun activation is consistent with the absence of neuronal loss in both cortex and hippocampal CA1 at 12 months. The JNK activation was localized to amyloid deposits, within neurites containing phosphorylated tau. Synaptophysin was quantified biochemically as a measure of synaptic integrity and was significantly reduced in an age-dependent manner in the Tg2576/PS1(P264L) cortex but not in either PS1(P264L) or Tg2576 cortex. Stress-responsive MAP kinase pathways were activated in the brain of the Tg2576/PS1(P264L) AD model, and this activation was coincident with the age-dependent increase in amyloid deposition, tau phosphorylation, and loss of synaptophysin.

Synaptic vesicle alterations in rod photoreceptors of synaptophysin-deficient mice.

The abundance of the integral membrane protein synaptophysin in synaptic vesicles and its multiple possible functional contributions to transmitter exocytosis and synaptic vesicle formation stand in sharp contrast to the observed lack of defects in synaptophysin knockout mice. Assuming that deficiencies are compensated by the often coexpressed synaptophysin isoform synaptoporin, we now show that retinal rod photoreceptors, which do not synthesize synaptoporin either in wild-type or in knockout mice, are affected by the loss of synaptophysin. Multiple pale-appearing photoreceptors, as seen by electron microscopy, possess reduced cytoplasmic electron density, swollen mitochondria, an enlarged cell surface area, and, most importantly, a significantly reduced number of synaptic vesicles with an unusually bright interior. Quantification of the number of synaptic vesicles per unit area, not only in these, but also in all other rod terminals of knockout animals, reveals a considerable reduction in vesicles that is even more pronounced during the dark period, i.e., at times of highest synaptic activity. Moreover, activity-dependent reduction in synaptic vesicle diameter, typically occurring in wild-type mice, is not detected in knockout animals. The large number of clathrin-coated pits and vesicles in dark-adapted synaptophysin knockout mice is taken as an indication of compensatory usage of synaptophysin-independent pathway(s), and, conversely, in view of the overall reduction in the number of synaptic vesicles, as an indication for the presence of another synaptophysin-dependent synaptic vesicle recycling pathway. Our results provide in vivo evidence for the importance of the integral membrane protein synaptophysin for synaptic vesicle recycling and formation.

Increased susceptibility to fatigue of slow- and fast-twitch muscles from mice lacking the MG29 gene.

Mitsugumin 29 (MG29), a major protein component of the triad junction in skeletal muscle, has been identified to play roles in the formation of precise junctional membrane structures important for efficient signal conversion in excitation-contraction (E-C) coupling. We carried out several experiments to not only study the role of MG29 in normal muscle contraction but also to determine its role in muscle fatigue. We compared the in vitro contractile properties of three muscles types, extensor digitorum longus (EDL) (fast-twitch muscle), soleus (SOL) (slow-twitch muscle), and diaphragm (DPH) (mixed-fiber muscle), isolated from mice lacking the MG29 gene and wild-type mice prior to and after fatigue. Our results indicate that the mutant EDL and SOL muscles, but not DPH, are more susceptible to fatigue than the wild-type muscles. The mutant muscles not only fatigued to a greater extent but also recovered significantly less than the wild-type muscles. Following fatigue, the mutant EDL and SOL muscles produced lower twitch forces than the wild-type muscles; in addition, fatiguing produced a downward shift in the force-frequency relationship in the mutant mice compared with the wild-type controls. Our results indicate that fatiguing affects the E-C components of the mutant EDL and SOL muscles, and the effect of fatigue in these mutant muscles could be primarily due to an alteration in the intracellular Ca homeostasis.

Abnormal features in skeletal muscle from mice lacking mitsugumin29.

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca(2+) release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca(2+)-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.

Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin I.

We have generated mice lacking synaptogyrin I and synaptophysin I to explore the functions of these abundant tyrosine-phosphorylated proteins of synaptic vesicles. Single and double knockout mice were alive and fertile without significant morphological or biochemical changes. Electrophysiological recordings in the hippocampal CA1 region revealed that short-term and long-term synaptic plasticity were severely reduced in the synaptophysin/synaptogyrin double knockout mice. LTP was decreased independent of the induction protocol, suggesting that the defect in LTP was not caused by insufficient induction. Our data show that synaptogyrin I and synaptophysin I perform redundant and essential functions in synaptic plasticity without being required for neurotransmitter release itself.

Age-related cognitive deficits, impaired long-term potentiation and reduction in synaptic marker density in mice lacking the beta-amyloid precursor protein.

Mutations in the beta-amyloid precursor protein are strongly associated with some cases of familial Alzheimer's disease. The normal physiological role of beta-amyloid precursor protein in the brain was evaluated in a cross-sectional analysis of mice deficient in beta-amyloid precursor protein. Compared with wild-type control mice the beta-amyloid precursor protein-null mice developed age-dependent deficits in cognitive function and also had impairments in long-term potentiation. In addition, the brains of the beta-amyloid precursor protein-null mice had marked reactive gliosis in many areas, especially in the cortex and hippocampus. A subpopulation of mice (n = 15) died prematurely (between three and 18 months of age). Analysis of another six mice from the same population that were showing weight loss and hypolocomotor activity exhibited a marked reactive gliosis as detected by immunoreactivity for glial fibrillary acidic protein and a profound loss of immunoreactivities for the presynaptic terminal vesicle marker proteins synaptophysin and synapsin and the dendritic marker microtubule-associated protein-2 in many brain areas, but most predominantly in the cortex and hippocampus. These results suggest that normal beta-amyloid precursor protein may serve an essential role in the maintenance of synaptic function during ageing. A compromise of this function of the beta-amyloid precursor protein may contribute to the progression of the memory decline and the neurodegenerative changes seen in Alzheimer's disease.

Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release.

Synaptophysin (syp I) is a synaptic vesicle membrane protein that constitutes approximately 7% of the total vesicle protein. Multiple lines of evidence implicate syp I in a number of nerve terminal functions. To test these, we have disrupted the murine Syp I gene. Mutant mice lacking syp I were viable and fertile. No changes in the structure and protein composition of the mutant brains were observed except for a decrease in synaptobrevin/VAMP II. Synaptic transmission was normal with no detectable changes in synaptic plasticity or the probability of release. Our data demonstrate that one of the major synaptic vesicle membrane proteins is not essential for synaptic transmission, suggesting that its function is either redundant or that it has a more subtle function not apparent in the assays used.

Mice lacking synaptophysin reproduce and form typical synaptic vesicles.

Synaptophysin is one of the major integral membrane proteins of the small (30-50nm diameter) electron-translucent transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells. Since its expression is tightly linked to the occurrence of these vesicle types, we mutated the X-chromosomally located synaptophysin gene in embryonic stem cells for the generation of synaptophysin-deficient mice in order to study the consequence of synaptophysin ablation for the formation and function of such vesicles in vivo. The behavior and appearance of mice lacking synaptophysin was indistinguishable from that of their litter mates and reproductive capacity was comparable to normal mice. Furthermore, no drastic compensatory changes were noted in the expression of several other neuronal polypeptides or in the mRNA levels of synaptophysin isoforms, the closely related neuronal synaptoporin/synaptophysinII, and the ubiquitous pantophysin. Immunofluorescence microscopy of several neuronal and neuroendocrine tissues showed that overall tissue architecture was maintained in the absence of synaptophysin, and that the distribution of other synaptic vesicle components was not visibly affected. In electron-microscopic preparations, large numbers of vesicles with a diameter of 39.9nm and an electron-translucent interior were seen in synaptic regions of synaptophysin-deficient mice; these vesicles could be labeled by antibodies against synaptic vesicle proteins, such as synaptobrevin 2.