The hybrid between the ABC domains of synapsin and the B subunit of Escherichia coli heat-labile toxin ameliorates experimental autoimmune encephalomyelitis.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) acts as efficient mucosal carrier for conjugated antigens. We expressed two heterologous proteins using E. coli as a host: a hybrid consisting of LTB and the A, B and C domain of synapsin (LTBABC) and the separated ABC peptide of this synaptic protein. Refolded LTBABC and LTB bound to the GM1 receptor and internalized into CHO-K1(GM1+) cells. LTBABC showed enhanced solubility and cell binding ability respect to the former hybrid LTBSC. Several oral doses of LTBABC were administered to rats with experimental autoimmune encephalomyelitis (EAE) from induction to the acute stage of the disease. This treatment decreased disease severity, delayed type hypersensitivity reaction and lymph node cell proliferation stimulated by myelin basic protein. Amelioration of EAE was also associated with modulation of the Th1/Th2 cytokine ratio, increased TGF-ß secretion in mesenteric lymph nodes as well as expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cell population. These results indicate that the fusion protein LTBABC is suitable for further exploration of its therapeutic effect on EAE development.

Myogenesis in the thoracic limbs of the American lobster.

Newly hatched lobster larvae have biramous thoracic limbs composed of an endopodite, which is used for walking in the adult, and an exopodite used for swimming. Several behavioural and physiological aspects of larval locomotion as well the ontogeny of the neuromuscular system have been examined in developing decapod crustaceans. Nevertheless, the cellular basis of embryonic muscle formation in these animals is poorly understood. Therefore, the present report analyses muscle formation in embryos of the American lobster Homarus americanus Milne Edwards, 1837 (Malacostraca, Eucarida, Decapoda, Homarida) using the monoclonal antibody 016C6 that recognizes an isoform of myosin heavy chain. 016C6 labelling at 25% of embryonic development (E25%) revealed that syncytial muscle precursor cells establish the muscles in the endopodites. During subsequent embryogenesis, these muscle precursors subdivide into several distinct units thereby giving rise to pairs of antagonistic primordial muscles in each of the successive podomeres, the layout of which at E45% already resembles the arrangement in the adult thoracopods. The pattern of primordial muscles was also mapped in the exopodites of thoracic limbs three to eight. Immunohistochemistry against acetylated α-tubulin and against presynaptic vesicle-associated phosphoproteins at E45% demonstrated the existence of characteristic neural tracts within the developing limbs as well as putative neuromuscular synapses in both the embryonic exo- and endopodites. The results are compared to muscle development in other Crustacea.

The conserved protein kinase-A target motif in synapsin of Drosophila is effectively modified by pre-mRNA editing.

BACKGROUND: Synapsins are abundant synaptic vesicle associated phosphoproteins that are involved in the fine regulation of neurotransmitter release. The Drosophila member of this protein family contains three conserved domains (A, C, and E) and is expressed in most or all synaptic terminals. Similar to mouse mutants, synapsin knock-out flies show no obvious structural defects but are disturbed in complex behaviour, notably learning and memory. RESULTS: We demonstrate that the N-terminal phosphorylation consensus motif RRxS that is conserved in all synapsins investigated so far, is modified in Drosophila by pre-mRNA editing. In mammals this motif represents the target site P1 of protein kinase A (PKA) and calcium/calmodulin dependent protein kinase I/IV. The result of this editing, by which RRFS is modified to RGFS, can be observed in cDNAs of larvae and adults and in both isolated heads and bodies. It is also seen in several newly collected wild-type strains and thus does not represent an adaptation to laboratory culture conditions. A likely editing site complementary sequence is found in a downstream intron indicating that the synapsin pre-mRNA can form a double-stranded RNA structure that is required for editing by the adenosine deaminase acting on RNA (ADAR) enzyme. A deletion in the Drosophila Adar gene generated by transposon remobilization prevents this modification, proving that the ADAR enzyme is responsible for the pre-mRNA editing described here. We also provide evidence for a likely function of synapsin editing in Drosophila. The N-terminal synapsin undeca-peptide containing the genomic motif (RRFS) represents an excellent substrate for in-vitro phosphorylation by bovine PKA while the edited peptide (RGFS) is not significantly phosphorylated. Thus pre-mRNA editing by ADAR could modulate the function of ubiquitously expressed synapsin in a cell-specific manner during development and adulthood. CONCLUSION: Similar to several other neuronal proteins of Drosophila, synapsin is modified by ADAR-mediated recoding at the pre-mRNA level. This editing likely reduces or abolishes synapsin phosphorylation by PKA. Since synapsin in Drosophila is required for various forms of behavioural plasticity, it will be fascinating to investigate the effect of this recoding on learning and memory.

Phosphorylation of Numb family proteins. Possible involvement of Ca2+/calmodulin-dependent protein kinases.

To search for the substrates of Ca2+/calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr177) GST-fused CaM-KI catalytic domain (residues 1-293, K49E) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr177-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser264 in Numb and Ser304 in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numbl antibody, we observed the phosphorylation of Numb family proteins in various rat tissue extracts, and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser264 in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl-blocked dephosphorylation of Ser304. Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein.

A subtractive cDNA library from an identified regenerating neuron is enriched in sequences up-regulated during nerve regeneration.

We have constructed a subtractive cDNA library from regenerating Retzius cells of the leech, Hirudo medicinalis. It is highly enriched in sequences up-regulated during nerve regeneration. Sequence analysis of selected recombinants has identified both novel sequences and sequences homologous to molecules characterised in other species. Homologies include alpha-tubulin, a calmodulin-like protein, CAAT/enhancer-binding protein (C/EBP), protein 4.1 and synapsin. These types of proteins are exactly those predicted to be associated with axonal growth and their identification confirms the quality of the library. Most interesting, however, is the isolation of 5 previously uncharacterised cDNAs which appear to be up-regulated during regeneration. Their analysis is likely to provide new information on the molecular mechanisms of neuronal regeneration.

Re-innervation patterns of chick auditory sensory epithelium after acoustic overstimulation.

There is evidence from several studies showing that sensory cells which are destroyed by trauma in the chick auditory epithelium are replaced by new cells. The fate of neurons that innervate the injured and degenerating sensory cells in the lesion, and the temporal sequence of re-innervation of regenerated hair cells are not well understood. This study examined efferent terminals in the chick auditory sensory epithelium following acoustic overstimulation using synapsin-specific immunocytochemistry. Chicks were exposed to an octave band noise (1.5 kHz center frequency, 116 dB SPL, 16 h) and killed on each day from 0 to 9 days postexposure. In the proximal half of control whole mounts of the basilar papillae, synapsin-specific immunoreactivity stained efferent terminals throughout the abneural portion of the sensory epithelium (the short hair cell region). In this area, the labeling appeared as 2-3 bouton-shaped clusters along the abneural edge of each hair cell. After acoustic overstimulation, a lesion was observed at the abneural edge of the papilla where many short hair cells were lost. The center of the lesion was located at 40% distance from the proximal end of each traumatized papilla. Synapsin-specific labeling was not found in sites where expanded supporting cells had replaced missing hair cells. Hair cells which survived the trauma exhibited a shrunken apical area, and synapsin-labeled boutons were observed near their basal domains. New hair cells, which first appeared in the papilla 4 days after trauma, were not initially associated with synapsin-labeled boutons. Regenerated hair cells first displayed contacts with synapsin-labeled boutons 7 days after trauma. Nine days after acoustic overstimulation, most new hair cells appeared to be associated with synapsin-labeled boutons which resembled the normal horseshoe configuration of efferent terminals. The data suggest that direct contact with functional efferent synapses is not necessary for the generation and differentiation of new hair cells.

Interactions of synapsin I with phospholipids: possible role in synaptic vesicle clustering and in the maintenance of bilayer structures.

Synapsin I is a synaptic vesicle-specific phosphoprotein composed of a globular and hydrophobic head and of a proline-rich, elongated and basic tail. Synapsin I binds with high affinity to phospholipid and protein components of synaptic vesicles. The head region of the protein has a very high surface activity, strongly interacts with acidic phospholipids and penetrates the hydrophobic core of the vesicle membrane. In the present paper, we have investigated the possible functional effects of the interaction between synapsin I and vesicle phospholipids. Synapsin I enhances both the rate and the extent of Ca(2+)-dependent membrane fusion, although it has no detectable fusogenic activity per se. This effect, which appears to be independent of synapsin I phosphorylation and localized to the head region of the protein, is attributable to aggregation of adjacent vesicles. The facilitation of Ca(2+)-induced liposome fusion is maximal at 50-80% of vesicle saturation and then decreases steeply, whereas vesicle aggregation does not show this biphasic behavior. Association of synapsin I with phospholipid bilayers does not induce membrane destabilization. Rather, 31P-nuclear magnetic resonance spectroscopy demonstrated that synapsin I inhibits the transition of membrane phospholipids from the bilayer (L alpha) to the inverted hexagonal (HII) phase induced either by increases in temperature or by Ca2+. These properties might contribute to the remarkable selectivity of the fusion of synaptic vesicles with the presynaptic plasma membrane during exocytosis.