Presynaptic Active Zone Density during Development and Synaptic Plasticity

Neural circuits transmit information through synapses, and the efficiency of synaptic transmission is closely related to the density of presynaptic active zones, where synaptic vesicles are released. The goal of this review is to highlight recent insights into the molecular mechanisms that control the number of active zones per presynaptic terminal (active zone density) during developmental and stimulus-dependent changes in synaptic efficacy. At the neuromuscular junctions (NMJs), the active zone density is preserved across species, remains constant during development, and is the same between synapses with different activities. However, the NMJ active zones are not always stable, as exemplified by the change in active zone density during acute experimental manipulation or as a result of aging. Therefore, a mechanism must exist to maintain its density. In the central nervous system (CNS), active zones have restricted maximal size, exist in multiple numbers in larger presynaptic terminals, and maintain a constant density during development. These findings suggest that active zone density in the CNS is also controlled. However, in contrast to the NMJ, active zone density in the CNS can also be increased, as observed in hippocampal synapses in response to synaptic plasticity. Although the numbers of known active zone proteins and protein interactions have increased, less is known about the mechanism that controls the number or spacing of active zones. The following molecules are known to control active zone density and will be discussed herein: extracellular matrix laminins and voltage-dependent calcium channels, amyloid precursor proteins, the small GTPase Rab3, an endocytosis mechanism including synaptojanin, cytoskeleton protein spectrins and β-adducin, and a presynaptic web including spectrins. The molecular mechanisms that organize the active zone density are just beginning to be elucidated

Spotted substrates for focal presentation of proteins to cells

Neurites and other cell processes encounter localized deposits of signaling factors as they grow. The difficulty in generating patterned artificial substrates has hindered the analysis of these instructive factors in vitro. Here we report a simple method for presenting cultured cells with small spots of protein on an otherwise uniform substrate. We use a biolistic device called a gene gun to deposit 0.1-5 microm fluorescent dots of pure protein on or beneath a growth-promoting substrate. Using this technique, we demonstrate local presynaptic differentiation of motoneurons in response to dots of a polycation. We also show that biotin-avidin and antibody-antigen interactions can be used to prepare spots from more dilute, more labile, or less abundant proteins. This method should prove useful for analyzing extracellular signaling molecules that act focally on neurons or other cell types

Dual-color STED microscopy reveals a sandwich structure of Bassoon and Piccolo in active zones of adult and aged mice

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author's publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Presynaptic active zones play a pivotal role as synaptic vesicle release sites for synaptic transmission, but the molecular architecture of active zones in mammalian neuromuscular junctions (NMJs) at sub-diffraction limited resolution remains unknown. Bassoon and Piccolo are active zone specific cytosolic proteins essential for active zone assembly in NMJs, ribbon synapses, and brain synapses. These proteins are thought to colocalize and share some functions at active zones. Here, we report an unexpected finding of non-overlapping localization of these two proteins in mouse NMJs revealed using dual-color stimulated emission depletion (STED) super resolution microscopy. Piccolo puncta sandwiched Bassoon puncta and aligned in a Piccolo-Bassoon-Piccolo structure in adult NMJs. P/Q-type voltage-gated calcium channel (VGCC) puncta colocalized with Bassoon puncta. The P/Q-type VGCC and Bassoon protein levels decreased significantly in NMJs from aged mouse. In contrast, the Piccolo levels in NMJs from aged mice were comparable to levels in adult mice. This study revealed the molecular architecture of active zones in mouse NMJs at sub-diffraction limited resolution, and described the selective degeneration mechanism of active zone proteins in NMJs from aged mice. Interestingly, the localization pattern of active zone proteins described herein is similar to active zone structures described using electron microscope tomography

Molecular cloning of haploid germ cell-specific tektin cDNA and analysis of the protein in mouse testis

AbstractTektins are a class of proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules. We report here the molecular cloning of a new member of the tektin family, tektin-t, identified from a mouse haploid germ cell-specific cDNA library. Tektin-t mRNA encodes a protein of 430 deduced amino acids possessing RSNVELCRD, the conserved sequence of tektin family proteins. Western blotting showed a single band having a molecular weight of 86 kDa in the mouse testis. Immunohistochemistry of the testis showed that tektin-t is localized in the flagella of elongating spermatids from developmental step 15 to maturity

Multiple roles of integrin-α3 at the neuromuscular junction

The neuromuscular junction (NMJ) is the synapse between motoneurons and skeletal muscle, and is responsible for eliciting muscle contraction. Neurotransmission at synapses depends on the release of synaptic vesicles at sites called active zones (AZs). Various proteins of the extracellular matrix are crucial for NMJ development; however, little is known about the identity and functions of the receptors that mediate their effects. Using genetically modified mice, we find that integrin-α3 (encoded by Itga3), an adhesion receptor at the presynaptic membrane, is involved in the localisation of AZ components and efficient synaptic vesicle release. Integrin-α3 also regulates integrity of the synapse – mutant NMJs present with progressive structural changes and upregulated autophagy, features commonly observed during ageing and in models of neurodegeneration. Unexpectedly, we find instances of nerve terminal detachment from the muscle fibre; to our knowledge, this is the first report of a receptor that is required for the physical anchorage of pre- and postsynaptic elements at the NMJ. These results demonstrate multiple roles of integrin-α3 at the NMJ, and suggest that alterations in its function could underlie defects that occur in neurodegeneration or ageing

River Bed Form in a Granitic Mountainous Catchment : A Case of the Source Area in Kurose River, Hiroshima Prefecture

In order to clarify spatial variations in a river bed form in a granitic mountainous catchment, we surveyed topographically a steep river on a source area of Kurose River in which the debris flow occurred in 1999. Firstly, we found that the spatial variation in erosion-deposition process on the steep river. Both of the lower stream and upper one held much sediments with large gravels (φ>2m). On the other hand, the middle stream exposed mainly the bedrock. Secondary, we confirmed spatial distribution of river falls. The height of river fall on the upper stream rose with the increase of river gradient. On the contray, the frequency of falls was constant in spite of inclination

cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis

Promoters, including neither TATA box nor initiator, have been frequently found in testicular germ cell-specific genes in mice. These investigations imply that unique forms of the polymerase II transcription initiation machinery play a role in selective activation of germ cell-specific gene expression programs during spermatogenesis. However, there is little information about testis-specific core promoters, because useful germ cell culture system is not available. In this study, we characterize the regulatory region of the haploid-specific Oxct2b gene in detail by using in vivo transient transfection assay in combination with a transgenic approach, with electrophoretic mobility shift and chromatin immunoprecipitation assays. Expression studies using mutant constructs demonstrate that a 34 bp region, which extends from −49 to −16, acts as a core promoter in an orientation-dependent manner. This promoter region includes the cAMP-responsive element (CRE)-like sequence TGACGCAG, but contains no other motifs, such as a TATA box or initiator. The CRE-like element is indispensable for the core promoter activity, but not for activator in testicular germ cells, through the binding of a testis-specific CRE modulator transcription factor. These results indicate the presence of alternative transcriptional initiation machinery for cell-type-specific gene expression in testicular germ cells

Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction

A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that laminins play an autocrine role in promoting this transformation. Laminins containing the α4, α5, and β2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking laminin α5 and arrested in mutants lacking both α4 and α5. Analysis of chimeric laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated laminin receptor, Bcam, is dispensable. Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation

A novel non-canonical Notch signaling regulates expression of synaptic vesicle proteins in excitatory neurons

Notch signaling plays crucial roles for cellular differentiation during development through γ-secretase-dependent intramembrane proteolysis followed by transcription of target genes. Although recent studies implicate that Notch regulates synaptic plasticity or cognitive performance, the molecular mechanism how Notch works in mature neurons remains uncertain. Here we demonstrate that a novel Notch signaling is involved in expression of synaptic proteins in postmitotic neurons. Levels of several synaptic vesicle proteins including synaptophysin 1 and VGLUT1 were increased when neurons were cocultured with Notch ligands-expressing NIH3T3 cells. Neuron-specific deletion of Notch genes decreased these proteins, suggesting that Notch signaling maintains the expression of synaptic vesicle proteins in a cell-autonomous manner. Unexpectedly, cGMP-dependent protein kinase (PKG) inhibitor, but not γ-secretase inhibitor, abolished the elevation of synaptic vesicle proteins, suggesting that generation of Notch intracellular domain is dispensable for this function. These data uncover a ligand-dependent, but γ-secretase-independent, non-canonical Notch signaling involved in presynaptic protein expression in postmitotic neurons