Jan Pielage

Presynaptic Spectrin Is Essential for Synapse Stabilization

BACKGROUND Precise neural circuitry is established and maintained through a regulated balance of synapse stabilization and disassembly. Currently, little is known about the molecular mechanisms that specify synapse stability versus disassembly. RESULTS Here, we demonstrate that presynaptic spectrin is an essential scaffold that is required to maintain synapse stability at the Drosophila neuromuscular junction (NMJ). Loss of presynaptic spectrin leads to synapse disassembly and ultimately to the elimination of the NMJ. Synapse elimination is documented through light-level, ultrastructural, and electrophysiological assays. These combined assays reveal that impaired neurotransmission is secondary to synapse retraction. We demonstrate that loss of presynaptic, but not postsynaptic, spectrin leads to the disorganization and elimination of essential synaptic cell-adhesion molecules. In addition, we provide evidence of altered axonal transport and disrupted synaptic microtubules as events that contribute to synapse retraction in animals lacking presynaptic spectrin. CONCLUSIONS Our data suggest that presynaptic spectrin functions as an essential presynaptic scaffold that may link synaptic cell adhesion with the stabilization of the underlying microtubule cytoskeleton.

A Presynaptic Homeostatic Signaling System Composed of the Eph Receptor, Ephexin, Cdc42 and CaV2.1 Calcium Channels

The molecular mechanisms underlying the homeostatic modulation of presynaptic neurotransmitter release remain largely unknown. In a screen, we isolated mutations in Drosophila ephexin (Rho-type guanine nucleotide exchange factor) that disrupt the homeostatic enhancement of presynaptic release following impairment of postsynaptic glutamate receptor function at the Drosophila neuromuscular junction. We show that Ephexin is sufficient presynaptically for synaptic homeostasis and localizes in puncta throughout the nerve terminal. However, ephexin mutations do not alter other aspects of neuromuscular development, including morphology or active zone number. We then show that, during synaptic homeostasis, Ephexin functions primarily with Cdc42 in a signaling system that converges upon the presynaptic CaV2.1 calcium channel. Finally, we show that Ephexin binds the Drosophila Eph receptor (Eph) and Eph mutants disrupt synaptic homeostasis. Based on these data, we propose that Ephexin/Cdc42 couples synaptic Eph signaling to the modulation of presynaptic CaV2.1 channels during the homeostatic enhancement of presynaptic release.

A Presynaptic Giant Ankyrin Stabilizes the NMJ through Regulation of Presynaptic Microtubules and Transsynaptic Cell Adhesion

In a forward genetic screen for mutations that destabilize the neuromuscular junction, we identified a novel long isoform of Drosophila ankyrin2 (ank2-L). We demonstrate that loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. We provide genetic evidence that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that we demonstrate can directly bind and organize synaptic microtubules. We propose that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal.

A postsynaptic Spectrin scaffold defines active zone size, spacing, and efficacy at the Drosophila neuromuscular junction

Synaptic connections are established with characteristic, cell type–specific size and spacing. In this study, we document a role for the postsynaptic Spectrin skeleton in this process. We use transgenic double-stranded RNA to selectively eliminate α-Spectrin, β-Spectrin, or Ankyrin. In the absence of postsynaptic α- or β-Spectrin, active zone size is increased and spacing is perturbed. In addition, subsynaptic muscle membranes are significantly altered. However, despite these changes, the subdivision of the synapse into active zone and periactive zone domains remains intact, both pre- and postsynaptically. Functionally, altered active zone dimensions correlate with an increase in quantal size without a change in presynaptic vesicle size. Mechanistically, β-Spectrin is required for the localization of α-Spectrin and Ankyrin to the postsynaptic membrane. Although Ankyrin is not required for the localization of the Spectrin skeleton to the neuromuscular junction, it contributes to Spectrin-mediated synapse development. We propose a model in which a postsynaptic Spectrin–actin lattice acts as an organizing scaffold upon which pre- and postsynaptic development are arranged.

Hts/Adducin controls synaptic elaboration and elimination.

Neural development requires both synapse elaboration and elimination, yet relatively little is known about how these opposing activities are coordinated. Here, we provide evidence Hts/Adducin can serve this function. We show that Drosophila Hts/Adducin is enriched both pre- and postsynaptically at the NMJ. We then demonstrate that presynaptic Hts/Adducin is necessary and sufficient to control two opposing processes associated with synapse remodeling: (1) synapse stabilization as determined by light level and ultrastructural and electrophysiological assays and (2) the elaboration of actin-based, filopodia-like protrusions that drive synaptogenesis and growth. Synapse remodeling is sensitive to Hts/Adducin levels, and we provide evidence that the synaptic localization of Hts/Adducin is controlled via phosphorylation. Mechanistically, Drosophila Hts/Adducin protein has actin-capping activity. We propose that phosphorylation-dependent regulation of Hts/Adducin controls the level, localization, and activity of Hts/Adducin, influencing actin-based synapse elaboration and spectrin-based synapse stabilization. Hts/Adducin may define a mechanism to switch between synapse stability and dynamics.

The Drosophila Cell Survival Gene discs lost Encodes a Cytoplasmic Codanin-1-like Protein, Not a Homolog of Tight Junction PDZ Protein Patj

The Drosophila gene discs lost (dlt) has been reported to encode a homolog of the vertebrate tight junction PDZ protein Patj, and was thought to play a role in cell polarity. Using rescue experiments and sequence analyses, we show that dlt mutations disrupt the Drosophila Codanin-1 homolog, a cytoplasmic protein, and not the PDZ protein. Mutations in human Codanin-1 are associated with congenital dyserythropoietic anemia type I (CDA I). In Drosophila, the genomic organization of dlt is unusual. dlt shares its first untranslated exon with alpha-spectrin, and both genes are coexpressed throughout development. We show that dlt is not required for cell polarity but is needed for cell survival and cell cycle progression. Finally, we present evidence that the PDZ protein previously thought to be encoded by dlt is not required for viability. We propose to rename this PDZ protein after its vertebrate homolog, Patj (Pals-associated tight junction protein).

Molecular mechanisms that enhance synapse stability despite persistent disruption of the spectrin/ankyrin/microtubule cytoskeleton

Neuromuscular junctions crippled by a disrupted microtubule cytoskeleton are rescued by stress-induced activation of MAPK-JNK-Fos signaling.

Transsynaptic Coordination of Synaptic Growth, Function, and Stability by the L1-Type CAM Neuroglian

Experiments in peripheral and central synapses reveal the regulatory mechanisms that enable trans-synaptic control of synapse development and maintenance by the L1-type CAM Neuroglian.

Agrin regulates CLASP2-mediated capture of microtubules at the neuromuscular junction synaptic membrane

Agrin regulates acetylcholine receptors at the neuromuscular junction by locally stabilizing microtubules through the plus end tracking proteins CLASP2 and CLIP-170.

Distinct functions of α-Spectrin and β-Spectrin during axonal pathfinding

Cell-shape changes during development require a precise coupling of the cytoskeleton with proteins situated in the plasma membrane. Important elements controlling the shape of cells are the Spectrin proteins that are expressed as a subcortical cytoskeletal meshwork linking specific membrane receptors with F-actin fibers. Here, we demonstrate that Drosophila karussell mutations affect β-spectrin and lead to distinct axonal patterning defects in the embryonic CNS. karussell mutants display a slit-sensitive axonal phenotype characterized by axonal looping in stage-13 embryos. Further analyses of individual, labeled neuroblast lineages revealed abnormally structured growth cones in these animals. Cell-type-specific rescue experiments demonstrate that β-Spectrin is required autonomously and non-autonomously in cortical neurons to allow normal axonal patterning. Within the cell, β-Spectrin is associated withα -Spectrin. We show that expression of the two genes is tightly regulated by post-translational mechanisms. Loss of β-Spectrin significantly reduces levels of neuronal α-Spectrin expression, whereas gain of β-Spectrin leads to an increase in α-Spectrin protein expression. Because the loss of α-spectrin does not result in an embryonic nervous system phenotype, β-Spectrin appears to act at least partially independent of α-Spectrin to control axonal patterning.