Bruchpilot in Ribbon-Like Axonal Agglomerates, Behavioral Defects, and Early Death in SRPK79D Kinase Mutants of Drosophila

Abstract

Defining the molecular structure and function of synapses is a central theme in brain research. In Drosophila the Bruchpilot (BRP) protein is associated with T-shaped ribbons (“T-bars”) at presynaptic active zones (AZs). BRP is required for intact AZ structure and normal evoked neurotransmitter release. By screening for mutations that affect the tissue distribution of Bruchpilot, we have identified a P-transposon insertion in gene CG11489 (location 79D) which shows high homology to mammalian genes for SR protein kinases (SRPKs). SRPKs phosphorylate serine-arginine rich splicing factors (SR proteins). Since proteins expressed from CG11489 cDNAs phosphorylate a peptide from a human SR protein in vitro, we name CG11489 the Drosophila Srpk79D gene. We have characterized Srpk79D transcripts and generated a null mutant. Mutation of the Srpk79D gene causes conspicuous accumulations of BRP in larval and adult nerves. At the ultrastructural level, these correspond to extensive axonal agglomerates of electron-dense ribbons surrounded by clear vesicles. Basic synaptic structure and function at larval neuromuscular junctions appears normal, whereas life expectancy and locomotor behavior of adult mutants are significantly impaired. All phenotypes of the mutant can be largely or completely rescued by panneural expression of SRPK79D isoforms. Isoform-specific antibodies recognize panneurally overexpressed GFP-tagged SRPK79D-PC isoform co-localized with BRP at presynaptic active zones while the tagged -PB isoform is found in spots within neuronal perikarya. SRPK79D concentrations in wild type apparently are too low to be revealed by these antisera. We propose that the Drosophila Srpk79D gene characterized here may be expressed at low levels throughout the nervous system to prevent the assembly of BRP containing agglomerates in axons and maintain intact brain function. The discovery of an SR protein kinase required for normal BRP distribution calls for the identification of its substrate and the detailed analysis of SRPK function for the maintenance of nervous system integrity.

Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons

“…This kind of accumulation is rarely observed in wild type, but is also most notably associated with loss of SRPK79D (Figure 7C), a conserved serine-arginine protein kinase that localizes to NMJ terminals and negatively regulates premature active zone assembly before Bruchpilot reaches the fly NMJ (Johnson et al, 2009; Nieratschker et al, 2009). In both lrp4 and srpk79D mutants, Brp accumulation was not accompanied by focal accumulations of Synaptotagmin I, indicating that axonal trafficking is not generally impaired (Figure 7A–C) (Gindhart et al, 1998; Johnson et al, 2009; Nieratschker et al, 2009). Because of the similarity in the transverse nerve phenotypes and the role of SRPK79D at peripheral synapses, we hypothesized that LRP4 and SRPK79D could operate together in the CNS to regulate synapse number.

10.7554/eLife.27347.017Figure 7.LRP4 is required for normal synaptic SRPK79D localization in the CNS.( A–C ) Representative images of larval transverse nerves stained with antibodies to Bruchpilot (Brp, green), Synaptotagmin I (Syt I, red), and HRP (blue).

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Cooperation of Syd-1 with Neurexin synchronizes pre- with postsynaptic assembly

“…Such a cooperative scheme might be optimized for the integration of regulatory elements and protect the system from untimely and aberrant assembly. In fact, the active zone component BRP has been shown to be under constitutive phosphorylation to avoid premature assembly 35,36 .…”

Regulation of synaptic development and function by the Drosophila PDZ protein Dyschronic

“…Interestingly, in addition to the axon, BK channels are also localized at the synapse near to the active zone (AZ) (Hu et al, 2001), a crucial structural determinant of the probability of vesicle fusion (Sigrist and Schmitz, 2011). In Drosophila, the AZ is an electron-dense macromolecular structure and functions in part to tether synaptic vesicles in close proximity to VGCCs (Atwood et al, 1993;Koenig and Ikeda, 1999); recent genetic studies have uncovered a number of core constituents of the Drosophila AZ as well as proteins involved in its assembly (Johnson et al, 2009;Kaufmann et al, 2002;Kittel et al, 2006;Liu et al, 2011;Nieratschker et al, 2009;Wagh et al, 2006). For example, Bruchpilot (BRP) is a key component of T-bars, dense bodies that project intracellularly from the AZ core and connect to a subpopulation of synaptic vesicles (Atwood et al, 1993;Koenig and Ikeda, 1999;Reist et al, 1998;Wagh et al, 2006).…”