Cloning and characterization of AASPs: novel axon-associated SH3 binding-like proteins.

Two cDNAs encoding closely related proteins were isolated from a crayfish nervous system lambdagtl0 cDNA library with a rat synapsin Ia cDNA probe. These proteins were expressed exclusively in neurons, were highly enriched in axons of the crayfish, and contained multiple, overlapping, putative Src homology 3 (SH3) binding sites. In concert with other proteins containing Src homology domains, SH3 binding proteins are thought to mediate protein-protein interactions in receptor signaling processes and with the cytoskeleton. We have named these proteins axon-associated SH3 binding-like proteins (AASPs). Except for these SH3 binding regions, which are also found in synapsins, AASPs were unlike any proteins in the database. AASPs were differentially expressed among motoneuron populations in crayfish and were found in growing axons and growth cones in culture. Affinity purified polyclonal antibodies to AASP-168 recognized immunoreactive proteins in rat and Xenopus, suggesting that AASPs may be conserved across species. Although the cellular function of AASPs is unclear at this time, they appear to be novel members of a neuron-specific SH3 binding protein family, which includes the synapsins.

Microinjection of mRNA encoding rat synapsin Ia alters synaptic physiology in identified motoneurons of the crayfish, Procambarus clarkii.

Studies of identified neurons have made important contributions to our understanding of cellular neurophysiology. We have developed a technique for modifying gene expression in identified motoneurons of the crayfish Procambarus clarkii in the isolated nervous system as well as in the intact animal through the injection of exogenously synthesized RNAs. mRNA suitable for injection was transcribed in vitro from cDNA templates cloned into a plasmid, pSEM. Initially, mRNAs encoding green fluorescent protein (GFP) and beta-galactosidase were injected into the soma of the motor giant neuron (MoG) to determine whether these mRNAs could be successfully translated into protein. Both proteins were expressed. Measurements of GFP fluorescence increase indicated that GFP mRNA was stable and translated into protein for at least 3 days postinjection. We then examined the effects of expression of GFP, AASP-168 (an endogenous crayfish axonal protein), and rat synapsin Ia on MoG synaptic physiology. The mRNA injection procedure did not appear to directly influence synaptic physiology based on the results of the AASP-168 and GFP injections. Injection of mRNA encoding rat synapsin Ia resulted in a significant increase in peak excitatory postsynaptic potential (EPSP) amplitude during repetitive stimulation. These data are consistent with previous studies that have shown that synapsin deficiency reduces synaptic vesicle numbers. The translation of mRNAs with diverse functions and species of origin suggests that this approach will prove useful for studying the function of a wide variety of endogenous and exogenous genes in identified neurons.

Experimental superficial siderosis of the central nervous system: biochemical correlates.

The pathogenesis of superficial siderosis of the central nervous system (CNS) may be examined by the repeated intracisternal injection of washed autologous red blood cells (RBC). In rabbits, the injections cause the accumulation of iron in the cytoplasm of microglial cells and astrocytes of cerebellar and cerebral cortices. Immunocytochemistry for ferritin reveals enhanced reaction product mainly in microglia but hemosiderin occurs only after extending the injections to 6 months. In an effort to determine the biochemical correlates of these morphological changes, iron, ferritin, ferritin subunits and the ferritin repressor protein (FRP) were quantitated. There was no increase of total iron or ferritin in the exposed cortical areas. However, the injections of RBC caused dramatic shifts of the relative contributions by heavy (H-) and light (L-) ferritin subunits. The initial response was a prompt increase of the H/L ratio to over 4.0 from the normal ratio near 1.0. Extended injections caused the ratio to drop to below unity, and the predominance of L-ferritin at 6 months coincided with the appearance of granular hemosiderin. This investigation also confirmed the presence of FRP in rabbit brain cytosols but the induction of experimental superficial siderosis did not change its levels or in vitro affinity for the iron-responsive element in ferritin messenger ribonucleic acid. It is proposed that the incrustation by hemosiderin which characterizes superficial siderosis of the CNS in humans occurs when prolonged exposure to hemoglobin produces persistent shifts of the H/L-ratios by accumulation of L-ferritin.

Myelin deficiency in female rats due to a mutation in the PLP gene.

Myelin deficiency (md) in female rats due to a mutation in the X-linked proteolipid protein (PLP) gene is caused by X-chromosome monosomy. Cytogenetic analysis revealed a single X karyotype [41,X(md/0)]. An immunocytochemical, electron microscopic, and biochemical study was performed on male and female md rats. The central nervous system (CNS) of the female md rat [41,X(md/0)] revealed the same total lack of PLP as the CNS of the affected male littermate [42,XY(md/Y)]. Immunocytochemistry for myelin basic protein (MBP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP) revealed "islands" of myelin sheath-like reaction product in both. Electron microscopy showed great paucity of compact myelin sheaths in 41,X(md/0) and 42,XY(md/Y). Reduced levels of MPB, MAG, and CNP were confirmed for both sexes but MAG and CNP were substantially higher in 41,X(md/0). Sexual differentiation of the brain may account for the observed differences since normal female reproductive organs are present in the md female rat.