Corticosteroid effects on blood gene expression in Duchenne muscular dystrophy.

Though Deflazacort and prednisone improve clinical endpoints in Duchenne muscular dystrophy (DMD) patients, Deflazacort produces fewer side effects. As mechanisms of improvement and side effect differences remain unknown, we evaluated effects of corticosteroid administration on gene expression in blood of DMD patients. Whole blood was obtained from 14 children and adolescents with DMD treated with corticosteroids (DMD-STEROID) and 20 DMD children and adolescents naïve to corticosteroids (DMD). The DMD-STEROID group was further subdivided into Deflazacort and prednisone groups. Affymetrix U133 Plus 2.0 expression microarrays were used to evaluate mRNA expression. Expression of 524 probes changed with corticosteroids, including genes in iron trafficking and the chondroitin sulfate biosynthesis pathway. Deflazacort compared with prednisone yielded 508 regulated probes, including many involved in adipose metabolism. These genes and pathways help explain mechanisms of efficacy and side effects of corticosteroids, and could provide new treatment targets for DMD and other neuromuscular disorders.

Citron binds to PSD-95 at glutamatergic synapses on inhibitory neurons in the hippocampus.

Synaptic NMDA-type glutamate receptors are anchored to the second of three PDZ (PSD-95/Discs large/ZO-1) domains in the postsynaptic density (PSD) protein PSD-95. Here, we report that citron, a protein target for the activated form of the small GTP-binding protein Rho, preferentially binds the third PDZ domain of PSD-95. In GABAergic neurons from the hippocampus, citron forms a complex with PSD-95 and is concentrated at the postsynaptic side of glutamatergic synapses. Citron is expressed only at low levels in glutamatergic neurons in the hippocampus and is not detectable at synapses onto these neurons. In contrast to citron, p135 SynGAP, an abundant synaptic Ras GTPase-activating protein that can bind to all three PDZ domains of PSD-95, and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) are concentrated postsynaptically at glutamatergic synapses on glutamatergic neurons. CaM kinase II is not expressed and p135 SynGAP is expressed in less than half of hippocampal GABAergic neurons. Segregation of citron into inhibitory neurons does not occur in other brain regions. For example, citron is expressed at high levels in most thalamic neurons, which are primarily glutamatergic and contain CaM kinase II. In several other brain regions, citron is present in a subset of neurons that can be either GABAergic or glutamatergic and can sometimes express CaM kinase II. Thus, in the hippocampus, signal transduction complexes associated with postsynaptic NMDA receptors are different in glutamatergic and GABAergic neurons and are specialized in a way that is specific to the hippocampus.

Characterization of densin-180, a new brain-specific synaptic protein of the O-sialoglycoprotein family.

We purified an abundant protein of apparent molecular mass 180 kDa from the postsynaptic density fraction of rat forebrain and obtained amino acid sequences of three tryptic peptides generated from the protein. The sequences were used to design a strategy for cloning the cDNA encoding the protein by polymerase chain reaction. The open reading frame of the cDNA encodes a novel protein of predicted molecular mass 167 kDa. We have named the protein densin-180. Antibodies raised against the predicted amino and carboxyl sequences of densin-180 recognize a 180 kDa band on immunoblots that is enriched in the postsynaptic density fraction. Immunocytochemical localization of densin-180 in dissociated hippocampal neuronal cultures shows that the protein is highly concentrated at synapses along dendrites. The message encoding densin-180 is brain specific and is more abundant in forebrain than in cerebellum. The sequence of densin-180 contains 17 leucine-rich repeats, a sialomucin domain, an apparent transmembrane domain, and a PDZ domain. This arrangement of domains is similar to that of several adhesion molecules, in particular GPIbalpha, which mediates binding of platelets to von Willebrand factor. We propose that densin-180 participates in specific adhesion between presynaptic and postsynaptic membranes at glutamatergic synapses.

The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B.

The postsynaptic density (PSD) is a specialization of the submembranous cytoskeleton that is visible in the electron microscope on the cytoplasmic face of the postsynaptic membrane. A subcellular fraction enriched in structures with the morphology of PSDs contains signal-transduction molecules thought to regulate receptor localization and function in the central nervous system. We have purified a prominent tyrosine-phosphorylated glycoprotein of apparent molecular mass 180 kDa, termed PSD-gp180, that is highly enriched in the rat forebrain PSD fraction. The sequences of four tryptic peptides generated from the protein reveal that it is the 2B subunit of the N-methyl-D-aspartate (NMDA) type glutamate receptor. We have confirmed the identity of PSD-gp180 by showing that it reacts with antibodies raised against a unique fragment of the 2B subunit of the NMDA receptor. We also show that the 2B subunit is the most prominently tyrosine-phosphorylated protein in the PSD fraction based upon recognition by an anti-phosphotyrosine antibody. Two types of NMDA receptor subunits have been identified by molecular cloning [Nakanishi, S. (1992) Science 258, 597-603]. The single type 1 subunit is expressed throughout the brain and is necessary for formation of the receptor channel. The four type 2 subunits (2A, 2B, 2C, and 2D) are expressed in discrete brain regions, contain unusually long unique C termini, and confer distinct kinetic properties on NMDA receptors that contain them. Our findings suggest that, in the forebrain, NMDA receptor subunit 2B may serve to anchor NMDA receptors at the postsynaptic membrane through its interaction with the PSD. The prominent presence of tyrosine phosphate further suggests that the NMDA receptor may be regulated by tyrosine phosphorylation or that it may participate in signaling through tyrosine phosphorylation and through its ion channel.

A yeast protein, homologous to the proteolipid of the chromaffin granule proton-ATPase, is important for cell growth.

We have characterized a gene, PPA1, adjacent to the yeast MAS2 gene. DNA sequence analysis of PPA1 predicts a hydrophobic protein of 23 kDa. This protein is homologous to the proteolipid of the bovine chromaffin granule proton ATPase and to the proteolipid of the yeast vacuolar proton ATPase. Gene disruption experiments indicate that the PPA1 protein is essential for viability in three unrelated yeast strains and important for optimal growth in a fourth strain.