Hajime Kusano

Cobblestone (type II) lissencephaly and mental retardation are characteristic features of a subset of congenital muscular dystrophies that include Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama-type congenital muscular dystrophy. Although the majority of clinical cases are genetically undefined, several causative genes have been identified that encode known or putative glycosyltransferases in the biosynthetic pathway of dystroglycan. Here we test the effects of brain-specific deletion of dystroglycan, and show distinct functions for neuronal and glial dystroglycan. Deletion of dystroglycan in the whole brain produced glial/neuronal heterotopia resembling the cerebral cortex malformation in cobblestone lissencephaly. In wild-type mice, dystroglycan stabilizes the basement membrane of the glia limitans, thereby supporting the cortical infrastructure necessary for neuronal migration. This function depends on extracellular dystroglycan interactions, since the cerebral cortex developed normally in transgenic mice that lack the dystroglycan intracellular domain. Also, forebrain histogenesis was preserved in mice with neuron-specific deletion of dystroglycan, but hippocampal long-term potentiation was blunted, as is also the case in the Largemyd mouse, in which dystroglycan glycosylation is disrupted. Our findings provide genetic evidence that neuronal dystroglycan plays a role in synaptic plasticity and that glial dystroglycan is involved in forebrain development. Differences in dystroglycan glycosylation in distinct cell types of the CNS may contribute to the diversity of dystroglycan function in the CNS, as well as to the broad clinical spectrum of type II lissencephalies.

Myocyte enhancer factor 2 (MEF2) plays essential roles in transcriptional control of muscle development. However, signaling pathways acting downstream of MEF2 are largely unknown. Here, we performed a microarray analysis using Mef2c-null mouse embryos and identified a novel MEF2-regulated gene encoding a muscle-specific protein kinase, Srpk3, belonging to the serine arginine protein kinase (SRPK) family, which phosphorylates serine/arginine repeat-containing proteins. The Srpk3 gene is specifically expressed in the heart and skeletal muscle from embryogenesis to adulthood and is controlled by a muscle-specific enhancer directly regulated by MEF2. Srpk3-null mice display a new entity of type 2 fiber-specific myopathy with a marked increase in centrally placed nuclei; while transgenic mice overexpressing Srpk3 in skeletal muscle show severe myofiber degeneration and early lethality. We conclude that normal muscle growth and homeostasis require MEF2-dependent signaling by Srpk3.

Caspase-mediated proteolysis is a critical and central element of the apoptotic process; therefore, it is important to identify the downstream molecular targets of caspases. We established a method for cloning the genes of caspase substrates by two major modifications of the yeast two-hybrid system: (i) both large and small subunits of active caspases were expressed in yeast under ADH1 promoters and the small subunit was fused to the LexA DNA-binding domain; and (ii) a point mutation was introduced that substituted serine for the active site cysteine and thereby prevented proteolytic cleavage of the substrates, possibly stabilizing the enzyme-substrate complexes in yeast. After screening a mouse embryo cDNA expression library by using the bait plasmid for caspase-3, we obtained 13 clones that encoded proteins binding to caspase-3, and showed that 10 clones including gelsolin, an actin-regulatory protein implicated in apoptosis, were cleaved by recombinant caspase-3 in vitro. Using the same bait, we also isolated human gelsolin cDNA from a human thymus cDNA expression library. We showed that human gelsolin was cleaved during Fas-mediated apoptosis in vivo and that the caspase-3 cleavage site of human gelsolin was at D352 of DQTD352G, findings consistent with previous observations on murine gelsolin. In addition, we ascribed the antiapoptotic activity of gelsolin (which we previously reported) to prevention of a step leading to cytochrome c release from the mitochondria into the cytosol. Our results indicate that this cloning method is useful for identification of the substrates of caspases and possibly also of other enzymes.

α-Helical polypeptides containing a pair of L-1-pyrenylalanine and L-p-nitrophenylalanine that are separated by 0−8 amino acid units were synthesized. The rates of photoinduced electron transfer (ET) from the pyrenyl group to the nitrophenyl group were evaluated from the decay curves of pyrenyl fluorescence recorded at different temperatures from −58 to +30 °C. The rate constants showed a complex dependence on the number of spacer amino acids. In particular, recoveries of the ET rates with increasing the number of spacer amino acids from 1 to 2 and from 5 to 6 were found. The ET rate constants, however, exhibited a simple exponential dependence on the edge-to-edge distance between the two chromophores, with a distance decay factor β = 0.66 ± 0.1 (Å-1). The ET data on the α-helical polypeptides were analyzed on the basis of the tunneling pathway model. The optimum ET pathways from the pyrenyl group to the nitrophenyl group were searched, and the relative values of the ET matrix elements were evaluated for each polypeptide with different number of the spacer units. The calculated distance dependence was in reasonable agreement with the experimental one when jumps through hydrogen bonds were taken into account.