Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.

Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in global DNA repair but exhibit normal transcription-coupled repair and enhanced UV resistance.

We investigated whether mutations in the p53 tumor suppressor gene alter UV sensitivity and/or repair of UV-induced DNA damage in primary human skin fibroblasts from patients with Li-Fraumeni syndrome, heterozygous for mutations in one allele of the p53 gene (p53 wt/mut) and sublines expressing only mutant p53 (p53 mut). The p53 mut cells were more resistant than the p53 wt/mut cells to UV cytotoxicity and exhibited less UV-induced apoptosis. DNA repair analysis revealed reduced removal of cyclobutane pyrimidine dimers from overall genomic DNA in vivo in p53 mut cells compared with p53 wt/mut or normal cells. However, p53 mut cells retained the ability to preferentially repair damage in the transcribed strands of expressed genes (transcription-coupled repair). These results suggest that loss of p53 function may lead to greater genomic instability by reducing the efficiency of DNA repair but that cellular resistance to DNA-damaging agents may be enhanced through elimination of apoptosis.

Decreased atherosclerosis in mice deficient in both macrophage colony-stimulating factor (op) and apolipoprotein E.

To develop a murine model system to test the role of monocyte-derived macrophage in atherosclerosis, the osteopetrotic (op) mutation in the macrophage colony-stimulating factor gene was bred onto the apolipoprotein E (apoE)-deficient background. The doubly mutant (op/apoE-deficient) mice fed a low-fat chow diet had significantly smaller proximal aortic lesions at an earlier stage of progression than their apoE-deficient control littermates. These lesions in the doubly mutant mice were composed of macrophage foam cells. The op/apoE-deficient mice also had decreased body weights, decreased blood monocyte differentials, and increased mean cholesterol levels of approximately 1300 mg/dl. Statistical analysis determined that atherosclerosis lesion area was significantly affected by the op genotype and gender. The confounding variables of body weight, plasma cholesterol, and monocyte differential, which were all affected by op genotype, had no significant additional effect on lesion area once they were adjusted for the effects of op genotype and gender. Unexpectedly, there was a significant inverse correlation between plasma cholesterol and lesion area, implying that each may be the result of a common effect of macrophage colony-stimulating factor levels. The data support the hypothesis that macrophage colony-stimulating factor and its effects on macrophage development and function play a key role in atherogenesis.

Integration of transplanted hepatocytes into host liver plates demonstrated with dipeptidyl peptidase IV-deficient rats.

To analyze mechanisms of liver repopulation, we transplanted normal hepatocytes into syngeneic rats deficient in dipeptidyl peptidase IV activity. When isolated hepatocytes were injected into splenic pulp, cells promptly migrated into hepatic sinusoids. To examine whether transplanted hepatocytes entered liver plates and integrated with host hepatocytes, we analyzed sharing of hepatocyte-specific gap junctions and bile canaliculi. Colocalization studies showed gap junctions uniting adjacent transplanted and host hepatocytes in liver plates. Visualization of bile canalicular domains in transplanted and host hepatocytes with dipeptidyl peptidase IV and ATPase activities, respectively, demonstrated hybrid bile canaliculi, which excreted a fluorescent conjugated bile acid analogue. These results indicate that transplanted hepatocytes swiftly overcome mechanical barriers in hepatic sinusoids to enter liver plates and join host cells. Integration into liver parenchyma should physiologically regulate the function or disposition of transplanted hepatocytes and benefit applications such as gene therapy.

The protein deficient in Lowe syndrome is a phosphatidylinositol-4,5-bisphosphate 5-phosphatase.

Lowe syndrome, also known as oculocerebrorenal syndrome, is caused by mutations in the X chromosome-encoded OCRL gene. The OCRL protein is 51% identical to inositol polyphosphate 5-phosphatase II (5-phosphatase II) from human platelets over a span of 744 aa, suggesting that OCRL may be a similar enzyme. We engineered a construct of the OCRL cDNA that encodes amino acids homologous to the platelet 5-phosphatase for expression in baculovirus-infected Sf9 insect cells. This cDNA encodes aa 264-968 of the OCRL protein. The recombinant protein was found to catalyze the reactions also carried out by platelet 5-phosphatase II. Thus OCRL converts inositol 1,4,5-trisphosphate to inositol 1,4-bisphosphate, and it converts inositol 1,3,4,5-tetrakisphosphate to inositol 1,3,4-trisphosphate. Most important, the enzyme converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The relative ability of OCRL to catalyze the three reactions is different from that of 5-phosphatase II and from that of another 5-phosphatase isoenzyme from platelets, 5-phosphatase I. The recombinant OCRL protein hydrolyzes the phospholipid substrate 10- to 30-fold better than 5-phosphatase II, and 5-phosphatase I does not cleave the lipid at all. We also show that OCRL functions as a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in OCRL-expressing Sf9 cells. These results suggest that OCRL is mainly a lipid phosphatase that may control cellular levels of a critical metabolite, phosphatidylinositol 4,5-bisphosphate. Deficiency of this enzyme apparently causes the protean manifestations of Lowe syndrome.

A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription.

Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II has been suggested to be critical for transcription initiation, activation, or elongation. A kinase activity specific for CTD is a component of the general transcription factor TFIIH. Recently, a cyclin-dependent kinase-activator kinase (MO15 and cyclin H) was found to be associated with TFIIH preparations and was suggested to be the CTD kinase. TFIIH preparations containing mutant, kinase-deficient MO15 lack CTD kinase activity, indicating that MO15 is critical for polymerase phosphorylation. Nonetheless, these mutant TFIIH preparations were fully functional (in vitro) in both basal and activated transcription. These results indicate that CTD phosphorylation is not required for transcription with a highly purified system.