Two mouse insulin genes, Ins1 and Ins2, were disrupted and lacZ was inserted at the Ins2 locus by gene targeting. Double nullizygous insulin-deficient pups were growth-retarded. They did not show any glycosuria at birth but soon after suckling developed diabetes mellitus with ketoacidosis and liver steatosis and died within 48 h. Interestingly, insulin deficiency did not preclude pancreas organogenesis and the appearance of the various cell types of the endocrine pancreas. The presence of lacZ expressing ␤ cells and glucagon-positive ␣ cells was demonstrated by cytochemistry and immunocytochemistry. Reverse transcriptioncoupled PCR analysis showed that somatostatin and pancreatic polypeptide mRNAs were present, although at reduced levels, accounting for the presence also of ␦ and pancreatic polypeptide cells, respectively. Morphometric analysis revealed enlarged islets of Langherans in the pancreas from insulin-deficient pups, suggesting that insulin might function as a negative regulator of islet cell growth. Whether insulin controls the growth of specific islet cell types and the molecular basis for this action remain to be elucidated.Insulin is synthesized, stored, and secreted by the pancreatic islet ␤ cells in a highly regulated manner and plays a vital role in glucose homeostasis. Insulin action also results in several other pleiotropic effects that are less well documented. Embryonic insulin synthesis begins early in gestation, but fetal glycemia closely follows maternal blood glucose levels. The question, therefore, arises as to what function embryonic insulin might fulfill during development. For instance, one might ask whether insulin plays an autocrine or paracrine role in pancreatic islet cell growth and differentiation, since insulin is synthesized with other hormones in developing islet cell types (1-3). Recently, this question has been addressed in a few transgenic studies. For instance, the gene encoding PDX-1 (4, 5), a homeodomain transcription factor synthesized in adult ␤ cells and capable of transactivating insulin gene expression, has been inactivated by targeted disruption (6, 7). Agenesis of pancreas resulting from PDX-1 deficiency precluded from addressing the question of the possible role of insulin in islet cell growth and differentiation. Similarly, mice lacking the LIM homeodomain transcription factor ISL1, synthesized in all classes of islet cells in the adult, were arrested in development soon after embryonic day 9.5 (8). The requirement of ISL1 in pancreatic epithelium for the differentiation of all islet cell types was, however, demonstrated by in vitro culture of explants from ISL1-deficient embryonic day 9.5 embryos that gave rise to cells that were negative for glucagon, insulin, and somatostatin. In another study, transgenic mouse embryos expressing the gene encoding the diphteria toxin A chain under control of the rat Ins2 promoter were generated (9). The resulting genetic ablation of the insulin-producing cells did not appear to alter the development of the nontarget...

To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, ␤2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and ␤2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

Ten transgenic mouse lines harboring the -346/-103 fragment of the rat insulin I enhancer linked to a heterologous promoter and a reporter gene (Eins-Ptk-CAT construct) were produced. Expression of the hybrid transgene was essentially observed in pancreas and to a lesser extent in brain. These results indicate that the rat insulin I promoter is dispensable for pancreatic expression. This insulin gene sequence is the shortest fragment described as conferring tissue-specific expression in transgenic mice. Two short homologous sequences in the rat insulin I enhancer fragment used, IEB2 and IEB1, have been described as playing a dominant role in the regulation of HIT hamster insulinoma cell-specific transcription of the insulin gene (1). We investigated whether the combination of IEB2 and IEB1 sequences is sufficient to confer specific expression in transgenic mice to a IEB2-IEB1-Ptk-CAT gene construct. No CAT activity was observed neither in pancreas nor in any other organ examined in 19 different transgenic mice. Moreover in transient expression experiments in RIN2A rat insulinoma cells, the IEB sequences had a very weak or no enhancer activity. These observations contribute to the conclusion that DNA regulatory elements other than the IEB sequences are necessary for gene expression in vivo.

We have previously identified Scrg1, a gene with increased cerebral mRNA levels in transmissible spongiform encephalopathies (TSE) such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. In this study, Scrg1-immunoreactive cells, essentially neurons, were shown to be widely distributed throughout the brain of scrapie-infected mice, while only rare and weakly immunoreactive cells could be detected in the brain of non-infected normal mice. Induction of the protein was confirmed by Western blot analysis. At the ultrastructural level, Scrg1 protein was associated with dictyosomes of the Golgi apparatus and autophagic vacuoles in the central neurons of the scrapie-infected mice. These results suggested a role for Scrg1 in the pathological changes observed in TSE. We have generated transgenic mice specifically expressing Scrg1 in neurons. No significant differences in the time course of the disease were detected between transgenic and non-transgenic mice infected with scrapie prions. However, tight association of Scrg1 with autophagic vacuoles was again observed in brain neurons of infected transgenic mice. High levels of the protein were also detected in degenerating Purkinje cells of Ngsk Prnp 0/0 mice overexpressing the Prnd gene coding for doppel, a neurotoxic paralogue of the prion protein. Furthermore, induction of Scrg1 protein was observed in the brain of mice injured by canine distemper virus or gold thioglucose treatment. Taken together, our results indicate that Scrg1 is associated with neurodegenerative processes in TSE, but is not directly linked to dysregulation of prion protein.

We have recently described a novel mRNA denominated ScRG-1, the level of which is increased in the brains of Scrapie-infected mice (Dandoy-Dron, F., Guillo, F., Benboudjema, L., Deslys, J.-P., Lasmé zas, C., Dormont, D., Tovey, M. G., and Dron, M. (1998) J. Biol. Chem. 273, 7691-7697). The increase in ScRG-1 mRNA in the brain follows the accumulation of PrPSc , the proteinase K-resistant form of the prion protein (PrP), and precedes the widespread neuronal death that occurs in late stage disease. In the present study, we have isolated a cDNA encoding the human counterpart of ScRG-1. Comparison of the human and mouse transcripts firmly established that both sequences encode a highly conserved protein of 98 amino acids that contains a signal peptide, suggesting that the protein may be secreted. Examination of the distribution of human ScRG-1 mRNA in adult and fetal tissues revealed that the gene was expressed primarily in the central nervous system as a 0.7-kilobase message and was under strict developmental control.

Dysfunction of the endoplasmic reticulum associated protein degradation/proteasome system is believed to contribute to the initiation or aggravation of neurodegenerative disorders associated with protein misfolding, and there is some evidence to suggest that proteasome dysfunctions might be implicated in prion disease. This study investigated the effect of proteasome inhibitors on the biogenesis of both the cellular (PrPC) and abnormal (PrPSc) forms of prion protein in CAD neuronal cells, a newly introduced prion cell system. In uninfected cells, proteasome impairment altered the intracellular distribution of PrPC, leading to a strong accumulation in the Golgi apparatus. Moreover, a detergent-insoluble and weakly protease-resistant PrP species of 26 kDa, termed PrP26K, accumulated in the cells, whether they were prion-infected or not. However, no evidence was found that, in infected cells, this PrP26K species converts into the highly proteinase K-resistant PrPSc. In the infected cultures, proteasome inhibition caused an increased intracellular aggregation of PrPSc that was deposited into large aggresomes. These findings strengthen the view that, in neuronal cells expressing wild-type PrPC from the natural promoter, proteasomal impairment may affect both the process of PrPC biosynthesis and the subcellular sites of PrPSc accumulation, despite the fact that these two effects could essentially be disconnected.

No abstract

Scrapie responsive gene one (Scrg1) is a novel transcript discovered through identification of the genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies. Scrg1 mRNA is distributed principally in the central nervous system and the cDNA sequence predicts a small cysteine-rich protein 98 amino acids in length, with a N-terminal signal peptide. In this study, we have generated antibodies against the predicted protein and revealed expression of a predominant immunoreactive protein of 10 kDa in mouse brain by Western blot analysis. We have established CAD neuronal cell lines stably expressing Scrg1 to determine its subcellular localization. Several lines of evidence show that the protein is targeted to dense-core vesicles in these cells. (i) Scrg1 is detected by immunocytochemistry as very punctate signals especially in the Golgi apparatus and tips of neurites, suggesting a vesicular localization for the protein. Moreover, Scrg1 exhibits a high degree of colocalization with secretogranin II, a dense-core vesicle marker and a very limited colocalization with markers for small synaptic vesicles. (ii) Scrg1 immunoreactivity is associated with large secretory granules/dense-core vesicles, as indicated by immuno-electron microscopy. (iii) Scrg1 is enriched in fractions of sucrose density gradient where synaptotagmin V, a dense-core vesicle-associated protein, is also enriched. The characteristic punctate immunostaining of Scrg1 is observed in N2A cells transfected with Scrg1 and for the endogenous protein in cultured primary neurons, attesting to the generality of the observations. Our findings strongly suggest that Scrg1 is associated with the secretory pathway of neuronal cells.