Dapper Antagonist of Catenin-1 Cooperates with Dishevelled-1 during Postsynaptic Development in Mouse Forebrain GABAergic Interneurons

Synaptogenesis has been extensively studied along with dendritic spine development in glutamatergic pyramidal neurons, however synapse development in cortical interneurons, which are largely aspiny, is comparatively less well understood. Dact1, one of 3 paralogous Dact (Dapper/Frodo) family members in mammals, is a scaffold protein implicated in both the Wnt/β-catenin and the Wnt/Planar Cell Polarity pathways. We show here that Dact1 is expressed in immature cortical interneurons. Although Dact1 is first expressed in interneuron precursors during proliferative and migratory stages, constitutive Dact1 mutant mice have no major defects in numbers or migration of these neurons. However, cultured cortical interneurons derived from these mice have reduced numbers of excitatory synapses on their dendrites. We selectively eliminated Dact1 from mouse cortical interneurons using a conditional knock-out strategy with a Dlx-I12b enhancer-Cre allele, and thereby demonstrate a cell-autonomous role for Dact1 during postsynaptic development. Confirming this cell-autonomous role, we show that synapse numbers in Dact1 deficient cortical interneurons are rescued by virally-mediated re-expression of Dact1 specifically targeted to these cells. Synapse numbers in these neurons are also rescued by similarly targeted expression of the Dact1 binding partner Dishevelled-1, and partially rescued by expression of Disrupted in Schizophrenia-1, a synaptic protein genetically implicated in susceptibility to several major mental illnesses. In sum, our results support a novel cell-autonomous postsynaptic role for Dact1, in cooperation with Dishevelled-1 and possibly Disrupted in Schizophrenia-1, in the formation of synapses on cortical interneuron dendrites.     

Early Inhibition of Acetylcholinesterase and Choline Acetyltransferase Activity in Herpes simplex Virus Type 1 Infection of PC12 Cells

Abstract: Early in the course of productive Herpes simplex virus type 1 (HSV-1) infection of PC12 cells, activities of both acetylcholinesterase (AChE) and choline acetyltransferase (CAT) fell. Studies using metabolic inhibitors and a temperature-sensitive mutant of the virus suggested that the decline in activities of both enzymes was associated with events occurring early in the replicative cycle related to expression of the immediate-early (α) group of viral polypeptides. HSV-1 gene products thus may alter specialized cell functions well before the production of viral progeny and initiation of cell lysis. The early clinical manifestations of nervous system viral infection may reflect focal metabolic disturbance rather than, or in addition to, simple cell death.     

CoupTFI Interacts with Retinoic Acid Signaling during Cortical Development

We examined the role of the orphan nuclear hormone receptor CoupTFI in mediating cortical development downstream of meningeal retinoic acid signaling. CoupTFI is a regulator of cortical development known to collaborate with retinoic acid (RA) signaling in other systems. To examine the interaction of CoupTFI and cortical RA signaling we utilized Foxc1-mutant mice in which defects in meningeal development lead to alterations in cortical development due to a reduction of RA signaling. By analyzing CoupTFI^−/−;Foxc1^H/L double mutant mice we provide evidence that CoupTFI is required for RA rescue of the ventricular zone and the neurogenic phenotypes in Foxc1-mutants. We also found that overexpression of CoupTFI in Foxc1-mutants is sufficient to rescue the Foxc1-mutant cortical phenotype in part. These results suggest that CoupTFI collaborates with RA signaling to regulate both cortical ventricular zone progenitor cell behavior and cortical neurogenesis.     

Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins.

Antibody response in mice to scrapie-associated fibril proteins (protease-resistant proteins [PrPs]) was generated to different epitopes depending on the source of antigen. Mice responded differently to PrPs isolated from scrapie-infected animals of homologous (mouse) versus heterologous (hamster) species. An enzyme-linked immunosorbent assay established to monitor this antibody response in mice immunized with PrPs was unable to detect such a response in scrapie-infected mice. A monoclonal antibody (MAb), 263K 3F4, derived from a mouse immunized with hamster 263K PrPs reacted with hamster but not mouse PrPs. MAb 263K 3F4 also recognized normal host protein of 33 to 35 kilodaltons in brain tissue from hamsters and humans but not from bovine, mouse, rat, sheep, or rabbit brains. This is the first demonstration of epitope differences on this host protein in different species. The defining of various epitopes on PrP through the use of MAbs will lead to a better understanding of the relationship of PrPs to their host precursor protein and to the infectious scrapie agent.     

An islet amyloid peptide is derived from an 89-amino acid precursor by proteolytic processing

Amyloid deposits occurring in the islets of Langerhans in patients with noninsulin-dependent diabetes mellitus and some insulinomas contain a 37-amino acid peptide that is structurally related to calcitonin gene-related peptide. We have identified three cDNA clones encoding islet amyloid polypeptide (IAPP) or diabetes-associated peptide (DAP) by oligonucleotide screening of a lambda gt10 human insulinoma cDNA library. Two of the three cDNAs contained a domain encoding IAPP/DAP but had an intron-like sequence in their 5' region. The other cDNA contained an open reading frame encoding an 89-amino acid precursor having a typical signal peptide followed by a small prohormone-like sequence containing within it the IAPP/DAP peptide bracketed at its NH2 and COOH termini by Lys-Arg and Gly-Lys-Arg, respectively. These data indicate that this amyloid peptide is generated by proteolytic processing similar to that for proinsulin and other islet prohormones and also that the peptide may be carboxyamidated. The isolation of cDNA clones having 5'-unprocessed intron-like sequences suggests that inefficient or alternative splicing of this mRNA occurred in the insulinoma.     

Characterization of lipoprotein produced by the perfused rhesus monkey liver

Isolated livers from rhesus monkeys (Macaca mulatta) were perfused in order to asses the nature of newly synthesized hepatic lipoprotein. Perfusate containing [3H]leucine was recirculated for 1.5 hr, followed by an additional 2.5-hr perfusion with fresh perfusate. Equilibrium density gradient ultracentrifugation clearly separated VLDL from LDL. The apoprotein composition of VLDL secreted by the liver was similar to that of serum VLDL. The perfusate LDL contained some poorly radiolabeled, apoB-rich material, which appeared to be contaminating serum LDL. There was also some material of an LDL-like density, which was rich in radiolabeled apoE. Rate zonal density gradient ultracentrifugation fractionated HDL. All perfusate HDL fractions had a decreased cholesteryl ester/unesterified cholesterol ratio, compared to serum HDL. Serum HDL distributed in one symmetric peak near the middle of the gradient, with coincident peaks of apoA-I and apoA-II. The least dense fractions of the perfusate gradient were rich in radiolabeled apoE. The middle of the perfusate gradient contained particles rich in radiolabeled apoA-I and apoA-II. The peak of apoA-I was offset from the apoA-II peak towards the denser end of the gradient. The dense end of the HDL gradient contained lipoprotein-free apoA-I, apoE, and small amounts of apoA-II, probably resulting from the relative instability of nascent lipoprotein compared to serum lipoprotein. Perfusate HDL apoA-I isoforms were more basic than serum apoA-I isoforms. Preliminary experiments, using noncentrifugal methods, suggest that some hepatic apoA-I is secreted in a lipoprotein-free form. In conclusion, the isolated rhesus monkey liver produces VLDL similar to serum VLDL, but produces LDL and HDL which differ in several important aspects from serum LDL and HDL.     

Induction of vascular smooth muscle alpha-isoactin expression in BC3H1 cells

An isoactin analysis was performed on L-[35S]cysteine labeled BC3H1 cells to determine if these smooth muscle-like cells synthesize vascular smooth muscle actin. Three different NH2-terminal peptides were identified on thin layer electrophoretograms of DNase I-purified and trypsin-digested BC3H1 cell actin. Results obtained from secondary digestion with thermolysin or Staphylococcus aureus V8 protease showed that the most acidic NH2-terminal peptide was derived from vascular smooth muscle alpha-isoactin. Treatment of cell monolayers with serum-free medium caused a 3-fold increase in the level of alpha-isoactin expression and a concomitant decrease in the level of non-muscle beta- and gamma-isoactin. Cell-cell contact was required for induction of alpha-isoactin, and the effects of serum depletion on isoactin expression and cell growth were reversible. The intensity of about 11 out of 500 polypeptide spots on two-dimensional gels of BC3H1 cell polypeptides also was influenced by the culture conditions. The finding that smooth muscle isoactin expression was coupled to cell growth conditions indicate the potential usefulness of BC3H1 cells in studies of isoactin expression and utilization during vascular smooth muscle development.     

Peripheral glucose uptake in young men with myocardial infarction

Forearm glucose uptake (FGU) was studied during 100 g oral glucose tolerance tests (GTT) in nonobese, nondiabetic men who had suffered a myocardial infarction (MI) at or before the age of 40, and the results compared with the response in age-matched normal men. In the MI group the rise in both glucose and insulin concentrations after glucose loading was similar to that in normal subjects, although in the former, peak levels tended to be slightly higher. Concomitant FGU, however, was significantly greater in the MI group than in control subjects in the period 0-90 min and in the test as a whole (0-180 min). The results show that at least in some nondiabetics suffering MI at an early age hyperinsulinism is not a feature and peripheral tissue sensitivity is increased.