Presenilin-1 and -2 are molecular targets for gamma-secretase inhibitors.

Presenilins are integral membrane protein involved in the production of amyloid beta-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter gamma-secretase cleavage of the beta-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A beta, the 4-kDa beta-peptide. Here, we show that radiolabeled gamma-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A beta formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N- and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of gamma-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective gamma-secretase inhibitors block A beta formation by binding to presenilin-1 and -2.

Successful survival of grafted transgenic neural plate cells in adult central nervous system environment.

1. Accumulating evidence indicates that damaged brain functions can be ameliorated in a variety of animal models by the grafting of fetal neuronal cell or tissue into damaged brain. Clinical trials are under way to determine whether human fetal mesencephalic tissue can ameliorate motor functions in patients with Parkinson's disease. 2. Autopsy findings of parkinsonian patient implanted with human fetal mesencephalic tissue clearly revealed that the fetal neuronal graft can survive for an extended period of time in the human brain and densely reinnervate the surrounding host striatal tissue. 3. It is, however, still important to obtain more practical, effective, and ethically justifiable donor material for the future clinical application of the procedures. Desirable properties for the donor cells include long-term survival in the brain, neuronal cell type for the reconstruction of damaged neural circuits, and susceptibility to genetic manipulation for the practical use. 4. With the development of molecular biology techniques, genetic modification and transplantation of the donor neuronal cells might be a feasible way to cure many kinds of central nervous system diseases toward a "graft-gene therapy."

Transgenic neural plate contributes neuronal cells that survive greater than one year when transplanted into the adult mouse central nervous system.

Neural plate cells from the early embryo may have a number of important advantages as donor material for the delivery of foreign genes into the diseased adult central nervous system (CNS). Mesencephalic neural plate from transgenic GT4-2 mice was used as a source of marked donor cells to determine whether transgene-expressing embryonic CNS progenitor cells can be used as donor material for implantation into the adult mouse brain. Transgenic mouse embryos from this line express the Escherichia coli beta-galactosidase (beta-gal) gene throughout early CNS development. At the early somite stage (Embryonic Day 8.5), mesencephalic neural plate tissue from heterozygous embryos was dissected out and either transferred into culture for characterization or immediately implanted into the striatum or lateral ventricle of adult wild-type CD-1 mice. Explants of neural plate tissue possessed intense beta-gal activity and produced extensive outgrowth of neurofilament-positive processes after 6 days in vitro. Many beta-gal-positive cells migrated away from the explanted tissue mass. Grafts of transgenic neural plate tissue in the normal adult mouse striatum, sampled 2 weeks to 1 year after implantation, possessed healthy beta-gal-positive cells. More detailed analysis of grafts 3 months after implantation indicated that most beta-gal-positive cells were also immunoreactive for neurofilament and microtubule-associated proteins, two neuron-specific markers. In addition, extensive neurofilament-positive axonal tangles were evident within the grafts among the beta-gal-positive cells. Electron microscopic (EM) findings of implanted tissue stained with Bluo-Gal revealed many beta-gal-positive neurons received synaptic contacts from other cells. A few donor-derived astrocytes were also found in the grafts by EM analysis. No obvious signs of immunological rejection, or of significant decrease in graft volume, were observed at any age. Some beta-gal-positive cells were observed to lie up to 230 microns away from the main graft mass in both striatal and intraventricular implantations. These data suggest that the neural plate can contribute a long-surviving population of neuronal and astrocytic cells when transplanted into the adult CNS.

Biochemistry of information storage in the nervous system.

The use of molecular biological approaches has defined new mechanisms that store information in the mammalian nervous system. Environmental stimuli alter steady-state levels of messenger RNA species encoding neurotransmitters, thereby altering synaptic, neuronal, and network function over time. External or internal stimuli alter impulse activity, which alters membrane depolarization and selectively changes the expression of specific transmitter genes. These processes occur in diverse peripheral and central neurons, suggesting that information storage is widespread in the neuraxis. The temporal profile of any particular molecular mnemonic process is determined by specific kinetics of turnover and by the geometry of the neuron resulting in axonal transport of molecules to different synaptic arrays at different times. Generally, transmitters, the agents of millisecond-to-millisecond communication, are subject to relatively long-lasting changes in expression, ensuring that ongoing physiological function is translated into information storage.

DNA sequence coding for an antifreeze protein precursor from winter flounder.

A cDNA made to antifreeze protein mRNA of the winter flounder was cloned in the plasmid pBR322 and its sequence was determined by the method of Maxam and Gilbert. Its sequence codes for a precursor protein that is 82 amino acids in length. This precursor has both a signal polypeptide and a prosequence before the mature protein of 38 amino acid residues. The mature protein matches in composition one of the alanine-rich serum antifreeze proteins that was purified by ion-exchange and reverse-phase chromatography. The composition of the pro- sequence is similar to that of the native protein except that it contains five prolines. The mature protein, but not the pro- sequence, contains three of the 11-residue repeats previously observed [Lin, Y. & Gross, J. K. (1981) Proc. Natl. Acad. Sci. USA 78, 2825-2829] in two other antifreeze protein components.

Catfish vitellogenin and its messenger RNA are smaller than their chicken and Xenopus counterparts.

Injection of male bullheads (Ameiurus nebulosus) with estradiol induced the production of a major serum phosphoprotein of molecular weight 145,000. This protein was immunoprecipitable by antisera raised against lipovitellin from bullhead eggs and was absent from the serum of control males. Production of this serum protein coincided with changes in the liver mRNA population, which suggested that estradiol had induced the synthesis of additional mRNA sequences in the high-frequency class. Agarose gel electrophoresis in the presence of methyl mercury hydroxide showed that this mRNA population contained at least one species which was not present in the liver of uninjected males. This new RNA was the major polyadenylated species present in the total cellular RNA and its size relative to ribosomal RNAs and locust vitellogenin mRNA was estimated as 5800 nucleotides. When the liver total RNAs were translated in the mRNA-dependent rabbit reticulocyte lysate system the major translation product from the induced fish had the same molecular weight (145,000) as the serum phosphoprotein and was immunoprecipitable by antilipovitellin antisera. This translation product was not coded for by RNA from control fish. These observations are consistent with the induction of vitellogenesis by estradiol as reported in other egg-laying vertebrates and they show that bullhead vitellogenin and its mRNA are significantly smaller than their avian and amphibian counterparts.