ABSTRACT
The membrane-bound glycoprotein, amyloid precursor protein (APP), plays a central role in Alzheimer's disease (AD). The present paper investigates the neuritogenic-neurotoxic properties of this protein and relates them to possible aetiopathological mechanisms in AD. Marked differences in neuritic differentiation were detected when comparing untransfected tetraploid mouse neuroblastoma cells (or vector only cells) with transfected cells overexpressing APP(751). Transfected cells developed neurites quicker, and whereas all transfected cells differentiated, the degree of differentiation of untransfected cells was more variable. Fully differentiated transfected and untransfected cells had marked differences in neuritic morphology. Transfected cells had more neurites per cell, these being shorter and more branched than neurites on untransfected cells. The precocious differentiation of transfected cells was not maintained with neuritic process disintegration and cell death occurring from the seventh day onwards. Untransfected cells continued to extend their neuritic processes for up to five weeks. Membrane-associated forms of APP were responsible for these effects, rather than secreted APP or the beta/A4-peptide. Combined data from Western blot and immunocytochemical procedures showed a prominent accumulation of APP-C-terminal fragments in the perinuclear region, neuritic varicosities and growth cones of transfected cells, suggesting their involvement in the neuritogenic-neurotoxic process. Similar neuritogenic-neurotoxic mechanisms occurring in vivo, in association with compensatory synaptoplastic responses in the aged brain, may be part of the pathological process in AD.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/metabolism , Membrane Glycoproteins/metabolism , Neurons/metabolism , Neurons/pathology , Neurotoxins/metabolism , Alzheimer Disease/etiology , Amyloid beta-Protein Precursor/adverse effects , Amyloid beta-Protein Precursor/genetics , Animals , Blotting, Western , Cell Differentiation , Cell Division , Cell Line , Immunohistochemistry , In Vitro Techniques , Membrane Glycoproteins/adverse effects , Mice , Neuroblastoma/genetics , Neurotoxins/adverse effects , TransfectionABSTRACT
Gene expression in the mammalian brain is highly complex and requires an immensely powerful functional genomics tool to unravel it. Antisense has the potential to meet this requirement, but has always been plagued by biological and technological hurdles that have made the technology unreliable. With recent progress in developing potent, low-toxicity nucleic acid chemistries and novel drug delivery methods to cross the blood-brain barrier, the use of antisense is gathering momentum.
Subject(s)
Brain/physiology , Genetic Therapy/trends , Neurobiology/trends , RNA, Antisense/genetics , RNA, Antisense/therapeutic use , Animals , HumansABSTRACT
Adrenal chromaffin cells are commonly used in studies of exocytosis. Progress in characterizing the molecular mechanisms has been slow, because no simple, high-efficiency technique is available for introducing and expressing heterologous cDNA in chromaffin cells. Here we demonstrate that Semliki Forest virus (SFV) vectors allow high-efficiency expression of heterologous protein in chromaffin cells.
Subject(s)
Adrenal Medulla/cytology , Chromaffin Cells/virology , Gene Transfer Techniques , Semliki forest virus/genetics , Animals , Cattle , Chromaffin Cells/cytology , Culture Media , Electrophysiology , Exocytosis , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Microscopy, Confocal , Polymerase Chain ReactionABSTRACT
The normal function of the pathogenicity-related protein, PrP (or prion protein), is unknown. To shed light on functionally important domains, we have characterized a candidate marsupial PrP gene. The deduced marsupial PrP has an overall identity of about 80% to eutherian PrP at the amino acid (aa) level. This similarity is not equally distributed and two regions (aa 118-142 and 177-223) are particularly highly conserved. In contrast, a repeat region in the N-terminal half of the marsupial PrP shows dipeptide inserts not described in other PrP. Another particular feature of the marsupial gene is the lack of a continuous ORF on the antisense strand, as is found in most eutherian PrP. We propose that antisense ORFs found in other species are artefactual. Comparison with all known PrP argues that the molecule characterised is the true marsupial PrP orthologue.
Subject(s)
Marsupialia/genetics , Prions/genetics , Amino Acid Sequence , Animals , Base Sequence , Conserved Sequence , DNA, Antisense/genetics , Mammals/genetics , Molecular Sequence Data , Open Reading Frames/genetics , Restriction Mapping , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
A systematic study of 'prion protein' genotype in cases of sporadic Creutzfeldt-Jakob disease showing amyloid plaques staining with anti-prion protein antibody has been performed. This revealed a relative excess of cases with valine at position 129 of the gene's open reading frame. The observation emphasises the importance of this site of common polymorphism in influencing the neuropathological phenotype in human spongiform encephalopathy.
