leupeptins and Amyloidosis

Amyloid beta-protein, Abeta, is normally produced in brain and is cleared by unknown mechanisms. In Alzheimer's disease (AD), Abeta accumulates in plaque-like deposits and is implicated genetically in neurodegeneration. Here we investigate mechanisms for Abeta degradation and Abeta toxicity in vivo, focusing on the effects of Abeta40, which is the peptide that accumulates in apolipoprotein E4-associated AD. Chronic intraventricular infusion of Abeta40 into rat brain resulted in limited deposition and toxicity. Coinfusion of Abeta40 with the cysteine protease inhibitor leupeptin resulted in increased extracellular and intracellular Abeta immunoreactivity. Analysis of gliosis and TUNEL in neuron layers of the frontal and entorhinal cortex suggested that leupeptin exacerbated Abeta40 toxicity. This was supported further by the neuronal staining of cathepsin B in endosomes or lysosomes, colocalizing with intracellular Abeta immunoreactivity in pyknotic cells. Leupeptin plus Abeta40 caused limited but significant neuronal phospho-tau immunostaining in the entorhinal cortex. Intriguingly, Abeta40 plus leupeptin induced intracellular accumulation of the more toxic Abeta, Abeta42, in a small group of septal neurons. Leupeptin infusion previously has been reported to interfere with lysosomal proteolysis and to result in the accumulation of lipofuscin, dystrophic neurites, tau- and ubiquitin-positive inclusions, and structures resembling paired helical filaments. Coinfusion of Abeta40 with the serine protease inhibitor aprotinin also increased diffuse extracellular deposition but reduced astrocytosis and TUNEL and was not associated with intracellular Abeta staining. Collectively, these data suggest that an age or Alzheimer's-related defect in lysosomal/endosomal function could promote Abeta deposition and DNA fragmentation in neurons and glia similar to that found in Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Animals; Antibodies, Monoclonal; Aprotinin; Cathepsins; Female; Frontal Lobe; Glial Fibrillary Acidic Protein; Gliosis; In Situ Nick-End Labeling; Leupeptins; Lysosomes; Microscopy, Confocal; Neuroglia; Neurons; Neurotoxins; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Serine Proteinase Inhibitors; Thalamus

The principal neuropathological feature of Alzheimer's disease is extracellular deposition of approximately 4-kDa proteinous fragment, designated as beta-amyloid peptides (beta/A4 peptides) derived by proteolytic cleavage from amyloid precursor protein (APP), a large cell-surface receptor-like protein. There has been evidence that APP is proteolytically degraded in the secretory and endosomal/lysosomal pathways. The pathway in which APP is cleaved to generate beta/A4 peptides is still not identified. To clarify the intracellular processing of APP into the generation of beta/A4 peptides, we detected and characterized potentially amyloidogenic or non-amyloidogenic fragments using newly established monoclonal and polyclonal antibodies in the cultured cells with or without leupeptin, potent lysosomal protease inhibitor of lysosome. APP fragments of 50 and 20 kDa containing full-length beta/A4 peptides were identified in the cultured cells. Immunoblot analysis, biochemical study for specific marker enzyme activity of the fractions obtained from subcellular fractionation, sucrose density gradient centrifugation indicated that the 50-kDa APP fragment was produced in the compartment closely related to endosomal/lysosomal system. Our data suggest that the endosomal/lysosomal pathway is involved in the processing and generation of beta/A4 peptides.

Topics: Amyloid; Amyloidosis; Blotting, Western; Cell Line; Centrifugation, Density Gradient; Humans; Immunologic Techniques; Intracellular Membranes; Leupeptins; Molecular Weight; Peptide Fragments; Prion Proteins; Prions; Protein Precursors; Subcellular Fractions

Leupeptins, protease inhibitors, suppress the appearance of experimental amyloidosis in CBA mice induced by the injections with complete Freund's adjuvant. This substance, however, should be administered continuously from 1 week prior to amyloid induction to the end of the experiment. On the other hand, Trypan blue, inhibitors of lysosomal enzymes, accelerates experimental amyloidosis in the mouse model above-mentioned. Trypan blue is effective when given either prior to or at the same time of the initiation of amyloid induction. Organic Germanium has not been confirmed to be a potent suppressor of experimental murine amyloidosis but the experimental group administered this substance continuously from 1 week prior to the induction shows a rather low incidence of amyloidosis, and the average number of amyloidotic organs per affected mouse is about half of that of the control group. The suppression and acceleration of experimental murine amyloidosis presented here are a useful tool for investigating the pathogenesis of amyloidosis.

Topics: Amyloidosis; Animals; Freund's Adjuvant; Germanium; Leupeptins; Male; Mice; Mice, Inbred CBA; Mice, Inbred Strains; Oligopeptides; Trypan Blue