carbobenzoxycarbonyl-l-phenylalanyl-l-alanine-d-diazomethane and Alzheimer-Disease

The oligomerization of the amyloid-β protein (Aβ) is an important event in Alzheimer disease (AD) pathology. Developing small molecules that disrupt formation of early oligomeric states of Aβ and thereby reduce the effective amount of toxic oligomers is a promising therapeutic strategy for AD. Here, mass spectrometry and ion mobility spectrometry were used to investigate the effects of a small molecule, Z-Phe-Ala-diazomethylketone (PADK), on the Aβ42 form of the protein. The mass spectrum of a mixture of PADK and Aβ42 clearly shows that PADK binds directly to Aβ42 monomers and small oligomers. Ion mobility results indicate that PADK not only inhibits the formation of Aβ42 dodecamers, but also removes preformed Aβ42 dodecamers from the solution. Electron microscopy images show that PADK inhibits Aβ42 fibril formation in the solution. These results are consistent with a previous study that found that PADK has protective effects in an AD transgenic mouse model. The study of PADK and Aβ42 provides an example of small molecule therapeutic development for AD and other amyloid diseases.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Animals; Diazomethane; Dimerization; Disease Models, Animal; Drug Design; Humans; Mass Spectrometry; Mice; Microscopy, Electron; Peptide Fragments; Protein Folding; Protein Structure, Secondary; Solubility

Previous reports suggest that age-related lysosomal disturbances contribute to Alzheimer-type accumulations of protein species, blockage of axonal/dendritic transport, and synaptic decline. Here, we tested the hypothesis that lysosomal enzymes are upregulated as a compensatory response to pathogenic protein accumulation. In the hippocampal slice model, tau deposits and amyloidogenic fragments induced by the lysosomal inhibitor chloroquine were accompanied by disrupted microtubule integrity and by corresponding declines in postsynaptic glutamate receptors and the presynaptic marker synaptophysin. In the same slices, cathepsins B, D, and L, beta-glucuronidase, and elastase were upregulated by 70% to 135%. To address whether this selective activation of the lysosomal system represents compensatory signaling, N-Cbz-L-phenylalanyl-L-alanyl-diazomethylketone (PADK) was used to enhance the lysosome response, generating 4- to 8-fold increases in lysosomal enzymes. PADK-mediated lysosomal modulation was stable for weeks while synaptic components remained normal. When PADK and chloroquine were co-infused, chloroquine no longer increased cellular tau levels. To assess pre-existing pathology, chloroquine was applied for 6 days after which its removal resulted in continued degeneration. In contrast, enhancing lysosomal activation by replacing chloroquine after 6 days with PADK led to clearance of accumulated protein species and restored microtubule integrity. Transport processes lost during chloroquine exposure were consequently re-established, resulting in marked recovery of synaptic components. These data indicate that compensatory activation of lysosomes follows protein accumulation events, and that lysosomal modulation represents a novel approach for treating Alzheimer disease and other protein deposition diseases.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Cathepsins; Chloroquine; Culture Techniques; Cysteine Proteinase Inhibitors; Diazomethane; Disease Models, Animal; Enzyme Activation; Hippocampus; Hydrolases; Lysosomes; Rats; Rats, Sprague-Dawley; Synapses; tau Proteins

Lysosomal disturbances may be a contributing factor to Alzheimer's disease. We used novel compounds to test if suppression of the lysosomal protease cathepsin D blocks production of known precursors to neurofibrillary tangles. Partial lysosomal dysfunction was induced in cultured hippocampal slices with a selective inhibitor of cathepsins B and L. This led within 48 h to hyperphosphorylated tau protein fragments recognized by antibodies against human tangles. Potent nonpeptidic cathepsin D inhibitors developed using combinatorial chemistry and structure-based design blocked production of the fragments in a dose-dependent fashion. Threshold was in the submicromolar range, with higher concentrations producing complete suppression. The effects were selective and not accompanied by pathophysiology. Comparable results were obtained with three structurally distinct inhibitors. These results support the hypothesis that cathepsin D links lysosomal dysfunction to the etiology of Alzheimer's disease and suggest a new approach to treating the disease.

Topics: Alzheimer Disease; Animals; Cathepsin D; Diazomethane; Dose-Response Relationship, Drug; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Hippocampus; Lysosomes; Organ Culture Techniques; Peptide Fragments; Phosphoproteins; Phosphorylation; Rats; Rats, Sprague-Dawley; tau Proteins

Lysosomal protease inhibitors induce signs of human brain aging in rat hippocampal slices. The present studies tested if they (1) also cause neurofibrillary tangles and (2) reproduce regional patterns of pathology found in Alzheimer's disease (AD). Slices of hippocampus plus retrohippocampal cortex were prepared from rats at postnatal days 6-7 and maintained for 2-5 weeks. In agreement with earlier studies, 6- to 12-day infusions of selective (ZPAD) or generalized (chloroquine) inhibitors of lysosomal proteases generated meganeurites of the type found in aged human cortex. Surveys and quantitative analyses established that the meganeurites developed almost exclusively in AD vulnerable regions. Antibodies against the phosphorylated tau protein in neurofibrillary tangles labeled thick filaments running through neurons in the superficial layers of entorhinal cortex in 6-day ZPAD-treated slices. The general appearance of the stained structures resembled that of early stage tangles. More mature tangle-like profiles were found at a number of sites after longer incubations; these were threefold more frequent in the superficial (AD vulnerable) than in the deep layers of the entorhinal cortex. Immunoblots indicated that essentially all phosphorylated tau labeling in the slices involved approximately 29-kDa fragments of the native isoforms. These findings establish that lysosomal dysfunction triggers the parallel formation of meganeurites and tangles with the regional distribution of both effects reflecting that for AD vulnerability.

Topics: Aged; Alzheimer Disease; Animals; Cathepsin B; Cathepsin L; Cathepsins; Chloroquine; Cysteine Endopeptidases; Diazomethane; Endopeptidases; Entorhinal Cortex; Hippocampus; Humans; Lysosomes; Neurites; Neurofibrillary Tangles; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Serine Proteinase Inhibitors