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UBIQUITIN (Ub)-immunocytochemistry on Alzheimer's disease (AD) brain sections shows diverse Ub-associated deposits in the neuropil and senile plaques, elevated levels of Ub reactivity in hippocampal neurons and glia, and co-localization of Ub and β-amyloid precursor protein (βPP) epitope reactivity in dystrophic axons. These observations may suggest a role for Ub and stress-related mechanisms in AD pathogenesis. Here we show for the first time that Ub interacts avidly but non-covalently with βPP and such complexes, apparently formed in vivo, can be isolated from AD brain extracts by Ub-gel matrix affinity chromatography. Polyclonal antibodies specific to Ub and to different regions of βPP were employed to characterize these proteins. The implication of Ub-βPP complex formation is discussed in the context of βPP processing.

CHLOROQUINE (CHQ)-sensitive cellular compartments, identified as endosomes–lysosomes (ELs), have been implicated in the proteolysis of amyloid β precursor protein (AβPP) in Alzheimer's disease. Here we show using immunocytochemistry and immunogold electron microscopy that not only AβPP but also ubiquitin (Ub) co-localize to ELs in CHQ-treated human neuroblastoma (SK-N-SH) and glioblastoma (U-373). Immunoblotting analysis of cell lysates indicated a significant degree of CHQ-mediated interference in AβPP metabolism in a time- and concentration-dependent manner. The implication is that abnormal intracellular accumulation of AβPP and its C-terminal fragments beyond a certain threshold may trigger the Ub response. We hypothesize that Ub may play a role in AβPP processing and/or trafficking to ELs, particularly in stress-related conditions.

Ubiquitin (UB) covalently linked to abnormal proteins acts a signal for attack by proteinases; non-covalent binding of UB to cellular proteins has also been predicted. Here we show non-covalent binding of heat-denatured UB to an acute phase protein, serum amyloid A (SAA). Four solid-phase binding assays were used to determine UB-SAA interaction. Mouse SAA rich plasma was incubated with UB affinity matrix; SAA in the eluted proteins was identified by specific antibodies and N-terminal sequence analysis. The results show that both murine SAA1 and SAA2 isotypes bind avidly and selectively to UB indicating a role for UB in SAA processing.

Murine ApoSAA3 is synthesized and secreted by activated monocytoid cells. in contrast, these cells have been implicated in the endocytosis of exogenous murine apoSAA/SAA2 and in AA amyloid formation. The implication is that endocytosed apoSAA1/SAA2 may be processed in the endosomes-lysosomes (EL). Here we show the topographic relationship between apoSAA3 and apoSAA1/SAA2 and identify apoSAA1 /SAA2 and their derivatives in peritoneal macrophages from alveolar hydatid cyst infected mice undergoing amyloidosis. Confocal microscopy localized apoSAA1/SAA2 exclusively to the EL whereas apoSAA3 generally had a non-vesicular cytoplasmic distribution. Immunoblotting of the macrophage cytoplasmic fractions, regardless of the duration of the infection, identified pre-dominantly two ~5 and 12 kDa C-terminus cleaved apoSAA1/SAA2 derivatives which resembled in molecular mass the tissue AA Immunoblotting of the infected mouse sera did not reveal any apoSAA1/SAA2 derivatives. These data suggest that following endocytosis, apoSAA1/SAA2 is most likely partially degraded and retained in the EL. Thus, the possibility remains that during chronic inflammation, all or a portion of the C-terminus cleaved apoSAA/SAA2 under low pH conditions in the EL, may transform into “nascent” AA.