MORPHOLOGICAL-DIFFERENTIATION AND PROTEOGLYCAN SYNTHESIS REGULATE ALZHEIMER AMYLOID PRECURSOR PROTEIN PROCESSING IN PC-12 AND HUMAN ASTROCYTE CULTURES

A morphologically differentiated strain of rat pheochromocytoma (PC-12H) metabolically labeled with [S-35] methionine and incubated with a phorbol ester displayed reduced 140-kDa and increased 15 kDa bands relative to cells incubated without phorbol ester after immunoprecipitation with antisera elicited by the C-terminal peptide of the Alzheimer amyloid precursor protein (APP). These bands correspond to glycosylated full length APP and a C-terminal fragment previously reported by Anderson et al. (Neurosci. Lett. 120:126-128, 1991) to result from a cleavage within the amyloidotic A4 region of APP, which releases a 120 kDa extracellular fragment. The 15 kDa fragment, not immunoprecipitated with an antisera elicited by the N-terminal portion of A4 amyloid, is nonamyloidogenic. Incubation of these cells with p-nitrophenylxyloside, known to inhibit proteoglycan formation, also increased this nonamyloidogenic cleavage of APP. In contrast to these results, an undifferentiated low passage PC-12-L strain constitutively displayed rapid nonamyloidogenic APP cleavage. Incubation of PC-12-L with phorbol ester did not affect the relative abundance of 140 or 15 kDa bands. Growth of PC-12-L with 7 S NGF or dibutyryl cAMP resulted in increased morphological differentiation and decreased APP cleavage which was now phorbol-inducible. Similar analyses of dividing and senescent human astrocytes and normal and F-AD fibroblasts indicate 5-fold lower rates of mid-A4 APP cleavage. Phorbol esters decreased the 140 kDa APP band without affecting the intensity of the 15 kDa band in these cells. Phorbol esters similarly reduces the 140 kDa band in 1 of 5 human neuroblastomas without affecting a constitutive level of 15 kDa APP which was 10-fold lower than that seen in PC-12-L. These data indicate that (1) rapid mid-A4 APP cleavage occurs in additional cell types including CNS astrocytes; (2) nonamyloidogenic APP cleavage is inhibited in constitutively or chemically differentiated PC-12 cells; (3) phorbol ester inductions of mid-A4 APP cleavage product accumulations and reduced accumulations of products of APP maturation are not coupled in all cell types; and (4) inhibition of proteoglycan formation increases this APP cleavage. These results may be relevant to alterations in APP processing in AD-affected brain regions.