Student Theses and Dissertations

Date of Award

1993

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Greengard Laboratory

Abstract

Extracellular deposition of the (β)/A4 amyloid peptide is a characteristic feature of the brain in patients with Alzheimer disease. (β)/A4 amyloid is derived from the integral membrane amyloid precursor protein (APP). Secreted truncated forms of A P P found in blood plasma and cerebrospinal fluid arise by proteolytic cleavage of APP within the (β)/A4 amyloid domain, precluding the possibility of amyloidogenesis for that population of molecules. The routes of APP processing were examined in metabolically labeled PC 12 cells treated with agents known to affect specific cellular functions. Treatment with either monensin or brefeldin A (BFA) prevented normal APP maturation (A^- and 0-glycosylation and tyrosine sulfation) and abolished APP secretion. Phorbol ester produced a several-fold increase in APP secretion, indicating that protein phosphorylation regulates intra- (β)/A4 amyloid cleavage and APP secretion. The lysosomotropic drug chloroquine exerted inhibitory effects on the degradation of mature APP holoprotein and of the carboxyl-terminal fragment resulting from secretory cleavage, but exhibited no effects on APP secretion. The results suggest that a substantial proportion of APP is degraded in an intracellular acidic compartment, but that the coupled APP cleavage/secretion event occurs in a chloroquine-insensitive compartment. Direct evidence that APP is targeted to the endosomal/lysosomal system was provided by the identification of APP in clathrin-coated vesicles, which mediate the transport of many proteins to endosomes. The subcellular distribution of APP was examined by microscopy and correlated with its biochemical processing. As shown by immunofluorescence microscopy of rat brain sections, APP was concentrated in the Golgi complex and in proximal axon segments. By immunoelectron microscopy of rat brain tissue fragments, APP was found associated with Golgi elements and with medium-sized, invaginated vesicles in both axons and dendrites. Prominent localization of APP to the Golgi complex was also found in primary cultures of rat hippocampal neurons and in non-neuronal cell lines. When cultured cells were treated with BFA, APP immunoreactivity changed from a Golgi-like to an ER-like distribution. No APP was detected in the BFA-induced reticulum identified by the transferrin receptor, indicating that concentration of APP in the Golgi does not reflect recycling between the rran5-Golgi network and the early endosomal system. Although treatment with phorbol ester resulted in a marked elevation of APP secretion, no redistribution of APP immunoreactivity was apparent. Chloroquine induced APP co-localization with the lysosomal marker lgpl20, whereas no co-localization was seen in untreated cells. Taken together, these results support a scheme in which APP is concentrated in the Golgi complex as it travels through the central vacuolar system en route to the plasma membrane for secretion and/or to lysosomes for degradation.

Comments

A thesis submitted to the Faculty of The Rockefeller University in partial fulfillment of the requirement for the degree of Doctor of Philosophy

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