Morphologic and biochemical analysis of the intracellular trafficking of the Alzheimer beta/A4 amyloid precursor protein

Abstract

Abnormal metabolic processing of the beta/A4 amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease. Several aspects of normal APP processing have been elucidated, but the precise cellular trafficking of APP remains unclear. To investigate APP trafficking pathways further, we have examined the subcellular distribution of APP in rat brain tissue and a variety of cultured cell types, and correlated this distribution with the biochemical processing of APP. In immunofluorescence microscopy of rat brain sections, APP immunoreactivity was concentrated in the Golgi complex and in proximal axon segments. In addition, a lower level of punctate fluorescence was visible throughout the neuropil. 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 brefeldin A (BFA), APP immunoreactivity changed from a Golgi-like to an endoplasmic reticulum-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 trans-Golgi network and early endosomal system. In immunoblots of BFA-treated cells, there was an accumulation of full-length APP and inhibition of APP secretory processing. Treatment with phorbol ester resulted in a marked elevation of APP secretion, but no obvious redistribution of APP immunoreactivity was apparent at the light microscope level. The lysosomotropic drug chloroquine induced accumulation of APP in cell lysates, as seen by immunoblotting. Immunofluorescence microscopy of chloroquine-treated cells demonstrated a colocalization of APP with the lysosomal marker Igp 120, whereas no colocalization 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 of its amino-terminal domain and/or to lysosomes for degradation.

Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein

“…The central role of the Golgi in APP metabolism is well appreciated; it represents the major site of APP concentration in the cell (18). More importantly, initial processing of APP by ␣-and ␤-secretases is intimately associated with a post-Golgi compartment and requires efficient transition of the precursor through this organelle (19,20).…”

Phospholipase D1 corrects impaired βAPP trafficking and neurite outgrowth in familial Alzheimer’s disease-linked presenilin-1 mutant neurons

“…After incubation of cell-free systems, vesicle and membrane fractions were separated by centrifugation at 1.14 ϫ 10 4 ϫ g for 30 s at 4°C in a Brinkmann centrifuge. Vesicle (supernatant) and membrane (pellet) fractions were diluted with immunoprecipitation buffer (50 mM Tris⅐HCl, pH 8.8͞150 mM NaCl͞6 mM EDTA͞2.5% Triton X-100͞5 mM methionine͞5 mM cysteine͞1 mg/ml BSA), immunoprecipitated using anti-␤APP C-terminal antibody 369 (4,38), and analyzed by SDS͞PAGE. Each experiment was performed at least three times.…”

“…T he distribution of ␤-amyloid precursor protein (␤APP) between the trans-Golgi network (TGN) and the cell surface may determine the relative generation of s␤APP␣ versus ␤-amyloid (1)(2)(3)(4)(5). Using a cell-free vesicle-trafficking reconstitution system derived from neuroblastoma cells, we showed previously that presenilin-1 (PS1), a major component of ␥-secretase, selectively affects ␤APP budding from the TGN and the endoplasmic reticulum (ER) (6).…”

The Functions of the Amyloid Precursor Protein Gene and Its Derivative Peptides: I Molecular Biology and Metabolic Processing

“…The protein and mRNAs for APLP1 and 2 are found in a similar distribution in human brain as APP providing further evidence for similarity of func- APP is found in neuronal cell membranes [74]. APP is metabolized in the Golgi apparatus and progresses to the liposomes for degradation and to the plasma membrane for secretion [75][76][77]. The activation of protein kinase C regulates the production of sAPP and decreases A in Golgi secretory vesicles [78].…”