Extracellular deposition of the (3/A4 amyloid peptide is a characteristic feature of the brain in patients with Alzheimer disease. ,f/A4 amyloid is derived from the amyloid precursor protein (APP), an integral membrane protein that exists as three major isoforms (APP695, APP75l, and APP770).Secreted forms of APP found in blood plasma and cerebrospinal fluid arise by proteolytic cleavage of APP within the 13/A4 amyloid domain, precluding the possibility of amyloidogenesis for that population of molecules. In the present study, we have demonstrated that treatment of PC12 cells with phorbol ester produces a severalfold increase in secretion of APP6s, APP751, and APP770. This increase is augmented by simultaneous treatment with the protein phosphatase inhibitor okadaic acid. These data indicate that protein phosphorylation regulates intra-3/A4 amyloid cleavage and APP secretion. These and other results suggest that APP molecules can normally follow either of two processing pathways: regulated secretion or proteolytic degradation unassociated with secretion.A characteristic pathological feature of Alzheimer disease is the extracellular deposition of 8/A4 amyloid in parenchymal brain tissues and in the walls of cerebral and meningeal blood vessels (1-3). This -4-kDa peptide is derived from the amyloid precursor protein (APP), a transmembrane glycoprotein that exists as three major isoforms (APP695, APP751, and APP770; Fig. 1 A), which result from the alternative splicing of mRNA from a single gene (4-10). The larger APP751 and APP770 isoforms contain a Kunitz-type protease inhibitor (KPI) insert in their extracellular domains (8-10). Secreted amino-terminal fragments of APP have been found in blood plasma and cerebrospinal fluid (11)(12)(13). These secreted fragments result from cleavage of APP within the ,f/A4 amyloid domain (14,15). The process of APP secretion, therefore, precludes the cerebral amyloidogenesis associated with Alzheimer disease. We have previously demonstrated that activation of protein kinase C (PKC) in PC12 cells by treatment with phorbol ester results in more rapid intracellular turnover of APP and increased production of carboxylterminal APP fragments (16). In the present report, we describe the regulation by protein phosphorylation of APP secretion in PC12 cells. MATERIALS AND METHODSThe materials and methods used in this report, including the [35S]methionine pulse-chase procedure, have been reported (17). PDBu, 4a-PDBu, and okadaic acid were purchased from LC Services (Woburn, MA). When the effects of phorbol esters (1 ,uM) or okadaic acid (1 ,uM) were examined, drug lanes 1-5) or for 4 h in the presence of PDBu (lanes 6-9). Four immunoprecipitating antibodies were used to identify the various APP species. Lanes: 1 and 6, anti-amino-terminal monoclonal antibody 22C11; 2 and 7, anti-KPI domain monoclonal antibody 56.1; 3 and 8, anti-carboxyl-terminal affinity-purified polyclonal antibody 369A; 4, antibody 369A preincubated with synthetic peptide corresponding to the cytoplasmic domain ...

Myelin, made by oligodendrocytes, is essential for rapid information transfer in the central nervous system. Oligodendrocyte precursors (OPs) receive glutamatergic synaptic input from axons but how this affects their development is unclear. Murine OPs in white matter express AMPA receptor (AMPAR) subunits GluA2, GluA3 and GluA4. We generated mice in which OPs lack both GluA2 and GluA3, or all three subunits GluA2/3/4, which respectively reduced or abolished AMPAR-mediated input to OPs. In both double-and triple-knockouts OP proliferation and number were unchanged but ~25% fewer oligodendrocytes survived in the subcortical white matter during development. In triple knockouts, this shortfall persisted into adulthood. The oligodendrocyte deficit resulted in ~20% fewer myelin sheaths but the average length, number and thickness of myelin internodes made by individual oligodendrocytes appeared normal. Thus, AMPAR-mediated signalling from active axons stimulates myelin production in developing white matter by enhancing oligodendrocyte survival, without influencing myelin synthesis per se.

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.

The metabolic fate of the Alzheimer 8/A4amyloid precursor protein (APP) includes intraamyloid proteolysis that leads to the production of secreted N-terminal and cell-associated C-terminal fragments. The cellular sites at which this processing occurs are not known. We have examined the route ofAPP processing in metabolically labeled PC12 cells. The lysosomotropic drug chloroquine exerted inhibitory effects on the degradation of mature APP holoprotein. In addition, recovery of a C-terminal fragment resulting from normal intraamyloid cleavage was signIficantly increased in the presence of chloroquine, suggesting that further degradation of the C-terminal fragment was inhibited. Chloroquine had virtually no effect on APP maturation (N-and 0-glycosylation and tyrosine sulfation) or secretion. (4,5), an =4-kDa polypeptide arising from cleavage of the amyloid precursor protein (APP) (6-9). APP exists as three major transmembrane isoforms (APP695, APP751, and APP770) that result from alternative splicing of a single primary transcript (Fig. 1A) (7,(10)(11)(12). Secretory processing of APP (13) leads to cleavage within the /A4 domain ("normal" cleavage) and precludes amyloidogenesis (14, 15). The biochemical defect responsible for amyloid production in AD might, therefore, involve either a deficiency in this proteolytic pathway or excessive activity of an alternative pathway. It is noteworthy that two types of inherited cerebral amyloidoses-hereditary cerebral hemorrhage with amyloidosis (Dutch type) and familial early-onset AD-are associated with mutations in the coding sequence of APP near the f3/A4-amyloid domain (16-19).Although the events leading to abnormal APP processing and amyloidosis are not known, elucidation of normal processing pathways may provide insights into AD pathogenesis. Cultured mammalian cells provide a readily accessible and well-characterized system for studies of protein trafficking. In the present investigation, we have examined the intracellular trafficking and processing of APP by [35S]methionine' pulse-labeling of PC12 neuroendocrine cells in the presence of pharmacologic agents known to affect the function of specific intracellular organelles. MATERIALS AND METHODSChloroquine was purchased from Sigma, monensin was purchased from Calbiochem, and brefeldin A was purchased from Epicentre Technologies (Madison, WI). Ascites fluid from mice injected intraperitoneally with hybridoma cells producing the anti-N-terminal APP monoclonal antibody 22C11 (13) was the kind gift of T. V. Ramabhadran (our laboratory) and S. S. Sisodia (The Johns Hopkins University School of Medicine, Baltimore). Affinity-purified rabbit anti-C-terminal APP antibody, here termed 369A, has been described (20). Agarose-coupled anti-mouse and anti-rabbit secondary antibodies were purchased from HyClone. Protein A-Sepharose CL-4B was obtained from Pharmacia LKB. Undifferentiated PC12 cells were grown to confluency on three 10-cm culture dishes in Dulbecco's modified Eagle's medium containing 10%o (vol/vol) heat-inac...

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Alzheimer (3/A4 amyloid precursor protein (APP) has been suggested to play a central role in the pathogenesis of Alzheimer disease. We have measured the content of different species of APP holoprotein and carboxyl-terminal fragments in human brains from young individuals, nondemented aged individuals, and aged individuals with Alzheimer disease. By using an antibody directed against the cytoplasmic domain of APP, five species were resolved. Three of these, of molecular masses 106, 113, and 133 kDa, represent presumptive immature and mature isoforms of APP holoprotein. Two smaller proteins, of molecular masses 15 and 19 kDa, represent presumptive proteolytic carboxyl-terminal fragments of APP. The 133-, 113-, 106-, and 15-kDa species were found in both grey and white matter, whereas the 19-kDa species was found only in grey matter. Total APP immunoreactivity (sum of an five species) and the levels of the 113-, 106-, and 15-kDa species were not iantiy different in brain samples from young individuals, nomdemented aged individuals, and aged individuals with Mzelmer disease. In contrast, the levels of the 133-and 19-kDa species increased 2-to 3-fold with age. A correlation was observed between the levels of the 133-and 19-kDa species, suggesting a possible precursor-product relationship.

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