Inhibition of Energy Metabolism Alters the Processing of Amyloid Precursor Protein and Induces a Potentially Amyloidogenic Derivative*

The cellular mechanisms which lead to the generation and pathological deposition of p amyloid in Alzheimer's disease are unknown. In this report we describe the pro- teolytic processing of the amyloid precursor protein (APP) to an 11.5-kDa COOH-terminal derivative which contains the full-length fi amyloid sequence. This processing step normally occurs at low levels in parallel with APP maturation in the secretory pathway. Inhibition of oxidative energy metabolism by sodium azide or the mitochondrial uncoupler carbonyl cyanide m-chlorophe- nylhydrazone increased the proteolysis of APP to the 11.5-kDa derivative by about 80-fold with accumulation of this APP derivative in the Golgi complex. Agents which inhibit protein transport in the secretory path- way, including monensin and brefeldin A, also increased the production of the 11.5-kDa derivative. Inhibition of APP maturation demonstrated that the 11.5-kDa derivative could be produced by proteolysis of immature APP. These results demonstrate that APP processing to potentially amyloidogenic COOH-terminal derivatives oc- curs in either the endoplasmic reticulum or Golgi complex and can be modulated

The cellular site of generation of the 4-kDa p amyloid peptide is unresolved, but has been proposed to occur in lysosomes (Shoji et al., 1992) or in the secretory pathway (Busciglio et a l . , 1993;Haass et al., 1993;Seubert et al., 1993).
In this report, we describe the cellular pathway of APP proteolysis to 11.5-kDa COOH-terminal derivatives which are potential processing intermediates in the generation of p amyloid.
Inhibitors of oxidative energy metabolism and inhibitors of APP transport in the secretory pathway markedly increase the proteolytic cleavage of APP to the 11.5-kDa derivative. These results demonstrate that impairment of oxidative energy metabolism alters the proteolytic processing of APP in the secretory pathway and may predispose to increased amyloid production in the Alzheimer brain.
ATP Depletion Induces a Potentially Amyloidogenic Deriva-Immunofluorescence Microscopy-Transfected COS cells plated on tive of MP-A'J" depletion has been shown to inhibit antero-12-mm covershps were fixed with methanol at -20 "C, washed, and grade transpod from the endoplasmic reticulum to the Golgi incubated with primary antibody for 60 min a t 37 "C. After washing, and to disrupt &lgi vesicular transport (Lippincott-Schwafiz cells were incubated with a rhodamine-or fluorescein-conjugated secondary antibody for 30 min at 37 "c, mounted, and viewed with a Zeiss et al., 1988;Donaldson et al., 1991;Persson et al., 1992). The Immunoprecipitations were performed with antibodies to the APP COOH terminus a(APP676-695) (lanes I d ) , the p amyloid amino terminus a(APP511-608) (lane 4 ) , and the entire p amyloid sequence a(pl-40) (lane 5) followed by 10-20% Tris-Tricine SDS-PAGE. Preabsorption of the antibodies with the cognate peptides abolished immunoprecipitation of the 11.5-kDa fragment (Busciglio et al., 1993). Cell viability after treatment with sodium azideRdeoxy-wglucose was confirmed by trypan blue exclusion. B, nontransfected HTB148 neuroglioma cells (ATCC) were labeled with [3sSlmethionine for 30 min followed by a 2-h chase in the absence (lane 1 ) or presence (lane 2) of 0.02% sodium azide and 50 m 2-deoxy-~glucose in glucose-free medium. Shown are cell lysates immunoprecipitated with a(APP676-695) and resolved by 10-20% Tris-Tricine SDS-PAGE. APP ( Fig. I.€?). The 11.5-kDa, but not the g-kDa, COOH-terminal derivative was selectively immunoprecipitated by an antibody to full-length p amyloid (pl-40) or the first 12 residues of p amyloid (clApP511-608) (Fig. lA, lanes 4 and 5). Previous results demonstrated that the 11.5-kDa COOH-terminal derivative is recognized by antibodies to /3 amyloid residues 1-12, 8-17, and 28-40 (Busciglio et al., 1993). Thus, ATP depletion induces a COOH-terminal APP derivative which contains both the NH,-and COOH-terminal domains of p amyloid.
To confirm that the 11.5-kDa COOH-terminal derivative contains the entire /3 amyloid sequence, radiosequence analysis of the amino terminus was performed after metabolic labeling with [3Hlphenylalanine. The major radioactive peak occurred at sequencer cycles 21-23 ( Fig. 2A derivative shows a minor secondary species with a peak a t cycle 4. C, [35S]methionine labeling of the 9-kDa APP derivative shows a major species with a peak at cycle 19. Immunoprecipitated radiolabeled protein was resolved by 10-20% Tris-Tricine SDS-PAGE and electroblotted to polyvinylidene difluoride membrane (Millipore). The band was identified by autoradiography, excised, and sequenced (Matsudaira, 1987). The anilinothiozolinone amino acids were collected a t each sequencer cycle and the radioactivity determined by liquid scintillation counting.

Energy Metabolism and APP Processing 13625
minor species with a peak a t cycle 4 ( Fig. 2B) was observed in nal derivatives (Fig. LA) (Busciglio et al., 1993). Indirect imfour separate radiosequence analyses, indicating a species munofluorescence microscopy of transfected COS cells with the which begins at the Asp-1 residue of /3 amyloid. Radiosequence APP COOH-terminal antibody showed APP staining in a reanalysis of the 9-kDa COOH-terminal derivative after labeling ticular and dense perinuclear pattern characteristic of the enwith [35Slmethionine showed a primary peak at cycle 19 with doplasmic reticulum and Golgi, respectively (Fig. 4A). Double some NH,-terminal microheterogeneity (Fig. 2C), consistent staining with the Golgi-specific monoclonal antibody JE4 demwith the sequence of the constitutively produced 9-kDa frag-onstrated the perinuclear distribution of the Golgi complex ment described previously (Esch et al., 1990). DNA sequencing (Fig. a). Shining with the rx(/31-40) antibody which reacts of the APP expression vector confirmed the correct sequence of with the 11.5-kDa derivative was limited to the distribution of the region which spans residues 576-695 of APPeg5.
the Golgi marker (Fig. 4, B and C). Induction of the 11.5-kDa The 11.5-kDa COOH-terminal U P Derivative I S Generated derivative by treatment with sodium azide and 2&oxy-~-gluin the secretory Pathway-To determine if the 11.5-kDa COOH-cOSe (~i~. 1~) resulted in increased a l g i staining with the terminal derivative is Produced at an early stage of APP PrOC-(y(pl-40) antibody (Fig. 5, C and D ) . These results suggest that essing, pulse-chase kinetic analysis was performed. The gen-the 1 1 . 5 -k~~ derivative is generated in the secretory pathway eration of the 9-and 11.5-kDa COOH-terminal derivatives and accumulates in the a l g i complex after inhibition ofenergy paralleled the maturation of APP (Fig. 3, A and B ) . However, metabolism. both COOH-terminal derivatives were detected after only 5 Inhibitors of Dansp0l.t and Processing in the Secretory Pathmin of labeling, Prior to APp maturation (Fig. 3, A and B). way Induce the 11.5-kDa App Derivative-To determine if the Thus, both the 9-and 11.5-kDa COOH-terminal derivatives are induction of the 1 1 . 5 -k~~ derivative by inhibition of energy generated during the maturation of APP and can be detected at metabolism is due to inhibition of APP processing in the secrean early stage of APP processing. tory pathway, we examined the effects of agents which inhibit processing in the secretory pathway by different To confirm that the 11.5-kDa derivative is localized to the secretory pathway, the distribution was determined mechanisms. Brefeldin A prevents the transport of proteins to immunoc~ochemistry~ The rx(/31-40) antibody reacts the trans-Golgi by causing fusion of the proximal Golgi cornstrongly with the 11.5-kDa APP derivative but weakly with APp in transfected ' Os whereas the partments with the endoplasmic reticulum (Lippincott-Schwa& et al., 1989;Klausner et al., 1992). Treatment of bution ofAPP to a diffuse reticular pattern characteristic of the

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to the 11.5-kDa COOH-terminal derivative occurs earlier in the nal antibody reacts with both APp and CooH-temitransfected COS cells with brefeldin A resulted in the redistri- generation O f w p COOH-terminal derivatives.A, 11.5-and 9-kDa secretion (Fig. 6, B and c, lane 3 ) and increased the level ofthe AF' P, 8% SDS-PAGE. Transfected COS cells overexpressingAPP6g, were derivative by (Fig' 6AJ lane 3) , 1980;Woods and Lazarides, 1985). Treatment with CCCP inhibited the maturation and secretion ofAPP (Fig.  6, B and C, lane 5 ) and dramatically increased the level of the 11.5-kDa COOH-terminal derivative (Fig. 6 A , lane 5 ) . The effect of CCCP was similar in magnitude to that of sodium azide/ 2-deoxy-~-ghcose. These results demonstrate that agents which inhibit APP maturation and secretion by several different mechanisms all induce the 11.5-kDa COOH-terminal derivative. The role of the endosomaVlysosoma1 pathway in the metabolism of the 11.5-kDa species was examined using chloroquine, a weak base which inhibits degradation in lysosomes (de Duve et al., 1974). Treatment with chloroquine did not significantly affect APP maturation or secretion ( Fig. 6B and C, lane 6), but resulted in the accumulation of an array of COOH-terminal species (Fig. 6 A , lane 6), as reported previously (Haass et al., 1992a), and increased the level of the 11.5-kDa COOH-terminal derivative by about 3-fold (Fig. S A , lane 6). A similar effect was observed after treatment with ammonium chloride (Busciglio et  al., 1993). These results suggest that the 11.5-kDa COOHterminal derivative is degraded in a chloroquine-sensitive endosomal or lysosomal compartment following its generation in the secretory pathway.

Energy Metabolism and APP
Inhibition of Energy Metabolism Increases Proteolytic Cleavage of Immature APP to the 11.5-kDa Derivative-The mechanism of induction of the 11.5-kDa derivative by ATP depletion was further examined by kinetic analysis of APP processing in the presence of sodium azide and 2-deoxy-~-ghcose. Sodium azide/2-deoxy-~-glucose inhibited APP maturation and degradation, as evidenced by persistence of the lower molecular weight incompletely glycosylated form (Fig. 7A). Treatment with sodium azide/2-deoxy-~-glucose resulted in a 14-fold increase in the level of the 11.5-kDa COOH-terminal derivative, which occurred abruptly after 2 h (Fig. 7, B and C). After 4 h of treatment with the inhibitors, the level of the 11.5-kDa species was 80-fold higher than in untreated cells (Fig. 7, B and C).
Quantitative analysis showed that this increase was due to increased proteolytic cleavage of APP to the 11.5-kDa derivative (Fig. 7C). In contrast, sodium azide/2-deoxy-~-glucose had a biphasic effect on the 9-kDa constitutive COOH-terminal derivative, initially inhibiting its generation and then inhibiting its degradation (Fig. 7, B and C). Wash out of the energy inhibitors was followed by degradation of the 9-and 11.5-kDa COOH-terminal derivatives with a half-life for both of 40 to 50 min. Thus, inhibition of energy metabolism increases the proteolytic cleavage of APP to a stable 11.5-kDa derivative.
To determine if the 11.5-kDa derivative could be derived from immature APP, sodium azide/2-deoxy-~-glucose was maintained in the culture medium during the entire metabolic labeling period. This resulted in partial inhibition of APP synthesis and almost complete inhibition of APP maturation (Fig.  8A). Under these conditions, the 11.5-kDa derivative was still generated from immature APP and was the most abundant COOH-terminal derivative (Fig. 8B). These results suggest that the 11.5-kDa derivative can be generated from immature APP early in the secretory pathway.

DISCUSSION
These experiments demonstrate that a potentially amyloidogenic 11.5-kDa COOH-terminal derivative of APP is generated by proteolytic cleavage in the secretory pathway. This process- ing event is novel because it is markedly induced by ATP depletion. A similar APP COOH-terminal Fragment has been reported, and it was suggested that this fragment is formed by proteolytic cleavage at the /3 amyloid amino terminus (Busciglio et al., 1993;Cai et al., 1993). The sequence analysis reported here shows that the 11.5-kDa species contains at least two COOH-terminal APP derivatives, a major derivative which begins 19 residues amino-terminal to the first residue of p amyloid and a second derivative which begins at the Asp-1 residue of p amyloid. The co-migration of these two species may reflect different COOH termini or may be due to aberrant electrophoretic migration, as noted previously for other APP COOH-terminal fragments (Esch et al., 1990).
Pulse-chase kinetic analysis showed that the 11.5-kDa derivative is normally produced at low levels during the processing of APP in the secretory pathway. This was markedly increased by agents which inhibit transport and processing in the secretory pathway, including inhibitors of energy metabolism, brefeldin A, and monensin. Consistent with these findings was the observation that immature APP could undergo proteolytic cleavage to the 11.5-kDa derivative, localizing the site of cleavage to the endoplasmic reticulum or proximal Golgi. Inhibitors of protein transport in the secretory pathway may increase this proteolytic cleavage either by increasing the level of immature APP or by increasing the exposure of APP to a protease in the endoplasmic reticulum or Golgi. In contrast, monensin and brefeldin A inhibited the secretase cleavage of APP, suggesting that it occurs later in the secretory pathway than the 11.5-kDagenerating cleavage, most likely in the trans-Golgi network or at the cell surface (Sambamurti et al., 1992;Sisodia, 1992). Together, these results suggest that the proteolytic cleavage of APP occurs in at least two distinct compartments of the secretory pathway.
Previous studies have shown that proteolytic degradation of some proteins occurs early in the secretory pathway in an endoplasmic reticulum-associated compartment (Lippincott-Schwartz et al., 1988;Klausner and Sitia, 1990). Protein degradation in this pathway occurs prior to maturation in the Golgi and can be distinguished from lysosomal degradation by its resistance to brefeldin A, monensin, and inhibitors of energy Energy Metabolism and APP Processing metabolism (Lippincott-Schwartz et al., 1988;Klausner and Sitia, 1990). Proteolysis of APP to the 11.5-kDa fragment exhibits these characteristics, suggesting that it may occur in this degradative pathway. Complete degradation of proteins in this pathway has been described, but to our knowledge, this may be the first report of the generation of a stable protein fragment in this compartment. Whether the 11.5-kDa COOH-terminal APP derivative is processed directly to p amyloid remains to be determined. This possiblity is supported by the observation that the familial Swedish APP mutation markedly increases the production of both p amyloid and the 11.5-kDa COOH-terminal derivative (Cai et al., 1993;Citron et al., 1992). Our results suggest that the APP cleavage which generates the amino terminus of p amyloid can occur in the endoplasmic reticulum or proximal Golgi giving rise to the 11.5-kDa derivative. The second cleavage which generates the COOH terminus of p amyloid most likely occurs later in either the trans-Golgi network or in endosomes, consistent with the inhibition of p amyloid production by monensin and brefeldinA (Busciglio et al., 1993;Haass et al., 1993). Although COOH-terminal derivatives can be metabolized in lysosomes (Caporaso et al., 1992;Golde et al., 1992;Haass et al., 1992a1, this is a less likely site of p amyloid production based on the minimal effects of some lysosomal inhibitors. The transmembrane cleavage required for the generation of p amyloid is not expected to occur in the normal processing of proteins but may be due to proteolysis of incorrectly processed APP molecules in the secretory pathway, as recently suggested by Zhong et al. (1994). The dramatic effect of inhibition of oxidative energy metabolism on the proteolytic processing ofAPP may have significance for the pathogenesis of Alzheimer's disease. Recent findings suggest that there is a selective deficit in the azide-sensitive mitochondrial complex IV in the cerebral cortex of patients with Alzheimer's disease . Furthermore, deficits in oxidative energy metabolism have been shown to accompany normal aging and may be related to an age-dependent increase in mitochondrial DNA mutations (Corral-Debrinski et al., 1992;Bowling et al., 1993;Mecocci et al., 1993). Impairment of oxidative energy metabolism may therefore contribute to the pathogenesis of Alzheimer's disease by altering the metabolism of APP, leading to decreased APP secretion and increased generation of a potentially amyloidogenic derivative.