Presenilin-dependent gamma-secretase processing of beta-amyloid precursor protein at a site corresponding to the S3 cleavage of Notch.

The presenilin (PS)-dependent site 3 (S3) cleavage of Notch liberates its intracellular domain (NICD), which is required for Notch signaling. The similar gamma-secretase cleavage of the beta-amyloid precursor protein (betaAPP) results in the secretion of amyloid beta-peptide (Abeta). However, little is known about the corresponding C-terminal cleavage product (CTFgamma). We have now identified CTFgamma in brain tissue, in living cells, as well as in an in vitro system. Generation of CTFgamma is facilitated by PSs, since a dominant-negative mutation of PS as well as a PS gene knock out prevents its production. Moreover, gamma-secretase inhibitors, including one that is known to bind to PS, also block CTFgamma generation. Sequence analysis revealed that CTFgamma is produced by a novel gamma-secretase cut, which occurs at a site corresponding to the S3 cleavage of Notch.

Phosphorylation regulates intracellular trafficking of beta-secretase.

beta-Secretase (BACE) is a transmembrane aspartyl protease, which generates the N terminus of Alzheimer's disease amyloid beta-peptide. Here, we report that BACE can be phosphorylated within its cytoplasmic domain at serine residue 498 by casein kinase 1. Phosphorylation exclusively occurs after full maturation of BACE by propeptide cleavage and complex N-glycosylation. Phosphorylation/dephosphorylation affects the subcellular localization of BACE. BACE wild type and an S498D mutant that mimics phosphorylated BACE are predominantly located within juxtanuclear Golgi compartments and endosomes, whereas nonphosphorylatable BACE S498A accumulates in peripheral EEA1-positive endosomes. Antibody uptake assays revealed that reinternalization of BACE from the cell surface is independent of its phosphorylation state. After reinternalization, BACE wild type as well as BACE S498D are efficiently retrieved from early endosomal compartments and further targeted to later endosomal compartments and/or the trans-Golgi network. In contrast, nonphosphorylatable BACE S498A is retained within early endosomes. Our results therefore demonstrate regulated trafficking of BACE within the secretory and endocytic pathway.

Glycine 384 is required for presenilin-1 function and is conserved in bacterial polytopic aspartyl proteases.

Endoproteolysis of beta-amyloid precursor protein (betaAPP) and Notch requires conserved aspartate residues in presenilins 1 and 2 (PS1 and PS2). Although PS1 and PS2 have therefore been proposed to be aspartyl proteases, no homology to other aspartyl proteases has been found. Here we identify homology between the presenilin active site and polytopic aspartyl proteases of bacterial origin, thus supporting the hypothesis that presenilins are novel aspartyl proteases.

A loss of function mutant of the presenilin homologue SEL-12 undergoes aberrant endoproteolysis in Caenorhabditis elegans and increases abeta 42 generation in human cells.

The familial Alzheimer's disease-associated presenilins (PSs) occur as a dimeric complex of proteolytically generated fragments, which functionally supports endoproteolysis of Notch and the beta-amyloid precursor protein (betaAPP). A homologous gene, sel-12, has been identified in Caenorhabditis elegans. We now demonstrate that wild-type (wt) SEL-12 undergoes endoproteolytic cleavage in C. elegans similar to the PSs in human tissue. In contrast, SEL-12 C60S protein expressed from the sel-12(ar131) allele is miscleaved in C. elegans, resulting in a larger mutant N-terminal fragment. Neither SEL-12 wt nor C60S undergo endoproteolytic processing upon expression in human cells, suggesting that SEL-12 is cleaved by a C. elegans-specific endoproteolytic activity. The loss of function of sel-12 in C. elegans is not associated with a dominant negative activity in human cells, because SEL-12 C60S and the corresponding PS1 C92S mutation do not interfere with Notch1 cleavage. Moreover, both mutant variants increase the aberrant production of the highly amyloidogenic 42-amino acid version of amyloid beta-peptide similar to familial Alzheimer's disease-associated human PS mutants. Our data therefore demonstrate that the C60S mutation in SEL-12 is associated with aberrant endoproteolysis and a loss of function in C. elegans, whereas a gain of misfunction is observed upon expression in human cells.

Maturation and pro-peptide cleavage of beta-secretase.

Amyloid beta-peptide is generated by two sequential proteolytic cleavages mediated by beta-secretase (BACE) and gamma-secretase. BACE was recently identified as a membrane-associated aspartyl protease. We have now analyzed the maturation and pro-peptide cleavage of BACE. Pulse-chase experiments revealed that BACE is post-translationally modified during transport to the cell surface, which can be monitored by a significant increase in the molecular mass. The increase in molecular mass is caused by complex N-glycosylation. Treatment with tunicamycin and N-glycosidase F led to a BACE derivative with a molecular weight corresponding to an unmodified version. In contrast, the mature form of BACE was resistant to endoglycosidase H treatment. The cytoplasmic tail of BACE was required for efficient maturation and trafficking through the Golgi; a BACE variant lacking the cytoplasmic tail undergoes inefficient maturation. In contrast a soluble BACE variant that does not contain a membrane anchor matured more rapidly than full-length BACE. Pro-BACE was predominantly located within the endoplasmic reticulum. Pro-peptide cleavage occurred immediately before full maturation and trafficking through the Golgi.

Separation of presenilin function in amyloid beta-peptide generation and endoproteolysis of Notch.

Most of the genetically inherited Alzheimer's disease cases are caused by mutations in the presenilin genes, PS1 and PS2. PS mutations result in the enhanced production of the highly amyloidogenic 42/43 amino acid variant of amyloid beta-peptide (Abeta). We have introduced arbitrary mutations at position 286 of PS1, where a naturally occurring PS1 mutation has been described (L286V). Introduction of charged amino acids (L286E or L286R) resulted in an increase of Abeta42/43 production, which reached almost twice the level of the naturally occurring PS1 mutation. Although pathological Abeta production was increased, endoproteolysis of Notch and nuclear transport of its cytoplasmic domain was significantly inhibited. These results demonstrate that the biological function of PS proteins in the endoproteolysis of beta-amyloid precursor protein and Notch can be separated.

Presenilin-1 differentially facilitates endoproteolysis of the beta-amyloid precursor protein and Notch.

Mutations in the presenilin-1 (PS1) gene are associated with Alzheimer's disease and cause increased secretion of the neurotoxic amyloid-beta peptide (Abeta). Critical intramembraneous aspartates at residues 257 and 385 are required for the function of PS1 protein. Here we investigate the biological function of a naturally occurring PS1 splice variant (PS1 Deltaexon 8), which lacks the critical aspartate 257. Cell lines that stably express PS1 Deltaexon 8 or a PS1 protein in which aspartate residue 257 is mutated secrete significant levels of Abeta, whereas Abeta generation is severely reduced in cells transfected with PS1 containing a mutation of aspartate 385. In contrast, endoproteolytic processing of Notch is almost completely inhibited in cell lines expressing any of the PS1 variants that lack one of the critical aspartates. These data indicate that PS1 may differentially facilitate gamma-secretase-mediated generation of Abeta and endoproteolysis of Notch.

Rest and exercise hemodynamics before and after valve replacement--a combined Doppler/catheter study.

BACKGROUND: Hemodynamic improvement is a common finding following valve replacement. However, despite a normally functioning prosthesis and normal left ventricular ejection fraction, some patients may show an abnormal hemodynamic response to exercise. METHODS: In a combined catheter/Doppler study, rest and exercise hemodynamics were evaluated in 23 patients following aortic (n = 12) (Group 1) or mitral valve (n = 11) (Group 2) replacement and compared with preoperative findings. Patient selection was based on absence of coronary artery disease and left ventricular failure as shown by preoperative angiography. Cardiac output, pulmonary artery pressure, pulmonary capillary pressure, and pulmonary resistance were measured by right heart catheterization, whereas the gradient across the valve prosthesis was determined by Doppler echocardiography. Postoperative evaluation was done at rest and during exercise. The mean follow-up was 8.2 +/- 2.2 years in Group 1 and 4.2 +/- 1 years in Group 2. RESULTS: With exercise, there was a significant rise in cardiac output in both groups. In Group 1, mean pulmonary pressure/capillary pressure decreased from 24 +/- 9/18 +/- 9 mmHg preoperatively to 18 +/- 2/12 +/- 4 mmHg postoperatively (p < 0.05), and increased to 43 +/- 12/30 +/- 8 mmHg with exercise (p < 0.05). The corresponding values for Group 2 were 36 +/- 12/24 +/- 6 mmHg preoperatively, 24 +/- 7/17 +/- 6 mmHg postoperatively (p < 0.05), and 51 +/- 2/38 +/- 4 mmHg with exercise (p < 0.05). Pulmonary vascular resistance was 109 +/- 56 dyne.s.cm-5 preoperatively, 70 +/- 39 dyne.s.cm-5 postoperatively (p < 0.05), and 70 +/- 36 dyne.s.cm-5 with exercise in Group 1. The corresponding values for Group 2 were 241 +/- 155 dyne.s.cm-5, 116 +/- 39 dyne.s.cm-5 (p < 0.05), and 104 +/- 47 dyne.s.cm-5. There was a significant increase in the gradients across the valve prosthesis in both groups, showing a significant correlation between the gradient at rest and exercise. No correlation was found between valve prosthesis gradient and pulmonary pressures. CONCLUSION: Exercise-induced pulmonary hypertension and abnormal left ventricular filling pressures seem to be a frequent finding following aortic or mitral valve replacement. Both hemodynamic abnormalities seem not to be determined by obstruction to flow across the valve prosthesis and may be concealed, showing nearly normal values at rest but a pathologic response to physical stress.

A loss of function mutation of presenilin-2 interferes with amyloid beta-peptide production and notch signaling.

Presenilin-1 (PS1) facilitates gamma-secretase cleavage of the beta-amyloid precursor protein and the intramembraneous cleavage of Notch1. Although Alzheimer's disease-associated mutations in the homologous presenilin (PS2) gene elevate amyloid beta-peptide (Abeta42) production like PS1 mutations, here we demonstrate that a gene ablation of PS2 (unlike that of PS1) in mice does not result in a severe phenotype resembling that of Notch-ablated animals. To investigate the amyloidogenic function of PS2 more directly, we mutagenized a conserved aspartate at position 366 to alanine, because the corresponding residue of PS1 is known to be required for its amyloidogenic function. Cells expressing the PS2 D366A mutation exhibit significant deficits in proteolytic processing of beta-amyloid precursor protein indicating a defect in gamma-secretase activity. The reduced gamma-secretase activity results in the almost complete inhibition of Abeta and p3 production in cells stably expressing PS2 D366A, whereas cells overexpressing the wild-type PS2 cDNA produce robust levels of Abeta and p3. Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation not only blocks gamma-secretase activity but also inactivates PS2 activity in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain. These data suggest that PS2 is functionally involved in Abeta production and Notch signaling by facilitating similar proteolytic cleavages.

hFE65L influences amyloid precursor protein maturation and secretion.

The amyloid precursor protein (APP) is processed in the secretory and endocytic pathways, where both the neuroprotective alpha-secretase-derived secreted APP (APPs alpha) and the Alzheimer's disease-associated beta-amyloid peptide are generated. All three members of the FE65 protein family bind the cytoplasmic domain of APP, which contains two sorting signals, YTS and YENPTY. We show here that binding of APP to the C-terminal phosphotyrosine interaction domain of hFE65L requires an intact YENPTY clathrin-coated pit internalization sequence. To study the effects of the hFE65L/APP interaction on APP trafficking and processing, we performed pulse/chase experiments and examined APP maturation and secretion in an H4 neuroglioma cell line inducible for expression of the hFE65L protein. Pulse/chase analysis of endogenous APP in these cells showed that the ratio of mature to total cellular APP increased after the induction of hFE65L. We also observed a three-fold increase in the amount of APPs alpha recovered from conditioned media of cells overexpressing hFE65L compared with uninduced controls. The effect of hFE65L on the levels of APPs alpha secreted is due neither to a simple increase in the steady-state levels of APP nor to activation of the protein kinase C-regulated APP secretion pathway. We conclude that the effect of hFE65L on APP processing is due to altered trafficking of APP as it transits through the secretory pathway.

Genes and mechanisms involved in beta-amyloid generation and Alzheimer's disease.

Alzheimer's disease is characterized by the invariable accumulation of senile plaques that are predominantly composed of amyloid beta-peptide (Abeta). Abeta is generated by proteolytic processing of the beta-amyloid precursor protein (betaAPP) involving the combined action of beta- and gamma-secretase. Cleavage within the Abeta domain by alpha-secretase prevents Abeta generation. In some very rare cases of familial AD (FAD), mutations have been identified within the betaAPP gene. These mutations are located close to or at the cleavage sites of the secretases and pathologically effect betaAPP processing by increasing Abeta production, specifically its highly amyloidogenic 42 amino acid variant (Abeta42). Most of the mutations associated with FAD have been identified in the two presenilin (PS) genes, particularly the PS1 gene. Like the mutations identified within the betaAPP gene, mutations in PS1 and PS2 cause the increased generation of Abeta42. PS1 has been shown to be functionally involved in Notch signaling, a key process in cellular differentation, and in betaAPP processing. A gene knock out of PS1 in mice leads to an embryonic lethal phenotype similar to that of mice lacking Notch. In addition, absence of PS1 results in reduced gamma-secretase cleavage and leads to an accumulation of betaAPP C-terminal fragments and decreased amounts of Abeta. Recent work may suggest that PS1 could be the gamma-secretase itself, exhibiting the properties of a novel aspartyl protease. Mutagenesis of either of two highly conserved intramembraneous aspartate residues of PS1 leads to reduced Abeta production as observed in the PS1 knockout. A corresponding mutation in PS2 interfered with betaAPP processing and Notch signaling suggesting a functional redundancy of both presenilins. In this issue, some of the recent work on the molecular mechanisms involved in Alzheimer's disease (AD) as well as novel diagnostic approaches and risk factors for AD will be discussed. In the first article, we like to give an overview on mechanisms involved in the proteolytic generation of Amyloid beta-peptide (Abeta), the major pathological player of this devastating disease. In the second part of this article recent results will be described, which demonstrate an unexpected biological and pathological function of an AD associated gene.

Truncated presenilin 2 derived from differentially spliced mRNA does not affect the ratio of amyloid beta-peptide 1-42/1-40.

Numerous mutations in the presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD). Here we characterize the expression of two naturally occurring alternative PS2 transcripts which lack either exons 3 and 4 (PS2 deltaexon3,4) or exons 3, 4, and 8 (PS2 deltaexon3,4,8). These transcripts do not contain the natural initiation codon within exon 3. The transcripts are efficiently translated as N-terminal truncated proteins. These deleted proteins are still able to regulate formation of endogenous PS fragments, indicating that the C-terminal half of the PS2 protein is sufficient for this phenomenon. Although approximately 50% of the PS1 and both PS2 mutations occur within the N-terminal region lacking in the PS2 deltaexon3,4 and PS2 deltaexon3,4,8 proteins, expression of these truncated proteins does not affect pathological generation of amyloid beta-peptide (Abeta). This suggests that point mutations causing AD are gain of function mutations.

Expression of Alzheimer's disease-associated presenilin-1 is controlled by proteolytic degradation and complex formation.

Numerous mutations causing early onset Alzheimer's disease have been identified in the presenilin (PS) genes, particularly the PS1 gene. Like the mutations identified within the beta-amyloid precursor protein gene, PS mutations cause the increased generation of a highly neurotoxic variant of amyloid beta-peptide. PS proteins are proteolytically processed to an N-terminal approximately 30-kDa (NTF) and a C-terminal approximately 20-kDa fragment (CTF20) that form a heterodimeric complex. We demonstrate that this complex is resistant to proteolytic degradation, whereas the full-length precursor is rapidly degraded. Degradation of the PS1 holoprotein is sensitive to inhibitors of the proteasome. Formation of a heterodimeric complex is required for the stability of both PS1 fragments, since fragments that do not co-immunoprecipitate with the PS complex are rapidly degraded by the proteasome. Mutant PS fragments not incorporated into the heterodimeric complex lose their pathological activity in abnormal amyloid beta-peptide generation even after inhibition of their proteolytic degradation. The PS1 heterodimeric complex can be attacked by proteinases of the caspase superfamily that generate an approximately 10-kDa proteolytic fragment (CTF10) from CTF20. CTF10 is rapidly degraded most likely by a calpain-like cysteine proteinase. From these data we conclude that PS1 metabolism is highly controlled by multiple proteolytic activities indicating that subtle changes in fragment generation/degradation might be important for Alzheimer's disease-associated pathology.

Proteolytic processing of Alzheimer's disease associated proteins.

Amyloid beta-peptide (A beta), the major component of senile plaques, is generated by proteolytic processing from the beta-amyloid precursor protein (beta APP). Mutations within the beta APP gene cause early onset familial AD (FAD) by affecting A beta generation. Interestingly, the much more abundant mutations within the presenilin (PS) genes also result in the abnormal generation of a 42 residue A beta (A beta 42), thus clearly supporting a pivotal role of A beta for the pathology of AD. PS proteins are proteolytically processed into stable 30 kDa N-terminal fragments (NTF) and 20 kDa C-terminal fragments (CTF). Beside the conventional proteolytic pathway. PS proteins can also be cleaved further C-terminal by proteases of the caspase superfamily. PS proteins were localized within the endoplasmic reticulum (ER) and early Golgi, compartments which we have demonstrated to be involved in A beta 42 generation and intracellular accumulation. Using Caenorhabditis elegans as a simple animal model, we demonstrate that PS proteins are involved in NOTCH signaling FAD causing mutations interfere with the biological function of PS proteins in NOTCH signaling.

Mutant presenilin 2 transgenic mouse: effect on an age-dependent increase of amyloid beta-protein 42 in the brain.

The N141I missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease (AD) in the Volga German families. We have generated transgenic mouse lines overexpressing human wild-type or mutant PS2 under transcriptional control of the chicken beta-actin promoter. In the brains of transgenic mice, the levels of human PS2 mRNA were found to be five- to 15-fold higher than that of endogenous mouse PS2 mRNA. The amyloid beta-protein (Abeta) 42 levels in the brains of mutant PS2 transgenic mice were higher than those in wild-type PS2 transgenic mice at the age of 2, 5, or 8 months. In addition, the Abeta42 levels appeared to increase steadily in the mutant PS2 transgenic mouse brains from 2 to 8 months of age, whereas there was only a small increase in wild-type transgenic mice between the ages of 5 and 8 months. There was no definite difference in the levels of N-terminal and C-terminal fragments between wild-type and mutant PS2 transgenic mice at the age of 2, 5, or 8 months. These data show a definite effect of the PS2 mutation on an age-dependent increase of Abeta42 content in the brain.

The proteolytic fragments of the Alzheimer's disease-associated presenilin-1 form heterodimers and occur as a 100-150-kDa molecular mass complex.

Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease to two stable fragments of approximately 30 kDa (N-terminal fragment (NTF)) and approximately 20 kDa (C-terminal fragment (CTF)) (Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190). Here we show that the CTF and NTF of PS-1 bind to each other. Fractionating proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-extracted membrane preparations by velocity sedimentation reveal a high molecular mass SDS and Triton X-100-sensitive complex of approximately 100-150 kDa. To prove if both proteolytic fragments of PS-1 are bound to the same complex, we performed co-immunoprecipitations using multiple antibodies specific to the CTF and NTF of PS-1. These experiments revealed that both fragments of PS-1 occur as a tightly bound non-covalent complex. Upon overexpression, unclipped wild type PS-1 sediments at a lower molecular weight in glycerol velocity gradients than the endogenous fragments. In contrast, the non-cleavable, FAD-associated PS-1 Deltaexon 9 sediments at a molecular weight similar to that observed for the endogenous proteolytic fragments. This result may indicate that the Deltaexon 9 mutation generates a mutant protein that exhibits biophysical properties similar to the naturally occurring PS-1 fragments. This could explain the surprising finding that the Deltaexon 9 mutation is functionally active, although it cannot be proteolytically processed (Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grünberg, J., and Haass, C. (1997) Genes & Function 1, 149-159; Levitan, D., Doyle, T., Brousseau, D., Lee, M., Thinakaran, G., Slunt, H., Sisodia, S., and Greenwald, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14940-14944). Formation of a high molecular weight complex of PS-1 composed of both endogenous PS-1 fragments may also explain the recent finding that FAD-associated mutations within the N-terminal portion of PS-1 result in the hyperaccumulation not only of the NTF but also of the CTF (Lee, M. K., Borchelt, D. R., Kim, G., Thinakaran, G., Slunt, H. H., Ratovitski, T., Martin, L. J., Kittur, A., Gandy, S., Levey, A. I., Jenkins, N., Copeland, N., Price, D. L., and Sisodia, S. S. (1997) Nat. Med. 3, 756-760). Moreover, these results provide a model to understand the highly regulated expression and processing of PS proteins.

Cellular expression and proteolytic processing of presenilin proteins is developmentally regulated during neuronal differentiation.

We have determined the expression of the Alzheimer's disease-associated proteins presenilin-1 and presenilin-2 in primary cultures of rat hippocampal neurons. Neurons highly express presenilin-1 and presenilin-2, whereas both proteins were not detected in astrocytes. Further, we have analyzed the subcellular localization and expression in rat hippocampal neurons during development. Although presenilin proteins were localized predominantly to the endoplasmic reticulum in nonneuronal cells transfected with presenilin cDNAs, in neurons, presenilin proteins were also found in compartments not staining with antibodies to grp78(BiP). Presenilin-1 and presenilin-2 were predominantly detected in vesicular structures within the somatodendritic compartment with much less expression in axons. Polarized distribution of presenilin-1 and presenilin-2 differs slightly, with more presenilin-2 expressed in axons compared with presenilin-1. Presenilin expression was found to be developmentally regulated. Presenilin expression strongly increased during neuronal differentiation until full morphological polarization and then declined. No full-length presenilin-1 or presenilin-2 could be detected within cell lysates. At early developmental stages the expected approximately 34-kDa N-terminal proteolytic fragment of presenilin-1 and the approximately 38-kDa fragment of presenilin-2 were detected. Later during differentiation we predominantly detected a approximately 38-kDa fragment for presenilin-1 and a approximately 42-kDa fragment for presenilin-2. By epitope mapping, we show that these slower migrating peptides represent N-terminal proteolytic fragments, cleaved C-terminal to the conventional site of processing. It is noteworthy that both presenilin-1 and presenilin-2 undergo alternative proteolytic cleavage at the same stage of neuronal differentiation. Regulation of presenilin expression and proteolytic processing might have implications for the pathological as well as the biological function of presenilins during aging in the human brain.

Intracellular generation and accumulation of amyloid beta-peptide terminating at amino acid 42.

Amyloid beta-peptide (Abeta) is known to accumulate in senile plaques of Alzheimer's disease (AD) patients and is now widely believed to play a major role in the disease. Two populations of peptides occur terminating either at amino acid 40 or at amino acid 42 (Abeta1-40 and Abeta1-42). Alternative N-terminal cleavages produce additional heterogeneity (Abetax-40 and Abetax-42). Peptides terminating at amino acid 42 are believed to be the major player in sporadic AD as well as familial AD (FAD). Whereas the cellular mechanism for the generation of Abeta terminating at amino acid 40 is well understood, very little is known about the cleavage of Abeta after amino acid 42. By using two independent methods we demonstrate intracellular Abeta1-42 as well as Abetax-42 but less Abetax-40 and Abeta1-40 in kidney 293 cells stably transfected with wild type beta-amyloid precursor protein (betaAPP) or the FAD-associated Val/Gly mutation. Moreover, retention of betaAPP within the endoplasmic reticulum (ER) by treatment with brefeldin A does not block the cleavage at amino acid 42 but results in an increased production of all species of Abeta terminating at amino acid 42. This indicates that the cleavage after amino acid 42 can occur within the ER. Treatment of cells with monensin, which blocks transport of (betaAPP) within the Golgi causes a marked accumulation of intracellular Abetax-42 and Abetax-40. Therefore these experiments indicate that the gamma-secretase cleavage of Abeta after amino acid 42 can occur within the ER and later within the secretory pathway within the Golgi. Moreover inhibition of reinternalization by cytoplasmic deletions of betaAPP as well as inhibition of intracellular acidification by NH4Cl does not block intracellular Abeta1-42 or Abetax-42 production.

Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an in vivo substrate for protein kinase C.

The majority of familial Alzheimer disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). It was shown that the full-length PS-2 protein is phosphorylated constitutively within its N-terminal domain by casein kinases, whereas the PS-1 protein is not. Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of approximately 20-kDa C-terminal fragments (CTF) and approximately 30-kDa N-terminal fragments [Thinakaran, G., et al. (1996) Neuron 17, 181-190]. Here we describe the surprising finding that the CTF of PS-1 is phosphorylated by protein kinase C (PKC). Stimulation of PKC causes a 4- to 5-fold increase of the phosphorylation of the approximately 20-kDa CTF of PS-1 resulting in reduced mobility in SDS gels. PKC-stimulated phosphorylation occurs predominantly on serine residues and can be induced either by direct stimulation of PKC with phorbol-12,13-dibutyrate or by activation of the m1 acetylcholine receptor-signaling pathway with the muscarinic agonist carbachol. However, phosphorylation of full-length PS-1 and PS-2 is not altered upon PKC stimulation. In addition, a mutant form of PS-1 lacking exon 10, which does not undergo endoproteolytic cleavage [Thinakaran, G., et al. (1996) Neuron 17, 181-190] is not phosphorylated by PKC, although it still contains all PKC phosphorylation sites conserved between different species. These results show that PKC phosphorylates the PS-1 CTF. Therefore, endoproteolytic cleavage of full-length PS-1 results in the generation of an in vivo substrate for PKC. The selective phosphorylation of the PS-1 CTF indicates that the physiological and/or pathological properties of the CTF are regulated by PKC activity.