Proteolytic cleavage of the amyloid protein from the amyloid protein precursor (APP) by APP secretases is a key event in Alzheimer's disease (AD) pathogenesis. K K-Secretases cleave APP within the amyloid sequences, whereas L L-and Q Qsecretases cleave on the N-and C-terminal ends respectively. The transmembrane aspartyl protease BACE has been identified as L L-secretase and several proteases (ADAM-10, TACE, PC7) may be K K-secretases. A number of studies have suggested that presenilins could be Q Q-secretases, although this remains to be demonstrated conclusively. Inhibition of L L-and Q Q-secretase, or stimulation of K K-secretase, is a rational strategy for therapeutic intervention in AD. ß

Mutations in Cu/Zn superoxide dismutase (SOD1) are linked to motor neuron death in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism, although misfolded SOD1 aggregates are commonly associated with disease. Proteomic analysis of the transgenic SOD1 G93A ALS rat model revealed significant up-regulation of endoplasmic reticulum (ER)-resident protein-disulfide isomerase (PDI) family members in lumbar spinal cords. Expression of SOD1 mutants (mSOD1) led to an up-regulation of PDI in motor neuron-like NSC-34 cells but not other cell lines. Inhibition of PDI using bacitracin increased aggregate production, even in wild type SOD1 transfectants that do not readily form inclusions, suggesting PDI may protect SOD1 from aggregation. Moreover, PDI co-localized with intracellular aggregates of mSOD1 and bound to both wild type and mSOD1. SOD1 was also found in the microsomal fraction of cells despite being a predominantly cytosolic enzyme, confirming ER-Golgi-dependent secretion. In SOD1 G93A mice, a significant up-regulation of unfolded protein response entities was also observed during disease, including caspase-12, -9, and -3 cleavage. Our findings therefore implicate unfolded protein response and ER stress-induced apoptosis in the pathophysiologyoffamilialALS.ThepossibilitythatPDImaybeatherapeutic target to prevent SOD1 aggregation is also raised by this study.Mutations in the Cu/Zn-superoxide dismutase (SOD1) 2 gene are associated with 20% of familial amyotrophic lateral sclerosis (FALS) cases (1), and when these mutations are overexpressed in transgenic rodents (2, 3), motor neuron degeneration reminiscent of ALS results. Although SOD1 is thermally very stable (4), abnormal mutant SOD1 (mSOD1) aggregates are present in spinal cords of FALS patients and transgenic mice (5). The mechanism of mSOD1-mediated toxicity is unclear but is non-cell autonomous and involves apoptotic signaling (reviewed in Ref. 6). The selective toxicity for motor neurons also remains unresolved.SOD1 is an intracellular homodimeric metalloprotein that forms an unusually stable intrasubunit disulfide bond between two highly conserved cysteines, Cys 57 and Cys 146 . Recent evidence implicates the disulfide-reduced monomer as the aggregation-prone and common neurotoxic intermediate for over 100 mSOD1 proteins (7-11). Hence, modulation of disulfide bond formation may be important in mSOD1-linked toxicity.The disulfide status of proteins is largely regulated by ER stress-inducible enzymes. ER stress is triggered when misfolded proteins accumulate within the lumen, inducing the unfolded protein response (UPR) (12). The 78-kDa chaperone immunoglobulin-binding protein (BiP) controls activity of the three major UPR sensors: the kinase and endonuclease IRE1, the basic leucine-zipper transcription factor ATF6, and the PERK kinase (13). The combined effect of the activation of these three molecules is the up-regulation of genes encoding ER-resident chaperones and down-regulation of protein synthesis. Proteindisulfide isomerase (PDI) and en...

The b-amyloid protein (Ab) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by b-secretase generates a C-terminal fragment (APPCTFb), which is subsequently cleaved by g-secretase to produce Ab. The aim of this study was to examine the cleavage of APP-CTFb by g-secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTFb sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [ Approximately 30% of the human APP-CTFb (hAPPCTFb) was converted to human Ab (hAb), which was rapidly secreted. The remaining 70% of the hAPP-CTFb was degraded by an alternative pathway. The cleavage of hAPP-CTFb to produce hAb was inhibited by specific g-secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTFb and hAb levels, suggesting that the alternative pathway was proteasome-dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APP cyt ) directly. The study suggests that in primary cortical neurons, APP-CTFb is degraded by two distinct pathways, one involving g-secretase, which produces Ab, and a second major pathway involving direct cleavage of APP-CTFb within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Ab production in the AD brain.

The beta-amyloid protein (Abeta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APP-CTFbeta), which is subsequently cleaved by gamma-secretase to produce Abeta. Our previous studies have shown that the proteasome can cleave the C-terminal cytoplasmic domain of APP. To identify proteasome cleavage sites in APP, two peptides homologous to the C-terminus of APP were incubated with recombinant 20S proteasome. Cleavage of the peptides was monitored by reversed phase high-performance liquid chromatography and mass spectrometry. Proteasome cleaved the APP C-terminal peptides at several sites, including a region around the sequence YENPTY that interacts with several APP-binding proteins. To examine the effect of this cleavage on Abeta production, APP-CTFbeta and mutant forms of APP-CTFbeta terminating on either side of the YENPTY sequence were expressed in CHO cells. Truncation of APP-CTFbeta on the N-terminal side of the YENPTY sequence at residue 677 significantly decreased the amount of Abeta produced, whereas truncation on the C-terminal side of residue 690 had little effect. The results suggest that proteasomal cleavage of the cytosolic domain of APP at the YENPTY sequence decreases gamma-secretase processing, and consequently inhibits Abeta production. Therefore, the proteasome-dependent trafficking pathway of APP may be a valid therapeutic target for altering Abeta production in the Alzheimer's disease brain.