It is well established that the proteolytic processing of the ␤-amyloid precursor protein (APP) generates ␤-amyloid (A␤), which plays a central role in the pathogenesis of Alzheimer's disease (AD). In contrast, the physiological role of APP and of its numerous proteolytic fragments and the question of whether a loss of these functions contributes to AD are still unknown. To address this question, we replaced the endogenous APP locus by gene-targeted alleles and generated two lines of knock-in mice that exclusively express APP deletion variants corresponding either to the secreted APP ectodomain (APPs␣) or to a C-terminal (CT) truncation lacking the YENPTY interaction motif (APP⌬CT15). Interestingly, the ⌬CT15 deletion resulted in reduced turnover of holoAPP, increased cell surface expression, and strongly reduced A␤ levels in brain, likely because of reduced processing in the endocytic pathway. Most importantly, we demonstrate that in both APP knock-in lines the expression of APP N-terminal domains either grossly attenuated or completely rescued the prominent deficits of APP knock-out mice, such as reductions in brain and body weight, grip strength deficits, alterations in circadian locomotor activity, exploratory activity, and the impairment in spatial learning and long-term potentiation. Together, our data suggest that the APP C terminus is dispensable and that APPs␣ is sufficient to mediate the physiological functions of APP assessed by these tests.

Huntingtin proteolysis is implicated in Huntington diseasepathogenesis, yet, the nature of huntingtin toxic fragments remains unclear. Huntingtin undergoes proteolysis by calpains and caspases within an N-terminal region between amino acids 460 and 600. We have focused on proteolytic steps producing shorter N-terminal fragments, which we term cp-1 and cp-2 (distinct from previously described cp-A/cp-B). We used HEK293 cells to express the first 511 residues of huntingtin and further define the cp-1 and cp-2 cleavage sites. Based on epitope mapping with huntingtin-specific antibodies, we found that cp-1 cleavage occurs between residues 81 and 129 of huntingtin. Affinity and size exclusion chromatography were used to further purify huntingtin cleavage products and enrich for the cp-1/ cp-2 fragments. Using mass spectrometry, we found that the cp-2 fragment is generated by cleavage of huntingtin at position Arg 167 . This site was confirmed by deletion analysis and specific detection with a custom-generated cp-2 site neo-epitope antibody. Furthermore, alterations of this cleavage site resulted in a decrease in toxicity and an increase in aggregation of huntingtin in neuronal cells. These data suggest that cleavage of huntingtin at residue Arg 167 may mediate mutant huntingtin toxicity in Huntington disease.

The low density lipoprotein receptor-related protein 1 (LRP1) emerges to play fundamental roles in cellular signaling pathways in the brain. One of its prominent ligands is the serine proteinase tissue-type plasminogen activator (tPA), which has been shown to act as a key activator of neuronal mitogen-activated protein kinase pathways via the N-methyl-D-aspartate (NMDA) receptor. However, here we set out to examine whether LRP1 and the NMDA receptor might eventually act in a combined fashion to mediate tPA downstream signaling. By blocking tPA from binding to LRP1 using the receptor-associated protein, we were able to completely inhibit NMDA receptor activation. Additionally, inhibition of NMDA receptor calcium influx with MK-801 resulted in dramatic reduction of tPA-mediated downstream signaling. This indicates a functional interaction between the two receptors, since both experimental approaches resulted in strongly reduced calcium influx and Erk1/2 phosphorylation. Additionally, we were able to inhibit Erk1/2 activation by competing for the LRP1 C-terminal binding motif with a truncated PSD95 construct resembling its PDZ III domain. Furthermore, we identified the distal NPXY amino acid motif in the C terminus of LRP1 as the crucial element for LRP1-NMDA receptor interaction via the adaptor protein PSD95. These results provide new insights into the mechanism of a tPA-induced, LRP1-mediated gating mechanism for NMDA receptors.LRP1 (low density lipoprotein receptor-related protein) is a member of the low density lipoprotein receptor gene family with its highest expression in liver and brain. Following its synthesis in the endoplasmic reticulum as a 600-kDa type I transmembrane glycoprotein, LRP1 is cleaved in the Golgi compartment by furin, producing two subunits, 515 and 85 kDa in size. These two subunits remain noncovalently associated during their transport to the cell surface (1). Via a receptor-recycling pathway, LRP1 is responsible for the endocytosis of more than 30 different extracellular ligands (2, 3). In neurons, where it is highly expressed and predominantly localized in neuronal cell bodies and dendritic processes (4), LRP1 is a major receptor for apoE/lipoprotein-containing particles and tissue-type plasminogen activator (tPA) 2 (5). In addition to its role in endocytosis of various ligands, LRP1 has been implicated to play a crucial role in cell signaling. The observation that the LRP1 C terminus undergoes rapid tyrosine phosphorylation after tPA binding corroborates its function as a signal transmitter (6). Several adaptor proteins can bind to the LRP1 tail, some of which seem to play a role in neuronal calcium, platelet-derived growth factor, or MAPK signaling (7-9). Beyond its known function of lipid uptake, LRP1 has also been attributed to be the mediator of an LTP-enhancing effect of exogenously added tPA in hippocampal slices from tPA-deficient mice (5). Further evidence supporting the hypothesis of an involvement in synaptic plasticity results from neuron-specific LRP1 knock-out mice, which...

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The Swedish mutation within the amyloid precursor protein (APP) causes early‐onset Alzheimer’s disease due to increased cleavage of APP by BACE1. While β‐secretase shedding of Swedish APP (APPswe) largely results from an activity localized in the late secretory pathway, cleavage of wild‐type APP occurs mainly in endocytic compartments. However, we show that liberation of Aβ from APPswe is still dependent on functional internalization from the cell surface. Inspite the unchanged overall β‐secretase cleaved soluble APP released from APPswe secretion, mutations of the APPswe internalization motif strongly reduced C99 levels and substantially decreased Aβ secretion. We point out that α‐secretase activity‐mediated conversion of C99 to C83 is the main cause of this Aβ reduction. Furthermore, we demonstrate that α‐secretase cleavage of C99 even contributes to the reduction of Aβ secretion of internalization deficient wild‐type APP. Therefore, inhibition of α‐secretase cleavage increased Aβ secretion through diminished conversion of C99 to C83 in APP695, APP695swe or C99 expressing cells.

Presenilin 1 (PS1) is a critical component of the ␥-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and the Notch receptor. Recently, the low density receptor-related protein (LRP) has been shown to be cleaved by a ␥-secretase-like activity. We postulated that LRP may interact with PS1 and tested its role as a competitive substrate for ␥-secretase. In this report we show that LRP colocalizes and interacts with endogenous PS1 using coimmunoprecipitation and fluorescence lifetime imaging microscopy. In addition, we found that ␥-secretase active site inhibitors do not disrupt the interaction between LRP and PS1, suggesting that the substrate associates with a ␥-secretase docking site located in close proximity to PS1. This is analogous to APP-␥-secretase interactions. Finally, we show that LRP competes with APP for ␥-secretase activity. Overexpression of a truncated LRP construct consisting of the C terminus, the transmembrane domain, and a short extracellular portion leads to a reduction in the levels of the A␤ 40 , A␤ 42 , and p3 peptides without changing the total level of APP expression. In addition, transfection with the ␤-chain of LRP causes an increase in uncleaved APP C-terminal fragments and a concomitant decrease in the signaling effects of the APP intracellular domain. In conclusion, LRP is a PS1 interactor and can compete with APP for ␥-secretase enzymatic activity.The low density lipoprotein receptor-related protein (LRP) 1 is a ϳ600 kDa type I integral membrane protein that belongs to the ancient low density lipoprotein receptor gene family (1). LRP interacts with at least 30 different ligands that bind to the extracellular domain, including ␣2-macroglobulin (2, 3), lipases, plasminogen activators (4 -6), apolipoprotein E (7, 8), and the amyloid precursor protein (APP) (9, 10). The cytoplasmic tail of LRP contains two NPXY motifs that serve as docking sites for endocytosis machinery, scaffold proteins, and cytoplasmic adaptors involved in signaling events (11,12). These include the mammalian Disabled-1 (Dab1) (12), the postsynaptic density protein (PSD-95) (13), and the scaffold protein Fe65, which interacts with LRP through its phosphotyrosine binding domain (12).LRP is cleaved by furin in the trans-Golgi network, generating a 515-kDa ␣-subunit and a 85 kDa ␤-subunit that remain non-covalently associated as they are transported to the cell surface (14). LRP also undergoes proteolytic shedding of the extracellular domain by a metalloproteinase (15). It has recently been shown that the cytoplasmic tail of LRP can be processed intramembranously by a ␥-secretase activity that releases its intracellular domain (16). The ␥-secretase complex is a multiprotein complex that is composed of at least four members, namely presenilin 1 (PS1), which is believed to contain the catalytic site, nicastrin, Pen-2, and Aph1 (17). This complex is responsible for cleavage of at least 15 substrates, including APP and the Notch receptor. APP is a type-I...

N-terminal proteolysis of huntingtin is thought to be an important mediator of HD pathogenesis. The formation of short N-terminal fragments of huntingtin (cp-1/cp-2, cp-A/cp-B) has been demonstrated in cells and in vivo. We previously mapped the cp-2 cleavage site by mass spectrometry to position Arg 167 of huntingtin. The proteolytic enzymes generating short N-terminal fragments of huntingtin remain unknown. To search for such proteases, we conducted a genome-wide screen using an RNA-silencing approach and an assay for huntingtin proteolysis based on the detection of cp-1 and cp-2 fragments by Western blotting. The primary screen was carried out in HEK293 cells, and the secondary screen was carried out in neuronal HT22 cells, transfected in both cases with a construct encoding the N-terminal 511 amino acids of mutant huntingtin. For additional validation of the hits, we employed a complementary assay for proteolysis of huntingtin involving overexpression of individual proteases with huntingtin in two cell lines. The screen identified 11 enzymes, with two major candidates to carry out the cp-2 cleavage, bleomycin hydrolase (BLMH) and cathepsin Z, which are both cysteine proteases of a papain-like structure. Knockdown of either protease reduced cp-2 cleavage, and ameliorated mutant huntingtin induced toxicity, whereas their overexpression increased the cp-2 cleavage. Both proteases partially co-localized with Htt in the cytoplasm and within or in association with early and late endosomes, with some nuclear co-localization observed for cathepsin Z. BLMH and cathepsin Z are expressed in the brain and have been associated previously with neurodegeneration. Our findings further validate the cysteine protease family, and BLMH and cathepsin Z in particular, as potential novel targets for HD therapeutics.Huntington disease (HD) 3 is caused by an expansion of the CAG repeat coding for polyglutamine (polyQ) within the HD gene product huntingtin (Htt) (1, 2). It is not clear how mutant Htt causes neuronal cell death, but evidence is accumulating that N-terminal fragments of mutant Htt are important mediators of pathogenesis. N-terminal fragments of Htt are found in human postmortem brain (3-8). In cell model experiments, shorter N-terminal fragments of Htt are generally more toxic to cells than longer fragments, and mouse HD models expressing shorter fragments usually develop more severe pathology than the full-length Htt models (6, 9 -19). The proteolytic pathway for mutant Htt may include several cleavage events mediated by different enzymes involved in the generation of toxic fragments of various lengths. These enzymes may represent potential novel therapeutic targets for HD.One example of such an enzyme is caspase-6, which cleaves mutant Htt at residue 586 from its N terminus and generates a toxic fragment; YAC128 mice expressing Htt with an altered caspase-6 site have a substantially ameliorated HD phenotype (20), whereas transgenic mice expressing a caspase-6 fragment with an expanded polyQ repeat develop an HD phe...

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