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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