Presenilin-1 affects trafficking and processing of βAPP and is targeted in a complex with nicastrin to the plasma membrane

Amyloid β-peptide (Aβ) is generated by the consecutive cleavages of β- and γ-secretase. The intramembraneous γ-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases with γ-secretase activity, it remains controversial whether their subcellular localization overlaps with the cellular sites of Aβ production. We now demonstrate that biologically active GFP-tagged PS1 as well as endogenous PS1 are targeted to the plasma membrane (PM) of living cells. On the way to the PM, PS1 binds to nicastrin (Nct), an essential component of the γ-secretase complex. This complex is targeted through the secretory pathway where PS1-bound Nct becomes endoglycosidase H resistant. Moreover, surface-biotinylated Nct can be coimmunoprecipitated with PS1 antibodies, demonstrating that this complex is located to cellular sites with γ-secretase activity. Inactivating PS1 or PS2 function by mutagenesis of one of the critical aspartate residues or by γ-secretase inhibitors results in delayed reinternalization of the β-amyloid precursor protein and its accumulation at the cell surface. Our data suggest that PS is targeted as a biologically active complex with Nct through the secretory pathway to the cell surface and suggest a dual function of PS in γ-secretase processing and in trafficking

Expression of the Alzheimer protease BACE1 is suppressed via its 5′-untranslated region

The aspartyl protease BACE1 has a pivotal role in the pathogenesis of Alzheimer's disease. Recently, it was shown that in Alzheimer's disease patients, BACE1 levels were elevated although mRNA levels were not changed compared with controls. Here, we demonstrate that the 5′-untranslated region (5′UTR) of BACE1 controls the rate of BACE1 translation. In the presence of the 5′UTR, we observed more than 90% reduction of BACE1 protein levels in HEK293, COS7 and H4 cells, and a similar reduction of BACE1 activity in vitro. mRNA levels were not affected, demonstrating that the 5′UTR repressed the translation but not the transcription of BACE1. The 3′UTR did not affect BACE1 expression. An extensive mutagenesis analysis predicts that the GC-rich region of the 5′UTR forms a constitutive translation barrier, which may prevent the ribosome from efficiently translating the BACE1 mRNA. Our data therefore demonstrate translational repression as a new mechanism controlling BACE1 expression

Furin-, ADAM 10-, and γ-Secretase-Mediated Cleavage of a Receptor Tyrosine Phosphatase and Regulation of β-Catenin's Transcriptional Activity

Several receptor protein tyrosine phosphatases (RPTPs) are cell adhesion molecules involved in homophilic interactions, suggesting that RPTP outside-in signaling is coupled to cell contact formation. However, little is known about the mechanisms by which cell density regulates RPTP function. We show that the MAM family prototype RPTPκ is cleaved by three proteases: furin, ADAM 10, and γ-secretase. Cell density promotes ADAM 10-mediated cleavage and shedding of RPTPκ. This is followed by γ-secretase-dependent intramembrane proteolysis of the remaining transmembrane part to release the phosphatase intracellular portion (PIC) from the membrane, thereby allowing its translocation to the nucleus. When cells were treated with leptomycin B, a nuclear export inhibitor, PIC accumulated in nuclear bodies. PIC is an active protein tyrosine phosphatase that binds to and dephosphorylates β-catenin, an RPTPκ substrate. The expression of RPTPκ suppresses β-catenin's transcriptional activity, whereas the expression of PIC increases it. Notably, this increase required the phosphatase activity of PIC. Thus, both isoforms have acquired opposing roles in the regulation of β-catenin signaling. We also found that RPTPμ, another MAM family member, undergoes γ-secretase-dependent processing. Our results identify intramembrane proteolysis as a regulatory switch in RPTPκ signaling and implicate PIC in the activation of β-catenin-mediated transcription

Postnatal Disruption of the Disintegrin/Metalloproteinase ADAM10 in Brain Causes Epileptic Seizures, Learning Deficits, Altered Spine Morphology, and Defective Synaptic Functions

The metalloproteinase ADAM10 is of importance for Notch-dependent cortical brain development. The protease is tightly linked with α-secretase activity toward the amyloid precursor protein (APP) substrate. Increasing ADAM10 activity is suggested as a therapy to prevent the production of the neurotoxic amyloid β (Aβ) peptide in Alzheimer's disease. To investigate the function of ADAM10 in postnatal brain, we generated Adam10 conditional knock-out (A10cKO) mice using a CaMKIIα-Cre deleter strain. The lack of ADAM10 protein expression was evident in the brain cortex leading to a reduced generation of sAPPα and increased levels of sAPPβ and endogenous Aβ peptides. The A10cKO mice are characterized by weight loss and increased mortality after weaning associated with seizures. Behavioral comparison of adult mice revealed that the loss of ADAM10 in the A10cKO mice resulted in decreased neuromotor abilities and reduced learning performance, which were associated with altered in vivo network activities in the hippocampal CA1 region and impaired synaptic function. Histological and ultrastructural analysis of ADAM10-depleted brain revealed astrogliosis, microglia activation, and impaired number and altered morphology of postsynaptic spine structures. A defect in spine morphology was further supported by a reduction of the expression of NMDA receptors subunit 2A and 2B. The reduced shedding of essential postsynaptic cell adhesion proteins such as N-Cadherin, Nectin-1, and APP may explain the postsynaptic defects and the impaired learning, altered network activity, and synaptic plasticity of the A10cKO mice. Our study reveals that ADAM10 is instrumental for synaptic and neuronal network function in the adult murine brain.status: publishe

Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase

Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRNhaploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration