Eckman, Christopher B.

The accumulation of Aβ (amyloid β-protein) peptides in the brain is a pathological hallmark of all forms of AD (Alzheimer's disease) and reducing Aβ levels can prevent or reverse cognitive deficits in mouse models of the disease. Aβ is produced continuously and its concentration is determined in part by the activities ofseveral degradative enzymes, including NEP (neprilysin), IDE (insulin-degrading enzyme), ECE-1 (endothelinconverting enzyme 1) and ECE-2, and probably plasmin. Decreased activity of any of these enzymes due to genetic mutation, or age- or disease-related alterations in gene expression or proteolytic activity, may increase the risk for AD. Conversely, increased expression of these enzymes may confer a protective effect. Increasing Aβ degradation through gene therapy, transcriptional activation or even pharmacological activation of the Aβ-degrading enzymes represents a novel therapeutic strategy for the treatment of AD that is currently being evaluated in cell-culture and animal models. In this paper, we will review the roles of NEP, IDE, ECE and plasmin in determining endogenous Aβ concentration, highlighting recent results concerning the regulation of these enzymes and their potential as therapeutic targets.

Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with β-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide–receptor–arrestin complex is sequestered into endosomes. Although dissociation of β-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase endothelin-converting enzyme 1 (ECE-1), determines stability of the peptide–receptor–arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK 1 R) induced membrane translocation of β-arrestins followed by trafficking of the SP–NK 1 R–β-arrestin complex to early endosomes containing ECE-1a–d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; β-arrestins returned to the cytosol, and the NK 1 R, freed from β-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK 1 R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK 3 R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide–receptor–arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control β-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.

Proteolytic processing of the amyloid precursor protein (APP) by β-secretase, β-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid β (Aβ) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrP C ), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt–Jakob disease in humans, remains enigmatic. Because both APP and PrP C are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrP C in the proteolytic processing of APP. Cellular overexpression of PrP C inhibited the β-secretase cleavage of APP and reduced Aβ formation. Conversely, depletion of PrP C in mouse N2a cells by siRNA led to an increase in Aβ peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Aβ levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the β-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrP C on the β-secretase cleavage of APP required the localization of PrP C to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrP C via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Aβ is regulated by PrP C and may have implications for both Alzheimer's and prion diseases.

ABSTRACT Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a devastating, degenerative neurological disorder. It is inherited as an autosomal recessive trait caused by loss‐of‐function mutations in the galactocerebrosidase (GALC) gene. Previously, we have shown that peripheral injection of recombinant GALC, administered every other day, results in a substantial improvement in early clinical phenotype in the twitcher mouse model of GLD. While we did detect active enzyme in the brain following peripheral administration, most of the administered enzyme was localized to the periphery. Given the substantial central nervous system (CNS) involvement in this disease, we were interested in determining whether or not a single‐dose administration of the recombinant enzyme directly to the CNS, which could potentially be achieved clinically, would result in any substantial improvement. Following intracerebroventricu‐lar (icv) administration of GALC we noted a significant, 16.5%, reduction in the GALC substrate psychosine, the abnormal accumulation of which is believed to play a pivotal role in the CNS pathology observed in this disease. Moreover, recombinant GALC was found not only in periventricular regions but also at sites distant to the injection such as the cerebral cortex and cerebellum. Most importantly, animals receiving a single icv dose of the enzyme at postnatal day 20 survived up to 51 days, which compares favorably to the control twitcher animals, which normally only live to postnatal day 40/42. These results indicate that even a single icv administration of the recombinant enzyme can have significant clinical impact and suggests that other lysosomal storage disorders with significant CNS involvement may similarly benefit.—Lee, W.C., Tsoi, Y.K., Troendle, F.J., DeLucia, M.W., Ahmed, Z., Dicky, C.A., Dickson, D.W., Eckman, C.B. Single dose intracerebroventricular administration of galactocerebrosidase improves survival in a mouse model of globoid cell leukodystrophy. FASEB J. 21, 2520–2527 (2007)