Cathepsin D expression is decreased in Alzheimer's disease fibroblasts
Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative dementia characterized by two pathological hallmarks in the cerebral cortex and hippocampus: senile plaques, consisting of deposits of β-amyloid peptide (Aβ) and neurofibrillary tangles, composed of an abnormally phosphorylated form of the cytoskeleton-associated protein tau. Even if the majority of AD cases appears sporadically (Sporadic Alzheimer's Disease, SAD), a small percentage of AD cases is familial (Familial Alzheimer's Disease, FAD) and is linked to mutations in three different genes: the amyloid precursor protein (APP), the presenilin 1 (PS1) and the presenilin 2 (PS2). The mechanism by which mutations cause FAD is not completely clear, but it was shown they are associated with abnormal APP processing [3], [9], [19]. Information about AD pathogenesis arises mainly from the study of these genetically transmitted forms, however the majority of AD cases are not genetically transmitted and their pathogenesis is unknown [24].
Previous studies demonstrated that neuronal populations that degenerate in AD show an up-regulation of the lysosomal system [1], [5], [6], [7]. Lysosomal system is composed of a family of acidic vesicles including lysosomes, late endosomes and Golgi vesicles, containing recently synthesized enzymes, and residual bodies, containing undigested material. Many neurodegenerative diseases arise from lysosomal dysfunction, thus indicating the importance of these organelles for cell viability. Indeed, mutations that cause lysosomal enzyme deficiencies result in approximately 40 different syndromes, termed Lysosomal Storage Disorders (LSDs). Most of these diseases are associated with abnormal brain development and mental retardation. In addition, they are characterized by intracellular deposition and protein aggregation, events also found in age-related neurodegenerative disorders, including AD [2].
Cathepsin D (CTSD) (EC 3.4.23.5) is the main lysosomal aspartic protease. It is synthetized and translocated into Endoplasmic Reticulum (ER) as a 52 kDa inactive proenzyme, that is converted in the endosomal compartment in a single chain active form of 48 kDa [13], [39]. In humans and in most species, this single chain form undergoes further proteolytic processing in late endosomes and lysosomes, yielding the mature form, composed of the light (14 kDa) and heavy (32 kDa) chains, held together by disulphide bonds. Its mostly described function is intracellular proteolytic catabolism in the lysosomal compartment, but other physiological effects include antigen processing and activation or inactivation of different protein substrates, that results in their maturation and secretion [14], [18], [47].
A naturally occurring cathepsin D mutation causes a recessive form of Neuronal Ceroid Lipofuscinosis (NCL) in sheep. NCLs are a group of diseases, which encompasses the most common reason for progressive encefalopathies of children in humans [27]. Affected sheep show an extreme brain atrophy of the cerebral cortex accompanied by a marked accumulation of storage materials in neurons [45]. Disruption of cathepsin D gene in mice causes ileal atrophy and loss of lymphoid cells leading to death [40]. Moreover, affected mice show neuronal storage with typical features of neuronal ceroid lipofuscinosis [33].
Several lines of evidence suggest a role for cathepsin D in Alzheimer's disease. Initially, it was identified as a major APP-processing activity [15], [17], [22], [26], [32] and tau-degradating enzyme [30]. Accumulating evidence ruled out the involvement of cathepsin D as a critical component of α-, β- and γ-secretase system: amyloidogenic processing of APP is normal in mice neurons devoid of cathepsin D [41] and aspartic proteases were identified that are involved in the cleavage of APP: β-secretase seems to be due to BACE1 [28], [46], [49], while γ-secretase identification is still a matter of controversy [34], [48], but invalidation of presenilin genes was reported to impair γ-secretase-mediated Aβ production [43]. In addition, cathepsin D mRNA and enzyme activity are up-regulated in AD patients’ brain and the protein is present in amyloid plaques [1], [5], [7], [42]. Since up-regulation of cathepsin D is an early and specific observation in AD brain, it seems likely that cathepsin D may have a role in the neurodegenerative process [7].
There are emerging evidences of an altered balance of lysosomal enzymes activities in AD also at peripheral level. Whereas the expression of glycohydrolases, such as β-hexoaminidase, α-mannosidase, β-galactosidase, is increased in fibroblasts from skin biopsy of patients with familiar or sporadic AD [16], the activity of cathepsin D, as evaluated by flow cytometry, is decreased in peripheral blood lymphocytes of AD patients [44].
To better elucidate the involvement of the lysosomal system in AD and go more insight the role of this proteolytic enzyme, we characterized cathepsin D expression in AD fibroblasts at both transcriptional and translational level. Cathepsin D processing was also investigated. Among peripheral sources, fibroblasts are an easy and accessible source to study the pathogenic mechanisms that are responsible for Alzheimer's disease [9], [10]. Previous studies demonstrated that Ras expression in the frontal cortex of AD patients is elevated at early stages of the disease [20], [21] and that AD fibroblasts are characterized by an increased content of Ras protein and activation of p38 MAPK [16]. In addition, Ras is involved in the processing and subcellular localization of cathepsin D [12]. Here, we provide evidence of the direct involvement of Ras signalling cascades in cathepsin D down-regulation.
Section snippets
Materials
Nonidet P-40, H2O2, DMSO, PD098059, SB203580, anisomycin, trypan blue, Bovine Serum Albumin (BSA), mouse monoclonal anti-β-tubulin antibody, goat anti-mouse HRP-linked and rabbit anti-goat HRP-linked secondary antibodies were from Sigma Chemical Co. (St. Louis, MO, USA). Bio-Rad protein assay reagent was from Bio-Rad Laboratoires (Hercules, CA, USA). Leupeptin and pepstatin A were from Calbiochem (San Diego, CA, USA). RNeasy Mini kit and Taq were from Qiagen (Hilden, Germany), SuperScriptII™
Cathepsin D activity in AD patients fibroblasts
Cathepsin D activity was measured in fibroblasts from patients with FAD, carrying mutations in PS1, PS2 and APP genes, as well as in patients with SAD and in controls, matched for sex and age. Scatter plots (Fig. 1) show the distribution of cathepsin D activity in controls as well as in FAD and SAD patients. Statistical analysis demonstrates that average level of cathepsin D activity is decreased in FAD patients as compared to controls in a significant manner (P < 0.01). FAD patients considered
Discussion
There is an increasing evidence that disturbance of the normal balance of cathepsins contributes to neurodegeneration in Alzheimer's disease [36]. Up-regulation of the endosomal/lysosomal system was demonstrated in affected neurons of AD brain [6], [7]. Furthermore, in senile plaques cathepsin D is present at high levels [5] and co-localizes with β-amyloid peptide in cerebellum and striatum of post-mortem patients [1].
We studied the activity and expression of cathepsin D at peripheral level, in
Acknowledgements
Many thanks are due to Ms Hilary Giles for English language revision of the manuscript. This work was supported by “Ministero dell’Istruzione, Università e Ricerca” COFIN-PRIN 2004055300-002 and FIRB RBNE012LW8-001 grants (Al.O. and C.E.), by the “Ministero della Salute” (RA00.87, Al.O. - RF03.1840 and RF04.1250, An.O.) and by the “Comitato Telethon Fondazione Onlus” (E0980PG, Al.O. - GGP030368, An.O. - E0980, S.S.).
References (49)
- et al.
Up-regulation of the lysosomal system in experimental models of neuronal injury: implications for Alzheimer's disease
Neuroscience
(2000) - et al.
Familial Alzheimer's disease-linked presenilin 1 variants elevate Abeta1-42/1-40 ratio in vitro and in vivo
Neuron
(1996) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)- et al.
Gene expression and cellular content of cathepsin D in Alzheimer's disease brain: evidence for early up-regulation of the endosomal-lysosomal system
Neuron
(1995) - et al.
Oxidative stress and reduced antioxidant defenses in peripheral cells from familial Alzheimer's patients
Free Radic Biol Med
(2002) Fibroblast models of neurological disorders: fluorescence measurement studies
Trends Pharmacol Sci
(1998)- et al.
High yield synthesis and characterization of phosphorylated recombinant human procathepsin D expressed in mammalian cells
Protein Expr Purif
(2006) - et al.
Cleavage of parathyroid hormone in macrophage endosomes illustrates a novel pathway for intracellular processing of proteins
J Biol Chem
(1989) - et al.
Up-regulation of glycohydrolases in Alzheimer's Disease fibroblasts correlates with Ras activation
J Biol Chem
(2003) - et al.
Cathepsin D is a good candidate for the specific release of a stable hemorphin from hemoglobin in vivo: VV-hemorphin-7
Biochem Biophys Res Commun
(1998)