Up-regulation of Glycohydrolases in Alzheimer's Disease Fibroblasts Correlates with Ras Activation

Carla Emiliani,Lorena Urbanelli,Leda Racanicchi,Antonio Orlacchio,Giuliana Pelicci,Sandro Sorbi,Giorgio Bernardi,Aldo Orlacchio

doi:10.1074/jbc.m303030200

Abstract

The lysosomal system is up-regulated in the brain of patients with Alzheimer's Disease (AD), as demonstrated by previous experiments carried out in postmortem samples of brain patients. In this paper we provide evidence that an up-regulation of lysosomal glycohydrolases (alpha-D-mannosidase, beta-D-hexosaminidase, and beta-D-galactosidase) takes place in skin fibroblasts from AD patients affected either by sporadic or familial forms and is detectable also in presymptomatic subjects carrying the above mutations but healthy at the time of skin biopsy. This increase of enzyme activity is consequent to a transcriptional up-regulation. The oncogene Ras appears to be involved in the regulation of enzymatic activity. A parallel increase of Ras transcript and Ras protein, without an increase of p44/p42 MAPK activation was revealed in the same AD fibroblasts. An activation of p38 MAPK already described to occur in neurodegenerative diseases such as Alzheimer's, was also found in fibroblasts derived from AD patients. High levels of expression of the constitutively active form of Ras in normal or AD fibroblasts induced glycohydrolases up-regulation. Overall results demonstrated that glycohydrolases up-regulation, as well as Ras up-regulation, are early markers of AD, detectable at peripheral level, and good candidates to be exploited for diagnostic purposes. These data also provide the first proof for a role of Ras in regulating lysosomal glycohydrolases expression.

Highlights

The lysosomal system is up-regulated in the brain of patients with Alzheimer’s Disease (AD), as demonstrated by previous experiments carried out in postmortem samples of brain patients
We investigated on lysosomal glycohydrolases ␤-D-hexosaminidase (EC 3.2.1.52), ␤-D-galactosidase (EC 3.2.1.23), and ␣-D-mannosidase (EC 3.2.1.24) expression and demonstrated that they are up-regulated in fibroblasts from AD patients and that their expression is regulated by Ras
Levels of Glycohydrolases Activity in Fibroblasts from AD Patients—Specific activity of the glycohydrolases ␣-D-mannosidase, ␤-D-hexosaminidase, and ␤-D-galactosidase, were measured in fibroblast cell extracts from FAD patients carrying a mutation in the PS1, PS2, or APP genes as well as in presymptomatic subjects from the same families; from sporadic AD cases (SAD) patients; from healthy subjects matched for age and sex, from patients affected by non-AD dementia and from centenarians

Summary

Sporadic AD

PALL a At the time of skin biopsy. b Presymptomatic subjects: carrying the mutation but healthy at the time of skin biopsy. PALL a At the time of skin biopsy. B Presymptomatic subjects: carrying the mutation but healthy at the time of skin biopsy. There are evidences that Ras alters the processing and subcellular localization of the lysosomal protease cathepsin D [18]. Current knowledge on lysosomal dysfunction or on signaling pathways alteration on AD arises from studies on postmortem brain of AD patients or in FAD animal models. We used as cell models fibroblast cell lines established from FAD patients, mutated on PS1, PS2, or APP genes; presymptomatic subjects (carrying the mutation but healthy at the time of skin biopsy); sporadic AD cases (SAD). We investigated on lysosomal glycohydrolases ␤-D-hexosaminidase (EC 3.2.1.52), ␤-D-galactosidase (EC 3.2.1.23), and ␣-D-mannosidase (EC 3.2.1.24) expression and demonstrated that they are up-regulated in fibroblasts from AD patients and that their expression is regulated by Ras

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION