Fibroblasts from PS1 mutated pre-symptomatic subjects and Alzheimer's disease patients share a unique protein levels profile.

In Alzheimer's disease (AD), a major goal is to improve early detection, as the diagnosis cannot be made until patients exhibit a noticeable decline in cognition and the brain is irreversibly damaged. With this aim in mind, we performed proteome analysis of familial AD fibroblasts from both demented and pre-symptomatic subjects, using a 2D-PAGE based approach and then identifying proteins by mass spectrometry. We compared primary fibroblast cultures from skin biopsy of presenilin 1 (PS1) mutated patients, pre-symptomatic subjects carrying mutations in the PS1 gene but healthy at the time of skin biopsy, and age-matched individuals as control. 15 differentially expressed proteins were identified in PS1 mutated fibroblasts, related to cell adhesion and cytoskeleton, energy and glucose metabolism, stress response and ubiquitin-proteasome system, and signal transduction. Interestingly, many of these proteins have been previously associated with AD and neurodegeneration. Overall results indicated that a unique protein profile can be identified by peripheral cell analysis of PS1 mutated individuals, and showed that fibroblasts are a useful cell model for pathological investigations as well as identification of potential biomarkers for AD diagnosis at early stages.

Occurrence of an anomalous endocytic compartment in fibroblasts from Sandhoff disease patients.

Sandhoff disease (SD) is a lysosomal storage disorder due to mutations in the gene encoding for the beta-subunit of beta-hexosaminidase, that result in beta-hexosaminidase A (alphabeta) and beta-hexosaminidase B (betabeta) deficiency. This leads to the storage of GM2 ganglioside in endosomes and lysosomes, which ends in a progressive neurodegeneration. Currently, very little is known about the biochemical pathways leading from GM2 ganglioside accumulation to pathogenesis. Defects in transport and sorting by the endosomal-lysosomal system have been described for several lysosomal storage disorders. Here, we have investigated the endosomal-lysosomal compartment in fibroblasts from SD patients and observed that both late endosomes and lysosomes, but not early endosomes, have a higher density in comparison with normal fibroblasts. Moreover, Sandhoff fibroblasts have an intracellular distribution of terminal endocytic organelles that differs from the characteristic perinuclear punctate pattern observed in normal fibroblasts and endocytic vesicles also appear larger. These findings reveal the occurrence of an alteration in the terminal endocytic organelles of Sandhoff fibroblasts, suggesting an involvement of this compartment in the disruption of cell metabolic and signalling pathways and in the onset of the pathological state.

New perspectives for the diagnosis of Alzheimer's disease.

Alzheimer's disease is the most common cause of dementia in the elderly. Currently its clinical assessment is based on the exclusion of other forms of dementia and a definitive diagnosis requires a confirmation by examination of post-mortem brains. Therefore, there is a strong need to find easy measurable AD biomarkers that could facilitate the early diagnosis and monitoring the efficacy of the few therapies currently available. This would favor the development of further therapeutic approaches. Recently, dozens of biomarkers altered in peripheral tissues and body fluids have been patented by a variety of approaches, including transcriptomics, proteomics and peptidomics. However, assays for the routine laboratory diagnosis of AD are not available yet. The validation of these biomarkers is hindered by the fact that patient classification relies on clinical diagnosis that is not always accurate and this problem obstacles the enrollment of well characterized large patient cohorts needed for confirmation. This review provides an update of the status of research on AD peripheral biomarkers in the current post-genomic era, including recent patents in the field.

Identification and characterization of mature beta-hexosaminidases associated with human placenta lysosomal membrane.

Hex (beta-hexosaminidase) is a soluble glycohydrolase involved in glycoconjugate degradation in lysosomes, however its localization has also been described in the cytosol and PM (plasma membrane). We previously demonstrated that Hex associated with human fibroblast PM as the mature form, which is functionally active towards G(M2) ganglioside. In the present study, Hex was analysed in a lysosomal membrane-enriched fraction obtained by purification from highly purified human placenta lysosomes. These results demonstrate the presence of mature Hex associated with the lysosomal membrane and displaying, as observed for the PM-associated form, an acidic optimum pH. When subjected to sodium carbonate extraction, the enzyme behaved as a peripheral membrane protein, whereas Triton X-114 phase separation confirmed its partially hydrophilic nature, characteristics which are shared with the PM-associated form of Hex. Moreover, two-dimensional electrophoresis indicated a slight difference in the pI of beta-subunits in the membrane and the soluble forms of the lysosomal Hex. These results reveal a new aspect of Hex biology and suggest that a fully processed membrane-associated form of Hex is translocated from the lysosomal membrane to the PM by an as yet unknown mechanism. We present a testable hypothesis that, at the cell surface, Hex changes the composition of glycoconjugates that are known to be involved in intercellular communication and signalling.