Lorena Urbanelli

Signaling Pathways in Exosomes Biogenesis, Secretion and Fate

Exosomes are small extracellular vesicles (30–100 nm) derived from the endosomal system, which have raised considerable interest in the last decade. Several studies have shown that they mediate cell-to-cell communication in a variety of biological processes. Thus, in addition to cell-to-cell direct interaction or secretion of active molecules, they are now considered another class of signal mediators. Exosomes can be secreted by several cell types and retrieved in many body fluids, such as blood, urine, saliva and cerebrospinal fluid. In addition to proteins and lipids, they also contain nucleic acids, namely mRNA and miRNA. These features have prompted extensive research to exploit them as a source of biomarkers for several pathologies, such as cancer and neurodegenerative disorders. In this context, exosomes also appear attractive as gene delivery vehicles. Furthermore, exosome immunomodulatory and regenerative properties are also encouraging their application for further therapeutic purposes. Nevertheless, several issues remain to be addressed: exosome biogenesis and secretion mechanisms have not been clearly understood, and physiological functions, as well as pathological roles, are far from being satisfactorily elucidated.

Cathepsin D expression is decreased in Alzheimer's disease fibroblasts

Cathepsin D (CTSD), a protease detectable in different cell types whose primary function is to degrade proteins by bulk proteolysis in lysosomes, has been suggested to be involved in Alzheimers disease (AD). In fact, there is increasing evidence that disturbance of the normal balance and localization of cathepsins may contribute to neurodegeneration in AD [Nakanishi H. Neuronal and microglial cathepsins in aging and age-related diseases. Aging Res Rev 2003; 2(4):367-81]. Here, we provide evidence of an altered balance of CTSD in skin fibroblasts from patients affected either by sporadic or familial forms of AD. In particular, we demonstrate that CTSD is down regulated at both transcriptional and translational level and its processing is altered in AD fibroblasts. The oncogene Ras is involved in the regulation of CTSD, as high expression level of the constitutively active form of Ras in normal or AD fibroblasts induces CTSD down-regulation. p38 MAPK signalling pathway also appears to down-modulate CTSD level. Overall results reinforce the hypothesis that a lysosomal impairment may be involved in AD pathogenesis and can be detected not only in the CNS but also at a peripheral level.

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

The lysosomal system is up-regulated in the brain of patients with Alzheimers 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 (α-d-mannosidase, β-d-hexosaminidase, and β-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 Alzheimers, 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.

Exosome-based strategies for Diagnosis and Therapy

Exosomes are small extracellular vesicles (30-120 nm) of endosomal origin, which are gaining the attention of the scientific community. Originally considered only a waste disposal system, they are now emerging as another class of signal mediators. Exosomes are secreted by any cell type and retrieved in every body fluid, such as blood, urine, saliva and amniotic liquid. Remarkably, their biochemical content includes not only lipids and proteins, but also nucleic acids, mainly miRNA and mRNA, with a few reports also indicating the presence of genomic and mitochondrial DNA. Their properties have stimulated extensive research to exploit them as a source of biomarkers for the diagnosis and the follow-up of several pathologies. Furthermore, exosomes are relatively robust and stable, so they appear attractive as gene and drug delivery vehicles. They have also revealed immunomodulatory and regenerative properties, which are encouraging their application for therapeutic purposes. Several issues remain to be addressed: exosome isolation is still time consuming and unsatisfactorily reproducible, making it difficult to compare results among laboratories, improve our knowledge of their physiological function and correlate their features with pathological outcomes. Nevertheless, the number of patents trying to address these problems is growing exponentially and many novelties will reach the scientific community in the next few years.

Extracellular Vesicles as New Players in Cellular Senescence

Cell senescence is associated with the secretion of many factors, the so-called “senescence-associated secretory phenotype”, which may alter tissue microenvironment, stimulating the organism to clean up senescent cells and replace them with newly divided ones. Therefore, although no longer dividing, these cells are still metabolically active and influence the surrounding tissue. Much attention has been recently focused not only on soluble factors released by senescent cells, but also on extracellular vesicles as conveyors of senescence signals outside the cell. Here, we give an overview of the role of extracellular vesicles in biological processes and signaling pathways related to senescence and aging.

Recent developments in therapeutic approaches for lysosomal storage diseases.

Genetic mutations that cause specific lysosomal protein deficiencies account for more than 45 Lysosomal Storage Diseases (LSDs), mostly pre-adult disorders which are associated with neurological symptoms and mental retardation. Interestingly, such diseases are often characterized by intracellular deposition and protein aggregation, events also found in age-related neurodegenerative diseases. During the past twenty years, different approaches have been introduced for the treatment of these disorders, several of which are now in routine clinical use or clinical trials. Among them, enzyme replacement therapy (ERT) represented a major progress. However, the usefulness of ERT is limited due to the fact that enzyme distribution is insufficient and treatment costs are very high. A further novel therapeutic option for LSDs is based on the use of small molecules, that can either inhibit a key enzyme which is responsible for substrate synthesis (substrate reduction) or act as a chaperone to increase the residual activity of the lysosomal enzyme (pharmacological chaperones). In addition, recently various gene therapy approaches have been developed, mostly based on adeno-associated and lentiviral vectors, and strategies based on stem cells administration are beginning their route. This review provides an update of the status of research on LSDs therapeutic approaches, including recent patents in the field.

Raman micro-spectroscopy: A powerful tool for the monitoring of dynamic supramolecular changes in living cells

Cellular imaging techniques have become powerful tools in cell biology. With respect to others, the techniques based on vibrational spectroscopy present a clear advantage: the molecular composition and the modification of subcellular compartments can be obtained in label-free conditions. In fact, from the evolution of positions, intensities and line widths of Raman and infrared bands in the cell spectra, characteristic information on cellular activities can be achieved, and particularly, cellular death can be investigated. In this work we present the time evolution of the Raman spectra of single live Jurkat cells (T-lymphocyte) by looking at the high frequency part of their Raman spectra, that is the CH stretching region, around 3000cm(-1). In particular, investigation into the composition or rearrangement of CH bounds, markers of cellular membrane fatty acids, can represent an important method to study and to recognize cell death. The experimental procedure we used, together with the analysis of these high frequency vibrational bands, may represent a new, improved and advantageous approach to this kind of study.

Spectroscopic Investigation of Interactions of New Potential Anticancer Drugs with DNA and Non-Ionic Micelles

Photophysical properties of some azinium iodides in aqueous solution of nanostructured systems as DNA and nonionic micelles were investigated using steady-state and ultrafast time-resolved spectroscopy. Spectrophotometric and fluorimetric titrations of the investigated compounds with salmon testes DNA supplied evidence of a good interaction between the salts and DNA with binding constants of 10(4)-10(6) M(-1), making them interesting for pharmaceutical applications. The interaction with DNA also changes the photobehavior of the compounds, increasing the radiative deactivation pathway to the detriment of internal conversion and slowing down the excited state dynamics. The interaction of the azinium salts with the nonionic surfactant Triton X-100 from premicellar to postmicellar concentration was studied by spectrophotometric and fluorimetric titrations evidencing the ability of the micelles to associate the studied salts in their hydrophobic portion and to release them in the presence of DNA, acting as promising drug carriers. Also transient absorption spectroscopy with femtosecond resolution demonstrated the insertion of the investigated compounds into micellar aggregates. Preliminary measurements by confocal fluorescence microscopy on MCF-7 cells in the presence of the studied azinium salts showed that they are able to cross the cellular membrane and that their cytotoxicity can be expressed through interaction with DNA (RNA). In fact, they showed a significant fluorescence signal in all cell compartments, particularly (for 2 and 3) into punctuate structures within the nuclei compatible with a localization into the nucleoli.

New Perspectives for the Diagnosis of Alzheimers Disease

Alzheimers 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.

Abnormal cortical lysosomal β-hexosaminidase and β-galactosidase activity at post-synaptic sites during Alzheimer's disease progression

A critical role of endosomal-lysosomal system alteration in neurodegeneration is supported by several studies. Dysfunction of the lysosomal compartment is a common feature also in Alzheimers disease. Altered expression of lysosomal glycohydrolases has been demonstrated not only in the brain and peripheral tissues of Alzheimers disease patients, but also in presymptomatic subjects before degenerative phenomenon becomes evident. Moreover, the presence of glycohydrolases associated to the plasma membrane have been widely demonstrated and their alteration in pathological conditions has been documented. In particular, lipid microdomains-associated glycohydrolases can be functional to the maintenance of the proper glycosphingolipids pattern, especially at cell surface level, where they are crucial for the function of cell types such as neurons. In this study we investigated the localization of β-hexosaminidase and β-galactosidase glycohydrolases, both involved in step by step degradation of the GM1 to GM3 gangliosides, in lipid microdomains from the cortex of both an early and advanced TgCRND8 mouse model of Alzheimers disease. Throughout immunoprecipitation experiments of purified cortical lipid microdomains, we demonstrated for the first time that β-hexosaminidase and β-galactosidase are associated with post-synaptic vesicles and that their activities are increased at both the early and the advanced stage of Alzheimers disease. The early increase of lipid microdomain-associated β-hexosaminidase and β-galactosidase activities could have relevant implications for the pathophysiology of the disease since their possible pharmacological manipulation could shed light on new reliable targets and biological markers of Alzheimers disease.