The ovarian cancer cell lines A2780 (wild-type p53) and NIHOVCAR3 (mutated p53) showed, respectively, sensitivity and resistance towards several chemotherapy drugs. We hypothesized that the two cell lines differ in their ability to activate the intrinsic death pathway and have, therefore, dissected the lysosome-mitochondrion signalling pathway by pharmacological inhibition or genetic manipulation of key regulators and executioners. Biochemical and morphological confocal fluorescence studies showed that: (1) In A2780 cells bcl-2 is expressed at an undetectable level, whereas Bax is expressed at a rather high level; by contrast, bcl-2 is highly expressed and Bax is expressed at extremely low levels in NIHOVCAR3 cells; (2) Chemotherapy treatment reduced the expression of bcl-2 in NIHOVCAR3 cells, yet these cells resisted to drug toxicity; (3) Cathepsin D (CD), not cathepsin B or L, mediates the activation of the mitochondrial intrinsic death pathway in A2780 cells; (4) Lysosome leakage and cytosolic relocation of CD occurs in the chemosensitive A2780 cells, not in the chemoresistant NIHOVCAR3 cells; (5) Bax is essential for the permeabilization of both lysosomes and mitochondria in A2780 cells exposed to chemotherapy drugs; (6) CD activity is mandatory for the oligomerization of Bax on both mitochondrial and lysosomal membranes; (7) Bax activation did not occur in the resistant NIHOVCAR3 cells despite their high content in CD. The present data are consistent with a model in which on treatment with a cytotoxic drug the activation of a CD-Bax loop leads to the generalized permeabilization of lysosomes and eventually of mitochondria, thus reaching the point of no return, and culminates with the activation of the caspase cascade. Our data also imply that dysfunctional permeabilization of lysosomes contributes to the development of chemoresistance.
Mesoporous silica nanoparticles are being explored as versatile tools for various biomedical and biotechnological applications including disease diagnosis, drug delivery, and intracellular imaging. In this paper, the synthesis and characterization of a fluorescent hybrid mesoporous silica nanomaterial, which is noncytotoxic and shows great potential for "in-cell" bioimaging applications, will be described. The hybrid mesoporous material has been obtained by confining highly fluorescent organic dyes, belonging to the indocyanine family, within the channels of mesoporous MCM-41. To explore the dispersion of the dye inside the mesoporous channels and the formation of dye aggregates, several hybrid samples with increasing dye/MCM-41 loading (up to 100 mg/g) were prepared. A uniform distribution of monomeric 1,1'-diethyl-3,3,3',3'-tetramethylindocarbocyanine iodide has been achieved at low dye loading (1 mg/g), as evidenced by photoluminescence spectra and lifetime, while a progressive formation of J-aggregates is induced by an increase in the dye loading. To elucidate the properties of the dye immobilized in mesoporous MCM-41, a detailed physical chemical characterization by structural (X-ray diffraction), volumetric and optical (Fourier transform infrared, diffuse-reflectance UV-vis and photoluminescence) techniques has been performed. By ultrasonication of the bulk material, nanoparticles of 2-20 nm diameter were obtained. Biocompatibility, endocytic uptake, and intracellular compartmentalization of such fluorescent nanoparticles were investigated in mammalian cultured cells.
Plex-B1, the receptor of Sema4D, has been implicated in tumour growth, angiogenesis and metastasis. The binding of Sema4D to Plex-B1 can trigger the activation of Met tyrosine kinase, thereby promoting cell dissociation and invasive growth. We tested the hypothesis that the expression of Plex-B1, either alone or in association with Met, can be of predictive value for tumour progression.
Autophagy is the mechanism through which cells degrade oxidized membranes-organelles and mis/unfolded proteins, in this latter function cooperating with the ubiquitin-proteasome system (UP system). Although autophagy has been known for a long time, its involvement in the pathogenesis of neurodegenerative diseases has been investigated only recently. The most fascinating data are very recent and show an impressive connection between proteins that are mutated in different forms of familial Parkinson's Disease (PD) and the critical role that these proteins play in the physiology of the Autophagy (ATG) pathway. This evidence is supported by neuropathological data showing at the ultrastructural level, the occurrence of an altered ATG in the dopamine (DA) neurons of the Substantia Nigra of patients affected by PD. Accordingly, by using experimental models of PD the involvement of ATG is documented as well. In particular, administration of the DA neurotoxin methamphetamine produces damage to DA-containing cells which is exacerbated and results in neuronal cell death when the ATG pathway is inhibited, thus confirming ATG as a critical pathway for the survival of DA neurons. In the present manuscript, after describing the general molecular and cellular features of ATG, we give a short overview of the most relevant aspects concerning the involvement of ATG in the pathogenesis of PD. We further propose that the ATG and the UP systems might converge in the formation of a so-called "autophagoproteasome" which might represent an early ultrastructure witnessing the presence of an ongoing degeneration within DA cells.
Methamphetamine causes nigrostriatal denervation and striatal dopamine loss, while sparing nigral cell bodies. Nigral dopamine neurons feature autophagic vacuoles and cytoplasmic alpha-synuclein-, ubiquitin- and parkin-positive inclusion-like bodies. On that basis, autophagy was considered essential in methamphetamine-induced neurotoxicity, but its neurotoxic or protective role has never been addressed. Here we review the gap between the descriptive evidence on activation of autophagy and the lack of knowledge about its role during methamphetamine intoxication. Our preliminary findings rule out a detrimental role for autophagy; this represents the first step in understanding the consequence of activation of autophagy in methamphetamine toxicity.
Mutations in the arginine vasopressin (AVP)-neurophysin II (NP-II) gene that affect the folding and transport of the prohormone result in loss of secretion of the anti-diuretic hormone AVP from pituitary nerve terminals and cause autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI). One such mutation consists of the replacement of a Cys residue at position 98 with a stop codon (C98X) in the AVP precursor (corresponding to C67X in NP domain). In neuroblastoma cells over-expressing this truncated AVP precursor autophagy, a macromolecular degradation process, was shown to be essential for assuring cell survival. In the present study, we investigated the role of the Akt pro-survival signalling in the regulation of autophagy and of apoptosis linked with the handling of C98X AVP. Impairing autophagy-lysosomal sequestration or cathepsin D (CD)-mediated proteolysis triggered the activation of the intrinsic death pathway of apoptosis in C98X-expressing cells, but not in the wild-type -AVP-expressing cells. This was shown by the expression of a Vps34 dominant negative, which down-regulates the PI3k class III-dependent signalling needed for autophagosome (APH) formation, by genetic silencing as a result of RNA interference (RNAi) of Lamp2, a protein indispensable for the fusion of APHs with lysosomes, and by RNAi silencing of the lysosomal protease CD. Ectopic expression of either the wild-type or the mutated C98X AVP altered neither the expression nor the phosphorylation of the pro-survival signalling molecule Akt. Strikingly, the ectopic adenoviral-directed expression of a constitutively active Akt, instead of preserving cell survival, resulted in the suppression of autophagy, and precipitated Bax-mediated cell death. The present data demonstrate the need for autophagy-mediated degradation of mutated C98X peptides, which otherwise become toxic to the cell, and suggest that, in the presence of mis-folded proteins, the stimulation of the Akt signalling counteracts the beneficial effects of autophagy and precipitates cell death. It follows that growth factors impinging on the Akt pathway may have deleterious effect in neurones expressing mutant neuropeptides. This can provide an explanation for the late onset and progressive neuronal cell loss observed in hypothalamic magnocellular neurones of adFNDI patients.
Methamphetamine abuse is toxic to dopaminergic neurons, causing nigrostriatal denervation and striatal dopamine loss. Following methamphetamine exposure, the number of nigral cell bodies is generally preserved, but their cytoplasm features autophagic-like vacuolization and cytoplasmic accumulation of alpha-synuclein-, ubiquitin- and parkin-positive inclusion-like bodies. Whether autophagy is epiphenomenal or it plays a role in the mechanism of methamphetamine toxicity and, in the latter case, whether its role consists of counteracting or promoting the neurotoxic effect remains obscure. We investigated the signaling pathway and the significance (protective vs. toxic) of autophagy activation and the convergence of the autophagic and the ubiquitin-proteasome pathways at the level of the same intracellular bodies in a simple cell model of methamphetamine toxicity. We show that autophagy is rapidly up-regulated in response to methamphetamine. Confocal fluorescence microscopy and immuno-electron microscopy studies demonstrated the presence of alpha-synuclein aggregates in autophagy-lysosomal structures in cells exposed to methamphetamine, a condition compatible with cell survival. Inhibition of autophagy either by pharmacologic or genetic manipulation of the class III Phosphatidylinositol-3 kinase-mediated signaling prevented the removal of alpha-synuclein aggregates and precipitated a bax-mediated mitochondrial apoptosis pathway.
Cathepsin D (CD), a lysosomal aspartic protease present in mammary tissue and milk in various molecular forms, is also found in the incubation medium of mammary acini in molecular forms that are proteolytically active on prolactin at a physiological pH. Because prolactin controls the vesicular traffic in mammary cells, we studied, in vivo and in vitro, its effects on the polarized transport and secretion of various forms of CD in the rat mammary gland. CD accumulated in vesicles not involved in endocytosis in the basal region of cells. Prolactin increased this accumulation and the release of endosomal active single-chain CD at the basal side of acini. The CD-mediated proteolysis of prolactin, leading to the antiangiogenic 16-kDa form, at a physiological pH, was observed only in conditioned medium but not milk. These data support the novel concept that an active molecular form of CD, secreted at the basal side of the mammary epithelium, participates in processing blood-borne prolactin outside the cell, this polarized secretion being controlled by prolactin itself.
In a pilot clinical study that we recently published we found that lithium administration slows the progression of Amyotrophic Lateral Sclerosis (ALS) in human patients. This clinical study was published in addition with basic (in vitro) and pre-clinical (in vivo) data demonstrating a defect of autophagy as a final common pathway in the genesis of ALS. In fact, lithium was used as an autophagy inducer. In detailing the protective effects of lithium we found for the first time that this drug stimulates the biogenesis of mitochondria in the central nervous system and, uniquely in the spinal cord, it induces neuronogenesis and neuronal differentiation. In particular, the effects induced by lithium can be summarized as follows: (i) the removal of altered mitochondria and protein aggregates; (ii) the biogenesis of well-structured mitochondria; (iii) the suppression of glial proliferation; (iv) the differentiation of newly formed neurons in the spinal cord towards a specific phenotype. In this addendum we focus on defective autophagy as a "leit motif" in ALS and the old and novel features of lithium which bridge autophagy activation to concomitant effects that may be useful for the treatment of a variety of neurodegenerative disorders. In particular, the biogenesis of mitochondria and the increase of calbindin D 28K-positive neurons, which are likely to support powerful neuroprotection towards autophagy failure, mitochondriopathy and neuronal loss in the spinal cord.
ALS is a devastating neurodegenerative disorder with no effective treatment. In the present study, we found that daily doses of lithium, leading to plasma levels ranging from 0.4 to 0.8 mEq/liter, delay disease progression in human patients affected by ALS. None of the patients treated with lithium died during the 15 months of the follow-up, and disease progression was markedly attenuated when compared with age-, disease duration-, and sex-matched control patients treated with riluzole for the same amount of time. In a parallel study on a genetic ALS animal model, the G93A mouse, we found a marked neuroprotection by lithium, which delayed disease onset and duration and augmented the life span. These effects were concomitant with activation of autophagy and an increase in the number of the mitochondria in motor neurons and suppressed reactive astrogliosis. Again, lithium reduced the slow necrosis characterized by mitochondrial vacuolization and increased the number of neurons counted in lamina VII that were severely affected in saline-treated G93A mice. After lithium administration in G93A mice, the number of these neurons was higher even when compared with saline-treated WT. All these mechanisms may contribute to the effects of lithium, and these results offer a promising perspective for the treatment of human patients affected by ALS.