BackgroundMesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation.Methods and Principal FindingsPaclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth.ConclusionsThese data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.

J. Neurochem. (2010) 115, 247–258. Abstract Dysfunction of the microtubule (MT) system is an emerging theme in the pathogenesis of Parkinson’s disease. This study was designed to investigate the putative role of MT dysfunction in dopaminergic neuron death induced by the neurotoxin 1‐methyl‐4‐phenylpiridinium (MPP+). In nerve growth factor‐differentiated PC12 cells, we have analyzed post‐translational modifications of tubulin known to be associated with differently dynamic MTs and show that MPP+ causes a selective loss of dynamic MTs and a concomitant enrichment of stable MTs. Through a direct live cell imaging approach, we show a significant reduction of MT dynamics following exposure to MPP+ and a reorientation of MTs. Furthermore, these alterations precede the impairment of intracellular transport as revealed by changes in mitochondria movements along neurites and their accumulation into varicosities. We have also analyzed activation of caspase 3 and mitochondrial injury, well‐known alterations induced by MPP+, and found that they are noticeable only when MT dysfunction is already established. These data provide the first evidence that axonal transport impairment and mitochondrial damage might be a consequence of MT dysfunction in MPP+‐induced neurodegeneration, lending support to the concept that alterations of MT organization and dynamics could play a pivotal role in neuronal death in Parkinson’s disease.

The role of microtubule (MT) dysfunction in Parkinson's disease is emerging. It is still unknown whether it is a cause or a consequence of neurodegeneration. Our objective was to assess whether alterations of MT stability precede or follow axonal transport impairment and neurite degeneration in experimental parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57Bl mice. MPTP induced a time- and dose-dependent increase in fibres with altered mitochondria distribution, and early changes in cytoskeletal proteins and MT stability. Indeed, we observed significant increases in neuron-specific βIII tubulin and enrichment of deTyr tubulin in dopaminergic neurons. Finally, we showed that repeated daily administrations of the MT stabilizer Epothilone D rescued MT defects and attenuated nigrostriatal degeneration induced by MPTP. These data suggest that alteration of ΜΤs is an early event specifically associated with dopaminergic neuron degeneration. Pharmacological stabilization of MTs may be a viable strategy for the management of parkinsonism.

Hereditary spastic paraplegia (HSP) is characterized by weakness and spasticity of the lower limbs, owing to degeneration of corticospinal axons. The most common form is due to heterozygous mutations in the SPG4 gene, encoding spastin, a microtubule (MT)‐severing protein. Here, we show that neurite growth in immortalized and primary neurons responds in pleiotropic ways to changes in spastin levels. Spastin depletion alters the development of primary hippocampal neurons leading to abnormal neuron morphology, dystrophic neurites, and axonal growth defects. By live imaging with End‐Binding Protein 3‐Fluorescent Green Protein (EB3‐GFP), a MT plus‐end tracking protein, we ascertained that the assembly rate of MTs is reduced when spastin is down‐regulated. Spastin over‐expression at high levels strongly suppresses neurite maintenance, while slight spastin up‐regulation using an endogenous promoter enhances neurite branching and elongation. Spastin severing activity is exerted preferentially on stable acetylated and detyrosinated MTs. We further show that SPG4 nonsense or splice site mutations found in hereditary spastic paraplegia patients result in reduced spastin levels, supporting haploinsufficiency as the molecular cause of the disease. Our study reveals that SPG4 is a dosage‐sensitive gene, and broadens the understanding of the role of spastin in neurite growth and MT dynamics.

α-Synuclein is a presynaptic protein associated to Parkinson’s disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson’s disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics.

Microtubules are polymeric structures formed by the self-assembly of tubulin dimers. The growth and shrinkage of these dynamic arrays have a key role during the cell-proliferation process. This makes tubulin the molecular target of many anticancer drugs currently in use or under clinical trial. Their impressive success is limited by the onset of resistant tumour cells during the treatment, so new resistance-proof molecules need to be developed. Here we use molecular dynamics and free-energy calculations to study the network of interactions that allow microtubule formation. Modelling the protein-protein interface allows us to identify the amino acids responsible for tubulin-tubulin binding and thus to design peptides, which correspond to tubulin subsequences, that interfere with microtubule formation. We show that the application of molecular modelling techniques leads to the identification of peptides that exhibit antitubulin activity both in vitro and in cultured cells.

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Data from both toxin-based and gene-based models suggest that dysfunction of the microtubule system contributes to the pathogenesis of Parkinson’s disease, even if, at present, no evidence of alterations of microtubules in vivo or in patients is available. Here we analyze cytoskeleton organization in primary fibroblasts deriving from patients with idiopathic or genetic Parkinson’s disease, focusing on mutations in parkin and leucine-rich repeat kinase 2. Our analyses reveal that genetic and likely idiopathic pathology affects cytoskeletal organization and stability, without any activation of autophagy or apoptosis. All parkinsonian fibroblasts have a reduced microtubule mass, represented by a higher fraction of unpolymerized tubulin in respect to control cells, and display significant changes in microtubule stability-related signaling pathways. Furthermore, we show that the reduction of microtubule mass is so closely related to the alteration of cell morphology and behavior that both pharmacological treatment with microtubule-targeted drugs, and genetic approaches, by transfecting the wild type parkin or leucine-rich repeat kinase 2, restore the proper microtubule stability and are able to rescue cell architecture. Taken together, our results suggest that microtubule destabilization is a point of convergence of genetic and idiopathic forms of parkinsonism and highlight, for the first time, that microtubule dysfunction occurs in patients and not only in experimental models of Parkinson’s disease. Therefore, these data contribute to the knowledge on molecular and cellular events underlying Parkinson’s disease and, revealing that correction of microtubule defects restores control phenotype, may offer a new therapeutic target for the management of the disease.