Daniele Cartelli

Microtubule dysfunction precedes transport impairment and mitochondria damage in MPP+ -induced neurodegeneration.

J. Neurochem. (2010) 115, 247–258.

Mesenchymal Stromal Cells Primed with Paclitaxel Provide a New Approach for Cancer Therapy

Background Mesenchymal 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 Findings Paclitaxel (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. Conclusions These 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.

Microtubule Alterations Occur Early in Experimental Parkinsonism and The Microtubule Stabilizer Epothilone D Is Neuroprotective

The role of microtubule (MT) dysfunction in Parkinsons 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.

Pleiotropic effects of spastin on neurite growth depending on expression levels

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.

Inhibitors of tubulin polymerization: synthesis and biological evaluation of hybrids of vindoline, anhydrovinblastine and vinorelbine with thiocolchicine, podophyllotoxin and baccatin III.

A series of novel hybrid compounds obtained by the attachment of anhydrovinblastine, vinorelbine, and vindoline to thiocolchicine, podophyllotoxin, and baccatin III are described. Two types of diacyl spacers are introduced. The influence of the hybrid compounds on tubulin polymerization is reported. The results highlight the importance of the length of the spacer. Immunofluorescence microscopy and flow cytometry measurements that compound with the best in vitro activity could disrupt microtubule networks in cell and prevent the formation of the proper spindle apparatus, thereby causing cell cycle arrest in the G2/M phase. The newly synthesized compounds were tested in the human lung cancer cell line A549.

Synthesis and biological evaluation of novel thiocolchicine–podophyllotoxin conjugates

The synthesis and biological evaluation of 9 dimeric compounds obtained by condensation of thiocolchicine and/or podophyllotoxin with 6 different dicarboxylic acids is described. In particular, tubulin assembly assay and immunofluorescence analysis results are reported. The biological data highlighted three compounds as being more active than the others, having a marked ability to inhibit the polymerization of tubulin in vitro and causing significant disruption to the microtubule network in vivo. The spacer unit was found to have a significant effect on biological activity, reinforcing the importance of the design of conjugate compounds to create new biologically active molecules in which the spacer could be useful to improve the solubility and to modulate the efficacy of well known anticancer drugs.

Microtubule Destabilization Is Shared by Genetic and Idiopathic Parkinson’s Disease Patient Fibroblasts

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.

In silico design of tubulin-targeted antimitotic peptides

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.

Investigation of in vitro cytotoxicity of the redox state of ionic iron in neuroblastoma cells

Background: there is an intimate relation between transition metals and cell homeostasis due to the physiological necessity of metals in vivo. Particularly, iron (ferrous and ferric state) is utilized in many physiological processes of the cell but in excess has been linked with negative role contributing in many neurodegenerative processes. Objective: the aim of this study was to investigate which oxidation state of ionic iron (Ferrous (II) versus Ferric (III)) is more toxic to neuronal cells (SHSY5Y). Materials and Methods: The neuroblastoma (SHSY5Y) cells were exposed to varying concentration of ferric and ferrous iron. Morphological studies using immunofluorescence staining and microscopic analysis as confirmed by intracellular glutathione (GSH) test demonstrated oxidative stress to cells in iron microenvironment. In addition, MTT assay was performed to evaluate the viability and metabolic state of the cells. Results: the results showed that ferrous form has significantly higher toxicity compared to the ferric ionic state of higher concentration. In addition, microscopic analysis shows cell fenestration at higher concentrations and swelling at intermediate ferric dosages as demonstrated by atomic force microscopy (AFM). Interestingly, the addition of a differentiation inducing factor, trans-retinoic rcid (RA) retains significant viability and morphological features of the cells irrespective of the ionic state of the iron. AFM images revealed clustered aggregates arising from iron chelation with RA. Conclusions: the results indicate that Fe (II) has more toxic effects on cells. In addition, it could be an interesting finding with respect to the antioxidant properties of RA as a chelating agent for the neurodegenerative therapeutics.

α-Synuclein is a Novel Microtubule Dynamase

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.