1,721,018 research outputs found
Insight into microtubule dynamics: from purified protein to cell
No full textMicrotubule-targeting agents have received considerable interest as potential tumour-selective anti-angiogenic and vascular-disrupting agents. To improve targeting of these agents, a detailed characterization of their interaction with tubulin/microtubule system is required and includes the analysis of stabilisation and depolymerisation of microtubules as well as imbalance in microtubule dynamics. We performed in vitro assays using purified tubulin to investigate the effects of microtubule-targeting agents on microtubule assembly in terms of polymer mass, polymerization kinetics, microtubule structure.and dynamics1. Moving from purified protein to cultured cells, we investigated the effects of microtubule-targeting agents on microtubule network by immunofluorescence and confocal analyses. Finally, microtubule dynamics in live cell was investigated by time lapse imaging of cells expressing the microtubule-associated protein EB3-GFP, a protein that binds specifically to the plus end of growing microtubules
Microtubule destabilization paves the way to Parkinson’s disease
No full textMicrotubules are dynamic structures normally associated to the cell division, during which they form the mitotic spindle, as well as to the initial phases of specification and polarization of various cell type, including neurons. Although microtubules could have a role in the death of many cells and tissues, the microtubule-based degenerative mechanisms have been poorly investigated; nevertheless, during the last two decades, many clues have been accumulated suggesting the importance of microtubule system during neurodegeneration. Thus, the aim of this review is to analyse how the changes of the microtubule cytoskeleton, in terms of organization and dynamics, as well as the failure of the microtubule-dependent neuronal processes, as axonal transport, may play a pivotal role in the chain of events leading to Parkinson’s disease. Last but not least, since disease-modifying or neuroprotective strategies are a clinical priority in Parkinson’s disease, we will also present the hints about the concrete possibility of a microtubule-targeted therapy, which would have the potentiality to block the running degenerative events and to prompt the regeneration of the lost tissues
Acetylation of tubulin: a feasible protective target from neurodevelopment to neurodegeneration.
No full textMicrotubules are dynamic polymers essential in the proper development and maintenance of a healthy nervous system. Previously increasing evidence linked the defective regulation of microtubules to a spectrum of disorders from neurodevelopmental to neurodegenerative diseases. Acetylation of tubulin determines the biochemical and biophysical diversity of microtubules, regulates their function, and has been recently related to the pathogenic events in different disorders including Schizophrenia, Autism and Alzheimer’s disease. Here we critically look at the experimental data coming from in vitro to in vivo disease models and from patients with the aim of understanding whether targeting tubulin acetylation could be a promising strategy for neuroprotection. We conclude that this is a feasible road but, in the future, a more comprehensive analysis of molecular details at the base of tubulin acetylation and, most important, the consequent determination of novel compounds targeting this process, are the condition sine qua non for moving towards therapeutic interventions
Microtubule dysfunction in experimental model of Parkinson's disease
No full textParkinson’s disease (PD) is the most common motor neurodegenerative disorder, affecting 1% of people at age of 65 and, and its cardinal symptoms are resting tremors, rigidity and bradykinesia. Now, it is well established that the clinical features of PD are due to the degeneration dopaminergic neurons residing in the substantia nigra and projecting to the striatum. There is no a conclusive demonstration that PD is an environmental or a genetic disease, but there is agreement about the fact that PD could be a multifactorial disorder and that the greater risk factor is the age. So, in attempt to elucidate molecular mechanisms of dopaminergic cell death, useful are both toxin-based and gene-based models. At the present time, there are several hypothesis about the molecular pathways leading to PD. Just to have a brief overview, toxin-induced model opened the way to the concept that mitochondrial dysfunction are the initial insult, because all toxins induce the formation of ROS, reduce ATP production and for some of them is clearly demonstrated an inhibition of the complex I of the respiratory chain. Two are the main hypothesis coming from genetic models of PD: ubiquitin-proteasome system (UPS) dysfunction, that acting in concert with the reduction of autophagy, seems to overload the cell with damaged proteins; oligomerization and aggregation of misfolded and/or unfolded proteins, that could act subtracting useful monomers, or through the formation of toxic oligomers. Flanking these large accepted mechanisms of degeneration in PD, is emerging a new possible cause of neuron dysfunction: microtubule (MT) dysfunction. MTs are a common target of PD-inducing neurotoxins, as rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and mutated proteins in PD, as synuclein and parkin. MT dysfunction could trigger cell death by axonal transport impairment, a common mechanism described in many neurodegenerative diseases.
The present study was designed to investigate the putative role of MT dysfunction in diverse models of PD, ranging from neurotoxic model represented by MPTP, to genetic model, i.e. synuclein and parkin. The implication of MT alterations have been investigated at different levels, from molecular level using purified protein system, to complex system as an animal model is, passing through cell cultures.
Particularly, we analyzed MT dysfunctions in PC12 cells treated with 1-methyl-4-phenyl-pyridinium (MPP+), the toxic metabolite of MPTP, and their relationship with known alterations induced by the neurotoxin as complex I block and impairment of axonal transport. In NGF-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 mitochondria transport along neurites. We have also analyzed activation of caspase-3 and mitochondrial injury, well known alterations induced by MPP+; in our experimental conditions, we found that they are noticeable only when MT dysfunction is already established. Furthermore, analysis of striatal lysates and sections revealed that alteration of MT stability is the first noticeable alterations in MPTP-treated mice, preceding mitochondrial accumulation and tyrosine hydroxylase (TH) depletion, a well accepted marker of dopaminergic degeneration progression. These data provide the first evidence that axonal transport impairment and mitochondrial damage are tightly associated with, and might be a consequence of MT dysfunction in MPTP-induced neurodegeneration.
Further, we investigated the effect of wild type and mutated synucleins on the polymerization kinetics of purified tubulin, and on MT structure. Wild type synuclein seems to stabilize MT structure promoting tubulin polymerization, whereas the pathologic mutants of synuclein elicit the opposite effects. In fact, they tend to broke the MT lattice and inhibit tubulin polymerization. The effect on tubulin polymerization kinetics that we observed, can suggest a physiological role of synuclein in regulating MT dynamic behavior and could be relevant from a pathogenic point of view. The MT destabilizing effects of mutant synucleins could interfere with the correct assembly of neuronal architecture, but also can impair axonal transport by breaking the railways along organelles move, leading to dopaminergic degeneration.
Finally, we investigated MT stability in mice lacking parkin. Using western blotting and immunofluorescence analysis, we show variations of tubulin post-translation modification in parkin null mice, both in striatum and substantia nigra. Analysis of mitochondria distribution and TH expression revealed that axonal transport seems to be blocked. Another one time, MT dysfunction is suggested as pivotal alteration in the chain of event leading to dopaminergic neuron death.
Putting all the pieces together, the data reported here show MTs as a common target of MPTP, synuclein and parkin. All these data make MT dysfunction an acceptable culprit of neurodegenerative process. Furthermore, the relationship between MPTP-induced MT dysfunction and the known alterations of mitochondria and axonal transport, and the fact that changes in MT stability are noticeable before any other signs of neurodegeneration in mice lacking parkin, strongly suggest a central and pivotal role of MT system in triggering neuronal death in PD.
Under this light, MTs could be a good “druggable” candidate for future prevention and PD therapies, that not only ameliorate the symptoms but that also restore the damaged tissue, improving the lifespan and the quality of life of PD patient
Microtubule dysfunction in experimental model of Parkinson's disease
No full textDysfunction of axonal transport is a common theme in many neurodegenerative disease, and recent data show that MPP+, a well known parkinsonism-inducing neurotoxin, causes an early defect of fast axonal transport. Alterations of axonal transport could be easily explained by microtubule (MT) dysfunctions, because MTs are the railways along proteins and organelles move. Starting from the notion that MT are a common target of toxins and mutated proteins inducing Parkinson’s disease, and knowing that MPP+ alters MT polymerization and dynamics in vitro, we investigated the relationship between MT dynamics and axonal transport in PC12 cells exposed to MPP+ and in mice treated with MPTP, the metabolic precursor of MPP+.
Combining Fluorescence Recovery After Photobleaching (FRAP) analyses and live cell imaging, we observed that MPP+ causes a reduction of MT dynamics and a reorientation of MTs before axonal transport impairment occurs. By using indirect approaches, as western blotting and immunofluorescence analyses, we found that in mice striata MPTP leads to an unbalance of dynamic and stable MTs, before that mitochondria accumulations became noticeable.
Taken together, live cell imaging and in vivo analyses show that MT dysfunctions precede axonal transport impairment in the MPTP model of Parkinson’s disease
Parkin absence impacts microtubule stability and axonal transport in knockout mice
No full textParkin is responsible for the vast majority of the Autosomal Recessive Juvenile Parkinsonism (Kitada et al., 1998), an atypical form of Parkinson’s Disease (PD) with early onset. Parkin is an ubiquitin E3 ligase (Shimura et al., 2000), catalyzing the addition of ubiquitin to target proteins, and therefore it is involved in the protein quality control and in the cellular cleaning system.
Furthermore, it is already reported that parkin is able to associate with (Ren et al., 2003) and to modulate the stability (Yang et al., 2005) of microtubules (MTs), cytoskeletal elements involved in cell shape control and axonal transport processes. Nevertheless, nothing is known about the capacity of parkin to regulate -tubulin PTMs, that are usually used as marker of MTs with different stability and that, recently, have been directly linked to neurodegenerative processes (Rogowski et al., 2010). Moreover, until now there are no evidences that parkin is involved in the regulation of axonal transport, a MT-dependent function implicated in neurodegenerative processes (Morfini et al., 2009).
Therefore, here we analyze -tubulin PTMs and axonal transport in mice lacking parkin, and we observe that alteration of MT stability is present in knockout mice and it is associated to defects of organelle transport. These data suggest a new physiological role for parkin, and because many PD-linked proteins, as parkin, -synuclein and leucine-rich repeat kinase 2, impact MT organization and stability, the pivotal role of MT dysfunction in PD etiopathogenesis is a well sustained and concrete hypothesis
Nitric oxide stabilizes microtubules during neuronal differentiation
No full textNitric oxide (NO) is a signalling molecule in the nervous system playing a role in neurotransmitter release, synaptic plasticity, excitability, learning, differentiation and development. The signalling pathway triggered by NO in physiological processes involves the activation of soluble guanylate cyclase, S-nitrosylation and nitration of proteins. Focusing on neuronal differentiation and development, NO-induced axonal retraction is involved in the refinement of neuronal projections during brain development and modulated by a S-nitrosylation-dependent signal –transduction pathway leading to the reconfiguration of axonal microtubules. On the other hand, NO donors have been reported to enhance neurite outgrowth suggesting the positive effects of NO on neuritogenesis.
Our previous results showed that nitrated proteins accumulate during neuronal differentiation and the cytoskeleton becomes the main cellular fraction containing nitrated proteins, being alpha-tubulin and tau two of the main targets, and that nitration correlates with increased microtubule stability.
Here we have addressed the question of the possible role played by protein tyrosine nitration and microtubules during neuronal differentiation and neuritogenesis. We modulated the level of intracellular NO by donors and investigated the effects on nitration of proteins, neuritogenesis, arrangement and dynamics of microtubules. Our results show that low-dose NO exposure inducing an increase in nitrated proteins stimulates neurite elongation, microtubule growth and stabilization as shown by indirect immunofluorescence and live cell imaging. On the contrary, high-dose NO exposure induces axonal retraction, causes the accumulation of nitrated proteins at growth cone and destabilizes microtubules. We suggest that protein nitration plays a dual role during neuronal differentiation and modulates NO signalling to the microtubular cytoskeleton
Protein tyrosine nitration: a beneficial or detrimental cue during neuronal differentiation?
No full textNitric oxide (NO) is well established as an intracellular and transcellular signalling molecule in the nervous system playing a role in neurotransmitter release, synaptic plasticity, excitability, learning, differentiation and development. The majority of NO actions under physiological conditions occur through the activation of soluble guanylate cyclase, leading to the intracellular increase of cGMP. However, NO can also produce cGMP-independent effects in living cells through protein modification including S-nitrosylation and tyrosine nitration. Focusing on neuronal differentiation and development, NO-induced axonal retraction is involved in the refinement of neuronal projections during brain development and modulated by an S-nitrosylation-dependent signal –transduction pathway leading to the reconfiguration of axonal microtubules. On the other hand, NO donors have been reported to enhance neurite outgrowth suggesting a positive effects of NO on neuritogenesis. We have addressed the question of the possible role played by protein tyrosine nitration in the signalling pathway triggered by NO during neuronal differentiation and neuritogenesis. Our previous results showed that nitrated proteins accumulate during NGF-induced differentiation of PC12 cells, the cytoskeleton becomes the main cellular fraction containing nitrated proteins, and that nitration correlates with increased microtubule stability.
Here we have modulated the level of intracellular NO by donors and investigated the effects on nitration of proteins, neuritogenesis, arrangement and dynamics of the microtubular cytoskeleton.
Our results show that NO donor (Glyco-SNAP-2) significantly affects neuritogenesis in NGF-differentiating PC12 cells. Low-dose NO exposure inducing an increase in nitrated protein stimulates neurite elongation and causes microtubule stabilization as shown by indirect immunofluorescence and live cell imaging analyses.
In conclusion, our results reveal a dual role of protein nitration during neuronal differentiation and neuritogenesis, with a beneficial or cytotoxic actions depending on the NO concentration, and suggest the involvement of this posttranslational modification in modulation of NO signalling to the cytoskeleton
Alpha-Synuclein nucleates short microtubules
No full textAlpha-Synuclein (alpha-syn) is the major component of Lewy body, the cytosolic inclusions associated to Parkinson’s disease, and its mutation have been linked to genetic forms of the disorder. Although largely studied, alpha-syn physiological role is still unclear. In vitro, -syn is unfolded, but it acquires a secondary structure upon interaction with the partners. It is also reported the capacity of alpha-syn to induce microtubule (MT) polymerization, but a detailed analysis of kinetic parameters still lacks.
Here, we decided to undertake an in depth evaluation of the effects of alpha-syn on tubulin polymerization kinetics. Because standard protocol failed in reproducibility, we decided to incubate alpha-syn in the presence of tubulin, just to allow alpha-syn folding. Circular dichroism spectra show that upon pre-incubation alpha-syn acquires a -helix conformation. Thus, we performed tubulin polymerization following pre-incubation with WT and mutated alpha-syns, and the analyses of kinetic parameters suggest that synucleins promote MT nucleation, but they seem to reduce MT mass at the end of polymerization. By using fluorescence microscopy, we evaluated the number and the length of MTs polymerized in presence of alpha-syns, at the very beginning and at the end of the polymerization. Accordingly to the already published data, mutated alpha-syn causes tubulin aggregation instead of MT polymerization. On the contrary, at both time points, WT alpha-syn promotes the polymerization of a higher number of MTs, confirming its capacity in nucleating MTs. Moreover, MTs polymerized in the presence of WT alpha-syn are shorter in respect to control MTs, reconciling the apparent reduction of MT mass due to the physics of the system.
Our results underscore the ability of alpha-syn to nucleate short MTs, as usually are MTs present at synaptic terminal, the cell compartment where alpha-syn resides. Thus, we shed light on a new physiological role for alpha-syn as a feasible axonal MT nucleating structures
Microtubule dynamics imbalance leads to axonal transport impairment in MPP+-treated PC12 cells
No full textMany neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and Amyotrophic lateral sclerosis, show axonal pathologies with abnormal accumulation of proteins and organelles. This can be easily explained with the impairment of axonal transport, that, in turn, can lead to the dying-back degeneration of neurons in many of the disorders mentioned above.
At the present days the question is: which is the trigger event leading to the block of axonal transport? Disruption to transport can occur via damage to cargoes, reduction of the ATP supply, dysfunction of motor proteins and alteration of microtubules (De Vos et al., 2008). Recently, it has been also reported that in isolated squid axoplasm, the administration of MPP+, a well known parkinsonism-inducing neurotoxin, leads to the alterations in fast axonal transport through the activation of caspase and protein kinase C (Morfini et al., 2007).
Starting from the notion that microtubules are a common target of toxins and mutated proteins inducing Parkinson’s disease (Feng, 2006), and knowing that MPP+ alters microtubule dynamics in vitro (Cappelletti et al., 2005), we have investigated if the imbalance of microtubule dynamics could be the triggering event that leads to axonal transport impairment in PC12 cells treated with MPP+.
Here we show that accumulations of mitochondria in axonal swellings, a typical marker of impaired axonal transport, are noticeable in a condition where the only alteration that could induce disruption of transport is microtubule dynamics imbalance. Using low dose of MPP+ we observed the increase of stable microtubules (acetylated and detyrosinated tubulin) without any sign of dysfunction of motor proteins (kinesin and dynein), damage to mitochondria or reduction of ATP supply. Further, in our model, the inhibition of caspase and PKC does not protect the cells from the neurotoxin.
However, all the alterations that are supposed to be causative for the block of axonal transport are observed with higher dose of MPP+, suggesting that they are secondary events.
These data clearly suggest the pivotal role of microtubule dynamics alteration in the chain of event that through the impairment of axonal transport leads to the degeneration of neurons in Parkinson’s disease
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