Parkinson’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 patients

Microtubule dysfunction in experimental model of Parkinson's disease / D. Cartelli ; tutor: G. Cappelletti ; coordinatore: M. Bolognesi. DIPARTIMENTO DI BIOLOGIA, 2010 May 07. 22. ciclo, Anno Accademico 2008/2009.

Microtubule dysfunction in experimental model of Parkinson's disease

D. Cartelli
2010

Abstract

Parkinson’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 patients
7-mag-2010
microtubule ; Axonal transport ; neurodegeneration ; Parkinson's disease
Settore BIO/06 - Anatomia Comparata e Citologia
CAPPELLETTI, GRAZIELLA
BOLOGNESI, MARTINO
Doctoral Thesis
Microtubule dysfunction in experimental model of Parkinson's disease / D. Cartelli ; tutor: G. Cappelletti ; coordinatore: M. Bolognesi. DIPARTIMENTO DI BIOLOGIA, 2010 May 07. 22. ciclo, Anno Accademico 2008/2009.
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