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Find video protocols related to scientific articles indexed in Pubmed.
Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation.
PLoS Genet.
PUBLISHED: 11-01-2014
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Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.
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Rab11 modulates ?-synuclein-mediated defects in synaptic transmission and behaviour.
Hum. Mol. Genet.
PUBLISHED: 10-09-2014
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A central pathological hallmark of Parkinson's disease (PD) is the presence of proteinaceous depositions known as Lewy bodies, which consist largely of the protein ?-synuclein (aSyn). Mutations, multiplications and polymorphisms in the gene encoding aSyn are associated with familial forms of PD and susceptibility to idiopathic PD. Alterations in aSyn impair neuronal vesicle formation/transport, and likely contribute to PD pathogenesis by neuronal dysfunction and degeneration. aSyn is functionally associated with several Rab family GTPases, which perform various roles in vesicle trafficking. Here, we explore the role of the endosomal recycling factor Rab11 in the pathogenesis of PD using Drosophila models of aSyn toxicity. We find that aSyn induces synaptic potentiation at the larval neuromuscular junction by increasing synaptic vesicle (SV) size, and that these alterations are reversed by Rab11 overexpression. Furthermore, Rab11 decreases aSyn aggregation and ameliorates several aSyn-dependent phenotypes in both larvae and adult fruit flies, including locomotor activity, degeneration of dopaminergic neurons and shortened lifespan. This work emphasizes the importance of Rab11 in the modulation of SV size and consequent enhancement of synaptic function. Our results suggest that targeting Rab11 activity could have a therapeutic value in PD.
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Interplay between Sumoylation and Phosphorylation for Protection against ?-Synuclein Inclusions.
J. Biol. Chem.
PUBLISHED: 09-17-2014
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Parkinson disease is associated with the progressive loss of dopaminergic neurons from the substantia nigra. The pathological hallmark of the disease is the accumulation of intracytoplasmic inclusions known as Lewy bodies that consist mainly of post-translationally modified forms of ?-synuclein. Whereas phosphorylation is one of the major modifications of ?-synuclein in Lewy bodies, sumoylation has recently been described. The interplay between ?-synuclein phosphorylation and sumoylation is poorly understood. Here, we examined the interplay between these modifications as well as their impact on cell growth and inclusion formation in yeast. We found that ?-synuclein is sumoylated in vivo at the same sites in yeast as in human cells. Impaired sumoylation resulted in reduced yeast growth combined with an increased number of cells with inclusions, suggesting that this modification plays a protective role. In addition, inhibition of sumoylation prevented autophagy-mediated aggregate clearance. A defect in ?-synuclein sumoylation could be suppressed by serine 129 phosphorylation by the human G protein-coupled receptor kinase 5 (GRK5) in yeast. Phosphorylation reduced foci formation, alleviated yeast growth inhibition, and partially rescued autophagic ?-synuclein degradation along with the promotion of proteasomal degradation, resulting in aggregate clearance in the absence of a small ubiquitin-like modifier. These findings suggest a complex interplay between sumoylation and phosphorylation in ?-synuclein aggregate clearance, which may open new horizons for the development of therapeutic strategies for Parkinson disease.
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Modulation of alpha-synuclein toxicity in yeast using a novel microfluidic-based gradient generator.
Lab Chip
PUBLISHED: 08-29-2014
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Parkinson's disease (PD) is a common age-associated neurodegenerative disorder. The protein ?-synuclein (aSyn) is a key factor in PD both due to its association with familial and sporadic cases and because it is the main component of the pathological protein aggregates known as Lewy bodies. However, the precise cellular effects of aSyn aggregation are still elusive. Here, we developed an elastomeric microfluidic device equipped with a chemical gradient generator and 9 chambers containing cell traps to study aSyn production and aggregation in Saccharomyces cerevisiae. This study involved capturing single cells, exposing them to specific chemical environments and imaging the expression of aSyn by means of a GFP fusion (aSyn-GFP). Using a galactose (GAL) gradient we modulated aSyn expression and, surprisingly, by tracking the behavior of single cells, we found that the response of individual cells in a population to a given stimulus can differ widely. To study the combined effect of environmental factors and aSyn expression levels, we exposed cells to a gradient of FeCl3. We found a dramatic increase in the percentage of cells displaying aSyn inclusions from 27% to 96%. Finally, we studied the effects of ascorbic acid, an antioxidant, on aSyn aggregation and found a significant reduction in the percentage of cells bearing aSyn inclusions from 87% to 37%. In summary, the device developed here offers a powerful way of studying aSyn biology with single-cell resolution and high throughput using genetically modified yeast cells.
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Mutant huntingtin alters Tau phosphorylation and subcellular distribution.
Hum. Mol. Genet.
PUBLISHED: 08-20-2014
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Tau abnormalities play a central role in several neurodegenerative diseases, collectively known as tauopathies. In the present study, we examined whether mutant huntingtin (mHtt), which causes Huntington's disease (HD), modifies Tau phosphorylation and subcellular localization using cell and mouse HD models. Initially, we used novel bimolecular fluorescence complementation assays in live cells to evaluate Tau interactions with either wild type (25QHtt) or mutant huntingtin (103QHtt). While 25QHtt and Tau interacted at the level of the microtubule network, 103QHtt and Tau interacted and formed 'ring-like' inclusions localized in the vicinity of the microtubular organizing center (MTOC). Fluorescence recovery after photobleaching experiments also indicated that, whereas homomeric 103QHtt/103QHtt pairs rapidly re-entered into inclusions, heteromeric 103QHtt/Tau pairs remained excluded from the 'ring-like' inclusions. Interestingly, in vitro Tau relocalization was associated to Tau hyperphosphorylation. Consistent with this observation, we found strong Tau hyperphosphorylation in brain samples from two different mouse models of HD, R6/2 and 140CAG knock-in. This was associated with a significant reduction in the levels of Tau phosphatases (PP1, PP2A and PP2B), with no apparent involvement of major Tau kinases. Thus, the present study strongly suggests that expression of mHtt leads to Tau hyperphosphorylation, relocalization and sequestration through direct protein-protein interactions in inclusion-like compartments in the vicinity of the MTOC. Likewise, our data also suggest that Tau alterations may also contribute to HD pathogenesis.
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Linking alpha-synuclein phosphorylation to reactive oxygen species formation and mitochondrial dysfunction in SH-SY5Y cells.
Mol. Cell. Neurosci.
PUBLISHED: 08-07-2014
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Alpha-synuclein (?-syn) is a soluble protein highly enriched in presynaptic terminals of neurons. Accumulation of ?-syn as intracellular filamentous aggregates is a pathological feature of sporadic and familial forms of Parkinson's disease (PD). Changes in ?-syn post-translational modifications, as well as mitochondrial dysfunction and oxidative stress constitute key pathogenic events of this disorder. Here we assessed the correlation between ?-syn phosphorylation at serine 129 (Ser129), the formation of reactive oxygen species (ROS) and mitochondrial dysfunction in SH-SY5Y cells expressing A53T mutant or wild-type (WT) ?-syn, exposed to ferrous iron (FeSO4) and rotenone (complex I inhibitor). Under basal conditions, prolonged expression of A53T mutant ?-syn altered mitochondria morphology, increased superoxide formation and phosphorylation at Ser129, which was linked to decreased activity of protein phosphatase 2A (PP2A). Exposure to FeSO4 or rotenone enhanced intracellular ROS levels, including superoxide anions, in both types of cells, along with ?-syn Ser129 phosphorylation and mitochondrial depolarization. Most of these changes were largely evident in A53T mutant ?-syn expressing cells. Overall, the data suggest that stimuli that promote ROS formation and mitochondrial alterations highly correlate with mutant ?-syn phosphorylation at Ser129, which may precede cell degeneration in PD.
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The small GTPase Rab11 co-localizes with ?-synuclein in intracellular inclusions and modulates its aggregation, secretion and toxicity.
Hum. Mol. Genet.
PUBLISHED: 08-04-2014
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Alpha-synuclein (aSyn) misfolding and aggregation are pathological features common to several neurodegenerative diseases, including Parkinson's disease (PD). Mounting evidence suggests that aSyn can be secreted and transferred from cell to cell, participating in the propagation and spreading of pathological events. Rab11, a small GTPase, is an important regulator in both endocytic and secretory pathways. Here, we show that Rab11 is involved in regulating aSyn secretion. Rab11 knockdown or overexpression of either Rab11a wild-type (Rab11a WT) or Rab11a GDP-bound mutant (Rab11a S25N) increased secretion of aSyn. Furthermore, we demonstrate that Rab11 interacts with aSyn and is present in intracellular inclusions together with aSyn. Moreover, Rab11 reduces aSyn aggregation and toxicity. Our results suggest that Rab11 is involved in modulating the processes of aSyn secretion and aggregation, both of which are important mechanisms in the progression of aSyn pathology in PD and other synucleinopathies.
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Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease.
PLoS Genet.
PUBLISHED: 05-01-2014
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Alpha-synuclein (aSyn) is the main component of proteinaceous inclusions known as Lewy bodies (LBs), the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Although aSyn is phosphorylated at low levels under physiological conditions, it is estimated that ? 90% of aSyn in LBs is phosphorylated at S129 (pS129). Nevertheless, the significance of pS129 in the biology of aSyn and in PD pathogenesis is still controversial. Here, we harnessed the power of budding yeast in order to assess the implications of phosphorylation on aSyn cytotoxicity, aggregation and sub-cellular distribution. We found that aSyn is phosphorylated on S129 by endogenous kinases. Interestingly, phosphorylation reduced aSyn toxicity and the percentage of cells with cytosolic inclusions, in comparison to cells expressing mutant forms of aSyn (S129A or S129G) that mimic the unphosphorylated form of aSyn. Using high-resolution 4D imaging and fluorescence recovery after photobleaching (FRAP) in live cells, we compared the dynamics of WT and S129A mutant aSyn. While WT aSyn inclusions were very homogeneous, inclusions formed by S129A aSyn were larger and showed FRAP heterogeneity. Upon blockade of aSyn expression, cells were able to clear the inclusions formed by WT aSyn. However, this process was much slower for the inclusions formed by S129A aSyn. Interestingly, whereas the accumulation of WT aSyn led to a marked induction of autophagy, cells expressing the S129A mutant failed to activate this protein quality control pathway. The finding that the phosphorylation state of aSyn on S129 can alter the ability of cells to clear aSyn inclusions provides important insight into the role that this posttranslational modification may have in the pathogenesis of PD and other synucleinopathies, opening novel avenues for investigating the molecular basis of these disorders and for the development of therapeutic strategies.
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Yeast DJ-1 superfamily members are required for diauxic-shift reprogramming and cell survival in stationary phase.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-02-2014
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The yeast Hsp31 minifamily proteins (Hsp31, Hsp32, Hsp33, Hsp34) belong to the highly conserved DJ-1 superfamily. The human DJ-1 protein is associated with cancer and neurodegenerative disorders, such as Parkinson disease. However, the precise function of human and yeast DJ-1 proteins is unclear. Here we show that the yeast DJ-1 homologs have a role in diauxic-shift (DS), characterized by metabolic reprogramming because of glucose limitation. We find that the Hsp31 genes are strongly induced in DS and in stationary phase (SP), and that deletion of these genes reduces chronological lifespan, impairs transcriptional reprogramming at DS, and impairs the acquisition of several typical characteristics of SP, including autophagy induction. In addition, under carbon starvation, the HSP31 family gene-deletion strains display impaired autophagy, disrupted target of rapamycin complex 1 (TORC1) localization to P-bodies, and caused abnormal TORC1-mediated Atg13 phosphorylation. Repression of TORC1 by rapamycin in the gene-deletion strains completely reversed their sensitivity to heat shock. Taken together, our data indicate that Hsp31 minifamily is required for DS reprogramming and cell survival in SP, and plays a role upstream of TORC1. The enhanced understanding of the cellular function of these genes sheds light into the biological role of other members of the superfamily, including DJ-1, which is an attractive target for therapeutic intervention in cancer and in Parkinson disease.
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Challenges and promises in the development of neurotrophic factor-based therapies for Parkinson's disease.
Drugs Aging
PUBLISHED: 03-11-2014
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Parkinson's disease (PD) is a chronic movement disorder typically coupled to progressive degeneration of dopaminergic neurons in the substantia nigra (SN). The treatments currently available are satisfactory for symptomatic management, but the efficacy tends to decrease as neuronal loss progresses. Neurotrophic factors (NTFs) are endogenous proteins known to promote neuronal survival, even in degenerating states. Therefore, the use of these factors is regarded as a possible therapeutic approach, which would aim to prevent PD or to even restore homeostasis in neurodegenerative disorders. Intriguingly, although favorable results in in vitro and in vivo models of the disease were attained, clinical trials using these molecules have failed to demonstrate a clear therapeutic benefit. Therefore, the development of animal models that more closely reproduce the mechanisms known to underlie PD-related neurodegeneration would be a major step towards improving the capacity to predict the clinical usefulness of a given NTF-based approach in the experimental setting. Moreover, some adjustments to the design of clinical trials ought to be considered, which include recruiting patients in the initial stages of the disease, improving the efficacy of the delivery methods, and combining synergetic NTFs or adding NTF-boosting drugs to the already available pharmacological approaches. Despite the drawbacks on the road to the use of NTFs as pharmacological tools for PD, very relevant achievements have been reached. In this article, we review the current status of the potential relevance of NTFs for treating PD, taking into consideration experimental evidence, human observational studies, and data from clinical trials.
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?-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner.
Neurobiol. Dis.
PUBLISHED: 03-05-2014
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Alpha-synuclein (?S) misfolding is associated with Parkinson's disease (PD) but little is known about the mechanisms underlying ?S toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with ?S in rodent brain. NMR spectroscopy reveals that the C-terminus of ?S binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/?S interaction, Rab8a enhanced ?S aggregation and reduced ?S-induced cellular toxicity. In addition, Rab8 - the Drosophila ortholog of Rab8a - ameliorated ?S-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the ?S-Rab8a interaction, phosphorylation of ?S at S129 enhanced binding to Rab8a, increased formation of insoluble ?S aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and ?S cytotoxicity, and underscores the therapeutic potential of targeting this interaction.
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Copy-number variation of the neuronal glucose transporter gene SLC2A3 and age of onset in Huntington's disease.
Hum. Mol. Genet.
PUBLISHED: 01-22-2014
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Huntington's disease (HD) is a devastating neurodegenerative disorder which is inherited in an autosomal dominant manner. HD is caused by a trinucleotide CAG repeat expansion that encodes a polyglutamine stretch in the huntingtin (HTT) protein. Mutant HTT expression leads to a myriad of cellular dysfunctions culminating in neuronal loss and consequent motor, cognitive and psychiatric disturbances in HD patients. The length of the CAG repeat is inversely correlated with age of onset (AO) in HD patients, while environmental and genetic factors can further modulate this parameter. Here, we explored whether the recently described copy-number variation (CNV) of the gene SLC2A3-which encodes the neuronal glucose transporter GLUT3-could modulate AO in HD. Strikingly, we found that increased dosage of SLC2A3 delayed AO in an HD cohort of 987 individuals, and that this correlated with increased levels of GLUT3 in HD patient cells. To our knowledge this is the first time that CNV of a candidate gene has been found to modulate HD pathogenesis. Furthermore, we found that increasing dosage of Glut1-the Drosophila melanogaster homologue of this glucose transporter-ameliorated HD-relevant phenotypes in fruit flies, including neurodegeneration and life expectancy. As alterations in glucose metabolism have been implicated in HD pathogenesis, this study may have important therapeutic relevance for HD.
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Protein phosphorylation in neurodegeneration: friend or foe?
Front Mol Neurosci
PUBLISHED: 01-01-2014
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Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and fronto-temporal dementia (FTD). In these disorders, the misfolding and aggregation of specific proteins occurs alongside neuronal degeneration in somewhat specific brain areas, depending on the disorder and the stage of the disease. However, we still do not fully understand the mechanisms governing protein aggregation, and whether this constitutes a protective or detrimental process. In PD, alpha-synuclein (aSyn) forms protein aggregates, known as Lewy bodies, and is phosphorylated at serine 129. Other residues have also been shown to be phosphorylated, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. In AD and in FTD, hyperphosphorylation of tau protein causes its misfolding and aggregation. Again, our understanding of the precise consequences of tau phosphorylation in the biology and pathophysiology of the protein is still limited. Through the use of a variety of model organisms and technical approaches, we are now gaining stronger insight into the effects of phosphorylation in the behavior of these proteins. In this review, we cover recent findings in the field and discuss how targeting phosphorylation events might be used for therapeutic intervention in these devastating diseases of the nervous system.
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MeCP2: a novel Huntingtin interactor.
Hum. Mol. Genet.
PUBLISHED: 10-08-2013
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Transcriptional dysregulation has been proposed to play a major role in the pathology of Huntingtons disease (HD). However, the mechanisms that cause selective downregulation of target genes remain unknown. Previous studies have shown that mutant huntingtin (Htt) protein interacts with a number of transcription factors thereby altering transcription. Here we report that Htt directly interacts with methyl-CpG binding protein 2 (MeCP2) in mouse and cellular models of HD using complimentary biochemical and Fluorescent Lifetime Imaging to measure Förster Resonance Energy Transfer approaches. Htt-MeCP2 interactions are enhanced in the presence of the expanded polyglutamine (polyQ) tract and are stronger in the nucleus compared with the cytoplasm. Furthermore, we find increased binding of MeCP2 to the promoter of brain-derived neurotrophic factor (BDNF), a gene that is downregulated in HD, in the presence of mutant Htt. Finally, decreasing MeCP2 levels in mutant Htt-expressing cells using siRNA increases BDNF levels, suggesting that MeCP2 downregulates BDNF expression in HD. Taken together, these findings suggest that aberrant interactions between Htt and MeCP2 contribute to transcriptional dysregulation in HD.
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DJ-1 modulates aggregation and pathogenesis in models of Huntingtons disease.
Hum. Mol. Genet.
PUBLISHED: 09-26-2013
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The oxidation-sensitive chaperone protein DJ-1 has been implicated in several human disorders including cancer and neurodegenerative diseases. During neurodegeneration associated with protein misfolding, such as that observed in Alzheimers disease and Huntingtons disease (HD), both oxidative stress and protein chaperones have been shown to modulate disease pathways. Therefore, we set out to investigate whether DJ-1 plays a role in HD. We found that DJ-1 expression and its oxidation state are abnormally increased in the human HD brain, as well as in mouse and cell models of HD. Furthermore, overexpression of DJ-1 conferred protection in vivo against neurodegeneration in yeast and Drosophila. Importantly, the DJ-1 protein directly interacted with an expanded fragment of huntingtin Exon 1 (httEx1) in test tube experiments and in cell models and accelerated polyglutamine aggregation and toxicity in an oxidation-sensitive manner. Our findings clearly establish DJ-1 as a potential therapeutic target for HD and provide the basis for further studies into the role of DJ-1 in protein misfolding diseases.
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Off-pathway ?-synuclein oligomers seem to alter ?-synuclein turnover in a cell model but lack seeding capability in vivo.
Amyloid
PUBLISHED: 09-20-2013
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Aggregated ?-synuclein is the major component of Lewy bodies, protein inclusions observed in the brain in neurodegenerative disorders such as Parkinsons disease and dementia with Lewy bodies. Experimental evidence indicates that ?-synuclein potentially can be transferred between cells and act as a seed to accelerate the aggregation process. Here, we investigated in vitro and in vivo seeding effects of ?-synuclein oligomers induced by the reactive aldehyde 4-oxo-2-nonenal (ONE). As measured by a Thioflavin-T based fibrillization assay, there was an earlier onset of aggregation when ?-synuclein oligomers were added to monomeric ?-synuclein. In contrast, exogenously added ?-synuclein oligomers did not induce aggregation in a cell model. However, cells overexpressing ?-synuclein that were treated with the oligomers displayed reduced ?-synuclein levels, indicating that internalized oligomers either decreased the expression or accelerated the degradation of transfected ?-synuclein. Also in vivo there were no clear seeding effects, as intracerebral injections of ?-synuclein oligomers into the neocortex of ?-synuclein transgenic mice did not induce formation of proteinase K resistant ?-synuclein pathology. Taken together, we could observe a seeding effect of the ONE-induced ?-synuclein oligomers in a fibrillization assay, but neither in a cell nor in a mouse model.
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Inhibition of formation of ?-synuclein inclusions by mannosylglycerate in a yeast model of Parkinsons disease.
Biochim. Biophys. Acta
PUBLISHED: 04-09-2013
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Protein aggregation in the brain is a central hallmark in many neurodegenerative diseases. In Parkinsons disease, ?-synuclein (?-Syn) is the major component of the intraneuronal inclusions found in the brains of patients. Current therapeutics is merely symptomatic, and there is a pressing need for developing novel therapies. Previously we showed that mannosylglycerate (MG), a compatible solute typical of marine microorganisms thriving in hot environments, is highly effective in protecting a variety of model proteins against thermal denaturation and aggregation in vitro.
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PLK2 modulates ?-synuclein aggregation in yeast and mammalian cells.
Mol. Neurobiol.
PUBLISHED: 03-15-2013
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Phosphorylation of ?-synuclein (aSyn) on serine 129 is one of the major post-translation modifications found in Lewy bodies, the typical pathological hallmark of Parkinsons disease. Here, we found that both PLK2 and PLK3 phosphorylate aSyn on serine 129 in yeast. However, only PLK2 increased aSyn cytotoxicity and the percentage of cells presenting cytoplasmic foci. Consistently, in mammalian cells, PLK2 induced aSyn phosphorylation on serine 129 and induced an increase in the size of the inclusions. Our study supports a role for PLK2 in the generation of aSyn inclusions by a mechanism that does not depend directly on serine 129 phosphorylation.
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Harnessing the power of yeast to unravel the molecular basis of neurodegeneration.
J. Neurochem.
PUBLISHED: 03-09-2013
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Several neurodegenerative diseases, such as Parkinsons disease (PD), Alzheimers disease (AD), Huntingtons disease (HD), amyotrophic lateral sclerosis (ALS), or prion diseases, are known for their intimate association with protein misfolding and aggregation. These disorders are characterized by the loss of specific neuronal populations in the brain and are highly associated with aging, suggesting a decline in proteostasis capacity may contribute to pathogenesis. Nevertheless, the precise molecular mechanisms that lead to the selective demise of neurons remain poorly understood. As a consequence, appropriate therapeutic approaches and effective treatments are largely lacking. The development of cellular and animal models that faithfully reproduce central aspects of neurodegeneration has been crucial for advancing our understanding of these diseases. Approaches involving the sequential use of different model systems, starting with simpler cellular models and ending with validation in more complex animal models, resulted in the discovery of promising therapeutic targets and small molecules with therapeutic potential. Within this framework, the simple and well-characterized eukaryote Saccharomyces cerevisiae, also known as budding yeast, is being increasingly used to study the molecular basis of several neurodegenerative disorders. Yeast provides an unprecedented toolbox for the dissection of complex biological processes and pathways. Here, we summarize how yeast models are adding to our current understanding of several neurodegenerative disorders.
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?-synuclein aggregates and induces neurodegeneration in dopaminergic neurons.
Ann. Neurol.
PUBLISHED: 03-07-2013
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Whereas the contribution of ?-synuclein to neurodegeneration in Parkinson disease is well accepted, the putative impact of its close homologue, ?-synuclein, is enigmatic. ?-Synuclein is widely expressed throughout the central nervous system, as is ?-synuclein, but the physiological functions of both proteins remain unknown. Recent findings have supported the view that ?-synuclein can act as an ameliorating regulator of ?-synuclein-induced neurotoxicity, having neuroprotective rather than neurodegenerative capabilities, and being nonaggregating due to the absence of most of the aggregation-promoting NAC domain. However, a mutation of ?-synuclein linked to dementia with Lewy bodies rendered the protein neurotoxic in transgenic mice, and fibrillation of ?-synuclein has been demonstrated in vitro.
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Heat-mediated enrichment of ?-synuclein from cells and tissue for assessing post-translational modifications.
J. Neurochem.
PUBLISHED: 02-27-2013
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?-Synuclein (?-syn) is the major component of Lewy bodies, a pathological hallmark of Parkinsons disease and other synucleinopathies. The characterization of ?-syn post-translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogenous protein from cells and tissues, and by the availability of antibodies toward ?-syn PTMs. Here, by taking advantage of ?-syn thermostability, we applied a method to achieve high enrichment of soluble ?-syn both from cultured cells and brain tissues followed by proteomics analysis. Using this approach, we obtained 98% ?-syn sequence coverage in a variety of model systems, including a transgenic mouse model of PD, and validated the strategy by identifying previously described PTMs such as phosphorylation and N-terminal acetylation. Our findings demonstrate that this procedure overcomes existing technical limitations and can be used to facilitate the systematic study of ?-syn PTMs, thereby enabling the clarification of their role under physiological and pathological conditions. Ultimately, this approach may enable the development of novel biomarkers and strategies for therapeutic intervention in synucleinopathies.
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Structural basis of kynurenine 3-monooxygenase inhibition.
Nature
PUBLISHED: 02-22-2013
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Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (that is, kynurenine pathway), leads to amelioration of Huntingtons-disease-relevant phenotypes in yeast, fruitfly and mouse models, as well as in a mouse model of Alzheimers disease. KMO is a flavin adenine dinucleotide (FAD)-dependent monooxygenase and is located in the outer mitochondrial membrane where it converts l-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders, as well as cancer and several peripheral inflammatory conditions. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained unknown. Here we report the first crystal structure of Saccharomyces cerevisiae KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active-site structure, preventing productive binding of the substrate l-kynurenine. Functional assays and targeted mutagenesis reveal that the active-site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO-UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood-brain barrier in targeted therapies against neurodegenerative diseases such as Huntingtons, Alzheimers and Parkinsons diseases.
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Oxidative and nitrative alpha-synuclein modifications and proteostatic stress: implications for disease mechanisms and interventions in synucleinopathies.
J. Neurochem.
PUBLISHED: 02-21-2013
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Alpha-synuclein (ASYN) is a major constituent of the typical protein aggregates observed in several neurodegenerative diseases that are collectively referred to as synucleinopathies. A causal involvement of ASYN in the initiation and progression of neurological diseases is suggested by observations indicating that single-point (e.g., A30P, A53T) or multiplication mutations of the gene encoding for ASYN cause early onset forms of Parkinsons disease (PD). The relative regional specificity of ASYN pathology is still a riddle that cannot be simply explained by its expression pattern. Also, transgenic over-expression of ASYN in mice does not recapitulate the typical dopaminergic neuronal death observed in PD. Thus, additional factors must contribute to ASYN-related toxicity. For instance, synucleinopathies are usually associated with inflammation and elevated levels of oxidative stress in affected brain areas. In turn, these conditions favor oxidative modifications of ASYN. Among these modifications, nitration of tyrosine residues, formation of covalent ASYN dimers, as well as methionine sulfoxidations are prominent examples that are observed in post-mortem PD brain sections. Oxidative modifications can affect ASYN aggregation, as well as its binding to biological membranes. This would affect neurotransmitter recycling, mitochondrial function and dynamics (fission/fusion), ASYNs degradation within a cell and, possibly, the transfer of modified ASYN to adjacent cells. Here, we propose a model on how covalent modifications of ASYN link energy stress, altered proteostasis, and oxidative stress, three major pathogenic processes involved in PD progression. Moreover, we hypothesize that ASYN may act physiologically as a catalytically regenerated scavenger of oxidants in healthy cells, thus performing an important protective role prior to the onset of disease or during aging.
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Alpha-synuclein and intracellular trafficking: impact on the spreading of Parkinsons disease pathology.
J. Mol. Med.
PUBLISHED: 01-25-2013
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Parkinsons disease is characterized by intracellular proteinaceous depositions known as Lewy bodies. These largely consist of the protein ?-synuclein, whose physiological function remains unclear, but mutations and overexpression of the protein have been shown to cause early onset cases of Parkinsons disease. Deregulation of ?-synuclein biology causes neurodegeneration and impaired neuronal trafficking, hinting at a possible contribution to the pathological mechanism. Recent studies produced some evidence hinting at the involvement of several regulators of the transport machinery such as Rab GTPases and SNARE proteins, but also shown that ?-synuclein can be propagated between cells. Here, we discuss the molecular interplay of ?-synuclein with the intracellular transport machinery, its consequences, and the implications for disease mechanisms.
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The NAD-dependent deacetylase sirtuin 2 is a suppressor of microglial activation and brain inflammation.
EMBO J.
PUBLISHED: 01-22-2013
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Deleterious sustained inflammation mediated by activated microglia is common to most of neurologic disorders. Here, we identified sirtuin 2 (SIRT2), an abundant deacetylase in the brain, as a major inhibitor of microglia-mediated inflammation and neurotoxicity. SIRT2-deficient mice (SIRT2(-/-)) showed morphological changes in microglia and an increase in pro-inflammatory cytokines upon intracortical injection of lipopolysaccharide (LPS). This response was associated with increased nitrotyrosination and neuronal cell death. Interestingly, manipulation of SIRT2 levels in microglia determined the response to Toll-like receptor (TLR) activation. SIRT2 overexpression inhibited microglia activation in a process dependent on serine 331 (S331) phosphorylation. Conversely, reduction of SIRT2 in microglia dramatically increased the expression of inflammatory markers, the production of free radicals, and neurotoxicity. Consistent with increased NF-?B-dependent transcription of inflammatory genes, NF-?B was found hyperacetylated in the absence of SIRT2, and became hypoacetylated in the presence of S331A mutant SIRT2. This finding indicates that SIRT2 functions as a gatekeeper, preventing excessive microglial activation through NF-?B deacetylation. Our data uncover a novel role for SIRT2 opening new perspectives for therapeutic intervention in neuroinflammatory disorders.
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The causative role and therapeutic potential of the kynurenine pathway in neurodegenerative disease.
J. Mol. Med.
PUBLISHED: 01-14-2013
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Metabolites of the kynurenine pathway (KP), which arise from the degradation of tryptophan, have been studied in detail for over a century and garnered the interest of the neuroscience community in the late 1970s and early 1980s with work uncovering the neuromodulatory potential of this pathway. Much research in the following decades has found that perturbations in the levels of KP metabolites likely contribute to the pathogenesis of several neurodegenerative diseases. More recently, it has become apparent that targeting KP enzymes, in particular kynurenine 3-monooxygenase (KMO), may hold substantial therapeutic potential for these disorders. Here we provide an overview of the KP, the neuroactive properties of KP metabolites and their role in neurodegeneration. We also discuss KMO as a therapeutic target for these disorders, and our recent resolution of the crystallographic structure of KMO, which will permit the development of new and improved KMO inhibitors which may ultimately expedite clinical application of these compounds.
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Assessing the subcellular dynamics of alpha-synuclein using photoactivation microscopy.
Mol. Neurobiol.
PUBLISHED: 01-08-2013
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Alpha-synuclein (aSyn) is implicated in Parkinsons disease and several other neurodegenerative disorders. To date, the function and intracellular dynamics of aSyn are still unclear. Here, we tracked the dynamics of aSyn using photoactivatable green fluorescent protein as a reporter. We found that the availability of the aSyn N terminus modulates its shuttling into the nucleus. Interestingly, familial aSyn mutations altered the dynamics at which the protein distributes throughout the cell. Both the A30P and A53T aSyn mutations increase the speed at which the protein moves between the nucleus and cytoplasm, respectively. We also found that specific kinases potentiate the shuttling of aSyn between nucleus and cytoplasm. A mutant aSyn form that blocks S129 phosphorylation, S129A, results in the formation of cytoplasmic inclusions, suggesting phosphorylation modulates aggregation in addition to modulating aSyn intracellular dynamics. Finally, we found that the molecular chaperone HSP70 accelerates the entry of aSyn into the nuclear compartment.
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SIRT1 and SIRT2: emerging targets in neurodegeneration.
EMBO Mol Med
PUBLISHED: 01-04-2013
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Sirtuins are NAD-dependent protein deacetylases known to have protective effects against age-related diseases such as cancer, diabetes, cardiovascular and neurodegenerative diseases. In mammals, there are seven sirtuins (SIRT1-7), which display diversity in subcellular localization and function. While SIRT1 has been extensively investigated due to its initial connection with lifespan extension and involvement in calorie restriction, important biological and therapeutic roles of other sirtuins have only recently been recognized. Here, we review the potential roles and effects of SIRT1 and SIRT2 in neurodegenerative diseases. We discuss different functions and targets of SIRT1 and SIRT2 in a variety of neurodegenerative diseases including Alzheimers disease (AD), Parkinsons disease (PD) and Huntingtons Disease (HD). We also cover the role of SIRT1 in neuronal differentiation due to the possible implications in neurodegenerative conditions, and conclude with an outlook on the potential therapeutic value of SIRT1 and SIRT2 in these disorders.
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?-Synuclein modifies huntingtin aggregation in living cells.
FEBS Lett.
PUBLISHED: 10-26-2011
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Several neurodegenerative disorders are characterized by the accumulation of proteinaceous inclusions in the central nervous system. These inclusions are frequently composed of a mixture of aggregation-prone proteins. Here, we used a bimolecular fluorescence complementation assay to study the initial steps of the co-aggregation of huntingtin (Htt) and ?-synuclein (?-syn), two aggregation-prone proteins involved in Huntingtons disease (HD) and Parkinsons disease (PD), respectively. We found that Htt (exon 1) oligomerized with ?-syn and sequestered it in the cytosol. In turn, ?-syn increased the number of cells displaying aggregates, decreased the number of aggregates per cell and increased the average size of the aggregates. Our results support the idea that co-aggregation of aggregation-prone proteins can contribute to the histopathology of neurodegenerative disorders.
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Aggresome formation and segregation of inclusions influence toxicity of ?-synuclein and synphilin-1 in yeast.
Biochem. Soc. Trans.
PUBLISHED: 09-23-2011
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PD (Parkinsons disease) is a neurodegenerative disorder, caused by a selective loss of dopaminergic neurons in the substantia nigra, which affects an increasing number of the elderly population worldwide. One of the major hallmarks of PD is the occurrence of intracellular protein deposits in the dying neurons, termed Lewy bodies, which contain different proteins, including aggregated ?-synuclein and its interacting protein synphilin-1. During the last decade, a number of groups developed yeast models that reproduced important features of PD and allowed the deciphering of pathways underlying the cytotoxicity triggered by ?-synuclein. Here, we review the recent contributions obtained with yeast models designed to study the presumed pathobiology of synphilin-1. These models pointed towards a crucial role of the sirtuin Sir2 and the chaperonin complex TRiC (TCP-1 ring complex)/CCT (chaperonin containing TCP-1) in handling misfolded and aggregated proteins.
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Impaired TrkB receptor signaling contributes to memory impairment in APP/PS1 mice.
Neurobiol. Aging
PUBLISHED: 07-19-2011
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Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal plasticity, learning, and memory. Levels of BDNF and its main receptor TrkB (TrkB.TK) have been reported to be decreased while the levels of the truncated TrkB (TrkB.T1) are increased in Alzheimers disease. We show here that incubation with amyloid-? increased TrkB.T1 receptor levels and decreased TrkB.TK levels in primary neurons. In vivo, APPswe/PS1dE9 transgenic mice (APdE9) showed an age-dependent relative increase in cortical but not hippocampal TrkB.T1 receptor levels compared with TrkB.TK. To investigate the role of TrkB isoforms in Alzheimers disease, we crossed AP mice with mice overexpressing the truncated TrkB.T1 receptor (T1) or the full-length TrkB.TK isoform. Overexpression of TrkB.T1 in APdE9 mice exacerbated their spatial memory impairment while the overexpression of TrkB.TK alleviated it. These data suggest that amyloid-? changes the ratio between TrkB isoforms in favor of the dominant-negative TrkB.T1 isoform both in vitro and in vivo and supports the role of BDNF signaling through TrkB in the pathophysiology and cognitive deficits of Alzheimers disease.
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Synthesis and in vitro evaluation of fluorinated styryl benzazoles as amyloid-probes.
Bioorg. Med. Chem.
PUBLISHED: 07-07-2011
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The formation of proteinaceous aggregates is a pathognomonic hallmark of several neurodegenerative disorders such as Alzheimers and Parkinsons diseases. To date, the final diagnostic for these diseases can only be achieved by immunostaining of post-mortem brain tissues with the commonly used congo red and Thioflavin T/S amyloid-dyes. The interest in developing amyloid-avid radioprobes to be used for protein aggregates imaging by positron emission tomography has grown substantialy, due to the promise in assisting diagnosis of these disorders. To this purpose, the present work describes the synthesis and characterization of four novel fluorinated styryl benzazole derivatives 1-4 by means of the Wittig reaction, as well as their in vitro evaluation as amyloid-probing agents. All compounds were obtained as mixtures of geometric E and Z isomers, with the preferable formation of the E isomer. Photoisomerization reactions allowed for the maximization of the minor Z isomers. The authentic 1-4E/Z isomers were isolated after purification by column chromatography under dark conditions. Profiting from the fluorescence properties of the different geometric isomers of 1-4, their binding affinities towards amyloid fibrils of insulin, ?-synuclein and ?-amyloid peptide were also measured. These compounds share similarities with Thioflavin T, interacting specifically with fibrillary species with a red-shift in the excitation wavelengths along with an increase in the fluorescence emission intensity. Apparent binding constants were determined and ranged between 1.22 and 23.96 ?M(-1). The present data suggest that the novel fluorinated styryl benzazole derivatives may prove useful for the design of (18)F-labeled amyloid radioprobes.
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Idebenone and resveratrol extend lifespan and improve motor function of HtrA2 knockout mice.
PLoS ONE
PUBLISHED: 07-06-2011
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Heterozygous loss-of-function mutation of the human gene for the mitochondrial protease HtrA2 has been associated with increased risk to develop mitochondrial dysfunction, a process known to contribute to neurodegenerative disorders such as Huntingtons disease (HD) and Parkinsons disease (PD). Knockout of HtrA2 in mice also leads to mitochondrial dysfunction and to phenotypes that resemble those found in neurodegenerative disorders and, ultimately, lead to death of animals around postnatal day 30. Here, we show that Idebenone, a synthetic antioxidant of the coenzyme Q family, and Resveratrol, a bioactive compound extracted from grapes, are both able to ameliorate this phenotype. Feeding HtrA2 knockout mice with either compound extends lifespan and delays worsening of the motor phenotype. Experiments conducted in cell culture and on brain tissue of mice revealed that each compound has a different mechanism of action. While Idebenone acts by downregulating the integrated stress response, Resveratrol acts by attenuating apoptosis at the level of Bax. These activities can account for the delay in neuronal degeneration in the striata of these mice and illustrate the potential of these compounds as effective therapeutic approaches against neurodegenerative disorders such as HD or PD.
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Antibodies against alpha-synuclein reduce oligomerization in living cells.
PLoS ONE
PUBLISHED: 06-08-2011
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Recent research implicates soluble aggregated forms of ?-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinsons disease and related disorders. The pathway by which ?-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing ?-synuclein fused to hemi:GFP constructs to study the effects of ?-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective ?-synuclein antibodies reduced the extent of ?-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower ?-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of ?-synuclein, thus providing further support for passive immunization against diseases with ?-synuclein pathology.
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Convergence of miRNA expression profiling, ?-synuclein interacton and GWAS in Parkinsons disease.
PLoS ONE
PUBLISHED: 05-18-2011
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miRNAs were recently implicated in the pathogenesis of numerous diseases, including neurological disorders such as Parkinsons disease (PD). miRNAs are abundant in the nervous system, essential for efficient brain function and play important roles in neuronal patterning and cell specification. To further investigate their involvement in the etiology of PD, we conducted miRNA expression profiling in peripheral blood mononuclear cells (PBMCs) of 19 patients and 13 controls using microarrays. We found 18 miRNAs differentially expressed, and pathway analysis of 662 predicted target genes of 11 of these miRNAs revealed an over-representation in pathways previously linked to PD as well as novel pathways. To narrow down the genes for further investigations, we undertook a parallel approach using chromatin immunoprecipitation-sequencing (ChIP-seq) analysis to uncover genome-wide interactions of ?-synuclein, a molecule with a central role in both monogenic and idiopathic PD. Convergence of ChIP-seq and miRNomics data highlighted the glycosphingolipid biosynthesis and the ubiquitin proteasome system as key players in PD. We then tested the association of target genes belonging to these pathways with PD risk, and identified nine SNPs in USP37 consistently associated with PD susceptibility in three genome-wide association studies (GWAS) datasets (0.46?OR?0.63) and highly significant in the meta-dataset (3.36×10??

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Tau enhances ?-synuclein aggregation and toxicity in cellular models of synucleinopathy.
PLoS ONE
PUBLISHED: 05-02-2011
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The simultaneous accumulation of different misfolded proteins in the central nervous system is a common feature in many neurodegenerative diseases. In most cases, co-occurrence of abnormal deposited proteins is observed in different brain regions and cell populations, but, in some instances, the proteins can be found in the same cellular aggregates. Co-occurrence of tau and ?-synuclein (?-syn) aggregates has been described in neurodegenerative disorders with primary deposition of ?-syn, such as Parkinsons disease and dementia with Lewy bodies. Although it is known that tau and ?-syn have pathological synergistic effects on their mutual fibrillization, the underlying biological effects remain unclear.
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Impaired proteostasis contributes to renal tubular dysgenesis.
PLoS ONE
PUBLISHED: 03-23-2011
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Protein conformational disorders are associated with the appearance, persistence, accumulation, and misprocessing of aberrant proteins in the cell. The etiology of renal tubular dysgenesis (RTD) is linked to mutations in the angiotensin-converting enzyme (ACE). Here, we report the identification of a novel ACE mutation (Q1069R) in an RTD patient. ACE Q1069R is found sequestered in the endoplasmic reticulum and is also subject to increased proteasomal degradation, preventing its transport to the cell surface and extracellular fluids. Modulation of cellular proteostasis by temperature shift causes an extension in the processing time and trafficking of ACE Q1069R resulting in partial rescue of the protein processing defect and an increase in plasma membrane levels. In addition, we found that temperature shifting causes the ACE Q1069R protein to be secreted in an active state, suggesting that the mutation does not affect the enzymes catalytic properties.
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Dopamine-depletion and increased ?-synuclein load induce degeneration of cortical cholinergic fibers in mice.
J. Neurol. Sci.
PUBLISHED: 03-15-2011
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Cognitive dysfunction can be common among Parkinsons disease (PD) patients, and multiplication of the gene ?-synuclein (?syn) increases the risk of dementia. Here, we studied the role of dopamine-depletion and increased ?syn load and aggregation on cholinergic structures in vivo. Wild-type (WT) and mice with A30P ?syn overexpression were treated subacutely with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and the number of cholinergic cells in their nucleus basalis magnocellularis-substantia innominata (NBM-SI), their cortical fiber density and their expression of different genes 1day or 90 days after the last MPTP-injection were measured. Long-term dopamine depletion decreased the expression of choline acetyl transferase (ChAT) in the NBM-SI of WT mice, but no neuron loss was observed. In contrast, cortical cholinergic fiber density was decreased three months after MPTP-injection. Increased brain-derived neurotrophic factor expression could maintain cholinergic functions under these conditions. Expression of A30P ?syn in six-months-old transgenic mice resulted in decreased tyrosine receptor kinase B expression, and lower cortical cholinergic fiber density. Dopamine-depletion by MPTP induced cholinergic cell loss in the NBM-SI and increased cortical fiber loss. Our findings may explain why cholinergic cells are more vulnerable in PD, leading to an increased probability of dementia.
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Assessment of the efficacy of solutes from extremophiles on protein aggregation in cell models of Huntingtons and Parkinsons diseases.
Neurochem. Res.
PUBLISHED: 03-02-2011
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Protein misfolding and deposition in the brain are implicated in the etiology of numerous neurodegenerative disorders. Here, organic solutes characteristic of microorganisms adapted to hot environments, were tested on experimental cell models of Huntingtons and Parkinsons diseases. Diglycerol phosphate, di-myo-inositol phosphate, mannosylglycerate, and mannosylglyceramide were not toxic to the cells, at 10 mM concentration, but caused a decrease in cell density, which suggested an effect on proliferation. In contrast, mannosyl-lactate, an artificial analogue of mannosylglycerate, had a negative impact on cell viability. Concerning protein aggregation, inclusions of mutant huntingtin were reduced in the presence of diglycerol phosphate and di-myo-inositol phosphate, increased with mannosylglycerate, while mannosyl-lactate and mannosylglyceramide had no significant effect. ?-Synuclein aggregation was not affected by the solutes tested, except for di-myo-inositol phosphate that led to a slight increased percentage of cells displaying visible aggregates. These solutes might be useful in the development of therapies for protein misfolding diseases.
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Visualization of cell-to-cell transmission of mutant huntingtin oligomers.
PLoS Curr
PUBLISHED: 02-04-2011
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We developed a new cell model for the visualization of toxic huntingtin oligomers in living cells. Huntingtin exon 1 (25Q or 103Q) was fused to non-fluorescent halves of the Venus protein. When huntingtin dimerizes inside the cells, Venus becomes functionally reconstituted and emits fluorescence. Oligomerization, aggregation and toxicity of mutant huntingtin were assessed by several procedures. We also present evidence that the transmission of huntingtin between cells can be determined in a quantitative manner with our model. Thus, this model can be a powerful screening tool for the identification of modifiers of oligomerization and cell-to-cell traffic of mutant huntingtin.
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?-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells.
J. Clin. Invest.
PUBLISHED: 01-18-2011
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Post-mortem analyses of brains from patients with Parkinson disease who received fetal mesencephalic transplants show that ?-synuclein-containing (?-syn-containing) Lewy bodies gradually appear in grafted neurons. Here, we explored whether intercellular transfer of ?-syn from host to graft, followed by seeding of ?-syn aggregation in recipient neurons, can contribute to this phenomenon. We assessed ?-syn cell-to-cell transfer using microscopy, flow cytometry, and high-content screening in several coculture model systems. Coculturing cells engineered to express either GFP- or DsRed-tagged ?-syn resulted in a gradual increase in double-labeled cells. Importantly, ?-syn-GFP derived from 1 neuroblastoma cell line localized to red fluorescent aggregates in other cells expressing DsRed-?-syn, suggesting a seeding effect of transmitted ?-syn. Extracellular ?-syn was taken up by cells through endocytosis and interacted with intracellular ?-syn. Next, following intracortical injection of recombinant ?-syn in rats, we found neuronal uptake was attenuated by coinjection of an endocytosis inhibitor. Finally, we demonstrated in vivo transfer of ?-syn between host cells and grafted dopaminergic neurons in mice overexpressing human ?-syn. In summary, intercellularly transferred ?-syn interacts with cytoplasmic ?-syn and can propagate ?-syn pathology. These results suggest that ?-syn propagation is a key element in the progression of Parkinson disease pathology.
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Functional gene expression profiling in yeast implicates translational dysfunction in mutant huntingtin toxicity.
J. Biol. Chem.
PUBLISHED: 11-02-2010
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Huntington disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (htt) protein. To uncover candidate therapeutic targets and networks involved in pathogenesis, we integrated gene expression profiling and functional genetic screening to identify genes critical for mutant htt toxicity in yeast. Using mRNA profiling, we have identified genes differentially expressed in wild-type yeast in response to mutant htt toxicity as well as in three toxicity suppressor strains: bna4?, mbf1?, and ume1?. BNA4 encodes the yeast homolog of kynurenine 3-monooxygenase, a promising drug target for HD. Intriguingly, despite playing diverse cellular roles, these three suppressors share common differentially expressed genes involved in stress response, translation elongation, and mitochondrial transport. We then systematically tested the ability of the differentially expressed genes to suppress mutant htt toxicity when overexpressed and have thereby identified 12 novel suppressors, including genes that play a role in stress response, Golgi to endosome transport, and rRNA processing. Integrating the mRNA profiling data and the genetic screening data, we have generated a robust network that shows enrichment in genes involved in rRNA processing and ribosome biogenesis. Strikingly, these observations implicate dysfunction of translation in the pathology of HD. Recent work has shown that regulation of translation is critical for life span extension in Drosophila and that manipulation of this process is protective in Parkinson disease models. In total, these observations suggest that pharmacological manipulation of translation may have therapeutic value in HD.
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Simple is good: yeast models of neurodegeneration.
FEMS Yeast Res.
PUBLISHED: 06-29-2010
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The budding yeast, Saccharomyces cerevisiae, is the best-studied eukaryotic cell, at both genetic and physiological levels. As a eukaryote, yeast shares highly conserved molecular and cellular mechanisms with human cells. Thus, this simple fungus is an invaluable model to study the fundamental molecular mechanisms involved in several human diseases. In the particular case of neurodegenerative disorders, yeast models have been able to recapitulate several important features of complex and devastating disorders, such as Huntingtons and Parkinsons diseases. Once validated, these models have also been used to accelerate the identification of both novel therapeutic targets and compounds with therapeutic potential. Here, we review the recent contributions of this simple, but powerful model organism toward our understanding of neurodegeneration.
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Epigenetics in neurodegeneration: a new layer of complexity.
Prog. Neuropsychopharmacol. Biol. Psychiatry
PUBLISHED: 03-30-2010
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Several diseases are known to have a multifactorial origin, depending not only on genetic but also on environmental factors. They are called "complex disorders" and include cardiovascular disease, cancer, diabetes, and neuropsychiatric and neurodegenerative diseases. In the latter class, Alzheimers (AD) and Parkinsons diseases (PD) are by far the most common in the elderly and constitute a tremendous social and economical problem. Both disorders present familial and sporadic forms and although some polymorphisms and risk factors have been associated with AD and PD, the precise way by which the environment contributes to neurodegeneration is still unclear. Recent studies suggest that environmental factors may contribute for neurodegeneration through induction of epigenetic modifications, such as DNA methylation, and chromatin remodeling, which may induce alterations in gene expression programs. Epigenetics, which refers to any process that alters gene activity without changing the actual DNA sequence, and leads to modifications that can be transmitted to daughter cells, is a relatively novel area of research that is currently attracting a high level of interest. Epigenetic modulation is present since the prenatal stages, and the aging process is now accepted to be associated with a loss of phenotypic plasticity to epigenetic modifications. Since aging is the most important risk factor for idiopathic AD and PD, it is expected that epigenetic alterations on DNA and/or chromatin structure may also accumulate in neurodegeneration, accounting at least in part to the etiology of these disorders.
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Synphilin-1 enhances ?-synuclein aggregation in yeast and contributes to cellular stress and cell death in a Sir2-dependent manner.
PLoS ONE
PUBLISHED: 03-10-2010
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Parkinsons disease is characterized by the presence of cytoplasmic inclusions, known as Lewy bodies, containing both aggregated ?-synuclein and its interaction partner, synphilin-1. While synphilin-1 is known to accelerate inclusion formation by ?-synuclein in mammalian cells, its effect on cytotoxicity remains elusive.
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The sour side of neurodegenerative disorders: the effects of protein glycation.
J. Pathol.
PUBLISHED: 02-27-2010
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Neurodegenerative diseases are associated with the misfolding and deposition of specific proteins, either intra- or extracellularly in the nervous system. Although familial mutations play an important role in protein misfolding and aggregation, the majority of cases of neurodegenerative diseases are sporadic, suggesting that other factors must contribute to the onset and progression of these disorders. Post-translational modifications are known to influence protein structure and function. Some of these modifications might affect proteins in detrimental ways and lead to their misfolding and accumulation. Reducing sugars play important roles in modifying proteins, forming advanced glycation end-products (AGEs) in a non-enzymatic process named glycation. Several proteins linked to neurodegenerative diseases, such as amyloid beta, tau, prions and transthyretin, were found to be glycated in patients, and this is thought to be associated with increased protein stability through the formation of crosslinks that stabilize protein aggregates. Moreover, glycation may be responsible, via the receptor for AGE (RAGE), for an increase in oxidative stress and inflammation through the formation of reactive oxygen species and the induction of NF-kappaB. Therefore, it is essential to unravel the molecular mechanisms underlying protein glycation to understand their role in neurodegeneration. Here, we reviewed the role of protein glycation in the major neurodegenerative disorders and highlight the potential value of protein glycation as a biomarker or target for therapeutic intervention.
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Zooming into protein oligomerization in neurodegeneration using BiFC.
Trends Biochem. Sci.
PUBLISHED: 02-23-2010
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Several neurodegenerative diseases are characterized by the accumulation of misfolded and aggregated proteins, which lead to neurotoxicity. However, the nature of those toxic species is controversial. Developments in optical microscopy and live-cell imaging are essential in providing crucial insight into the molecular mechanisms involved. In particular, the technique of bimolecular fluorescence complementation (BiFC) represents a remarkable improvement for observing protein-protein interactions within living cells. Unlike other techniques, BiFC provides spatial and temporal resolution and can be carried out in a physiological environment. Among other applications, BiFC has been used to study molecular determinants of oligomerization in neurodegenerative disorders, thereby promising to unveil novel targets for therapeutics. We review the applicability of BiFC for investigating the molecular basis of neurodegenerative diseases associated with protein misfolding and aggregation.
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Increased serum HSP70 levels are associated with the duration of diabetes.
Cell Stress Chaperones
PUBLISHED: 01-21-2010
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The evolutionary conserved family of heat shock proteins (HSP) is responsible for protecting cells against different types of stress, including oxidative stress. Although the levels of HSPs can be readily measured in blood serum, the levels of HSP70 in patients with different durations of diabetes have not been studied before. We quantified serum HSP70 levels in a healthy control group (n?=?36) and two groups of type 2 diabetic patients, defined as newly diagnosed diabetes (n?=?36) and patients with diabetes duration of more than 5 years (n?=?37). The clinical characteristics and biochemical parameters were evaluated in the studied population. We found that serum HSP70 levels were significantly higher in patients with diabetes when compared with controls (p?
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Sirtuins: common targets in aging and in neurodegeneration.
Curr Drug Targets
PUBLISHED: 01-18-2010
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Aging has been a subject of interest since primordial times. More recently, it became clear that aging is the major known risk factor for several neurodegenerative disorders, such as Alzheimers disease, Parkinsons disease and Huntingtons disease. A major focus in the field of aging is to examine whether the genetic regulators of lifespan also regulate the trigger and/or progression of age-related disorders. Sirtuins, which belong to the Sir2 family of NAD(+)-dependent deacetylases, are known to regulate longevity in yeast, worms, and flies. In mammals, there are seven homologs of the yeast Sir2, Sirt1-7. Therefore, the challenge now is to unravel howthe seven mammalian Sir2 proteins communicate to regulate the cross talk between aging and the onset and progression of age-related disorders. Here, we review how sirtuins contribute for aging and, in particular, for neurodegeneration and how they are becoming attractive targets for therapeutic intervention.
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Neurotrophic factors as a protective strategy in Parkinsons disease.
CNS Neurol Disord Drug Targets
PUBLISHED: 01-04-2010
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Neurodegenerative disorders are devastating human diseases that include Parkinsons, Huntingtons, Alzheimers, amyotrophic lateral sclerosis, and the frontal temporal dementias. Although the clinical manifestations of these disorders have been known for quite some time, our understanding of the molecular underpinnings is only starting to emerge. Protein misfolding and aggregation is a common hallmark among these diseases, and produce a number of cellular and functional alterations. The loss of dopaminergic neurons in the substantia nigra justified the use of dopaminergic therapies in patients. However, these strategies do not appear to confer disease-modifying effects, and do not prevent progression. The idea that neurotrophic factors might promote cell survival is an attractive one. Existing evidence from clinical trials is currently inconclusive, but some patients display clear clinical benefits. Thus, the current challenge is to develop novel strategies that make the use of neurotrophic factors more consistent.
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Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinsons disease models.
Dis Model Mech
PUBLISHED: 12-28-2009
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alpha-Synuclein (alpha-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because alpha-syn dysfunction is associated with several neurodegenerative disorders, including Parkinsons disease (PD). We previously created a yeast model of alpha-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to alpha-syn expression. We also uncovered a core group of proteins with diverse activities related to alpha-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of alpha-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress alpha-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of alpha-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced alpha-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of alpha-syn foci, re-established ER-to-Golgi trafficking and ameliorated alpha-syn-mediated damage to mitochondria. They also corrected the toxicity of alpha-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of alpha-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.
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Current and future therapeutic strategies for Parkinsons disease.
Curr. Pharm. Des.
PUBLISHED: 06-25-2009
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The heterogeneity of symptoms and disease progression observed in synucleinopathies, of which Parkinsons disease (PD) is the most common representative, poses large problems for its treatment and for the discovery of novel therapeutics. The molecular basis for pathology is currently unclear, both in familial and in sporadic cases. While the therapeutic effects of L-DOPA and dopamine receptor agonists are still the gold standards for symptomatic treatment in PD, the development of neuroprotective and/or neurorestorative treatments for these disorders faces significant challenges due to the poor knowledge of the putative targets involved. Recent experimental evidence strongly suggests a central role for neurotoxic alpha-synuclein oligomeric species in neurodegeneration. The events leading to protein oligomerization, as well as the oligomeric species themselves, are likely amenable to modulation by small molecules, which are beginning to emerge in high throughput compound screens in a variety of model organisms. The therapeutic potential of small molecule modulators of oligomer formation demands further exploration and validation in cellular and animal disease models in order to accelerate human drug development.
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Dopamine-induced conformational changes in alpha-synuclein.
PLoS ONE
PUBLISHED: 01-30-2009
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Oligomerization and aggregation of alpha-synuclein molecules play a major role in neuronal dysfunction and loss in Parkinsons disease [1]. However, alpha-synuclein oligomerization and aggregation have mostly been detected indirectly in cells using detergent extraction methods [2], [3], [4]. A number of in vitro studies showed that dopamine can modulate the aggregation of alpha-synuclein by inhibiting the formation of or by disaggregating amyloid fibrils [5], [6], [7].
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Live-cell imaging of p53 interactions using a novel Venus-based bimolecular fluorescence complementation system.
Biochem. Pharmacol.
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p53 plays an important role in regulating a wide variety of cellular processes, such as cell cycle arrest and/or apoptosis. Dysfunction of p53 is frequently associated with several pathologies, such as cancer and neurodegenerative diseases. In recent years substantial progress has been made in developing novel p53-activating molecules. Importantly, modulation of p53 interaction with its main inhibitor, Mdm2, has been highlighted as a promising therapeutic target. In this regard, bimolecular fluorescence complementation (BiFC) analysis, by providing direct visualization of protein interactions in living cells, offers a straightforward method to identify potential modulators of protein interactions. In this study, we developed a simple and robust Venus-based BiFC system to screen for modulators of p53-p53 and p53-Mdm2 interactions in live mammalian cells. We used nutlin-3, a well-known disruptor of p53-Mdm2 interaction, to validate the specificity of the assay. The reduction of BiFC signal mediated by nutlin-3 was correlated with an increase in Puma transactivation, PARP cleavage, and cell death. Finally, this novel BiFC approach was exploited to identify potential modulators of p53-Mdm2 complex formation among a commercially available chemical library of 33 protein phosphatase inhibitors. Our results constitute "proof-of-concept" that this model has strong potential as an alternative to traditional target-based drug discovery strategies. Identification of new modulators of p53-p53 and p53-Mdm2 interactions will be useful to achieve synergistic drug efficacy with currently used anti-tumor therapies.
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LRRK2 interactions with ?-synuclein in Parkinsons disease brains and in cell models.
J. Mol. Med.
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Mutations in the genes encoding leucine-rich repeat kinase 2 (LRRK2) and ?-synuclein are associated with both autosomal dominant and idiopathic forms of Parkinsons disease (PD). ?-Synuclein is the main protein in Lewy bodies, hallmark inclusions present in both sporadic and familial PD. We show that in PD brain tissue, the levels of LRRK2 are positively related to the increase in ?-synuclein phosphorylation and aggregation in affected brain regions (amygdala and anterior cingulate cortex), but not in the unaffected visual cortex. In disease-affected regions, we show co-localization of these two proteins in neurons and Lewy body inclusions. Further, in vitro experiments show a molecular interaction between ?-synuclein and LRRK2 under endogenous and over-expression conditions. In a cell culture model of ?-synuclein inclusion formation, LRRK2 co-localizes with the ?-synuclein inclusions, and knocking down LRRK2 increases the number of smaller inclusions. In addition to providing strong evidence for an interaction between LRRK2 and ?-synuclein, our results shed light on the complex relationship between these two proteins in the brains of patients with PD and the underlying molecular mechanisms of the disease.
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Parkinsons disease-associated mutations in DJ-1 modulate its dimerization in living cells.
J. Mol. Med.
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Mutations in the protein DJ-1 cause recessive forms of early onset familial Parkinsons disease (PD). To date, most of the causative mutations studied destabilize formation of DJ-1 homodimers, which appears to be closely linked to its normal function in oxidative stress and other cellular processes. Despite the importance of understanding the dimerization dynamics of this protein, this aspect of DJ-1 biology has not previously been directly studied in living cells. Here, we use bimolecular fluorescence complementation to study DJ-1 dimerization and find not only that DJ-1 forms homodimers in living cells but that most PD causative DJ-1 mutations disrupt this process, including the L166P, M26I, L10P, and P158? mutations. Interestingly, the E64D mutant form of DJ-1 retains the ability to form homodimers. However, while wild-type DJ-1 dimers are stabilized under oxidative stress conditions, we find that the E64D mutation blocks this stabilization. Furthermore, our data show that the E64D mutation potentiates the formation of aggresomes containing DJ-1. We also observe that while the widely studied L166P mutation prevents DJ-1 from forming homodimers or heterodimers with wild-type protein, the mutant protein is able to partially disrupt formation of wild-type homodimers. In summary, by investigating DJ-1 dimerization in living cells, we have uncovered several novel properties of PD causative mutations in DJ-1, which may ultimately provide novel insight into PD pathogenesis and possible therapeutic options.
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The zebrafish homologue of Parkinsons disease ATP13A2 is essential for embryonic survival.
Brain Res. Bull.
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ATP13A2 is a lysosome-specific transmembrane ATPase protein of unknown function. This protein was initially linked to Kufor-Rakeb syndrome where it is absent or mutated. More recently, point mutations in ATP13A2 were linked to familial cases of Parkinsons disease. Zebrafish is commonly used as a vertebrate model for the study of different neurodegenerative diseases and has homologues of several Parkinsons disease associated proteins. Here, we describe for the first time the zebrafish homologue of human ATP13A2, demonstrating the homology between the protein sequences, which supports a conserved biological role. Furthermore, the spatial pattern of protein expression was studied and the lethality of the knockdown of ATP13A2 suggests it plays a crucial role during embryonic development. Our findings bring new insight into the biology of ATP13A2 and open novel opportunities for its study using zebrafish as a model organism.
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Extracellular alpha-synuclein oligomers modulate synaptic transmission and impair LTP via NMDA-receptor activation.
J. Neurosci.
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Parkinsons disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of ?-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.
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SNCA (?-synuclein)-induced toxicity in yeast cells is dependent on sirtuin 2 (Sir2)-mediated mitophagy.
Autophagy
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SNCA (?-synuclein) misfolding and aggregation is strongly associated with both idiopathic and familial forms of Parkinson disease (PD). Evidence suggests that SNCA has an impact on cell clearance routes and protein quality control systems such as the ubiquitin-proteasome system (UPS) and autophagy. Recent advances in the key role of the autosomal recessive PARK2/PARKIN and PINK1 genes in mitophagy, highlighted this process as a prominent new pathogenic mechanism. Nevertheless, the role of autophagy/mitophagy in the pathogenesis of sporadic and autosomal dominant familial forms of PD is still enigmatic. The yeast Saccharomyces cerevisiae is a powerful "empty room" model that has been exploited to clarify different molecular aspects associated with SNCA toxicity, which combines the advantage of being an established system for aging research. The contribution of autophagy/mitophagy for the toxicity induced by the heterologous expression of the human wild-type SNCA gene and the clinical A53T mutant during yeast chronological life span (CLS) was explored. A reduced CLS together with an increase of autophagy and mitophagy activities were observed in cells expressing both forms of SNCA. Impairment of mitophagy by deletion of ATG11 or ATG32 resulted in a CLS extension, further implicating mitophagy in the SNCA toxicity. Deletion of SIR2, essential for SNCA toxicity, abolished autophagy and mitophagy, thereby rescuing cells. These data show that Sir2 functions as a regulator of autophagy, like its mammalian homolog, SIRT1, but also of mitophagy. Our work highlights that increased mitophagy activity, mediated by the regulation of ATG32 by Sir2, is an important phenomenon linked to SNCA-induced toxicity during aging.
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Aggregate clearance of ?-synuclein in Saccharomyces cerevisiae depends more on autophagosome and vacuole function than on the proteasome.
J. Biol. Chem.
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Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated ?-synuclein. The molecular mechanism of clearance of ?-synuclein aggregates was addressed using the bakers yeast Saccharomyces cerevisiae as the model. Overexpression of wild type ?-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The ?-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an ?atg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1?atg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for ?-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of ?-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.
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High-throughput study of alpha-synuclein expression in yeast using microfluidics for control of local cellular microenvironment.
Biomicrofluidics
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Microfluidics is an emerging technology which allows the miniaturization, integration, and automation of fluid handling processes. Microfluidic systems offer low sample consumption, significantly reduced processing time, and the prospect of massive parallelization. A microfluidic platform was developed for the control of the soluble cellular microenvironment of Saccharomyces cerevisiae cells, which enabled high-throughput monitoring of the controlled expression of alpha-synuclein (aSyn), a protein involved in Parkinsons disease. Y-shaped structures were fabricated using particle desorption mass spectrometry-based soft-lithography techniques to generate biomolecular gradients along a microchannel. Cell traps integrated along the microchannel allowed the positioning and monitoring of cells in precise locations, where different, well-controlled chemical environments were established. S. cerevisiae cells genetically engineered to encode the fusion protein aSyn-GFP (green fluorescent protein) under the control of GAL1, a galactose inducible promoter, were loaded in the microfluidic structure. A galactose concentration gradient was established in the channel and a time-dependent aSyn-GFP expression was obtained as a function of the positioning of cells along the galactose gradient. Our results demonstrate the applicability of this microfluidic platform to the spatiotemporal control of cellular microenvironment and open a range of possibilities for the study of cellular processes based on single-cell analysis.
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Highlights of the Keystone Symposium: sirtuins in metabolism, aging and disease.
EMBO Mol Med
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From February 12-16, 2012, leading members of the sirtuin scientific community assembled in Tahoe, CA to attend the Keystone Symposium "Sirtuins in Aging, Metabolism, and Disease." It was a vibrant and lively meeting, and in the spirit of Keystone Symposia, both established sirtuin researchers and those new to the field enjoyed a unique opportunity to interact and exchange ideas.
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Impairment of the septal cholinergic neurons in MPTP-treated A30P ?-synuclein mice.
Neurobiol. Aging
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Dementia in Parkinsons disease (PDD) and dementia with Lewy bodies (DLB) are characterized by loss of acetylcholine (ACh) from cortical areas. Clinical studies report positive effects of acetylcholine esterase (AChE) inhibitors in PDD and dementia with Lewy bodies. We here report that the number of neurons expressing a cholinergic marker in the medial septum-diagonal band of Broca complex decreases in A30P ?-synuclein-expressing mice during aging, paralleled by a lower AChE fiber density in the dentate gyrus and in the hippocampal CA1 field. After inducing dopamine depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), no acute but a delayed loss of cholinergic neurons and AChE-positive fibers was observed, which was attenuated by L-3,4-dihydroxyphenylalanine (DOPA) treatment. Expression of nerve growth factor (NGF) and tyrosine receptor kinase A (TrkA) genes was upregulated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride-treated wild type mice, but not in A30P ?-synuclein expressing animals. In contrast, upregulation of sortilin and p75(NTR) genes was found in the A30P ?-synuclein-expressing mice. These results suggest that dopamine deficiency may contribute to the impairment of the septohippocampal system in patients with PDD and that L-3,4-dihydroxyphenylalanine may not only result in symptomatic treatment of the akinetic-rigid syndrome but may also alleviate the degeneration of basal forebrain cholinergic system and the cognitive decline.
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SIRT2 as a Therapeutic Target for Age-Related Disorders.
Front Pharmacol
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Sirtuin proteins are conserved regulators of aging that have recently emerged as important modifiers of several diseases which commonly occur later in life such as cancer, diabetes, cardiovascular, and neurodegenerative diseases. In mammals, there are seven sirtuins (SIRT1-7), which display diversity in subcellular localization and function. SIRT1 has received much of attention due to its possible impact on longevity, while important biological and therapeutic roles of other sirtuins have been underestimated and just recently recognized. Here we focus on SIRT2, a member of the sirtuin family, and discuss its role in cellular and tissue-specific functions. This review summarizes the main scientific advances on SIRT2 protein biology and explores its potential as a therapeutic target for treatment of age-related disorders.
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Suppression of ?-synuclein toxicity and vesicle trafficking defects by phosphorylation at S129 in yeast depends on genetic context.
Hum. Mol. Genet.
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The aggregation of ?-synuclein (?Syn) is a neuropathologic hallmark of Parkinsons disease and other synucleinopathies. In Lewy bodies, ?Syn is extensively phosphorylated, predominantly at serine 129 (S129). Recent studies in yeast have shown that, at toxic levels, ?Syn disrupts Rab homeostasis, causing an initial endoplasmic reticulum-to-Golgi block that precedes a generalized trafficking collapse. However, whether ?Syn phosphorylation modulates trafficking defects has not been evaluated. Here, we show that constitutive expression of ?Syn in yeast impairs late-exocytic, early-endocytic and/or recycling trafficking. Although members of the casein kinase I (CKI) family phosphorylate ?Syn at S129, they attenuate ?Syn toxicity and trafficking defects by an S129 phosphorylation-independent mechanism. Surprisingly, phosphorylation of S129 modulates ?Syn toxicity and trafficking defects in a manner strictly determined by genetic background. Abnormal endosome morphology, increased levels of the endosome marker Rab5 and co-localization of mammalian CKI with ?Syn aggregates are observed in brain sections from ?Syn-overexpressing mice and human synucleinopathies. Our results contribute to evidence that suggests ?Syn-induced defects in endocytosis, exocytosis and/or recycling of vesicles involved in these cellular processes might contribute to the pathogenesis of synucleinopathies.
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Imaging protein oligomerization in neurodegeneration using bimolecular fluorescence complementation.
Meth. Enzymol.
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Neurodegenerative disorders such as Alzheimers, Parkinsons, Huntingtons, or Prion diseases belong to a superfamily of pathologies known as protein misfolding disorders. The hallmark of these pathologies is the aberrant accumulation of specific proteins in beta sheet-rich amyloid aggregates either inside or outside cells. Current evidence suggests that oligomeric species, rather than mature protein aggregates, are the most toxic forms of the pathogenic proteins. This is due, at least in part, to their greater solubility and ability to diffuse between intracellular and extracellular compartments. Understanding how oligomerization occurs is essential for the development of new treatments for this group of diseases. Bimolecular fluorescence complementation assays (BiFC) have proved to be excellent systems to study aberrant protein-protein interactions, including those involved in neurodegenerative diseases. Here, we provide a detailed description of the rationale to develop and validate BiFC assays for the visualization of oligomeric species in living cells in the context of neurodegeneration. These systems could constitute powerful tools for the identification of genetic and pharmacological modifiers of protein misfolding and aggregation.
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