Hugo Vicente Miranda | Universidade Nova de Lisboa (original) (raw)

Papers by Hugo Vicente Miranda

Journal of Heterocyclic Chemistry, Dec 29, 2015

Here, we describe the synthesis and preliminary biological evaluation of novel N-unsubstituted an... more Here, we describe the synthesis and preliminary biological evaluation of novel N-unsubstituted and N-methylated 2-aryl benzimidazole derivatives that contain fluorinated or hydroxylated alkyl substituents in the 4-N-aryl position and different substitution patterns (H vs Br vs I) in the benzimidazole ring. For the selected compounds and for comparison purposes, the congener benzothiazoles were also tested. The cytotoxic effect of 11 benzazole derivatives was evaluated in a panel of human cancer cell lines, such as breast (MCF7), melanoma (A375), cervix (HeLa), and glioblastoma (U87). In general, the compounds exerted a moderate cytotoxic activity against all cells tested. In particular, for the A375 and HeLa cells, the N-unsubstituted benzimidazoles 2 and 3 displayed a better cytotoxic profile than the respective N-methylated benzimidazole congeners (5 and 7). The biodistribution of compound 2, which has shown the highest cytotoxic activity active in the U87 glioblastoma cells (IC50...

FEBS Journal, Oct 25, 2006

Protein glycation is a post-translational modification whereby amino groups in arginine and lysin... more Protein glycation is a post-translational modification whereby amino groups in arginine and lysine side chains react irreversibly with carbonyl molecules, forming advanced glycation end-products (AGEs). Glycation is equivalent to a point mutation, exerting profound effects on protein structure, stability and function. AGE formation in proteins is associated with the clinical complications of diabetes mellitus [1], cataracts [2], uremia [3], atherosclerosis [4] and agerelated disorders [5]. Glycated proteins are present in b-amyloid deposits and s deposits in Alzheimer's disease [6-8], in Lewy inclusion bodies of a-sinuclein in

Brain, 2021

The publisher apologizes for providing incomplete Supplementary material for awx056. The missing ... more The publisher apologizes for providing incomplete Supplementary material for awx056. The missing files have been uploaded.

Journal of Parkinson's disease, 2018

Parkinson's disease (PD) is a neurodegenerative disorder with complex etiology and variable ... more Parkinson's disease (PD) is a neurodegenerative disorder with complex etiology and variable pathology. While a subset of cases is associated with single-gene mutations, the majority originates from a combination of factors we do not fully understand. Thus, understanding the underlying causes of PD is indispensable for the development of novel therapeutics. Glycation, the non-enzymatic reaction between reactive dicarbonyls and amino groups, gives rise to a variety of different reaction products known as advanced glycation end products (AGEs). AGEs accumulate over a proteins life-time, and increased levels of glycation reaction products play a role in diabetic complications. It is now also becoming evident that PD patients also display perturbed sugar metabolism and protein glycation, including that of alpha-synuclein, a key player in PD. Here, we hypothesize that anti-diabetic drugs targeting the levels of glycation precursors, or promoting the clearance of glycated proteins may...

Scientific reports, Jan 20, 2017

Parkinson's disease (PD) is a progressive neurodegenerative disorder known for the typical mo... more Parkinson's disease (PD) is a progressive neurodegenerative disorder known for the typical motor features associated. Pathologically, it is characterized by the intracellular accumulation of alpha-synuclein (aSyn) in Lewy bodies and Lewy neurites. Currently, there are no established biochemical markers for diagnosing or for following disease progression, a major limitation for the clinical practice. Posttranslational modifications (PTMs) in aSyn have been identified and implicated on its pathobiology. Since aSyn is abundant in blood erythrocytes, we aimed to evaluate whether PTMs of aSyn in the blood might hold value as a biomarker for PD. We examined 58 patients with PD and 30 healthy age-matched individuals. We found that the levels of Y125 phosphorylated, Y39 nitrated, and glycated aSyn were increased in PD, while those of SUMO were reduced. A combinatory analysis of the levels of these PTMs resulted in an increased sensitivity, with an area under curve (AUC) of 0.843 for PD ...

PLoS biology, Mar 1, 2017

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways.... more Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation a...

Scientific Reports, 2016

Protein glycation is an age-dependent posttranslational modification associated with several neur... more Protein glycation is an age-dependent posttranslational modification associated with several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. By modifying amino-groups, glycation interferes with folding of proteins, increasing their aggregation potential. Here, we studied the effect of pharmacological and genetic manipulation of glycation on huntingtin (HTT), the causative protein in Huntington’s disease (HD). We observed that glycation increased the aggregation of mutant HTT exon 1 fragments associated with HD (HTT72Q and HTT103Q) in yeast and mammalian cell models. We found that glycation impairs HTT clearance thereby promoting its intracellular accumulation and aggregation. Interestingly, under these conditions autophagy increased and the levels of mutant HTT released to the culture medium decreased. Furthermore, increased glycation enhanced HTT toxicity in human cells and neurodegeneration in fruit flies, impairing eclosion and decreasing life span. O...

Movement disorders : official journal of the Movement Disorder Society, Jun 4, 2016

Glycation is a spontaneous age-dependent posttranslational modification that can impact the struc... more Glycation is a spontaneous age-dependent posttranslational modification that can impact the structure and function of several proteins. Interestingly, glycation can be detected at the periphery of Lewy bodies in the brain in Parkinson's disease. Moreover, α-synuclein can be glycated, at least under experimental conditions. In Alzheimer's disease, glycation of amyloid β peptide exacerbates its toxicity and contributes to neurodegeneration. Recent studies establish diabetes mellitus as a risk factor for several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. However, the mechanisms underlying this connection remain unclear. We hypothesize that hyperglycemia might play an important role in the development of these disorders, possibly by also inducing protein glycation and thereby dysfunction, aggregation, and deposition. Here, we explore protein glycation as a common player in Parkinson's and Alzheimer's diseases and propose it may c...

npj Parkinson's Disease

Alpha-synuclein (aSyn) is a central player in the pathogenesis of synucleinopathies due to its ac... more Alpha-synuclein (aSyn) is a central player in the pathogenesis of synucleinopathies due to its accumulation in typical protein aggregates in the brain. However, it is still unclear how it contributes to neurodegeneration. Type-2 diabetes mellitus is a risk factor for Parkinson’s disease (PD). Interestingly, a common molecular alteration among these disorders is the age-associated increase in protein glycation. We hypothesized that glycation-induced neuronal dysfunction is a contributing factor in synucleinopathies. Here, we dissected the impact of methylglyoxal (MGO, a glycating agent) in mice overexpressing aSyn in the brain. We found that MGO-glycation potentiates motor, cognitive, olfactory, and colonic dysfunction in aSyn transgenic (Thy1-aSyn) mice that received a single dose of MGO via intracerebroventricular injection. aSyn accumulates in the midbrain, striatum, and prefrontal cortex, and protein glycation is increased in the cerebellum and midbrain. SWATH mass spectrometry a...

Journal of Neurology, Neurosurgery & Psychiatry, 2016

Protein glycation is an age-dependent posttranslational modification associated with several neur... more Protein glycation is an age-dependent posttranslational modification associated with several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. By modifying amino-groups, glycation interferes with protein folding, increasing their aggregation potential. Here, we studied the effect of pharmacological and genetic manipulation of glycation on huntingtin (HTT), the causative protein in Huntington’s disease (HD). We observed that glycation increased the aggregation of mutant HTT exon 1 fragments associated with HD (HTT72Q and HTT103Q) in yeast and mammalian cell models. We showed that glycation impairs HTT clearance thereby promoting its intracellular accumulation and aggregation. Interestingly, autophagy increased and the levels of HTT released to the culture medium decreased. Glycation enhanced HTT toxicity in human cells and neurodegeneration in fruit flies, impairing eclosion and decreasing life span. Overall, our study provides evidence that glycation modulates HTT exon-1 aggregation and toxicity, and suggests it may constitute a novel target for therapeutic intervention in HD.

The Journal of Pathology, 2009

Neurodegenerative diseases are associated with the misfolding and deposition of specific proteins... more Neurodegenerative diseases are associated with the misfolding and deposition of specific proteins, either intraor 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 β, 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-κB. 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.

The FASEB Journal

This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmer... more This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Alpha-synuclein (aSyn) is assumed to be a central player in the pathogenesis of synucleinopathies... more Alpha-synuclein (aSyn) is assumed to be a central player in the pathogenesis of synucleinopathies due to its accumulation in typical protein aggregates in the brain. However, it is still unclear how it contributes to neurodegeneration. Type-2 diabetes mellitus is a risk factor for Parkinson’s disease and, one common molecular alteration among these disorders is an age-associated increase in protein glycation. Thus, we hypothesized that glycation-induced dysfunction of neuronal pathways might be an underlying molecular cause of synucleinopathies. Here, we evaluated if increased brain glycation modulated motor and/or non-motor phenotypes in a mouse model of synucleinopathies. In addition, we dissected the specific impact of methylglyoxal (MGO, a glycating agent) in mice overexpressing aSyn in the brain, and unveiled the major molecular pathways altered. Age-matched (16 weeks old) male aSyn transgenic (Thy1-aSyn) or WT mice received a single dose of MGO or vehicle via intracerebroventr...

Frontiers in Pharmacology

Scientific Reports

Cell-to-cell propagation of aggregated alpha synuclein (aSyn) has been suggested to play an impor... more Cell-to-cell propagation of aggregated alpha synuclein (aSyn) has been suggested to play an important role in the progression of alpha synucleinopathies. A critical step for the propagation process is the accumulation of extracellular aSyn within recipient cells. Here, we investigated the trafficking of distinct exogenous aSyn forms and addressed the mechanisms influencing their accumulation in recipient cells. The aggregated aSyn species (oligomers and fibrils) exhibited more pronounced accumulation within recipient cells than aSyn monomers. In particular, internalized extracellular aSyn in the aggregated forms was able to seed the aggregation of endogenous aSyn. Following uptake, aSyn was detected along endosome-to-lysosome and autophagosome-to-lysosome routes. Intriguingly, aggregated aSyn resulted in lysosomal activity impairment, accompanied by the accumulation of dilated lysosomes. Moreover, analysis of autophagy-related protein markers suggested decreased autophagosome clearance. In contrast, the endocytic pathway, proteasome activity, and mitochondrial homeostasis were not substantially affected in recipient cells. Our data suggests that extracellularly added aggregated aSyn primarily impairs lysosomal activity, consequently leading to aSyn accumulation within recipient cells. Importantly, the autophagy inducer trehalose prevented lysosomal alterations and attenuated aSyn accumulation within aSyn-exposed cells. Our study underscores the importance of lysosomes for the propagation of aSyn pathology, thereby proposing these organelles as interventional targets. Alpha synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy, are characterized by intracellular deposition of alpha synuclein (aSyn) 1-3. It is widely accepted that abnormal aggregation of aSyn, a physiologically soluble protein with a molecular weight of 14 kDa, contributes to the neurodegeneration in alpha synucleinopahties. Current knowledge about aSyn aggregation suggests that aSyn monomers are first assembled into oligomers and subsequently into β-sheet-rich amyloid fibrils 2,4. Amyloid fibrils are finally deposited along with other components, forming inclusions, such as the Lewy bodies. In addition to pathological aSyn aggregation, mitochondrial dysfunction and impaired protein degradation pathways, including the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system, have been linked to the neurodegeneration in alpha synucleinopathies 5-7. Moreover, cell-to-cell propagation of pathogenic aSyn was recently suggested to be a mechanism contributing to the progression of alpha synucleinopathies. The propagation hypothesis was initially based on the clinical and neuropathological findings that (1) aSyn was detected in blood plasma and cerebrospinal fluid 8,9 ; (2) the distribution of aggregated aSyn in postmortem brains

Nature Neuroscience

Synucleinopathies, such as Parkinson&... more Synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, are neurodegenerative disorders that are characterized by the accumulation of α-synuclein (aSyn) in intracellular inclusions known as Lewy bodies. Prefibrillar soluble aSyn oligomers, rather than larger inclusions, are currently considered to be crucial species underlying synaptic dysfunction. We identified the cellular prion protein (PrP(C)) as a key mediator in aSyn-induced synaptic impairment. The aSyn-associated impairment of long-term potentiation was blocked in Prnp null mice and rescued following PrP(C) blockade. We found that extracellular aSyn oligomers formed a complex with PrP(C) that induced the phosphorylation of Fyn kinase via metabotropic glutamate receptors 5 (mGluR5). aSyn engagement of PrP(C) and Fyn activated NMDA receptor (NMDAR) and altered calcium homeostasis. Blockade of mGluR5-evoked phosphorylation of NMDAR in aSyn transgenic mice rescued synaptic and cognitive deficits, supporting the hypothesis that a receptor-mediated mechanism, independent of pore formation and membrane leakage, is sufficient to trigger early synaptic damage induced by extracellular aSyn.

PLoS biology, 2017

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways.... more Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with ageassociated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.

Metabolism, the set of all chemical transformations inside a living cell, comprises nonenzymatic ... more Metabolism, the set of all chemical transformations inside a living cell, comprises nonenzymatic processes that generate toxic products such as reactive oxygen species and 2-oxoaldehydes. Methylglyoxal, a highly reactive 2-oxoaldehyde by-product of glycolysis, is able to react irreversibly and nonenzymatically with proteins, forming methylglyoxal advanced glycation end-products, which alter protein structure, stability and function. Therefore, protein glycation may influence cell metabolism and its physiology in a way beyond what can be predicted based on the implicit codification used in systems biology. Genome-wide approaches and transcriptomics, two mainstays of systems biology, are powerless to tackle the problems caused by nonenzymatic reactions that are part of cell metabolism and biochemistry. The effects of methylglyoxal-derived protein glycation and the cell's response to this unspecific posttranslational modification were investigated in Saccharomyces cerevisiae as a model organism. Specific protein glycation phenotypes were identified using yeast null-mutants for methylglyoxal catabolism and the existence of specific protein glycation targets by peptide mass fingerprint was discovered. Enolase, the major target, endures a glycation-dependent activity loss caused by dissociation of the active dimer upon glycation at a specific arginine residue, identified using the hidden information of peptide mass fingerprint. Once glycation occurs, a cellular response involving heat shock proteins from the refolding chaperone pathway is elicited and Hsp26p is activated by glycation.

Neurobiology of Aging, 2012

Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal plasticity, learning... more 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 Alzheimer's disease. We show here that incubation with amyloid-β 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 Alzheimer's 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-β 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 Alzheimer's disease.

Brain : a journal of neurology, Jan 10, 2017

α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several ot... more α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes...

Journal of Heterocyclic Chemistry, Dec 29, 2015

Here, we describe the synthesis and preliminary biological evaluation of novel N-unsubstituted an... more Here, we describe the synthesis and preliminary biological evaluation of novel N-unsubstituted and N-methylated 2-aryl benzimidazole derivatives that contain fluorinated or hydroxylated alkyl substituents in the 4-N-aryl position and different substitution patterns (H vs Br vs I) in the benzimidazole ring. For the selected compounds and for comparison purposes, the congener benzothiazoles were also tested. The cytotoxic effect of 11 benzazole derivatives was evaluated in a panel of human cancer cell lines, such as breast (MCF7), melanoma (A375), cervix (HeLa), and glioblastoma (U87). In general, the compounds exerted a moderate cytotoxic activity against all cells tested. In particular, for the A375 and HeLa cells, the N-unsubstituted benzimidazoles 2 and 3 displayed a better cytotoxic profile than the respective N-methylated benzimidazole congeners (5 and 7). The biodistribution of compound 2, which has shown the highest cytotoxic activity active in the U87 glioblastoma cells (IC50...

FEBS Journal, Oct 25, 2006

Protein glycation is a post-translational modification whereby amino groups in arginine and lysin... more Protein glycation is a post-translational modification whereby amino groups in arginine and lysine side chains react irreversibly with carbonyl molecules, forming advanced glycation end-products (AGEs). Glycation is equivalent to a point mutation, exerting profound effects on protein structure, stability and function. AGE formation in proteins is associated with the clinical complications of diabetes mellitus [1], cataracts [2], uremia [3], atherosclerosis [4] and agerelated disorders [5]. Glycated proteins are present in b-amyloid deposits and s deposits in Alzheimer's disease [6-8], in Lewy inclusion bodies of a-sinuclein in

Brain, 2021

The publisher apologizes for providing incomplete Supplementary material for awx056. The missing ... more The publisher apologizes for providing incomplete Supplementary material for awx056. The missing files have been uploaded.

Journal of Parkinson's disease, 2018

Parkinson's disease (PD) is a neurodegenerative disorder with complex etiology and variable ... more Parkinson's disease (PD) is a neurodegenerative disorder with complex etiology and variable pathology. While a subset of cases is associated with single-gene mutations, the majority originates from a combination of factors we do not fully understand. Thus, understanding the underlying causes of PD is indispensable for the development of novel therapeutics. Glycation, the non-enzymatic reaction between reactive dicarbonyls and amino groups, gives rise to a variety of different reaction products known as advanced glycation end products (AGEs). AGEs accumulate over a proteins life-time, and increased levels of glycation reaction products play a role in diabetic complications. It is now also becoming evident that PD patients also display perturbed sugar metabolism and protein glycation, including that of alpha-synuclein, a key player in PD. Here, we hypothesize that anti-diabetic drugs targeting the levels of glycation precursors, or promoting the clearance of glycated proteins may...

Scientific reports, Jan 20, 2017

Parkinson's disease (PD) is a progressive neurodegenerative disorder known for the typical mo... more Parkinson's disease (PD) is a progressive neurodegenerative disorder known for the typical motor features associated. Pathologically, it is characterized by the intracellular accumulation of alpha-synuclein (aSyn) in Lewy bodies and Lewy neurites. Currently, there are no established biochemical markers for diagnosing or for following disease progression, a major limitation for the clinical practice. Posttranslational modifications (PTMs) in aSyn have been identified and implicated on its pathobiology. Since aSyn is abundant in blood erythrocytes, we aimed to evaluate whether PTMs of aSyn in the blood might hold value as a biomarker for PD. We examined 58 patients with PD and 30 healthy age-matched individuals. We found that the levels of Y125 phosphorylated, Y39 nitrated, and glycated aSyn were increased in PD, while those of SUMO were reduced. A combinatory analysis of the levels of these PTMs resulted in an increased sensitivity, with an area under curve (AUC) of 0.843 for PD ...

PLoS biology, Mar 1, 2017

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways.... more Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation a...

Scientific Reports, 2016

Protein glycation is an age-dependent posttranslational modification associated with several neur... more Protein glycation is an age-dependent posttranslational modification associated with several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. By modifying amino-groups, glycation interferes with folding of proteins, increasing their aggregation potential. Here, we studied the effect of pharmacological and genetic manipulation of glycation on huntingtin (HTT), the causative protein in Huntington’s disease (HD). We observed that glycation increased the aggregation of mutant HTT exon 1 fragments associated with HD (HTT72Q and HTT103Q) in yeast and mammalian cell models. We found that glycation impairs HTT clearance thereby promoting its intracellular accumulation and aggregation. Interestingly, under these conditions autophagy increased and the levels of mutant HTT released to the culture medium decreased. Furthermore, increased glycation enhanced HTT toxicity in human cells and neurodegeneration in fruit flies, impairing eclosion and decreasing life span. O...

Movement disorders : official journal of the Movement Disorder Society, Jun 4, 2016

Glycation is a spontaneous age-dependent posttranslational modification that can impact the struc... more Glycation is a spontaneous age-dependent posttranslational modification that can impact the structure and function of several proteins. Interestingly, glycation can be detected at the periphery of Lewy bodies in the brain in Parkinson's disease. Moreover, α-synuclein can be glycated, at least under experimental conditions. In Alzheimer's disease, glycation of amyloid β peptide exacerbates its toxicity and contributes to neurodegeneration. Recent studies establish diabetes mellitus as a risk factor for several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. However, the mechanisms underlying this connection remain unclear. We hypothesize that hyperglycemia might play an important role in the development of these disorders, possibly by also inducing protein glycation and thereby dysfunction, aggregation, and deposition. Here, we explore protein glycation as a common player in Parkinson's and Alzheimer's diseases and propose it may c...

npj Parkinson's Disease

Alpha-synuclein (aSyn) is a central player in the pathogenesis of synucleinopathies due to its ac... more Alpha-synuclein (aSyn) is a central player in the pathogenesis of synucleinopathies due to its accumulation in typical protein aggregates in the brain. However, it is still unclear how it contributes to neurodegeneration. Type-2 diabetes mellitus is a risk factor for Parkinson’s disease (PD). Interestingly, a common molecular alteration among these disorders is the age-associated increase in protein glycation. We hypothesized that glycation-induced neuronal dysfunction is a contributing factor in synucleinopathies. Here, we dissected the impact of methylglyoxal (MGO, a glycating agent) in mice overexpressing aSyn in the brain. We found that MGO-glycation potentiates motor, cognitive, olfactory, and colonic dysfunction in aSyn transgenic (Thy1-aSyn) mice that received a single dose of MGO via intracerebroventricular injection. aSyn accumulates in the midbrain, striatum, and prefrontal cortex, and protein glycation is increased in the cerebellum and midbrain. SWATH mass spectrometry a...

Journal of Neurology, Neurosurgery & Psychiatry, 2016

Protein glycation is an age-dependent posttranslational modification associated with several neur... more Protein glycation is an age-dependent posttranslational modification associated with several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. By modifying amino-groups, glycation interferes with protein folding, increasing their aggregation potential. Here, we studied the effect of pharmacological and genetic manipulation of glycation on huntingtin (HTT), the causative protein in Huntington’s disease (HD). We observed that glycation increased the aggregation of mutant HTT exon 1 fragments associated with HD (HTT72Q and HTT103Q) in yeast and mammalian cell models. We showed that glycation impairs HTT clearance thereby promoting its intracellular accumulation and aggregation. Interestingly, autophagy increased and the levels of HTT released to the culture medium decreased. Glycation enhanced HTT toxicity in human cells and neurodegeneration in fruit flies, impairing eclosion and decreasing life span. Overall, our study provides evidence that glycation modulates HTT exon-1 aggregation and toxicity, and suggests it may constitute a novel target for therapeutic intervention in HD.

The Journal of Pathology, 2009

Neurodegenerative diseases are associated with the misfolding and deposition of specific proteins... more Neurodegenerative diseases are associated with the misfolding and deposition of specific proteins, either intraor 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 β, 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-κB. 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.

The FASEB Journal

This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmer... more This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Alpha-synuclein (aSyn) is assumed to be a central player in the pathogenesis of synucleinopathies... more Alpha-synuclein (aSyn) is assumed to be a central player in the pathogenesis of synucleinopathies due to its accumulation in typical protein aggregates in the brain. However, it is still unclear how it contributes to neurodegeneration. Type-2 diabetes mellitus is a risk factor for Parkinson’s disease and, one common molecular alteration among these disorders is an age-associated increase in protein glycation. Thus, we hypothesized that glycation-induced dysfunction of neuronal pathways might be an underlying molecular cause of synucleinopathies. Here, we evaluated if increased brain glycation modulated motor and/or non-motor phenotypes in a mouse model of synucleinopathies. In addition, we dissected the specific impact of methylglyoxal (MGO, a glycating agent) in mice overexpressing aSyn in the brain, and unveiled the major molecular pathways altered. Age-matched (16 weeks old) male aSyn transgenic (Thy1-aSyn) or WT mice received a single dose of MGO or vehicle via intracerebroventr...

Frontiers in Pharmacology

Scientific Reports

Cell-to-cell propagation of aggregated alpha synuclein (aSyn) has been suggested to play an impor... more Cell-to-cell propagation of aggregated alpha synuclein (aSyn) has been suggested to play an important role in the progression of alpha synucleinopathies. A critical step for the propagation process is the accumulation of extracellular aSyn within recipient cells. Here, we investigated the trafficking of distinct exogenous aSyn forms and addressed the mechanisms influencing their accumulation in recipient cells. The aggregated aSyn species (oligomers and fibrils) exhibited more pronounced accumulation within recipient cells than aSyn monomers. In particular, internalized extracellular aSyn in the aggregated forms was able to seed the aggregation of endogenous aSyn. Following uptake, aSyn was detected along endosome-to-lysosome and autophagosome-to-lysosome routes. Intriguingly, aggregated aSyn resulted in lysosomal activity impairment, accompanied by the accumulation of dilated lysosomes. Moreover, analysis of autophagy-related protein markers suggested decreased autophagosome clearance. In contrast, the endocytic pathway, proteasome activity, and mitochondrial homeostasis were not substantially affected in recipient cells. Our data suggests that extracellularly added aggregated aSyn primarily impairs lysosomal activity, consequently leading to aSyn accumulation within recipient cells. Importantly, the autophagy inducer trehalose prevented lysosomal alterations and attenuated aSyn accumulation within aSyn-exposed cells. Our study underscores the importance of lysosomes for the propagation of aSyn pathology, thereby proposing these organelles as interventional targets. Alpha synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy, are characterized by intracellular deposition of alpha synuclein (aSyn) 1-3. It is widely accepted that abnormal aggregation of aSyn, a physiologically soluble protein with a molecular weight of 14 kDa, contributes to the neurodegeneration in alpha synucleinopahties. Current knowledge about aSyn aggregation suggests that aSyn monomers are first assembled into oligomers and subsequently into β-sheet-rich amyloid fibrils 2,4. Amyloid fibrils are finally deposited along with other components, forming inclusions, such as the Lewy bodies. In addition to pathological aSyn aggregation, mitochondrial dysfunction and impaired protein degradation pathways, including the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system, have been linked to the neurodegeneration in alpha synucleinopathies 5-7. Moreover, cell-to-cell propagation of pathogenic aSyn was recently suggested to be a mechanism contributing to the progression of alpha synucleinopathies. The propagation hypothesis was initially based on the clinical and neuropathological findings that (1) aSyn was detected in blood plasma and cerebrospinal fluid 8,9 ; (2) the distribution of aggregated aSyn in postmortem brains

Nature Neuroscience

Synucleinopathies, such as Parkinson&... more Synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, are neurodegenerative disorders that are characterized by the accumulation of α-synuclein (aSyn) in intracellular inclusions known as Lewy bodies. Prefibrillar soluble aSyn oligomers, rather than larger inclusions, are currently considered to be crucial species underlying synaptic dysfunction. We identified the cellular prion protein (PrP(C)) as a key mediator in aSyn-induced synaptic impairment. The aSyn-associated impairment of long-term potentiation was blocked in Prnp null mice and rescued following PrP(C) blockade. We found that extracellular aSyn oligomers formed a complex with PrP(C) that induced the phosphorylation of Fyn kinase via metabotropic glutamate receptors 5 (mGluR5). aSyn engagement of PrP(C) and Fyn activated NMDA receptor (NMDAR) and altered calcium homeostasis. Blockade of mGluR5-evoked phosphorylation of NMDAR in aSyn transgenic mice rescued synaptic and cognitive deficits, supporting the hypothesis that a receptor-mediated mechanism, independent of pore formation and membrane leakage, is sufficient to trigger early synaptic damage induced by extracellular aSyn.

PLoS biology, 2017

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways.... more Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with ageassociated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.

Metabolism, the set of all chemical transformations inside a living cell, comprises nonenzymatic ... more Metabolism, the set of all chemical transformations inside a living cell, comprises nonenzymatic processes that generate toxic products such as reactive oxygen species and 2-oxoaldehydes. Methylglyoxal, a highly reactive 2-oxoaldehyde by-product of glycolysis, is able to react irreversibly and nonenzymatically with proteins, forming methylglyoxal advanced glycation end-products, which alter protein structure, stability and function. Therefore, protein glycation may influence cell metabolism and its physiology in a way beyond what can be predicted based on the implicit codification used in systems biology. Genome-wide approaches and transcriptomics, two mainstays of systems biology, are powerless to tackle the problems caused by nonenzymatic reactions that are part of cell metabolism and biochemistry. The effects of methylglyoxal-derived protein glycation and the cell's response to this unspecific posttranslational modification were investigated in Saccharomyces cerevisiae as a model organism. Specific protein glycation phenotypes were identified using yeast null-mutants for methylglyoxal catabolism and the existence of specific protein glycation targets by peptide mass fingerprint was discovered. Enolase, the major target, endures a glycation-dependent activity loss caused by dissociation of the active dimer upon glycation at a specific arginine residue, identified using the hidden information of peptide mass fingerprint. Once glycation occurs, a cellular response involving heat shock proteins from the refolding chaperone pathway is elicited and Hsp26p is activated by glycation.

Neurobiology of Aging, 2012

Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal plasticity, learning... more 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 Alzheimer's disease. We show here that incubation with amyloid-β 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 Alzheimer's 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-β 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 Alzheimer's disease.

Brain : a journal of neurology, Jan 10, 2017

α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several ot... more α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes...