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Funding: This study was supported by Fundação para a Ciência e Tecnologia (FCT) PTDC/NEU-OSD/5644/2014 and EXPL/NEU-OSD/0606/2012. Authors were supported by: HVM (FCT, SFRH/BPD/109347/2015); MO (FCT, EXPL/ NEU-OSD/0606/2012); AC (FCT, PD/BD/136863/2018; ProRegeM—PhD program, mechanisms of disease and regenerative medicine); BFG (PTDC/NEU-OSD/5644/2014). TFO is supported an EU Joint Program—Neurodegenerative Disease Research (JPND) project (aSynProtec). The project is supported through the following funding organizations under the aegis of JPND—www.jpnd.edu (BMBF). α-synuclein (aSyn) is a major player in Parkinson's disease and a group of other disorders collectively known as synucleinopathies, but the precise molecular mechanisms involved are still unclear. aSyn, as virtually all proteins, undergoes a series of posttranslational modifications during its lifetime, which can affect its biology and pathobiology. We recently showed that glycation of aSyn by methylglyoxal (MGO) potentiates its oligomerization and toxicity, induces dopaminergic neuronal cell loss in mice, and affects motor performance in flies. Small heat-shock proteins (sHsps) are molecular chaperones that facilitate the folding of proteins or target misfolded proteins for clearance. Importantly, sHsps were shown to prevent aSyn aggregation and cytotoxicity. Upon treating cells with increasing amounts of methylglyoxal, we found that the levels of Hsp27 decreased in a dose-dependent manner. Therefore, we hypothesized that restoring the levels of Hsp27 in glycating environments could alleviate the pathogenicity of aSyn. Consistently, we found that Hsp27 reduced MGO-induced aSyn aggregation in cells, leading to the formation of nontoxic aSyn species. Remarkably, increasing the levels of Hsp27 suppressed the deleterious effects induced by MGO. Our findings suggest that in glycating environments, the levels of Hsp27 are important for modulating the glycation-associated cellular pathologies in synucleinopathies. publishersversion published

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 intracerebroventricular (ICV) injection. Behavioural phenotypes were analysed 4 weeks post-treatment, and, at the end of the tests, biochemical and histological studies were conducted on brain tissue. We found that glycation potentiates motor dysfunction, assessed by vertical pole, rotarod and hindlimb clasping tests in Thy1-aSyn mice. In addition, it induces cognitive impairment (Y maze test), olfactory disturbances (block test), and colonic dysfunction. These behavioural changes were accompanied by the accumulation of aSyn in the midbrain, striatum, and prefrontal cortex, and by an overall increase in glycation in the midbrain and cerebellum. Furthermore, MGO induced neuronal and dopaminergic cell loss in the midbrain of Thy1-aSyn mice. Quantitative proteomic analysis revealed that, in Thy1-aSyn mice, MGO mainly impacts on glutamatergic proteins in the midbrain, but not in the prefrontal cortex, where it mainly affects the electron transport chain. Among the altered proteins in the midbrain, we found an upregulation of N-Methyl-D-Aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors levels, glutaminase, vesicle glutamate transporter (VGLUT), and the excitatory amino acid transporter (EAAT1), suggesting potentiation of glutamatergic signalling. Overall, we demonstrated that MGO-induced glycation accelerates Parkinsonian-like sensorimotor and cognitive alterations. The increase in glutamatergic-related proteins in the midbrain may represent a compensatory mechanism to the MGO-induced dopaminergic neurodegeneration. Our study sheds light into the enhanced vulnerability of the midbrain in Parkinson’s disease-related synaptic dysfunction that, ultimately leads to cell loss, and provides molecular insight into the observation that glycation suppressors and anti-glutamatergic drugs hold promise as disease-modifying therapies for synucleinopathies.

α-synuclein (aSyn) is a major player in Parkinson’s disease (PD) and a group of other disorders collectively known as synucleinopathies, but the precise molecular mechanisms involved are still unclear. aSyn, as virtually all proteins, undergoes a series of posttranslational modifications (PTMs) during its lifetime which can affect its biology and pathobiology. We recently showed that glycation of aSyn by methylglyoxal (MGO) potentiates its oligomerization and toxicity, induces dopaminergic neuronal cell loss in mice, and affects motor performance in flies. Small heat-shock proteins (sHsps) are molecular chaperones that facilitate the folding of proteins or target misfolded proteins for clearance. Importantly, sHsps were shown to prevent aSyn aggregation and cytotoxicity. Upon treating cells with increasing amounts of methylglyoxal, we found that the levels of Hsp27 decreased in a dose-dependent manner. Therefore, we hypothesized that restoring the levels of Hsp27 in glycating environments could alleviate the pathogenicity of aSyn. Consistently, we found that Hsp27 reduced MGO-induced aSyn aggregation in cells, leading to the formation of non-toxic aSyn species. Remarkably, increasing the levels of Hsp27 suppressed the deleterious effects induced by MGO. Our findings suggest that in glycating environments, the levels of Hsp27 are important for modulating the glycation-associated cellular pathologies in synucleinopathies.