Tese de Doutoramento Mecanismos de Doença e Medicina Regenerativa 2022 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Parkinson’s disease (PD) is the most common movement disorder and the second most common neurodegenerative disorder, affecting 7 to 10 million people worldwide and is an age-related disease. PD is characterized by a progressive motor decline that includes bradykinesia and tremor, and by non-motor features such as mood and cognitive changes and belongs to a wide group of neurodegenerative diseases known as synucleinopathies. The neuropathological hallmarks of PD are the presence of intracellular proteinaceous aggregates primarily composed of alpha-synuclein (aSyn), known as Lewy bodies (LBs) and Lewy neurites (LNs), and the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) within the midbrain. The degeneration of dopaminergic neurons in the SNpc leads to the depletion of dopamine in the striatum that underlies the cardinal motor features of parkinsonism observed in PD. Parkinsonism is the clinical syndrome characterized by resting tremors, bradykinesia, muscular rigidity, postural instability, and gait impairment. Although the dopaminergic system in the SNpc is highly affected, several other neurotransmission and neuromodulatory systems are also implicated in PD including the glutamatergic, GABAergic, and cholinergic pathways. The imbalance of these neurotransmission systems impacts both motor and non-motor features of PD. Although several genetic mutations and polymorphisms have been associated with familial forms of PD, about 90% of the cases are sporadic or idiopathic. Currently, PD lacks curative, restorative, preventive, or disease-modifying therapies and this is one of the major challenges in the field. The available therapeutic approaches and tools for PD are restricted to the symptomatic management of motor features and other non-motor signs. aSyn is a small presynaptic protein, abundant in the brain and in several other tissues. aSyn has been reported to participate in synaptic vesicles (SV) dynamics and in different steps of neurotransmitters release. aSyn is a protein that is natively unfolded and highly prone to aggregation. The oligomerization and aggregation of aSyn is believed to contribute for the neurodegenerative process in PD and related synucleinopathies. Although valuable information about the mechanisms governing aSyn pathogenicity and aggregation have been collected from the study of PD-linked mutations, the pathological mechanisms in PD remain largely elusive as most cases are sporadic. Our poor understanding of the molecular mechanisms underlying neurodegeneration in PD has been one of the major obstacles for the development of effective therapies. Although ageing is the major risk factor for neurodegenerative diseases, such as PD, it is imperative to identify and better understand the role and contribution of different risk factors for the pathogenesis of PD, as well as to disclose the mechanisms driving to protein abnormalities and aggregation. In the last 20 years, diabetes mellitus (DM) emerged as a relevant risk factor for neurodegenerative diseases such as PD. Epidemiological studies revealed that type 2 diabetes mellitus (T2DM) can increase up to 40% the risk of developing PD in the general population, and this risk rises to 380% for young diabetic individuals. T2DM is a chronic metabolic disease characterized by glucose metabolism imbalance, hyperglycaemia, insulin resistance, and impaired glucose tolerance. Hyperglycaemia is a hallmark of T2DM and is the major responsible for the deleterious accumulation of the highly reactive reducing sugars, such as methylglyoxal (MGO), that are unavoidably formed as by-products of glycolysis. MGO, one of the most powerful reducing sugars, reacts and covalently bind to biomolecules such as proteins in a non-enzymatic process named glycation, leading to the formation of the irreversible advanced glycation end products (AGEs). Compelling evidence support a key role of glycation in the aetiology, pathogenesis, and progression of PD. Studies reported the presence of AGEs at the periphery of LBs, increased levels of glycation in the brains of patients with PD, and of glycated aSyn in the brains of patients with PD and FTD. In vitro studies showed that glycation accelerates the formation of aSyn oligomers by impairing both the proteasome and lysosome activity, contributing to the loss of proteostasis, and promoting neuronal cell death. In a transgenic mouse model of aSyn pathology, MGO injection in the substantia nigra or striatum induced neurodegeneration, particularly of dopaminergic neurons. Considering the prior findings suggesting a pivotal role of glycation in potentiating aSyn pathology, our main hypothesis is that glycation triggers or exacerbates PD-like phenotypes. Furthermore, we also hypothesize that strategies aimed at suppressing glycation may prove effective in preventing PD onset and progression. To unveil the brain proteome response to aSyn pathology and the role of protein glycation on aSyn pathobiology in the brain, age-matched male transgenic Thy1-aSyn and WT littermates mice received a single dose of MGO through intracerebroventricular (ICV) injection. Three weeks after the procedure, mice were weighed, handled, and the Shirpa protocol performed. Behavioural phenotyping was performed four weeks after surgery to evaluate motor, cognitive, olfactory, and anxiety-related function. Here, we reported that 20 weeks Thy1-aSyn mice exhibited impaired motor performance comparing to WT littermates. In this time-point, we did not observe colonic dysfunction, olfactory alterations, cognitive impairment, neither anxiety-related alterations. Thy1-aSyn mice at 20 weeks of age did not exhibit dopaminergic or non-dopaminergic neuronal degeneration in the SNpc. By using a quantitative SWATH proteomics approach, we disclosed the pathological impact of aSyn overexpression in the brain of the transgenic Thy1-aSyn mouse model by defining the molecular targets and associated dysregulated pathways. We found that aSyn overexpression in the midbrain mostly impacts proteins associated with oxidative phosphorylation, endocytosis and intracellular vesicular trafficking, and synaptic vesicles dynamics. Although this work does not unveil new pathways implicated in aSyn pathology, it clearly identifies the molecular targets that are dysregulated upon aSyn overexpression in the midbrain. By determining the profile of proteins responding to aSyn pathology, we can unveil potential molecular targets that might hold potential as aSyn pathology-modifiers, which can disclose new therapeutic avenues for synucleinopathies. In this work, we also hypothesized that glycation-induced neuronal dysfunction might be a contributing factor in synucleinopathies such as PD. We reported that MGO-glycation in the brain potentiates motor, cognitive, olfactory, and colonic dysfunction in transgenic Thy1-aSyn mice that received a single dose of MGO by ICV injection. In these mice, aSyn accumulates in the midbrain, striatum, and prefrontal cortex, and protein glycation is increased in the cerebellum and midbrain. We also observed increased aSyn phosphorylation at S129 and aSyn insolubility in the midbrain. MGO triggered a general neuronal loss at the vicinity of SNpc within the midbrain. SWATH mass spectrometry analysis disclosed that glycation mainly increase glutamatergic signalling associated proteins in the midbrain. By contrast, in the prefrontal cortex the most affected proteins correspond to the electron transport chain. These findings suggest that MGO differently impacts the proteome depending on the brain region. Furthermore, glycated proteins in the midbrain of MGO-injected Thy1-aSyn mice strongly correlate with PD and dopaminergic pathways. Altogether, our study demonstrated that MGO-induced glycation accelerates PD-like sensorimotor and cognitive alterations, pointing that an increased glutamatergic signalling may underly the exacerbated behaviour phenotype. Our study not only sheds new light into the enhanced vulnerability of the midbrain in PD-related synaptic dysfunction, but also defines a link between PD and T2DM. Moreover, our results support that glycation suppressors and anti-glutamatergic drugs may hold promise as disease-modifying therapies for PD and related synucleinopathies. Considering the pivotal role of glycation on aSyn pathobiology and in the pathogenesis and progression of PD, we hypothesize that strategies aimed at blocking or suppressing glycation could be used as novel preventive or disease-modifying therapies for PD and other synucleinopathies. To test this hypothesis, we used both a genetic strategy and a drug-based screening of compounds that target glycation. Following the observation that a MGO insult in H4 neuroglioma cells decreases the levels of heat shock protein 27 (Hsp27) in a dose-dependent manner, we hypothesized that compensating Hsp27 loss in glycating environments could alleviate the glycation-induced pathogenicity of aSyn. In this cellular system, we co- expressed aSyn and Hsp27 and after challenging these cells to MGO glycation we found that Hsp27 overexpression prevented MGO-induced aSyn cytotoxicity, accumulation, and aggregation. Our work uncovers the importance of Hsp27 in suppressing the pathological conditions induced by glycation, suggesting that Hsp27 may constitute a suitable target for intervention in PD and related synucleinopathies. By using a drug-based screening, H4 neuroglioma cells overexpressing aSyn or SynT under glycating conditions were treated with different compounds. From the 11 compounds tested we found that carnosine, metformin, and penicillamine prevented MGO-induced aSyn pathology. Metformin prevented MGO- associated aSyn cytotoxicity, SynT insolubility and aggregation, and the impairment of aSyn clearance. Carnosine prevented MGO-associated aSyn cytotoxicity, SynT insolubility and inclusions formation. Penicillamine prevented MGO-induced cytotoxicity, the increase in the levels of aSyn and SynT levels, SynT insolubility and aggregation and improved aSyn clearance. Our findings suggest that carnosine, metformin, and penicillamine are the most promising and lead therapeutic compounds requiring further validation in pre-clinical models of synucleinopathies. There is an urgent demand for preventive or disease-modifying therapies for PD and other neurodegenerative diseases. Our work highlights the pivotal role of glycation in aSyn pathobiology and in the exacerbation of PD-like features, narrowing the bridge between PD and DM. Our data suggests decreasing glycation and targeting the glutamatergic system hold preventive or disease-modifying potential therapies for PD and other synucleinopathies. Our research opens novel therapeutic avenues for synucleinopathies that needs to be properly exploited in both pre-clinical and clinical studies.