A glyco approach for the treatment of Parkinson’s disease: From the glycobiological characterisation to the development of glyco-modulatory collagen-based hydrogel systems
Rebelo, Ana Lúcia
Rebelo, Ana Lúcia
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Publication Date
2021-12-20
Type
Thesis
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Abstract
Parkinson’s disease (PD) is the most common motor-related progressive neurodegenerative condition, mainly caused by the loss of dopaminergic neurons in the substantia nigra. Despite the increased knowledge regarding its cellular and molecular signature, the importance of glycosylation is still unknown in this pathology. The lack of disease-modifying treatments makes it even more important to explore new mechanisms that may play a role in the disease pathophysiology to develop therapeutic approaches. Glycosylation is one of the most-common post-translational modifications, which determines the proteins’ arrangement. Since most neurodegenerative disorders are associated with aberrant aggregation of proteins (including PD), and as glycosylation affects proteins’ structure and function, studying this feature becomes even more attractive. The development of biomaterial-based therapies that can modulate such dysregulation of glycosylation will be of interest to explore how this molecular feature might impact not only the disease progression but also its improvement. Therefore, the work described in this thesis is focused on glycosylation and biomaterials in the context of Parkinson’s disease. The project's overall aim was to characterise the glycomic profile upon PD in humans and in an animal model of neuroinflammation, and to analyse the impact that functionalised collagen-based biomaterials have on modulating glycosylation in vitro and in vivo. The first step was to characterise in-depth the N-glycomic profile of the human Parkinsonian brain in two brain regions (substantia nigra and striatum) through a multidimensional approach. By using liquid-chromatography and mass spectrometry-based methods, the full N-glycome was deciphered and its changes with PD were assessed. Since glycosylation is non-template driven and depends on other molecular players such as glycosylation enzymes and organelle physiology, the expression of such enzymes and of markers for endoplasmic reticulum stress (ER stress) was also investigated. This showed region-specific alterations in all glycosylation traits as well as some specific enzymes. Since PD is very complex and the N-glyco-phenotype described is influenced by different pathophysiological pathways and players, a pre-clinical rodent model of neuroinflammation (LPS-induced) was then used to analyse the N-glycome in a scenario where there is mainly one phenomenon of single-origin taking place. Spatial analysis of the N-glycome confirmed that the alterations seen are restricted to the affected area and decrease with increasing distance to the core of the lesion. This allowed to establish a N-glycophenotype for neuroinflammation, emphasising the role of mannosylation and fucosylation in such scenario. In an attempt to modulate glycosylation, two different collagen-based biomaterials were developed and fabricated. Functionalisation of biomaterials can occur either through the attachment or the encapsulation of active ingredients. In this thesis, both options were explored. Firstly, collagen was functionalised with different disaccharides through reductive amination, and hydrogels were fabricated. These neo-glycofunctionalised collagen hydrogels were tested in vitro using a primary culture of ventral mesencephalic cells. Their impact on the cellular glycoprofile was determined, showing differences depending on the functionalisation performed. This highlighted how distinct glycoenvironments can influence the cellular glycophenotype differently. The other biomaterial-based platform optimised was a collagen hydrogel loaded with a neurotrophic factor that is selective for dopaminergic neurons and acts mainly on the ER stress – cerebral dopamine neurotrophic factor (CDNF). The rationale behind the use of CDNF was related to the intrinsic tight relationship between ER stress and dysregulations in Nglycosylation. This system was administered in an in vivo PD model (6-hydroxydopamine injected rats) and the impact on the behaviour, brain neurocircuitry/neuroinflammation and Nglycosylation was evaluated. Injection of a high dose of CDNF led to improvements in motor function and neuroregeneration, accompanied by changes in glycosylation, which were dose and time-dependent (further enhanced by using the biomaterial as a vehicle). Nonetheless, the specific mechanism through which CDNF and collagen led to the specific glyco-changes seen will have to be further explored in future studies. To summarise, this thesis attempts to address some significant clinical gaps in the PD field of research by employing transversal multifaceted methods to investigate clinical and preclinical samples, emphasising glycosylation. The impact of biomaterials on the modulation of glycosylation was also assessed in vitro and in vivo, highlighting the importance of investigating this piece in the molecular signature of PD; however, further studies are required to understand the mechanistic response that occurs and how these materials can be further tuned to achieve a healthy glyco-phenotype.
Publisher
NUI Galway