Fibrin-in-fibrin intervention for enhanced dopaminergic graft survival in Parkinson’s disease: Special focus on brain N-glycans
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Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disorder caused due to the selective loss of dopaminergic neurons in substantia nigra pars compacta (SN) translating into the classical motor symptoms of tremors, rigidity, akinesia and postural instability. Most of the available therapeutic strategies in PD focus on the management of the motor and non-motor symptoms and primarily rely on dopamine replacement strategies. However, there is a major unmet clinical need to address the underlying cause of PD. i.e. the loss of dopaminergic neurons. Comprehensive pre-clinical and clinical assessment have proven the potential of primary dopaminergic neurons to survive, integrate with the host system, re-equilibrate the striatal dopamine content and promote functional recovery. However, their routine use as a clinical therapy is hindered by the poor graft survival after transplantation and the subsequent ethical concerns with the use of multiple fetal donors for each patient. An injectable biomaterial intervention has the potential to provide a solution to this problem in a multi-faceted way by improving the engraftment of encapsulated cells through the provision of a supportive and growth factor-rich environment which effectively shields the cells from the host immune response. To this end, the overall aim of this project was to assess the effect of a multimodal, glial-derived neurotrophic factor (GDNF)-loaded fibrin-in fibrin intervention on the survival dopaminergic neurons and the functional recovery after transplantation into the Parkinsonian brain. The first step in the project was to fabricate and characterise in vivo the fibrin-based hollow microspheric reservoirs for the controlled release of neurotrophic factors. Following this, the fibrin-in-fibrin platform was optimised for the intra-cranial delivery of primary dopaminergic neurons through an exhaustive in vitro analysis. In vitro studies showed that altering the macromolecular, cell and microspheric concentration can tune the paracrine responses of the encapsulated cells. The success of biomaterial therapy is usually governed by the careful investigation and manipulation of inter-cellular and cell-matrix signalling, wherein the cell surface N-glycans play a consequential role. To this end, investigation of the variations in the glycosylation patterns of proteins at cellular and matrix level in brain could lead to the identification of molecular targets for devising efficient therapeutic targets and their modulation with biomaterial therapies. This lead to the next stage of the project which dealt with the complete spatial resolution of N-glycans in striatum and SN of the healthy and diseased brains. To the best of our knowledge, this was the first study elucidating the spatio-temporal patterns of N-glycosylation modulation in PD brains. This study holds tremendous potential in deciphering the glycan cues implicated in PD pathophysiology and to develop viable biomaterial therapies harnessing the ‘glyco-code’ elucidated in this study. Further, to test for therapeutic potential of the fibrin-in-fibrin intervention, 6-hydroxydopamine (6-OHDA) rodent model was used as a relevant pre-clinical animal model as it is the most suited model to validate the experimental therapies. In summary, it was demonstrated that the optimised fibrin-in-fibrin intervention is well tolerated in the brain and successfully augmented the engrafted cell survival along with the neurite outgrowth. The intervention acted as an effective shield to attenuate the host response against the implanted cells. Building on this, a pilot analysis showed that the encapsulation of embryonic day (E) 14 VM cells in a GDNF-loaded fibrin-in-fibrin platform mediates a dramatic five- fold increase in the survival, which translated into a significant improvement in the functional recovery in the rotational behaviour and limb placement asymmetry. Additionally, this study represented the dynamic re-modelling of the brain glyco-environment using the fibrin-in-fibrin intervention away from the diseased phenotype and towards the heathy brain glyco-phenotype using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI). To summarise, this thesis attempts to address some major biological and clinical lacunae in the field by the application of a multidimensional approach through the investigation of the implication of glycosylation in a relevant model of PD, the application of an ECM-inspired fibrin-based, brain-targeted intervention and elucidating the mechanistic story.