Characterization of a novel Glucocerebrosidase pharmacological chaperone in cellular and animal models of α-synuclein neurotoxicity

Parkinson's disease (PD) is a condition characterized by the progressive loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc) and the formation of α-synuclein (α-syn) protein aggregates, known as Lewy bodies, in neuronal somata. The most evident clinical signs of the disease include resting tremor, bradykinesia (slowness of movement), rigidity of the trunk and limbs, abnormal postural reflexes, and amimia. These characteristic symptoms are due to a reduction of dopamine in the brain, caused by the degeneration of dopaminergic neurons in the SNpc. The loss of dopaminergic neurons results in a dramatic striatal dopaminergic denervation and triggers a cascade of functional modifications that affect all components of the basal ganglia circuit. It is well established that mutations in the GBA1 gene (which encodes the lysosomal enzyme Glucocerebrosidase - GCase) predispose individuals to a higher risk of developing PD, pointing to a mechanistic link between lysosomal/autophagosome function and the aggregation and toxicity of α-syn. Based on this evidence, it has been suggested that increasing the stability and activity of lysosomal GCase could represent a novel therapeutic approach to block α-syn accumulation in PD and other synucleinopathies. As alterations in GCase activity have also been observed in non-GBA1 PD cases, functional GCase enhancers, such as pharmacological chaperones (PC), could represent a useful therapeutic approach for PD patients. PCs are small molecules, available orally, that can enter the central nervous system (CNS), bind to and stabilize their target protein, thereby increasing GCase activity in the brain (Boyd et al. 2013; Pereira et al. 2018). Within this class of compounds, the mucolytic ambroxol is being repositioned as a PD therapy in a Phase II study, supporting the validity of the chaperone strategy in PD. Due to their drug-like properties, iminosugars, which are glycomimetics where a nitrogen atom replaces the endocyclic oxygen of sugars, are excellent candidates as PCs for lysosomal enzymes. In this context, the present work has focused on the potential neuroprotective effect of the molecular chaperone CF30 in preclinical models of PD and PD-GD, including SH-SY5Y neuroblastoma cells, cultured cortico-striatal slices and GBA1L444P/WT mice. First, the effective enhancement of glucocerebrosidase activity by molecular chaperones was evaluated in normal SHSY5Y cell cultures and SHSH5Y cells transfected with two GCase forms: WT and L444P. Chaperone pharmacodynamics, the effective enzymatic activity enhancement of GCase, and biochemical analyses were conducted to assess protein levels and key autophagic markers. Next, cortico-striatal organotypic slices were used to recreate an in vitro model of PD, where the effects of α-syn aggregates and subsequent CF30 treatment were studied. In vivo, a GBA1 mouse model was used for pharmacokinetic and pharmacodynamic analyses. The compound significantly enhanced the activity of GCase WT in multiple experimental models, simultaneously reducing the accumulation of phosphorylated α-syn and LAMP-1, oxidative stress and internalization of OB*. Furthermore, it showed good in vivo tolerability and ability to cross the blood-brain barrier making. In vivo, the candidate chaperone was able to increase GCase enzyme activity both in a mouse model with a GCase WT (C57BL/6N), showing a trend in the same direction in GBA1-L444P/WT mice. In conclusion, the experimental evidence I gathered during my doctoral work suggests that CF30 has the potential to become a promising therapeutic agent for the treatment of neurodegenerative diseases, especially in the presence of a GBA1 genotype.