Mitochondrial alterations in Parkinson's disease human samples and cellular models

Abstract Mitochondrial impairment is one of the most important hallmarks of Parkinson's disease (PD) pathogenesis. In this work, we wanted to verify the molecular basis of altered mitochondrial dynamics and disposal in Substantia nigra specimens of sporadic PD patients, by the comparison with two cellular models of PD. Indeed, SH-SY5Y cells were treated with either dopamine or 1-methyl-4-phenylpyridinium (MPP + ) in order to highlight the effect of altered dopamine homeostasis and of complex I inhibition, respectively. As a result, we found that fusion impairment of the inner mitochondrial membrane is a common feature of both PD human samples and cellular models. However, the effects of dopamine and MPP + treatments resulted to be different in terms of the mitochondrial damage induced. Opposite changes in the levels of two mitochondrial protein markers (voltage-dependent anion channels (VDACs) and cytochrome c oxidase subunit 5β (COX5β)) were observed. In this case, dopamine treatment better recapitulated the molecular picture of patients' samples. Moreover, the accumulation of PTEN-induced putative kinase 1 (PINK1), a mitophagy marker, was not observed in both PD patients samples and cellular models. Eventually, in transmission electron microscopy images, small electron dense deposits were observed in mitochondria of PD subjects, which are uniquely reproduced in dopamine-treated cells. In conclusion, our study suggests that the mitochondrial molecular landscape of Substantia nigra specimens of PD patients can be mirrored by the impaired dopamine homeostasis cellular model, thus supporting the hypothesis that alterations in this process could be a crucial pathogenetic event in PD

CFTR interactome mapping using the mammalian membrane two-hybrid high-throughput screening system

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein-protein interactions of wild-type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient-specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient-derived intestinal organoids has a significant effect on CFTR functional expression

Discovering mithocondrial alterations in Parkinson's disease: the role of mitophagy impairment.

Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders, characterized by the loss of dopaminergic neurons in the Substantia nigra pars compacta. Although the cause of PD is currently unknown, strong evidences indicate that a complex interplay between several factors including genetic susceptibility, environmental factors, abnormal protein handling and oxidative stress could be involved. Many of the molecular pathways implicated in PD etiology converge on mitochondria, resulting in their dysfunction, which could impact on neuronal survival. Given the importance of mitochondrial dysfunctions in PD, we decided to investigate the missed removal of damaged mitochondria due to mitophagy impairment as the possible trigger of pathogenesis. To this purpose, we characterized specific mitochondrial alterations in Substantia nigra specimens from PD patients, comparing them to what observed by inducing mitochondrial impairment in human neuroblastoma SH-SY5Y cells using dopamine or MPP+. Since mitophagy impairment seems to play a central role in the development of this pathology, the second part of this thesis focused on mitochondrial alterations that occur in skin fibroblasts obtained from PARK2-mutated patients. As a result, we found that mutations in the PARK2 gene do not cause any significant morphological alterations in the mitochondrial network shape. Moreover, the proteomics analysis revealed an impairment of proteins involved in several mitochondrial functions. Overall, this project contributes to a complete definition of the PARK2-related molecular signature, that will be crucial for providing new insights into disease mechanisms and identifying new therapeutic targets for this pathology