Desirèe Ficarra

I obtained my Bachelor’s degree in Biotechnology from the University of Milano-Bicocca in 2018 and my Master’s degree in Medical Biotechnology in 2021, with a thesis based on the preliminary characterization of extracellular vesicle populations derived from preconditioned mesenchymal stem cells, supervised by Dr. Silvia Coco.

Since November 2021, I have been a PhD student in Experimental Neuroscience in the laboratory of Professor Carlo Ferrarese. My research project focuses on the study of Parkinson’s Disease, investigating the mechanisms of action of Ambroxol drug in autophagy pathways and ita neuroprotective effect.

PhD research project

The neuroprotective potential effect of Ambroxol and its role in autophagy pathways in an in vitro models of Parkinson’s disease

The neurodegenerative Parkinson’s Disease (PD) is characterized by a gradual loss of dopaminergic neurons in the substantia nigra, due to the presence of neurites and Lewy bodies, mainly composed by the pathological hallmark alpha-synuclein (asyn), within the degenerating neurons.

Motor- (tremor, rigidity, etc.) and non-motor-related (neuropsychiatric, behavior and mood alterations, etc.) symptoms of PD negatively affect daily PD-patients life. Currently, diagnosis is only possible at an advanced stage of the disease and PD can’t be cured, but several medications can help control the symptoms.

According to the literature, the presence of misfolded proteins aggregates into the neurons is a basic requirement for the neurodegenerative process and dysfunctions of the protein catabolic systems play a cruciale role in PD.

Recently in vitro and in vivo studies have reported that Ambroxol, a drug used as treatment for respiratory diseases, is able to increase beta-glucocerebrosidase lysosomal enzime activity and to reduce asyn levels.

Our aims are to investigate alterations of asyn catabolic pathways (macroautophagy and chaperone-mediated autopagy CMA) and to test the potential neuroprotective effect of Ambroxol, in order to correct these dysfunctions.

Human neuroblastoma SH-SY5Y cells were exposed to Ambroxol alone or co-treated with the lysosomal inhibitor NH4Cl (10mM) for the last 24h. Protein and gene levels of asyn and targets involved in asyn clearance were quantified by Western blot (WB) and RT-qPCR, and extracellular protein release by dot-blot.

We found that Ambroxol: enhances the intracellular level of both monomeric and oligomeric asyn forms starting from 48h-exposure; increases protein levels of LAMP1, a lysosomal marker, and p62, a macroautophagy substrate, with no changes in other macroautophagy (beclin-1, LC3II) and CMA (LAMP2A, hsc70) effectors; doesn’t affect gene expression of all investigated parameters and extracellular asyn release; does not modify the NH4Cl-induced increase of asyn protein levels.

The previously described protocol was applied to non-tumoral cells represented by neonatal rat neurons, to validate the results obtained in neuroblastoma cells. Obtained results from WB analyses denote that 72h Ambroxol exposure significantly increases the monomeric form of asyn and enahances the level of macroautophagic targets p62 and LC3II, both under basal conditions and especially in the presence of NH4Cl.

The obtained data suggest that Ambroxol under basal conditions improves cellular homeostasis by increasing levels of physiological asyn and the number of lysosomes. Further experiments under pathological-like conditions in the same cells are needed to understand the effect of Ambroxol on asyn and its clearance.