• Ph.D., Molecular Biology and Biochemistry, Universita' della Calabria, Italy

• B.S., Pharmaceutical Chemistry & Technology, Universita' della Calabria, Italy

Mutations of Beta-Propeller Protein Associated Neurodegeneration (BPAN) leads to formation of exosomes and increased secretory autophagy

Mutations linked to WDR45 gene, coding for a beta-propeller protein and located on the X chromosome, are associated with Beta-Propeller Protein Associated Neurodegeneration (BPAN) (Haack et al., 2013; Hayflick et al., 2013). This is a neurodevelopmental and neurodegenerative disease with onset in infancy determined by an iron accumulation in the basal ganglia (Ingrassia et al 2017). BPAN is sporadic with a female preponderance. In females, the phenotype is more variable while in males, even though less predominant, is more severe (Adang et al 2020).

Phenotypes show seizures and developmental delay with attention deficit, sleep disruptions and intellectual and motos deficit in childhood. Disease is static until early adulthood when patients develop progressive dystonia, parkinsonism and dementia (Haack et all 2012). BPAN is often lethal in mid-adulthood. At this point there is no treatment for BPAN only symptomatic relief for movement and seizure symptom (Wilson et al, 2021).

WDR45, also known as WIPI4, is a member of the WD40 repeat protein family (Ingrassia et al, 2017). WD40 protein family presents 40 conversed amino acids with a consensus repeat of tryptophan-aspartic acid (WD) residues crucial for binding phosphatidylinositol 3-phosphate (PtdIns3P) (Ingrassia et al, 2017). WDR45 is the mammalian ortholog of the yeast autophagy gene ATG18, overexpressed during autophagy and a proposed biomarker of autophagome formation (Tsuyuki et all 2014).

Autophagy is an intracellular degradation process with a central role in normal physiology and pathophysiology highly conversed from yeast to mammalian (Lu et al, 2011). This catabolic process involves the formation of a closed double membrane structure, called autophagosome, that fuses with a lysosome where the sequestered material is finally degraded (Tsuyuki et al, 2014). In yeast, 20 ATG genes have been identified that are essential for autophagosome formation (Nakatogawa et al, 2009) and WDR45 is the mammalian ortholog of the yeast autophagy gene ATG18, overexpressed during autophagy and a proposed biomarker of autophagome formation (Tsuyuki et all 2014).

Extracellular vesicles (EVs) comprise a varied and heterogeneous group of particles released from cells originating largely from endosomes and/or the plasma membrane (Madeo et al, 2018). Initially described as the disposal mechanisms cells use to discard unwanted material, EVs are now considered mediators of intercellular communication in normal physiology and pathophysiology (Sedgwick et al, 2018). Recent studies have shown that EVs contain various proteins, lipids, glycolipids, glycoproteins and nucleic acids including DNA, mRNA and noncoding RNAs (5). Tumor released exosomes mediate axonogenesis in cancer and that this innervation is sensory in nature (Madeo et al, 2018). Consequentially, EVs have the potential to deliver multiplexed information to surrounding tissues and through the body (Madeo et al, 2018).

Preliminary BioID data from the Pilaz Lab, our collaborator in this project, shows that WDR45 interacts with exosomal protein. My laboratory will explore the packaging of WDR45 in exosomes and its role in the autophagic process. Exosomes, the best characterized of the EV subtypes, are formed first as intraluminal vesicles within a multivesicular body (MVB), which are released into the extracellular space upon MVB fusion with the plasma membrane. We will isolate exosome from mouse neuroblastoma cell lines N2A (Neuro-2a) cell line at different growth conditions, able to induce autophagy. Cells will be cultured under normal conditions (DMEM supplemented with 10% FBS), and under pharmacological-Induced autophagy by using two different drugs, Rapamycin (1µM) and Torin1 (100 nM). We will also induce autophagy in starvation condition, depriving the condition media of the FBS. We will isolate cell lysates and exosomes form those cells. We will verify the induction of autophagy in cell lysate by looking at the expression of LC3, a well-known protein overexpressed during such process. We will quantify exosome particles by Nanosight and we will be also looking if there is any change in size. Exosomes will be characterized by Western Blot analysis using exosomal marker, i.e. flotillin-2, CD-9 and CD-81 (Madeo et al, 2018). Packaging or WDR45 in the different growing conditions will be tested by Western Blotting Analysis.

Our finding will deepen the knowledge of the role of WDR45 protein in the autophagic process leading to the study of its interaction with the other ATG proteins and its role in the formation of the autophagosome.