Activation of ATP13A2 as therapeutic strategy for neurodegenerative disorders

Neurodegenerative diseases result from progressive loss of structure and/or function of the neurons leading to motor and cognitive dysfunction. Parkinson’s disease (PD) is one of the most common neurological diseases caused by a complex interplay between genetic and environmental factors. The ATP13A2/PARK9 gene encodes an endo- and lysosomal membrane-associated protein belonging to the superfamily of P-type ATPases (type 5B) and is associated with Kufor-Rakeb syndrome: an early-onset, autosomal recessive form of parkinsonism with dementia. Although its exact function remains elusive, loss of its function interferes with the lysosomal autophagic pathway and affects lysosomal degradation, whereas its activation protects neurons against α-synuclein, mitochondrial and heavy metal (Mn2+, Zn2+, Fe3+) toxicity. In other words, ATP13A2 seems to play a key role in parkinsonism, as fail of mitochondrial quality control and impaired lysosomal function have recently emerged as important players in the pathogenesis of PD. The activity of ATP13A2 depends on the consummation of ATP. Under basal conditions, ATP13A2 undergoes autophosphorylation, which can be stimulated by two membrane-associated signaling lipids, phosphatidic acid (PA) and phosphatidyl inositol (3,5) bisphosphate [PI(3,5)P2], that bind to the N-terminus of ATP13A2. In a cellular model of PD, ATP13A2 activity confers protection against rotenone- and MPP+ (complex I inhibitors)- induced mitochondrial stress. Along the N-terminus, three putative lipid binding sites (LBSs) were previously identified. Of interest, ATP13A2 containing mutations in the LBSs tends to form more dimers and does not longer autophosphorylate or provide protection against mitochondrial stress in SHSY5Y cells, pointing out the significance of this region in dimerization and mediating activation of the protein. Moreover, this suggests that the N-terminus of ATP13A2 may serve as an autoinhibitory domain that under basal conditions prevents its activation and is unlocked by the N-terminal lipid interactions under stress conditions. The aim of this research project is to explore the role of the N-terminus in the activation mechanism of ATP13A2. To this end, peptides corresponding to the N-terminus will be GST-purified and their activating potential will be tested using radiometric phosphorylation assays and ATPase activity assays. Secondly, monoclonal antibodies directed to the autoinhibitory domain will be generated in mouse and evaluated in a similar way. In the next step, the potential interaction sites of the N-terminal peptides with the cytosolic domains will be identified using immunoprecipitation (pulldown experiments). Finally, the most interesting N-terminal peptides and/or antibodies will be overexpressed using lentiviral vectors and their protective effects will be assessed in cellular (SHSY5Y cells) and animal (rat) models of PD. Targeting ATP13A2 and the lysosomal degradation pathway may provide a new therapeutic strategy in the treatment of PD.