FUNCTIONAL CHARACTERIZATION OF NEURONAL LYSOSOMAL ION CHANNELS AND THEIR ROLE IN THE PATHOPHYSIOLOGY OF NEURODEGENERATIVE DISEASES

The patch-clamp technique, developed by Neher and Sakmann in the late 1970s, enabled studying of ion channel activity in biological membranes and revolutionized our understanding of excitable cells. Originally developed for studying whole-cell and single-channel currents in cellular membranes, this method has been later adapted for intracellular organelles, including nuclei and, more recently, lysosomes.
Lysosomes are critical organelles involved in cellular homeostasis, signaling, and neurodegeneration. Disruption in lysosome function directly effects the protein balance and energy metabolism. Up to date little is known about ionic channels in neuronal lysosomes, their role in lysosome function and, consequentially, in the development of neurological disorders. PARK9 is a lysosomal ATPase that presumably regulates cation transport. Mutation in the PARK9 gene leads to a rapidly progressing form of Parkinson's disease (PD). We hypothesize that defective PARK9 alters lysosomal ion channel activity, thereby contributing to the etiology of PD.
In this study we adapted the patch-clamp technique to study neuronal lysosomes, enabling the direct measurement of ion channel activity (TPC2, TRPML) at the subcellular level. We first aim to characterize neuronal lysosomal ion conductances and their voltage-dependent properties.
Preliminary analysis demonstrates activation of lysosomal ionic currents upon stimulation by specific ion channel agonists, e.g. PI(3,5)P2.
To further explore the implications of lysosomal dysfunction in neurodegenerative diseases, we use PARK9 shRNA knockdown and assess the resulting effects on ion channel activity. This approach will provide insights into how alterations in lysosomal ion channel activity can lead to impaired neuronal function.