Cardiac glycoside-mediated turnover of Na, K-ATPases as a rational approach to reducing cell surface levels of the cellular prion protein

Abstract

It is widely anticipated that a reduction of brain levels of the cellular prion protein (PrPC) can prolong survival in a group of neurodegenerative diseases known as prion diseases. To date, efforts to decrease steady-state PrPC levels by targeting this protein directly with small molecule drug-like compounds have largely been unsuccessful. Recently, we reported Na,K-ATPases to reside in immediate proximity to PrPC in the brain, unlocking an opportunity for an indirect PrPC targeting approach that capitalizes on the availability of potent cardiac glycosides (CGs). Here, we report that exposure of human co-cultures of neurons and astrocytes to non-toxic nanomolar levels of CGs causes profound reductions in PrPC levels. The mechanism of action underpinning this outcome relies primarily on a subset of CGs engaging the ATP1A1 isoform, one of three α subunits of Na,K-ATPases expressed in brain cells. Upon CG docking to ATP1A1, the ligand receptor complex, and PrPC along with it, is internalized by the cell. Subsequently, PrPC is channeled to the lysosomal compartment where it is digested in a manner that can be rescued by silencing the cysteine protease cathepsin B. These data signify that the repurposing of CGs may be beneficial for the treatment of prion disorders.

Identification of a cardiac glycoside exhibiting favorable brain bioavailability and potency for reducing levels of the cellular prion protein

“…To evaluate this characteristic experimentally in a relevant model, immortalized human cells (ReN VM cells) that we had differentiated to acquire neural or astrocytic characteristics were exposed for a duration of one week to low nanomolar levels of oleandrin or 15 KDC203. Following cell lysis, steady-state ATP1A1 levels were assessed by western blot analysis to determine if the ligand-receptor interactions had caused the previously observed reduction in ATP1A1 levels [18]. This analysis established that KDC203 had indeed similar potency as oleandrin on the basis that both drugs achieved a similar reduction in ATP1A1 levels when added at 4 nM concentration to the cell culture medium (Fig 6A).…”

“…This analysis established that KDC203 had indeed similar potency as oleandrin on the basis that both drugs achieved a similar reduction in ATP1A1 levels when added at 4 nM concentration to the cell culture medium ( Fig 6A ). However, whereas oleandrin was toxic at concentrations exceeding 4 nM [18], KDC203 was tolerated by the cells at all concentrations tested in this initial pilot experiment. Remarkably, exposure of ReN VM cells for one week to 8 or 16 nM levels of this compound led to additional reductions in ATP1A1 levels.…”

“…Intrigued by our observation that KDC203 suppressed steady-state protein levels of ATP1A1 and ATP1A2 yet increased ATP1A3 levels when ReN VM cells were exposed to concentrations exceeding 16 nM, we wondered if the levels of these α subunits were independently or interdependently affected by KDC203. We had previously observed that the CG-dependent reduction in the steady-state protein levels of ATP1A1 and PrP C could be rescued by rendering the human ATP1A1 protein resistant to high-affinity CG docking [18], thereby establishing the need for CG and NKAs forming a ligand-receptor interaction to achieve this outcome, as opposed to other, less defined effects of CG on the cell. To obtain this result we had mutated the ATP1A1 allele in two positions such that two amino acid residues known to contribute to CG binding are instead coding for the corresponding amino acids present in mouse Atp1a1 [55] ( Fig 7C ).…”

“…A replacement of key amino acid residues in the CG-docking site of 6 ATP1A1 in the human ReN VM cell model prevented the CG-dependent PrP C degradation, indicating that the latter is dependent on CG ligand-ATP1A1 receptor engagement in this paradigm, as opposed to some other non-specific effect of CGs on the cell. Finally, we learned that the degradation of PrP C under these circumstances relies on the endo-lysosomal system and specifically involves the cysteine protease cathepsin B [18].…”

Identification of a Cardiac Glycoside Exhibiting Favorable Brain Bioavailability and Potency for Reducing Levels of the Cellular Prion Protein

“…We recently discovered that PrP C is surrounded by Na,K-ATPases (NKAs) in the brain [ 17 ], which led us to propose an indirect targeting approach [ 18 ]. We showed that when cardiac glycosides (CGs), a class of compounds also known as cardiotonic steroids, bind to NKAs, it causes their internalization and degradation.…”