ATP P2X4 receptor in ALS pathogenesis and biomarker – P2XforALS

Context- Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative motoneuron (MN) disease. ALS is considered as a protein misfolding disorder, accompanied by neuroinflammation that accelerates MN cell death and disease progression. To date, the absence of both biomarker discovery and key cellular mechanism identification impairs the elaboration of effective treatments of ALS. Alterations of the purinergic signaling are involved in the pathogenesis of several neurological diseases with a pivotal role for P2X4 receptors. P2X4 is an ATP-gated cation expressed in CNS neurons and glial cells as well as in peripheral tissues such as macrophages, monocytes in mammals including humans. In the healthy organism, P2X4 is constitutively internalized and as a result, is found preferentially in intracellular compartments where it may have intracellular functions. In contrast, in various diseases such as chronic pain, ischemia and neurodegenerative diseases including Alzheimer Disease (AD) and ALS, surface P2X4 receptors are upregulated by mobilization of intracellular pools and/or de novo expression in glia or neurons. We have recently shown using P2X4 knock-out or our novel upregulated P2X4 knock-in mice, that P2X4 are instrumental for ALS pathogenesis and lifespan in a murine model of ALS, the SOD1 mice. In addition, we found that P2X4 surface trafficking is increased in both spinal MNs and microglia but also in peripheral macrophages in SOD1 mice compared to wild-type animals at pre- and symptomatic phases.
Hypotheses- Our data suggest that ATP and P2X4 receptors are attractive novel targets for understanding and fighting the ALS disease. Our innovative hypothesis, validated by our data is that misfolded protein accumulation in ALS such as mutant SOD1 proteins interferes with the internalization of P2X4 and leads to an increase in surface P2X4, first in spinal MNs and second in microglia during ALS. The increase in surface P2X4 in peripheral macrophages of SOD1 mice at pre-symptomatic phase leads to our second hypothesis: aberrant P2X4 expression in blood cells may represent a marker of ALS before the symptom onset. Our data show that surface/total ratio of human P2X4 can be measured by FACS analysis from peripheral blood of ALS patients.
Objectives- We propose using innovative transgenic mice and blood samples from ALS patients by behavioral, cellular, functional and biochemical approaches to:
1) unravel the cell-specific function of P2X4 in ALS pathogenesis, vulnerability of MNs, and neuroinflammation in SOD1 mice expressing conditional either knock-in non-internalized P2X4 (cP2X4KI) or knock-out for P2X4 selectively in macrophage/microglia or neurons and to determine how and when P2X4 can serve as therapeutic targets.
2) to make the proof of concept that detection of aberrant surface expression of human P2X4 in serum-derived macrophages from patients with ALS can serve as an early biomarker of the disease.
3) generalize the mechanism of P2X4 upregulation in several ALS models.
4) screen by functional assay in vitro the impact of several nanobodies on the function of human P2X4 or murine P2X4. Nanobodies modulating human P2X4 function may represent novel innovative drugs that could improve the patients’ quality of life by delaying the progression of ALS.
Impact- Our project fits very well within the orientation of CE 17- translational research in Health and the priority axis towards rare diseases. The medical and scientific complementary expertise of our consortium belonging to Bordeaux Neurocampus and the ALS reference center of Bordeaux should allow us to i) provide attractive novel strategy and cellular targets to fight against this devastating neurodegenerative disorder; ii), define P2X4 as a useful biomarker for ALS patients and develop a method for P2X4 detection from human peripheral blood; iii) identify new innovative drugs with the screening of camelid nanobodies.