Increased surface P2X4 receptors by mutant SOD1 proteins contribute to ALS pathogenesis in SOD1-G93A mice.

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron (MN) disease characterized by protein misfolding and aggregation leading to cellular degeneration. So far neither biomarker, nor effective treatment has been found. ATP signaling and P2X4 receptors (P2X4) are upregulated in various neurodegenerative diseases. Here we show that several ALS-related misfolded proteins including mutants of SOD1 or TDP-43 lead to a significant increase in surface P2X4 receptor density and function in vitro. In addition, we demonstrate in the spinal the cord of SOD1-G93A (SOD1) mice that misfolded SOD1-G93A proteins directly interact with endocytic adaptor protein-2 (AP2); thus, acting as negative competitors for the interaction between AP2 and P2X4, impairing constitutive P2X4 endocytosis. The higher P2X4 surface density was particularly observed in peripheral macrophages of SOD1 mice before the onset and during the progression of ALS symptoms positioning P2X4 as a potential early biomarker for ALS. P2X4 expression was also upregulated in spinal microglia of SOD1 mice during ALS and affect microglial inflammatory responses. Importantly, we report using double transgenic SOD1 mice expressing internalization-defective P2X4mCherryIN knock-in gene or invalidated for the P2X4 gene that P2X4 is instrumental for motor symptoms, ALS progression and survival. This study highlights the role of P2X4 in the pathophysiology of ALS and thus its potential for the development of biomarkers and treatments. We also decipher the molecular mechanism by which misfolded proteins related to ALS impact P2X4 trafficking at early pathological stage in cells expressing-P2X4.

Increased surface P2X4 receptor regulates anxiety and memory in P2X4 internalization-defective knock-in mice.

ATP signaling and surface P2X4 receptors are upregulated selectively in neurons and/or glia in various CNS disorders including anxiety, chronic pain, epilepsy, ischemia, and neurodegenerative diseases. However, the cell-specific functions of P2X4 in pathological contexts remain elusive. To elucidate P2X4 functions, we created a conditional transgenic knock-in P2X4 mouse line (Floxed P2X4mCherryIN) allowing the Cre activity-dependent genetic swapping of the internalization motif of P2X4 by the fluorescent mCherry protein to prevent constitutive endocytosis of P2X4. By combining molecular, cellular, electrophysiological, and behavioral approaches, we characterized two distinct knock-in mouse lines expressing noninternalized P2X4mCherryIN either exclusively in excitatory forebrain neurons or in all cells natively expressing P2X4. The genetic substitution of wild-type P2X4 by noninternalized P2X4mCherryIN in both knock-in mouse models did not alter the sparse distribution and subcellular localization of P2X4 but increased the number of P2X4 receptors at the surface of the targeted cells mimicking the pathological increased surface P2X4 state. Increased surface P2X4 density in the hippocampus of knock-in mice altered LTP and LTD plasticity phenomena at CA1 synapses without affecting basal excitatory transmission. Moreover, these cellular events translated into anxiolytic effects and deficits in spatial memory. Our results show that increased surface density of neuronal P2X4 contributes to synaptic deficits and alterations in anxiety and memory functions consistent with the implication of P2X4 in neuropsychiatric and neurodegenerative disorders. Furthermore, these conditional P2X4mCherryIN knock-in mice will allow exploring the cell-specific roles of P2X4 in various physiological and pathological contexts.

Dynamic Uni- and Multicellular Patterns Encode Biphasic Activity in Pancreatic Islets.

Biphasic secretion is an autonomous feature of many endocrine micro-organs to fulfill physiological demands. The biphasic activity of islet ß-cells maintains glucose homeostasis and is altered in type 2 diabetes. Nevertheless, underlying cellular or multicellular functional organizations are only partially understood. High-resolution noninvasive multielectrode array recordings permit simultaneous analysis of recruitment, of single-cell, and of coupling activity within entire islets in long-time experiments. Using this unbiased approach, we addressed the organizational modes of both first and second phase in mouse and human islets under physiological and pathophysiological conditions. Our data provide a new uni- and multicellular model of islet ß-cell activation: during the first phase, small but highly active ß-cell clusters are dominant, whereas during the second phase, electrical coupling generates large functional clusters via multicellular slow potentials to favor an economic sustained activity. Postprandial levels of glucagon-like peptide 1 favor coupling only in the second phase, whereas aging and glucotoxicity alter coupled activity in both phases. In summary, biphasic activity is encoded upstream of vesicle pools at the micro-organ level by multicellular electrical signals and their dynamic synchronization between ß-cells. The profound alteration of the electrical organization of islets in pathophysiological conditions may contribute to functional deficits in type 2 diabetes.

Implication of Neuronal Versus Microglial P2X4 Receptors in Central Nervous System Disorders.

The P2X4 receptor (P2X4) is an ATP-gated cation channel that is highly permeable to Ca2+ and widely expressed in neuronal and glial cell types throughout the central nervous system (CNS). A growing body of evidence indicates that P2X4 plays key roles in numerous central disorders. P2X4 trafficking is highly regulated and consequently in normal situations, P2X4 is present on the plasma membrane at low density and found mostly within intracellular endosomal/lysosomal compartments. An increase in the de novo expression and/or surface density of P2X4 has been observed in microglia and/or neurons during pathological states. This review aims to summarize knowledge on P2X4 functions in CNS disorders and provide some insights into the relative contributions of neuronal and glial P2X4 in pathological contexts. However, determination of the cell-specific functions of P2X4 along with its intracellular and cell surface roles remain to be elucidated before its potential as a therapeutic target in multiple disorders can be defined.

P2X Electrophysiology and Surface Trafficking in Xenopus Oocytes.

Xenopus oocytes serve as a standard heterologous expression system for the study of various ligand-gated ion channels including ATP P2X receptors. Here we describe the whole-cell two-electrode voltage clamp and biotinylation/Western blotting techniques to investigate the functional properties and surface trafficking from P2X-expressing oocytes.