A Molecular Mechanism for Endocytic Recycling of the M5 Muscarinic Acetylcholine Receptor

Muscarinic acetylcholine receptors (MRs), a family of five G protein-coupled receptors (GPCRs), play an essential role in the regulation of mammalian physiology. In the brain, MR-mediated neurotransmission is required for the control of movement and motivated behavior by the basal ganglia, and MR dysfunction may contribute to schizophrenia, Alzheimerʼs disease, and motor disorders. Functional studies of the muscarinic receptors have been hampered by a lack of selective pharmacology, poor receptor immunoreactivity and a wide, overlapping pattern of expression. MRs are characterized by the presence of a large third intracellular loop domain (i3), the sequence of which is divergent between MR subtypes. The i3 is known to determine signaling and trafficking characteristics of GPCRs by binding to defined subsets of regulatory and effector proteins. In an effort to discover novel, subtype-specific muscarinic receptor regulatory mechanisms, we performed yeast two-hybrid proteinprotein interaction screens with the five MR i3 regions. An interaction between M5 and the Arf GAP protein AGAP1 was detected, and was observed to be specific to the M5 subtype. This interaction was confirmed in vitro, and was shown to mediate the binding of the AP-3 adaptor complex to the M5 i3. Immunocytochemical and live cell imaging of primary rat hippocampal neurons revealed co-localization of M5 and AGAP1- or AP-3- positive vesicles after treatment with a muscarinic agonist. Activity-induced receptor trafficking studies demonstrated that interaction with AGAP1 and activity of AP-3 were required for the endocytic recycling of M5 in neurons, the lack of which resulted in downregulation of cell surface receptor density. M5 has been shown to be expressed in the dopaminergic neurons of the ventral midbrain and to function in the presynaptic modulation of dopamine release in the striatum. Results from dopamine release studies suggest that the abrogation of AGAP1-mediated recycling decreases the magnitude of presynaptic M5-mediated release potentiation. Our study demonstrates a novel, neuronspecific trafficking function for AGAP1 and AP-3, and suggests the presence of a previously unknown receptor recycling pathway that may underlie mechanisms of sustained sensitivity of GPCRs

alpha-Synuclein promotes dilation of the exocytotic fusion pore

The protein α-synuclein has a central role in the pathogenesis of Parkinson's disease. Like that of other proteins that accumulate in neurodegenerative disease, however, the function of α-synuclein remains unknown. Localization to the nerve terminal suggests a role in neurotransmitter release, and overexpression inhibits regulated exocytosis, but previous work has failed to identify a clear physiological defect in mice lacking all three synuclein isoforms. Using adrenal chromaffin cells and neurons, we now find that both overexpressed and endogenous synuclein accelerate the kinetics of individual exocytotic events, promoting cargo discharge and reducing pore closure ('kiss-and-run'). Thus, synuclein exerts dose-dependent effects on dilation of the exocytotic fusion pore. Remarkably, mutations that cause Parkinson's disease abrogate this property of α-synuclein without impairing its ability to inhibit exocytosis when overexpressed, indicating a selective defect in normal function

A mouse model of autism implicates endosome pH in the regulation of presynaptic calcium entry.

Psychoactive compounds such as chloroquine and amphetamine act by dissipating the pH gradient across intracellular membranes, but the physiological mechanisms that normally regulate organelle pH remain poorly understood. Interestingly, recent human genetic studies have implicated the endosomal Na+/H+ exchanger NHE9 in both autism spectrum disorders (ASD) and attention deficit hyperactivity disorder (ADHD). Plasma membrane NHEs regulate cytosolic pH, but the role of intracellular isoforms has remained unclear. We now find that inactivation of NHE9 in mice reproduces behavioral features of ASD including impaired social interaction, repetitive behaviors, and altered sensory processing. Physiological characterization reveals hyperacidic endosomes, a cell-autonomous defect in glutamate receptor expression and impaired neurotransmitter release due to a defect in presynaptic Ca2+ entry. Acute inhibition of synaptic vesicle acidification rescues release but without affecting the primary defect due to loss of NHE9

Loss of α-Synuclein Does Not Affect Mitochondrial Bioenergetics in Rodent Neurons.

Increased α-synuclein (αsyn) and mitochondrial dysfunction play central roles in the pathogenesis of Parkinson's disease (PD), and lowering αsyn is under intensive investigation as a therapeutic strategy for PD. Increased αsyn levels disrupt mitochondria and impair respiration, while reduced αsyn protects against mitochondrial toxins, suggesting that interactions between αsyn and mitochondria influences the pathologic and physiologic functions of αsyn. However, we do not know if αsyn affects normal mitochondrial function or if lowering αsyn levels impacts bioenergetic function, especially at the nerve terminal where αsyn is enriched. To determine if αsyn is required for normal mitochondrial function in neurons, we comprehensively evaluated how lowering αsyn affects mitochondrial function. We found that αsyn knockout (KO) does not affect the respiration of cultured hippocampal neurons or cortical and dopaminergic synaptosomes, and that neither loss of αsyn nor all three (α, β and γ) syn isoforms decreased mitochondria-derived ATP levels at the synapse. Similarly, neither αsyn KO nor knockdown altered the capacity of synaptic mitochondria to meet the energy requirements of synaptic vesicle cycling or influenced the localization of mitochondria to dopamine (DA) synapses in vivo. Finally, αsyn KO did not affect overall energy metabolism in mice assessed with a Comprehensive Lab Animal Monitoring System. These studies suggest either that αsyn has little or no significant physiological effect on mitochondrial bioenergetic function, or that any such functions are fully compensated for when lost. These results implicate that αsyn levels can be reduced in neurons without impairing (or improving) mitochondrial bioenergetics or distribution

Cooperation, Norms, and Revolutions: A Unified Game-Theoretical Approach

Cooperation is of utmost importance to society as a whole, but is often challenged by individual self-interests. While game theory has studied this problem extensively, there is little work on interactions within and across groups with different preferences or beliefs. Yet, people from different social or cultural backgrounds often meet and interact. This can yield conflict, since behavior that is considered cooperative by one population might be perceived as non-cooperative from the viewpoint of another. To understand the dynamics and outcome of the competitive interactions within and between groups, we study game-dynamical replicator equations for multiple populations with incompatible interests and different power (be this due to different population sizes, material resources, social capital, or other factors). These equations allow us to address various important questions: For example, can cooperation in the prisoner's dilemma be promoted, when two interacting groups have different preferences? Under what conditions can costly punishment, or other mechanisms, foster the evolution of norms? When does cooperation fail, leading to antagonistic behavior, conflict, or even revolutions? And what incentives are needed to reach peaceful agreements between groups with conflicting interests? Our detailed quantitative analysis reveals a large variety of interesting results, which are relevant for society, law and economics, and have implications for the evolution of language and culture as well

The Function of α-Synuclein

Human genetics has indicated a causal role for the protein α-synuclein in the pathogenesis of familial Parkinson's disease (PD), and the aggregation of synuclein in essentially all patients with PD suggests a central role for this protein in the sporadic disorder. Indeed, the accumulation of misfolded α-synuclein now defines multiple forms of neural degeneration. Like many of the proteins that accumulate in other neurodegenerative disorders, however, the normal function of synuclein remains poorly understood. In this article, we review the role of synuclein at the nerve terminal and in membrane remodeling. We also consider the prion-like propagation of misfolded synuclein as a mechanism for the spread of degeneration through the neuraxis