AMPA Receptors Commandeer an Ancient Cargo Exporter for Use as an Auxiliary Subunit for Signaling

Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs). AMPARs are protein complexes of the pore-lining α-subunits GluA1-4 and auxiliary β-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstitution experiments, CNIH-2 promotes surface expression of GluAs and modulates their biophysical properties. However, its relevance in native AMPAR physiology remains controversial. Here, we have studied the role of CNIH-2 in GluA processing both in heterologous cells and primary rat neurons. Our data demonstrate that CNIH-2 serves an evolutionarily conserved role as a cargo exporter from the endoplasmic reticulum (ER). CNIH-2 cycles continuously between ER and Golgi complex to pick up cargo protein in the ER and then to mediate its preferential export in a coat protein complex (COP) II dependent manner. Interaction with GluA subunits breaks with this ancestral role of CNIH-2 confined to the early secretory pathway. While still taking advantage of being exported preferentially from the ER, GluAs recruit CNIH-2 to the cell surface. Thus, mammalian AMPARs commandeer CNIH-2 for use as a bona fide auxiliary subunit that is able to modify receptor signaling

TLR-4 deficiency protects against focal cerebral ischemia and axotomy-induced neurodegeneration

The pattern recognition receptor toll-like receptor (TLR)-4 mediates innate danger signaling in the brain, being activated in response to lipopolysaccharide. Until now, its role in the degenerating brain remained unknown. We here examined effects of a loss-of-function mutation of TLR-4 in mice submitted to transient focal cerebral ischemia and retinal ganglion cell (RGC) axotomy, which are highly reproducible and clinically relevant in vivo models of acute and subacute neuronal degeneration. We show that TLR-4 deficiency protects mice against ischemia and axotomy-induced RGC degeneration. Decreased phosphorylation levels of the mitogen-activated kinases ERK-1/-2, JNK-1/-2 and p38 together with reduced inducible NO synthase levels in injured neurons of TLR-4 mutant mice suggests that TLR-4 deficiency downscales parenchymal stress responses, thereby enhancing neuronal survival. At the same time, densities of MPO+ neutrophils and Iba1+ microglial cells were increased in the brains of TLR-4 mutant animals, pointing towards a futile inflammatory response aiming to compensate lost functions. Our data indicate that innate immunity may represent an attractive target for neuroprotective treatments in stroke and neurodegeneration

Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting

Multidimensional time‐correlated single photon counting (TCSPC) is based on the excitation of the sample by a high‐repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterized by its arrival time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two‐photon laser scanning microscope and a pulsed laser, multidimensional TCSPC makes a fluorescence lifetime technique with multiwavelength capability, near‐ideal counting efficiency, and the capability to resolve multiexponential decay functions. We show that the same technique and the same hardware can be used for precision fluorescence decay analysis and fluorescence correlation spectroscopy (FCS) in selected spots of a sample