The Endoplasmic Reticulum Resident Protein AGR3. Required for Regulation of Ciliary Beat Frequency in the Airway.

Protein disulfide isomerase (PDI) family members regulate protein folding and calcium homeostasis in the endoplasmic reticulum (ER). The PDI family member anterior gradient (AGR) 3 is expressed in the airway, but the localization, regulation, and function of AGR3 are poorly understood. Here we report that AGR3, unlike its closest homolog AGR2, is restricted to ciliated cells in the airway epithelium and is not induced by ER stress. Mice lacking AGR3 are viable and develop ciliated cells with normal-appearing cilia. However, ciliary beat frequency was lower in airways from AGR3-deficient mice compared with control mice (20% lower in the absence of stimulation and 35% lower after ATP stimulation). AGR3 deficiency had no detectable effects on ciliary beat frequency (CBF) when airways were perfused with a calcium-free solution, suggesting that AGR3 is required for calcium-mediated regulation of ciliary function. Decreased CBF was associated with impaired mucociliary clearance in AGR3-deficient airways. We conclude that AGR3 is a specialized member of the PDI family that plays an unexpected role in the regulation of CBF and mucociliary clearance in the airway.

AGR2 is induced in asthma and promotes allergen-induced mucin overproduction.

Mucins are gel-forming proteins that are responsible for the characteristic viscoelastic properties of mucus. Mucin overproduction is a hallmark of asthma, but the cellular requirements for airway mucin production are poorly understood. The endoplasmic reticulum (ER) protein anterior gradient homolog 2 (AGR2) is required for production of the intestinal mucin MUC2, but its role in the production of the airway mucins MUC5AC and MUC5B is not established. Microarray data were analyzed to examine the relationship between AGR2 and MUC5AC expression in asthma. Immunofluorescence was used to localize AGR2 in airway cells. Coimmunoprecipitation was used to identify AGR2-immature MUC5AC complexes. Agr2(-/-) mice were used to determine the role of AGR2 in allergic airway disease. AGR2 localized to the ER of MUC5AC- and MUC5B-producing airway cells and formed a complex with immature MUC5AC. AGR2 expression increased together with MUC5AC expression in airway epithelium from "Th2-high" asthmatics. Allergen-challenged Agr2(-/-) mice had greater than 50% reductions in MUC5AC and MUC5B proteins compared with allergen-challenged wild-type mice. Impaired mucin production in Agr2(-/-) mice was accompanied by an increase in the proportion of mucins contained within the ER and by evidence of ER stress in airway epithelium. This study shows that AGR2 increases with mucin overproduction in individuals with asthma and in mouse models of allergic airway disease. AGR2 interacts with immature mucin in the ER and loss of AGR2 impairs allergen-induced MUC5AC and MUC5B overproduction.

Fear learning induces persistent facilitation of amygdala synaptic transmission.

In the maintenance phase of fear memory, synaptic transmission is potentiated and the stimulus requirements and signalling mechanisms are altered for long-term potentiation (LTP) in the cortico-lateral amygdala (LA) pathway. These findings link amygdala synaptic plasticity to the coding of fear memories. Behavioural experiments suggest that the amygdala serves to store long-term fear memories. Here we provide electrophysiological evidence showing that synaptic alterations in rats induced by fear conditioning are evident in vitro 10 days after fear conditioning. We show that synaptic transmission was facilitated and that high-frequency stimulation dependent LTP (HFS-LTP) of the cortico-lateral amygdala pathway remained attenuated 10 days following fear conditioning. Additionally, we found that the low-frequency stimulation dependent LTP (LFS-LTP) measured 24 h after fear conditioning was absent 10 days post-training. The persistent facilitation of synaptic transmission and occlusion of HFS-LTP suggests that, unlike hippocampal coding of contextual fear memory, the cortico-lateral amygdala synapse is involved in the storage of long-term fear memories. However, the absence of LFS-LTP 10 days following fear conditioning suggests that amygdala physiology 1 day following fear learning may reflect a dynamic state during memory stabilization that is inactive during the long-term storage of fear memory. Results from these experiments have significant implications regarding the locus of storage for maladaptive fear memories and the synaptic alterations induced by these memories.

Fear memories induce a switch in stimulus response and signaling mechanisms for long-term potentiation in the lateral amygdala.

Activity-dependent modification of synapses is fundamental for information storage in the brain and underlies behavioral learning. Fear conditioning is a model of emotional memory and anxiety that is expressed as an enduring increase in synaptic strength in the lateral amygdala (LA). Here we analysed synaptic plasticity in the rat cortico-LA pathway during maintenance of fear memory. We show for the first time that the stimulus frequency for synaptic potentiation is switched during maintenance of fear memory, and the underlying signaling mechanisms are altered in the cortico-LA pathway. In slices from fear-conditioned animals, high-frequency stimulation-induced (HFS) long-term potentiation (LTP) was attenuated, whereas low-frequency stimulation (LFS) elicited a long-lasting potentiation. HFS generates robust LTP that is dependent on N-methyl-d-aspartate receptor (NMDAR) and L-type voltage-gated calcium channel (VGCC) activation in control animals, whereas in fear-conditioned animals HFS LTP is NMDAR- and VGCC-independent. LFS-LTP is partially NMDAR-dependent, but VGCCs are necessary for potentiation in fear memory. Collectively, these results show that during maintenance of fear memory the stimulus requirements for amygdala afferents and critical signaling mechanisms for amygdala synaptic potentiation are altered, suggesting that cue-engaged synaptic mechanisms in the amygdala are dramatically affected as a result of emotional learning.