Regulatory role of endoplasmic reticulum resident chaperone protein ERp29 in anti-murine ß-coronavirus host cell response.

Gap junctional intercellular communication (GJIC) involving astrocytes is important for proper CNS homeostasis. As determined in our previous studies, trafficking of the predominant astrocyte GJ protein, Connexin43 (Cx43), is disrupted in response to infection with a neurotropic murine ß-coronavirus (MHV-A59). However, how host factors are involved in Cx43 trafficking and the infection response is not clear. Here, we show that Cx43 retention due to MHV-A59 infection was associated with increased ER stress and reduced expression of chaperone protein ERp29. Treatment of MHV-A59-infected astrocytes with the chemical chaperone 4-sodium phenylbutyrate increased ERp29 expression, rescued Cx43 transport to the cell surface, increased GJIC, and reduced ER stress. We obtained similar results using an astrocytoma cell line (delayed brain tumor) upon MHV-A59 infection. Critically, delayed brain tumor cells transfected to express exogenous ERp29 were less susceptible to MHV-A59 infection and showed increased Cx43-mediated GJIC. Treatment with Cx43 mimetic peptides inhibited GJIC and increased viral susceptibility, demonstrating a role for intercellular communication in reducing MHV-A59 infectivity. Taken together, these results support a therapeutically targetable ERp29-dependent mechanism where ß-coronavirus infectivity is modulated by reducing ER stress and rescuing Cx43 trafficking and function.

Microtubule-assisted altered trafficking of astrocytic gap junction protein connexin 43 is associated with depletion of connexin 47 during mouse hepatitis virus infection.

Gap junctions (GJs) are important for maintenance of CNS homeostasis. GJ proteins, connexin 43 (Cx43) and connexin 47 (Cx47), play a crucial role in production and maintenance of CNS myelin. Cx43 is mainly expressed by astrocytes in the CNS and forms gap junction intercellular communications between astrocytes-astrocytes (Cx43-Cx43) and between astrocytes-oligodendrocytes (Cx43-Cx47). Mutations of these connexin (Cx) proteins cause dysmyelinating diseases in humans. Previously, it has been shown that Cx43 localization and expression is altered due to mouse hepatitis virus (MHV)-A59 infection both in vivo and in vitro; however, its mechanism and association with loss of myelin protein was not elaborated. Thus, we explored potential mechanisms by which MHV-A59 infection alters Cx43 localization and examined the effects of viral infection on Cx47 expression and its association with loss of the myelin marker proteolipid protein. Immunofluorescence and total internal reflection fluorescence microscopy confirmed that MHV-A59 used microtubules (MTs) as a conduit to reach the cell surface and restricted MT-mediated Cx43 delivery to the cell membrane. Co-immunoprecipitation experiments demonstrated that Cx43-ß-tubulin molecular interaction was depleted due to protein-protein interaction between viral particles and MTs. During acute MHV-A59 infection, oligodendrocytic Cx47, which is mainly stabilized by Cx43 in vivo, was down-regulated, and its characteristic staining remained disrupted even at chronic phase. The loss of Cx47 was associated with loss of proteolipid protein at the chronic stage of MHV-A59 infection.

Association of connexin36 with adherens junctions at mixed synapses and distinguishing electrophysiological features of those at mossy fiber terminals in rat ventral hippocampus.

BACKGROUND: Granule cells in the hippocampus project axons to hippocampal CA3 pyramidal cells where they form large mossy fiber terminals. We have reported that these terminals contain the gap junction protein connexin36 (Cx36) specifically in the stratum lucidum of rat ventral hippocampus, thus creating morphologically mixed synapses that have the potential for dual chemical/electrical transmission. METHODOLOGY: Here, we used various approaches to characterize molecular and electrophysiological relationships between the Cx36-containing gap junctions at mossy fiber terminals and their postsynaptic elements and to examine molecular relationships at mixed synapses in the brainstem. RESULTS: In rat and human ventral hippocampus, many of these terminals, identified by their selective expression of vesicular zinc transporter-3 (ZnT3), displayed multiple, immunofluorescent Cx36-puncta representing gap junctions, which were absent at mossy fiber terminals in the dorsal hippocampus. In rat, these were found in close proximity to the protein constituents of adherens junctions (i.e., N-cadherin and nectin-1) that are structural hallmarks of mossy fiber terminals, linking these terminals to the dendritic shafts of CA3 pyramidal cells, thus indicating the loci of gap junctions at these contacts. Cx36-puncta were also associated with adherens junctions at mixed synapses in the brainstem, supporting emerging views of the structural organization of the adherens junction-neuronal gap junction complex. Electrophysiologically induced long-term potentiation (LTP) of field responses evoked by mossy fiber stimulation was greater in the ventral than dorsal hippocampus. CONCLUSIONS: The electrical component of transmission at mossy fiber terminals may contribute to enhanced LTP responses in the ventral hippocampus.

Connexin36 localization along axon initial segments in the mammalian CNS.

Electrical synapses formed by gap junctions occur at a variety of neuronal subcellular sites in the mammalian central nervous system (CNS), including at somatic, dendritic and axon terminal compartments. Numerous electrophysiological studies using mice and rats, as well as computer modelling approaches, have predicted the additional occurrence of electrical synapses between axons near their emergence from neuronal somata. Here, we used immunofluorescence methods to search for localization of the neuronal gap junction-forming protein connexin36 (Cx36) along axon initial segments (AISs) labelled for the AIS marker ankyrinG. Immunofluorescent Cx36-puncta were found to be associated with AISs in several CNS regions of mice, including the spinal cord, inferior olive and cerebral cortex. Localization of Cx36-puncta at AISs was confirmed by confocal single scan and 3D imaging, immunofluorescence intensity profiling and high resolution structured illumination microscopy (SIM). AISs measuring up to 30 µm in length displayed typically a single Cx36-punctum and the incidence of these long AISs displaying Cx36-puncta ranged from 3% to 7% in the inferior olive and in various layers of the cerebral cortex. In the inferior olive, the gap junction associated protein zonula occludens-1 (ZO-1) was found to be co-localized with Cx36-puncta on AISs, indicating that these puncta have some of the molecular constituents of gap junctions. Our results add to the neuronal subcellular locations at which Cx36 is deployed, and raise possibilities for its involvement in novel functions in the AIS compartment.

Perception of Consonants in Speech-Shaped Noise among Young and Middle-Aged Adults.

OBJECTIVE: The present study was carried out to compare the consonant perception of young and middle-aged adults in quiet and noisy listening conditions. MATERIALS AND METHODS: Twenty-nine adults aged between 18 and 55 years old participated in the study, and were separated into two groups based on their age: Group I, comprising 15 young adults aged between 18 and 40 years old, and Group II, comprising 14 middle-aged adults aged between 41 and 55 years old. All the participants had normal hearing sensitivity in both ears. RESULTS: Consonant perception was better in favorable listening conditions for both young and middle-aged adults. Comparison of the consonant identification scores of young and middle-aged adults showed significantly poorer scores among middle-aged adults in both quiet and noisy listening conditions. CONCLUSION: The findings of the present study reveal that middle-aged adults have small but significant consonant perception difficulties compared to younger adults in quiet and noisy listening conditions.