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Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.

ABSTRACT: We employed a pH-sensitive GFP analog, superecliptic phluorin, to observe aspects of nicotinic acetylcholine receptor (nAChR) trafficking to the plasma membrane (PM) in cultured mouse cortical neurons. The experiments exploit differences in the pH among endoplasmic reticulum (ER), trafficking vesicles, and the extracellular solution. The data confirm that few ?4?4 nAChRs, but many ?4?2 nAChRs, remain in neutral intracellular compartments, mostly the ER. We observed fusion events between nAChR-containing vesicles and PM; these could be quantified in the dendritic processes. We also studied the ?4R348C polymorphism, linked to amyotrophic lateral sclerosis (ALS). This mutation depressed fusion rates of ?4?4 receptor-containing vesicles with the PM by ?2-fold, with only a small decrease in the number of nAChRs per vesicle. The mutation also decreased the number of ER exit sites, showing that the reduced receptor insertion results from a change at an early stage in trafficking. We confirm the previous report that the mutation leads to reduced agonist-induced currents; in the cortical neurons studied, the reduction amounts to 2-3-fold. Therefore, the reduced agonist-induced currents are caused by the reduced number of ?4?4-containing vesicles reaching the membrane. Chronic nicotine exposure (0.2 ?M) did not alter the PM insertion frequency or trafficking behavior of ?4?4-laden vesicles. In contrast, chronic nicotine substantially increased the number of ?4?2-containing vesicle fusions at the PM; this stage in ?4?2 nAChR up-regulation is presumably downstream from increased ER exit. Superecliptic phluorin provides a tool to monitor trafficking dynamics of nAChRs in disease and addiction.

SUBMITTER: Richards CI

PROVIDER: S-EPMC3173132 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.

Richards Christopher I CI Srinivasan Rahul R Xiao Cheng C Mackey Elisha D W ED Miwa Julie M JM Lester Henry A HA

The Journal of biological chemistry 20110718 36

We employed a pH-sensitive GFP analog, superecliptic phluorin, to observe aspects of nicotinic acetylcholine receptor (nAChR) trafficking to the plasma membrane (PM) in cultured mouse cortical neurons. The experiments exploit differences in the pH among endoplasmic reticulum (ER), trafficking vesicles, and the extracellular solution. The data confirm that few α4β4 nAChRs, but many α4β2 nAChRs, remain in neutral intracellular compartments, mostly the ER. We observed fusion events between nAChR-co ...[more]

PMID: 21768117

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Project description:Rationale: The anatomical substrate of skeletal muscle autonomic innervation has remained underappreciated since it was described many decades ago. As such, the structural and functional features of muscle sympathetic innervation are largely undetermined in both physiology and pathology, mainly due to methodological limitations in the histopathological analysis of small neuronal fibers in tissue samples. Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease which mainly targets motor neurons, and despite autonomic symptoms occurring in a significant fraction of patients, peripheral sympathetic neurons (SNs) are generally considered unaffected and, as such, poorly studied. Purpose: In this research, we compared sympathetic innervation of normal and ALS muscles, through structural analysis of the sympathetic network in human and murine tissue samples. Methods and Results: We first refined tissue processing to circumvent methodological limitations interfering with the detection of muscle sympathetic innervation. The optimized "Neuro Detection Protocol" (NDP) was validated in human muscle biopsies, demonstrating that SNs innervate, at high density, both blood vessels and skeletal myofibers, independent of the fiber metabolic type. Subsequently, NDP was exploited to analyze sympathetic innervation in muscles of SOD1G93A mice, a preclinical ALS model. Our data show that ALS murine muscles display SN denervation, which has already initiated at the early disease stage and worsened during aging. SN degeneration was also observed in muscles of MLC/SOD1G93A mice, with muscle specific expression of the SOD1G93A mutant gene. Notably, similar alterations in SNs were observed in muscle biopsies from an ALS patient, carrying the SOD1G93A mutation. Conclusion: We set up a protocol for the analysis of murine and, more importantly, human muscle sympathetic innervation. Our results indicate that SNs are additional cell types compromised in ALS and suggest that dysfunctional SOD1G93A muscles affect their sympathetic innervation.

Dataset Information

Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.

Publications

Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.

Similar Datasets

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