As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D 1 and D 2 dopamine receptors. These proteinprotein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D 1 -green fluorescent protein and D 2 -yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D 1 and D 2 receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D 1 and D 2 receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.

Symptom onset in amyotrophic lateral sclerosis (ALS) may occur in the muscles of the limbs (spinal onset) or those of the head and neck (bulbar onset). Most preclinical studies have focused on spinal symptoms, despite the prevalence of and increased morbidity and mortality associated with bulbar disease. We measured lick rhythm and tongue force to evaluate bulbar disease in the SOD1-G93A rat model of familial ALS. Body weight and grip strength were measured concomitantly. Testing spanned the early (maturation), middle (pre-symptomatic), and late (symptomatic and end-stage) phases of the disease. We measured a persistent tongue motility deficit that became apparent in the early phase of the disease, providing behavioral evidence of bulbar pathology. At end-stage, however, cytochrome oxidase (CO) activity was normal in the hypoglossal nucleus, and in the tongue, neuromuscular innervation, citrate synthase (CS) protein levels and activity, and uncoupling protein 3 (UCP3) protein levels remained unchanged. Interestingly, significant denervation and atrophy were evident in the end-stage sternomastoid muscle, providing peripheral anatomical evidence of bulbar pathology. Changes in body weight and grip strength occurred in the late phase of the disease. Extensive atrophy and denervation were observed in the end-stage gastrocnemius muscle. In contrast to our findings in the tongue, CS protein levels were decreased in the extensor digitorum longus (EDL) and soleus, although CS activity was maintained or increased. UCP3 protein was decreased also in the EDL. These data provide evidence of differential effects in muscles that were more or less affected by disease.

Most preclinical studies of amyotrophic lateral sclerosis (ALS) have focused on spinal symptoms, despite the importance of bulbar deficits in progression of the disease. We sought to determine how bulbar deficits related to spinal deficits and survival in the SOD1-G93A rat model of ALS. We examined forelimb and hindlimb grip force and tongue motility in SOD1-G93A rats using statistical cluster analysis. Decrements in forelimb grip force, hindlimb grip force, and tongue motility were used to cluster affected rats into groups. The analysis clustered one group that exhibited primarily forelimb deficits (forelimb group) and a second group that exhibited forelimb and tongue motility deficits (forelimb + bulbar group). The analysis did not identify a distinct hindlimb phenotype group because all SOD1-G93A rats exhibited deficits in hindlimb grip force. Rats in the forelimb + bulbar group exhibited earlier and greater forelimb deficits, and earlier mortality than rats without bulbar deficits. Hindlimb deficits were similar in both groups. There was a significant correlation between forelimb grip force and tongue motility deficits, but not between forelimb and hindlimb deficits. These data extend clinical findings of a more rapid disease progression in individuals with bulbar symptoms to the SOD1-G93A rat model of ALS.

Dopamine receptors (DARs) do not exist as singular independent units within the synaptic membrane, but are part of large macromolecular complexes of interacting proteins. These interacting proteins influence the receptor in a variety of ways. Our current studies employ a co‐immunoprecipitation assay for DARs from transfected cell lines, coupled with mass spectrometry sequencing to identify interacting partners. One protein identified this way was sorting nexin‐25 (SNX25). Mammalian SNXs have been suggested to be involved in intracellular trafficking, internalization, and endosomal recycling or sorting. The physiological role of SNX25 is unknown. Using radioligand binding assays, we found that increasing the expression levels of SNX25 in HEK293 cells increased the amount of D1 and D2 receptor expressed. This increase in receptor expression was accompanied by an alteration in the subcellular distribution of the receptors as seen using confocal microscopy (D1 DAR) and intact cell binding assays (D2 DAR). SNX25 over‐expression also caused an increase in both D1 and D2 receptor‐mediated signaling in HEK293 cells. In contrast, SNX25 over‐expression does not appear to affect D1 DAR desensitization. Overall, these data suggest that SNX25 regulates the intracellular trafficking of D1 and D2 DARs. The mechanisms underlying these effects are being investigated. Supported by The NIH intramural program.

Rationale Orolingual motor deficits, such as dysarthria and dysphagia, contribute to increased morbidity and mortality in the elderly. In preclinical studies we and others have reported age-related decreases in tongue motility in both F344 and F344/BN rats. The fact that these deficits are associated with nigrostriatal dopamine (DA) tissue measures suggests that increasing dopamine function might normalize tongue motility. Objective The purpose of the current study was to determine whether two indirect dopamine agonists with locomotor-enhancing effects, d-amphetamine (amphetamine; 1 & 2 mg/kg) and GBR-12909 (5, 10, and 20 mg/kg), can improve tongue motility in aged F344/BN rats. Methods Young (6 months) and aged (30 months) F344/BN rats licked water from an isometric force disc so that tongue motility (licks/second) and tongue force could be measured as a function of age and drug dose. Results Consistent with our previous studies, tongue force was greater and tongue motility was lower in the aged group. Tongue motility was increased by amphetamine but not by GBR-12909. Amphetamine decreased peak tongue force, primarily in the young group. GBR-12909 did not affect tongue force. GBR-12909 increased the number of licks/session in the young group but not in the aged group, while amphetamine increased this measure in both groups. Conclusion These results demonstrate differential effects of these drugs on orolingual motor function and suggest that blocking DA uptake is insufficient to increase tongue motility in aging.

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