The possibility that clathrin plays a role in the agonist-mediated sequestration of muscarinic cholinergic receptors in human SH-SY5Y neuroblastoma cells has been investigated by the application of ...experimental paradigms previously established to perturb clathrin distribution and receptor cycling events. Preincubation of SH-SY5Y cells under hypertonic conditions resulted in a pronounced inhibition of agonist-induced muscarinic receptor sequestration (70-80% at 550 mOsm), which was reversed when cells were returned to isotonic medium. Depletion of intracellular K+ or acidification of the cytosol also resulted in > 80% inhibition of muscarinic receptor sequestration. Under conditions of hypertonicity, depletion of intracellular K+, or acidification of cytosol, muscarinic receptor-stimulated phosphoinositide hydrolysis and Ca2+ signaling events were either unaffected or markedly less inhibited than receptor sequestration. That these same experimental conditions did perturb clathrin distribution was verified by immunofluorescence studies. Hypertonicity and depletion of intracellular K+ resulted in a pronounced accumulation of clathrin in the perinuclear region, whereas acidification of the cytosol resulted in the appearance of microaggregates of clathrin throughout the cytoplasm and at the plasma membrane. The results are consistent with the possibility that muscarinic receptors in SH-SY5Y cells are endocytosed via a clathrin-dependent mechanism.
The feasibility of using a permeabilized preparation of human SH-SY-5Y neuroblastoma cells for studies of muscarinic acetylcholine receptor (mAChR) sequestration has been evaluated. Exposure of cells ...permeabilized with digitonin, streptolysin-O, or the alpha-toxin from Staphylococcus aureus to oxotremorine-M (Oxo-M) for 30 min resulted in a 25-30% reduction in the number of cell surface mAChRs, as monitored by the loss of N3Hmethylscopolamine (3HNMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 microM digitonin, the Oxo-M-mediated reduction in 3HNMS binding was time (t1/2 approximately 5 min) and concentration (EC50 approximately 10 microM) dependent and was agonist specific (Oxo-M > bethanechol = arecoline = pilocarpine). In contrast, no reduction in total mAChR number, as monitored by the binding of 3Hquinuclidinyl benzilate, occurred following Oxo-M treatment. The loss of 3HNMS sites observed in the presence of Oxo-M was unaffected by omission of either ATP or Ca2+, both of which are required for stimulated phosphoinositide hydrolysis, but could be inhibited by the inclusion of guanosine 5'-O-(2-thiodiphosphate). mAChRs sequestered in response to Oxo-M addition were unmasked when the cells were permeabilized in the presence of higher concentrations of digitonin (80 microM).
Agonist occupancy of muscarinic cholinergic receptors in human SH-SY-5Y neuroblastoma cells elicited two kinetically distinct phases of phosphoinositide hydrolysis when monitored by either an ...increased mass of inositol 1,4,5-trisphosphate, or the accumulation of a total inositol phosphate fraction. Within 5s of the addition of the muscarinic agonist, oxotremorine-M, the phosphoinositide pool was hydrolyzed at a maximal rate of 9.5%/min. This initial phase of phosphoinositide hydrolysis was short-lived (t1/2 = 14s) and after 60s of agonist exposure, the rate of inositol lipid breakdown had declined to a steady state level of 3.4%/min which was then maintained for at least 5-10 min. This rapid, but partial, attenuation of muscarinic receptor stimulated phosphoinositide hydrolysis occurred prior to the agonist-induced internalization of muscarinic receptors.
To examine the possibility that NaF enhances phosphoinositide-specific phospholipase C (PIC) activity in neural tissues by a mechanism independent of a guanine nucleotide binding protein (Gp), we ...have evaluated the contribution of Gp activation to NaF-stimulated phosphoinositide hydrolysis in human SK-N-SH neuroblastoma cells. Addition of NaF to intact cells resulted in an increase in the release of inositol phosphates (450% of control values; EC50 of approximately 8 mM). Inclusion of U-73122, an aminosteroid inhibitor of guanine nucleotide-regulated PIC activity in these cells, resulted in a dose-dependent inhibition of NaF-stimulated inositol lipid hydrolysis (IC50 of approximately 3.5 microM). When added to digitonin-permeabilized cells, NaF or guanosine-5'-O-thiotriphosphate (GTP gamma S) resulted in a three- and sevenfold enhancement, respectively, of inositol phosphate release. In the combined presence of optimal concentrations of NaF and GTP gamma S, inositol phosphate release was less than additive, indicative of a common site of action. Inclusion of 2-5 mM concentrations of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) fully blocked phosphoinositide hydrolysis elicited by GTP gamma S, whereas that induced by NaF was partially inhibited (65%). However, preincubation of the cells with GDP beta S resulted in a greater reduction in the ability of NaF to stimulate inositol phosphate release (87% inhibition). Both GTP gamma S and NaF-stimulated inositol phosphate release were inhibited by inclusion of 10 microM U-73122 (54-71%). The presence of either NaF or GTP gamma S also resulted in a marked lowering of the Ca2+ requirement for activation of PIC in permeabilized cells. These results indicate that in SK-N-SH cells, little evidence exists for direct stimulation of PIC by NaF and that the majority of inositol phosphate release that occurs in the presence of NaF can be attributed to activation of Gp.
Huntington's disease (HD) is a rare neurodegenerative disease characterized by cognitive, behavioral and motor symptoms that progressively worsen with time. Cognitive and behavioral signs of HD are ...generally present in the years prior to a diagnosis; however, manifest HD is typically assessed by genetic confirmation and/or the presence of unequivocal motor symptoms. Nevertheless, there is a large variation in symptom severity and rate of progression among individuals with HD.
In this retrospective study, longitudinal natural history of disease progression was modeled in individuals with manifest HD from the global, observational Enroll-HD study (NCT01574053). Unsupervised machine learning (k-means; km3d) was used to jointly model clinical and functional disease measures simultaneously over time, based on one-dimensional clustering concordance such that individuals with manifest HD (
= 4,961) were grouped into three clusters: rapid (Cluster A; 25.3%), moderate (Cluster B; 45.5%) and slow (Cluster C; 29.2%) progressors. Features that were considered predictive of disease trajectory were then identified using a supervised machine learning method (XGBoost).
The cytosine adenine guanine-age product score (a product of age and polyglutamine repeat length) at enrollment was the top predicting feature for cluster assignment, followed by years since symptom onset, medical history of apathy, body mass index at enrollment and age at enrollment.
These results are useful for understanding factors that affect the global rate of decline in HD. Further work is needed to develop prognostic models of HD progression as these could help clinicians with individualized clinical care planning and disease management.
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