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MSTPublications: November 2023

Posted by on Wednesday, November 29, 2023 in New Publications .

Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of raptor and this contributes to the weight loss effect of liraglutide.
Le TDV, Liu D, Besing GK, Raghavan R, Ellis BJ, Ceddia RP, Collins S, Ayala JE.
Elife. 2023 Nov 6;12:e80944. doi: 10.7554/eLife.80944. Online ahead of print.

The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser791 following β-adrenergic stimulation. The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.


Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles.
Richardson TM*, Pettway YD*, Walker JT, Nelson HA, Ishahak M, Poffenberger G, Aramandla R, Reihsmann C, Agarwal A, Powers AC, Brissova M.
J Vis Exp. 2023 Nov 3;(201). doi: 10.3791/65259.

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.


Cervical spinal cord susceptibility-weighted MRI at 7T: Application to multiple sclerosis.
Clarke MA*, Witt AA*, Robison RK, Fleishman S, Combes AJE, Houston D, Prock LE, Sweeney G, O’Grady KP, McKnight CD, Smith SA.
Neuroimage. 2023 Nov 17;284:120460. doi: 10.1016/j.neuroimage.2023.120460. Online ahead of print.

Background: Susceptibility-weighted imaging (SWI) has been extensively studied in the brain and in diseases of the central nervous system such as multiple sclerosis (MS) providing unique opportunities to visualize cerebral vasculature and disease-related pathology, including the central vein sign (CVS) and paramagnetic rim lesions (PRLs). However, similar studies evaluating SWI in the spinal cord of patients with MS remain severely limited.
Purpose: Based on our previous findings of enlarged spinal vessels in MS compared to healthy controls (HCs), we developed high-field SWI acquisition and processing methods for the cervical spinal cord with application in people with MS (pwMS) and HCs. Here, we demonstrate the vascular variability between the two cohorts and unique MS lesion features in the cervical cord.
Methods: In this retrospective, exploratory pilot study conducted between March 2021 and March 2022, we scanned 12 HCs and 9 pwMS using an optimized non-contrast 2D T2*-weighted gradient echo sequence at 7 tesla. The overall appearance of the white and gray matter as well as tissue vasculature were compared between the two cohorts and areas of MS pathology in the patient group were assessed using both the magnitude and processed SWI images.
Results: We show improved visibility of vessels and more pronounced gray and white matter contrast in the MS group compared to HCs, hypointensities surrounding the cord in the MS cohort, and identify signal changes indicative of the CVS and paramagnetic rims in 66 % of pwMS with cervical spinal lesions.
Conclusion: In this first study of SWI at 7T in the human spinal cord, SWI holds promise in advancing our understanding of disease processes in the cervical cord in MS.

Targeting hedgehog-driven mechanisms of drug-resistant cancers.
Miller JS, Bennett NE, Rhoades JA.
Front Mol Biosci. 2023 Oct 23;10:1286090. doi: 10.3389/fmolb.2023.1286090. eCollection 2023. Review.

​​Time-resolved live-cell spectroscopy reveals EphA2 multimeric assembly.
Shi X, Lingerak R, Herting CJ, Ge Y, Kim S, Toth P, Wang W, Brown BP, Meiler J, Sossey-Alaoui K, Buck M, Himanen J, Hambardzumyan D, Nikolov DB, Smith AW, Wang B.
Science. 2023 Nov 16:eadg5314. doi: 10.1126/science.adg5314. Online ahead of print.

Longer Screws Decrease the Risk of Radiographic Pseudarthrosis Following Elective Anterior Cervical Discectomy and Fusion.
Chanbour H, Bendfeldt GA, Johnson GW, Peterson K, Ahluwalia R, Younus I, Longo M, Abtahi AM, Stephens BF, Zuckerman SL.
Global Spine J. 2023 Nov 11:21925682231214361. doi: 10.1177/21925682231214361. Online ahead of print.

Jedi-1/MEGF12-mediated phagocytosis controls the pro-neurogenic properties of microglia in the ventricular-subventricular zone.
Morrison V, Houpert M, Trapani J, Brockman A, Kingsley P, Katdare K, Layden H, Nguena-Jones G, Trevisan A, Maguire-Zeiss K, Marnett L, Bix G, Ihrie R, Carter B.
Cell Rep. 2023 Nov 10;42(11):113423. doi: 10.1016/j.celrep.2023.113423. Online ahead of print.