Parkinson's disease (PD) is a basal ganglia movement disorder characterized by progressive degeneration of the nigrostriatal dopaminergic system. Immunohistochemical methods have been widely used for characterization of dopaminergic neuronal injury in animal models of PD, including the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model. However, conventional immunohistochemical techniques applied to tissue sections have inherent limitations with respect to loss of 3D resolution, yielding insufficient information on the architecture of the dopaminergic system. To provide a more comprehensive and non-biased map of MPTP-induced changes in central dopaminergic pathways, we used iDISCO immunolabeling, light-sheet fluorescence microscopy (LSFM) and deep-learning computational methods for whole-brain three-dimensional visualization and automated quantitation of tyrosine hydroxylase (TH)-positive neurons in the adult mouse brain. Mice terminated 7 days after acute MPTP administration demonstrated widespread alterations in TH expression. Compared to vehicle controls, MPTP-dosed mice showed a significant loss of TH-positive neurons in the substantia nigra pars compacta and ventral tegmental area. Also, MPTP dosing reduced overall TH signal intensity in basal ganglia nuclei, i.e. the substantia nigra, caudate-putamen, globus pallidus and subthalamic nucleus. In contrast, increased TH signal intensity was predominantly observed in limbic regions, including several subdivisions of the amygdala and hypothalamus. In conclusion, mouse whole-brain 3D imaging is ideal for unbiased automated counting and densitometric analysis of TH-positive cells. The LSFM–deep learning pipeline tracked brain-wide changes in catecholaminergic pathways in the MPTP mouse model of PD, and may be applied for preclinical characterization of compounds targeting dopaminergic neurotransmission.

Objective The development of effective anti-obesity therapeutics relies heavily on the ability to target specific brain homeostatic and hedonic mechanisms controlling body weight. To obtain further insight into neurocircuits recruited by anti-obesity drug treatment, the present study aimed to determine whole-brain activation signatures of six different weight-lowering drug classes. Methods Chow-fed C57BL/6J mice (n = 8 per group) received acute treatment with lorcaserin (7 mg/kg; i.p.), rimonabant (10 mg/kg; i.p.), bromocriptine (10 mg/kg; i.p.), sibutramine (10 mg/kg; p.o.), semaglutide (0.04 mg/kg; s.c.) or setmelanotide (4 mg/kg; s.c.). Brains were sampled two hours post-dosing and whole-brain neuronal activation patterns were analysed at single-cell resolution using c-Fos immunohistochemistry and automated quantitative three-dimensional (3D) imaging. Results The whole-brain analysis comprised 308 atlas-defined mouse brain areas. To enable fast and efficient data mining, a web-based 3D imaging data viewer was developed. All weight-lowering drugs demonstrated brain-wide responses with notable similarities in c-Fos expression signatures. Overlapping c-Fos responses were detected in discrete homeostatic and non-homeostatic feeding centres located in the dorsal vagal complex and hypothalamus with concurrent activation of several limbic structures as well as the dopaminergic system. Conclusions Whole-brain c-Fos expression signatures of various weight-lowering drug classes point to a discrete set of brain regions and neurocircuits which could represent key neuroanatomical targets for future anti-obesity therapeutics.

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Objective Obesity-linked type 2 diabetes (T2D) is a worldwide health concern and many novel approaches are being considered for its treatment and subsequent prevention of serious comorbidities. Co-administration of glucagon like peptide 1 (GLP-1) and peptide YY 3-36 (PYY 3-36 ) renders a synergistic decrease in energy intake in obese men. However, mechanistic details of the synergy between these peptide agonists and their effects on metabolic homeostasis remain relatively scarce. Methods In this study, we utilized long-acting analogues of GLP-1 and PYY 3-36 (via Fc-peptide conjugation) to better characterize the synergistic pharmacological benefits of their co-administration on body weight and glycaemic regulation in obese and diabetic mouse models. Hyperinsulinemic-euglycemic clamps were used to measure weight-independent effects of Fc-PYY 3-36 + Fc-GLP-1 on insulin action. Fluorescent light sheet microscopy analysis of whole brain was performed to assess activation of brain regions. Results Co-administration of long-acting Fc-IgG/peptide conjugates of Fc-GLP-1 and Fc-PYY 3-36 (specific for PYY receptor-2 (Y2R)) resulted in profound weight loss, restored glucose homeostasis, and recovered endogenous β-cell function in two mouse models of obese T2D. Hyperinsulinemic-euglycemic clamps in C57BLKS/J db/db and diet-induced obese Y2R-deficient (Y2RKO) mice indicated Y2R is required for a weight-independent improvement in peripheral insulin sensitivity and enhanced hepatic glycogenesis. Brain cFos staining demonstrated distinct temporal activation of regions of the hypothalamus and hindbrain following Fc-PYY 3-36 + Fc-GLP-1R agonist administration. Conclusions These results reveal a therapeutic approach for obesity/T2D that improved insulin sensitivity and restored endogenous β-cell function. These data also highlight the potential association between the gut–brain axis in control of metabolic homeostasis.

Prolactin-releasing peptide (PrRP) is an endogenous neuropeptide involved in appetite regulation and energy homeostasis. PrRP binds with high affinity to G-protein coupled receptor 10 (GPR10) and with lesser activity towards the neuropeptide FF receptor type 2 (NPFF2R). The present study aimed to develop long-acting PrRP31 analogues with potent anti-obesity efficacy. A comprehensive series of C18 lipidated PrRP31 analogues was characterized in vitro and analogues with various GPR10 and NPFF2R activity profiles were profiled for bioavailability and metabolic effects following subcutaneous administration in diet-induced obese (DIO) mice. PrRP31 analogues acylated with a C18 lipid chain carrying a terminal acid (C18 diacid) were potent GPR10-selective agonists and weight-neutral in DIO mice. In contrast, acylation with aliphatic C18 lipid chain (C18) resulted in dual GPR10-NPFF2R co-agonists that suppressed food intake and promoted a robust weight loss in DIO mice, which was sustained for at least one week after last dosing. Rapid in vivo degradation of C18 PrRP31 analogues gave rise to circulating lipidated PrRP metabolites maintaining dual GPR10-NPFF2R agonist profile and long-acting anti-obesity efficacy in DIO mice. Combined GPR10 and NPFF2R activation may therefore be a critical mechanism for obtaining robust anti-obesity efficacy of PrRP31 analogues.

Background: The development of effective anti-obesity therapeutics relies heavily on the ability to target CNS signaling mechanisms critically involved in the homeostatic control of body weight. To get insight into neurocircuits recruited by anti-obesity drug treatment, the present study aimed to determine whole-brain activation signatures of six different weight-lowering drug classes with documented efficacy in humans. Methods: Chow-fed mice received a single dose of lorcaserin (7 mg/kg, i.p.), rimonabant (10 mg/kg, i.p.), bromocriptine (D2 receptor agonist, 10 mg/kg, i.p.), sibutramine (10 mg/kg, p.o.), semaglutide (0.04 mg/kg, s.c.) or setmelanotide (4 mg/kg, s.c.). Brains were sampled two hours post-dosing, immunolabelled using c-Fos expression as a proxy for neuronal stimulation. Cleared whole-brains were imaged using light sheet fluorescence microscopy-deep learning pipeline enabling fully automated 3D mapping and quantitation of brain activation patterns at single-cell resolution. Results: The whole-brain analysis comprised 308 atlas-defined mouse brain areas. To enable fast and efficient data mining, an open-access web-based 3D imaging data viewer (NeuroPedia.dk) was developed. All weight-lowering drugs demonstrated brain-wide responses with notable similarities in c-Fos expression signatures. Overlapping c-Fos responses were detected in discrete homeostatic and non-homeostatic feeding centres located in the dorsal vagal complex and hypothalamus with concurrent activation of several limbic structures as well as the dopaminergic system. Conclusion: We pinpoint several overlapping brain activation signatures of various weight-lowering drugs. This shared feature suggests that weight-lowering drugs stimulate distinct homeostatic and non-homeostatic feeding centres. Future centrally acting anti-obesity compounds may be specifically designed to target key components of this neurocircuitry framework to provide more effective and sustained weight loss in obese patients. Disclosure L.S.Dalboege: Employee; Gubra aps. J.Hecksher-sørensen: Employee; Gubra ApS. M.Tozzi: None. C.G.Salinas: Employee; Gubra. T.Porsgaard: Employee; Gubra. D.D.Thorbek: Employee; Gubra APS. J.L.Skytte: Employee; Gubra ApS. U.Roostalu: Employee; Gubra. H.H.Hansen: None. J.Perens: Employee; Gubra ApS.

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