Epigenetic silencing of triple negative breast cancer hallmarks by Withaferin A Epigenetic silencing of triple negative breast cancer hallmarks by Withaferin A

G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR–GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein–protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR–GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR–GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays.

INTRODUCTION: The locus coeruleus (LC), the brain's primary source of noradrenaline (NA), undergoes early neurodegeneration in Parkinson's disease (PD), Alzheimer's diseases (AD), and Down syndrome (DS); however, differences have not been examined in parallel.METHODS: Post mortem brains (n = 67) from individuals with AD, DS‐AD, and PD without and with dementia (PD‐D) and controls were analyzed for amyloid beta (Aβ), phosphorylated tau (pTau), α‐synuclein, endo‐lysosomal alterations, biogenic amines, and selective biomarkers.RESULTS: LC degeneration correlated with age, peaking in AD and PD‐D, while NA and dopaminergic metabolites were significantly reduced only in PD‐D. DS‐AD, the youngest group, showed the highest Aβ and pTau levels but the least noradrenergic neuron loss. We demonstrated for the first time that endosomal alterations were present in AD, lysosomal changes were present in PD‐D/DS‐AD, and DYRK1A, a key protein from chromosome 21, was elevated only in DS‐AD.DISCUSSION: Loss of noradrenergic neurons may occur independently of amyloid and tau pathologies.Highlights: We provide the first analysis of neuropathological and biochemical features including biogenic amines of the LC in AD, DS, and PD. Loss of noradrenergic neurons was most severe in AD and PD. Only in DS, levels of DYRK1A – a kinase encoded on chromosome 21 and implicated in neurodegenerative processes – were elevated and negatively correlated to biogenic amine levels. Although individuals with DS having AD were the youngest group, they had the highest levels of amyloid and tau pathologies, but less noradrenergic neurons loss compared to other disease groups.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor control deficits, which is associated with the loss of striatal dopaminergic neurons from the substantia nigra. In parallel to dopaminergic denervation, there is an increase of acetylcholine within the striatum, resulting in a striatal dopaminergic–cholinergic neurotransmission imbalance. Currently, available PD pharmacotherapy (e.g., prodopaminergic drugs) does not reinstate the altered dopaminergic–cholinergic balance. In addition, it can eventually elicit cholinergic-related adverse effects. Here, we investigated the interplay between dopaminergic and cholinergic systems by assessing the physical and functional interaction of dopamine D₂ and muscarinic acetylcholine M₁ receptors (D₂R and M₁R, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of D₂R–M₁R complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET¹ and NanoBiT^®) assays, performed in transiently transfected HEK293T cells. Subsequently, a D₂R–M₁R co-distribution in the mouse striatum was observed through double-immunofluorescence staining and AlphaLISA^® immunoassay. Finally, we evaluated the functional interplay between both receptors via behavioral studies, by implementing the classical acute reserpine pharmacological animal model of experimental parkinsonism. Reserpinized mice were administered with a D₂R-selective agonist (sumanirole) and/or an M₁R-selective antagonist (VU0255035), and alterations in PD-related behavioral tasks (i.e., locomotor activity) were evaluated. Importantly, VU0255035 (10 mg/kg) potentiated the antiparkinsonian-like effects (i.e., increased locomotor activity and decreased catalepsy) of an ineffective sumanirole dose (3 mg/kg). Altogether, our data suggest the existence of putative striatal D₂R/M₁R heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects.

The adenosine A_2A receptor (A_2AR), dopamine D₂ receptor (D₂R) and metabotropic glutamate receptor type 5 (mGluR₅) form A_2AR-D₂R-mGluR₅ heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A_2AR protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D₂R-mGluR₅ heteromeric component of the A_2AR-D₂R-mGluR₅ complex in vitro and in vivo. The A_2AR and mGluR₅ protomers interact and modulate D₂R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A_2AR in HEK293T cells co-expressing D₂R and mGluR₅ resulted in a significant and marked increase in the formation of the D₂R-mGluR₅ heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D₂R (D2R_{Ki High)} values was found upon cotreatment with the mGluR₅ and A_2AR agonists in the cells expressing A_2AR, D₂R and mGluR₅ with a significant effect observed also with the mGluR₅ agonist alone compared to cells expressing only D₂R and mGluR₅. In cells co-expressing A_2AR, D₂R and mGluR₅, stimulation of the cells with an mGluR₅ agonist like or D₂R antagonist fully counteracted the D₂R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR₅ and D₂R. In agreement, the mGluR₅-negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A_2AR knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR₅ and A_2AR in enhancing D₂R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes.