Characterization of MK6240, a tau PET tracer, in autopsy brain tissue from Alzheimer’s disease cases

Purpose MK6240 is a second-generation tau PET tracer designed to detect the neurofibrillary tangles in the brains of patients with Alzheimer’s disease (AD). The aim of the study was to characterize 3H-MK6240 in AD and control brain tissue and to compare its binding properties with those of first-generation tau PET tracers. Methods Saturation binding assays with 3H-MK6240 were carried out in the temporal and parietal cortices of AD brains to determine the maximum number of binding sites (Bmax) and the dissociation constants (Kd) at these sites. Competitive binding assays were carried out between 3H-MK6240 and unlabelled MK6240, AV-1451 (aka T807, flortaucipir) and THK5117, and between 3H-THK5351 and unlabelled MK6240. Regional binding studies with 3H-MK6240 were carried out in homogenates from six AD and seven control brains and, using autoradiography, on large frozen sections from two AD brains and one control brain. Results The saturation binding assays gave Bmax and Kd values of 59.2 fmol/mg and 0.32 nM in the temporal cortex and 154.7 fmol/mg and 0.15 nM in the parietal cortex. The competitive binding assays revealed two binding sites with affinities in the picomolar and nanomolar range shared by 3H-MK6240 and all the tested unlabelled compounds. There were no binding sites in common between 3H-THK5351 and unlabelled MK6240. Regional binding of 3H-MK6240 was significantly higher in AD brain tissue than in controls. Binding in brain tissue from AD patients with early-onset AD was significantly higher than in brain tissue from patients with late-onset AD. Binding of 3H-MK6240 was not observed in off-target regions. Autoradiography showed high regional cortical binding in the two AD brains and very low binding in the control brain. Conclusions 3H-MK6240 has a high binding affinity for tau deposits in AD brain tissue but also has different binding characteristics from those of the first-generation tau tracers. This confirms the complexity of tau tracer binding on tau deposits with different binding affinities for different binding sites.

Adenosinergic System Involvement in Ischemic Stroke Patients’ Lymphocytes

Adenosine modulates many physiological processes through the interaction with adenosine receptors (ARs) named as A1, A2A, A2B, and A3ARs. During ischemic stroke, adenosine mediates neuroprotective and anti-inflammatory effects through ARs activation. One of the dominant pathways generating extracellular adenosine involves the dephosphorylation of ATP by ecto-nucleotidases CD39 and CD73, which efficiently hydrolyze extracellular ATP to adenosine. The aim of the study is to assess the presence of ARs in lymphocytes from ischemic stroke patients compared to healthy subjects and to analyze changes in CD39 and CD73 expression in CD4+ and CD8+ lymphocytes. Saturation binding experiments revealed that A2AARs affinity and density were significantly increased in ischemic stroke patients whilst no differences were found in A1, A2B, and A3ARs. These results were also confirmed in reverse transcription (RT)-polymerase chain reaction (PCR) assays where A2AAR mRNA levels of ischemic stroke patients were higher than in control subjects. In flow cytometry experiments, the percentage of CD73+ cells was significantly decreased in lymphocytes and in T-lymphocyte subclasses CD4+ and CD8+ obtained from ischemic stroke patients in comparison with healthy individuals. These data corroborate the importance of the adenosinergic system in ischemic stroke and could open the way to more targeted therapeutic approaches and biomarker development for ischemic stroke.

Binding affinities of oxytocin, vasopressin, and Manning Compound at oxytocin and V1a receptors in Syrian hamster brains

ABSTRACTOxytocin (OT) and arginine vasopressin (AVP), as well as synthetic ligands targeting their receptors (OTR, V1aR), are used in a wide variety of research contexts, but typically their pharmacological properties are determined in only a few species. Syrian hamsters (Mesocricetus auratus) have a long history of use as a behavioral and biomedical model for the study of oxytocin and vasopressin, and more recently, hamsters have been used to investigate behavioral consequences of OT-mediated activation of V1aRs. We sought to determine the binding affinities of OT, AVP, and the selective V1aR antagonist, Manning compound, in OTRs and V1aRs found in hamster brains. We performed saturation binding asays to determine the Kd values for the selective OTR and V1aR radioligands, [125I]OVTA and [125I]LVA in hamster brains. We then performed competition binding assays to determine Ki values for OT, AVP, and Manning compond at both the OTR and V1aR. We found that OT and AVP each had the highest affinity for their canonical receptors (OT-OTR Ki=4.28 nM; AVP-V1ar Ki=4.70 nM), and had the lowest affinity for their non-canonical ligands (OT-V1aR=495.2nM; AVP-OTR Ki=36.1 nM). Manning compound had the highest affinity for the V1aR (MC-V1aR Ki=6.87 nM; MC-OTR Ki=213.8 nM), but Manning compound was not as selective for the V1aR as has been reported in rat receptor. When comparing these data to previously published work, we found that the promiscuity of the V1aR in hamsters with respect to oxytocin and vasopressin binding is more similar to the promiscuity of the human V1aR than the rat V1aR receptor. Moreover, the selectivity of oxytocin at hamster receptors is more similar to the selectivity of oxytocin at human receptors than the selectivity of oxytocin at rat receptors. These data highlight the importance of determining the pharmacological properties of behaviorally relevant compounds in diverse models species.

Differently fluorescence-labelled dibenzodiazepinone-type muscarinic acetylcholine receptor ligands with high M2R affinity

New fluorescent molecular tools for the muscarinic acetylcholine M2 receptor, bearing various fluorescent dyes, showed high M2 receptor affinity in flow cytometric saturation binding studies at CHO–hM2R cells (pKd > 8.3).

Usf1, a suppressor of the circadian Clock mutant, reveals the nature of the DNA-binding of the CLOCK:BMAL1 complex in mice

Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the ClockΔ19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the ClockΔ19 mutation to a ∼900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCKΔ19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals.