M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss.

The current frontline symptomatic treatment for Alzheimer's disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR-selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.

An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.

Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning.

Histone deacetylase 1 and 2 are essential for normal T-cell development and genomic stability in mice.

Histone deacetylase 1 and 2 (HDAC1/2) regulate chromatin structure as the catalytic core of the Sin3A, NuRD and CoREST co-repressor complexes. To better understand the key pathways regulated by HDAC1/2 in the adaptive immune system and inform their exploitation as drug targets, we have generated mice with a T-cell specific deletion. Loss of either HDAC1 or HDAC2 alone has little effect, while dual inactivation results in a 5-fold reduction in thymocyte cellularity, accompanied by developmental arrest at the double-negative to double-positive transition. Transcriptome analysis revealed 892 misregulated genes in Hdac1/2 knock-out thymocytes, including down-regulation of LAT, Themis and Itk, key components of the T-cell receptor (TCR) signaling pathway. Down-regulation of these genes suggests a model in which HDAC1/2 deficiency results in defective propagation of TCR signaling, thus blocking development. Furthermore, mice with reduced HDAC1/2 activity (Hdac1 deleted and a single Hdac2 allele) develop a lethal pathology by 3-months of age, caused by neoplastic transformation of immature T cells in the thymus. Tumor cells become aneuploid, express increased levels of c-Myc and show elevated levels of the DNA damage marker, γH2AX. These data demonstrate a crucial role for HDAC1/2 in T-cell development and the maintenance of genomic stability.

Differential G-protein-coupled receptor phosphorylation provides evidence for a signaling bar code.

G-protein-coupled receptors are hyper-phosphorylated in a process that controls receptor coupling to downstream signaling pathways. The pattern of receptor phosphorylation has been proposed to generate a "bar code" that can be varied in a tissue-specific manner to direct physiologically relevant receptor signaling. If such a mechanism existed, receptors would be expected to be phosphorylated in a cell/tissue-specific manner. Using tryptic phosphopeptide maps, mass spectrometry, and phospho-specific antibodies, it was determined here that the prototypical G(q/11)-coupled M(3)-muscarinic receptor was indeed differentially phosphorylated in various cell and tissue types supporting a role for differential receptor phosphorylation in directing tissue-specific signaling. Furthermore, the phosphorylation profile of the M(3)-muscarinic receptor was also dependent on the stimulus. Full and partial agonists to the M(3)-muscarinic receptor were observed to direct phosphorylation preferentially to specific sites. This hitherto unappreciated property of ligands raises the possibility that one mechanism underlying ligand bias/functional selectivity, a process where ligands direct receptors to preferred signaling pathways, may be centered on the capacity of ligands to promote receptor phosphorylation at specific sites.

Improving outpatient warfarin use for hospitalized patients with atrial fibrillation

Atrial fibrillation affects an estimated 5 million Americans and accounts for approximately 15% of all strokes. Few studies have successfully addressed patient screening, assessment, and introduction of appropriate antithrombotic therapy in patients with atrial fibrillation. Objective: To assess whether an intervention improved planned antithrombotic prescribing at the time of discharge in hospitalized patients. Methods: The study was a prospectively designed, retrospectively evaluated, non-blinded, historical control study of a pharmacist-initiated intervention. The intervention, consisting of pharmacist review and assessment of antithrombotic prescribing in patients with non-valvular atrial fibrillation, was conducted in an urban teaching hospital. Results: Although antithrombotic prescribing was not significantly higher at discharge in the 252 enrolled subjects (control 67.3% vs. intervention 70.8%; p = 0.58), a significantly greater number of patients had a written discharge plan for antithrombotic therapy (control 73.5% vs. intervention 88.3%; p < 0.01). The adjusted odds ratio that the study group was associated with an improvement in planned or actual warfarin use was 2.46 (95% CI 1.63-3.74). In addition, clinicians adhered to guidelines for antithrombotic therapy in patients with atrial fibrillation more frequently in the intervention group (control 70.4% vs. intervention 88.2%; p < 0.01). Conclusion: A program designed to identify hospitalized patients with non-valvular atrial fibrillation, assess their need for stroke prophylaxis, and initiate appropriate antithrombotic therapy led to an increase in planned antithrombotic, and most importantly, warfarin use upon discharge from the hospital. Confirmation that an increase in planned antithrombotic use upon discharge results in an actual increase in use after discharge is needed to determine the true effectiveness of this intervention (AU)

La fibrilación auricular afecta aproximadamente a 5 millones de norteamericanos y aparece en el aproximadamente el 15% de todos los infartos. Pocos estudios han afrontado con éxito el cribado de pacientes, la evaluación y la introducción de tratamiento antitrombótico apropiado en pacientes con fibrilación auricular. Objetivo: Evaluar si una intervención mejoraba la prescripción antitrombótica planeada en el alta en pacientes hospitalizados. Métodos: El estudio fue diseñado prospectivamente y evaluado retrospectivamente, no ciego, con control histórico de una intervención iniciada por el farmacéutico. La intervención, que consistía en la revisión y evaluación por un farmacéutico de la prescripción antitrómbótica de pacientes con fibrilación auricular no valvular, se llevó a cabo en un hospital universitario urbano. Resultados: Aunque la prescripción antitrombótica no fuie significativamente mayor en el alta en los 252 individuos (control 67.3% vs. intervención 70.8%; p = 0.58), un número significativamente mayor de pacientes tuvo un plan escrito al alta para el tratamiento antitrombótico (control 73.5% vs. intervención 88.3%; p < 0.01). El odds ratio ajustado de que el grupo de estudio estaba asociado a un mejor uso de la warfarina planificada o real fue 2,46 (CI95% 1.63-3.74). Además, los clínicos se adhirieron a las guías de tratamiento antitrombótico en pacientes con fibrilación auricular más frecuentemente en el grupo intervención (control 70.4% vs. intervención 88.2%; p < 0.01). Conclusión: Un programa diseñado para identificar pacientes hospitalizados con fibrilación auricular, evaluar su necesidad de prevención de infarto e iniciar tratamiento antitrombótico llevó a un aumento de antitrombóticos planificados, y más importante, al uso de warfarina al alta del hospital. Se necesita la confirmación de que un aumento en los antitrombóticos planificados lleve a un aumento en el uso real para determinar la verdadera efectividad de esta intervención (AU)

Improving outpatient warfarin use for hospitalized patients with atrial fibrillation.

UNLABELLED: Atrial fibrillation affects an estimated 5 million Americans and accounts for approximately 15% of all strokes. Few studies have successfully addressed patient screening, assessment, and introduction of appropriate antithrombotic therapy in patients with atrial fibrillation. OBJECTIVE: To assess whether an intervention improved planned antithrombotic prescribing at the time of discharge in hospitalized patients. METHODS: The study was a prospectively designed, retrospectively evaluated, non-blinded, historical control study of a pharmacist-initiated intervention. The intervention, consisting of pharmacist review and assessment of antithrombotic prescribing in patients with non-valvular atrial fibrillation, was conducted in an urban teaching hospital. RESULTS: Although antithrombotic prescribing was not significantly higher at discharge in the 252 enrolled subjects (control 67.3% vs. intervention 70.8%; p = 0.58), a significantly greater number of patients had a written discharge plan for antithrombotic therapy (control 73.5% vs. intervention 88.3%; p < 0.01). The adjusted odds ratio that the study group was associated with an improvement in planned or actual warfarin use was 2.46 (95% CI 1.63-3.74). In addition, clinicians adhered to guidelines for antithrombotic therapy in patients with atrial fibrillation more frequently in the intervention group (control 70.4% vs. intervention 88.2%; p < 0.01). CONCLUSION: A program designed to identify hospitalized patients with non-valvular atrial fibrillation, assess their need for stroke prophylaxis, and initiate appropriate antithrombotic therapy led to an increase in planned antithrombotic, and most importantly, warfarin use upon discharge from the hospital. Confirmation that an increase in planned antithrombotic use upon discharge results in an actual increase in use after discharge is needed to determine the true effectiveness of this intervention.