In vivo detection of GPCR-dependent signaling using fiber photometry and FRET-based biosensors.

Genetically encoded fluorescent biosensors allow intracellular signaling dynamics to be tracked in live cells and tissues using optical detection. Many such biosensors are based on the principle of Förster resonance energy transfer (FRET), and we have recently developed a simple approach for in vivo detection of FRET-based biosensor signals using fiber photometry. By combining fiber photometry with FRET-based biosensors, we were able to track GPCR-dependent signaling pathways over time, and in response to drug treatments in freely-moving adult rats. Recording from specific neuronal populations, we can quantify intracellular signaling while simultaneously measuring behavioral responses. Our approach, described in detail here, uses adeno-associated viruses infused intracerebrally in order to express genetically-encoded FRET-based biosensors. After several weeks to allow biosensor expression, fiber photometry is used in order to record drug responses in real time from freely-moving adult rats. This methodology would be compatible with other mammalian species and with many biosensors. Hence, it has wide applicability across a spectrum of neuroscience research, ranging from the study of neural circuits and behavior, to preclinical drug development and screening.

The Signaling and Pharmacology of the Dopamine D1 Receptor.

The dopamine D1 receptor (D1R) is a Gαs/olf-coupled GPCR that is expressed in the midbrain and forebrain, regulating motor behavior, reward, motivational states, and cognitive processes. Although the D1R was initially identified as a promising drug target almost 40 years ago, the development of clinically useful ligands has until recently been hampered by a lack of suitable candidate molecules. The emergence of new non-catechol D1R agonists, biased agonists, and allosteric modulators has renewed clinical interest in drugs targeting this receptor, specifically for the treatment of motor impairment in Parkinson's Disease, and cognitive impairment in neuropsychiatric disorders. To develop better therapeutics, advances in ligand chemistry must be matched by an expanded understanding of D1R signaling across cell populations in the brain, and in disease states. Depending on the brain region, the D1R couples primarily to either Gαs or Gαolf through which it activates a cAMP/PKA-dependent signaling cascade that can regulate neuronal excitability, stimulate gene expression, and facilitate synaptic plasticity. However, like many GPCRs, the D1R can signal through multiple downstream pathways, and specific signaling signatures may differ between cell types or be altered in disease. To guide development of improved D1R ligands, it is important to understand how signaling unfolds in specific target cells, and how this signaling affects circuit function and behavior. In this review, we provide a summary of D1R-directed signaling in various neuronal populations and describe how specific pathways have been linked to physiological and behavioral outcomes. In addition, we address the current state of D1R drug development, including the pharmacology of newly developed non-catecholamine ligands, and discuss the potential utility of D1R-agonists in Parkinson's Disease and cognitive impairment.

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors.

As with many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell type-specific, and can change in the face of disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinson's disease (PD), alterations in this pathway lead to functional upregulation of extracellular regulated kinases 1/2 (ERK1/2), contributing to L-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Förster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with L-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.

Cluster of Differentiation 96 in Children with Acute Leukemia: A Single Center Cohort Study.

Cluster of differentiation 96 (CD96) is an important leukemic stem cells (LSCs) surface marker. We evaluated CD96 expression in children with acute leukemia (AL) and described its relation with treatment response. We conducted a prospective cohort study in Mansoura University Children's Hospital, Egypt during the period from 2014 to 2016. We studied 96 children with AL and 96 controls at clinical, laboratory and radiological levels. We assessed CD96% in LSCs using flow cytometry. AL group included 59 acute lymphoblastic leukemia (ALL) and 37 acute myeloid leukemia (AML) patients. ALL subgroup involved 44 B-ALL and 15 T-ALL patients while AML subgroup included 17 M2, 12 M4 and 8 M5 patients. CD96% was higher in AL group [57.63 (21.18-89.93)] than control [34.12 (16.15-39.51)] (P < 0.001). CD96% was higher in AML [68.25 (31.1-89.86)] than ALL [54.18 (21.18-89.93] (P < 0.001). CD96% in AML was M4 > M2 > M5 (P = 0.04) while within ALL subgroup, no significant difference was found between B-ALL and T-ALL (P = 0.807). CD96% in patients with non-complete remission was higher than those with complete remission (P = 0.004). CD96 is a reliable diagnostic marker for AL mainly AML and could be used as a prognostic marker for treatment response.

Perampanel but Not Amantadine Prevents Behavioral Alterations and Epileptogenesis in Pilocarpine Rat Model of Status Epilepticus.

Pilocarpine-induced status epilepticus (SE), which results in the development of spontaneous recurrent seizures (SRSs) activates glutamatergic receptors that contribute to seizure sustenance and neuronal cell death. In the current study, we evaluate whether the exposure to perampanel, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker, or amantadine, a N-methyl-D-aspartic acid (NMDA) receptor blocker would reduce the SE-induced long-term consequences. SE was induced in adult male Sprague Dawley rats with pilocarpine. Perampanel or amantadine was injected 10 or 60 min after SE onset. The efficacy of either, in overcoming pilocarpine-induced SE was assessed using electroencephalogram (EEG) recordings. In addition, alterations in cognitive function, development of spontaneous recurrent seizures (SRSs), and hippocampal damage that are generally encountered after SE were also assessed at 72 h and 5 weeks after the induction of SE. Our results indicate that both early and late treatment with perampanel but not amantadine significantly reduced seizure activity. Furthermore, perampanel but not amantadine, reversed the memory deficits in Y-maze and novel object recognition (NOR) tests and retarded the appearance of SRSs. Moreover, perampanel treatment led to reduced SE-induced caspase-3 activation in the hippocampal lysates. Taken together, the data obtained from the study reveals that blocking AMPA receptors by perampanel can modify SE-induced long-term consequences. Our results may provide a proof of principle for the potential therapeutic application of perampanel in clinical use for status epilepticus in future.

Retinopathy in newly diagnosed type 2 diabetes mellitus.

OBJECTIVE: To study the prevalence of diabetic retinopathy and its association with risk factors in newly diagnosed type 2 diabetic patients in two primary care diabetic clinics in Kuwait. SUBJECTS AND METHODS: All newly diagnosed type 2 diabetes mellitus patients attending Ferdous and Yarmouk Health Care Diabetic Clinics in Farwaniya and Asma Health Areas, respectively, during the period of January 2002 to July 2004 were examined for the presence of diabetic retinopathy by color fundus photographs. Risk factors such as sex, age, hypertension, nephropathy, hyperglycemia, level of glycated hemoglobin, microalbuminuria and hyperlipidemia were evaluated by statistical analysis for their possible association with the presence of diabetic retinopathy at the time of diagnosis of diabetes. RESULTS: Of the 92 newly diagnosed type 2 diabetes mellitus patients studied, diabetic retinopathy was present in 7 (7.6%) patients. Two of the 7 patients had maculopathy that needed treatment by photocoagulation. High glycated hemoglobin in 51 patients (55.4%), microalbuminuria in 25 (27.2%), hypertension in 29 (315%), hyperlipidemia in 69 (75%), obesity in 45 (48.9%) and overweight in 37(40.2%) were noted in the study population. Microalbuminuria was weakly associated with the presence of retinopathy (p = 0.08) at the time of diagnosis of diabetes mellitus, but other risk factors were not statistically significant. CONCLUSIONS: Prevalence of diabetic retinopathy was 7.6% among the study population. Maculopathy was present in 2 of the 7 patients needing photocoagulation.