Involvement of cyclin-dependent kinase-5 in the kainic acid-mediated degeneration of glutamatergic synapses in the rat hippocampus

Increased levels of glutamate causing excitotoxic damage accompany neurological disorders such as ischemia/stroke, epilepsy and some neurodegenerative diseases. Cyclin-dependent kinase-5 (Cdk5) is important for synaptic plasticity and is deregulated in neurodegenerative diseases. However, the mechanisms by which kainic acid (KA)-induced excitotoxic damage involves Cdk5 in neuronal injury are not fully understood. In this work, we have thus studied involvement of Cdk5 in the KA-mediated degeneration of glutamatergic synapses in the rat hippocampus. KA induced degeneration of mossy fiber synapses and decreased glutamate receptor (GluR)6/7 and post-synaptic density protein 95 (PSD95) levels in rat hippocampus in vivo after intraventricular injection of KA. KA also increased the cleavage of Cdk5 regulatory protein p35, and Cdk5 phosphorylation in the hippocampus at 12\u2003h after treatment. Studies with hippocampal neurons in\u2003vitro showed a rapid decline in GluR6/7 and PSD95 levels after KA treatment with the breakdown of p35 protein and phosphorylation of Cdk5. These changes depended on an increase in calcium as shown by the chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N\u200a',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and glycol-bis (2-aminoethylether)-N,N,N\u200a',N\u200a'-tetra-acetic acid. Inhibition of Cdk5 using roscovitine or employing dominant-negative Cdk5 and Cdk5 silencing RNA constructs counteracted the decreases in GluR6/7 and PSD95 levels induced by KA in hippocampal neurons. The dominant-negative Cdk5 was also able to decrease neuronal degeneration induced by KA in cultured neurons. The results show that Cdk5 is essentially involved in the KA-mediated alterations in synaptic proteins and in cell degeneration in hippocampal neurons after an excitotoxic injury. Inhibition of pathways activated by Cdk5 may be beneficial for treatment of synaptic degeneration and excitotoxicity observed in various brain diseases

Autocrine Endocannabinoid Signaling through CB 1 Receptors Potentiates OX 1 Orexin Receptor Signaling s

ABSTRACT It has been proposed that OX 1 orexin receptors and CB 1 cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX 1 receptors and potentiate OX 1 receptor signaling to extracellular signal-regulated kinase (ERK). We have recently shown that OX 1 receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX 1 -CB 1 receptor interaction in signaling, for instance, in retrograde synaptic transmission. In the current study, we set out to investigate this possibility utilizing recombinant Chinese hamster ovary K1 cells. 2-AG released from OX 1 receptor-expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB 1 receptor-expressing cells. When OX 1 and CB 1 receptors were expressed in the same cells, OX 1 stimulation-induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX 1 response as such was fully abolished by specific inhibition of CB 1 receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX 1 -CB 1 heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX 1 -CB 1 synergism and thus suggest a functional rather than a molecular interaction of OX 1 and CB 1 receptors

Autocrine endocannabinoid signaling through CB 1 receptors potentiates OX 1 orexin receptor signaling MOL #80523 2 Running title: CB 1 receptor signaling potentiates OX 1 receptor signaling

Although the results do not exclude the previously proposed OX 1 -CB 1 heteromerization, they nevertheless unequivocally identify diacylglycerol lipase-dependent 2-AG generation as the pivotal determinant of the OX 1 -CB 1 synergism and thus suggest rather a functional than a molecular interaction of OX 1 and CB 1 receptors

Tumor-independent Detection of Inherited Mismatch Repair Deficiency for the Diagnosis of Lynch Syndrome with High Specificity and Sensitivity

Lynch syndrome (LS) is the most common hereditary cancer syndrome. Early diagnosis improves prognosis and reduces health care costs, through existing cancer surveillance methods. The problem is finding and diagnosing the cancer predisposing genetic condition. The current workup involves a complex array of tests that combines family cancer history and clinical phenotypes with tumor characteristics and sequencing data, followed by a challenging task to interpret the found variant(s). On the basis of the knowledge that an inherited mismatch repair (MMR) deficiency is a hallmark of LS, we have developed and validated a functional MMR test, DiagMMR, that detects inherited MMR deficiency directly from healthy tissue without need of tumor and variant information. The validation included 119 skin biopsies collected from clinically pathogenic MMR variant carriers (MSH2, MSH6) and controls, and was followed by a small clinical pilot study. The repair reaction was performed on proteins extracted from primary fibroblasts and the interpretation was based on the MMR capability of the sample in relation to cutoff, which distinguishes MMR proficient (non-LS) from MMR deficient (LS) function. The results were compared with the reference standard (germline NGS). The test was shown to have exceptional specificity (100%) with high sensitivity (89%) and accuracy (97%). The ability to efficiently distinguish LS carriers from controls was further shown with a high area under the receiving operating characteristic (AUROC) value (0.97). This test offers an excellent tool for detecting inherited MMR deficiency linked to MSH2 or MSH6 and can be used alone or with conventional tests to recognize genetically predisposed individuals.Clinical validation of DiagMMR shows high accuracy in distinguishing individuals with hereditary MSH2 or MSH6 MMR deficiency (i.e., LS). The method presented overcomes challenges faced by the complexity of current methods and can be used alone or with conventional tests to improve the ability to recognize genetically predisposed individuals.Peer reviewe