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Rescue of Cyclic AMP Mediated Long Term Potentiation Impairment in the Hippocampus of Mecp2 Knockout (Mecp2(-/y) ) Mice by Rolipram.

ABSTRACT: Rett syndrome (RTT) patients experience learning difficulties and memory loss. Analogous deficits of hippocampal plasticity are reported in mouse models of RTT. To elucidate the underlying pathophysiology, we studied long term potentiation (LTP) at the CA3 to CA1 synapses in the hippocampus in acute brain slices from WT and Mecp2(-/y) mice, by either activating cAMP dependent pathway or using high frequency stimulation, by means of patch clamp. We have observed that, the NMDA channel current characteristics remain unchanged in the Mecp2(-/y) mice. The adenylyl cyclase (AC) agonist forskolin evoked a long lasting potentiation of evoked EPSCs in WT CA1 neurons, but only minimally enhanced the EPSCs in the Mecp2(-/y) mice. This weaker potentiation in Mecp2 (-/) (y) mice was ameliorated by application of phosphodiesterase 4 inhibitor rolipram. The hyperpolarization activated cyclic nucleotide gated channel current (I h) was potentiated to similar extent by forskolin in both phenotypes. Multiple tetanus induced cAMP-dependent plasticity was also impaired in the Mecp2 (-/) (y) mice, and was also partially rescued by rolipram. Western blot analysis of CA region of Mecp2 (-/) (y) mice hippocampus revealed more than twofold up-regulation of protein kinase A (PKA) regulatory subunits, while the expression of the catalytic subunit remained unchanged. We hypothesize that the overexpressed PKA regulatory subunits buffer cAMP and restrict the PKA mediated phosphorylation of target proteins necessary for LTP. Blocking the degradation of cAMP, thereby saturating the regulatory subunits alleviated this defect.

SUBMITTER: Balakrishnan S

PROVIDER: S-EPMC4737891 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Rescue of Cyclic AMP Mediated Long Term Potentiation Impairment in the Hippocampus of Mecp2 Knockout (Mecp2(-/y) ) Mice by Rolipram.

Balakrishnan Saju S Niebert Marcus M Richter Diethelm W DW

Frontiers in cellular neuroscience 20160203

Rett syndrome (RTT) patients experience learning difficulties and memory loss. Analogous deficits of hippocampal plasticity are reported in mouse models of RTT. To elucidate the underlying pathophysiology, we studied long term potentiation (LTP) at the CA3 to CA1 synapses in the hippocampus in acute brain slices from WT and Mecp2(-/y) mice, by either activating cAMP dependent pathway or using high frequency stimulation, by means of patch clamp. We have observed that, the NMDA channel current cha ...[more]

PMID: 26869885

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Project description:The stereospecificity of rolipram inhibition of particulate cyclic AMP-specific phosphodiesterase (PDE IV) from guinea-pig eosinophils has been investigated. (-)-Rolipram (IC50 = 0.22 +/- 0.08 microM) was 2.5-fold more potent than (+)-rolipram (IC50 = 0.58 +/- 0.05 microM) in inhibiting membrane-bound PDE IV. Solubilization of PDE IV with deoxycholate (0.5%) and NaCl (100 mM) increased rolipram stereospecificity [IC50 (-)-rolipram = 0.020 +/- 0.002 microM; IC50 (+)-rolipram = 0.33 +/- 0.07 microM]. Partial purification of this solubilized PDE IV by DEAE-trisacryl anion-exchange chromatography reduced the enantiomeric potency difference compared with the pre-chromatographed activity, with (-)-rolipram (IC50 = 0.20 +/- 0.02 microM) being only 2.9-fold more potent than (+)-rolipram (IC50 = 0.57 +/- 0.14 microM). Vanadate-glutathione complex (V-GSH) stimulated membrane-bound PDE IV activity and increased the potency of (-)-rolipram (IC50 = 0.014 +/- 0.006 microM) but not (+)-rolipram (IC50 = 0.32 +/- 0.07 microM). In intact eosinophils, (-)-rolipram (EC50 = 0.19 +/- 0.02 microM) was 10-fold more potent than (+)-rolipram (EC50 = 1.87 +/- 0.09 microM) in enhancing isoprenaline (10 microM)-stimulated cyclic AMP accumulation. Strong correlations were demonstrated for displacement of [3H]rolipram binding to brain membranes by several PDE inhibitors and their inhibition of solubilized PDE IV (r = 0.98, P < 0.001, n = 7) and stimulation of cyclic AMP accumulation in intact cells (r = 0.98, P < 0.001, n = 6). Rolipram was a relatively weak inhibitor of partially purified pig aortic PDE IV and only slight stereospecificity was exhibited [IC50 (-)-rolipram = 1.47 +/- 0.09 microM; IC50 (+)-rolipram = 2.73 +/- 0.38 microM]. The results indicate the presence of a partially concealed stereospecific site (Sr) on eosinophil PDE IV possibly similar to the high-affinity rolipram-binding site in brain through which rolipram can potently inhibit enzyme activity. This site, which apparently is not present on partially purified pig aortic PDE IV, is concealed in freshly prepared eosinophil membranes but is exposed by solubilization or V-GSH treatment and is important in regulating intracellular cyclic AMP accumulation in intact cells.

Project description:Long-term potentiation (LTP) is an activity-dependent and persistent increase in synaptic transmission. Currently available techniques to measure LTP are time-intensive and require highly specialized expertise and equipment, and thus are not well suited for screening of multiple candidate treatments, even in animal models. To expand and facilitate the analysis of LTP, here we use a flow cytometry-based method to track chemically induced LTP by detecting surface AMPA receptors in isolated synaptosomes: fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). First, we demonstrate that FASS-LTP is simple, sensitive, and models electrically induced LTP recorded in intact circuitries. Second, we conducted FASS-LTP analysis in two well-characterized Alzheimer's disease (AD) mouse models (3xTg and Tg2576) and, importantly, in cryopreserved human AD brain samples. By profiling hundreds of synaptosomes, our data provide the first direct evidence to support the idea that synapses from AD brain are intrinsically defective in LTP. Third, we used FASS-LTP for drug evaluation in human synaptosomes. Testing a panel of modulators of cAMP and cGMP signaling pathways, FASS-LTP identified vardenafil and Bay-73-6691 (phosphodiesterase-5 and -9 inhibitors, respectively) as potent enhancers of LTP in synaptosomes from AD cases. These results indicate that our approach could provide the basis for protocols to study LTP in both healthy and diseased human brains, a previously unattainable goal. SIGNIFICANCE STATEMENT:Learning and memory depend on the ability of synapses to strengthen in response to activity. Long-term potentiation (LTP) is a rapid and persistent increase in synaptic transmission that is thought to be affected in Alzheimer's disease (AD). However, direct evidence of LTP deficits in human AD brain has been elusive, primarily due to methodological limitations. Here, we analyze LTP in isolated synapses from AD brain using a novel approach that allows testing LTP in cryopreserved brain. Our analysis of hundreds of synapses supports the idea that AD-diseased synapses are intrinsically defective in LTP. Further, we identified pharmacological agents that rescue LTP in AD, thus opening up a new avenue for drug screening and evaluation of strategies for alleviating memory impairments.

Dataset Information

Rescue of Cyclic AMP Mediated Long Term Potentiation Impairment in the Hippocampus of Mecp2 Knockout (Mecp2(-/y) ) Mice by Rolipram.

Publications

Rescue of Cyclic AMP Mediated Long Term Potentiation Impairment in the Hippocampus of Mecp2 Knockout (Mecp2(-/y) ) Mice by Rolipram.

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