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Tau is a protein that normally participates in the assembly and stability of microtubules. However, it can form intraneuronal hyperphosphorylated aggregates that are hallmarks of Alzheimer's disease and other neurodegenerative disorders known as tauopathies. Tau can be phosphorylated by multiple kinases at several sites. Among such kinases, the cAMP‐dependent protein kinase A (PKA) phosphorylates tau at Ser214 (pTAU‐S214), an event that was shown to reduce the pathological assembly of the protein. Given that the neuronal cAMP/PKA‐activated cascade is involved in synaptic plasticity and memory, and that cAMP‐enhancing strategies demonstrated promising therapeutic potential for the treatment of cognitive deficits, we investigated the impact of cAMP on pTAU‐S214 in N2a cells and rat hippocampal slices. Our results confirm that the activation of adenylyl cyclase increases pTAU‐S214 in both model systems and, more interestingly, this effect is mimicked by GEBR‐7b, a phosphodiesterase 4D inhibitor with proven pro‐cognitive efficacy in rodents. [ABSTRACT FROM AUTHOR]

Intraneuronal accumulation of hyperphosphorylated tau is a pathological hallmark of several neurodegenerative disorders, including Alzheimer's disease. Phosphorylation plays a crucial role in modulating the tau‐microtubule interaction and the ability of the protein to aggregate, but despite efforts during the past decades, the real identity of the kynases involved in vivo remains uncertain. Here, for the first time, we demonstrate that the cGMP‐dependent protein kinase G (PKG) phosphorylates tau in both in vitro and in vivo models. More intriguingly, we provide evidence that PKG phosphorylates tau at Ser214 but not at Ser202, a condition that could reduce the pathological aggregation of the protein shifting tau from a pro‐aggregant to a neuroprotective anti‐aggregant conformation. [ABSTRACT FROM AUTHOR]

Leucine‐rich repeat kinase 2 (LRRK2) is a large multidomain scaffolding protein with kinase and GTPase activities involved in synaptic vesicle (SV) dynamics. While its role in Parkinson's disease has been largely investigated, little is known about LRRK2 physiological role and until now few proteins have been described as substrates. We have previously demonstrated that LRRK2 through its WD40 domain interacts with synapsin I, an important SV‐associated phosphoprotein involved in neuronal development and in the regulation of neurotransmitter release. To test whether synapsin I is substrate for LRRK2 and characterize the properties of its phosphorylation, we used in vitro kinase and binding assays as well as cellular model and site‐direct mutagenesis. Using synaptosomes in superfusion, patch‐clamp recordings in autaptic WT and synapsin I KO cortical neurons and SypHy assay on primary cortical culture from wild‐type and BAC human LRRK2 G2019S mice we characterized the role of LRRK2 kinase activity on glutamate release and SV trafficking. Here we reported that synapsin I is phosphorylated by LRRK2 and demonstrated that the interaction between LRRK2 WD40 domain and synapsin I is crucial for this phosphorylation. Moreover, we showed that LRRK2 phosphorylation of synapsin I at threonine 337 and 339 significantly reduces synapsin I‐SV/actin interactions. Using complementary experimental approaches, we demonstrated that LRRK2 controls glutamate release and SV dynamics in a kinase activity and synapsin I‐dependent manner. Our findings show that synapsin I is a LRRK2 substrate and describe a novel mechanisms of regulation of glutamate release by LRRK2 kinase activity. [ABSTRACT FROM AUTHOR]

Abstract: Glucagon‐like peptide‐1 receptors (GLP‐1Rs) have been shown to mediate cognitive‐enhancing and neuroprotective effects in the central nervous system. However, little is known about their physiological roles on central neurotransmission, especially at the presynaptic level. Using purified synaptosomal preparations and immunofluorescence techniques, here we show for the first time that GLP‐1Rs are localized on mouse cortical and hippocampal synaptic boutons, in particular on glutamatergic and GABAergic nerve terminals. Their activation by the selective agonist exendin‐4 (1–100 nM) was able to increase the release of either [3H] d‐aspartate or [3H]GABA. These effects were abolished by 10 nM of the selective GLP1‐R antagonist exendin‐3 (9–39) and were prevented by the selective adenylyl cyclase inhibitor 2′,5′‐dideoxyadenosine (10 µM), indicating the involvement of classic GLP‐1Rs coupled to Gs protein stimulating cAMP synthesis. Our data demonstrate the existence and activity of presynaptic receptors for GLP‐1 that could represent additional mechanisms by which this neurohormone exerts its effects in the CNS. © 2017 BioFactors, 44(2):148–157, 2018 [ABSTRACT FROM AUTHOR]

Cyclic adenosine monophosphate (cAMP) is one of the second messengers critically involved in the molecular mechanisms underlying memory formation. In the CNS, the availability of cAMP is tightly controlled by phosphodiesterase 4 (PDE4), a family of enzymes that degrades the cyclic nucleotide to inactive AMP. Among the different PDE4 isoforms, in the last few years PDE4D has been hogging the limelight due to accumulating evidence for its crucial role in cognitive processes, which makes this enzyme a promising target for therapeutic interventions in a variety of pathological conditions characterized by memory impairment, such as Alzheimer's disease. In this article, we review the role of the cAMP signal transduction pathway in memory formation with a particular focus on the recent progress in PDE4D research. [ABSTRACT FROM AUTHOR]

Cyclic adenosine monophosphate (cAMP) regulates long-term potentiation (LTP) and ameliorates memory in healthy and diseased brain. Increasing evidence shows that, under physiological conditions, low concentrations of amyloid β (Aβ) are necessary for LTP expression and memory formation. Here, we report that cAMP controls amyloid precursor protein (APP) translation and Aβ levels, and that the modulatory effects of cAMP on LTP occur through the stimulation of APP synthesis and Aβ production. Ann Neurol 2014;75:602-607 [ABSTRACT FROM AUTHOR]

Besides playing a pathogenic role in Alzheimer disease, amyloid-beta peptides are normally produced in low amounts in the brain, and several lines of evidence suggest that they can modulate synaptic plasticity and memory. As cyclic adenosine monophosphate (cAMP) is known to be involved in the same processes and the blockade of its degradation by phosphodiesterase 4 inhibitors has consistently shown beneficial effects on cognition, we investigated the possible correlation between this second messenger and Aβ peptides in neuronal N2a cells overexpressing the amyloid-β precursor protein (APP). We herein report that the elevation of endogenous cAMP by rolipram increased APP protein expression and both its amyloidogenic and nonamyloidogenic processing. The effects of rolipram were reproduced by both the cAMP membrane-permeant analog 8Br-cAMP and the forskolin-induced activation of adenylyl cyclase but were not affected by the PKA inhibitor H-89. Our results demonstrate that, in neuronal cells, APP metabolism is physiologically modulated by cAMP and suggest that this might represent an additional mechanism through which the second messenger could influence memory functions. © 2013 IUBMB Life, 65(2)127-133, 2013 [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 115, 343-352. Abstract We have investigated the spontaneous and the depolarisation-induced release of [3H] d-aspartate ([3H] d-ASP), a non-metabolisable analogue of glutamate, in spinal cord slices, synaptosomes and gliosomes from mice with experimental autoimmune encephalomyelitis (EAE) at 13, 21 and 55 days post-immunisation (d.p.i.), representing onset, peak and chronic phases of the pathology. At 13 and 21 d.p.i., the KCl-evoked, calcium-dependent overflow of [3H] d-ASP in spinal cord slices was significantly lower (30-40%), whereas at 55 d.p.i. it was significantly higher (30%), than that elicited in matched controls. When the release was measured from spinal cord synaptosomes and gliosomes in superfusion, a different picture emerged. The spontaneous and the KCl(15 mM)-induced release of [3H] d-ASP were significantly increased both in synaptosomes (17% and 45%, respectively) and gliosomes (26% and 25%, respectively) at 21, but not at 13, d.p.i. At 55 d.p.i., the KCl-induced [3H] d-ASP release was significantly increased (40%) only in synaptosomes. Finally, uptake of [3H] d-ASP was markedly (50-60%) increased in spinal cord synaptosomes, but not in gliosomes, obtained from EAE mice at 21 d.p.i., whereas no differences could be detected at 13 d.p.i. Our data indicate that glutamatergic neurotransmission is altered in the spinal cord of EAE mice. [ABSTRACT FROM AUTHOR]

Pathological oscillations characterize the firing discharge of different basal ganglia (BG) stations in rat models of Parkinson's disease. Most recent literature focused on the prominence of the beta frequency band in awake rats. Yet, in 6-hydroxydopamine-lesioned animals, the firing discharge of the globus pallidus (GP) and the substantia nigra reticulata are in phase with urethane-induced slow wave cortical activity. The neuronal basis of this pathological synergy at low frequency is widely debated. In order to understand the role of substantia nigra pars compacta (SNc) signalling in the development of pathological synchronization, we performed a pharmacological inactivation of the medial forebrain bundle (MFB) through tetrodotoxin (TTX), which led to a dramatic, but reversible, reduction of the dopamine content in the striatum. This procedure caused a significant contralateral akinesia, detectable as soon as anaesthesia vanished, and lasting about 3–4 h. We sought to determine the electrophysiological counterpart of this transient Parkinsonian-like hypokinetic syndrome. Hence, we obtained the electrocorticogram (ECoG) and single unit recordings from GP and subthalamic nucleus (STN) in normal rats before and after the TTX injection in MFB. Intriguingly, the TTX-mediated inactivation of MFB induced a fast developing coherence between cortex and GP and a significant increase of the cortex/STN synchronization. The intra-GP iontophoretic delivery of haloperidol or the GABAA receptor antagonist bicuculline induced a short term cortex/GP synchronization. Strikingly, STN inactivation by local muscimol reversed both haloperidol- and TTX-mediated coherence between cortex and GP. Our data show that an abnormal cortical/BG synchronization, at low frequency, can be reproduced also without SNc neuronal loss and striatal cytoarchitectonic alterations. In addition, our results, which represent an acute and reversible Parkinsonism based upon impaired cable properties, seem compatible with the interpretation of acute changes of the functional interplay between cortex and the STN–GP pathway as a key factor mechanism underlying the fast deep brain stimulation-induced acute Off–On transitions. [ABSTRACT FROM AUTHOR]

The role ofl-aspartate as a classical neurotransmitter of the CNS has been a matter of great debate. In this study, we have characterized the main mechanisms of its depolarization-induced release from rat purified cerebrocortical synaptosomes in superfusion and compared them with those of the well-known excitatory neurotransmitterl-glutamate. High KCl and 4-aminopyridine were used as depolarizing agents. At 15 mM KCl, the overflows of both transmitters were almost completely dependent on external Ca2+. At 35 and 50 mM KCl, the overflows ofl-aspartate, but not those ofl-glutamate, became sensitive todl-threo-β-benzyloxyaspartic acid (dl-TBOA), an excitatory amino acid transporter inhibitor. In the presence ofdl-TBOA, the 50 mM KCl-evoked release ofl-aspartate was still largely external Ca2+-dependent. Thedl-TBOA insensitive, external Ca2+-independent component of the 50 mM KCl-evoked overflows ofl-aspartate andl-glutamate was significantly decreased by the mitochondrial Na+/Ca2+ exchanger blocker CGP 37157. The Ca2+-dependent, KCl-evoked overflows ofl-aspartate andl-glutamate were diminished by botulinum neurotoxin C, although to a significantly different extent. The 4-aminopyridine-inducedl-aspartate andl-glutamate release was completely external Ca2+-dependent and never affected bydl-TBOA. Superimposable results have been obtained by pre-labeling synaptosomes with [3H]d-aspartate and [3H]l-glutamate. Therefore, our data showing thatl-aspartate is released from nerve terminals by calcium-dependent, exocytotic mechanisms support the neurotransmitter role of this amino acid. [ABSTRACT FROM AUTHOR]

Extracellular [K+] can increase during some pathological conditions, resulting into excessive glutamate release through multiple mechanisms. We here investigate the overflow of [3H]d-aspartate ([3H]d-ASP) and of endogenous glutamate elicited by increasing [K+] from purified rat cerebrocortical synaptosomes. Depolarization with [K+] ≤ 15 mmol/L provoked [3H]d-ASP and glutamate overflows almost totally dependent on external Ca2+. Consistent with release by exocytosis, the overflow of [3H]d-ASP evoked by 12 mmol/L K+ was sensitive to clostridial toxins. The overflows evoked by 35/50 mmol/L K+ remained external Ca2+-dependent by more than 50%. The Ca2+-independent components of the [3H]d-ASP overflows evoked by [K+] > 15 mmol/L were prevented by the glutamate transporter inhibitorsdl- threo-beta-benzyloxyaspartate (dl-TBOA) and dihydrokainate. Differently, the overflows of endogenous glutamate provoked by [K+] > 15 mmol/L were insensitive to both inhibitors; the external Ca2+-independent glutamate overflow caused by 50 mmol/L KCl was prevented by bafilomycin, by chelating intraterminal Ca2+, by blocking the mitochondrial Na+/Ca2+ exchanger and, for a small portion, by blocking anion channels. In contrast to purified synaptosomes, the 50 mmol/L K+-evoked release of endogenous glutamate or [3H]D-ASP was inhibited bydl-TBOA in crude synaptosomes; moreover, it was external Ca2+-insensitive and blocked bydl-TBOA in purified gliosomes, suggesting that carrier-mediated release of endogenous glutamate provoked by excessive [K+] in CNS tissues largely originates from glia. [ABSTRACT FROM AUTHOR]

To understand the events underlying the clinical efficacy of deep brain stimulation (DBS) of the subthalamic nucleus (STN), electrophysiological recordings and microdialysis evaluations were carried out in the substantia nigra pars reticulata (SNr), one of the two basal ganglia (BG) nuclei targeted by STN output, in patients with Parkinson's disease (PD). Clinically effective STN-DBS caused a significant increase of the SNr firing rate. The poststimulus histogram (PSTH) showed an excitation peak at 1.92–3.85 ms after the STN stimulus. The spontaneous discharge of SNr neurons was driven at the frequency of the stimulation (130 Hz), as shown in the autocorrelograms (AutoCrl). The fast Fourier transform (FFT) analysis showed a peak at 130 Hz, and a less pronounced second one at 260 Hz. Accordingly, in the distribution of the interspike intervals (ISIs), the mode was earlier, and skewness more asymmetric. Biochemically, the increased excitatory driving from the STN was reflected by a clear-cut increase in cyclic guanosine 3',5'-monophosphate (cGMP) levels in the SNr. These results indicate that the beneficial effect of DBS in PD patients is paralleled with a stimulus-synchronized activation of the STN target, SNr. Our findings suggest that, during STN-DBS, a critical change towards a high-frequency oscillatory discharge occurs. [ABSTRACT FROM AUTHOR]

Using intracerebral microdialysis, we reported previously that acutein vivoactivation of NMDA glutamate receptors triggers rapid and transient releases of prostaglandin E2 (PGE2) andF2-isoprostane 15-F2t-IsoP in the hippocampus of freely moving rats. The formation of the two metabolites– produced through cyclo-oxygenase (COX) enzymatic activity and free radical-mediated peroxidation of arachidonic acid (AA), respectively,– was prevented by the specific NMDA antagonist MK-801, and was largely dependent on COX-2 activity. Here, we demonstrate that besides COX-2, which is the prominent COX isoform in the brain and particularly in the hippocampus, the constitutive isoform, COX-1 also contributes to prostaglandin (PG) synthesis and oxidative damage followingin vivoacute activation of hippocampal NMDA glutamate receptors. The relative contribution of the two isoforms is dynamically regulated, as the COX-2 selective inhibitor NS398 immediately prevented PGE2 and 15-F2t-IsoP formation during the application of NMDA, whereas the COX-1 selective inhibitor SC560 was effective only 1 h after agonist infusion. Our data suggest that, although COX-2 is the prominent isoform, COX-1 activity may significantly contribute to excitotoxicity, particularly when considering the amount of lipid peroxidation associated with its catalytic cycle. We suggest that both isoforms should be considered as possible therapeutic targets to prevent brain damage caused by excitotoxicity. [ABSTRACT FROM AUTHOR]

Recently, a new system of astrocyte–neurone glutamatergic signalling has been identified. It is started in astrocytes by ectocellular, CD38-catalysed conversion of NAD+ to the calcium mobilizer cyclic ADP-ribose (cADPR). This is then pumped by CD38 itself into the cytosol where the resulting free intracellular Ca2+ concentration [Ca2+ i transients elicit an increased release of glutamate, which can induce an enhanced Ca2+ response in neighbouring neurones. Here, we demonstrate that co-culture of either cortical or hippocampal astrocytes with neurones results in a significant overexpression of astrocyte CD38 both on the plasma membrane and intracellularly. The causal role of neurone-released glutamate in inducing overexpression of astrocyte CD38 is demonstrated by two observations: first, in the absence of neurones, induction of CD38 in pure astrocyte cultures can be obtained with glutamate and second, it can be prevented in co-cultures by glutamate receptor antagonists. The neuronal glutamate-mediated effect of neurones on astrocyte CD38 expression is paralleled by increased intracellular cADPR and [Ca2+ i levels, both findings indicating functionality of overexpressed CD38. These results reveal a new neurone-to-astrocyte glutamatergic signalling based on the CD38/cADPR system, which affects the [Ca2+ i in both cell types, adding further complexity to the bi-directional patterns of communication between astrocytes and neurones. [ABSTRACT FROM AUTHOR]

In the cerebellum, infusion of NMDA (200 μM) for 20 min evoked a marked (200%) increase of extracellular cyclic GMP (cGMP) levels. The selective GABAA receptor agonist muscimol (0.01-100 μM) was able to counteract the NMDA effect with an EC50 of 0.65 μM ; the inhibitory effect of muscimol (10 μM) was prevented by bicuculline (50 μM). Diazepam (10 μM) significantly potentiated the muscimol (1 μM) inhibition ; furthermore, when coinfused with 0.1 μM muscimol (a concentration not affecting, on its own, the cGMP response to NMDA), diazepam (10 μM) reduced the NMDA effect. Similar results were obtained with zolpidem (0.1-1 μM). Finally, local infusion of the benzodiazepine site antagonist flumazenil (10 μM), together with muscimol and diazepam, almost completely restored the effect of NMDA on extracellular cGMP levels. It is concluded that GABAA receptors potently control the NMDA/nitric oxide/cGMP pathway in the cerebellum in vivo. In terms of the α subunit composition, we can deduce that the cerebellar GABAA receptor does not contain α6 or β4 subunits because it is diazepam-sensitive. Moreover, the observation that zolpidem is active at a rather low concentration, in combination with localization studies present in the literature, tend to exclude the presence of α5 subunits in the receptor composition and suggest the involvement of an α1 subunit. [ABSTRACT FROM AUTHOR]

Abstract Dopamine (DA) autoreceptors expressed at DA nerve terminals regulate DA release. Considerable evidence has indicated that, in rodents, these autoreceptors belong to the D2 type of the DA receptor family, which, in turn, comprises the D2, D3 and D4 subtypes. We investigated here, for the first time, the subclassification of native human DA autoreceptors by studying the release of [3H]DA evoked by electrical stimulation in fresh human neocortical slices. The results have been compared with those obtained in three animal systems: rat neocortical and striatal slices and rat mesencephalic neuronal cultures. In human neocortical slices, the D2/D3 receptor agonist quinpirole (1 nm–10 μm) inhibited tritium release with a calculated EC50 of 17 nm and a maximal inhibition of ≈ 75% reached at 1 μm. In the presence of the D2/D3 receptor antagonist (–)-sulpiride (0.1 and 1 μm), the concentration–response curve of quinpirole was shifted to the right, and the apparent pA2 mean value was 8.5 (8.14–8.77); on the other hand, the inhibitory effects of quinpirole were not affected by the D3 receptor-selective antagonist [7-N,N-dipropylamino-5,6,7,8-tetrahydro-naphtho(2,3b) dihydro,2,3-furane] (S 14297) and the D4 receptor-selective antagonist 3-(4-[4-chlorophenyl]piperazin-1-yl)-methyl-1H-pyrrolo [2,3-b]pyridine (L-745,870) (0.01–1 μm in each case). Superimposable results have been obtained when the release was elicited from rat striatal slices or dopamine mesencephalic neurons in culture, whereas quantitative differences emerged in the case of rat cortical slices. It is concluded that in human brain, as well as in rat brain, the release of DA in the terminal region of midbrain dopaminergic neurons is regulated through autoreceptors of the D2 subtype. [ABSTRACT FROM AUTHOR]