Publications by authors named "Ernesto Fedele"

45 Publications

Protein kinase G phosphorylates the Alzheimer's disease-associated tau protein at distinct Ser/Thr sites.

Biofactors 2021 Jan 19;47(1):126-134. Epub 2021 Jan 19.

Section of General Pathology, Department of Experimental Medicine, University of Genoa, Genoa, Italy.

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.
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http://dx.doi.org/10.1002/biof.1705DOI Listing
January 2021

Memory Enhancers for Alzheimer's Dementia: Focus on cGMP.

Pharmaceuticals (Basel) 2021 Jan 13;14(1). Epub 2021 Jan 13.

IRCCS Ospedale Policlinico San Martino, 16132 Genova, Italy.

Cyclic guanosine-3',5'-monophosphate, better known as cyclic-GMP or cGMP, is a classical second messenger involved in a variety of intracellular pathways ultimately controlling different physiological functions. The family of guanylyl cyclases that includes soluble and particulate enzymes, each of which comprises several isoforms with different mechanisms of activation, synthesizes cGMP. cGMP signaling is mainly executed by the activation of protein kinase G and cyclic nucleotide gated channels, whereas it is terminated by its hydrolysis to GMP operated by both specific and dual-substrate phosphodiesterases. In the central nervous system, cGMP has attracted the attention of neuroscientists especially for its key role in the synaptic plasticity phenomenon of long-term potentiation that is instrumental to memory formation and consolidation, thus setting off a "gold rush" for new drugs that could be effective for the treatment of cognitive deficits. In this article, we summarize the state of the art on the neurochemistry of the cGMP system and then review the pre-clinical and clinical evidence on the use of cGMP enhancers in Alzheimer's disease (AD) therapy. Although preclinical data demonstrates the beneficial effects of cGMP on cognitive deficits in AD animal models, the results of the clinical studies carried out to date are not conclusive. More trials with a dose-finding design on selected AD patient's cohorts, possibly investigating also combination therapies, are still needed to evaluate the clinical potential of cGMP enhancers.
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http://dx.doi.org/10.3390/ph14010061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828493PMC
January 2021

Acute and Chronic Dopaminergic Depletion Differently Affect Motor Thalamic Function.

Int J Mol Sci 2020 Apr 15;21(8). Epub 2020 Apr 15.

Laboratory for Biomedical Neurosciences, Neurocenter of Southern Switzerland, 6900 Taverne, Switzerland.

The motor thalamus (MTh) plays a crucial role in the basal ganglia (BG)-cortical loop in motor information codification. Despite this, there is limited evidence of MTh functionality in normal and Parkinsonian conditions. To shed light on the functional properties of the MTh, we examined the effects of acute and chronic dopamine (DA) depletion on the neuronal firing of MTh neurons, cortical/MTh interplay and MTh extracellular concentrations of glutamate (GLU) and gamma-aminobutyric acid (GABA) in two states of DA depletion: acute depletion induced by the tetrodotoxin (TTX) and chronic denervation obtained by 6-hydroxydopamine (6-OHDA), both infused into the medial forebrain bundle (MFB) in anesthetized rats. The acute TTX DA depletion caused a clear-cut reduction in MTh neuronal activity without changes in burst content, whereas the chronic 6-OHDA depletion did not modify the firing rate but increased the burst firing. The phase correlation analysis underscored that the 6-OHDA chronic DA depletion affected the MTh-cortical activity coupling compared to the acute TTX-induced DA depletion state. The TTX acute DA depletion caused a clear-cut increase of the MTh GABA concentration and no change of GLU levels. On the other hand, the 6-OHDA-induced chronic DA depletion led to a significant reduction of local GABA and an increase of GLU levels in the MTh. These data show that MTh is affected by DA depletion and support the hypothesis that a rebalancing of MTh in the chronic condition counterbalances the profound alteration arising after acute DA depletion state.
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http://dx.doi.org/10.3390/ijms21082734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215393PMC
April 2020

cGMP favors the interaction between APP and BACE1 by inhibiting Rab5 GTPase activity.

Sci Rep 2020 Jan 28;10(1):1358. Epub 2020 Jan 28.

Department of Experimental Medicine, University of Genoa, Genoa, Italy.

We previously demonstrated that cyclic guanosine monophosphate (cGMP) stimulates amyloid precursor protein (APP) and beta-secretase (BACE1) approximation in neuronal endo-lysosomal compartments, thus boosting the production of amyloid-β (Aβ) peptides and enhancing synaptic plasticity and memory. Here, we further investigated the mechanism by which cGMP regulates the subcellular localization of APP and BACE1, finding that the cyclic nucleotide inhibits the activity of Rab5, a small GTPase associated with the plasma membrane and early endosomes. Accordingly, we also found that expression of a dominant-negative Rab5 mutant increases both APP-BACE1 approximation and Aβ extracellular levels, therefore mimicking the effects induced by cGMP. These results reveal a functional correlation between the cGMP/Aβ pathway and the activity of Rab5 that may contribute to the understanding of Alzheimer's disease pathophysiology.
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http://dx.doi.org/10.1038/s41598-020-58476-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6987147PMC
January 2020

The Novel Phosphodiesterase 9A Inhibitor BI 409306 Increases Cyclic Guanosine Monophosphate Levels in the Brain, Promotes Synaptic Plasticity, and Enhances Memory Function in Rodents.

J Pharmacol Exp Ther 2019 12 2;371(3):633-641. Epub 2019 Oct 2.

Boehringer Ingelheim International GmbH, Biberach an der Riss, Germany (H.R., R.G., G.S., E.K., F.R., H.F., A.M., C.D.-C.); Leibniz Institute for Neurobiology, Magdeburg, Germany (K.G.R., U.H.S.); and Department of Pharmacy, Centre of Excellence for Biomedical Research, University of Genova, Genova, Italy (E.F.).

-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) is an established cellular model underlying learning and memory, and involves intracellular signaling mediated by the second messenger cyclic guanosine monophosphate (cGMP). As phosphodiesterase (PDE)9A selectively hydrolyses cGMP in areas of the brain related to cognition, PDE9A inhibitors may improve cognitive function by enhancing NMDA receptor-dependent LTP. This study aimed to pharmacologically characterize BI 409306, a novel PDE9A inhibitor, using in vitro assays and in vivo determination of cGMP levels in the brain. Further, the effects of BI 409306 on synaptic plasticity evaluated by LTP in ex vivo hippocampal slices and on cognitive performance in rodents were also investigated. In vitro assays demonstrated that BI 409306 is a potent and selective inhibitor of human and rat PDE9A with mean concentrations at half-maximal inhibition (IC) of 65 and 168 nM. BI 409306 increased cGMP levels in rat prefrontal cortex and cerebrospinal fluid and attenuated a reduction in mouse striatum cGMP induced by the NMDA-receptor antagonist MK-801. In ex vivo rat brain slices, BI 409306 enhanced LTP induced by both weak and strong tetanic stimulation. Treatment of mice with BI 409306 reversed MK-801-induced working memory deficits in a T-maze spontaneous-alternation task and improved long-term memory in an object recognition task. These findings suggest that BI 409306 is a potent and selective inhibitor of PDE9A. BI 409306 shows target engagement by increasing cGMP levels in brain, facilitates synaptic plasticity as demonstrated by enhancement of hippocampal LTP, and improves episodic and working memory function in rodents. SIGNIFICANCE STATEMENT: This preclinical study demonstrates that BI 409306 is a potent and selective PDE9A inhibitor in rodents. Treatment with BI 409306 increased brain cGMP levels, promoted long-term potentiation, and improved episodic and working memory performance in rodents. These findings support a role for PDE9A in synaptic plasticity and cognition. The potential benefits of BI 409306 are currently being investigated in clinical trials.
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http://dx.doi.org/10.1124/jpet.119.260059DOI Listing
December 2019

Enhanced Function and Overexpression of Metabotropic Glutamate Receptors 1 and 5 in the Spinal Cord of the SOD1 Mouse Model of Amyotrophic Lateral Sclerosis during Disease Progression.

Int J Mol Sci 2019 Sep 13;20(18). Epub 2019 Sep 13.

Department of Pharmacy, Unit of Pharmacology and Toxicology and Center of Excellence for Biomedical Research (CEBR), University of Genoa, 16132 Genova, Italy.

Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) and our previous work highlighted that abnormal Glu release may represent a leading mechanism for excessive synaptic Glu. We demonstrated that group I metabotropic Glu receptors (mGluR1, mGluR5) produced abnormal Glu release in SOD1 mouse spinal cord at a late disease stage (120 days). Here, we studied this phenomenon in pre-symptomatic (30 and 60 days) and early-symptomatic (90 days) SOD1 mice. The mGluR1/5 agonist ()-3,5-Dihydroxyphenylglycine (3,5-DHPG) concentration dependently stimulated the release of [H]d-Aspartate ([H]d-Asp), which was comparable in 30- and 60-day-old wild type mice and SOD1 mice. At variance, [H]d-Asp release was significantly augmented in 90-day-old SOD1 mice and both mGluR1 and mGluR5 were involved. The 3,5-DHPG-induced [H]d-Asp release was exocytotic, being of vesicular origin and mediated by intra-terminal Ca release. mGluR1 and mGluR5 expression was increased in Glu spinal cord axon terminals of 90-day-old SOD1 mice, but not in the whole axon terminal population. Interestingly, mGluR1 and mGluR5 were significantly augmented in total spinal cord tissue already at 60 days. Thus, function and expression of group I mGluRs are enhanced in the early-symptomatic SOD1 mouse spinal cord, possibly participating in excessive Glu transmission and supporting their implication in ALS. Please define all abbreviations the first time they appear in the abstract, the main text, and the first figure or table caption.
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http://dx.doi.org/10.3390/ijms20184552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774337PMC
September 2019

Antagonizing α7 nicotinic receptors with methyllycaconitine (MLA) potentiates receptor activity and memory acquisition.

Cell Signal 2019 10 5;62:109338. Epub 2019 Jun 5.

Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, 6200, MD, Maastricht, The Netherlands. Electronic address:

α7 nicotinic acetylcholine receptors (α7nAChRs) have been targeted to improve cognition in different neurological and psychiatric disorders. Nevertheless, no α7nAChR activating ligand has been clinically approved. Here, we investigated the effects of antagonizing α7nAChRs using the selective antagonist methyllycaconitine (MLA) on receptor activity in vitro and cognitive functioning in vivo. Picomolar concentrations of MLA significantly potentiated receptor responses in electrophysiological experiments mimicking the in vivo situation. Furthermore, microdialysis studies showed that MLA administration substantially increased hippocampal glutamate efflux which is related to memory processes. Accordingly, pre-tetanus administration of low MLA concentrations produced longer lasting potentiation (long-term potentiation, LTP) in studies examining hippocampal plasticity. Moreover, low doses of MLA improved acquisition, but not consolidation memory processes in rats. While the focus to enhance cognition by modulating α7nAChRs lies on agonists and positive modulators, antagonists at low doses should provide a novel approach to improve cognition in neurological and psychiatric disorders.
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http://dx.doi.org/10.1016/j.cellsig.2019.06.003DOI Listing
October 2019

Leucine-rich repeat kinase 2 phosphorylation on synapsin I regulates glutamate release at pre-synaptic sites.

J Neurochem 2019 08 10;150(3):264-281. Epub 2019 Jul 10.

Department of Experimental Medicine, University of Genova, Genova, Italy.

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.
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http://dx.doi.org/10.1111/jnc.14778DOI Listing
August 2019

cAMP, cGMP and Amyloid β: Three Ideal Partners for Memory Formation.

Trends Neurosci 2018 05 28;41(5):255-266. Epub 2018 Feb 28.

Department of Pharmacy, Section of Pharmacology and Toxicology, University of Genoa, Genoa, Italy; Center of Excellence for Biomedical Research, University of Genoa, Genoa, Italy. Electronic address:

cAMP and cGMP are well established second messengers required for long-term potentiation (LTP) and memory formation/consolidation. By contrast, amyloid β (Aβ), mostly known as one of the main culprits for Alzheimer's disease (AD), has received relatively little attention in the context of plasticity and memory. Of note, however, low physiological concentrations of Aβ seem necessary for LTP induction and for memory formation. This should come as no surprise, since hormesis emerged as a central dogma in biology. Additionally, recent evidence indicates that Aβ is one of the downstream effectors for cAMP and cGMP to trigger synaptic plasticity and memory. We argue that these emerging findings depict a new scenario that should change the general view on the amyloidogenic pathway, and that could have significant implications for the understanding of AD and its pharmacological treatment in the future.
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http://dx.doi.org/10.1016/j.tins.2018.02.001DOI Listing
May 2018

Presynaptic GLP-1 receptors enhance the depolarization-evoked release of glutamate and GABA in the mouse cortex and hippocampus.

Biofactors 2018 Mar 19;44(2):148-157. Epub 2017 Dec 19.

Department of Pharmacy, Section of Pharmacology and Toxicology, University of Genova, Italy.

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 [ H]d-aspartate or [ H]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 G 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.
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http://dx.doi.org/10.1002/biof.1406DOI Listing
March 2018

Investigating the amyloid-beta enhancing effect of cGMP in neuro2a cells.

Mech Ageing Dev 2017 09 5;166:1-5. Epub 2017 Aug 5.

Department of Experimental Medicine, University of Genoa, Genoa, Italy. Electronic address:

Long-term potentiation (LTP) and the process of memory formation require activation of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) pathways. Notably, recent evidence indicated that both cyclic nucleotides boost the production of amyloid-beta (Aβ) peptides. In particular, cAMP was shown to favor hippocampal LTP by stimulating the synthesis of the amyloid precursor protein APP, whereas cGMP was found to enhance LTP and to improve memory by increasing Aβ levels without affecting the expression of APP. The results of the present study substantiate that cGMP has a role in the endocytic pathway of APP and suggest a scenario where the cyclic nucleotide enhances the production of Aβ by favoring the trafficking of APP from the cell cortex to the endolysosomal compartment.
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http://dx.doi.org/10.1016/j.mad.2017.08.001DOI Listing
September 2017

Amyloid-β Peptide Is Needed for cGMP-Induced Long-Term Potentiation and Memory.

J Neurosci 2017 07 16;37(29):6926-6937. Epub 2017 Jun 16.

Department of Biomedical and Biotechnological Sciences, Section of Physiology, University of Catania, 95123 Catania, Italy,

High levels of amyloid-β peptide (Aβ) have been related to Alzheimer's disease pathogenesis. However, in the healthy brain, low physiologically relevant concentrations of Aβ are necessary for long-term potentiation (LTP) and memory. Because cGMP plays a key role in these processes, here we investigated whether the cyclic nucleotide cGMP influences Aβ levels and function during LTP and memory. We demonstrate that the increase of cGMP levels by the phosphodiesterase-5 inhibitors sildenafil and vardenafil induces a parallel release of Aβ due to a change in the approximation of amyloid precursor protein (APP) and the β-site APP cleaving enzyme 1. Moreover, electrophysiological and behavioral studies performed on animals of both sexes showed that blocking Aβ function, by using anti-murine Aβ antibodies or APP knock-out mice, prevents the cGMP-dependent enhancement of LTP and memory. Our data suggest that cGMP positively regulates Aβ levels in the healthy brain which, in turn, boosts synaptic plasticity and memory. Amyloid-β (Aβ) is a key pathogenetic factor in Alzheimer's disease. However, low concentrations of endogenous Aβ, mimicking levels of the peptide in the healthy brain, enhance hippocampal long-term potentiation (LTP) and memory. Because the second messenger cGMP exerts a central role in LTP mechanisms, here we studied whether cGMP affects Aβ levels and function during LTP. We show that cGMP enhances Aβ production by increasing the APP/BACE-1 convergence in endolysosomal compartments. Moreover, the cGMP-induced enhancement of LTP and memory was disrupted by blockade of Aβ, suggesting that the physiological effect of the cyclic nucleotide on LTP and memory is dependent upon Aβ.
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http://dx.doi.org/10.1523/JNEUROSCI.3607-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518421PMC
July 2017

Memory-enhancing effects of GEBR-32a, a new PDE4D inhibitor holding promise for the treatment of Alzheimer's disease.

Sci Rep 2017 04 12;7:46320. Epub 2017 Apr 12.

Department of Pharmacy, Section of Pharmacology and Toxicology, School of Medical and Pharmaceutical Sciences, University of Genoa, Viale Cembrano, 4, 16148 Genoa, Italy.

Memory loss characterizes several neurodegenerative disorders, including Alzheimer's disease (AD). Inhibition of type 4 phosphodiesterase (PDE4) and elevation of cyclic adenosine monophosphate (cAMP) has emerged as a promising therapeutic approach to treat cognitive deficits. However, PDE4 exists in several isoforms and pan inhibitors cannot be used in humans due to severe emesis. Here, we present GEBR-32a, a new PDE4D full inhibitor that has been characterized both in vitro and in vivo using biochemical, electrophysiological and behavioural analyses. GEBR-32a efficiently enhances cAMP in neuronal cultures and hippocampal slices. In vivo pharmacokinetic analysis shows that GEBR-32a is rapidly distributed within the central nervous system with a very favourable brain/blood ratio. Specific behavioural tests (object location and Y-maze continuous alternation tasks) demonstrate that this PDE4D inhibitor is able to enhance memory in AD transgenic mice and concomitantly rescues their hippocampal long-term potentiation deficit. Of great relevance, our preliminary toxicological analysis indicates that GEBR-32a is not cytotoxic and genotoxic, and does not seem to possess emetic-like side effects. In conclusion, GEBR-32a could represent a very promising cognitive-enhancing drug with a great potential for the treatment of Alzheimer's disease.
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http://dx.doi.org/10.1038/srep46320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389348PMC
April 2017

Homovanillic acid in CSF of mild stage Parkinson's disease patients correlates with motor impairment.

Neurochem Int 2017 May 18;105:58-63. Epub 2017 Jan 18.

Movement Disorders Centre, Department of Systems Medicine, University of Rome "Tor Vergata", Rome, Italy.

In Parkinson's disease (PD), several efforts have been spent in order to find biochemical parameters able to identify the progression of the pathological processes at the basis of the disease. It is already known that advanced PD patients manifesting dyskinesia are featured by the high homovanillic acid (HVA)/dopamine (DA) ratio, suggesting the increased turnover of DA in these patients. Less clear is whether similar changes affect mild and moderate stages of the disease (between 1 and 2.5 of Hoehn & Yahr -H&Y- stage). Hence, here we tested whether cerebrospinal fluid (CSF) concentrations of DA and its major metabolites, either 3,4-dihydroxyphenylacetic acid (DOPAC) or HVA, correlate with motor performance in mild and moderate PD patients. CSF samples were collected after 2 days of anti-PD drugs washout, via lumbar puncture (LP) performed 130 min following administration of oral levodopa (LD) dose (200 mg). LP timing was determined in light of our previous tests clarifying that 2 h after oral LD administration CSF DA concentration reaches a plateau, which was un-respective of PD stage or duration. DA, DOPAC and HVA were assayed by high performance liquid chromatography in a group of 19 patients, distributed in two groups on the basis of the H&Y stage with a cut-off of 1.5. In these PD patients, HVA was correlated with DOPAC (R = 0,56, p < 0,01) and both HVA and DOPAC CSF levels increased in parallel with the motor impairment. More importantly, HVA correlated with motor impairment measured by the Unified Parkinson's Disease Score -III (UPDRS) (R = 0.61; p < 0.0001). The present findings showed the early alteration of the DA pre-synaptic machinery, as documented by the progressive increase of CSF HVA concentrations, which also correlated with PD motor impairment. Therefore, we suggest the potential use of measuring the CSF HVA level as a possible biomarker of PD stage changes in order to monitor the effectiveness of PD-modifying pharmacological therapies.
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http://dx.doi.org/10.1016/j.neuint.2017.01.007DOI Listing
May 2017

The Amyloid Cascade Hypothesis in Alzheimer's Disease: It's Time to Change Our Mind.

Curr Neuropharmacol 2017 ;15(6):926-935

Department of Pharmacy, Section of Pharmacology and Toxicology, University of Genova, Genova, Italy.

Since its discovery in 1984, the beta amyloid peptide has treaded the boards of neurosciences as the star molecule in Alzheimer's disease pathogenesis. In the last decade, however, this vision has been challenged by evidence-based medicine showing the almost complete failure of clinical trials that experimented anti-amyloid therapies with great hopes. Moreover, data have accumulated which clearly indicate that this small peptide plays a key role in the physiological processes of memory formation. In the present review, we will discuss the different aspects of the amyloid cascade hypothesis, highlighting its pros and cons, and we will analyse the results of the therapeutic approaches attempted to date that should change the direction of Alzheimer's disease research in the future.
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http://dx.doi.org/10.2174/1570159X15666170116143743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652035PMC
April 2018

Altered fronto-striatal functions in the Gdi1-null mouse model of X-linked Intellectual Disability.

Neuroscience 2017 03 3;344:346-359. Epub 2017 Jan 3.

Molecular Genetics of Intellectual Disability Unit, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy. Electronic address:

RAB-GDP dissociation inhibitor 1 (GDI1) loss-of-function mutations are responsible for a form of non-specific X-linked Intellectual Disability (XLID) where the only clinical feature is cognitive impairment. GDI1 patients are impaired in specific aspects of executive functions and conditioned response, which are controlled by fronto-striatal circuitries. Previous molecular and behavioral characterization of the Gdi1-null mouse revealed alterations in the total number/distribution of hippocampal and cortical synaptic vesicles as well as hippocampal short-term synaptic plasticity, and memory deficits. In this study, we employed cognitive protocols with high translational validity to human condition that target the functionality of cortico-striatal circuitry such as attention and stimulus selection ability with progressive degree of complexity. We previously showed that Gdi1-null mice are impaired in some hippocampus-dependent forms of associative learning assessed by aversive procedures. Here, using appetitive-conditioning procedures we further investigated associative learning deficits sustained by the fronto-striatal system. We report that Gdi1-null mice are impaired in attention and associative learning processes, which are a key part of the cognitive impairment observed in XLID patients.
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http://dx.doi.org/10.1016/j.neuroscience.2016.12.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315088PMC
March 2017

New insights into selective PDE4D inhibitors: 3-(Cyclopentyloxy)-4-methoxybenzaldehyde O-(2-(2,6-dimethylmorpholino)-2-oxoethyl) oxime (GEBR-7b) structural development and promising activities to restore memory impairment.

Eur J Med Chem 2016 Nov 13;124:82-102. Epub 2016 Aug 13.

Department of Pharmacy, Section of Medicinal Chemistry, School of Medical and Pharmaceutical Sciences, University of Genoa, Viale Benedetto XV, 3, 16132 Genoa, Italy. Electronic address:

Phosphodiesterase type 4D (PDE4D) has been indicated as a promising target for treating neurodegenerative pathologies such as Alzheimer's Disease (AD). By preventing cAMP hydrolysis, PDE4 inhibitors (PDE4Is) increase the cAMP response element-binding protein (CREB) phosphorylation, synaptic plasticity and long-term memory formation. Pharmacological and behavioral studies on our hit GEBR-7b demonstrated that selective PDE4DIs could improve memory without causing emesis and sedation. The hit development led to new molecule series, herein reported, characterized by a catechol structure bonded to five member heterocycles. Molecular modeling studies highlighted the pivotal role of a polar alkyl chain in conferring selective enzyme interaction. Compound 8a showed PDE4D3 selective inhibition and was able to increase intracellular cAMP levels in neuronal cells, as well as in the hippocampus of freely moving rats. Furthermore, 8a was able to readily cross the blood-brain barrier and enhanced memory performance in mice without causing any emetic-like behavior. These data support the view that PDE4D is an adequate molecular target to restore memory deficits in different neuropathologies, including AD, and also indicate compound 8a as a promising candidate for further preclinical development.
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http://dx.doi.org/10.1016/j.ejmech.2016.08.018DOI Listing
November 2016

Amyloid β: Walking on the dark side of the moon.

Mech Ageing Dev 2015 Dec 8;152:1-4. Epub 2015 Sep 8.

Department of Experimental Medicine, University of Genoa, Genoa, Italy. Electronic address:

For some decades, amyloid β (Aβ) has only been considered as a cytotoxic peptide, putative cause and marker of Alzheimer's disease (AD). Today, however, a considerable amount of evidence goes against the classical amyloid hypothesis and illustrates a new picture in which the Aβ loss of function, rather than its accumulation, has a pathogenic role in AD. In this concise review, we summarize some highlights of a collection of research pointing to the physiological function of Aβ and its role in the mechanisms of memory formation.
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http://dx.doi.org/10.1016/j.mad.2015.09.001DOI Listing
December 2015

Phosphodiesterase 4D: an enzyme to remember.

Br J Pharmacol 2015 Oct 22;172(20):4785-9. Epub 2015 Sep 22.

Department of Pharmacy, Section of Pharmacology and Toxicology, Center of Excellence for Biomedical Research, University of Genoa, Genoa, Italy.

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.
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http://dx.doi.org/10.1111/bph.13257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621991PMC
October 2015

Synthesis, biological activities and pharmacokinetic properties of new fluorinated derivatives of selective PDE4D inhibitors.

Bioorg Med Chem 2015 Jul 16;23(13):3426-35. Epub 2015 Apr 16.

Department of Pharmacy, Section of Medicinal Chemistry, School of Medical and Pharmaceutical Sciences, University of Genoa, Viale Benedetto XV, 3, 16132 Genoa, Italy. Electronic address:

A new series of selective PDE4D inhibitors has been designed and synthesized by replacing 3-methoxy group with 3-difluoromethoxy isoster moiety in our previously reported cathecolic structures. All compounds showed a good PDE4D3 inhibitory activity, most of them being inactive toward other PDE4 isoforms (PDE4A4, PDE4B2 and PDE4C2). Compound 3b, chosen among the synthesized compounds as the most promising in terms of inhibitory activity, selectivity and safety, showed an improved pharmacokinetic profile compared to its non fluorinated analogue. Spontaneous locomotor activity, assessed in an open field apparatus, showed that, differently from rolipram and diazepam, selective PDE4D inhibitors, such as compounds 3b, 5b and 7b, did not affect locomotion, whereas compound 1b showed a tendency to reduce the distance traveled and to prolong the immobility period, possibly due to a poor selectivity.
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http://dx.doi.org/10.1016/j.bmc.2015.04.027DOI Listing
July 2015

Isolation of hydroxyoctaprenyl-1',4'-hydroquinone, a new octaprenylhydroquinone from the marine sponge Sarcotragus spinosulus and evaluation of its pharmacological activity on acetylcholine and glutamate release in the rat central nervous system.

Nat Prod Commun 2014 Nov;9(11):1581-4

Three polyprenyl-1',4'-hydroquinone derivatives, heptaprenyl-1',4'-hydroquinone (1), octaprenyl-1',4'-hydroquinone (2), and hydroxyoctaprenyl-1',4'- hydroquinone (3) were isolated from the marine sponge Sarcotragus spinosulus collected at Baia di Porto Conte, Alghero (Italy). Our findings indicate that the compounds isolated from S. spinosulus can significantly modulate the release of glutamate and acetylcholine in the rat hippocampus and cortex and might, therefore, represent the prototype of a new class of drugs regulating glutamatergic and cholinergic transmission in the mammalian central nervous system.
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November 2014

Exocytosis regulates trafficking of GABA and glycine heterotransporters in spinal cord glutamatergic synapses: a mechanism for the excessive heterotransporter-induced release of glutamate in experimental amyotrophic lateral sclerosis.

Neurobiol Dis 2015 Feb 10;74:314-24. Epub 2014 Dec 10.

Department of Pharmacy, Unit of Pharmacology and Toxicology and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy. Electronic address:

The impact of synaptic vesicle endo-exocytosis on the trafficking of nerve terminal heterotransporters was studied by monitoring membrane expression and function of the GABA transporter-1 (GAT-1) and of type-1/2 glycine (Gly) transporters (GlyT-1/2) at spinal cord glutamatergic synaptic boutons. Experiments were performed by inducing exocytosis in wild-type (WT) mice, in amphiphysin-I knockout (Amph-I KO) mice, which show impaired endocytosis, or in mice expressing high copy number of mutant human SOD1 with a Gly93Ala substitution (SOD1(G93A)), a model of human amyotrophic lateral sclerosis showing constitutively excessive Glu exocytosis. Exposure of spinal cord synaptosomes from WT mice to a 35mM KCl pulse increased the expression of GAT-1 at glutamatergic synaptosomal membranes and enhanced the GAT-1 heterotransporter-induced [(3)H]d-aspartate ([(3)H]d-Asp) release. Similar results were obtained in the case of GlyT-1/2 heterotransporters. Preventing depolarization-induced exocytosis normalized the excessive GAT-1 and GlyT-1/2 heterotransporter-induced [(3)H]d-Asp release in WT mice. Impaired endocytosis in Amph-I KO mice increased GAT-1 membrane expression and [(3)H]GABA uptake in spinal cord synaptosomes. Also the GAT-1 heterotransporter-evoked release of [(3)H]d-Asp was augmented in Amph-I KO mice. The constitutively excessive Glu exocytosis in SOD1(G93A) mice resulted in augmented GAT-1 expression at glutamatergic synaptosomal membranes and GAT-1 or GlyT-1/2 heterotransporter-mediated [(3)H]d-Asp release. Thus, endo-exocytosis regulates the trafficking of GAT-1 and GlyT-1/2 heterotransporters sited at spinal cord glutamatergic nerve terminals. As a consequence, it can be hypothesized that the excessive GAT-1 and GlyT-1/2 heterotransporter-mediated Glu release, in the spinal cord of SOD1(G93A) mice, is due to the heterotransporter over-expression at the nerve terminal membrane, promoted by the excessive Glu exocytosis.
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http://dx.doi.org/10.1016/j.nbd.2014.12.004DOI Listing
February 2015

Synthesis, biological evaluation, and molecular modeling of new 3-(cyclopentyloxy)-4-methoxybenzaldehyde O-(2-(2,6-dimethylmorpholino)-2-oxoethyl) Oxime (GEBR-7b) related phosphodiesterase 4D (PDE4D) inhibitors.

J Med Chem 2014 Aug 15;57(16):7061-72. Epub 2014 Aug 15.

Department of Pharmacy, Section of Medicinal Chemistry, School of Medical and Pharmaceutical Sciences, University of Genoa , Viale Benedetto XV, 3, 16132 Genoa, Italy.

A new series of 3-(cyclopentyloxy)-4-methoxyphenyl derivatives, structurally related to our hit GEBR-4a (1) and GEBR-7b (2), has been designed by changing length and functionality of the chain linking the catecholic moiety to the terminal cycloamine portion. Among the numerous molecules synthesized, compounds 8, 10a, and 10b showed increased potency as PDE4D enzyme inhibitors with respect to 2 and a good selectivity against PDE4A4, PDE4B2, and PDE4C2 enzymes, without both cytotoxic and genotoxic effects. The ability to enhance cAMP level in neuronal cells was assessed for compound 8. SAR considerations, also confirmed by in silico docking simulations, evidenced that both chain and amino terminal function characterized by higher hydrophilicity are required for a good and selective inhibitor-catalytic pocket interaction.
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http://dx.doi.org/10.1021/jm500855wDOI Listing
August 2014

PDE4D inhibitors: a potential strategy for the treatment of memory impairment?

Neuropharmacology 2014 Oct 2;85:290-2. Epub 2014 Jun 2.

Department of Pharmacy, Section of Pharmacology and Toxicology, University of Genoa, Genoa, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.neuropharm.2014.05.038DOI Listing
October 2014

LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex.

Front Mol Neurosci 2014 27;7:49. Epub 2014 May 27.

Division of Neuroscience, San Raffaele Scientific Institute and Vita-Salute University Milan, Italy ; Department of Molecular and Cellular Pharmacology, National Research Council, Neuroscience Institute Milan, Italy.

Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex.
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http://dx.doi.org/10.3389/fnmol.2014.00049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4034499PMC
June 2014

A novel mechanism for cyclic adenosine monophosphate-mediated memory formation: Role of amyloid beta.

Ann Neurol 2014 Apr 14;75(4):602-7. Epub 2014 Apr 14.

Department of Experimental Medicine, Section of General Pathology, University of Genoa, Genoa, Italy.

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.
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http://dx.doi.org/10.1002/ana.24130DOI Listing
April 2014

Neuropeptide S inhibits release of 5-HT and glycine in mouse amygdala and frontal/prefrontal cortex through activation of the neuropeptide S receptor.

Neurochem Int 2013 Mar 11;62(4):360-6. Epub 2013 Feb 11.

Department of Pharmacy, Pharmacology and Toxicology Unit, University of Genoa, Genoa, Italy.

Neuropeptide S (NPS) is a neurotransmitter/neuromodulator that has been identified as the natural ligand of G protein-coupled receptors termed NPS receptors (NPSRs). The NPS-NPSR system is involved in the control of numerous centrally-mediated behaviours, including anxiety. As several classical transmitters play a role in fear/anxiety, we here investigated the regulation by NPS of the exocytotic release of 5-hydroxytryptamine (5-HT) and glycine in nerve terminals isolated from mouse frontal/prefrontal cortex and amygdala. Synaptosomes, prelabelled with the tritiated neurotransmitters, were depolarized in superfusion with 12-15 mM KCl and exposed to varying concentrations of NPS. The evoked release of [(3)H]5-HT in frontal/prefrontal cortex was potently inhibited by NPS (maximal effect about 25% at 0.1 nM). Differently, the neuropeptide exhibited higher efficacy but much lower potency in amygdala (maximal effect about 40% at 1 μM). NPS was an extremely potent inhibitor of the K(+)-evoked release of [(3)H]glycine in frontal/prefrontal nerve endings (maximal effect about 25% at 1 pM). All the inhibitory effects observed were counteracted by the NPSR antagonist SHA 68, indicating that the neuropeptide acted at NPSRs. In conclusion, NPS can inhibit the exocytosis of 5-HT and of glycine through the activation of presynaptic NPSRs situated on serotonergic and glycinergic terminals in areas involved in fear/anxiety behaviours. The possibility exists that the NPSRs in frontal/prefrontal cortex are high-affinity receptors involved in non-synaptic transmission, whereas the NPSRs on amygdala serotonergic terminals are low-affinity receptors involved in axo-axonic synaptic communication.
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http://dx.doi.org/10.1016/j.neuint.2013.02.003DOI Listing
March 2013

Cyclic adenosine monophosphate as an endogenous modulator of the amyloid-β precursor protein metabolism.

IUBMB Life 2013 Feb 8;65(2):127-33. Epub 2013 Jan 8.

Department of Experimental Medicine, University of Genoa, Genoa, Italy.

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.
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http://dx.doi.org/10.1002/iub.1109DOI Listing
February 2013

Reduced GABA Content in the Motor Thalamus during Effective Deep Brain Stimulation of the Subthalamic Nucleus.

Front Syst Neurosci 2011 5;5:17. Epub 2011 Apr 5.

Istituto di Ricovero e Cura a Carattere Scientifico Fondazione S. Lucia, Roma, Italy.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN), in Parkinson's disease (PD) patients, is a well established therapeutic option, but its mechanisms of action are only partially known. In our previous study, the clinical transitions from OFF- to ON-state were not correlated with significant changes of GABA content inside GPi or substantia nigra reticulata. Here, biochemical effects of STN-DBS have been assessed in putamen (PUT), internal pallidus (GPi), and inside the antero-ventral thalamus (VA), the key station receiving pallidothalamic fibers. In 10 advanced PD patients undergoing surgery, microdialysis samples were collected before and during STN-DBS. cGMP, an index of glutamatergic transmission, was measured in GPi and PUT by radioimmunoassay, whereas GABA from VA was measured by HPLC. During clinically effective STN-DBS, we found a significant decrease in GABA extracellular concentrations in VA (-30%). Simultaneously, cGMP extracellular concentrations were enhanced in PUT (+200%) and GPi (+481%). These findings support a thalamic dis-inhibition, in turn re-establishing a more physiological corticostriatal transmission, as the source of motor improvement. They indirectly confirm the relevance of patterning (instead of mere changes of excitability) and suggest that a rigid interpretation of the standard model, at least when it indicates the hyperactive indirect pathway as key feature of hypokinetic signs, is unlikely to be correct. Finally, given the demonstration of a key role of VA in inducing clinical relief, locally administration of drugs modulating GABA transmission in thalamic nuclei could become an innovative therapeutic strategy.
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http://dx.doi.org/10.3389/fnsys.2011.00017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078559PMC
July 2011