Altered glucose catabolism in the presynaptic and perisynaptic compartments of SOD1G93A mouse spinal cord and motor cortex indicates that mitochondria are the site of bioenergetic imbalance in ALS.

Amyotrophic lateral sclerosis is an adult-onset neurodegenerative disease that develops because of motor neuron death. Several mechanisms occur supporting neurodegeneration, including mitochondrial dysfunction. Recently, we demonstrated that the synaptosomes from the spinal cord of SOD1G93A mice, an in vitro model of presynapses, displayed impaired mitochondrial metabolism at early pre-symptomatic stages of the disease, whereas perisynaptic astrocyte particles, or gliosomes, were characterized by mild energy impairment only at symptomatic stages. This work aimed to understand whether mitochondrial impairment is a consequence of upstream metabolic damage. We analyzed the critical pathways involved in glucose catabolism at presynaptic and perisynaptic compartments. Spinal cord and motor cortex synaptosomes from SOD1G93A mice displayed high activity of hexokinase and phosphofructokinase, key glycolysis enzymes, and of citrate synthase and malate dehydrogenase, key Krebs cycle enzymes, but did not display high lactate dehydrogenase activity, the key enzyme in lactate fermentation. This enhancement was evident in the spinal cord from the early stages of the disease and in the motor cortex at only symptomatic stages. Conversely, an increase in glycolysis and lactate fermentation activity, but not Krebs cycle activity, was observed in gliosomes from the spinal cord and motor cortex of SOD1G93A mice although only at the symptomatic stages of the disease. The cited enzymatic activities were enhanced in spinal cord and motor cortex homogenates, paralleling the time-course of the effect observed in synaptosomes and gliosomes. The observed metabolic modifications might be considered an attempt to restore altered energetic balance and indicate that mitochondria represent the ultimate site of bioenergetic impairment.

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

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.

Enhanced Function and Overexpression of Metabotropic Glutamate Receptors 1 and 5 in the Spinal Cord of the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis during Disease Progression.

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 SOD1G93A 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) SOD1G93A mice. The mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) concentration dependently stimulated the release of [3H]d-Aspartate ([3H]d-Asp), which was comparable in 30- and 60-day-old wild type mice and SOD1G93A mice. At variance, [3H]d-Asp release was significantly augmented in 90-day-old SOD1G93A mice and both mGluR1 and mGluR5 were involved. The 3,5-DHPG-induced [3H]d-Asp release was exocytotic, being of vesicular origin and mediated by intra-terminal Ca2+ release. mGluR1 and mGluR5 expression was increased in Glu spinal cord axon terminals of 90-day-old SOD1G93A 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 SOD1G93A 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.

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

High levels of amyloid-ß peptide (Aß) have been related to Alzheimer's disease pathogenesis. However, in the healthy brain, low physiologically relevant concentrations of Aß 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 Aß 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 Aß due to a change in the approximation of amyloid precursor protein (APP) and the ß-site APP cleaving enzyme 1. Moreover, electrophysiological and behavioral studies performed on animals of both sexes showed that blocking Aß function, by using anti-murine Aß antibodies or APP knock-out mice, prevents the cGMP-dependent enhancement of LTP and memory. Our data suggest that cGMP positively regulates Aß levels in the healthy brain which, in turn, boosts synaptic plasticity and memory.SIGNIFICANCE STATEMENT Amyloid-ß (Aß) is a key pathogenetic factor in Alzheimer's disease. However, low concentrations of endogenous Aß, 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 Aß levels and function during LTP. We show that cGMP enhances Aß 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 Aß, suggesting that the physiological effect of the cyclic nucleotide on LTP and memory is dependent upon Aß.

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.

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.

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

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.

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

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 [3 H]d-aspartate or [3 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 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.

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

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.

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.

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.