Rosaria Romano

Persistent increase of d-aspartate in d-aspartate oxidase mutant mice induces a precocious hippocampal age-dependent synaptic plasticity and spatial memory decay

The atypical amino acid d-aspartate (d-Asp) occurs at considerable amounts in the developing brain of mammals. However, during postnatal life, d-Asp levels diminish following the expression of d-aspartate oxidase (DDO) enzyme. The strict control of DDO over its substrate d-Asp is particularly evident in the hippocampus, a brain region crucially involved in memory, and highly vulnerable to age-related deterioration processes. Herein, we explored the influence of deregulated higher d-Asp brain content on hippocampus-related functions during aging of mice lacking DDO (Ddo(-/-)). Strikingly, we demonstrated that the enhancement of hippocampal synaptic plasticity and cognition in 4/5-month-old Ddo(-/-) mice is followed by an accelerated decay of basal glutamatergic transmission, NMDAR-dependent LTP and hippocampus-related reference memory at 13/14 months of age. Therefore, the precocious deterioration of hippocampal functions observed in mutants highlights for the first time a role for DDO enzyme in controlling the rate of brain aging process in mammals.

Higher free d-aspartate and N-methyl-d-aspartate levels prevent striatal depotentiation and anticipate l-DOPA-induced dyskinesia

In Parkinsons disease (PD) progressive alteration of striatal N-methyl-D-aspartate receptors (NMDARs) signaling has emerged as a considerable factor for the onset of the adverse motor effects of long-term levodopa (l-DOPA) treatment. In this regard, the NMDAR channel blocker amantadine is so far the only drug available for clinical use that attenuates L-DOPA-induced dyskinesia (LID). In this study, we examined the influence of a basal corticostriatal hyper-glutamatergic transmission in the appearance of dyskinesia, using a genetic mouse model lacking D-Aspartate Oxidase (DDO) enzyme (Ddo(-/-) mice). We found that, in Ddo(-/-) mice, non-physiological, high levels of the endogenous free D-amino acids D-aspartate (D-Asp) and NMDA, known to stimulate NMDAR transmission, resulted in the loss of corticostriatal synaptic depotentiation and precocious expression of LID. Interestingly, the block of depotentiation precedes any change in dopaminergic transmission associated to 6-OHDA lesion and l-DOPA treatment. Indeed, lesioned mutant mice display physiological L-DOPA-dependent enhancement of striatal D1 receptor/PKA/protein phosphatase-1 and ERK signaling. Moreover, in line with synaptic rearrangements of NMDAR subunits occurring in dyskinetic animal models, a short L-DOPA treatment produces a dramatic and selective reduction of the NR2B subunit in the striatal post-synaptic fraction of Ddo(-/-) lesioned mutants but not in controls. These data indicate that a preexisting hyper-glutamatergic tone at NMDARs in Ddo(-/-) mice produce abnormal striatal synaptic changes that, in turn, facilitate the onset of LID.

Genetic deletion of monoacylglycerol lipase leads to impaired cannabinoid receptor CB1R signaling and anxiety‐like behavior

Endocannabinoids (eCB) are key regulators of excitatory/inhibitory neurotransmission at cannabinoid‐1‐receptor (CB1R)‐expressing axon terminals. The most abundant eCB in the brain, that is 2‐arachidonoylglycerol (2‐AG), is hydrolyzed by the enzyme monoacylglycerol lipase (MAGL), whose chronic inhibition in the brain was reported to cause CB1R desensitization. We employed the MAGL knock‐out mouse (MAGL−/−), a genetic model of congenital and sustained elevation of 2‐AG levels in the brain, to provide morphological and biochemical evidence for β‐arrestin2‐mediated CB1R desensitization in brain regions involved in the control of emotional states, that is, the prefrontal cortex (PFC), amygdala, hippocampus and cerebellar cortex. We found a widespread CB1R/β‐arrestin2 co‐expression in the mPFC, amygdala and hippocampus accompanied by impairment of extracellular signal‐regulated kinase signaling and elevation of vesicular glutamate transporter (VGluT1) at CB1R‐positive excitatory terminals in the mPFC, or vesicular GABA transporter (VGAT) at CB1R‐positive inhibitory terminals in the amygdala and hippocampus. The impairment of CB1R signaling in MAGL−/− mice was also accompanied by enhanced excitatory drive in the basolateral amygdala (BLA)‐mPFC circuit, with subsequent elevation of glutamate release to the mPFC and anxiety‐like and obsessive‐compulsive behaviors, as assessed by the light/dark box and marble burying tests, respectively. Collectively, these data provide evidence for a β‐arrestin2‐mediated desensitization of CB1R in MAGL−/− mice, with impact on the synaptic plasticity of brain circuits involved in emotional functions.

Palmitoylethanolamide induces microglia changes associated with increased migration and phagocytic activity: involvement of the CB2 receptor

The endogenous fatty acid amide palmitoylethanolamide (PEA) has been shown to exert anti-inflammatory actions mainly through inhibition of the release of pro-inflammatory molecules from mast cells, monocytes and macrophages. Indirect activation of the endocannabinoid (eCB) system is among the several mechanisms of action that have been proposed to underlie the different effects of PEA in vivo. In this study, we used cultured rat microglia and human macrophages to evaluate whether PEA affects eCB signaling. PEA was found to increase CB2 mRNA and protein expression through peroxisome proliferator-activated receptor-α (PPAR-α) activation. This novel gene regulation mechanism was demonstrated through: (i) pharmacological PPAR-α manipulation, (ii) PPAR-α mRNA silencing, (iii) chromatin immunoprecipitation. Moreover, exposure to PEA induced morphological changes associated with a reactive microglial phenotype, including increased phagocytosis and migratory activity. Our findings suggest indirect regulation of microglial CB2R expression as a new possible mechanism underlying the effects of PEA. PEA can be explored as a useful tool for preventing/treating the symptoms associated with neuroinflammation in CNS disorders.

MMPIP, an mGluR7-selective negative allosteric modulator, alleviates pain and normalizes affective and cognitive behavior in neuropathic mice.

Abstract This study investigated the effects of a single administration of 6-(4-methoxyphenyl)-5-methyl-3-pyridinyl-4-isoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), a negative allosteric modulator (NAM) of metabotropic glutamate receptor 7 (mGluR7), on pain and on affective and cognitive behavior in neuropathic mice. The activity of pyramidal neurons in the prelimbic cortex (PLC), which respond to stimulation of the basolateral amygdala (BLA) with either excitation or inhibition, was also investigated. The spared nerve injury (SNI) of the sciatic nerve induced, 14 days after surgery, thermal hyperalgesia and mechanical allodynia, reduced open-arm choice in the elevated plus-maze, increased time of immobility in the tail suspension, and increased digging and burying in the marble burying test. Cognitive performance was also significantly compromised in the SNI mice. Spared nerve injury induced phenotypic changes on pyramidal neurons of the PLC; excitatory responses increased, whereas inhibitory responses decreased after BLA stimulation. mGluR7 expression, mainly associated with vesicular glutamate transporter, increased in the hippocampus and decreased in the BLA, PLC, and dorsal raphe in SNI mice. MMPIP increased thermal and mechanical thresholds and open-arm choice. It reduced the immobility in the tail suspension test and the number of marbles buried and of digging events in the marble burying test. MMPIP also improved cognitive performance and restored the balance between excitatory and inhibitory responses of PLC neurons in SNI mice. 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one, XAP044, another selective mGluR7 NAM, reproduced the effects of MMPIP on thermal hyperalgesia, mechanical allodynia, tail suspension, and marble burying test. Altogether, these findings show that mGluR7 NAMs reduce pain responses and affective/cognitive impairments in neuropathic pain conditions.

Loss of striatal cannabinoid CB1 receptor function in attention-deficit / hyperactivity disorder mice with point-mutation of the dopamine transporter.

Abnormal dopamine (DA) transmission in the striatum plays a pivotal role in attention‐deficit/hyperactivity disorder (ADHD). As striatal DA signalling modulates the endocannabinoid system (ECS), the present study was aimed at investigating cannabinoid CB1 receptor (CB1R) function in a model of ADHD obtained by triple point‐mutation in the dopamine transporter (DAT) gene in mice, making them insensitive to cocaine [DAT cocaine‐insensitive (DAT‐CI) mice]. DAT‐CI mice had a marked hyperactive phenotype, and neurophysiological recordings revealed that the sensitivity of CB1Rs controlling GABA‐mediated synaptic currents [CB1Rs(GABA)] in the striatum was completely lost. In contrast, CB1Rs modulating glutamate transmission [CB1Rs(Glu)], and GABAB receptors were not affected in this model of ADHD. In DAT‐CI mice, the blockade of CB1R(GABA) function was complete even after cocaine or environmental manipulations activating the endogenous DA‐dependent reward system, which are known to sensitize these receptors in control animals. Conversely, the hedonic property of sucrose was intact in DAT‐CI mice, indicating normal sweet perception in these animals. Our results point to CB1Rs as novel molecular players in ADHD, and suggest that therapeutic strategies aimed at interfering with the ECS might prove effective in this disorder.

A2A Adenosine Receptor Antagonism Enhances Synaptic and Motor Effects of Cocaine via CB1 Cannabinoid Receptor Activation

Background Cocaine increases the level of endogenous dopamine (DA) in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs) have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. Principal Findings Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. Conclusions The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine.

PC1, a non-peptide PKR1-preferring antagonist, reduces pain behavior and spinal neuronal sensitization in neuropathic mice.

Peripheral neuropathy is characterized by abnormal pain responses triggered by the release of several mediators and neuronal hyperexcitability at the spinal cord level. Emerging evidence indicates that the enhanced activity of dorsal horn neurons requires communication with glia and microglia, cells that are physiologically involved in neuronal wellbeing. Prokineticins (PKs), which include PK1 and PK2, represent a novel family of chemokines characterized by a unique structural motif comprising five disulfide bonds. They are expressed in the peripheral and central nervous system. PKs bind two G protein coupled receptors, PKR1 and PKR2, and participate in the regulation of several biological processes, including pain sensation. This study aimed to investigate the anti-nociceptive effect of PC1, a non-peptide PKR1-preferring antagonist, in a mouse model of neuropathic pain. To do this, we assessed the activity of spinal cord nociceptive neurons as well as astrocyte and microglia phenotypes after repeated administration of PC1 in vivo. PC1 treatment strongly delayed the development of thermal hyperalgesia and tactile and mechanical allodynia. It also reduced spinal microglial and glial activation 8 days post injury in spared nerve injury (SNI) mice. Neuropathic mice showed an increased level of PK2 protein in the spinal cord, mostly in astrocytes. PC1 treatment completely reversed the increased responsiveness to mechanical stimuli, the decreased threshold of neuronal activation, and the increased spontaneous activity that were observed in nociceptive specific (NS) neurons of SNI mice.

d-Aspartic acid ameliorates painful and neuropsychiatric changes and reduces β-amyloid Aβ1-42 peptide in a long lasting model of neuropathic pain

Depressive symptoms and other neuropsychiatric dysfunctions are common in neurodegenerative disorders, including chronic pain and dementia. A correlation between the β-amyloid protein accumulation and the development of depression has been suggested, however the underlying mechanisms are unknown. d-Aspartate (d-Asp) is a free d-amino acid found in the mammalian brain and involved in neurological and psychiatric processes, such as cognition and affective disorders. In this study we have investigated the effects of a repeated treatment with d-Asp in a long-lasting (12 months) model of neuropathic pain, the spared nerve injury (SNI), in mice. Specifically, we evaluated i) the pain sensitivity and related emotional/cognitive dysfunctions induced by SNI, ii) possible changes in the β-amyloid protein accumulation in specific brain regions involved in pain mechanisms ii) possible changes in steroids level in neuropathic animals with or without d-Asp in the same brain areas. SNI mice showed an increase of the insoluble form of Aβ1-42 at hippocampal level and displayed cognitive impairments, stereotypical and depressive-like behaviours. d-Asp treatment reduced abnormal behaviours and normalized the β-amyloid protein expression. Moreover, d-Asp dramatically increased steroids level measured in the prefrontal cortex and in the hippocampus. Our findings provide new insights into pain mechanisms and suggest a possible role of β-amyloid protein in neuropsychiatric dysfunctions associated with chronic pain.

Palmitoylethanolamide Reduces Neuropsychiatric Behaviors by Restoring Cortical Electrophysiological Activity in a Mouse Model of Mild Traumatic Brain Injury

Traumatic brain injury (TBI) represents a major public health problem, which is associated with neurological dysfunction. In severe or moderate cases of TBI, in addition to its high mortality rate, subjects may encounter diverse behavioral dysfunctions. Previous reports suggest that an association between TBI and chronic pain syndromes tends to be more common in patients with mild forms of brain injury. Despite causing minimal brain damage, mild TBI (mTBI) often leads to persistent psychologically debilitating symptoms, which can include anxiety, various forms of memory and learning deficits, and depression. At present, no effective treatment options are available for these symptoms, and little is known about the complex cellular activity affecting neuronal activity that occurs in response to TBI during its late phase. Here, we used a mouse model to investigate the effect of Palmitoylethanolamide (PEA) on both the sensorial and neuropsychiatric dysfunctions associated with mTBI through behavioral, electrophysiological, and biomolecular approaches. Fourteen-day mTBI mice developed anxious, aggressive, and reckless behavior, whilst depressive-like behavior and impaired social interactions were observed from the 60th day onward. Altered behavior was associated with changes in interleukin 1 beta (IL-1β) expression levels and neuronal firing activity in the medial prefrontal cortex. Compared with vehicle, PEA restored the behavioral phenotype and partially normalized the biochemical and functional changes occurring at the supraspinal level. In conclusion, our findings reveal some of the supraspinal modifications responsible for the behavioral alterations associated with mTBI and suggest PEA as a pharmacological tool to ameliorate neurological dysfunction induced by the trauma.