Rémi Quirion

Specific Plasma Membrane Binding Sites for Polyphenols, Including Resveratrol, in the Rat Brain

Using [3H]resveratrol (3,5,4′-trihydroxy-trans-stilbene) as radioligand, we investigated the possible existence of specific polyphenol binding sites at the level of the cellular plasma membrane in rat brain. Specific [3H]resveratrol binding sites were found to be enriched in the plasma membrane pellet with lower levels in the nuclear and cell debris fraction. Specific [3H]resveratrol binding to the plasma membrane fraction was sensitive to trypsin digestion and protein denaturation but not to DNase and RNase treatment. Saturation binding experiments revealed that specific [3H]resveratrol recognized a single class of sites with an apparent affinity (KD) of 220 ± 45 nM and a maximal capacity (Bmax) of 1060 ± 120 fmol/mg protein. Various polyphenols and resveratrol derivatives competed against specific [3H]resveratrol binding in rat brain plasma membrane homogenates with the tea catechin gallates (epigallocatechin gallate and epicatechin gallate) displaying the highest affinities (Ki = 25-45 nM) followed by resveratrol (Ki = 102 nM). Quantitative autoradiographic studies revealed that specific [3H]resveratrol binding sites are broadly distributed in the rat brain, with highest levels of labeling seen in the choroid plexus and subfornical organ. Finally, the potency of various polyphenols and resveratrol analogs in protecting hippocampal cells against β-amyloid-induced toxicity correlated well (r = 0.74) with their apparent affinity in the [3H]resveratrol binding assay. Taken together, these results suggest that the neuroprotective action of various polyphenols and resveratrol analogs could be mediated by the activation of common “receptor” binding sites particularly enriched at the level of the cellular plasma membrane in the rat brain.

Insulin-like growth factor-1 (IGF-1) induces the activation/phosphorylation of Akt kinase and cAMP response element-binding protein (CREB) by activating different signaling pathways in PC12 cells

BackgroundInsulin-like growth factor-1 (IGF-1) is a polypeptide growth factor with a variety of functions in both neuronal and non-neuronal cells. IGF-1 plays anti-apoptotic and other functions by activating multiple signaling pathways including Akt kinase, a serine/threonine kinase essential for cell survival. The nuclear transcription factor cAMP response element-binding protein (CREB) may also be involved although relationships between these two proteins in IGF-1 receptor signaling and protection is not clear, especially in neuronal cells.ResultsIGF-1, in a concentration- and time-dependent manner, induces the activation/phosphorylation of Akt and CREB in PC12 cells by activating different signaling pathways. IGF-1 induced a sustained phosphorylation of Akt while only a transient one was seen for CREB. The phosphorylation of Akt is mediated by the PI3 kinase pathway while that of CREB is dependent on the activation of both MAPK kinase and p38 MAPK. Moreover, the stimulation of PKC attenuated the phosphorylation of Akt induced by IGF-1 while enhancing that of CREB. Survival assays with various kinase inhibitors suggested that the activation/phosphorylation of both Akt and CREB contributes to IGF-1 mediated cell survival in PC12 cells.ConclusionThese data suggest that IGF-1 induced the activation of Akt and CREB using distinct pathways in PC12 cells.

Increased calcitonin gene‐related peptide in neuroma and invading macrophages is involved in the up‐regulation of interleukin‐6 and thermal hyperalgesia in a rat model of mononeuropathy

The pain related peptide, calcitonin gene‐related peptide (CGRP), plays an important role in inflammatory pain and immune responses. However, its role in neuropathic pain is not established. Following nerve injury, CGRP and pro‐inflammatory interleukin‐6 (IL‐6) are increased in injured nerves. The aim of this study was to determine if CGRP in injured nerves is involved in the up‐regulation of IL‐6 and in the maintenance of neuropathic pain. Perineural injection of a neutralizing IL‐6 antiserum or CGRP receptor antagonists (CGRP8‐37 and BIBN4096BS) effectively attenuated thermal hyperalgesia 4 weeks after partial sciatic nerve ligation. Perineural CGRP antagonists also dramatically reduced IL‐6 level in injured nerves. CGRP release from injured sites was dramatically increased and CGRP immunoreactivity was localized in both neuroma and invading macrophages. CGRP receptor markers (CRLR and RAMP1) were expressed in invading macrophages. Both CGRP antagonists significantly reduced IL‐6 release from injured nerve explants. In cell cultures derived from injured nerves, CGRP concentration‐dependently increased IL‐6 release, an effect also blocked by CGRP antagonists. Taken together, these data show that increased levels of CGRP in injured neuroma and invading macrophages are involved in the up‐regulation of IL‐6 in macrophages as well as in the maintenance of neuropathic pain.

A role for adrenomedullin as a pain-related peptide in the rat

Adrenomedullin (AM) belongs to the calcitonin gene-related peptide (CGRP) family and is a well known potent vasodilator. We show here that AM is a powerful pain-inducing neuropeptide. AM-like immunoreactivity is widely distributed in both CGRP-containing and lectin IB4-binding nociceptors in dorsal root ganglion and axon terminals in the superficial dorsal horn of the rat spinal cord. Specific binding sites for the radioligand, [125I]AM13–52 as well as immunoreactivity for receptor markers such as the calcitonin receptor-like receptor and three receptor-activity-modifying proteins are localized in the superficial dorsal horn, demonstrating the existence of AM/CGRP receptors in this region. Intrathecal injection of rat AM1–50, dose- and time-dependently, induced long-lasting heat hyperalgesia and increased the phosphorylation of Akt and GSK3β in the dorsal horn. Pre- and posttreatments with the AM receptor antagonist AM22–52 and PI3 kinase inhibitors (LY294002 and Wortmannin) significantly blocked or reversed AM-induced heat hyperalgesia. Pre- and posttreatments with AM22–52 and Wortmannin also significantly blocked or reversed intraplantar capsaicin-induced heat hyperalgesia. Taken together, our results demonstrate that AM acts as a pain-inducing peptide in the dorsal horn. By activating specific receptors (likely AM2) and the PI3K/Akt/GSK3β signaling pathway, AM could play a significant role in long-lasting heat hypersensitivity and inflammatory heat hyperalgesia.

Neuropeptide Y: Role in Emotion and Alcohol Dependence

Neuropeptide Y (NPY) is considered to be an important neuromodulator in the regulation of emotional behavior. For example, NPY is consistently involved in anxiety-related behaviors and there is increasing support for a role of this peptide in mood disorders such as depression. Furthermore, recent evidence suggests that NPY has a significant role in the neurobiological response to alcohol, including alcohol consumption, dependence, and withdrawal. In addition, NPY is beginning to emerge as an important modulator in the etiology of alcoholism that is independent from the addictive and reinforcing properties of the traditional system commonly associated with dopamine and instead, is strongly associated with innate emotionality. The recent developments elucidating the role of NPY in emotion and alcohol dependence are reviewed and the potential of the NPY system as a novel therapeutic strategy in the treatment of anxiety, depression and alcohol-related disorders is examined.

Expression of cortical and hippocampal apoptosis-inducing factor (AIF) in aging and Alzheimer's disease

Apoptosis-inducing factor (AIF) is a mitochondrial oxidoreductase originally identified for its role in caspase-independent programmed cell death (PCD). In this study, we investigated AIF protein expression levels in frontal and temporal cortices of normal subjects of various ages, as well as in subjects with Alzheimers disease (AD). AIF levels were also measured in the hippocampus of age-matched elderly and AD subjects. Amounts of all three AIF isoforms increased significantly with age in both cortical areas. Interestingly, AIF expression levels in the cortex (but not hippocampus) were consistently lower in AD compared to age-matched controls. The up-regulation of cortical AIF in normal aging is consistent with its previously hypothesized role as a free radical scavenger, and may thus represent an adaptive cellular response to compensate for the steady increase in oxidative stress occurring with age.

Emotional behavior in aged neuropeptide Y (NPY) Y2 knockout mice

Neuropeptide Y (NPY) was shown to modulate anxiety- and depression-related behaviors in various animal models. Previous studies demonstrated that NPY Y2 receptor knockout (KO) mice display an anxiolytic- and antidepressant-like phenotype compared with control animals. However, the long-term effect of the deletion of this receptor in aged animals is unknown. Thus, anxiety- and depression-related behaviors were investigated in 2-yr-old NPY Y2 KO mice. Aged NPY Y2 KO mice display an anxiolytic-like profile as assessed in the elevated plus-maze and open field, providing further support for a role for Y2 receptors in anxiety-related behaviors. Furthermore, aged NPY Y2 KO mice have significantly lower immobility scores in the forced swim test; supproting the role for this receptor in antidepressand-like behaviors. These data provide further evidence that modulators of the NPY Y2 receptor subtype are drug targets for the treatment of anxiety and mood disorders in human subjects.

Prototypical antipsychotic drugs protect hippocampal neuronal cultures against cell death induced by growth medium deprivation

BackgroundSeveral clinical studies suggested that antipsychotic-based medications could ameliorate cognitive functions impaired in certain schizophrenic patients. Accordingly, we investigated the effects of various dopaminergic receptor antagonists – including atypical antipsychotics that are prescribed for the treatment of schizophrenia – in a model of toxicity using cultured hippocampal neurons, the hippocampus being a region of particular relevance to cognition.ResultsHippocampal cell death induced by deprivation of growth medium constituents was strongly blocked by drugs including antipsychotics (10-10-10-6 M) that display nM affinities for D2 and/or D4 receptors (clozapine, haloperidol, (±)-sulpiride, domperidone, clozapine, risperidone, chlorpromazine, (+)-butaclamol and L-741,742). These effects were shared by some caspases inhibitors and were not accompanied by inhibition of reactive oxygen species. In contrast, (-)-raclopride and remoxipride, two drugs that preferentially bind D2 over D4 receptors were ineffective, as well as the selective D3 receptor antagonist U 99194. Interestingly, (-)-raclopride (10-6 M) was able to block the neuroprotective effect of the atypical antipsychotic clozapine (10-6 M).ConclusionTaken together, these data suggest that D2-like receptors, particularly the D4 subtype, mediate the neuroprotective effects of antipsychotic drugs possibly through a ROS-independent, caspase-dependent mechanism.

Cellular distribution of insulin-like growth factor-II/mannose-6-phosphate receptor in normal human brain and its alteration in Alzheimer's disease pathology.

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a multifunctional membrane glycoprotein, which binds different classes of ligands including IGF-II and M6P-bearing lysosomal enzymes. Besides participating in the process of endocytosis this receptor functions in the trafficking of lysosomal enzymes from the trans-Glogi network (TGN) or the cell surface to lysosomes. In Alzheimers disease (AD) brain, marked overexpression of certain lysosomal enzymes in vulnerable neuronal populations and their association to beta-amyloid (Abeta) containing neuritic plaques has been correlated to altered metabolic functions. In the present study, we measured the levels of IGF-II/M6P receptor and characterized its distribution profile in selected regions of AD and age-matched normal postmortem brains. Western blot analysis revealed no significant alteration in the levels of IGF-II/M6P receptor either in the hippocampus, frontal cortex or cerebellum between AD and age-matched control brains. However, a significant gene dose effect of apolipoprotein E (APOE) epsilon4 allele on IGF-II/M6P receptor levels was evident in the hippocampus of the AD brain. At the cellular level, immunoreactive IGF-II/M6P receptors were localized in the neurons of the frontal cortex, hippocampus and cerebellum of control brains. In AD brains, the labeling of the neurons was less intense in the frontal cortex and hippocampus than in the age-matched control brains. Additionally, IGF-II/M6P receptor immunoreactivity was observed in association with a subpopulation of Abeta-containing neuritic plaques as well as tau-positive neurofibrillary tangles both in the frontal cortex and the hippocampus. Reactive glial cells localized adjacent to the plaques also occasionally exhibited IGF-II/M6P receptor immunoreactivity. These results, when analyzed in context of the established role of the IGF-II/M6P receptor in the regulation of the intracellular trafficking of lysosomal enzymes, suggest that alterations in IGF-II/M6P receptor levels/distribution are possibly associated with altered functioning of the lysosomal enzymes and/or loss of neurons observed in AD brains, especially in patients carrying APOE epsilon4 alleles.

Characterization and effects on cAMP accumulation of adrenomedullin and calcitonin gene-related peptide (CGRP) receptors in dissociated rat spinal cord cell culture

1 Adrenomedullin (AM) and calcitonin gene‐related peptide (CGRP) have structural similarities, interact with each others receptors (calcitonin receptor‐like receptor (CLR)/receptor‐activity‐modifying proteins (RAMPs)) and show overlapping biological activities. AM and CGRP receptors are chiefly coupled to cAMP production. In this study, a method of primary dissociated cell culture was used to investigate the presence of AM and CGRP receptors and their effects on cAMP production in embryonic spinal cord cells. 2 Both neuronal and non‐neuronal CLR immunopositive cells were present in our model. 3 High affinity, specific [125I]‐AM binding sites (Kd 79±9 pM and Bmax 571±34 fmol mg−1 protein) were more abundant than specific [125I]‐CGRP binding sites (Kd 12±0.7 pM and Bmax 32±2 fmol mg−1 protein) in embryonic spinal cord cells. 4 Specific [125I]‐AM binding was competed by related molecules with a ligand selectivity profile of rAM>hAM22–52>rCGRPα>CGRP8–37≫[r‐(r*,s*)]‐N‐[2‐[[5‐amino‐1‐[[4‐(4‐pyridinyl)‐1‐piperazinyl]carbonyl]pentyl]amino]‐1‐[(3,5‐dibromo‐4‐hydroxyphenyl)methyl]‐2‐oxoethyl]‐4‐(1,4‐dihydro‐2‐oxo‐3(2H)‐quinazolinyl)‐,1‐piperidinecarboxamide (BIBN4096BS). 5 Specific [125I]‐CGRP binding was competed by rCGRPα>rAMCGRP8–37BIBN4096BS>hAM22–52. 6 Cellular levels of cAMP were increased by AM (pEC50 10.2±0.2) and less potently by rCGRPα (pEC50 8.9±0.4). rCGRPα‐induced cAMP accumulation was effectively inhibited by CGRP8–37 (pA2 7.63±0.44) and hAM22–52 (pA2 6.18±0.21) while AM‐stimulation of cAMP levels was inhibited by CGRP8–37 (pA2 7.41±0.15) and AM22–52 (pA2 7.26±0.18). BIBN4096BS only antagonized the effects of CGRP (pA2 8.40±0.30) on cAMP accumulation. 7 These pharmacological profiles suggest that effects of CGRP are mediated by the CGRP1 (CLR/RAMP1) receptor in our model while those of AM are related to the activation of the AM1 (CLR/RAMP2) receptor subtype.