Representative ratemeters showing the spontaneous activity and the responses to a noxious stimulation (von Frey filaments 97.8 mN/2 sec) of a single NS neuron both before and after spinal application of PMF_2α (16 nmol) alone, which increased the spontaneous activity and the evoked activity of NS neurons (A), or in combination with AGN 211336 (6 nmol) which did not alter either NS spontaneous activity or the noxious stimulation-evoked activity in normal knee mice (B).

<p>PMF_2α (16 nmol) was also administered both before and after knee joint injection of kaolin/λ-carrageenan, which alone increased the spontaneous activity and the noxious stimulation evoked activity of NS neurons (C), or in combination with AGN 211336 (6 nmol), which prevented the effect induced by inflammation on NS spontaneous activity or the noxious stimulation-evoked activity in inflamed knee mice (D). Scale grey bar indicates 5 min intervals for ratemeter records and small black arrows indicate the noxious stimulation on mouse hind paw.</p

Levels of PMF_2α in the spinal cord after the induction of inflammation with kaolin/λ-carrageenan (K/C) (A) and after the administration of COX inhibitors (B).

<p>Data are means ± SEM of separate determinations in N = 5 rats. ^# P<0.05 Control <i>vs.</i> K/C+vh; ** P<0.01 K/C+vh vs. K/C+indomethacin-K/C+SC560-K/C+NS398.</p

Effects of spinal application (microinjections) of vehicle (0.05% DMSO in ACSF), PGF_2α (0.5, 1 and 2 nmol) alone (A), or in combination with AGN 211336 (6 nmol) or AL 8810 (0.06 nmol) (B), and the effects of AL 8810 (0.06 nmol) alone (B), on mouse paw withdrawal latency (PWL).

<p>Vehicle or drugs were administered at time 0, as indicated by the black arrow, whereas AGN 211336 (6 nmol) or AL 8810 (0.06 nmol) were administered 10 min before (not shown). Each point represents the mean ± S.E.M 7 mice per group. * indicates statistically significant difference versus vehicle, and ° versus PGF_2α (2 nmol). P values<0.05 were considered statistically significant (one-way ANOVA). Representative ratemeters show the spontaneous activity and the responses to a noxious stimulation (von Frey filaments 97.8 mN/2 sec) of a single NS neuron both before and after spinal application of PGF_2α (2 nmol) alone, which increased the spontaneous activity and the noxious stimulation-evoked activity of NS neurons (C), or in combination with AL 8810 (0.06 nmol), which prevented the effect induced by PGF_2α (2 nmol) alone on NS spontaneous activity or on evoked activity (D). Scale grey bar indicates 5 min intervals for ratemeter records and small black arrows indicate the noxious stimulation on mouse hind paw.</p

Concentrations of anandamide (AEA) and 2-arachidonoylglycerol in the spinal cord of healthy and inflamed knee mice, and after treatment with COX inhibitors.

<p>Data are means ± SEMs of separate determinations in N = 5 mice. No statistically significant difference between groups was found (as assessed by ANOVA followed by Bonferroni's post-hoc test). K/C, kaolin/λ-carrageenan.</p

Effects of spinal application (microinjections) of vehicle (0.05% DMSO in ACSF), PMF_2α (4, 8 and 16 nmol) alone (A) or in combination with AGN 211336 (6 nmol) or AL 8810 (0.06 nmol) (B) on evoked activity of NS neurons in normal knee mice.

<p>Effects of spinal application of vehicle (0.05% DMSO in ACSF) and AGN 211336 (6 nmol) on evoked activity of NS neurons in normal knee (B), in sham and in inflamed knee mice (C). Vehicle or drugs were administered at time 0, as indicated by the black arrow, whereas AGN 211336 (6 nmol) or AL 8810 (0.06 nmol) were administered 10 min before (not shown). Each point represents the mean ± S.E.M of 6–7 neurons of different groups of mice. * indicates statistically significant difference versus vehicle (A and B) or versus sham/veh (C), and ° versus PMF_2α (16 nmol) (B) or versus kaolin/λ-carrageenan (C). P values<0.05 were considered statistically significant (one-way ANOVA).</p

Representative extracted ion chromatogram of a pre-purified lipid extract from a rat brain homogenate spiked with synthetic standards of prostamides and prostaglandin-glycerol esters (100 pmol each).

<p>LC parameters were optimized to ensure good separation among the analytes (PME₂, PMF_2α, PGE₂-GE and PGF_2α–GE). Shown in the bottom panel are an example of positive-ion electrospray mass spectrum of a major component of this spiked homogenate, the PMF_2α precursor ion (m/z 420.2671), as a sodium adduct, and the corresponding product ions for the CID of the fragment with m/z 420.2671 in the MS-MS spectra (m/z 402.2555), which in turn corresponds to the C₂₂H₃₇NO₄ sodiated fragment, after loss of water. Pre-purified rat brain lipid extracts do not contain measurable amount of endogenous prostamides (not shown). Instead, pre-purified mouse spinal cord extracts (not shown) only contain PMF_2α in measurable although much smaller amounts than those shown here (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031111#pone-0031111-g002" target="_blank">Fig. 2</a>).</p

Effects of spinal application (microinjections) of vehicle (0.05% DMSO in ACSF), PMF_2α (4, 8 and 16 nmol) alone (A), or in combination with AGN 211336 (6 nmol) or AL 8810 (0.06 nmol) (B), on the spontaneous firing of NS neurons in normal knee mice.

<p>Effects of spinal application of vehicle (0.05% DMSO in ACSF) and AGN 211336 (6 nmol) on the spontaneous firing of NS neurons in normal knee (B), in sham and in inflamed knee mice (C). Vehicle or drugs were administered at time 0 whereas AGN 211336 (6 nmol) or AL 8810 (0.06 nmol) were administered 10 min before. Black arrow indicates vehicle or agonist spinal application while white arrow indicates antagonist spinal injection. Each point represents the mean ± S.E.M of 6–7 neurons of different treated group of mice. * indicates statistically significant difference versus vehicle (A and B) or versus sham/veh (C), and ° versus PMF_2α (16 nmol) (B) or versus kaolin/λ-carrageenan (C). P values<0.05 were considered statistically significant (one-way ANOVA).</p

Effects of spinal application (microinjections) of vehicle (0.05% DMSO in ACSF), PGF_2α (0.5, 1 and 2 nmol) alone (A and C) or in combination with AGN 211336 (6 nmol) or AL 8810 (0.06 nmol) (B), and effects of AL 8810 (0.06 nmol) alone (B and D) on the spontaneous firing of NS neurons and on the evoked activity of NS neurons.

<p>Vehicle or drugs were administered at time 0, as indicated by the black arrow, whereas AGN 211336 (6 nmol) or AL 8810 (0.06 nmol) were administered 10 min before (not shown). Each point represents the mean ± S.E.M of 6–7 neurons of different treated group of mice. * indicates statistically significant difference versus vehicle, and ° versus PGF_2α (2 nmol). P values<0.05 were considered statistically significant (one-way ANOVA).</p

Effects of spinal application (microinjections) of vehicle (0.05% DMSO in ACSF), PMF_2α (4, 8 and 16 nmol) alone (A) or in combination with AGN 211336 (6 nmol) or AL 8810 (0.06 nmol) (B) on paw withdrawal thresholds (PWT) in normal knee mice.

<p>Effects of spinal application of vehicle (0.05% DMSO in ACSF) and AGN 211336 (6 nmol) on paw withdrawal latency (PWL) in normal knee (B), in sham and in inflamed knee mice (C). Vehicle or drugs were administered at time 0, as indicated by the black arrow, whereas AGN 211336 (6 nmol) or AL 8810 (0.06 nmol) were administered 10 min before (not shown). Each point represents the mean ± S.E.M of 6–7 neurons of different treated group of mice. * indicates statistically significant difference versus vehicle (A and B) or versus sham/veh (C), and ° versus PMF_2α (16 nmol) (B) or versus kaolin/λ-carrageenan (C). P values<0.05 were considered statistically significant (one-way ANOVA).</p

Data_Sheet_1_Positive association between plasmatic levels of orexin A and the endocannabinoid-derived 2-arachidonoyl lysophosphatidic acid in Alzheimer’s disease.pdf

A regular sleep-wake cycle plays a positive function that preserves synaptic plasticity and brain activity from neuropathological injuries. The hypothalamic neuropeptide orexin-A (OX-A) is central in sleep-wake regulation and has been found to be over-expressed in the cerebrospinal fluid (CSF) of patients with Alzheimer’s disease (AD) suffering from sleep disturbances. OX-A promotes the biosynthesis of 2-arachidonoylglycerol (2-AG), which, in turn, could be phosphorylated to 2-arachidonoyl lysophosphatidic acid (2-AGP). The reorganization of the actin cytoskeleton during neurite retraction is one of the best-characterized effects of lysophosphatidic acids. However, less information is available regarding the reorganization of the neuronal microtubule network in response to OX-A-induced 2-AG and, possibly consequent, 2-AGP production in AD patients. This is of special relevance also considering that higher 2-AG levels are reported in the CSF of AD patients. Here, we found a positive correlation between OX-A and 2-AGP concentrations in the plasma, and an increase of 2-AGP levels in the CSF of AD patients. Furthermore, a negative correlation between the plasmatic 2-AGP levels and the mini-mental state examination score is also revealed in AD patients. By moving from the human patients to in vitro and in vivo models of AD we investigated the molecular pathway linking OX-A, 2-AG and 2-AGP to the phosphorylation of pT231-Tau, which is a specific early plasma biomarker of this disorder. By LC-MS analysis we show that OX-A, via OX-1R, induces 2-AG biosynthesis via DAGLα, and in turn 2-AG is converted to 2-AGP in primary hippocampal neurons. By confocal microscopy and western blotting assay we found an OX-A- or 2-AGP-mediated phosphorylation of Tau at threonine 231 residue, in a manner prevented by LPA1R (2-AGP receptor) or OX1R (OX-A receptor) antagonism with AM095 or SB334867, respectively. Finally, by patch-clamp recording we documented that 2-AGP-mediated pT231-Tau phosphorylation impairs glutamatergic transmission in the mouse hippocampus. Although further additional research is still required to clarify the potential role of orexin signaling in neurodegeneration, this study provides evidence that counteraction of aberrant OX-A signaling, also via LPA-1R antagonism, may be beneficial in the mild-to-moderate age-related cognitive decline associated with sleep disturbances.</p