Specific Phospholipid Modulation by Muscarinic Signaling in a Rat Lesion Model of Alzheimer's Disease.

Alzheimer's disease (AD) represents the most common cause of dementia worldwide and has been consistently associated with the loss of basal forebrain cholinergic neurons (BFCNs) leading to impaired cholinergic neurotransmission, aberrant synaptic function, and altered structural lipid metabolism. In this sense, membrane phospholipids (PLs) can be used for de novo synthesis of choline (Ch) for the further obtaining of acetylcholine (ACh) when its availability is compromised. Specific lipid species involved in the metabolism of Ch have been identified as possible biomarkers of phenoconversion to AD. Using a rat model of BFCN lesion, we have evaluated the lipid composition and muscarinic signaling in brain areas related to cognitive processes. The loss of BFCN resulted in alterations of varied lipid species related to Ch metabolism at nucleus basalis magnocellularis (NMB) and cortical projection areas. The activity of muscarinic receptors (mAChR) was decreased in the NMB and increased in the hippocampus according to the subcellular distribution of M1/M2 mAChR which could explain the learning and memory impairment reported in this AD rat model. These results suggest that the modulation of specific lipid metabolic routes could represent an alternative therapeutic strategy to potentiate cholinergic neurotransmission and preserve cell membrane integrity in AD.

Endocannabinoid and Muscarinic Signaling Crosstalk in the 3xTg-AD Mouse Model of Alzheimer's Disease.

The endocannabinoid system, which modulates emotional learning and memory through CB1 receptors, has been found to be deregulated in Alzheimer's disease (AD). AD is characterized by a progressive decline in memory associated with selective impairment of cholinergic neurotransmission. The functional interplay of endocannabinoid and muscarinic signaling was analyzed in seven-month-old 3xTg-AD mice following the evaluation of learning and memory of an aversive stimulus. Neurochemical correlates were simultaneously studied with both receptor and functional autoradiography for CB1 and muscarinic receptors, and regulations at the cellular level were depicted by immunofluorescence. 3xTg-AD mice exhibited increased acquisition latencies and impaired memory retention compared to age-matched non-transgenic mice. Neurochemical analyses showed changes in CB1 receptor density and functional coupling of CB1 and muscarinic receptors to Gi/o proteins in several brain areas, highlighting that observed in the basolateral amygdala. The subchronic (seven days) stimulation of the endocannabinoid system following repeated WIN55,212-2 (1 mg/kg) or JZL184 (8 mg/kg) administration induced a CB1 receptor downregulation and CB1-mediated signaling desensitization, normalizing acquisition latencies to control levels. However, the observed modulation of cholinergic neurotransmission in limbic areas did not modify learning and memory outcomes. A CB1 receptor-mediated decrease of GABAergic tone in the basolateral amygdala may be controlling the limbic component of learning and memory in 3xTg-AD mice. CB1 receptor desensitization may be a plausible strategy to improve behavior alterations associated with genetic risk factors for developing AD.

Increase in cortical endocannabinoid signaling in a rat model of basal forebrain cholinergic dysfunction.

The basal forebrain cholinergic pathways progressively degenerate during the progression of Alzheimer's disease, leading to an irreversible impairment of memory and thinking skills. The stereotaxic lesion with 192IgG-saporin in the rat brain has been used to eliminate basal forebrain cholinergic neurons and is aimed at emulating the cognitive damage described in this disease in order to explore its effects on behavior and on neurotransmission. Learning and memory processes that are controlled by cholinergic neurotransmission are also modulated by the endocannabinoid (eCB) system. The objective of the present study is to evaluate the eCB signaling in relation to the memory impairment induced in adult rats following a specific cholinergic lesion of the basal forebrain. Therefore, CB1 receptor-mediated signaling was analyzed using receptor and functional autoradiography, and cellular distribution by immunofluorescence. The passive avoidance test and histochemical data revealed a relationship between impaired behavioral responses and a loss of approximately 75% of cholinergic neurons in the nucleus basalis magnocellularis (NBM), accompanied by cortical cholinergic denervation. The decrease in CB1 receptor density observed in the hippocampus, together with hyperactivity of eCB signaling in the NBM and cortex, suggest an interaction between the eCB and cholinergic systems. Moreover, following basal forebrain cholinergic denervation, the presynaptic GABAergic immunoreactivity was reduced in cortical areas. In conclusion, CB1 receptors present in presynaptic GABAergic terminals in the hippocampus are down regulated, but not those in cortical glutamatergic synapses.

Anatomical location of LPA1 activation and LPA phospholipid precursors in rodent and human brain.

Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors: LPA1 -LPA6 . LPA evokes several responses in the CNS, including cortical development and folding, growth of the axonal cone and its retraction process. Those cell processes involve survival, migration, adhesion proliferation, differentiation, and myelination. The anatomical localization of LPA1 is incompletely understood, particularly with regard to LPA binding. Therefore, we have used functional [(35) S]GTPγS autoradiography to verify the anatomical distribution of LPA1 binding sites in adult rodent and human brain. The greatest activity was observed in myelinated areas of the white matter such as corpus callosum, internal capsule and cerebellum. MaLPA1 -null mice (a variant of LPA1 -null) lack [(35) S]GTPγS basal binding in white matter areas, where the LPA1 receptor is expressed at high levels, suggesting a relevant role of the activity of this receptor in the most myelinated brain areas. In addition, phospholipid precursors of LPA were localized by MALDI-IMS in both rodent and human brain slices identifying numerous species of phosphatides and phosphatidylcholines. Both phosphatides and phosphatidylcholines species represent potential LPA precursors. The anatomical distribution of these precursors in rodent and human brain may indicate a metabolic relationship between LPA and LPA1 receptors. Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors (GPCR), LPA1 to LPA6 . LPA evokes several responses in the central nervous system (CNS), including cortical development and folding, growth of the axonal cone and its retraction process. We used functional [(35) S]GTPγS autoradiography to verify the anatomical distribution of LPA1 -binding sites in adult rodent and human brain. The distribution of LPA1 receptors in rat, mouse and human brains show the highest activity in white matter myelinated areas. The basal and LPA-evoked activities are abolished in MaLPA1 -null mice. The phospholipid precursors of LPA are localized by MALDI-IMS. The anatomical distribution of LPA precursors in rodent and human brain suggests a relationship with functional LPA1 receptors.

Anatomical distribution of lipids in human brain cortex by imaging mass spectrometry.

Molecular mass images of tissues will be biased if differences in the physicochemical properties of the microenvironment affect the intensity of the spectra. To address this issue, we have performed-by means of MALDI-TOF mass spectrometry-imaging on slices and lipidomic analysis in extracts of frontal cortex, both from the same postmortem tissue samples of human brain. An external calibration was used to achieve a mass accuracy of 10 ppm (1σ) in the spectra of the extracts, although the final assignment was based on a comparison with previously reported species. The spectra recorded directly from tissue slices (imaging) show excellent s/n ratios, almost comparable to those obtained from the extracts. In addition, they retain the information about the anatomical distribution of the molecular species present in autopsied frozen tissue. Further comparison between the spectra from lipid extracts devoid of proteins and those recorded directly from the tissue unambiguously show that the differences in lipid composition between gray and white matter observed in the mass images are not an artifact due to microenvironmental influences of each anatomical area on the signal intensity, but real variations in the lipid composition.

Profiling and imaging of lipids on brain and liver tissue by matrix-assisted laser desorption/ ionization mass spectrometry using 2-mercaptobenzothiazole as a matrix.

2-Mercaptobenzothiazole (MBT) is employed for the first time as a matrix for the analysis of lipids from tissue extracts using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We demonstrate that the performance of MBT is superior to that of the matrixes commonly employed for lipids, due to its low vapor pressure, its low acidity, and the formation of small crystals, although because of the strong background at low m/z, it precludes detection of species below approximately 500 Da. This inconvenience can be partly overcome with the formation of Cs adducts. Using a polymer-based dual calibration, a mass accuracy of approximately 10 ppm in lipid extracts and of approximately 80 ppm in tissues is achieved. We present spectra from liver and brain lipid extracts where a large amount of lipid species is identified, in both positive and negative ion modes, with high reproducibility. In addition, the above-mentioned special properties of MBT allow its employment for imaging mass spectrometry. In the present work, images of brain and liver tissues showing different lipid species are presented, demonstrating the advantages of the employment of MBT.