Iván Manuel

Distribution of lipids in human brain

The enormous abundance of lipid molecules in the central nervous system (CNS) suggests that their role is not limited to be structural and energetic components of cells. Over the last decades, some lipids in the CNS have been identified as intracellular signalers, while others are known to act as neuromodulators of neurotransmission through binding to specific receptors. Neurotransmitters of lipidic nature, currently known as neurolipids, are synthesized during the metabolism of phospholipid precursors present in cell membranes. Therefore, the anatomical identification of each of the different lipid species in human CNS by imaging mass spectrometry (IMS), in association with other biochemical techniques with spatial resolution, can increase our knowledge on the precise metabolic routes that synthesize these neurolipids and their localization. The present study shows the lipid distribution obtained by MALDI-TOF IMS in human frontal cortex, hippocampus, and striatal area, together with functional autoradiography of cannabinoid and LPA receptors. The combined application of these methods to postmortem human brain samples may be envisioned as critical to further understand neurological diseases, in general, and particularly, the neurodegeneration that accompanies Alzheimer’s disease.

Type-1 Cannabinoid Receptor Activity During Alzheimer's Disease Progression

The activity of CB1 cannabinoid receptors was studied in postmortem brain samples of Alzheimers disease (AD) patients during clinical deterioration. CB1 activity was higher at earlier AD stages in limited hippocampal areas and internal layers of frontal cortex, but a decrease was observed at the advanced stages. The pattern of modification appears to indicate initial hyperactivity of the endocannabinoid system in brain areas that lack classical histopathological markers at earlier stages of AD, indicating an attempt to compensate for the initial synaptic impairment, which is then surpassed by disease progression. These results suggest that initial CB1 stimulation might have therapeutic relevance.

Imaging mass spectrometry (IMS) of cortical lipids from preclinical to severe stages of Alzheimer's disease

Alzheimers disease (AD) is a progressive neurodegenerative disease affecting millions of patients worldwide. Previous studies have demonstrated alterations in the lipid composition of lipid extracts from plasma and brain samples of AD patients. However, there is no consensus regarding the qualitative and quantitative changes of lipids in brains from AD patients. In addition, the recent developments in imaging mass spectrometry methods are leading to a new stage in the in situ analysis of lipid species in brain tissue slices from human postmortem samples. The present study uses the matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS), permitting the direct anatomical analysis of lipids in postmortem brain sections from AD patients, which are compared with the intensity of the lipid signal in samples from matched subjects with no neurological diseases. The frontal cortex samples from AD patients were classified in three groups based on Braaks histochemical criteria, ranging from non-cognitively impaired patients to those severely affected. The main results indicate a depletion of different sulfatide lipid species from the earliest stages of the disease in both white and gray matter areas of the frontal cortex. Therefore, the decrease in sulfatides in cortical areas could be considered as a marker of the disease, but may also indicate neurochemical modifications related to the pathogenesis of the disease. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Lipid mapping of the rat brain for models of disease.

Lipids not only constitute the primary component of cellular membranes and contribute to metabolism but also serve as intracellular signaling molecules and bind to specific membrane receptors to control cell proliferation, growth and convey neuroprotection. Over the last several decades, the development of new analytical techniques, such as imaging mass spectrometry (IMS), has contributed to our understanding of their involvement in physiological and pathological conditions. IMS allows researchers to obtain a wide range of information about the spatial distribution and abundance of the different lipid molecules that is crucial to understand brain functions. The primary aim of this study was to map the spatial distribution of different lipid species in the rat central nervous system (CNS) using IMS to find a possible relationship between anatomical localization and physiology. The data obtained were subsequently applied to a model of neurological disease, the 192IgG-saporin lesion model of memory impairment. The results were obtained using a LTQ-Orbitrap XL mass spectrometer in positive and negative ionization modes and analyzed by ImageQuest and MSIReader software. A total of 176 different molecules were recorded based on the specific localization of their intensities. However, only 34 lipid species in negative mode and 51 in positive were assigned to known molecules with an error of 5ppm. These molecules were grouped by different lipid families, resulting in: Phosphatidylcholines (PC): PC (34: 1)+K+ and PC (32: 0)+K+ distributed primarily in gray matter, and PC (36: 1)+K+ and PC (38: 1)+Na+ distributed in white matter. Phosphatidic acid (PA): PA (38: 3)+K+ in white matter, and PA (38: 5)+K+ in gray matter and brain ventricles. Phosphoinositol (PI): PI (18: 0/20: 4)-H+ in gray matter, and PI (O-30: 1) or PI (P-30: 0)-H+ in white matter. Phosphatidylserines (PS): PS (34: 1)-H+ in gray matter, and PS (38: 1)-H+ in white matter. Sphingomyelin (SM) SM (d18: 1/16: 0)-H+ in ventricles and SM (d18: 1/18: 0)-H+ in gray matter. Sulfatides (ST): ST (d18: 1/24: 1)-H+ in white matter. The specific distribution of different lipids supports their involvement not only in structural and metabolic functions but also as intracellular effectors or specific receptor ligands and/or precursors. Moreover, the specific localization in the CNS described here will enable us to analyze lipid distribution to identify their physiological conditions in rat models of neurodegenerative pathologies, such as Alzheimers disease. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

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 [35S]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 [35S]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 [35S]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.

Activity of muscarinic, galanin and cannabinoid receptors in the prodromal and advanced stages in the triple transgenic mice model of Alzheimer’s disease

Neurochemical alterations in Alzheimers disease (AD) include cholinergic neuronal loss in the nucleus basalis of Meynert (nbM) and a decrease in densities of the M2 muscarinic receptor subtype in areas related to learning and memory. Neuromodulators present in the cholinergic pathways, such as neuropeptides and neurolipids, control these cognitive processes and have become targets of research in order to understand and treat the pathophysiological and clinical stages of the disease. This is the case of the endocannabinoid and galaninergic systems, which have been found to be up-regulated in AD, and could therefore have a neuroprotective role. In the present study, the functional coupling of Gi/o protein-coupled receptors to GalR1, and the CB1 receptor subtype for endocannabinoids were analyzed in the 3xTg-AD mice model of AD. In addition, the activity mediated by Gi/o protein-coupled M2/4 muscarinic receptor subtypes was also analyzed in brain areas involved in anxiety and cognition. Thus, male mice were studied at 4 and 15months of age (prodromal and advanced stages, respectively) and compared to age-matched non-transgenic (NTg) mice (adult and old, respectively). In 4-month-old 3xTg-AD mice, the [(35)S]GTPγS binding stimulated by galanin was significantly increased in the hypothalamus, but a decrease of functional M2/4 receptors was observed in the posterior amygdala. The CB1 cannabinoid receptor activity was up-regulated in the anterior thalamus at that age. In 15-month-old 3xTg-AD mice, muscarinic receptor activity was found to be increased in motor cortex, while CB1 activity was decreased in nbM. No changes were found in GalR1-mediated activity at this age. Our results provide further evidence of the relevance of limbic areas in the prodromal stage of AD, the profile of which is characterized by anxiety. The up-regulation of galaninergic and endocannabinoid systems support the hypothesis of their neuroprotective roles, and these are established prior to the onset of clear clinical cognitive symptoms of the disease.

Neurotransmitter receptor localization: from autoradiography to imaging mass spectrometry.

Autoradiography is used to determine the anatomical distribution of biological molecules in human tissue and experimental animal models. This method is based on the analysis of the specific binding of radiolabeled compounds to locate neurotransmitter receptors or transporters in fresh frozen tissue slices. The anatomical resolution obtained by quantification of the radioligands has allowed the density of receptor proteins to be mapped over the last 40 years. The data yielded by autoradiography identify the receptors at their specific microscopic localization in the tissues and also in their native microenvironment, the intact cell membrane. Furthermore, in functional autoradiography, the effects of small molecules on the activity of G protein-coupled receptors are evaluated. More recently, autoradiography has been combined with membrane microarrays to improve the high-throughput screening of compounds. These technical advances have made autoradiography an essential analytical method for the progress of drug discovery. We include the future prospects and some preliminary results for imaging mass spectrometry (IMS) as a useful new method in pharmacodynamic and pharmacokinetic studies, complementing autoradiographic studies. IMS results could also be presented as density maps of molecules, proteins, and metabolites in tissue sections that can be identified, localized, and quantified, with the advantage of avoiding any labeling of marker molecules. The limitations and future developments of these techniques are discussed here.

Regulation of subthalamic neuron activity by endocannabinoids

High levels of anandamide are located in the basal ganglia. The subthalamic nucleus (STN) is considered to be an important modulator of basal ganglia output. The present study aims at characterizing the modulation of the electrical activity of STN neurons by exogenous anandamide or endocannabinoids. Single‐unit extracellular recordings in anesthetized rats and patch‐clamp techniques in rat brain slices containing the STN were performed. Immunohistochemical assays were used. In vivo, anandamide administration produced two opposite effects (inhibition or stimulation) on STN neuron firing rates, depending of the precise location of the neuron within the nucleus. These effects were enhanced by prior inhibition of fatty acid amide hydrolase with URB597, but not by the inhibitor of carrier‐mediated anandamide transport AM404. Rimonabant, a specific CB1 receptor antagonist, also produced inhibition or stimulation of STN neuron activity when administered alone or after anandamide. These effects seem to be mediated by indirect mechanisms since: (1) STN neuron activity is not modified by the cannabinoid agonist Δ9‐tetrahydrocannabinol (Δ9‐THC) in vitro; (2) no depolarization‐induced suppression of inhibition phenomena were observed; and (3) CB1 receptor immunolabeling was not detected in the STN, but was abundant in areas which project efferents to this nucleus. Moreover, chemical lesion of the globus pallidus abolished the stimulatory effect of anandamide and microinfusion of anandamide into the prefrontal cortex led to inhibition of STN neuron activity. The present results show that endocannabinoids exert a tonic control on STN activity via receptors located outside the nucleus. These findings may contribute to enhance our understanding of the role of the endocannabinoid system in motor control. Synapse 64:682–698, 2010.

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 Alzheimers 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.

G protein coupling of galanin, muscarinic and cannabinoid receptors in areas related to the control of memory and cognitive functions of Alzheimer patients

was to determine the effects of beta amyloid 1-42 treatment on VDR expression in primary cortical neuron cultures. Methods: Cerebral cortex dissected from brains of Sprague Dawley rat embryos on the embryonic day 16 and cultured. The groups including 48 hours of 6 uM beta amyloid 1-42 treated group and control groups, were established. mRNA isolation and cDNA synthesis performed. The levels of VDR expressions were determined by RTqPCR. Localization of VDR was identified by immunofluorescent immunocytochemistry. Results: Expression of VDR in beta amyloid 1-42 treated group was found decreased when compared with control group. The immunolocalization of VDR was observed in both nucleus and cytoplasm of the primary cultured neurons of cerebral cortex. Conclusions: Relative attenuation in the levels of VDR expression by beta amyloid treatment in primary cortical neurons, might indicate potential role of beta amyloid to prevent probable neuroprotective effects of vitamin D in brain.