Frontal Cortex Lipid Alterations During the Onset of Alzheimer's Disease.

Background: Although sporadic Alzheimer's disease (AD) is a neurodegenerative disorder of unknown etiology, familial AD is associated with specific gene mutations. A commonality between these forms of AD is that both display multiple pathogenic events including cholinergic and lipid dysregulation. Objective: We aimed to identify the relevant lipids and the activity of their related receptors in the frontal cortex and correlating them with cognition during the progression of AD. Methods: MALDI-mass spectrometry imaging (MSI) and functional autoradiography was used to evaluate the distribution of phospholipids/sphingolipids and the activity of cannabinoid 1 (CB1), sphingosine 1-phosphate 1 (S1P1), and muscarinic M2/M4 receptors in the frontal cortex (FC) of people that come to autopsy with premortem clinical diagnosis of AD, mild cognitive impairment (MCI), and no cognitive impairment (NCI). Results: MALDI-MSI revealed an increase in myelin-related lipids, such as diacylglycerol (DG) 36:1, DG 38:5, and phosphatidic acid (PA) 40:6 in the white matter (WM) in MCI compared to NCI, and a downregulation of WM phosphatidylinositol (PI) 38:4 and PI 38:5 levels in AD compared to NCI. Elevated levels of phosphatidylcholine (PC) 32:1, PC 34:0, and sphingomyelin 38:1 were observed in discrete lipid accumulations in the FC supragranular layers during disease progression. Muscarinic M2/M4 receptor activation in layers V-VI decreased in AD compared to MCI. CB1 receptor activity was upregulated in layers V-VI, while S1P1 was downregulated within WM in AD relative to NCI. Conclusions: FC WM lipidomic alterations are associated with myelin dyshomeostasis in prodromal AD, suggesting WM lipid maintenance as a potential therapeutic target for dementia.

Lipid metabolism in dopaminergic neurons influences light entrainment.

Dietary lipids, particularly omega-3 polyunsaturated fatty acids, are speculated to impact behaviors linked to the dopaminergic system, such as movement and control of circadian rhythms. However, the ability to draw a direct link between dopaminergic omega-3 fatty acid metabolism and behavioral outcomes has been limited to the use of diet-based approaches, which are confounded by systemic effects. Here, neuronal lipid metabolism was targeted in a diet-independent manner by manipulation of long-chain acyl-CoA synthetase 6 (ACSL6) expression. ACSL6 performs the initial reaction for cellular fatty acid metabolism and prefers the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA). The loss of Acsl6 in mice (Acsl6-/- ) depletes neuronal membranes of DHA content and results in phenotypes linked to dopaminergic control, such as hyperlocomotion, impaired short-term spatial memory, and imbalances in dopamine neurochemistry. To investigate the role of dopaminergic ACSL6 on these outcomes, a dopaminergic neuron-specific ACSL6 knockout mouse was generated (Acsl6DA-/- ). Acsl6DA-/- mice demonstrated hyperlocomotion and imbalances in striatal dopamine neurochemistry. Circadian rhythms of both the Acsl6-/- and the Acsl6DA-/- mice were similar to control mice under basal conditions. However, upon light entrainment, a mimetic of jet lag, both the complete knockout of ACSL6 and the dopaminergic-neuron-specific loss of ACSL6 resulted in a longer recovery to entrainment compared to control mice. In conclusion, these data demonstrate that ACSL6 in dopaminergic neurons alters dopamine metabolism and regulation of light entrainment suggesting that DHA metabolism mediated by ACSL6 plays a role in dopamine neuron biology.

Mass Spectrometry for the Advancement of Lipid Analysis in Alzheimer's Research.

Recent technical advances in mass spectrometry, as applied to the analytical chemistry of lipid molecules, enable the simultaneous detection of the multiplicity of lipid complex species present in the human brain. This, in combination with quantitative studies carried out in plasma samples, helps to identify disease biomarkers including for Alzheimer's disease (AD). Mass spectrometry imaging (MSI) is particularly powerful for the anatomical localization of lipids in brain slices, identifying lipid modifications in postmortem frozen samples from AD patients.Human brain tissues are sectioned in a cryostat and then covered with a chemical matrix, such as mercaptobenzothiazole (MBT) or α-cyano-4-hydroxycinnamic acid (CHCA), to ionize the lipid molecules either by sublimation or by spraying. We describe the use of matrix-assisted laser desorption ionization (MALDI) in an LTQ-Orbitrap-XL mass spectrometer to scan brain tissue slices with high spatial resolution, analyzing 50 µm cell layers. The lipid spectra obtained for each pixel are transformed to color-coded intensity maps of hundreds of lipid species included those within a single tissue slice.

Cannabinoid Receptors and Glial Response Following a Basal Forebrain Cholinergic Lesion.

The endocannabinoid system modulates learning, memory, and neuroinflammatory processes, playing a key role in neurodegeneration, including Alzheimer's disease (AD). Previous results in a rat lesion model of AD showed modulation of endocannabinoid receptor activity in the basalo-cortical pathway following a specific lesion of basal forebrain cholinergic neurons (BFCNs), indicating that the glial neuroinflammatory response accompanying the lesion is related to endocannabinoid signaling. In this study, 7 days after the lesion, decreased astrocyte and increased microglia immunoreactivities (GFAP and Iba-1) were observed, indicating microglia-mediated neuroinflammation. Using autoradiographic studies, the density and functional coupling to G-proteins of endocannabinoid receptor subtypes were studied in tissue sections from different brain areas where microglia density increased, using CB1 and CB2 selective agonists and antagonists. In the presence of the specific CB1 receptor antagonist, SR141716A, [3H]CP55,940 binding (receptor density) was completely blocked in a dose-dependent manner, while the selective CB2 receptor antagonist, SR144528, inhibited binding to 25%, at best. [35S]GTPγS autoradiography (receptor coupling to Gi/0-proteins) evoked by CP55,940 (CB1/CB2 agonist) and HU308 (more selective for CB2) was abolished by SR141716A in all areas, while SR144528 blocked up to 51.8% of the coupling to Gi/0-proteins evoked by CP55,940 restricted to the nucleus basalis magnocellularis. Together, these results demonstrate that there are increased microglia and decreased astrocyte immunoreactivities 1 week after a specific deletion of BFCNs, which projects to cortical areas, where the CB1 receptor coupling to Gi/0-proteins is upregulated. However, at the lesion site, the area with the highest neuroinflammatory response, there is also a limited contribution of CB2.

Handling and novel object recognition modulate fear response and endocannabinoid signaling in nucleus basalis magnocellularis.

Storage of aversive memories is of utmost importance for survival, allowing animals to avoid upcoming similar stimuli. However, without reinforcement, the learned avoidance response gradually decreases over time. Although the molecular mechanisms controlling this extinction process are not well known, there is evidence that the endocannabinoid system plays a key role through CB1 receptor-mediated modulation of cholinergic signaling. In this study, we measured fear extinction throughout 7 months using naïve rats, assessed in passive avoidance (PA) test in a non-reinforced manner. Then, we evaluated the effect of gentle handling and non-aversive novel object recognition test (NORT) on the extinction and expression of fear memories by measuring passive avoidance responses. Neurochemical correlates were analyzed by functional autoradiography for cannabinoid, cholinergic, and dopaminergic receptors. Despite results showing a gradual decrease of passive avoidance response, it did not fully disappear even after 7 months, indicating the robustness of this process. Meanwhile, in rats that received gentle handling or performed NORT after receiving the PA aversive stimulus, extinction occurred within a week. In contrast, gentle handling performed before receiving the aversive stimulus exacerbated fear expression and triggered escape response in PA. The neurochemical analysis showed increased cannabinoid and cholinergic activity in the nucleus basalis magnocellularis (NBM) in rats that had performed only PA, as opposed to rats that received gentle handling before PA. Additionally, a correlation between CB1 mediated-signaling in the NBM and freezing in PA was found, suggesting that the endocannabinoid system might be responsible for modulating fear response induced by aversive memories.

Modulation of Neurolipid Signaling and Specific Lipid Species in the Triple Transgenic Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia in aging populations. Recently, the regulation of neurolipid-mediated signaling and cerebral lipid species was shown in AD patients. The triple transgenic mouse model (3xTg-AD), harboring ßAPPSwe, PS1M146V, and tauP301L transgenes, mimics many critical aspects of AD neuropathology and progressively develops neuropathological markers. Thus, in the present study, 3xTg-AD mice have been used to test the involvement of the neurolipid-based signaling by endocannabinoids (eCB), lysophosphatidic acid (LPA), and sphingosine 1-phosphate (S1P) in relation to the lipid deregulation. [35S]GTPγS autoradiography was used in the presence of specific agonists WIN55,212-2, LPA and CYM5442, to measure the activity mediated by CB1, LPA1, and S1P1 Gi/0 coupled receptors, respectively. Consecutive slides were used to analyze the relative intensities of multiple lipid species by MALDI Mass spectrometry imaging (MSI) with microscopic anatomical resolution. The quantitative analysis of the astrocyte population was performed by immunohistochemistry. CB1 receptor activity was decreased in the amygdala and motor cortex of 3xTg-AD mice, but LPA1 activity was increased in the corpus callosum, motor cortex, hippocampal CA1 area, and striatum. Conversely, S1P1 activity was reduced in hippocampal areas. Moreover, the observed modifications on PC, PA, SM, and PI intensities in different brain areas depend on their fatty acid composition, including decrease of polyunsaturated fatty acid (PUFA) phospholipids and increase of species containing saturated fatty acids (SFA). The regulation of some lipid species in specific brain regions together with the modulation of the eCB, LPA, and S1P signaling in 3xTg-AD mice indicate a neuroprotective adaptation to improve neurotransmission, relieve the myelination dysfunction, and to attenuate astrocyte-mediated neuroinflammation. These results could contribute to identify new therapeutic strategies based on the regulation of the lipid signaling in familial AD patients.

Acyl-CoA synthetase 6 is required for brain docosahexaenoic acid retention and neuroprotection during aging.

The omega-3 fatty acid docosahexaenoic acid (DHA) inversely relates to neurological impairments with aging; however, limited nondietary models manipulating brain DHA have hindered a direct linkage. We discovered that loss of long-chain acyl-CoA synthetase 6 in mice (Acsl6-/-) depletes brain membrane phospholipid DHA levels, independent of diet. Here, Acsl6-/- brains contained lower DHA compared with controls across the life span. The loss of DHA- and increased arachidonate-enriched phospholipids were visualized by MALDI imaging predominantly in neuron-rich regions where single-molecule RNA in situ hybridization localized Acsl6 to neurons. ACSL6 is also astrocytic; however, we found that astrocyte-specific ACSL6 depletion did not alter membrane DHA because astrocytes express a non-DHA-preferring ACSL6 variant. Across the life span, Acsl6-/- mice exhibited hyperlocomotion, impairments in working spatial memory, and increased cholesterol biosynthesis genes. Aging caused Acsl6-/- brains to decrease the expression of membrane, bioenergetic, ribosomal, and synaptic genes and increase the expression of immune response genes. With age, the Acsl6-/- cerebellum became inflamed and gliotic. Together, our findings suggest that ACSL6 promotes membrane DHA enrichment in neurons, but not in astrocytes, and is important for neuronal DHA levels across the life span. The loss of ACSL6 impacts motor function, memory, and age-related neuroinflammation, reflecting the importance of neuronal ACSL6-mediated lipid metabolism across the life span.

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.