International Journal of Molecular Sciences Article Modulation of Neurolipid Signaling and Specific Lipid Species in the Triple Transgenic Mouse Model of Alzheimer’s Disease Estibaliz González de San Román 1 , Alberto Llorente-Ovejero 1 , Jonatan Martínez-Gardeazabal 1 , Marta Moreno-Rodríguez 1 , Lydia Giménez-Llort 2 , Iván Manuel 1,3 and Rafael Rodríguez-Puertas 1,3, * 1 Department of Pharmacology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain; estibaliz.gonzalezdesanroman@ehu.eus (E.G.d.S.R.); alberto.llorente@ehu.eus (A.L.-O.); jonatan.martinez@ehu.eus (J.M.-G.); marta.morenor@ehu.eus (M.M.-R.); ivan.manuel@ehu.eus (I.M.) 2 Department of Psychiatry and Forensic Medicine, Institute of Neuroscience, Faculty of Medicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; lidia.gimenez@uab.cat 3 Neurodegenerative Diseases, BioCruces Bizkaia Health Research Institute, 48903 Barakaldo, Spain * Correspondence: rafael.rodriguez@ehu.es; Tel.: +34-946-012-739; Fax: +34-946-013-220 Abstract: 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 βAPPSwe , PS1M146V , and tauP301L transgenes, mimics many critical aspects of  AD neuropathology and progressively develops neuropathological markers. Thus, in the present Citation: González de San Román, study, 3xTg-AD mice have been used to test the involvement of the neurolipid-based signaling E.; Llorente-Ovejero, A.; Martínez- by endocannabinoids (eCB), lysophosphatidic acid (LPA), and sphingosine 1-phosphate (S1P) in Gardeazabal, J.; Moreno-Rodríguez, relation to the lipid deregulation. [35 S]GTPγS autoradiography was used in the presence of specific M.; Giménez-Llort, L.; Manuel, I.; agonists WIN55,212-2, LPA and CYM5442, to measure the activity mediated by CB1 , LPA1 , and Rodríguez-Puertas, R. Modulation of S1P1 Gi/0 coupled receptors, respectively. Consecutive slides were used to analyze the relative Neurolipid Signaling and Specific intensities of multiple lipid species by MALDI Mass spectrometry imaging (MSI) with microscopic Lipid Species in the Triple Transgenic anatomical resolution. The quantitative analysis of the astrocyte population was performed by Mouse Model of Alzheimer’s Disease. Int. J. Mol. Sci. 2021, 22, 12256. immunohistochemistry. CB1 receptor activity was decreased in the amygdala and motor cortex of https://doi.org/10.3390/ijms222212256 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 Academic Editor: observed modifications on PC, PA, SM, and PI intensities in different brain areas depend on their Giuseppe Lazzarino 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 Received: 21 October 2021 specific brain regions together with the modulation of the eCB, LPA, and S1P signaling in 3xTg-AD Accepted: 10 November 2021 mice indicate a neuroprotective adaptation to improve neurotransmission, relieve the myelination Published: 12 November 2021 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 Publisher’s Note: MDPI stays neutral AD patients. with regard to jurisdictional claims in published maps and institutional affil- Keywords: Alzheimer’s disease; functional autoradiography; cannabinoid receptors; LPA receptors; iations. sphingosine 1-phosphate; ligand binding; G protein; MALDI-MSI; [35 S]GTPγS autoradiography; 3xTg-AD mice Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and The progressive and irreversible deterioration of cognitive functions present in conditions of the Creative Commons Alzheimer’s disease (AD) include a chronic neurodegeneration characterized by patho- Attribution (CC BY) license (https:// logical hallmarks including the loss of synapses, the intracellular neurofibrillary tangles creativecommons.org/licenses/by/ (NFT) (mostly composed by hyperphosphorylated tau protein) [1], and extracellular neu- 4.0/). ritic plaques (enriched in Aβ) [2,3]. AD often has comorbidities with other severe human Int. J. Mol. Sci. 2021, 22, 12256. https://doi.org/10.3390/ijms222212256 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 12256 2 of 22 diseases, for example, type 2 diabetes. Some common pathophysiological alterations and signaling pathways may be involved in the association between these two disorders [4]. AD can be classified into sporadic AD, which accounts for the majority of the cases, and familial early-onset form, accounting for 1–5% of all cases, in which mutations of genes, for example, amyloid β precursor protein (APP) [5], and presenilin-1 and -2 have been suggested to underlie the development of the disease [6–9]. Aβ deposition has been related to neuroinflammatory responses, in which astrocytes and microglia play a key role [10,11]. Furthermore, the presence of NFTs has been accepted and used for postmortem diagnostic criteria [12]. The AD is a complex neurodegenerative disease specific to humans involving multiple factors, such as inflammation [13]. Thus, the best way to study the AD should be directly in patients. However, some animal models have been developed and must be compared to AD patients for their validation. In addition, the translational research based on “omics” technologies (including lipidomics) are increasing our knowledge of AD for the identification of early AD biomarkers [14]. The triple transgenic mouse model, 3xTg-AD, mimics many critical aspects of AD neuropathology, harboring βAPPSwe , PS1M146V , and tauP301L transgenes [15–18]. These mice progressively develop the neuropathological markers of AD [19]. At 6 months of age, 3xTg-AD mice are characterized by diffuse amyloid plaques in neocortex, and in- traneuronal Aβ accumulation in pyramidal neurons of the hippocampus, cortex, and amygdala [20]. Other authors have described tau phosphorylation at pyramidal layers of hippocampus [21]. This evidence suggests that tau protein dysfunction contributes to AD-related pathophysiology in 3xTg-AD mice at early ages [19]. Moreover, synaptic dysfunction and long-term potentiation deficits are already apparent at this age, although no extracellular Aβ deposits are localized at the hippocampal region. Moreover, behavioral age is an important factor in this 3xTg-AD mouse model [22,23]. Regarding the neuro- chemical alterations observed in AD patients, cholinergic neurotransmission seems to be one of the most characteristic alterations, including the loss of cholinergic neurons at the basal nucleus of Meynert in AD patients [24]. However, neurodegeneration is not limited to a specific neurotransmitter system; the glutamatergic [25], serotonergic [26], noradrenergic [27], and peptidergic (e.g., galanin) [28] neurotransmitter systems are also deregulated in AD [29]. Furthermore, we have recently demonstrated, in a rat lesion model of basal forebrain cholinergic neurons that shows learning and memory impairment, the specific regula- tion of phospholipids which is controlled by muscarinic receptor signaling [30]. Lipid molecules dynamics, finely tuned by neurotransmitter systems, may play pivotal roles in AD development. Thus, different changes occur in the composition of neural membrane glycerophospholipid, sphingolipid, and cholesterol during neurodegeneration (e.g., AD, Parkinson’s disease, and amyotrophic lateral sclerosis) [31–35]. These changes lead to significant increases in glycerophospholipid, sphingolipid, and cholesterol-derived lipid mediators at the plasma membrane and nuclear levels [36]. Lipid mediators are involved in neural cell proliferation, migration, cell cycle, and angiogenesis, promoting cell survival in physiological conditions [37–39]. As mentioned above, the study of brain lipids by lipidomic techniques, together with the analysis of neurolipid-based signaling has emerged with important neuromodulatory properties on different neurotransmitter systems in AD [30,40]. The main neurolipid sys- tems identified so far are the endocannabinoid (eCB), and the lysophospholipid signaling systems, including the lysophosphatidic acid (LPA) and the sphingosine 1-phosphate (S1P) [41–43]. The endocannabinoid signaling is also modified during AD progression. Some authors suggest reduced CB1 densities in the entorhinal cortex, hippocampus, and caudate nucleus in advanced stages of AD patients [44]. Moreover, using immunohisto- chemical techniques, decreased expression of CB1 receptor protein was demonstrated in frontal cortex samples in AD patients [45]. Furthermore, CB1 receptors are up-regulated in the early stages of AD, followed by a diminution of its availability during the progress of the disease [40,46]. In contrast, other studies found no alteration of CB1 densities in Int. J. Mol. Sci. 2021, 22, 12256 3 of 22 Int. J. Mol. Sci. 2021, 22, 12256 3 of 24 AD brains including demonstrated several in frontal cortical cortex samples and in hippocampal AD patientsareas [45].[47,48]. CB1 receptors Furthermore, play a CB1 receptors fundamental role in neuroprotection, controlling excitotoxicity events are up-regulated in the early stages of AD, followed by a diminution of its availability related to neurode- generative during the and inflammatory progress processes of the disease [15,49,50]. [40,46]. In contrast,Regarding lysophospholipid other studies found no alterationsystems,of in vivo and in vitro studies have indicated that the neurophysiology CB1 densities in AD brains including several cortical and hippocampal areas [47,48]. of the LPA and S1P CB1 is relevant for AD. For example, autotaxin, an enzyme involved in LPA production, with receptors play a fundamental role in neuroprotection, controlling excitotoxicity events antioxidative properties, is up-regulated in AD patients [51,52]. Moreover, LPA has been related to neurodegenerative and inflammatory processes [15,49,50]. Regarding lyso- implicated in the up-regulation of BACE1 expression [53]. LPA also activates the phospho- phospholipid systems, in vivo and in vitro studies have indicated that the neurophysi- rylation of GSK-3 enzyme; LPA-mediated activation of GSK-3 occurs in the Rho pathway ology of the LPA and S1P is relevant for AD. For example, autotaxin, an enzyme involved and may represent an important link between microtubule and microfilament dynamics in in LPA production, with antioxidative properties, is up-regulated in AD patients [51,52]. AD [54,55]. In addition, LPA has been described as a novel potential mediator in myeli- Moreover, LPA has been implicated in the up-regulation of BACE1 expression [53]. LPA nation [56] which is disrupted in AD [57]. Finally, the neuroprotective effects of LPA on also activates the phosphorylation of GSK-3 enzyme; LPA-mediated activation of GSK-3 Aβ 31–35 induced apoptosis in cultured cortical neurons have been reported [16]. occurs in the Rho pathway and may represent an important link between microtubule The knowledge about the role of S1P-mediated signaling in AD is more recent, but and microfilament dynamics in AD [54,55]. In addition, LPA has been described as a indicates decreased levels of its endogenous ligand in cortical and subcortical areas in novel potential mediator in myelination [56] which is disrupted in AD [57]. Finally, the brain samples from AD patients [58] which, at least in the cortex, may be the consequence neuroprotective of effects oforLPA aberrant functionality alteredon expression Aβ 31–35 induced apoptosis in cultured of the S1P-synthesizing and/or cortical degradingneu- rons have been reported [16]. enzymes [59]. The S1P1 receptors have also been localized in astrocytes [60]. The knowledge Thus, the presentabout study the role ofthe analyzed S1P-mediated signaling activities mediated by CBin AD is more recent, but 1 , LPA1 , and S1P1 recep- indicates decreased levels of its endogenous ligand in cortical and tors, respectively, in 6-month-old 3xTg-AD transgenic mice using the functional [35 S]GTPγS subcortical areas in brain samples from autoradiography AD patients method. [58] which, The receptor at least specificity in the was cortex,atmay localized be the consequence the subcellular level by of aberrant functionality or altered expression of the S1P-synthesizing microscopic immunofluorescent localization. These results are compared to the anatomical and/or degrading enzymes [59]. distribution The S1Pof1 receptors patterns have also been the lipid distribution localized in the brain of in 3xTg-AD astrocytesmice, [60]. obtained by Thus, the present study analyzed MALDI mass spectrometry imaging (MALDI-MSI). the activities mediated by CB 1, LPA 1, and S1P1 re- ceptors, respectively, in 6-month-old 3xTg-AD transgenic mice using the functional 2. Results autoradiography method. The receptor specificity was localized at the sub- [35S]GTPγS cellular level by Binding 2.1. [35 S]GTPγS microscopic Assayimmunofluorescent in 3xTg-AD Mice Brain localization. Sections These results are compared to the anatomical distribution patterns of the lipid distribution in the brain of 3xTg-AD In order to assess changes between 3xTg-AD mice and age-matched wild type (WT) mice, obtained by MALDI mass spectrometry imaging (MALDI-MSI). mice, firstly, we examined the basal G protein activity in whole brain, which was quite similar in 3xTg-AD and WT mice. The functional coupling induced by WIN55,212-2, 2. Results accounting mainly for CB1 -mediated activity, was decreased in transgenic mice compared 2.1.WT to [35S]GTPγS mice in the Binding amygdala Assay in 3xTg-AD (3xTg-AD Mice 112.8 Brain Sections ± 28.9% vs. WT 295.5 ± 41.7%; p < 0.01). This CB1 activity In orderwas also decreased to assess changes in the VI layer between of themice 3xTg-AD motor andcortex (3xTg-ADwild age-matched 238.4 ± 22.9% type (WT) vs. WT 435.4 ± 58.2%; p < 0.01) (Figure 1, Table 1). mice, firstly, we examined the basal G protein activity in whole brain, which was quite Onin the other hand, 35 S]GTPγS binding induced by LPA was increased in trans- similar 3xTg-AD and the WT[ mice. The functional coupling induced by WIN55,212-2, ac- genic countingmice at thefor mainly striatum (3xTg-AD CB1-mediated 23.1 ±was activity, 3.8% vs. WT in decreased ± 7.2%; pmice 3.3transgenic < 0.05), motor compared cortex to WT (3xTg-AD mice in the26.1 ± 7.6%(3xTg-AD amygdala vs. WT 6.2 ± 12.4%; 112.8 ± 28.9% p