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.

Enzyme Therapy: Current Challenges and Future Perspectives.

In recent years, enzymes have risen as promising therapeutic tools for different pathologies, from metabolic deficiencies, such as fibrosis conditions, ocular pathologies or joint problems, to cancer or cardiovascular diseases. Treatments based on the catalytic activity of enzymes are able to convert a wide range of target molecules to restore the correct physiological metabolism. These treatments present several advantages compared to established therapeutic approaches thanks to their affinity and specificity properties. However, enzymes present some challenges, such as short in vivo half-life, lack of targeted action and, in particular, patient immune system reaction against the enzyme. For this reason, it is important to monitor serum immune response during treatment. This can be achieved by conventional techniques (ELISA) but also by new promising tools such as microarrays. These assays have gained popularity due to their high-throughput analysis capacity, their simplicity, and their potential to monitor the immune response of patients during enzyme therapies. In this growing field, research is still ongoing to solve current health problems such as COVID-19. Currently, promising therapeutic alternatives using the angiotensin-converting enzyme 2 (ACE2) are being studied to treat COVID-19.

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.

Pharmacological ablation of astrocytes reduces Aß degradation and synaptic connectivity in an ex vivo model of Alzheimer's disease.

BACKGROUND: Astrocytes provide a vital support to neurons in normal and pathological conditions. In Alzheimer's disease (AD) brains, reactive astrocytes have been found surrounding amyloid plaques, forming an astrocytic scar. However, their role and potential mechanisms whereby they affect neuroinflammation, amyloid pathology, and synaptic density in AD remain unclear. METHODS: To explore the role of astrocytes on Aß pathology and neuroinflammatory markers, we pharmacologically ablated them in organotypic brain culture slices (OBCSs) from 5XFAD mouse model of AD and wild-type (WT) littermates with the selective astrocytic toxin L-alpha-aminoadipate (L-AAA). To examine the effects on synaptic circuitry, we measured dendritic spine number and size in OBCSs from Thy-1-GFP transgenic mice incubated with synthetic Aß42 or double transgenics Thy-1-GFP/5XFAD mice treated with LAAA or vehicle for 24 h. RESULTS: Treatment of OBCSs with L-AAA resulted in an increased expression of pro-inflammatory cytokine IL-6 in conditioned media of WTs and 5XFAD slices, associated with changes in microglia morphology but not in density. The profile of inflammatory markers following astrocytic loss was different in WT and transgenic cultures, showing reductions in inflammatory mediators produced in astrocytes only in WT sections. In addition, pharmacological ablation of astrocytes led to an increase in Aß levels in homogenates of OBCS from 5XFAD mice compared with vehicle controls, with reduced enzymatic degradation of Aß due to lower neprilysin and insulin-degrading enzyme (IDE) expression. Furthermore, OBSCs from wild-type mice treated with L-AAA and synthetic amyloid presented 56% higher levels of Aß in culture media compared to sections treated with Aß alone, concomitant with reduced expression of IDE in culture medium, suggesting that astrocytes contribute to Aß clearance and degradation. Quantification of hippocampal dendritic spines revealed a reduction in their density following L-AAA treatment in all groups analyzed. In addition, pharmacological ablation of astrocytes resulted in a decrease in spine size in 5XFAD OBCSs but not in OBCSs from WT treated with synthetic Aß compared to vehicle control. CONCLUSIONS: Astrocytes play a protective role in AD by aiding Aß clearance and supporting synaptic plasticity.

Microarray and Mass Spectrometry-Based Methodology for Lipid Profiling of Tissues and Cell Cultures.

The recent developments in mass spectrometry have revealed the importance of lipids as biomarkers in the context of different diseases and as indicators of the cell's homeostasis. However, further advances are required to unveil the complex relationships between lipid classes and lipid species with proteins. Here, we present a new methodology that combines microarrays with mass spectrometry to obtain the lipid fingerprint of samples of a different nature in a standardized and fast way, with minimal sample consumption. As a proof of concept, we use the methodology to obtain the lipid fingerprint of 20 rat tissues and to create a lipid library for tissue classification. Then, we combine those results with immunohistochemistry and enzymatic assays to unveil the relationship between some lipid species and two enzymes. Finally, we demonstrate the performance of the methodology to explore changes in lipid composition of the nucleus accumbens from mice subjected to two lipid diets.

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.

PPARγ-coactivator-1α gene transfer reduces neuronal loss and amyloid-ß generation by reducing ß-secretase in an Alzheimer's disease model.

Current therapies for Alzheimer's disease (AD) are symptomatic and do not target the underlying Aß pathology and other important hallmarks including neuronal loss. PPARγ-coactivator-1α (PGC-1α) is a cofactor for transcription factors including the peroxisome proliferator-activated receptor-γ (PPARγ), and it is involved in the regulation of metabolic genes, oxidative phosphorylation, and mitochondrial biogenesis. We previously reported that PGC-1α also regulates the transcription of ß-APP cleaving enzyme (BACE1), the main enzyme involved in Aß generation, and its expression is decreased in AD patients. We aimed to explore the potential therapeutic effect of PGC-1α by generating a lentiviral vector to express human PGC-1α and target it by stereotaxic delivery to hippocampus and cortex of APP23 transgenic mice at the preclinical stage of the disease. Four months after injection, APP23 mice treated with hPGC-1α showed improved spatial and recognition memory concomitant with a significant reduction in Aß deposition, associated with a decrease in BACE1 expression. hPGC-1α overexpression attenuated the levels of proinflammatory cytokines and microglial activation. This effect was accompanied by a marked preservation of pyramidal neurons in the CA3 area and increased expression of neurotrophic factors. The neuroprotective effects were secondary to a reduction in Aß pathology and neuroinflammation, because wild-type mice receiving the same treatment were unaffected. These results suggest that the selective induction of PGC-1α gene in specific areas of the brain is effective in targeting AD-related neurodegeneration and holds potential as therapeutic intervention for this disease.

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 Alzheimer's 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.

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.