PCSK9 ablation attenuates Aß pathology, neuroinflammation and cognitive dysfunctions in 5XFAD mice.

BACKGROUND: Increasing evidence highlights the importance of novel players in Alzheimer's disease (AD) pathophysiology, including alterations of lipid metabolism and neuroinflammation. Indeed, a potential involvement of Proprotein convertase subtilisin/kexin type 9 (PCSK9) in AD has been recently postulated. Here, we first investigated the effects of PCSK9 on neuroinflammation in vitro. Then, we examined the impact of a genetic ablation of PCSK9 on cognitive performance in a severe mouse model of AD. Finally, in the same animals we evaluated the effect of PCSK9 loss on Aß pathology, neuroinflammation, and brain lipids. METHODS: For in vitro studies, U373 human astrocytoma cells were treated with Aß fibrils and human recombinant PCSK9. mRNA expression of the proinflammatory cytokines and inflammasome-related genes were evaluated by q-PCR, while MCP-1 secretion was measured by ELISA. For in vivo studies, the cognitive performance of a newly generated mouse line - obtained by crossing 5XFADHet with PCSK9KO mice - was tested by the Morris water maze test. After sacrifice, immunohistochemical analyses were performed to evaluate Aß plaque deposition, distribution and composition, BACE1 immunoreactivity, as well as microglia and astrocyte reactivity. Cholesterol and hydroxysterols levels in mouse brains were quantified by fluorometric and LC-MS/MS analyses, respectively. Statistical comparisons were performed according to one- or two-way ANOVA, two-way repeated measure ANOVA or Chi-square test. RESULTS: In vitro, PCSK9 significantly increased IL6, IL1B and TNFΑ mRNA levels in Aß fibrils-treated U373 cells, without influencing inflammasome gene expression, except for an increase in NLRC4 mRNA levels. In vivo, PCSK9 ablation in 5XFAD mice significantly improved the performance at the Morris water maze test; these changes were accompanied by a reduced corticohippocampal Aß burden without affecting plaque spatial/regional distribution and composition or global BACE1 expression. Furthermore, PCSK9 loss in 5XFAD mice induced decreased microgliosis and astrocyte reactivity in several brain regions. Conversely, knocking out PCSK9 had minimal impact on brain cholesterol and hydroxysterol levels. CONCLUSIONS: In vitro studies showed a pro-inflammatory effect of PCSK9. Consistently, in vivo data indicated a protective role of PCSK9 ablation against cognitive impairments, associated with improved Aß pathology and attenuated neuroinflammation in a severe mouse model of AD. PCSK9 may thus be considered a novel pharmacological target for the treatment of AD.

Mechanisms of Hydrogen Sulfide against the Progression of Severe Alzheimer's Disease in Transgenic Mice at Different Ages.

Backgroud: Alzheimer disease is an age-related severe neurodegenerative pathology. The level of the third endogenous gas, hydrogen sulfide (H2S), is decreased in the brain of Alzheimer's disease (AD) patients compared with the brain of the age-matched normal individuals; also, plasma H2S levels are negatively correlated with the severity of AD. Recently, we have demonstrated that systemic H2S injections are neuroprotective in an early phase of preclinical AD. OBJECTIVES: This study focuses on the possible neuroprotection of a chronic treatment with an H2S donor and sulfurous water (rich of H2S) in a severe transgenic 3×Tg-AD mice model. METHOD: 3×Tg-AD mice at 2 different ages (6 and 12 months) were daily treated intraperitoneally with an H2S donor and sulfurous water (rich of H2S) for 3 months consecutively. We investigated the cognitive ability, brain morphological alterations, amyloid/tau cascade, excitotoxic, inflammatory and apoptotic responses. RESULTS: Three months of treatments with H2S significantly protected against impairment in learning and memory in a severe 3×Tg-AD mice model, at both ages studied, and reduced the size of Amyloid ß plaques with preservation of the morphological picture. This neuroprotection appeared mainly in the cortex and hippocampus, associated with reduction in activity of c-jun N-terminal kinases, extracellular signal-regulated kinases and p38, which have an established role not only in the phosphorylation of tau protein but also in the inflammatory and excitotoxic response. CONCLUSION: Our findings indicate that appropriate treatments with various sources of H2S, might represent an innovative approach to counteract early and severe AD progression in humans.

Effects of COX1-2/5-LOX blockade in Alzheimer transgenic 3xTg-AD mice.

OBJECTIVE AND DESIGN: Alzheimer's disease (AD) is associated with amyloid plaques (Aß) and hyperphosphorylated tau protein tangles in the brain. We investigated the possible neuroprotective role of flavocoxid, a dual inhibitor of cyclooxygenases-1/2 (COX-1/2) and 5-Lipoxygenase (5-LOX), in triple-transgenic (3xTg-AD) mice. SUBJECTS: Mice were 3 months at the beginning of the study. TREATMENT: Animals received once daily for 3-month saline solution or flavocoxid (20 mg/kg/ip). METHODS: Morris water maze was used to assess learning and memory. Histology was performed to evidence Aß plaques and neuronal loss, while inflammatory proteins were determined by western blot analysis. RESULTS: Saline-treated 3xTg-AD mice showed an impairment in spatial learning and memory (assessed at 6 months of age), and increased expression of inflammatory and apoptotic molecules. Treatment of 3xTg-AD mice with flavocoxid reduced: (1) learning and memory loss; (2) the increased eicosanoid production and the phosphorylation level of amyloid precursor protein (APP-pThr668), Aß 1-42, p-tau (pThr181), pERK, and the activation of the NLRP3 inflammasome; (3) Aß plaques; and (4) neuronal loss, compared to saline-treated animals. CONCLUSIONS: Pharmacological blockade of both COX-1/2 and 5-LOX was able to counteract the progression of AD by targeting pathophysiological mechanisms up- and downstream of Aß and tau.

NDP-α-MSH induces intense neurogenesis and cognitive recovery in Alzheimer transgenic mice through activation of melanocortin MC4 receptors.

Melanocortins exert neuroprotection in a variety of experimental neurodegenerative disorders, including Alzheimer's disease (AD). Further, in previous research we showed that these endogenous peptides stimulate neurogenesis in an acute neurodegenerative disorder such as ischemic stroke. In the present research, we investigated the potential neurogenic effect of melanocortins in AD using APPSwe transgenic mice (Tg2576). To this purpose, 24week-old animals were prepared for 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells on days 1-11 of the study. Treatment of Tg2576 mice with nanomolar doses of the melanocortin analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH), administered once daily from day 1 to 50, improved brain histology and cognitive functions relative to saline-treated Tg2576 animals. No signs of toxicity were observed. Immunohistochemical examination of the hippocampus at the end of the study (day 50) showed that NDP-α-MSH-treated Tg2576 mice had a greater number of BrdU immunoreactive cells colocalized with NeuN (an indicator of mature neurons) and Zif268 (an indicator of functionally integrated neurons) in the dentate gyrus, relative to saline-treated Tg2576 animals; no newly formed astrocytes were found. Animal pretreatment with the selective melanocortin MC4 receptor antagonist HS024 before each NDP-α-MSH administration prevented all the beneficial effects of the peptide. The present data indicate that MC4 receptor stimulation by a melanocortin prevents cognitive decline in experimental AD, this effect being associated not only with neuroprotection but also with an intense neurogenesis. MC4 receptor agonists could be innovative and safe candidates to counteract AD progression in humans.

Melanocortin receptor 4 as a new target in melanoma therapy: Anticancer activity of the inhibitor ML00253764 alone and in association with B-raf inhibitor vemurafenib.

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor. The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed. Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity. Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies.

Melanocortin receptor agonist NDP-α-MSH improves cognitive deficits and microgliosis but not amyloidosis in advanced stages of AD progression in 5XFAD and 3xTg mice.

Introduction: Alzheimer's disease (AD) is the most frequent cause of dementia and still lacks effective therapy. Clinical signs of AD include low levels of endogenous melanocortins (MCs) and previous studies have shown that treatment with MC analogs induces neuroprotection in the early stages of AD. Methods: We investigated the neuroprotective role of MCs in two transgenic mouse models of severe AD using 5 and 7 month-old (mo) 5XFAD mice and 9 and 12 mo 3xTg mice. These mice were subjected to a chronic stimulation of MC receptors (MCRs) with MC analogue Nle4-D-Phe7-α-melanocyte stimulating hormone (NDP-α-MSH, 340 µg/kg, i.p.). Mouse behavior and ex-vivo histological and biochemical analyses were performed after 50 days of treatment. Results: Our analysis demonstrated an improvement in cognitive abilities of AD mice at late stage of AD progression. We also showed that these protective effects are associated with decreased levels of hyperphosphorylated Tau but not with Aß burden, that was unaffected in the hippocampus and in the cortex of AD mice. In addition, an age-dependent NDP effect on glial reactivity was observed only in 3xTg mice whereas a global downregulation of p38 mitogen-activated protein kinase was selectively observed in 7 mo 5XFAD and 14 mo 3xTg mice. Conclusion: Our results suggest that MCR stimulation by NDP-α-MSH could represent a promising therapeutic strategy in managing cognitive decline also at late stage of AD, whereas the effects on neuroinflammation may be restricted to specific stages of AD progression.

Bioresorbable Nanostructured Chemical Sensor for Monitoring of pH Level In Vivo.

Here, the authors report on the manufacturing and in vivo assessment of a bioresorbable nanostructured pH sensor. The sensor consists of a micrometer-thick porous silica membrane conformably coated layer-by-layer with a nanometer-thick multilayer stack of two polyelectrolytes labeled with a pH-insensitive fluorophore. The sensor fluorescence changes linearly with the pH value in the range 4 to 7.5 upon swelling/shrinking of the polymer multilayer and enables performing real-time measurements of the pH level with high stability, reproducibility, and accuracy, over 100 h of continuous operation. In vivo studies carried out implanting the sensor in the subcutis on the back of mice confirm real-time monitoring of the local pH level through skin. Full degradation of the pH sensor occurs in one week from implant in the animal model, and its biocompatibility after 2 months is confirmed by histological and fluorescence analyses. The proposed approach can be extended to the detection of other (bio)markers in vivo by engineering the functionality of one (at least) of the polyelectrolytes with suitable receptors, thus paving the way to implantable bioresorbable chemical sensors.

Identification of a Thyroid Hormone Derivative as a Pleiotropic Agent for the Treatment of Alzheimer's Disease.

The identification of effective pharmacological tools for Alzheimer's disease (AD) represents one of the main challenges for therapeutic discovery. Due to the variety of pathological processes associated with AD, a promising route for pharmacological intervention involves the development of new chemical entities that can restore cellular homeostasis. To investigate this strategy, we designed and synthetized SG2, a compound related to the thyroid hormone thyroxine, that shares a pleiotropic activity with its endogenous parent compound, including autophagic flux promotion, neuroprotection, and metabolic reprogramming. We demonstrate herein that SG2 acts in a pleiotropic manner to induce recovery in a C. elegans model of AD based on the overexpression of Aß42 and improves learning abilities in the 5XFAD mouse model of AD. Further, in vitro ADME-Tox profiling and toxicological studies in zebrafish confirmed the low toxicity of this compound, which represents a chemical starting point for AD drug development.

Oxidative Stress in Alzheimer's Disease: In Vitro Therapeutic Effect of Amniotic Fluid Stem Cells Extracellular Vesicles.

Alzheimer's disease (AD) is characterized by abnormal protein aggregation, deposition of extracellular ß-amyloid proteins (Aß), besides an increase of oxidative stress. Amniotic fluid stem cells (AFSCs) should have a therapeutic potential for neurodegenerative disorders, mainly through a paracrine effect mediated by extracellular vesicles (EV). Here, we examined the effect of EV derived from human AFSCs (AFSC-EV) on the disease phenotypes in an AD neuron primary culture. We observed a positive effect of AFSC-EV on neuron morphology, viability, and Aß and phospho-Tau levels. This could be due to the apoptotic and autophagic pathway modulation derived from the decrease in oxidative stress. Indeed, reactive oxygen species (ROS) were reduced, while GSH levels were enhanced. This modulation could be ascribed to the presence of ROS regulating enzymes, such as SOD1 present into the AFSC-EV themselves. This study describes the ROS-modulating effects of extracellular vesicles alone, apart from their deriving stem cell, in an AD in vitro model, proposing AFSC-EV as a therapeutic tool to stop the progression of AD.

NDP-α-MSH attenuates heart and liver responses to myocardial reperfusion via the vagus nerve and JAK/ERK/STAT signaling.

Melanocortin peptides afford cardioprotection during myocardial ischemia/reperfusion via janus kinases (JAK), extracellular signal-regulated kinases (ERK) and signal transducers/activators of transcription (STAT) pathways. Here we investigated whether melanocortin-induced modulation of the JAK/ERK/STAT signaling occurs via the cholinergic anti-inflammatory pathway, focusing our study on cardiac and hepatic responses to prolonged myocardial ischemia/reperfusion. Ischemia was produced in rats by ligature of the left anterior descending coronary artery for 30min; effects of ischemia/reperfusion were evaluated using Western blot of heart and liver proteins. Intravenous treatment, during coronary artery occlusion, with the melanocortin analog (Nle(4), D-Phe(7))α-melanocyte-stimulating hormone (NDP-α-MSH) induced a left ventricle up-regulation of the cardioprotective transcription factors pJAK2, pERK1/2 and pTyr-STAT3 (JAK-dependent), and a reduction in the levels of the inflammatory mediators tumor necrosis factor-α (TNF-α) and pJNK (a transcription factor also involved in apoptosis), as assessed at the end of the 2-h reperfusion period. Further, these beneficial effects of NDP-α-MSH were associated with heart over-expression of the pro-survival proteins heme oxygenase-1 (HO-1) and Bcl-XL, and decrease of ventricular arrhythmias and infarct size. In the liver NDP-α-MSH induced a decrease in the pJAK2 and pTyr-STAT3 levels, and strongly reduced pERK1/2 expression. In the liver of ischemic rats NDP-α-MSH also blunted pJNK activity and TNF-α expression, and up-regulated Bcl-XL. Bilateral cervical vagotomy prevented all effects of NDP-α-MSH, both in the heart and liver. These results indicate that melanocortins inhibit heart and liver damage triggered by prolonged myocardial ischemia/reperfusion likely, as main mechanism, via the vagus nerve-mediated modulation of the JAK/STAT/ERK signaling pathways.

Melanocortins protect against brain damage and counteract cognitive decline in a transgenic mouse model of moderate Alzheimer׳s disease.

We previously reported that melanocortins induce neuroprotection in experimental acute and chronic neurodegenerative conditions, including Alzheimer׳s disease (AD) of mild severity. Here we investigated whether melanocortins afford neuroprotection and counteract cognitive decline in AD with a medium level of severity by using 24 week-old (at the start of the study) APPSwe transgenic mice (Tg2576). Saline-treated (days 1-50) control Tg2576 mice showed an impairment in spatial learning and memory, associated (at day 50, end of the study) with hippocampus at low levels of the synaptic activity-dependent gene Zif268, relevant brain changes such as cerebral cortex/hippocampus increased level of ß-amyloid (Aß) deposit, and neuronal loss, in comparison with wild-type animals. Treatment of Tg2576 mice (once daily at days 1-50) with a nanomolar dose of the melanocortin analog [Nle4,D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) reduced cerebral cortex/hippocampus level of Aß deposit, decreased neuronal loss, increased hippocampus Zif268 expression and improved cognitive functions, relative to saline-treated Tg2576 mice. Pharmacological blockade of melanocortin MC4 receptors with the MC4 receptor antagonist HS024 prevented all favorable effects of NDP-α-MSH. Our data indicate that MC4 receptor-stimulating melanocortins are able to counteract cognitive decline in experimental AD of medium severity through induction of neuroprotection and improvement of synaptic transmission. After further studies, these agents could gain a role as disease modifying therapeutics for AD.