Carnosine Protects Macrophages against the Toxicity of Aβ1-42 Oligomers by Decreasing Oxidative Stress
TL;DR: In this paper, the effects of carnosine on macrophages and oxidative stress associated with AD were investigated using a combination of methods including trypan blue exclusion, microchip electrophoresis with laser-induced fluorescence, flow cytometry, fluorescence microscopy, and high-throughput quantitative real-time PCR.
Abstract: Carnosine (β-alanyl-L-histidine) is a naturally occurring endogenous peptide widely distributed in excitable tissues such as the brain. This dipeptide has well-known antioxidant, anti-inflammatory, and anti-aggregation activities, and it may be useful for treatment of neurodegenerative disorders such as Alzheimer’s disease (AD). In this disease, peripheral infiltrating macrophages play a substantial role in the clearance of amyloid beta (Aβ) peptides from the brain. Correspondingly, in patients suffering from AD, defects in the capacity of peripheral macrophages to engulf Aβ have been reported. The effects of carnosine on macrophages and oxidative stress associated with AD are consequently of substantial interest for drug discovery in this field. In the present work, a model of stress induced by Aβ1-42 oligomers was investigated using a combination of methods including trypan blue exclusion, microchip electrophoresis with laser-induced fluorescence, flow cytometry, fluorescence microscopy, and high-throughput quantitative real-time PCR. These assays were used to assess the ability of carnosine to protect macrophage cells, modulate oxidative stress, and profile the expression of genes related to inflammation and pro- and antioxidant systems. We found that pre-treatment of RAW 264.7 macrophages with carnosine counteracted cell death and apoptosis induced by Aβ1-42 oligomers by decreasing oxidative stress as measured by levels of intracellular nitric oxide (NO)/reactive oxygen species (ROS) and production of peroxynitrite. This protective activity of carnosine was not mediated by modulation of the canonical inflammatory pathway but instead can be explained by the well-known antioxidant and free-radical scavenging activities of carnosine, enhanced macrophage phagocytic activity, and the rescue of fractalkine receptor CX3CR1. These new findings obtained with macrophages challenged with Aβ1-42 oligomers, along with the well-known multimodal mechanism of action of carnosine in vitro and in vivo, substantiate the therapeutic potential of this dipeptide in the context of AD pathology.
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TL;DR: In this article, the authors provide evidence that different nutraceuticals, such as Hypericum perforatum (hypericin and hyperforin), flavonoids such as hesperidin, omega-3, and carnosine, can target TGFβ1 signaling and increase TGF-β1 production in the central nervous system as well as cognitive function.
Abstract: Rescue of cognitive function represents an unmet need in the treatment of neurodegenerative disorders such as Alzheimer's disease (AD). Nutraceuticals deliver a concentrated form of a presumed bioactive(s) agent(s) that can improve cognitive function alone or in combination with current approved drugs for the treatment of cognitive disorders. Nutraceuticals include different natural compounds such as flavonoids and their subclasses (flavan-3-ols, catechins, anthocyanins, and flavonols), omega-3, and carnosine that can improve synaptic plasticity and rescue cognitive deficits through multiple molecular mechanisms. A deficit of transforming growth factor-β1 (TGF-β1) pathway is an early event in the pathophysiology of cognitive impairment in different neuropsychiatric disorders, from depression to AD. In the present review, we provide evidence that different nutraceuticals, such as Hypericum perforatum (hypericin and hyperforin), flavonoids such as hesperidin, omega-3, and carnosine, can target TGF-β1 signaling and increase TGF-β1 production in the central nervous system as well as cognitive function. The bioavailability of these nutraceuticals, in particular carnosine, can be significantly improved with novel formulations (nanoparticulate systems, nanoliposomes) that increase the efficacy and stability of this peptide. Overall, these studies suggest that the synergism between nutraceuticals targeting the TGF-β1 pathway and current approved drugs might represent a novel pharmacological approach for reverting cognitive deficits in AD patients.
16 citations
TL;DR: The data suggest that C. sativa phytocomplex and its multitarget mechanism could represent a novel therapeutic strategy for neuroinflammatory‐related diseases.
Abstract: Cannabis sativa L. is increasingly emerging for its protective role in modulating neuroinflammation, a complex process orchestrated among others by microglia, the resident immune cells of the central nervous system. Phytocannabinoids, especially cannabidiol (CBD), terpenes, and other constituents trigger several upstream and downstream microglial intracellular pathways. Here, we investigated the molecular mechanisms of a CBD‐ and terpenes‐enriched C. sativa extract (CSE) in an in vitro model of neuroinflammation. We evaluated the effect of CSE on the inflammatory response induced by exposure to lipopolysaccharide (LPS) in BV‐2 microglial cells, compared with CBD and β‐caryophyllene (CAR), CB2 receptors (CB2r) inverse and full agonist, respectively. The LPS‐induced upregulation of the pro‐inflammatory cytokines IL‐1β, IL‐6, and TNF‐α was significantly attenuated by CSE and only partially by CBD, whereas CAR was ineffective. In BV‐2 cells, these anti‐inflammatory effects exerted by CSE phytocomplex were only partially dependent on CB2r modulation and they were mediated by the regulation of enzymes responsible for the endocannabinoids metabolism, by the inhibition of reactive oxygen species release and the modulation of JNK/p38 cascade with consequent NF‐κB p65 nuclear translocation suppression. Our data suggest that C. sativa phytocomplex and its multitarget mechanism could represent a novel therapeutic strategy for neuroinflammatory‐related diseases.
14 citations
TL;DR: The role of microglia in AD is discussed and their role in AD iron homeostasis is emphasized, providing a basis for better regulation of iron metabolism disorders in AD and the discovery of new potential therapeutic targets for AD.
Abstract: Alzheimer’s disease (AD), the most common type of senile dementia, includes the complex pathogenesis of abnormal deposition of amyloid beta-protein (Aβ), phosphorylated tau (p-tau) and neuroimmune inflammatory. The neurodegenerative process of AD triggers microglial activation, and the overactivation of microglia produces a large number of neuroimmune inflammatory factors. Microglia dysfunction can lead to disturbances in iron metabolism and enhance iron-induced neuronal degeneration in AD, while elevated iron levels in brain areas affect microglia phenotype and function. In this manuscript, we firstly discuss the role of microglia in AD and then introduce the role of microglia in the immune-inflammatory pathology of AD. Their role in AD iron homeostasis is emphasized. Recent studies on microglia and ferroptosis in AD are also reviewed. It will help readers better understand the role of microglia in iron metabolism in AD, and provides a basis for better regulation of iron metabolism disorders in AD and the discovery of new potential therapeutic targets for AD.
13 citations
TL;DR: The existing works that study alterations in carnosine metabolism in Alzheimer's disease and Parkinson’s disease are summarized and the beneficial effect that Carnosine supplementation presents in models of such diseases as well as in aging-related neurodegeneration is reviewed.
Abstract: Neurodegenerative diseases (NDs) constitute a global challenge to human health and an important social and economic burden worldwide, mainly due to their growing prevalence in an aging population and to their associated disabilities. Despite their differences at the clinical level, NDs share fundamental pathological mechanisms such as abnormal protein deposition, intracellular Ca2+ overload, mitochondrial dysfunction, redox homeostasis imbalance and neuroinflammation. Although important progress is being made in deciphering the mechanisms underlying NDs, the availability of effective therapies is still scarce. Carnosine is a natural endogenous molecule that has been extensively studied during the last years due to its promising beneficial effects for human health. It presents multimodal mechanisms of action, being able to exert antioxidant, anti-inflammatory and anti-aggregate activities, among others. Interestingly, most NDs exhibit oxidative and nitrosative stress, protein aggregation and inflammation as molecular hallmarks. In this review, we discuss the neuroprotective functions of carnosine and its implications as a therapeutic strategy in different NDs. We summarize the existing works that study alterations in carnosine metabolism in Alzheimer’s disease and Parkinson’s disease, the two most common NDs. In addition, we review the beneficial effect that carnosine supplementation presents in models of such diseases as well as in aging-related neurodegeneration.
13 citations
TL;DR: It is demonstrated, at chemical and biochemical levels, that insulin-degrading enzyme plays a pivotal role in carnosine neuroprotection.
Abstract: l-Carnosine is an endogenous dipeptide that has high potential for therapeutic purposes, being an antioxidant with metal chelating, anti-aggregating, anti-inflammatory, and neuroprotective properties. Despite its potential therapeutic values, the biomolecular mechanisms involved in neuroprotection are not fully understood. Here, we demonstrate, at chemical and biochemical levels, that insulin-degrading enzyme plays a pivotal role in carnosine neuroprotection.
9 citations
References
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TL;DR: The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which opens up the possibility of studying the biological relevance of small expression differences.
Abstract: Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.
18,261 citations
TL;DR: A novel, innovative, and robust strategy to identify stably expressed genes among a set of candidate normalization genes, rooted in a mathematical model of gene expression, that provides a direct measure for the estimated expression variation, enabling the user to evaluate the systematic error introduced when using the gene.
Abstract: Accurate normalization is an absolute prerequisite for correct measurement of gene expression. For quantitative real-time reverse transcription-PCR (RT-PCR), the most commonly used normalization strategy involves standardization to a single constitutively expressed control gene. However, in recent years, it has become clear that no single gene is constitutively expressed in all cell types and under all experimental conditions, implying that the expression stability of the intended control gene has to be verified before each experiment. We outline a novel, innovative, and robust strategy to identify stably expressed genes among a set of candidate normalization genes. The strategy is rooted in a mathematical model of gene expression that enables estimation not only of the overall variation of the candidate normalization genes but also of the variation between sample subgroups of the sample set. Notably, the strategy provides a direct measure for the estimated expression variation, enabling the user to evaluate the systematic error introduced when using the gene. In a side-by-side comparison with a previously published strategy, our model-based approach performed in a more robust manner and showed less sensitivity toward coregulation of the candidate normalization genes. We used the model-based strategy to identify genes suited to normalize quantitative RT-PCR data from colon cancer and bladder cancer. These genes are UBC, GAPD, and TPT1 for the colon and HSPCB, TEGT, and ATP5B for the bladder. The presented strategy can be applied to evaluate the suitability of any normalization gene candidate in any kind of experimental design and should allow more reliable normalization of RT-PCR data.
6,007 citations
University Hospital Bonn1, University of California, Riverside2, Harvard University3, Case Western Reserve University4, University of Illinois at Chicago5, European Institute6, VA Palo Alto Healthcare System7, Stanford University8, Spanish National Research Council9, Cleveland Clinic Lerner Research Institute10, Hong Kong University of Science and Technology11, University of California, Los Angeles12, University of Southern Denmark13, University of Cambridge14, University of Manchester15, Ikerbasque16, University of the Basque Country17, RIKEN Brain Science Institute18, University of Eastern Finland19, University of Massachusetts Medical School20, University of Bonn21, Center of Advanced European Studies and Research22, University of Southern California23, University of South Florida24, Duke University25, Southampton General Hospital26, Moorgreen Hospital27, University of Southampton28, Louisiana State University29, Imperial College London30, Centre national de la recherche scientifique31, Karolinska Institutet32, Max Planck Society33, University of Tübingen34, University of Groningen35, University of Colorado Denver36, Douglas Mental Health University Institute37
TL;DR: Genome-wide analysis suggests that several genes that increase the risk for sporadic Alzheimer's disease encode factors that regulate glial clearance of misfolded proteins and the inflammatory reaction.
Abstract: Increasing evidence suggests that Alzheimer's disease pathogenesis is not restricted to the neuronal compartment, but includes strong interactions with immunological mechanisms in the brain. Misfolded and aggregated proteins bind to pattern recognition receptors on microglia and astroglia, and trigger an innate immune response characterised by release of inflammatory mediators, which contribute to disease progression and severity. Genome-wide analysis suggests that several genes that increase the risk for sporadic Alzheimer's disease encode factors that regulate glial clearance of misfolded proteins and the inflammatory reaction. External factors, including systemic inflammation and obesity, are likely to interfere with immunological processes of the brain and further promote disease progression. Modulation of risk factors and targeting of these immune mechanisms could lead to future therapeutic or preventive strategies for Alzheimer's disease.
3,947 citations
TL;DR: The cellular sources of these cytokines, receptor signaling pathways, and induced markers and gene signatures are reviewed and the concept of macrophage activation in the context of the immune response is revisit.
Abstract: Macrophages are innate immune cells with well-established roles in the primary response to pathogens, but also in tissue homeostasis, coordination of the adaptive immune response, inflammation, resolution, and repair. These cells recognize danger signals through receptors capable of inducing specialized activation programs. The classically known macrophage activation is induced by IFN-gamma, which triggers a harsh proinflammatory response that is required to kill intracellular pathogens. Macrophages also undergo alternative activation by IL-4 and IL-13, which trigger a different phenotype that is important for the immune response to parasites. Here we review the cellular sources of these cytokines, receptor signaling pathways, and induced markers and gene signatures. We draw attention to discrepancies found between mouse and human models of alternative activation. The evidence for in vivo alternative activation of macrophages is also analyzed, with nematode infection as prototypic disease. Finally, we revisit the concept of macrophage activation in the context of the immune response.
2,515 citations
TL;DR: The unexpected identification of the 4K (Mr 4,000) Aβ and a truncated form of Aβ in media from cultures of primary cells and untransfected and β-APP-transfected cell lines grown under normal conditions provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A β, the primary protein constituent of the senile plaques of Alzheimer's disease.
Abstract: Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.
1,943 citations