Tracking neuroinflammatory biomarkers in Alzheimer's disease: a strategy for individualized therapeutic approaches?

BACKGROUND: Recent trials of anti-amyloid-ß (Aß) monoclonal antibodies, including lecanemab and donanemab, in early Alzheimer disease (AD) showed that these drugs have limited clinical benefits and their use comes with a significant risk of serious adverse events. Thus, it seems crucial to explore complementary therapeutic approaches. Genome-wide association studies identified robust associations between AD and several AD risk genes related to immune response, including but not restricted to CD33 and TREM2. Here, we critically reviewed the current knowledge on candidate neuroinflammatory biomarkers and their role in characterizing the pathophysiology of AD. MAIN BODY: Neuroinflammation is recognized to be a crucial and contributing component of AD pathogenesis. The fact that neuroinflammation is most likely present from earliest pre-stages of AD and co-occurs with the deposition of Aß reinforces the need to precisely define the sequence and nature of neuroinflammatory events. Numerous clinical trials involving anti-inflammatory drugs previously yielded unfavorable outcomes in early and mild-to-moderate AD. Although the reasons behind these failures remain unclear, these may include the time and the target selected for intervention. Indeed, in our review, we observed a stage-dependent neuroinflammatory process in the AD brain. While the initial activation of glial cells counteracts early brain Aß deposition, the downregulation in the functional state of microglia occurs at more advanced disease stages. To address this issue, personalized neuroinflammatory modulation therapy is required. The emergence of reliable blood-based neuroinflammatory biomarkers, particularly glial fibrillary acidic protein, a marker of reactive astrocytes, may facilitate the classification of AD patients based on the ATI(N) biomarker framework. This expands upon the traditional classification of Aß ("A"), tau ("T"), and neurodegeneration ("N"), by incorporating a novel inflammatory component ("I"). CONCLUSIONS: The present review outlines the current knowledge on potential neuroinflammatory biomarkers and, importantly, emphasizes the role of longitudinal analyses, which are needed to accurately monitor the dynamics of cerebral inflammation. Such a precise information on time and place will be required before anti-inflammatory therapeutic interventions can be considered for clinical evaluation. We propose that an effective anti-neuroinflammatory therapy should specifically target microglia and astrocytes, while considering the individual ATI(N) status of patients.

Low TGF-ß1 plasma levels are associated with cognitive decline in Down syndrome.

Almost all individuals with Down's syndrome (DS) show the characteristic neuropathological features of Alzheimer's disease (AD) by the age of 40, yet not every individual with DS experiences symptoms of AD later in life. Similar to neurotypical developing subjects, AD in people with DS lasts for a long preclinical phase in which biomarkers follow a predictable order of changes. Hence, a prolonged asymptomatic period precedes the onset of dementia, underscoring the importance of identifying new biomarkers for the early detection and monitoring of cognitive decline in individuals with DS. Blood-based biomarkers may offer an alternative non-invasive strategy for the detection of peripheral biological alterations paralleling nervous system pathology in an early phase of the AD continuum. In the last few years, a strong neurobiological link has been demonstrated between the deficit of transforming growth factor-ß1 (TGF-ß1) levels, an anti-inflammatory cytokine endowed with neuroprotective activity, and early pro-inflammatory processes in the AD brain. In this clinical prospective observational study, we found significant lower plasma TGF-ß1 concentrations at the first neuropsychological evaluation (baseline = T0) both in young adult DS individuals (19-35 years) and older DS subjects without AD (35-60 years) compared to age- and sex-matched healthy controls. Interestingly, we found that the lower TGF-ß1 plasma concentrations at T0 were strongly correlated with the following cognitive decline at 12 months. In addition, in young individuals with DS, we found, for the first time, a negative correlation between low TGF-ß1 concentrations and high TNF-α plasma concentrations, a pro-inflammatory cytokine that is known to be associated with cognitive impairment in DS individuals with AD. Finally, adopting an ex vivo approach, we found that TGF-ß1 concentrations were reduced in parallel both in the plasma and in the peripheral blood mononuclear cells (PBMCs) of DS subjects, and interestingly, therapeutic concentrations of fluoxetine (FLX) applied to cultured PBMCs (1 µM for 24 h) were able to rescue TGF-ß1 concentrations in the culture media from DS PBMCs, suggesting that FLX, a selective serotonin reuptake inhibitor (SSRI) endowed with neuroprotective activity, might rescue TGF-ß1 concentrations in DS subjects at higher risk to develop cognitive decline.

Microfluidic/HPLC combination to study carnosine protective activity on challenged human microglia: Focus on oxidative stress and energy metabolism.

Carnosine (ß-alanyl-L-histidine) is a naturally occurring endogenous peptide widely distributed in excitable tissues such as the brain. This dipeptide possesses well-demonstrated antioxidant, anti-inflammatory, and anti-aggregation properties, and it may be useful for treatment of pathologies characterized by oxidative stress and energy unbalance such as depression and Alzheimer's disease (AD). Microglia, the brain-resident macrophages, are involved in different physiological brain activities such synaptic plasticity and neurogenesis, but their dysregulation has been linked to the pathogenesis of numerous diseases. In AD brain, the activation of microglia towards a pro-oxidant and pro-inflammatory phenotype has found in an early phase of cognitive decline, reason why new pharmacological targets related to microglia activation are of great importance to develop innovative therapeutic strategies. In particular, microglia represent a common model of lipopolysaccharides (LPS)-induced activation to identify novel pharmacological targets for depression and AD and numerous studies have linked the impairment of energy metabolism, including ATP dyshomeostasis, to the onset of depressive episodes. In the present study, we first investigated the toxic potential of LPS + ATP in the absence or presence of carnosine. Our studies were carried out on human microglia (HMC3 cell line) in which LPS + ATP combination has shown the ability to promote cell death, oxidative stress, and inflammation. Additionally, to shed more light on the molecular mechanisms underlying the protective effect of carnosine, its ability to modulate reactive oxygen species production and the variation of parameters representative of cellular energy metabolism was evaluated by microchip electrophoresis coupled to laser-induced fluorescence and high performance liquid chromatography, respectively. In our experimental conditions, carnosine prevented LPS + ATP-induced cell death and oxidative stress, also completely restoring basal energy metabolism in human HMC3 microglia. Our results suggest a therapeutic potential of carnosine as a new pharmacological tool in the context of multifactorial disorders characterize by neuroinflammatory phenomena including depression and AD.

Molecular Connections between DNA Replication and Cell Death in ß-Amyloid-Treated Neurons.

BACKGROUND: Ectopic cell cycle reactivation in neurons is associated with neuronal death in Alzheimer's disease. In cultured rodent neurons, synthetic ß-amyloid (Aß) reproduces the neuronal cell cycle re-entry observed in the Alzheimer's brain, and blockade of the cycle prevents Aß-induced neurodegeneration. DNA polymerase-ß, whose expression is induced by Aß, is responsible for the DNA replication process that ultimately leads to neuronal death, but the molecular mechanism(s) linking DNA replication to neuronal apoptosis are presently unknown. AIM: To explore the role of a conserved checkpoint pathway started by DNA replication stress, namely the ATM-ATR/Claspin/Chk-1 pathway, in switching the neuronal response from DNA replication to apoptosis. METHODS: Experiments were carried out in cultured rat cortical neurons challenged with toxic oligomers of Aß protein. RESULTS: Small inhibitory molecules of ATM/ATR kinase or Chk-1 amplified Aß-induced neuronal DNA replication and apoptosis, as they were permissive to the DNA polymerase-ß activity triggered by Aß oligomers. Claspin, i.e., the adaptor protein between ATM/ATR kinase and the downstream Chk-1, was present on DNA replication forks of neurons early after Aß challenge, and decreased at times coinciding with neuronal apoptosis. The caspase-3/7 inhibitor I maintained overtime the amount of Claspin loaded on DNA replication forks and, concomitantly, reduced neuronal apoptosis by holding neurons in the S phase. Moreover, a short phosphopeptide mimicking the Chk-1-binding motif of Claspin was able to prevent Aß-challenged neurons from entering apoptosis. CONCLUSION: We speculate that, in the Alzheimer's brain, Claspin degradation by intervening factors may precipitate the death of neurons engaged into DNA replication.

Carnosine Counteracts the Molecular Alterations Aß Oligomers-Induced in Human Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD) has been described as a progressive eye disease characterized by irreversible impairment of central vision, and unfortunately, an effective treatment is still not available. It is well-known that amyloid-beta (Aß) peptide is one of the major culprits in causing neurodegeneration in Alzheimer's disease (AD). The extracellular accumulation of this peptide has also been found in drusen which lies under the retinal pigment epithelium (RPE) and represents one of the early signs of AMD pathology. Aß aggregates, especially in the form of oligomers, are able to induce pro-oxidant (oxidative stress) and pro-inflammatory phenomena in RPE cells. ARPE-19 is a spontaneously arising human RPE cell line validated for drug discovery processes in AMD. In the present study, we employed ARPE-19 treated with Aß oligomers, representing an in vitro model of AMD. We used a combination of methods, including ATPlite, quantitative real-time PCR, immunocytochemistry, as well as a fluorescent probe for reactive oxygen species to investigate the molecular alterations induced by Aß oligomers. In particular, we found that Aß exposure decreased the cell viability of ARPE-19 cells which was paralleled by increased inflammation (increased expression of pro-inflammatory mediators) and oxidative stress (increased expression of NADPH oxidase and ROS production) along with the destruction of ZO-1 tight junction protein. Once the damage was clarified, we investigated the therapeutic potential of carnosine, an endogenous dipeptide that is known to be reduced in AMD patients. Our findings demonstrate that carnosine was able to counteract most of the molecular alterations induced by the challenge of ARPE-19 with Aß oligomers. These new findings obtained with ARPE-19 cells challenged with Aß1-42 oligomers, along with the well-demonstrated multimodal mechanism of action of carnosine both in vitro and in vivo, able to prevent and/or counteract the dysfunctions elicited by Aß oligomers, substantiate the neuroprotective potential of this dipeptide in the context of AMD pathology.

Characterization of Carnosine Effect on Human Microglial Cells under Basal Conditions.

The activity of microglia is fundamental for the regulation of numerous physiological processes including brain development, synaptic plasticity, and neurogenesis, and its deviation from homeostasis can lead to pathological conditions, including numerous neurodegenerative disorders. Carnosine is a naturally occurring molecule with well-characterized antioxidant and anti-inflammatory activities, able to modulate the response and polarization of immune cells and ameliorate their cellular energy metabolism. The better understanding of microglia characteristics under basal physiological conditions, as well as the possible modulation of the mechanisms related to its response to environmental challenges and/or pro-inflammatory/pro-oxidant stimuli, are of utmost importance for the development of therapeutic strategies. In the present study, we assessed the activity of carnosine on human HMC3 microglial cells, first investigating the effects of increasing concentrations of carnosine on cell viability. When used at a concentration of 20 mM, carnosine led to a decrease of cell viability, paralleled by gene expression increase and decrease, respectively, of interleukin 6 and heme oxygenase 1. When using the maximal non-toxic concentration (10 mM), carnosine decreased nitric oxide bioavailability, with no changes in the intracellular levels of superoxide ion. The characterization of energy metabolism of HMC3 microglial cells under basal conditions, never reported before, demonstrated that it is mainly based on mitochondrial oxidative metabolism, paralleled by a high rate of biosynthetic reactions. The exposure of HMC3 cells to carnosine seems to ameliorate microglia energy state, as indicated by the increase in the adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio and energy charge potential. The improvement of cell energy metabolism mediated by 10 mM carnosine could represent a useful protective weapon in the case of human microglia undergoing stressing conditions.

Prenatal stress induces a depressive-like phenotype in adolescent rats: The key role of TGF-ß1 pathway.

Stressful experiences early in life, especially in the prenatal period, can increase the risk to develop depression during adolescence. However, there may be important qualitative and quantitative differences in outcome of prenatal stress (PNS), where some individuals exposed to PNS are vulnerable and develop a depressive-like phenotype, while others appear to be resilient. PNS exposure, a well-established rat model of early life stress, is known to increase vulnerability to depression and a recent study demonstrated a strong interaction between transforming growth factor-ß1 (TGF-ß1) gene and PNS in the pathogenesis of depression. Moreover, it is well-known that the exposure to early life stress experiences induces brain oxidative damage by increasing nitric oxide levels and decreasing antioxidant factors. In the present work, we examined the role of TGF-ß1 pathway in an animal model of adolescent depression induced by PNS obtained by exposing pregnant females to a stressful condition during the last week of gestation. We performed behavioral tests to identify vulnerable or resilient subjects in the obtained litters (postnatal day, PND > 35) and we carried out molecular analyses on hippocampus, a brain area with a key role in the pathogenesis of depression. We found that female, but not male, PNS adolescent rats exhibited a depressive-like behavior in forced swim test (FST), whereas both male and female PNS rats showed a deficit of recognition memory as assessed by novel object recognition test (NOR). Interestingly, we found an increased expression of type 2 TGF-ß1 receptor (TGFß-R2) in the hippocampus of both male and female resilient PNS rats, with higher plasma TGF-ß1 levels in male, but not in female, PNS rats. Furthermore, PNS induced the activation of oxidative stress pathways by increasing inducible nitric oxide synthase (iNOS), NADPH oxidase 1 (NOX1) and NOX2 levels in the hippocampus of both male and female PNS adolescent rats. Our data suggest that high levels of TGF-ß1 and its receptor TGFß-R2 can significantly increase the resiliency of adolescent rats to PNS, suggesting that TGF-ß1 pathway might represent a novel pharmacological target to prevent adolescent depression in rats.

The Multimodal MOPr/DOPr Agonist LP2 Reduces Allodynia in Chronic Constriction Injured Rats by Rescue of TGF-ß1 Signalling.

Neuropathic pain is one of the most disabling forms of chronic pain and it is characterized by hyperalgesia and allodynia linked to an aberrant processing of pain transmission and to neuroinflammation. Transforming growth factor-ß1 (TGF-ß1) is an anti-inflammatory cytokine, which protects against neuroinflammation. It has been demonstrated that TGF-ß1 and opioid receptors signalling crosstalk results in an improvement of endogenous opioid analgesia, but it is not known whether mu opioid peptide receptor (MOPr) or delta opioid peptide receptor (DOPr) agonists can positively modulate TGF-ß1 pathway. In the present study, we examined the correlation between anti-allodynic effect of LP2, a dual-target MOPr/DOPr agonist, and TGF-ß1 signalling in the chronic constriction injury (CCI) model. We detected a significant decrease of active TGF-ß1 and of its type II receptor TGFß-R2 levels in the spinal cord from CCI rats and a selective deficit of TGF-ß1 in microglia cells both at days 11 and 21 post-ligature, as assessed by immunofluorescence analysis. LP2, when administered from the 11 days post-ligature to 21 days, was able to reduce CCI-induced mechanical allodynia by rescue of TGF-ß1 and TGFß-R2 levels. Our data suggest that the rescue of TGF-ß1 signalling by dual-target MOPr/DOPr agonist LP2 could be mediated by DOPr activation in spinal microglia, thus the dual-target approach could represent a novel pharmacological approach to increase the analgesic efficacy of MOPr agonists.

Carnosine Protects Macrophages against the Toxicity of Aß1-42 Oligomers by Decreasing Oxidative Stress.

Carnosine (ß-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 (Aß) peptides from the brain. Correspondingly, in patients suffering from AD, defects in the capacity of peripheral macrophages to engulf Aß 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 Aß1-42 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 Aß1-42 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 Aß1-42 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.

Antioxidant Activity of Fluoxetine and Vortioxetine in a Non-Transgenic Animal Model of Alzheimer's Disease.

Depression is a risk factor for the development of Alzheimer's disease (AD). A neurobiological and clinical continuum exists between AD and depression, with neuroinflammation and oxidative stress being involved in both diseases. Second-generation antidepressants, in particular selective serotonin reuptake inhibitors (SSRIs), are currently investigated as neuroprotective drugs in AD. By employing a non-transgenic AD model, obtained by intracerebroventricular (i.c.v.) injection of amyloid-ß (Aß) oligomers in 2-month-old C57BL/6 mice, we recently demonstrated that the SSRI fluoxetine (FLX) and the multimodal antidepressant vortioxetine (VTX) reversed the depressive-like phenotype and memory deficits induced by Aß oligomers rescuing the levels of transforming growth factor-ß1 (TGF-ß1). Aim of our study was to test FLX and VTX for their ability to prevent oxidative stress in the hippocampus of Aß-injected mice, a brain area strongly affected in both depression and AD. The long-term intraperitoneal (i.p.) administration of FLX (10 mg/kg) or VTX (5 and 10 mg/kg) for 24 days, starting 7 days before Aß injection, was able to prevent the over-expression of inducible nitric oxide synthase (iNOS) and NADPH oxidase 2 (Nox2) induced by Aß oligomers. Antidepressant pre-treatment was also able to rescue the mRNA expression of glutathione peroxidase 1 (Gpx1) antioxidant enzyme. FLX and VTX also prevented Aß-induced neurodegeneration in mixed neuronal cultures treated with Aß oligomers. Our data represent the first evidence that the long-term treatment with the antidepressants FLX or VTX can prevent the oxidative stress phenomena related to the cognitive deficits and depressive-like phenotype observed in a non-transgenic animal model of AD.

Antioxidant Properties of Second-Generation Antipsychotics: Focus on Microglia.

Recent studies suggest a primary role of oxidative stress in an early phase of the pathogenesis of schizophrenia and a strong neurobiological link has been found between dopaminergic system dysfunction, microglia overactivation, and oxidative stress. Different risk factors for schizophrenia increase oxidative stress phenomena raising the risk of developing psychosis. Oxidative stress induced by first-generation antipsychotics such as haloperidol significantly contributes to the development of extrapyramidal side effects. Haloperidol also exerts neurotoxic effects by decreasing antioxidant enzyme levels then worsening pro-oxidant events. Opposite to haloperidol, second-generation antipsychotics (or atypical antipsychotics) such as risperidone, clozapine, and olanzapine exert a strong antioxidant activity in experimental models of schizophrenia by rescuing the antioxidant system, with an increase in superoxide dismutase and glutathione (GSH) serum levels. Second-generation antipsychotics also improve the antioxidant status and reduce lipid peroxidation in schizophrenic patients. Interestingly, second-generation antipsychotics, such as risperidone, paliperidone, and in particular clozapine, reduce oxidative stress induced by microglia overactivation, decreasing the production of microglia-derived free radicals, finally protecting neurons against microglia-induced oxidative stress. Further, long-term clinical studies are needed to better understand the link between oxidative stress and the clinical response to antipsychotic drugs and the therapeutic potential of antioxidants to increase the response to antipsychotics.

Dihydrotanshinone, a Natural Diterpenoid, Preserves Blood-Retinal Barrier Integrity via P2X7 Receptor.

Activation of P2X7 signaling, due to high glucose levels, leads to blood retinal barrier (BRB) breakdown, which is a hallmark of diabetic retinopathy (DR). Furthermore, several studies report that high glucose (HG) conditions and the related activation of the P2X7 receptor (P2X7R) lead to the over-expression of pro-inflammatory markers. In order to identify novel P2X7R antagonists, we carried out virtual screening on a focused compound dataset, including indole derivatives and natural compounds such as caffeic acid phenethyl ester derivatives, flavonoids, and diterpenoids. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring and structural fingerprint clustering of docking poses from virtual screening highlighted that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567. A human-based in vitro BRB model made of retinal pericytes, astrocytes, and endothelial cells was used to assess the potential protective effect of DHTS against HG and 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP), a P2X7R agonist, insult. We found that HG/BzATP exposure generated BRB breakdown by enhancing barrier permeability (trans-endothelial electrical resistance (TEER)) and reducing the levels of ZO-1 and VE-cadherin junction proteins as well as of the Cx-43 mRNA expression levels. Furthermore, HG levels and P2X7R agonist treatment led to increased expression of pro-inflammatory mediators (TLR-4, IL-1ß, IL-6, TNF-α, and IL-8) and other molecular markers (P2X7R, VEGF-A, and ICAM-1), along with enhanced production of reactive oxygen species. Treatment with DHTS preserved the BRB integrity from HG/BzATP damage. The protective effects of DHTS were also compared to the validated P2X7R antagonist, JNJ47965567. In conclusion, we provided new findings pointing out the therapeutic potential of DHTS, which is an inhibitor of P2X7R, in terms of preventing and/or counteracting the BRB dysfunctions elicited by HG conditions.

A New Human Blood-Retinal Barrier Model Based on Endothelial Cells, Pericytes, and Astrocytes.

Blood-retinal barrier (BRB) dysfunction represents one of the most significant changes occurring during diabetic retinopathy. We set up a high-reproducible human-based in vitro BRB model using retinal pericytes, retinal astrocytes, and retinal endothelial cells in order to replicate the human in vivo environment with the same numerical ratio and layer order. Our findings showed that high glucose exposure elicited BRB breakdown, enhanced permeability, and reduced the levels of junction proteins such as ZO-1 and VE-cadherin. Furthermore, an increased expression of pro-inflammatory mediators (IL-1ß, IL-6) and oxidative stress-related enzymes (iNOS, Nox2) along with an increased production of reactive oxygen species were observed in our triple co-culture paradigm. Finally, we found an activation of immune response-regulating signaling pathways (Nrf2 and HO-1). In conclusion, the present model mimics the closest human in vivo milieu, providing a valuable tool to study the impact of high glucose in the retina and to develop novel molecules with potential effect on diabetic retinopathy.

Modulation of Pro-Oxidant and Pro-Inflammatory Activities of M1 Macrophages by the Natural Dipeptide Carnosine.

Carnosine is a natural endogenous dipeptide widely distributed in mammalian tissues, existing at particularly high concentrations in the muscles and brain and possesses well-characterized antioxidant and anti-inflammatory activities. In an in vitro model of macrophage activation, induced by lipopolysaccharide + interferon-gamma (LPS + IFN-γ), we here report the ability of carnosine to modulate pro-oxidant and pro-inflammatory activities of macrophages, representing the primary cell type that is activated as a part of the immune response. An ample set of parameters aimed to evaluate cytotoxicity (MTT assay), energy metabolism (HPLC), gene expressions (high-throughput real-time PCR (qRT-PCR)), protein expressions (western blot) and nitric oxide production (qRT-PCR and HPLC), was used to assess the effects of carnosine on activated macrophages challenged with a non cytotoxic LPS (100 ng/mL) + IFN-γ (600 U/mL) concentration. In our experimental model, main carnosine beneficial effects were: (1) the modulation of nitric oxide production and metabolism; (2) the amelioration of the macrophage energy state; (3) the decrease of the expressions of pro-oxidant enzymes (Nox-2, Cox-2) and of the lipid peroxidation product malondialdehyde; (4) the restoration and/or increase of the expressions of antioxidant enzymes (Gpx1, SOD-2 and Cat); (5) the increase of the transforming growth factor-ß1 (TGF-ß1) and the down-regulation of the expressions of interleukins 1ß and 6 (IL-1ß and IL-6) and 6) the increase of the expressions of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). According to these results carnosine is worth being tested in the treatment of diseases characterized by elevated levels of oxidative stress and inflammation (atherosclerosis, cancer, depression, metabolic syndrome, and neurodegenerative diseases).

Ocular Pharmacological Profile of Hydrocortisone in Dry Eye Disease.

To investigate the ocular pharmacological profile of hydrocortisone (HC) using in vitro and in vivo models of dry eye disease. Rabbit corneal epithelial cells (SIRCs) were used to assess the effect of HC in two paradigms of corneal damage: hyperosmotic stress and scratch-wound assay. Dry eye was induced in albino rabbits by topical administration of atropine sulfate or by injection of concanavalin A (ConA) into the lacrimal gland. TNFα, TNF-related apoptosis-inducing ligand (TRAIL), IL-1ß, and IL-8 were determined by ELISA or western blot in a corneal damage hyperosmotic in vitro model, with or without HC treatment. Inflammatory biomarkers, such as TNFα, IL-8, and MMP-9, were evaluated in tears of rabbit eye injected with ConA and treated with HC. Tear volume and tear film integrity, in both in vivo models, were evaluated by the Schirmer test and tear break-up time (TBUT). Ocular distribution of four formulations containing HC (0.001%, 0.003%, 0.005%, and 0.33%) was performed in the rabbit eye. Aqueous humor samples were collected after 15, 30, 60, and 90 min from instillation and then detected by LC-MS/MS. Hyperosmotic insult significantly activated protein expression of inflammatory biomarkers, which were significantly modulated by HC treatment. HC significantly enhanced the re-epithelialization of scratched SIRCs. Treatment with HC eye drops significantly reduced the tear concentrations of TNF-α, IL-8, and MMP-9 vs. vehicle in the ConA dry eye model. Moreover, HC significantly restored the tear volume and tear film integrity to levels of the control eyes, both in ConA- and atropine-induced dry eye paradigms. Finally, we demonstrated that HC crossed, in a dose-dependent manner, the corneal barrier when the eyes were topically treated with HC formulations (dose range 0.003-0.33%). No trace of HC was detected in the aqueous humor after ocular administration of eye drops containing the lowest dose of the drug (0.001%), indicating that, at this very low concentration, the drug did not pass the corneal barrier avoiding potential side effects such as intraocular pressure rise. Altogether, these data suggest that HC, at very low concentrations, has an important anti-inflammatory effect both in vitro and in vivo dry eye paradigms and a good safety profile.

Carnosine Decreases PMA-Induced Oxidative Stress and Inflammation in Murine Macrophages.

Carnosine is an endogenous dipeptide composed of ß-alanine and L-histidine. This naturally occurring molecule is present at high concentrations in several mammalian excitable tissues such as muscles and brain, while it can be found at low concentrations in a few invertebrates. Carnosine has been shown to be involved in different cellular defense mechanisms including the inhibition of protein cross-linking, reactive oxygen and nitrogen species detoxification as well as the counteraction of inflammation. As a part of the immune response, macrophages are the primary cell type that is activated. These cells play a crucial role in many diseases associated with oxidative stress and inflammation, including atherosclerosis, diabetes, and neurodegenerative diseases. In the present study, carnosine was first tested for its ability to counteract oxidative stress. In our experimental model, represented by RAW 264.7 macrophages challenged with phorbol 12-myristate 13-acetate (PMA) and superoxide dismutase (SOD) inhibitors, carnosine was able to decrease the intracellular concentration of superoxide anions (O2-•) as well as the expression of Nox1 and Nox2 enzyme genes. This carnosine antioxidant activity was accompanied by the attenuation of the PMA-induced Akt phosphorylation, the down-regulation of TNF-α and IL-6 mRNAs, and the up-regulation of the expression of the anti-inflammatory mediators IL-4, IL-10, and TGF-ß1. Additionally, when carnosine was used at the highest dose (20 mM), there was a generalized amelioration of the macrophage energy state, evaluated through the increase both in the total nucleoside triphosphate concentrations and the sum of the pool of intracellular nicotinic coenzymes. Finally, carnosine was able to decrease the oxidized (NADP+)/reduced (NADPH) ratio of nicotinamide adenine dinucleotide phosphate in a concentration dependent manner, indicating a strong inhibitory effect of this molecule towards the main source of reactive oxygen species in macrophages. Our data suggest a multimodal mechanism of action of carnosine underlying its beneficial effects on macrophage cells under oxidative stress and inflammation conditions.

Fluoxetine and Vortioxetine Reverse Depressive-Like Phenotype and Memory Deficits Induced by Aß1-42 Oligomers in Mice: A Key Role of Transforming Growth Factor-ß1.

Depression is a risk factor for the development of Alzheimer's disease (AD), and the presence of depressive symptoms significantly increases the conversion of mild cognitive impairment (MCI) into AD. A long-term treatment with antidepressants reduces the risk to develop AD, and different second-generation antidepressants such as selective serotonin reuptake inhibitors (SSRIs) are currently being studied for their neuroprotective properties in AD. In the present work, the SSRI fluoxetine and the new multimodal antidepressant vortioxetine were tested for their ability to prevent memory deficits and depressive-like phenotype induced by intracerebroventricular injection of amyloid-ß (1-42) (Aß1-42) oligomers in 2-month-old C57BL/6 mice. Starting from 7 days before Aß injection, fluoxetine (10 mg/kg) and vortioxetine (5 and 10 mg/kg) were intraperitoneally injected daily for 24 days. Chronic treatment with fluoxetine and vortioxetine (both at the dose of 10 mg/kg) was able to rescue the loss of memory assessed 14 days after Aß injection by the passive avoidance task and the object recognition test. Both antidepressants reversed the increase in immobility time detected 19 days after Aß injection by forced swim test. Vortioxetine exerted significant antidepressant effects also at the dose of 5 mg/kg. A significant deficit of transforming growth factor-ß1 (TGF-ß1), paralleling memory deficits and depressive-like phenotype, was found in the hippocampus of Aß-injected mice in combination with a significant reduction of the synaptic proteins synaptophysin and PSD-95. Fluoxetine and vortioxetine completely rescued hippocampal TGF-ß1 levels in Aß-injected mice as well as synaptophysin and PSD-95 levels. This is the first evidence that a chronic treatment with fluoxetine or vortioxetine can prevent both cognitive deficits and depressive-like phenotype in a non-transgenic animal model of AD with a key contribution of TGF-ß1.

Blood-retinal barrier protection against high glucose damage: The role of P2X7 receptor.

Blood retinal barrier (BRB) breakdown is a hallmark of diabetic retinopathy, whose occurrence in early or later phases of the disease has not yet been completely clarified. Recent evidence suggests that hyperglycemia induces activation of the P2X7 receptor (P2X7R) leading to pericyte cell death. We herein investigated the role of P2X7R on retinal endothelial cells viability and expression of tight- and adherens-junctions following high glucose (HG) exposure. We found that HG elicited P2X7R activation and expression and release of the pro-inflammatory cytokine IL-1ß in human retinal endothelial cells (HRECs). Furthermore, HG exposure caused a decrease in HRECs viability and a damage of the BRB. JNJ47965567, a P2X7R antagonist, protected HRECs from HG-induced damage (LDH release) and preserved the BRB, as shown by transendothelial electrical resistance and cell junction morphology (ZO-1, claudin-5 and VE-cadherin). Moreover, JNJ47965567 treatment significantly decreased IL-1ß expression and release, elicited by HG. These data indicate that P2X7R plays an important role to regulate BRB integrity, in particular the block of this receptor was useful to counteract the damage elicited by HG in HRECs, and warranting further clinical evaluation of P2X7R antagonists for the treatment of diabetic macular edema.

Aflibercept regulates retinal inflammation elicited by high glucose via the PlGF/ERK pathway.

Diabetic retinopathy (DR) is a secondary complication of diabetes. DR can cause irreversible blindness, and its pathogenesis is considered multifactorial. DR can progress from non-proliferative DR to proliferative DR, characterized by retinal neovascularization. The main cause of vision loss in diabetic patients is diabetic macular edema, caused by vessel leakage and blood retinal barrier breakdown. Currently, aflibercept is an anti-VEGF approved for diabetic macular edema. Aflibercept can bind several members of vascular permeability factors, namely VEGF-A, B, and PlGF. We analyzed the aflibercept-PlGF complex at molecular level, through an in silico approach. In order to explore the role of PlGF in DR, we treated primary human retinal endothelial cells (HRECs) and mouse retinal epithelial cells (RPEs) with aflibercept and an anti-PlGF antibody. We explored the hypothesis that aflibercept has anti-inflammatory action through blocking of PlGF signaling and the ERK axis in an in vitro and in vivo model of DR. Both aflibercept and the anti-PlGF antibody exerted protective effects on retinal cells, by inhibition of the ERK pathway. Moreover, aflibercept significantly decreased (p < 0.05) the expression of TNF-α in an in vitro and in vivo model of DR. Therefore, our data suggest that inhibition of PlGF signaling, or a selective blocking, may be useful in the management of early phases of DR when the inflammatory process is largely involved.

Novel ophthalmic formulation of myriocin: implications in retinitis pigmentosa.

Myriocin is an antibiotic derived from Mycelia sterilia, and is a potent inhibitor of serine palmitoyltransferase, the enzyme involved in the first step of sphingosine synthesis. Myriocin, inhibiting ceramide synthesis, has a great potential for treatment of diseases characterized by high ceramide levels in affected tissues, such as retinitis pigmentosa (RP). Drug delivery to the retina is a challenging task, which is generally by-passed through intravitreal injection, that represents a risky invasive procedure. We, therefore, developed and characterized an ophthalmic topical nanotechnological formulation based on a nanostructured lipid carrier (NLC) and containing myriocin. The ocular distribution of myriocin in the back of the eye was assessed both in rabbits and mice using LC-MS/MS. Moreover, rabbit retinal sphingolipid and ceramides levels, after myriocin-NLC (Myr-NLC) eye drops treatment, were assessed. The results demonstrated that Myr-NLC formulation is well tolerated and provided effective levels of myriocin in the back of the eye both in rabbits and mice. We found that Myr-NLC eye drops treatment was able to significantly decrease retinal sphingolipid levels. In conclusion, these data suggest that the Myr-NLC ophthalmic formulation is suitable for pharmaceutical development and warrants further clinical evaluation of this eye drops for the treatment of RP.