Daniele De Filippis

The spice sage and its active ingredient rosmarinic acid protect PC12 cells from amyloid-β peptide-induced neurotoxicity

Traditional use and clinical reports suggest that the culinary herb sage (Salvia officinalis) may be effective for patients with mild to moderate Alzheimers disease (AD). In this study, we evaluated the effect of a standardized extract from the leaves of S. officinalis (SOE) and its active ingredient rosmarinic acid on Alzheimer amyloid-β peptide (Aβ)-induced toxicity in cultured rat pheochromocytoma (PC12) cells. Incubation of PC12 cells with Aβ (fragment 1–42) for 24 h caused cell death, and this effect was reduced by SOE and its active ingredient, rosmarinic acid. Rosmarinic acid reduced a number of events induced by Aβ. These include reactive oxygen species formation, lipid peroxidation, DNA fragmentation, caspase-3 activation, and tau protein hyperphosphorylation. Moreover, rosmarinic acid inhibited phosphorylated p38 mitogen-activated protein kinase but not glycogen synthase kinase 3β activation. These data show the neuroprotective effect of sage against Aβ-induced toxicity, which could validate the traditional use of this spice in the treatment of AD. Rosmarinic acid could contribute, at least in part, for sage-induced neuroprotective effect.

Cannabidiol inhibits inducible nitric oxide synthase protein expression and nitric oxide production in β-amyloid stimulated PC12 neurons through p38 MAP kinase and NF-κB involvement

In view of the pro-inflammatory scenario observed in Alzheimers disease, in the recent years anti-inflammatory drugs have been proposed as potential therapeutic agents. We have previously shown that cannabidiol, the main non-psychotropic component from Cannabis sativa, possess a variegate combination of anti-oxidant and anti-apoptotic effects that protect PC12 cells from Abeta toxicity. In parallel, cannabidiol has been described to have anti-inflammatory properties in acute models of inflammation but the possible inhibitory effect of cannabidiol on iNOS protein expression and nitrite production in the nitrosative stress induced by Abeta in neuronal cell-line is un-investigated. Stimulation of differentiated PC12 cells with Abeta (1-42) (1 microg/mL) for 36 h caused a significant increase of nitrite production, compared to un-stimulated cells, that was inhibited in a concentration-dependent manner by both the non-selective iNOS inhibitor, L-NAME (0.3-30 microM), and, at higher extent, by the selective iNOS inhibitor SMT (0.3-30 microM). CBD (10(-6) to 10(-4) M) inhibited both nitrite production and iNOS protein expression induced by Abeta (1-42). Cannabidiol effect was mediated through the inhibition of phosphorylated form of p38 MAP kinase and transcription factor nuclear factor-kappaB activation in a concentration-dependent manner. The here reported data increases our knowledge about the possible neuroprotective mechanism of cannabidiol, highlighting the importance of this compound to inhibit beta-amyloid induced neurodegeneration, in view of its low toxicity in humans.

The marijuana component cannabidiol inhibits β-amyloid-induced tau protein hyperphosphorylation through Wnt/β-catenin pathway rescue in PC12 cells

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. A massive accumulation of β-amyloid (Aβ) peptide aggregates has been proposed as pivotal event in AD. Aβ-induced toxicity is accompanied by a variegated combination of events including oxidative stress. The Wnt pathway has multiple actions in the cascade of events triggered by Aβ, and drugs that rescue Wnt activity may be considered as novel therapeutics for AD treatment. Cannabidiol, a non-psychoactive marijuana component, has been recently proposed as an antioxidant neuroprotective agent in neurodegenerative diseases. Moreover, it has been shown to rescue PC12 cells from toxicity induced by Aβ peptide. However, the molecular mechanism of cannabidiol-induced neuroprotective effect is still unknown. Here, we report that cannabidiol inhibits hyperphosphorylation of tau protein in Aβ-stimulated PC12 neuronal cells, which is one of the most representative hallmarks in AD. The effect of cannabidiol is mediated through the Wnt/β-catenin pathway rescue in Aβ-stimulated PC12 cells. These results provide new molecular insight regarding the neuroprotective effect of cannabidiol and suggest its possible role in the pharmacological management of AD, especially in view of its low toxicity in humans.

Cannabidiol: A Promising Drug for Neurodegenerative Disorders?

Neurodegenerative diseases represent, nowadays, one of the main causes of death in the industrialized country. They are characterized by a loss of neurons in particular regions of the nervous system. It is believed that this nerve cell loss underlies the subsequent decline in cognitive and motor function that patients experience in these diseases. A range of mutant genes and environmental toxins have been implicated in the cause of neurodegenerative disorders but the mechanism remains largely unknown. At present, inflammation, a common denominator among the diverse list of neurodegenerative diseases, has been implicated as a critical mechanism that is responsible for the progressive nature of neurodegeneration. Since, at present, there are few therapies for the wide range of neurodegenerative diseases, scientists are still in search of new therapeutic approaches to the problem. An early contribution of neuroprotective and antiinflammatory strategies for these disorders seems particularly desirable because isolated treatments cannot be effective. In this contest, marijuana derivatives have attracted special interest, although these compounds have always raised several practical and ethical problems for their potential abuse. Nevertheless, among Cannabis compounds, cannabidiol (CBD), which lacks any unwanted psychotropic effect, may represent a very promising agent with the highest prospect for therapeutic use.

Cannabidiol reduces Aβ-induced neuroinflammation and promotes hippocampal neurogenesis through PPARγ involvement

Peroxisome proliferator-activated receptor-γ (PPARγ) has been reported to be involved in the etiology of pathological features of Alzheimers disease (AD). Cannabidiol (CBD), a Cannabis derivative devoid of psychomimetic effects, has attracted much attention because of its promising neuroprotective properties in rat AD models, even though the mechanism responsible for such actions remains unknown. This study was aimed at exploring whether CBD effects could be subordinate to its activity at PPARγ, which has been recently indicated as its putative binding site. CBD actions on β-amyloid-induced neurotoxicity in rat AD models, either in presence or absence of PPAR antagonists were investigated. Results showed that the blockade of PPARγ was able to significantly blunt CBD effects on reactive gliosis and subsequently on neuronal damage. Moreover, due to its interaction at PPARγ, CBD was observed to stimulate hippocampal neurogenesis. All these findings report the inescapable role of this receptor in mediating CBD actions, here reported.

Cannabidiol in Medicine : A Review of its Therapeutic Potential in CNS Disorders

Cannabidiol (CBD) is the main non‐psychotropic component of the glandular hairs of Cannabis sativa. It displays a plethora of actions including anticonvulsive, sedative, hypnotic, antipsychotic, antiinflammatory and neuroprotective properties. However, it is well established that CBD produces its biological effects without exerting significant intrinsic activity upon cannabinoid receptors. For this reason, CBD lacks the unwanted psychotropic effects characteristic of marijuana derivatives, so representing one of the bioactive constituents of Cannabis sativa with the highest potential for therapeutic use.

S100B induces tau protein hyperphosphorylation via Dickopff‐1 up‐regulation and disrupts the Wnt pathway in human neural stem cells

Previous studies suggest that levels of the astrocyte‐derived S100B protein, such as those occurring in brain extra‐cellular spaces consequent to persistent astroglial activation, may have a pathogenetic role in Alzheimers disease (AD). Although S100B was reported to promote β amyloid precursor protein overexpression, no clear mechanistic relationship between S100B and formation of neurofibrillary tangles (NFTs) is established. This in vitro study has been aimed at investigating whether S100B is able to disrupt Wnt pathway and lead to tau protein hyperphosphorylation. Utilizing Western blot, electrophoretic mobility shift assay, supershift and reverse transcriptase‐polymerase chain reaction techniques, it has been demonstrated that micromolar S100B concentrations stimulate c‐Jun N‐terminal kinase (JNK) phosphorylation through the receptor for advanced glycation ending products, and subsequently activate nuclear AP‐1/cJun transcription, in cultured human neural stem cells. In addition, as revealed by Western blot, small interfering RNA and immunofluorescence analysis, S100B‐induced JNK activation increased expression of Dickopff‐1 that, in turn, promoted glycogen synthase kinase 3β phosphorylation and β‐catenin degradation, causing canonical Wnt pathway disruption and tau protein hyperphosphorylation. These findings propose a previously unrecognized link between S100B and tau hyperphosphorylation, suggesting S100B can contribute to NFT formation in AD and in all other conditions in which neuroinflammation may have a crucial role.

Opposing Control of Cannabinoid Receptor Stimulation on Amyloid-β-Induced Reactive Gliosis: In Vitro and in Vivo Evidence

Beside cytotoxic mechanisms impacting on neurons, amyloid β (Aβ)-induced astroglial activation is operative in Alzheimers disease brain, suggesting that persistent inflammatory response may have a role in the illness and that positive results may be achieved by curbing the astroglial reaction. Because the role of the endocannabinoid system could represent a promising field of research, the present study conducted in vitro and in vivo experiments to assess this system. C6 rat astroglioma cells were challenged with 1 μg/ml Aβ 1-42 in the presence or absence of selective agonists and antagonists of cannabinoid (CB)1 and CB2 receptors. Furthermore, rats were inoculated into the frontal cortex with 30 ng of Aβ 1-42 and were i.p. administered with 5 mg/kg of the same substances. Immunohistochemical and biochemical findings revealed that selective agonism at CB1 and antagonism at CB2 receptors was able to blunt Aβ-induced reactive astrogliosis with subsequent overexpression of glial fibrillary acidic protein and S100B protein. Moreover, Aβ provoked down-regulation of CB1 receptors together with a reduction of anandamide concentration, whereas CB2 receptors were up-regulated and 2-arachidonoyl glycerol concentration was increased. Finally, to our knowledge, the current study is the first showing that interactions at cannabinoid receptors result in a dual regulation of Aβ-induced reactive astrogliosis. The data support the assumption that compounds able to selectively block CB2 receptors may have therapeutic potential in controlling Aβ-related pathology, due to their beneficial effects devoid of psychotropic consequences.

Palmitoylethanolamide counteracts reactive astrogliosis induced by β-amyloid peptide

Emerging evidence indicates that astrogliosis is involved in the pathogenesis of neurodegenerative disorders. Our previous findings suggested cannabinoids and Autacoid Local Injury Antagonism Amides (ALIAmides) attenuate glial response in models of neurodegeneration. The present study was aimed at exploring palmitoylethanolamide (PEA) ability to mitigate β‐amyloid (Aβ)‐induced astrogliosis. Experiments were carried out to investigate PEA’s (10−7M) effects upon the expression and release of pro‐inflammatory molecules in rat primary astrocytes activated by soluble Aβ1–42 (1 μg/ml) as well as to identify mechanisms responsible for such actions. The effects of Aβ and exogenous PEA on the astrocyte levels of the endocannabinoidsand of endogenous ALIAmides were also studied. The peroxisome proliferator‐activated receptor (PPAR)‐α (MK886, 3 μM) or PPAR‐γ (GW9662, 9 nM) antagonists were co‐administered with PEA. Aβ elevated endogenous PEA and d5–2‐arachidonoylglycerol (2‐AG) levels. Exogenous PEA blunted the Aβ‐induced expression of pro‐inflammatory molecules. This effect was reduced by PPAR‐α antagonist. Moreover, this ALIAmide, like Aβ, increased 2‐AG levels. These results indicate that PEA exhibits anti‐inflammatory properties able to counteract Aβ‐induced astrogliosis, and suggest novel treatment for neuroinflammatory/ neurodegenerative processes.

Cannabidiol Reduces Intestinal Inflammation through the Control of Neuroimmune Axis

Enteric glial cells (EGC) actively mediate acute and chronic inflammation in the gut; EGC proliferate and release neurotrophins, growth factors, and pro-inflammatory cytokines which, in turn, may amplify the immune response, representing a very important link between the nervous and immune systems in the intestine. Cannabidiol (CBD) is an interesting compound because of its ability to control reactive gliosis in the CNS, without any unwanted psychotropic effects. Therefore the rationale of our study was to investigate the effect of CBD on intestinal biopsies from patients with ulcerative colitis (UC) and from intestinal segments of mice with LPS-induced intestinal inflammation. CBD markedly counteracted reactive enteric gliosis in LPS-mice trough the massive reduction of astroglial signalling neurotrophin S100B. Histological, biochemical and immunohistochemical data demonstrated that S100B decrease was associated with a considerable decrease in mast cell and macrophages in the intestine of LPS-treated mice after CBD treatment. Moreover the treatment of LPS-mice with CBD reduced TNF-α expression and the presence of cleaved caspase-3. Similar results were obtained in ex vivo cultured human derived colonic biopsies. In biopsies of UC patients, both during active inflammation and in remission stimulated with LPS+INF-γ, an increased glial cell activation and intestinal damage were evidenced. CBD reduced the expression of S100B and iNOS proteins in the human biopsies confirming its well documented effect in septic mice. The activity of CBD is, at least partly, mediated via the selective PPAR-gamma receptor pathway. CBD targets enteric reactive gliosis, counteracts the inflammatory environment induced by LPS in mice and in human colonic cultures derived from UC patients. These actions lead to a reduction of intestinal damage mediated by PPARgamma receptor pathway. Our results therefore indicate that CBD indeed unravels a new therapeutic strategy to treat inflammatory bowel diseases.