Untargeted Metabolomics in Forensic Toxicology: A New Approach for the Detection of Fentanyl Intake in Urine Samples.

The misuse of fentanyl, and novel synthetic opioids (NSO) in general, has become a public health emergency, especially in the United States. The detection of NSO is often challenged by the limited diagnostic time frame allowed by urine sampling and the wide range of chemically modified analogues, continuously introduced to the recreational drug market. In this study, an untargeted metabolomics approach was developed to obtain a comprehensive "fingerprint" of any anomalous and specific metabolic pattern potentially related to fentanyl exposure. In recent years, in vitro models of drug metabolism have emerged as important tools to overcome the limited access to positive urine samples and uncertainties related to the substances actually taken, the possible combined drug intake, and the ingested dose. In this study, an in vivo experiment was designed by incubating HepG2 cell lines with either fentanyl or common drugs of abuse, creating a cohort of 96 samples. These samples, together with 81 urine samples including negative controls and positive samples obtained from recent users of either fentanyl or "traditional" drugs, were subjected to untargeted analysis using both UHPLC reverse phase and HILIC chromatography combined with QTOF mass spectrometry. Data independent acquisition was performed by SWATH in order to obtain a comprehensive profile of the urinary metabolome. After extensive processing, the resulting datasets were initially subjected to unsupervised exploration by principal component analysis (PCA), yielding clear separation of the fentanyl positive samples with respect to both controls and samples positive to other drugs. The urine datasets were then systematically investigated by supervised classification models based on soft independent modeling by class analogy (SIMCA) algorithms, with the end goal of identifying fentanyl users. A final single-class SIMCA model based on an RP dataset and five PCs yielded 96% sensitivity and 74% specificity. The distinguishable metabolic patterns produced by fentanyl in comparison to other opioids opens up new perspectives in the interpretation of the biological activity of fentanyl.

Sleep fragmentation affects glymphatic system through the different expression of AQP4 in wild type and 5xFAD mouse models.

Alzheimer's disease (AD) is characterized by genetic and multifactorial risk factors. Many studies correlate AD to sleep disorders. In this study, we performed and validated a mouse model of AD and sleep fragmentation, which properly mimics a real condition of intermittent awakening. We noticed that sleep fragmentation induces a general acceleration of AD progression in 5xFAD mice, while in wild type mice it affects cognitive behaviors in particular learning and memory. Both these events may be correlated to aquaporin-4 (AQP4) modulation, a crucial player of the glymphatic system activity. In particular, sleep fragmentation differentially affects aquaporin-4 channel (AQP4) expression according to the stage of the disease, with an up-regulation in younger animals, while such change cannot be detected in older ones. Moreover, in wild type mice sleep fragmentation affects cognitive behaviors, in particular learning and memory, by compromising the glymphatic system through the decrease of AQP4. Nevertheless, an in-depth study is needed to better understand the mechanism by which AQP4 is modulated and whether it could be considered a risk factor for the disease development in wild type mice. If our hypotheses are going to be confirmed, AQP4 modulation may represent the convergence point between AD and sleep disorder pathogenic mechanisms.

ß-amyloid 1-42 induces physiological transcriptional regulation of BACE1.

The pathogenesis of Alzheimer's disease (AD) is only partially understood. ß-amyloid (Aß) is physiologically generated by sequential cleavage of its precursor protein by the ß- and the γ-secretase and it is normally disposed of. In Alzheimer's disease, Aß is excessively produced or less dismissed, but the hypothesis on its physiological and pathological role are heterogeneous and often discordant. It has been described a positive feedback loop from the γ- to the ß-secretase cleavages of Aß precursor protein, which is activated by mutations of Presenilin 1 (PS1), the catalytic core of the γ-secretase. These findings show that Aß precursor protein as well the activity of the γ-secretase are required to obtain the up-regulation of ß-secretase which is induced by Presenilin 1 mutations. Then, Aß 1-42 is the Aß precursor protein derivative that up-regulates the expression of ß-secretase, and c-jun N-terminal kinase (JNK)/c-Jun and ERK1/2 are involved. Here, we describe the activation of ß-secretase and c-jun N-terminal kinase related proteins by monomeric Aß 1-42, defining the conditions that most efficiently strike the described signaling without producing toxicity. Taken together these data imply that monomeric Aß 1-42, at non-toxic concentrations and time frames, are able to induce a signaling pathway that leads to transcriptional activation of ß-secretase.

Transcriptional and post-transcriptional regulation of ß-secretase.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder that results in loss of memory and cognitive function, eventually leading to dementia. A key neuropathological event in AD is the cerebral accumulation of senile plaques formed by aggregates of amyloid-ß-peptides (Aß). Aß results from two sequential endoproteolytic cleavages operated on the amyloid-ß precursor protein (AßPP), an integral membrane protein with a single-membrane spanning domain, a large extracellular N-terminus and a shorter, cytoplasmic C-terminus. First, ß-secretase (BACE1) cleaves AßPP at the N-terminal end of the Aß sequence to produce a secreted form of AßPP, named sAßPP, and a C-terminal membrane-bound 99-aminoacid fragment (C99). Then, γ-secretase cleaves C99 within the transmembrane domain to release the Aß peptides of different lengths, predominantly Aß1-40 and Aß1-42.

Dysregulation of SREBP2 induces BACE1 expression.

ß-Amyloid hyperproduction has been observed in response to alterations in neuronal intracellular cholesterol storage, efflux, and synthesis, induced in rats by a high-fat diet. It has been suggested that cholesterol homeostasis is altered in Alzheimer's disease resulting in higher ß- and γ-secretase activity. In the current study the neuronal activation status of sterol regulatory element binding protein 2 (SREBP2) as well as its involvement in ß-secretase BACE1 activity was investigated in high-fat fed rats (26% fat and 4% cholesterol for 20 weeks), and in SK-N-BE neuroblastoma cells exposed to 20 µM cholesterol. This work demonstrates that in the brain a hyperlipidic diet is able to induce a hyper-expression of BACE1 and determine an unbalance in cerebral cholesterol homeostasis so that SREBP2 is activated. In addition, we show for the first time the involvement of SREBP2 on expression of BACE1 in SK-N-BE cells exposed to high cholesterol. Although the enhanced risk of Alzheimer's disease in metabolic syndrome is related to several factors, our results suggest that SREBP2, which can be modulated by the impairment of cerebral cholesterol homeostasis, has a direct role on BACE1 expression and may be involved in Alzheimer's disease progression.

Intracellular reactive oxygen species are required for directional migration of resident and bone marrow-derived hepatic pro-fibrogenic cells.

BACKGROUND & AIMS: Liver fibrogenesis is sustained by myofibroblast-like cells originating from hepatic stellate cells (HSC/MFs), portal fibroblasts or bone marrow-derived cells, including mesenchymal stem cells (MSCs). Herein, we investigated the mechanistic role of intracellular generation of reactive oxygen species (ROS) and redox-sensitive signal transduction pathways in mediating chemotaxis, a critical profibrogenic response for human HSC/MFs and for MSC potentially engrafting chronically injured liver. METHODS: Intracellular generation of ROS and signal transduction pathways were evaluated by integrating morphological and molecular biology techniques. Chemokinesis and chemotaxis were evaluated by wound healing assay and modified Boyden's chamber assay, respectively. Additional in vivo evidence was obtained in human specimens from HCV-related cirrhosis. RESULTS: Human MSCs and HSC/MFs migrate in response to a panel of polypeptide chemoattractants and extracellularly generated superoxide anion. All polypeptides induced a NADPH-oxidase-dependent intracellular rise in ROS, resulting in activation of ERK1/2 and JNK1/2. Moreover, menadione or 2,3-dimethoxy-1,4-naphthoquinone, which generate intracellular superoxide anion or hydrogen peroxide, respectively, induced ERK1/2 and JNK1/2 activation and migration. JNK1 activation was predominant for migration as shown by specific silencing. Finally, activation of ERK1/2 and JNK1/2 was found in extracts obtained from HSC/MFs during the course of an oxidative stress-mediated model of liver injury and phosphorylated JNK1/2 isoforms were detected in α-smooth muscle actin-positive myofibroblasts lining fibrotic septa in human cirrhotic livers. CONCLUSIONS: Intracellular generation of ROS, through activation of specific signaling pathways, is a critical event for directional migration of HSC/MFs and MSCs.

Oxidative Stress and Beta Amyloid in Alzheimer's Disease. Which Comes First: The Chicken or the Egg?

The pathogenesis of Alzheimer's disease involves ß amyloid (Aß) accumulation known to induce synaptic dysfunction and neurodegeneration. The brain's vulnerability to oxidative stress (OS) is considered a crucial detrimental factor in Alzheimer's disease. OS and Aß are linked to each other because Aß induces OS, and OS increases the Aß deposition. Thus, the answer to the question "which comes first: the chicken or the egg?" remains extremely difficult. In any case, the evidence for the primary occurrence of oxidative stress in AD is attractive. Thus, evidence indicates that a long period of gradual oxidative damage accumulation precedes and results in the appearance of clinical and pathological AD symptoms, including Aß deposition, neurofibrillary tangle formation, metabolic dysfunction, and cognitive decline. Moreover, oxidative stress plays a crucial role in the pathogenesis of many risk factors for AD. Alzheimer's disease begins many years before its symptoms, and antioxidant treatment can be an important therapeutic target for attacking the disease.

Estrogens Inhibit Amyloid-ß-Mediated Paired Helical Filament-Like Conformation of Tau Through Antioxidant Activity and miRNA 218 Regulation in hTau Mice.

BACKGROUND: The risk of developing Alzheimer's disease as well as its progression and severity are known to be different in men and women, and cognitive decline is greater in women than in men at the same stage of disease and could be correlated at least in part on estradiol levels. OBJECTIVE: In our work we found that biological sex influences the effect of amyloid-ß42 (Aß42) monomers on pathological tau conformational change. METHODS: In this study we used transgenic mice expressing the wild-type human tau (hTau) which were subjected to intraventricular (ICV) injections of Aß peptides in nanomolar concentration. RESULTS: We found that Aß42 produces pathological conformational changes and hyperphosphorylation of tau protein in male or ovariectomized female mice but not in control females. The treatment of ovariectomized females with estradiol replacement protects against the pathological conformation of tau and seems to be mediated by antioxidant activity as well as the ability to modulate the expression of miRNA 218 linked to tau phosphorylation. CONCLUSION: Our study indicates that factors as age, reproductive stage, hormone levels, and the interplay with other risk factors should be considered in women, in order to identify the best appropriate therapeutic approach in prevention of cognitive impairment.

The up-regulation of BACE1 mediated by hypoxia and ischemic injury: role of oxidative stress and HIF1alpha.

While it is well established that stroke and cerebral hypoperfusion are both significant risk factors for Alzheimer's disease, the molecular link between ischemia and amyloid precursor protein processing has only been recently established. Specifically, hypoxia significantly increases beta-site APP cleaving enzyme (BACE1) gene transcription through the over-expression of hypoxia inducible factor 1alpha, resulting in increased BACE1 secretase activity and amyloid-beta production. In this study, we significantly extend these findings both in vitro, in differentiated SK-N-BE neuroblastoma cells, and in vivo, in rats subjected to cerebral ischemia, showing that hypoxia up-regulates BACE1 expression through a biphasic mechanism. The early post-hypoxic up-regulation of BACE1 depends on the production of reactive oxygen species mediated by the sudden interruption of the mitochondrial electron transport chain, while the later expression of BACE1 is caused by hypoxia inducible factor 1alpha activation. The involvement of reactive oxygen species released by mitochondria in the BACE1 up-regulation was confirmed by the complete protection exerted by complex I inhibitors such as rotenone and diphenyl-phenylen iodonium. Moreover, the oxidative stress-mediated up-regulation of BACE1 is mediated by c-jun N terminal kinase pathway as demonstrated by the protection exerted by the silencing of c-jun N-terminal kinase isoforms 1 and 2. Our study strengthens the hypothesis that oxidative stress is a basic common mechanism of amyloid-beta accumulation.

Oxidative stress and hypoxia contribute to Alzheimer's disease pathogenesis: two sides of the same coin.

While it is well established that stroke and cerebral hypoperfusion are risk factors for Alzheimer's disease (AD), the molecular link between ischemia/hypoxia and amyloid precursor protein (APP) processing has only been recently established. Here we review the role of the release of reactive oxygen species (ROS) by the mitochondrial electron chain in response to hypoxia, providing evidence that hypoxia fosters the amyloidogenic APP processing through a biphasic mechanism that up-regulates Beta-secretase activity, which involves an early release of ROS and an activation of HIF-1Alpha.

Stroke and Amyloid-ß Downregulate TREM-2 and Uch-L1 Expression that Synergistically Promote the Inflammatory Response.

Neuroinflammation is involved in the pathogenesis of Alzheimer's disease, and the transcription factor NF-κB is a player in this event. We found here that the ischemic damage alone or in association with Aß1-42 activates the NF-κB pathway, induces an increase of BACE1 and a parallel inhibition of Uch-L1 and TREM2, both in vitro and in vivo, in Tg 5XFAD and in human brains of sporadic AD. This mechanism creates a synergistic loop that fosters inflammation. We also demonstrated a significant protection exerted by the restoration of Uch-L1 activity. The rescue of the enzyme is able to abolish the decrease of TREM2 and the parameters of neuroinflammation.

Oxidative stress activates a positive feedback between the gamma- and beta-secretase cleavages of the beta-amyloid precursor protein.

Sequential cleavages of the beta-amyloid precursor protein cleaving enzyme 1 (BACE1) by beta-secretase and gamma-secretase generate the amyloid beta-peptides, believed to be responsible of synaptic dysfunction and neuronal cell death in Alzheimer's disease (AD). Levels of BACE1 are increased in vulnerable regions of the AD brain, but the underlying mechanism is unknown. Here we show that oxidative stress (OS) stimulates BACE1 expression by a mechanism requiring gamma-secretase activity involving the c-jun N-terminal kinase (JNK)/c-jun pathway. BACE1 levels are increased in response to OS in normal cells, but not in cells lacking presenilins or amyloid precursor protein. Moreover, BACE1 is induced in association with OS in the brains of mice subjected to cerebral ischaemia/reperfusion. The OS-induced BACE1 expression correlates with an activation of JNK and c-jun, but is absent in cultured cells or mice lacking JNK. Our findings suggest a mechanism by which OS induces BACE1 transcription, thereby promoting production of pathological levels of amyloid beta in AD.

The Unexpected Role of Aß1-42 Monomers in the Pathogenesis of Alzheimer's Disease.

Amyloid-ß (Aß) has been proposed as a biomarker and a drug target for the therapy of Alzheimer's disease (AD). The neurotoxic entity and relevance of each conformational form of Aß to AD pathology is still under debate; Aß oligomers are considered the major killer form of the peptide whereas monomers have been proposed to be involved in physiological process. Here we reviewed some different effects mediated by monomers and oligomers on mechanisms involved in AD pathogenesis such as autophagy and tau aggregation. Data reported in this review demonstrate that Aß monomers could have a major role in sustaining the pathogenesis of AD and that AD therapy should be focused not only in the removal of oligomers but also of monomers.

A silver lining for 24-hydroxycholesterol in Alzheimer's disease: The involvement of the neuroprotective enzyme sirtuin 1.

It is now established that cholesterol oxidation products (oxysterols) are involved in several events underlying Alzheimer's disease (AD) pathogenesis. Of note, certain oxysterols cause neuron dysfunction and degeneration but, recently, some of them have been shown also to have neuroprotective effects. The present study, which aimed to understand the potential effects of 24-hydroxycholesterol (24-OH) against the intraneuronal accumulation of hyperphosphorylated tau protein, stressed these latter effects. A beneficial effect of 24-OH was demonstrated in SK-N-BE neuroblastoma cells, and is due to its ability to modulate the deacetylase sirtuin 1 (SIRT1), which contributes to preventing the neurotoxic accumulation of the hyperphosphorylated tau protein. Unlike 24-OH, 7-ketocholesterol (7-K) did not modulate the SIRT1-dependent neuroprotective pathway. To confirm the neuroprotective role of 24-OH, in vivo experiments were run on mice that express human tau without spontaneously developing tau pathology (hTau mice), by means of the intracerebroventricular injection of 24-OH. 24-OH, unlike 7-K, was found to completely prevent the hyperphosphorylation of tau induced by amyloid ß monomers. These data highlight the importance of preventing the loss of 24-OH in the brain, and of maintaining high levels of the enzyme SIRT1, in order to counteract neurodegeneration.

Dual Mechanism of Toxicity for Extracellular Injection of Tau Oligomers versus Monomers in Human Tau Mice.

The mechanism of tau toxicity is still unclear. Here we report that recombinant tau oligomers and monomers, intraventricularly injected in mice with a pure human tau background, foster tau pathology through different mechanisms. Oligomeric forms of tau alter the conformation of tau in a paired helical filament-like manner. This effect occurs without tau hyperphosphorylation as well as activation of specific kinases, suggesting that oligomers of tau induce tau assembly through a nucleation effect. Monomers, in turn, induce neurodegeneration through a calpain-mediated tau cleavage that leads to accumulation of a 17 kDa neurotoxic peptide and induction of apoptotic cell death.

The Decrease of Uch-L1 Activity Is a Common Mechanism Responsible for Aß 42 Accumulation in Alzheimer's and Vascular Disease.

Alzheimer's disease (AD) is a multifactorial pathology causing common brain spectrum disorders in affected patients. These mixed neurological disorders not only include structural AD brain changes but also cerebrovascular lesions. The main aim of the present issue is to find the factors shared by the two pathologies. The decrease of ubiquitin C-terminal hydrolase L1 (Uch-L1), a major neuronal enzyme involved in the elimination of misfolded proteins, was observed in ischemic injury as well as in AD, but its role in the pathogenesis of AD is far to be clear. In this study we demonstrated that Uch-L1 inhibition induces BACE1 up-regulation and increases neuronal and apoptotic cell death in control as well as in transgenic AD mouse model subjected to Bengal Rose, a light-sensitive dye inducing that induces a cortical infarction through photo-activation. Under the same conditions we also found a significant activation of NF-κB. Thus, the restoration of Uch-L1 was able to completely prevent both the increase in BACE1 protein levels and the amount of cell death. Our data suggest that the Uch-L1-mediated BACE1 up-regulation could be an important mechanism responsible for Aß peptides accumulation in vascular injury and indicate that the modulation of the activity of this enzyme could provide new therapeutic strategies in AD.

Inhibition of autophagy delays motoneuron degeneration and extends lifespan in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous mutations or deletions in the telomeric survival motoneuron gene 1 (SMN1). SMA is characterized by motor impairment, muscle atrophy, and premature death following motor neuron (MN) degeneration. Emerging evidence suggests that dysregulation of autophagy contributes to MN degeneration. We here investigated the role of autophagy in the SMNdelta7 mouse model of SMA II (intermediate form of the disease) which leads to motor impairment by postnatal day 5 (P5) and to death by P13. We first showed by immunoblots that Beclin 1 and LC3-II expression levels increased in the lumbar spinal cord of the SMA pups. Electron microscopy and immunofluorescence studies confirmed that autophagic markers were enhanced in the ventral horn of SMA pups. To clarify the role of autophagy, we administered intracerebroventricularly (at P3) either an autophagy inhibitor (3-methyladenine, 3-MA), or an autophagy inducer (rapamycin) in SMA pups. Motor behavior was assessed daily with different tests: tail suspension, righting reflex, and hindlimb suspension tests. 3-MA significantly improved motor performance, extended the lifespan, and delayed MN death in lumbar spinal cord (10372.36 ± 2716 MNs) compared to control-group (5148.38 ± 94 MNs). Inhibition of autophagy by 3-MA suppressed autophagosome formation, reduced the apoptotic activation (cleaved caspase-3 and Bcl2) and the appearance of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive neurons, underlining that apoptosis and autophagy pathways are intricately intertwined. Therefore, autophagy is likely involved in MN death in SMA II, suggesting that it might represent a promising target for delaying the progression of SMA in humans as well.

The various aggregation states of beta-amyloid 1-42 mediate different effects on oxidative stress, neurodegeneration, and BACE-1 expression.

The amyloid cascade hypothesis suggests that the insoluble and fibrillar form of beta-amyloid (A beta) may play a primary pathogenic role in Alzheimer disease at the molecular level. However, neither the rate of dementia nor the extent of neuronal change seems to correlate with the levels of amyloidotic plaques (i.e., aggregated/fibrillar A beta). Recent evidence suggests, however, that neurotoxicity may be exerted also by rather small soluble aggregates of A beta, including oligomers. To characterize the mechanisms underlying toxicity mediated by the various aggregation states of A beta peptides is then a major goal of research. In this work we investigated the effects of fibrillar, prefibrillar, and oligomeric A beta(1-42) on the induction of oxidative stress, cell death, and BACE-1 expression in NT2 neuronal cells. We found that prefibrillar and oligomeric A beta(1-42) resulted in a more dramatic increase in the oxidative stress markers 4-hydroxynonenal and hydrogen peroxide compared to fibrillar A beta(1-42). Moreover, increased oxidative stress levels also resulted in a more rapid and significant induction of both apoptotic and necrotic neuronal cell death. Accordingly, fibrillar A beta(1-42), but not the soluble nonfibrillar forms, was the only condition able to up-regulate BACE-1 expression and activity.

Beta-amyloid 1-42 monomers, but not oligomers, produce PHF-like conformation of Tau protein.

The mechanistic relationship between amyloid ß1-42 (Aß1-42) and the alteration of Tau protein are debated. We investigated the effect of Aß1-42 monomers and oligomers on Tau, using mice expressing wild-type human Tau that do not spontaneously develop Tau pathology. After intraventricular injection of Aß1-42, mice were sacrificed after 3 h or 4 days. The short-lasting treatment with Aß monomers, but not oligomers, showed a conformational PHF-like change of Tau, together with hyperphosphorylation. The same treatment induced increase in concentration of GSK3 and MAP kinases. The inhibition of the kinases rescued the Tau changes. Aß monomers increased the levels of total Tau, through the inhibition of proteasomal degradation. Aß oligomers reproduced all the aforementioned alterations only after 4 days of treatment. It is known that Aß1-42 monomers foster synaptic activity. Our results suggest that Aß monomers physiologically favor Tau activity and dendritic sprouting, whereas their excess causes Tau pathology. Moreover, our study indicates that anti-Aß therapies should be targeted to Aß1-42 monomers too.