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.

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.

Extracellular Fragmented Self-DNA Is Involved in Plant Responses to Biotic Stress.

A growing body of evidence indicates that extracellular fragmented self-DNA (eDNA), by acting as a signaling molecule, triggers inhibitory effects on conspecific plants and functions as a damage-associated molecular pattern (DAMP). To evaluate early and late events in DAMP-dependent responses to eDNA, we extracted, fragmented, and applied the tomato (Solanum lycopersicum) eDNA to tomato leaves. Non-sonicated, intact self-DNA (intact DNA) was used as control. Early event analyses included the evaluation of plasma transmembrane potentials (Vm), cytosolic calcium variations (Ca2+ cy t), the activity and subcellular localization of both voltage-gated and ligand-gated rectified K+ channels, and the reactive oxygen species (ROS) subcellular localization and quantification. Late events included RNA-Seq transcriptomic analysis and qPCR validation of gene expression of tomato leaves exposed to tomato eDNA. Application of eDNA induced a concentration-dependent Vm depolarization which was correlated to an increase in (Ca2+)cyt; this event was associated to the opening of K+ channels, with particular action on ligand-gated rectified K+ channels. Both eDNA-dependent (Ca2+)cyt and K+ increases were correlated to ROS production. In contrast, application of intact DNA produced no effects. The plant response to eDNA was the modulation of the expression of genes involved in plant-biotic interactions including pathogenesis-related proteins (PRPs), calcium-dependent protein kinases (CPK1), heat shock transcription factors (Hsf), heat shock proteins (Hsp), receptor-like kinases (RLKs), and ethylene-responsive factors (ERFs). Several genes involved in calcium signaling, ROS scavenging and ion homeostasis were also modulated by application of eDNA. Shared elements among the transcriptional response to eDNA and to biotic stress indicate that eDNA might be a common DAMP that triggers plant responses to pathogens and herbivores, particularly to those that intensive plant cell disruption or cell death. Our results suggest the intriguing hypothesis that some of the plant reactions to pathogens and herbivores might be due to DNA degradation, especially when associated to the plant cell disruption. Fragmented DNA would then become an important and powerful elicitor able to trigger early and late responses to biotic stress.

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.

Elovl5 is required for proper action potential conduction along peripheral myelinated fibers.

Elovl5 elongates fatty acids with 18 carbon atoms and in cooperation with other enzymes guarantees the normal levels of very long-chain fatty acids, which are necessary for a proper membrane structure. Action potential conduction along myelinated axons depends on structural integrity of myelin, which is maintained by a correct amount of fatty acids and a proper interaction between fatty acids and myelin proteins. We hypothesized that in Elovl5-/- mice, the lack of elongation of Elovl5 substrates might cause alterations of myelin structure. The analysis of myelin ultrastructure showed an enlarged periodicity with reduced G-ratio across all axonal diameters. We hypothesized that the structural alteration of myelin might affect the conduction of action potentials. The sciatic nerve conduction velocity was significantly reduced without change in the amplitude of the nerve compound potential, suggesting a myelin defect without a concomitant axonal degeneration. Since Elovl5 is important in attaining normal amounts of polyunsaturated fatty acids, which are the principal component of myelin, we performed a lipidomic analysis of peripheral nerves of Elovl5-deficient mice. The results revealed an unbalance, with reduction of fatty acids longer than 18 carbon atoms relative to shorter ones. In addition, the ratio of saturated to unsaturated fatty acids was strongly increased. These findings point out the essential role of Elovl5 in the peripheral nervous system in supporting the normal structure of myelin, which is the key element for a proper conduction of electrical signals along myelinated nerves.

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 transcription factor Nurr1 is upregulated in amyotrophic lateral sclerosis patients and SOD1-G93A mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects both lower and upper motor neurons (MNs) in the central nervous system. ALS etiology is highly multifactorial and multifarious, and an effective treatment is still lacking. Neuroinflammation is a hallmark of ALS and could be targeted to develop new therapeutic approaches. Interestingly, the transcription factor Nurr1 has been demonstrated to have an important role in the inflammatory process in several neurological disorders, such as Parkinson's disease and multiple sclerosis. In the present paper, we demonstrate for the first time that Nurr1 expression levels are upregulated in the peripheral blood of ALS patients. Moreover, we investigated Nurr1 function in the SOD1-G93A mouse model of ALS. Nurr1 was strongly upregulated in the spinal cord during the asymptomatic and early symptomatic phases of the disease, where it promoted the expression of brain-derived neurotrophic factor mRNA and the repression of NFκB pro-inflammatory targets, such as inducible nitric oxide synthase. Therefore, we hypothesize that Nurr1 is activated in an early phase of the disease as a protective endogenous anti-inflammatory mechanism, although not sufficient to reverse disease progression. On the basis of these observations, Nurr1 could represent a potential biomarker for ALS and a promising target for future therapies.

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.

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.

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.

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 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.

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.

Preventive motor training but not progenitor grafting ameliorates cerebellar ataxia and deregulated autophagy in tambaleante mice.

Treatment options for degenerative cerebellar ataxias are currently very limited. A large fraction of such disorders is represented by hereditary cerebellar ataxias, whose familiar transmission facilitates an early diagnosis and may possibly allow to start preventive treatments before the onset of the neurodegeneration and appearance of first symptoms. In spite of the heterogeneous aetiology, histological alterations of ataxias often include the primary degeneration of the cerebellar cortex caused by Purkinje cells (PCs) loss. Thus, approaches aimed at replacing or preserving PCs could represent promising ways of disease management. In the present study, we compared the efficacy of two different preventive strategies, namely cell replacement and motor training. We used tambaleante (tbl) mice as a model for progressive ataxia caused by selective loss of PCs and evaluated the effectiveness of the preventive transplantation of healthy PCs into early postnatal tbl cerebella, in terms of PC replacement and functional preservation. On the other hand, we investigated the effects of motor training on PC survival, cerebellar circuitry and their behavioral correlates. Our results demonstrate that, despite a good survival rate and integration of grafted PCs, the adopted grafting protocol could not alleviate the ataxic symptoms in tbl mice. Conversely, preventive motor training increases PCs survival with a moderate positive impact on the motor phenotype.

Extracellular Self-DNA (esDNA), but Not Heterologous Plant or Insect DNA (etDNA), Induces Plasma Membrane Depolarization and Calcium Signaling in Lima Bean (Phaseolus lunatus) and Maize (Zea mays).

Extracellular self-DNA (esDNA) is produced during cell and tissue damage or degradation and has been shown to induce significant responses in several organisms, including plants. While the inhibitory effects of esDNA have been shown in conspecific individuals, little is known on the early events involved upon plant esDNA perception. We used electrophysiology and confocal laser scanning microscopy calcium localization to evaluate the plasma membrane potential (Vm) variations and the intracellular calcium fluxes, respectively, in Lima bean (Phaseolus lunatus) and maize (Zea mays) plants exposed to esDNA and extracellular heterologous DNA (etDNA) and to etDNA from Spodoptera littoralis larvae and oral secretions. In both species, esDNA induced a significant Vm depolarization and an increased flux of calcium, whereas etDNA was unable to exert any of these early signaling events. These findings confirm the specificity of esDNA to induce plant cell responses and to trigger early signaling events that eventually lead to plant response to damage.

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.

E2 Regulates Epigenetic Signature on Neuroglobin Enhancer-Promoter in Neuronal Cells.

Estrogens are neuroprotective factors in several neurological diseases. Neuroglobin (NGB) is one of the estrogen target genes involved in neuroprotection, but little is known about its transcriptional regulation. Estrogen genomic pathway in gene expression regulation is mediated by estrogen receptors (ERα and ERß) that bind to specific regulatory genomic regions. We focused our attention on 17ß-estradiol (E2)-induced NGB expression in human differentiated neuronal cell lines (SK-N-BE and NT-2). Previously, using bioinformatics analysis we identified a putative enhancer in the first intron of NGB locus. Therefore, we observed that E2 increased the enrichment of the H3K4me3 epigenetic marks at the promoter and of the H3K4me1 and H3K27Ac at the intron enhancer. In these NGB regulatory regions, we found estrogen receptor alpha (ERα) binding suggesting that ERα may mediate chromatin remodeling to induce NGB expression upon E2 treatment. Altogether our data show that NGB expression is regulated by ERα binding on genomic regulatory regions supporting hormone therapy applications for the neuroprotection against neurodegenerative diseases.

Aß1-42 monomers or oligomers have different effects on autophagy and apoptosis.

The role of autophagy and its relationship with apoptosis in Alzheimer disease (AD) pathogenesis is poorly understood. Disruption of autophagy leads to buildup of incompletely digested substrates, amyloid-ß (Aß) peptide accumulation in vacuoles and cell death. Aß, in turn, has been found to affect autophagy. Thus, Aß might be part of a loop in which it is both the substrate of altered autophagy and its cause. Given the relevance of different soluble forms of Aß1-42 in AD, we have investigated whether monomers and oligomers of the peptide have a differential role in causing altered autophagy and cell death. Using differentiated SK-N-BE neuroblastoma cells, we found that monomers hamper the formation of the autophagic BCL2-BECN1/Beclin 1 complex and activate the MAPK8/JNK1-MAPK9/JNK2 pathway phosphorylating BCL2. Monomers also inhibit apoptosis and allow autophagy with intracellular accumulation of autophagosomes and elevation of levels of BECN1 and LC3-II, resulting in an inhibition of substrate degradation due to an inhibitory action on lysosomal activity. Oligomers, in turn, favor the formation of the BCL2-BECN1 complex favoring apoptosis. In addition, they cause a less profound increase in BECN1 and LC3-II levels than monomers without affecting the autophagic flux. Thus, data presented in this work show a link for autophagy and apoptosis with monomers and oligomers, respectively. These studies are likely to help the design of novel disease modifying therapies.

Up-regulation of ß-amyloidogenesis in neuron-like human cells by both 24- and 27-hydroxycholesterol: protective effect of N-acetyl-cysteine.

An abnormal accumulation of cholesterol oxidation products in the brain of patients with Alzheimer's disease (AD) would further link an impaired cholesterol metabolism in the pathogenesis of the disease. The first evidence stemming from the content of oxysterols in autopsy samples from AD and normal brains points to an increase in both 27-hydroxycholesterol (27-OH) and 24-hydroxycholesterol (24-OH) in the frontal cortex of AD brains, with a trend that appears related to the disease severity. The challenge of differentiated SK-N-BE human neuroblastoma cells with patho-physiologically relevant amounts of 27-OH and 24-OH showed that both oxysterols induce a net synthesis of Aß1-42 by up-regulating expression levels of amyloid precursor protein and ß-secretase, as well as the ß-secretase activity. Interestingly, cell pretreatment with N-acetyl-cysteine (NAC) fully prevented the enhancement of ß-amyloidogenesis induced by the two oxysterols. The reported findings link an impaired cholesterol oxidative metabolism to an excessive ß-amyloidogenesis and point to NAC as an efficient inhibitor of oxysterols-induced Aß toxic peptide accumulation in the brain.