Paving Plant-Food-Derived Bioactives as Effective Therapeutic Agents in Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a neurodevelopmental disorder, where social and communication deficits and repetitive behaviors are present. Plant-derived bioactives have shown promising results in the treatment of autism. In this sense, this review is aimed at providing a careful view on the use of plant-derived bioactive molecules for the treatment of autism. Among the plethora of bioactives, curcumin, luteolin, and resveratrol have revealed excellent neuroprotective effects and can be effectively used in the treatment of neuropsychological disorders. However, the number of clinical trials is limited, and none of them have been approved for the treatment of autism or autism-related disorder. Further clinical studies are needed to effectively assess the real potential of such bioactive molecules.N.C.-M. acknowledges the Portuguese Foundation for Science and Technology under the Horizon 2020 Program (PTDC/PSI-GER/28076/2017)

Targeting quiescent leukemic stem cells using second generation autophagy inhibitors

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin−Sca-1+c-kit+CD48−CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence

Monitoring Antigen Processing for MHC Presentation via Macroautophagy

Macroautophagy has recently emerged as an important catabolic process involved not only in innate immunity but also in adaptive immunity. Initially described to deliver intracellular antigens to MHC class II loading compartments, its molecular machinery has now also been described to impact the delivery of extracellular antigens to MHC class II loading compartments through the noncanonical use of the macroautophagy machinery during LC3-associated phagocytosis (LAP). Therefore, in pathological situations (viral or bacterial infections, tumorigenesis) the pathway might be involved in shaping CD4$^{+}$ T cell responses.In this chapter we describe three basic experiments for the monitoring and manipulation of macroautophagic antigen processing toward MHC class II presentation through the canonical pathway. Firstly, we will discuss how to monitor autophagic flux and autophagosome fusion with MHC class II loading compartments. Secondly, we will show how to target proteins to autophagosomes in order to monitor macroautophagy dependent antigen processing via their enhanced presentation on MHC class II molecules to CD4$^{+}$ T cells. And finally, we will describe how macroautophagy can be silenced in antigen presenting cells, like human monocyte-derived dendritic cells (DCs)

Chemical Composition, Biological Activity, and Health-Promoting Effects of Withania somnifera for Pharma-Food Industry Applications

The Withania genus comes from the Solanaceae family and includes around 23 species, spread over some areas of the Mediterranean, Asia, and East Africa. Widely used in traditional medicine for thousands of years, these plants are rich in secondary metabolites, with special emphasis on steroidal lactones, named withanolides which are used as ingredients in numerous formulations for a plethora of diseases, such as asthma, diabetes, arthritis, impotence, amnesia, hypertension, anxiety, stress, cancer, neurodegenerative, and cardiovascular diseases, and many others. Among them, Withania somnifera (L.) Dunal is the most widely addressed species from a pharmacological and agroindustrial point of view. In this sense, this review provides an overview of the folk uses, phytochemical composition, and biological activity, such as antioxidant, antimicrobial, anti-inflammatory, and cytotoxic activity of W. somnifera, although more recently other species have also been increasingly investigated. In addition, their health-promoting effects, i.e., antistress, anxiolytic, adaptogenic, antirheumatoid arthritis, chemoprotective, and cardiorespiratory-enhancing abilities, along with safety and adverse effects are also discussed.N. C. -M. acknowledges the Portuguese Foundation for Science and Technology under the Horizon 2020 Program (PTDC/PSI-GER/28076/2017)

Anacardium plants: Chemical,nutritional composition and biotechnological applications

Anacardium plants are native to the American tropical regions, and Anacardium occidentale L. (cashew tree) is the most recognized species of the genus. These species contain rich secondary metabolites in their leaf and shoot powder, fruits and other parts that have shown diverse applications. This review describes the habitat and cultivation of Anacardium species, phytochemical and nutritional composition, and their industrial food applications. Besides, we also discuss the secondary metabolites present in Anacardium plants which display great antioxidant and antimicrobial effects. These make the use of Anacardium species in the food industry an interesting approach to the development of green foods.AK. Jugran acknowledges the partial funding from Uttarakhand council for Biotechnology, Pantnagar, Uttarakhand, India (File No. UCB/R&D Project/2018-311) for this work. M. Martorell would like to thank the support offered by CONICYT PIA/APOYO CCTE AFB170007. N. Martins would like to thank the Portuguese Foundation for Science and Technology (FCT-Portugal) for the Strategic project ref. UID/BIM/04293/2013 and ?NORTE2020 - Northern Regional Operational Program? (NORTE-01-0145-FEDER- 000012)

p70S6K is regulated by Focal Adhesion Kinase and is required for Src-selective autophagy

AbstractHere we report that focal adhesion kinase (FAK) is required for optimal signalling to the Akt-p70S6K-S6 pathway in squamous cell carcinoma (SCC) cells. Specifically, in SCCs that are genetically deficient for FAK, there is reduced phosphorylation of Akt, p70S6K and S6, and signalling to Akt-p70S6K-S6 is more sensitive to inhibition by multiple agents that suppress the pathway. By contrast, mTOR is unaffected. Indeed, pharmacological agents that inhibit the Akt-p70S6K-S6 pathway, and PDK1 that lies upstream of Akt, also impair the autophagic targeting of activated c-Src (p-Src) in FAK deficient cells. This is associated with loss of a complex between p-Src and the autophagy protein LC3, a biochemical surrogate of impaired Src-selective autophagy. In keeping with a vital role for p70S6K, inhibition by a selective inhibitor and specific siRNA also impaired Src-selective autophagy. Finally, components of the PDK1-Akt-p70S6K signalling pathway were co-located with p-Src at autophagosomes, and Src and p70S6K co-exist in the same biochemical complex. We therefore deduce that the FAK-regulated signalling module PDK1-Akt-p70S6K that controls Src's intracellular trafficking operates at Src-containing autophagosomes

Niclosamide Prevents the Formation of Large Ubiquitin-Containing Aggregates Caused by Proteasome Inhibition

Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes.To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates.Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity

SVIP Induces Localization of p97/VCP to the Plasma and Lysosomal Membranes and Regulates Autophagy

The small p97/VCP-interacting protein (SVIP) functions as an inhibitor of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. Here we show that overexpression of SVIP in HeLa cells leads to localization of p97/VCP at the plasma membrane, intracellular foci and juxtanuclear vacuoles. The p97/VCP-positive vacuolar structures colocalized or associated with LC3 and lamp1, suggesting that SVIP may regulate autophagy. In support of this possibility, knockdown of SVIP diminished, whereas overexpression of SVIP enhanced LC3 lipidation. Surprisingly, knockdown of SVIP reduced the levels of p62 protein at least partially through downregulation of its mRNA, which was accompanied by a decrease in starvation-induced formation of p62 bodies. Overexpression of SVIP, on the other hand, increased the levels of p62 protein and enhanced starvation-activated autophagy as well as promoted sequestration of polyubiquitinated proteins and p62 in autophagosomes. These results suggest that SVIP plays a regulatory role in p97 subcellular localization and is a novel regulator of autophagy

Regulation of Toll-like receptor signaling by NDP52-mediated selective autophagy is normally inactivated by A20

Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting ‘autophagy receptors’ in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF–TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling

Neuronopathic Gaucher disease in the mouse: viable combined selective saposin C deficiency and mutant glucocerebrosidase (V394L) mice with glucosylsphingosine and glucosylceramide accumulation and progressive neurological deficits

Gaucher disease is caused by defective acid β-glucosidase (GCase) function. Saposin C is a lysosomal protein needed for optimal GCase activity. To test the in vivo effects of saposin C on GCase, saposin C deficient mice (C−/−) were backcrossed to point mutated GCase (V394L/V394L) mice. The resultant mice (4L;C*) began to exhibit CNS abnormalities ∼30 days: first as hindlimb paresis, then progressive tremor and ataxia. Death occurred ∼48 days due to neurological deficits. Axonal degeneration was evident in brain stem, spinal cord and white matter of cerebellum accompanied by increasing infiltration of the brain stem, cortex and thalamus by CD68 positive microglial cells and activation of astrocytes. Electron microscopy showed inclusion bodies in neuronal processes and degenerating cells. Accumulation of p62 and Lamp2 were prominent in the brain suggesting the impairment of autophagosome/lysosome function. This phenotype was different from either V394L/V394L or C−/− alone. Relative to V394L/V394L mice, 4L;C* mice had diminished GCase protein and activity. Marked increases (20- to 30-fold) of glucosylsphingosine (GS) and moderate elevation (1.5- to 3-fold) of glucosylceramide (GC) were in 4L;C* brains. Visceral tissues had increases of GS and GC, but no storage cells were found. Neuronal cells in thick hippocampal slices from 4L;C* mice had significantly attenuated long-term potentiation, presumably resulting from substrate accumulation. The 4L;C* mouse mimics the CNS phenotype and biochemistry of some type 3 (neuronopathic) variants of Gaucher disease and is a unique model suitable for testing pharmacological chaperone and substrate reduction therapies, and investigating the mechanisms of neuronopathic Gaucher disease