Neuroprotective properties of queen bee acid by autophagy induction.

Autophagy is a conserved intracellular catabolic pathway that removes cytoplasmic components to contribute to neuronal homeostasis. Accumulating evidence has increasingly shown that the induction of autophagy improves neuronal health and extends longevity in several animal models. Therefore, there is a great interest in the identification of effective autophagy enhancers with potential nutraceutical or pharmaceutical properties to ameliorate age-related diseases, such as neurodegenerative disorders, and/or promote longevity. Queen bee acid (QBA, 10-hydroxy-2-decenoic acid) is the major fatty acid component of, and is found exclusively in, royal jelly, which has beneficial properties for human health. It is reported that QBA has antitumor, anti-inflammatory, and antibacterial activities and promotes neurogenesis and neuronal health; however, the mechanism by which QBA exerts these effects has not been fully elucidated. The present study investigated the role of the autophagic process in the protective effect of QBA. We found that QBA is a novel autophagy inducer that triggers autophagy in various neuronal cell lines and mouse and fly models. The beclin-1 (BECN1) and mTOR pathways participate in the regulation of QBA-induced autophagy. Moreover, our results showed that QBA stimulates sirtuin 1 (SIRT1), which promotes autophagy by the deacetylation of critical ATG proteins. Finally, QBA-mediated autophagy promotes neuroprotection in Parkinson's disease in vitro and in a mouse model and extends the lifespan of Drosophila melanogaster. This study provides detailed evidences showing that autophagy induction plays a critical role in the beneficial health effects of QBA.

The parkinsonian LRRK2 R1441G mutation shows macroautophagy-mitophagy dysregulation concomitant with endoplasmic reticulum stress.

Autophagy is a mechanism responsible for the degradation of cellular components to maintain their homeostasis. However, autophagy is commonly altered and compromised in several diseases, including neurodegenerative disorders. Parkinson's disease (PD) can be considered a multifactorial disease because environmental factors, genetic factors, and aging are involved. Several genes are involved in PD pathology, among which the LRRK2 gene and its mutations, inherited in an autosomal dominant manner, are responsible for most genetic PD cases. The R1441G LRRK2 mutation is, after G2019S, the most important in PD pathogenesis. Our results demonstrate a relationship between the R1441G LRRK2 mutation and a mechanistic dysregulation of autophagy that compromises cell viability. This altered autophagy mechanism is associated with organellar stress including mitochondrial (which induces mitophagy) and endoplasmic reticulum (ER) stress, consistent with the fact that patients with this mutation are more vulnerable to toxins related to PD, such as MPP+.

Molecular characterization of autophagic and apoptotic signaling induced by sorafenib in liver cancer cells.

Sorafenib is the unique accepted molecular targeted drug for the treatment of patients in advanced stage of hepatocellular carcinoma. The current study evaluated cell signaling regulation of endoplasmic reticulum (ER) stress, c-Jun-N-terminal kinase (JNK), Akt, and 5'AMP-activated protein kinase (AMPK) leading to autophagy and apoptosis induced by sorafenib. Sorafenib induced early (3-12 hr) ER stress characterized by an increase of Ser51 P-eIF2α/eIF2α, C/EBP homologous protein (CHOP), IRE1α, and sXBP1, but a decrease of activating transcription factor 6 expression, overall temporally associated with the increase of Thr183,Tyr185 P-JNK1/2/JNK1/2, Thr172 P-AMPKα, Ser413 P-Foxo3a, Thr308 P-AKt/AKt and Thr32 P-Foxo3a/Foxo3a ratios, and reduction of Ser2481 P-mammalian target of rapamycin (mTOR)/mTOR and protein translation. This pattern was related to a transient increase of tBid, Bim EL , Beclin-1, Bcl-xL, Bcl-2, autophagy markers, and reduction of myeloid cell leukemia-1 (Mcl-1) expression. The progressive increase of CHOP expression, and reduction of Thr308 P-AKt/AKt and Ser473 P-AKt/AKt ratios were associated with the reduction of autophagic flux and an additional upregulation of Bim EL expression and caspase-3 activity (24 hr). Small interfering-RNA (si-RNA) assays showed that Bim, but not Bak and Bax, was involved in the induction of caspase-3 in sorafenib-treated HepG2 cells. Sorafenib increased autophagic and apoptotic markers in tumor-derived xenograft model. In conclusion, the early sorafenib-induced ER stress and regulation of JNK and AMPK-dependent signaling were related to the induction of survival autophagic process. The sustained drug treatment induced a progressive increase of ER stress and PERK-CHOP-dependent rise of Bim EL , which was associated with the shift from autophagy to apoptosis. The kinetic of Bim EL expression profile might also be related to the tight balance between AKt- and AMPK-related signaling leading to Foxo3a-dependent BIM EL upregulation.

N370S-GBA1 mutation causes lysosomal cholesterol accumulation in Parkinson's disease.

BACKGROUND: Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme ß-glucocerebrosidase-1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S-GBA1 mutation on cellular homeostasis and vulnerability in a patient-specific cellular model of PD. METHODS: We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy-lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry. RESULTS: The N370S mutation produced a significant reduction in ß-glucocerebrosidase-1 protein and enzyme activity and ß-glucocerebrosidase-1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death. CONCLUSIONS: Our results support a connection between the loss of ß-glucocerebrosidase-1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1-PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1-PD shares common features with lipid-storage diseases. © 2017 International Parkinson and Movement Disorder Society.

Inhibition of autophagy by TAB2 and TAB3.

Autophagic responses are coupled to the activation of the inhibitor of NF-κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFß-activated kinase 1 (TAK1)-binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1-IKK signalling axis, constitutively interact with each other via their coiled-coil domains (CCDs). Upon autophagy induction, TAB2 and TAB3 dissociate from Beclin 1 and bind TAK1. Moreover, overexpression of TAB2 and TAB3 suppresses, while their depletion triggers, autophagy. The expression of the C-terminal domain of TAB2 or TAB3 or that of the CCD of Beclin 1 competitively disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3, hence stimulating autophagy through a pathway that requires endogenous Beclin 1, TAK1 and IKK to be optimally efficient. These results point to the existence of an autophagy-stimulatory 'switch' whereby TAB2 and TAB3 abandon inhibitory interactions with Beclin 1 to engage in a stimulatory liaison with TAK1.

Routine Western blot to check autophagic flux: cautions and recommendations.

At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible disadvantages and limitations that this method presents for a correct interpretation of the results according to the lysis buffer used for extracting proteins. Here, we tested the LC3 and p62 protein levels by WB in four cell models (mouse embryonic and human fibroblasts (MEFs and HFs, respectively), N27 rat mesencephalic dopaminergic neurons and SH-SY5Y human neuroblastoma cells). The cells were exposed to the autophagy inhibitor bafilomycin A1 (Baf. A1) in combination (or not) with nutrient deprivation to induce autophagy, and they were lysed by using four different buffers (nonyl phenoxypolyethoxylethanol (NP-40), radioimmunoprecipitation assay (RIPA), Triton X-100, and sample buffer (SB) 1×). Based on our observations, we want to highlight that this technique is not always appropriate for analyzing and monitoring autophagy. In this report, we show conflicting data that hinder the correct interpretation of the results, especially in relation to p62 protein levels, at least in the models studied in this work.

Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression.

Mutations of the PTEN-induced kinase 1 (PINK1) gene are a cause of autosomal recessive Parkinson's disease (PD). This gene encodes a mitochondrial serine/threonine kinase, which is partly localized to mitochondria, and has been shown to play a role in protecting neuronal cells from oxidative stress and cell death, perhaps related to its role in mitochondrial dynamics and mitophagy. In this study, we report that increased mitochondrial PINK1 levels observed in human neuroblastoma SH-SY5Y cells after carbonyl cyanide m-chlorophelyhydrazone (CCCP) treatment were due to de novo protein synthesis, and not just increased stabilization of full length PINK1 (FL-PINK1). PINK1 mRNA levels were significantly increased by 4-fold after 24h. FL-PINK1 protein levels at this time point were significantly higher than vehicle-treated, or cells treated with CCCP for 3h, despite mitochondrial content being decreased by 29%. We have also shown that CCCP dissipated the mitochondrial membrane potential (Δψm) and induced entry of extracellular calcium through L/N-type calcium channels. The calcium chelating agent BAPTA-AM impaired the CCCP-induced PINK1 mRNA and protein expression. Furthermore, CCCP treatment activated the transcription factor c-Fos in a calcium-dependent manner. These data indicate that PINK1 expression is significantly increased upon CCCP-induced mitophagy in a calcium-dependent manner. This increase in expression continues after peak Parkin mitochondrial translocation, suggesting a role for PINK1 in mitophagy that is downstream of ubiquitination of mitochondrial substrates. This sensitivity to intracellular calcium levels supports the hypothesis that PINK1 may also play a role in cellular calcium homeostasis and neuroprotection.

The LRRK2 G2019S mutant exacerbates basal autophagy through activation of the MEK/ERK pathway.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a major cause of familial Parkinsonism, and the G2019S mutation of LRRK2 is one of the most prevalent mutations. The deregulation of autophagic processes in nerve cells is thought to be a possible cause of Parkinson's disease (PD). In this study, we observed that G2019S mutant fibroblasts exhibited higher autophagic activity levels than control fibroblasts. Elevated levels of autophagic activity can trigger cell death, and in our study, G2019S mutant cells exhibited increased apoptosis hallmarks compared to control cells. LRRK2 is able to induce the phosphorylation of MAPK/ERK kinases (MEK). The use of 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a highly selective inhibitor of MEK1/2, reduced the enhanced autophagy and sensibility observed in G2019S LRRK2 mutation cells. These data suggest that the G2019S mutation induces autophagy via MEK/ERK pathway and that the inhibition of this exacerbated autophagy reduces the sensitivity observed in G2019S mutant cells.

The Role of Cardiolipin in Mitochondrial Function and Neurodegenerative Diseases.

Cardiolipin (CL) is a mitochondria-exclusive phospholipid synthesized in the inner mitochondrial membrane. CL plays a key role in mitochondrial membranes, impacting a plethora of functions this organelle performs. Consequently, it is conceivable that abnormalities in the CL content, composition, and level of oxidation may negatively impact mitochondrial function and dynamics, with important implications in a variety of diseases. This review concentrates on papers published in recent years, combined with basic and underexplored research in CL. We capture new findings on its biological functions in the mitochondria, as well as its association with neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. Lastly, we explore the potential applications of CL as a biomarker and pharmacological target to mitigate mitochondrial dysfunction.

Immunological aspects of central neurodegeneration.

The etiology of various neurodegenerative disorders that mainly affect the central nervous system including (but not limited to) Alzheimer's disease, Parkinson's disease and Huntington's disease has classically been attributed to neuronal defects that culminate with the loss of specific neuronal populations. However, accumulating evidence suggests that numerous immune effector cells and the products thereof (including cytokines and other soluble mediators) have a major impact on the pathogenesis and/or severity of these and other neurodegenerative syndromes. These observations not only add to our understanding of neurodegenerative conditions but also imply that (at least in some cases) therapeutic strategies targeting immune cells or their products may mediate clinically relevant neuroprotective effects. Here, we critically discuss immunological mechanisms of central neurodegeneration and propose potential strategies to correct neurodegeneration-associated immunological dysfunction with therapeutic purposes.

Heat shock and thermotolerance in Caenorhabditis elegans: An overview of laboratory techniques.

The soil nematode worm Caenorhabditis elegans is a simple and well-established model for the study of many biological processes. Heat shock and thermotolerance assays have been developed for this nematode, and have been used to decipher the molecular relationships between thermal stress and aging, among others. Nevertheless, a systematic and methodological comparison of the different approaches and tools utilized is lacking in the literature. Here, we aim to provide a comprehensive summary of the most commonly used strategies for carrying out heat shock and thermotolerance assays that have been reported, highlighting specific readouts and scientific questions that can be addressed. Furthermore, we offer examples of thermotolerance assays performed with wild type nematodes, that can serve as a gauge of the animal survival under diverse conditions of stress.

Role of TFEB in Huntington's Disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by an expansion of the CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. This expansion leads to a polyglutamine (polyQ) tract at the N-terminal end of HTT, which reduces the solubility of the protein and promotes its accumulation. Inefficient clearance of mutant HTT (mHTT) by the proteasome or autophagy-lysosomal system leads to accumulation of oligomers and toxic protein aggregates in neurons, resulting in impaired proteolytic systems, transcriptional dysregulation, impaired axonal transport, mitochondrial dysfunction and cellular energy imbalance. Growing evidence suggests that the accumulation of mHTT aggregates and autophagic and/or lysosomal dysfunction are the major pathogenic mechanisms underlying HD. In this context, enhancing autophagy may be an effective therapeutic strategy to remove protein aggregates and improve cell function. Transcription factor EB (TFEB), a master transcriptional regulator of autophagy, controls the expression of genes critical for autophagosome formation, lysosomal biogenesis, lysosomal function and autophagic flux. Consequently, the induction of TFEB activity to promote intracellular clearance may be a therapeutic strategy for HD. However, while some studies have shown that overexpression of TFEB facilitates the clearance of mHTT aggregates and ameliorates the disease phenotype, others indicate such overexpression may lead to mHTT co-aggregation and worsen disease progression. Further studies are necessary to confirm whether TFEB modulation could be an effective therapeutic strategy against mHTT-mediated toxicity in different disease models.

Vitamin D Receptor Polymorphisms in a Spanish Cohort of Parkinson's Disease Patients.

Background: Vitamin D receptor (VDR) is a nuclear hormone receptor widely expressed in the substantia nigra. Its association with an increased risk of Parkinson's disease (PD) is based on vitamin D deficiency and/or different polymorphisms in its gene receptor. This fact has been demonstrated by several case-control studies. Materials and Methods: Consequently, we investigated the association between VDR ApaI, BsmI, FokI, and TaqI gene polymorphisms and PD in a Spanish cohort that included 54 cases and 17 healthy controls. The detection of single nucleotide polymorphisms (SNPs) was performed using a polymerase chain reaction-restriction fragment length polymorphism. Results: Our data indicate that the SNPs were not associated with the age of onset of PD, nor with the occurrence of motor symptoms. However, only BsmI polymorphism was significantly associated with PD in this Spanish cohort. In fact, BsmI genotype was five times higher among PD patients than among controls, and the A allele was considered as a genetic risk for PD. Additionally, the combination of FokI and BsmI polymorphisms was significantly associated with PD and could represent a risk factor. Conclusion: We conclude that ApaI, TaqI, and FokI polymorphisms were not associated with PD, but BsmI could be a risk factor for PD in this randomized population.

Detection of rare genetic variations in the promoter regions of the ATG16L gene in Parkinson's patients.

Mutations in the ATG genes have been related to impair autophagic function, contributing to the sporadic onset of Parkinsons Disease (PD). However, scarce studies have been performed about ins/del within the regulatory domains of the autophagy genes in sporadic PD patients. This study was aimed to found ins/del within part of the crucial core autophagy promotor gene region of the ATG16L1 in a groups of sporadic PD patients. After developing a genetic marker to find ins/del using fragment size analysis, a rare mutation by insertion (0.45%) was reported in the patients. This mutation was characterized by sequencing. No others ins/del were found. As a results, the frequency of this insertion should be considered as a rare genetic variant. An in silico analysis also highlighting the usefulness of a search GDV which revealed multiples ins/del within ATG16L1 promoter. Furthermore, these genetic insertions could be found in patients with sporadic PD in the ATG161L promoter gene. When a breakpoint as deletions, insertions or tandem duplication are located within a functional gene interruption of the gene and a loss of function was expected but removing or altering in the regulatory sequence can influence the expression or the regulation of a nearby gene which may impair healthy due to dosage effects in sporadic diseases.

Comparative Genetic Analysis of the Promoters of the ATG16L1 and ATG5 Genes Associated with Sporadic Parkinson's Disease.

Sporadic Parkinson's disease, characterised by a decline in dopamine, usually manifests in people over 65 years of age. Although 10% of cases have a genetic (familial) basis, most PD is sporadic. Genome sequencing studies have associated several genetic variants with sporadic PD. Our aim was to analyse the promoter region of the ATG16L1 and ATG5 genes in sporadic PD patients and ethnically matched controls. Genotypes were obtained by using the Sanger method with primers designed by us. The number of haplotypes was estimated with DnaSP software, phylogeny was reconstructed in Network, and genetic divergence was explored with Fst. Seven and two haplotypes were obtained for ATG16L1 and ATG5, respectively. However, only ATG16L1 showed a significant contribution to PD and a significant excess of accumulated mutations that could influence sporadic PD disease. Of a total of seven haplotypes found, only four were unique to patients sharing the T allele (rs77820970). Recent studies using MAPT genes support the notion that the architecture of haplotypes is worthy of being considered genetically risky, as shown in our study, confirming that large-scale assessment in different populations could be relevant to understanding the role of population-specific heterogeneity. Finally, our data suggest that the architecture of certain haplotypes and ethnicity determine the risk of PD, linking haplotype variation and neurodegenerative processes.

Mitochondrial Dysfunction in Repeat Expansion Diseases.

Repeat expansion diseases are a group of neuromuscular and neurodegenerative disorders characterized by expansions of several successive repeated DNA sequences. Currently, more than 50 repeat expansion diseases have been described. These disorders involve diverse pathogenic mechanisms, including loss-of-function mechanisms, toxicity associated with repeat RNA, or repeat-associated non-ATG (RAN) products, resulting in impairments of cellular processes and damaged organelles. Mitochondria, double membrane organelles, play a crucial role in cell energy production, metabolic processes, calcium regulation, redox balance, and apoptosis regulation. Its dysfunction has been implicated in the pathogenesis of repeat expansion diseases. In this review, we provide an overview of the signaling pathways or proteins involved in mitochondrial functioning described in these disorders. The focus of this review will be on the analysis of published data related to three representative repeat expansion diseases: Huntington's disease, C9orf72-frontotemporal dementia/amyotrophic lateral sclerosis, and myotonic dystrophy type 1. We will discuss the common effects observed in all three repeat expansion disorders and their differences. Additionally, we will address the current gaps in knowledge and propose possible new lines of research. Importantly, this group of disorders exhibit alterations in mitochondrial dynamics and biogenesis, with specific proteins involved in these processes having been identified. Understanding the underlying mechanisms of mitochondrial alterations in these disorders can potentially lead to the development of neuroprotective strategies.

Changes in Liver Lipidomic Profile in G2019S-LRRK2 Mouse Model of Parkinson's Disease.

The identification of Parkinson's disease (PD) biomarkers has become a main goal for the diagnosis of this neurodegenerative disorder. PD has not only been intrinsically related to neurological problems, but also to a series of alterations in peripheral metabolism. The purpose of this study was to identify metabolic changes in the liver in mouse models of PD with the scope of finding new peripheral biomarkers for PD diagnosis. To achieve this goal, we used mass spectrometry technology to determine the complete metabolomic profile of liver and striatal tissue samples from WT mice, 6-hydroxydopamine-treated mice (idiopathic model) and mice affected by the G2019S-LRRK2 mutation in LRRK2/PARK8 gene (genetic model). This analysis revealed that the metabolism of carbohydrates, nucleotides and nucleosides was similarly altered in the liver from the two PD mouse models. However, long-chain fatty acids, phosphatidylcholine and other related lipid metabolites were only altered in hepatocytes from G2019S-LRRK2 mice. In summary, these results reveal specific differences, mainly in lipid metabolism, between idiopathic and genetic PD models in peripheral tissues and open up new possibilities to better understand the etiology of this neurological disorder.

Possible involvement of the relationship of LRRK2 and autophagy in Parkinson's disease.

PD (Parkinson's disease) is a neurodegenerative disorder caused by loss of dopamine-generating cells in the substantia nigra. The implication of genetic factors in the aetiology of PD has an essential importance in our understanding of the development of the disease. Mutations in the LRRK2 (leucine-rich repeat kinase 2) gene cause late-onset PD with a clinical appearance indistinguishable from idiopathic PD. Moreover, LRRK2 has been associated with the process of autophagy regulation. Autophagy is an intracellular catabolic mechanism whereby a cell recycles or degrades damaged proteins and cytoplasmic organelles. In the present paper, we discuss the role of LRRK2 in autophagy, and the importance of this relationship in the development of nigral degeneration in PD.

Neuroprotective and Anti-Inflammatory Effects of Linoleic Acid in Models of Parkinson's Disease: The Implication of Lipid Droplets and Lipophagy.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease after Alzheimer's disease. The principal pathological feature of PD is the progressive loss of dopaminergic neurons in the ventral midbrain. This pathology involves several cellular alterations: oxidative stress, mitochondrial dysfunction, loss of proteostasis, and autophagy impairment. Moreover, in recent years, lipid metabolism alterations have become relevant in PD pathogeny. The modification of lipid metabolism has become a possible way to treat the disease. Because of this, we analyzed the effect and possible mechanism of action of linoleic acid (LA) on an SH-SY5Y PD cell line model and a PD mouse model, both induced by 6-hydroxydopamine (6-OHDA) treatment. The results show that LA acts as a potent neuroprotective and anti-inflammatory agent in these PD models. We also observed that LA stimulates the biogenesis of lipid droplets and improves the autophagy/lipophagy flux, which resulted in an antioxidant effect in the in vitro PD model. In summary, we confirmed the neuroprotective effect of LA in vitro and in vivo against PD. We also obtained some clues about the novel neuroprotective mechanism of LA against PD through the regulation of lipid droplet dynamics.