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.

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+.

Metabolic alterations in plasma from patients with familial and idiopathic Parkinson's disease.

The research of new biomarkers for Parkinson's disease is essential for accurate and precocious diagnosis, as well as for the discovery of new potential disease mechanisms and drug targets. The main objective of this work was to identify metabolic changes that might serve as biomarkers for the diagnosis of this neurodegenerative disorder. For this, we profiled the plasma metabolome from mice with neurotoxin-induced Parkinson's disease as well as from patients with familial or sporadic Parkinson's disease. By using mass spectrometry technology, we analyzed the complete metabolome from healthy volunteers compared to patients with idiopathic or familial (carrying the G2019S or R1441G mutations in the LRRK2 gene) Parkinson's disease, as well as, from mice treated with 6-hydroxydopamine to induce Parkinson disease. Both human and murine Parkinson was accompanied by an increase in plasma levels of unconjugated bile acids (cholic acid, deoxycholic acid and lithocholic acid) and purine base intermediary metabolites, in particular hypoxanthine. The comprehensive metabolomic analysis of plasma from Parkinsonian patients underscores the importance of bile acids and purine metabolism in the pathophysiology of this disease. Therefore, plasma measurements of certain metabolites related to these pathways might contribute to the diagnosis of Parkinson's Disease.

Metabolic and psychiatric effects of acyl coenzyme A binding protein (ACBP)/diazepam binding inhibitor (DBI).

Acyl coenzyme A binding protein (ACBP), also known as diazepam binding inhibitor (DBI) is a multifunctional protein with an intracellular action (as ACBP), as well as with an extracellular role (as DBI). The plasma levels of soluble ACBP/DBI are elevated in human obesity and reduced in anorexia nervosa. Accumulating evidence indicates that genetic or antibody-mediated neutralization of ACBP/DBI has anorexigenic effects, thus inhibiting food intake and inducing lipo-catabolic reactions in mice. A number of anorexiants have been withdrawn from clinical development because of their side effects including an increase in depression and suicide. For this reason, we investigated the psychiatric impact of ACBP/DBI in mouse models and patient cohorts. Intravenously (i.v.) injected ACBP/DBI protein conserved its orexigenic function when the protein was mutated to abolish acyl coenzyme A binding, but lost its appetite-stimulatory effect in mice bearing a mutation in the γ2 subunit of the γ-aminobutyric acid (GABA) A receptor (GABAAR). ACBP/DBI neutralization by intraperitoneal (i.p.) injection of a specific mAb blunted excessive food intake in starved and leptin-deficient mice, but not in ghrelin-treated animals. Neither i.v. nor i.p. injected anti-ACBP/DBI antibody affected the behavior of mice in the dark-light box and open-field test. In contrast, ACBP/DBI increased immobility in the forced swim test, while anti-ACBP/DBI antibody counteracted this sign of depression. In patients diagnosed with therapy-resistant bipolar disorder or schizophrenia, ACBP/DBI similarly correlated with body mass index (BMI), not with the psychiatric diagnosis. Patients with high levels of ACBP/DBI were at risk of dyslipidemia and this effect was independent from BMI, as indicated by multivariate analysis. In summary, it appears that ACBP/DBI neutralization has no negative impact on mood and that human depression is not associated with alterations in ACBP/DBI concentrations.

Antibody-mediated neutralization of ACBP/DBI has anorexigenic and lipolytic effects.

We recently identified acyl coenzyme A-binding protein (ACBP)/diazepam binding inhibitor (DBI) as a novel 'hunger factor': a protein that is upregulated in human or murine obesity and that, if administered to mice, causes hyperphagy, adipogenesis and obesity. Conversely, neutralization of ACBP/DBI by systemic injection of neutralizing monoclonal antibodies or autoantibodies produced after auto-immunization against ACBP/DBI has anorexigenic and lipolytic effects. Thus, neutralization of ACBP/DBI results in reduced food intake subsequent to the activation of anorexigenic neurons and the inactivation of orexigenic neurons in the hypothalamus. Moreover, ACBP/DBI neutralization results into enhanced triglyceride lipolysis in white fat, a surge in free fatty acids in the plasma, enhanced incorporation of glycerol-derived carbon atoms into glucose, as well as an increase in ß-oxidation, resulting in a net reduction of fat mass. Importantly, ACBP/DBI neutralization also stimulated an increase in autophagy in various organs, suggesting that it might mediate anti-ageing effects.

Acyl-CoA-binding protein (ACBP): a phylogenetically conserved appetite stimulator.

Recently, we reported that, in mice, hunger causes the autophagy-dependent release of a protein called "acyl-CoA-binding protein" or "diazepam binding inhibitor" (ACBP/DBI) from cells, resulting in an increase in plasma ACBP concentrations. Administration of extra ACBP is orexigenic and obesogenic, while its neutralization is anorexigenic in mice, suggesting that ACBP is a major stimulator of appetite and lipo-anabolism. Accordingly, obese persons have higher circulating ACBP levels than lean individuals, and anorexia nervosa is associated with subnormal ACBP plasma concentrations. Here, we investigated whether ACBP might play a phylogenetically conserved role in appetite stimulation. We found that extracellular ACBP favors sporulation in Saccharomyces cerevisiae, knowing that sporulation is a strategy for yeast to seek new food sources. Moreover, in the nematode Caenorhabditis elegans, ACBP increased the ingestion of bacteria as well as the frequency pharyngeal pumping. These observations indicate that ACBP has a phylogenetically ancient role as a 'hunger factor' that favors food intake.

Acyl-CoA-Binding Protein Is a Lipogenic Factor that Triggers Food Intake and Obesity.

Autophagy facilitates the adaptation to nutritional stress. Here, we show that short-term starvation of cultured cells or mice caused the autophagy-dependent cellular release of acyl-CoA-binding protein (ACBP, also known as diazepam-binding inhibitor, DBI) and consequent ACBP-mediated feedback inhibition of autophagy. Importantly, ACBP levels were elevated in obese patients and reduced in anorexia nervosa. In mice, systemic injection of ACBP protein inhibited autophagy, induced lipogenesis, reduced glycemia, and stimulated appetite as well as weight gain. We designed three approaches to neutralize ACBP, namely, inducible whole-body knockout, systemic administration of neutralizing antibodies, and induction of antiACBP autoantibodies in mice. ACBP neutralization enhanced autophagy, stimulated fatty acid oxidation, inhibited appetite, reduced weight gain in the context of a high-fat diet or leptin deficiency, and accelerated weight loss in response to dietary changes. In conclusion, neutralization of ACBP might constitute a strategy for treating obesity and its co-morbidities.

Impaired Mitophagy and Protein Acetylation Levels in Fibroblasts from Parkinson's Disease Patients.

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder. While most PD cases are idiopathic, the known genetic causes of PD are useful to understand common disease mechanisms. Recent data suggests that autophagy is regulated by protein acetylation mediated by histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities. The changes in histone acetylation reported to be involved in PD pathogenesis have prompted this investigation of protein acetylation and HAT and HDAC activities in both idiopathic PD and G2019S leucine-rich repeat kinase 2 (LRRK2) cell cultures. Fibroblasts from PD patients (with or without the G2019S LRRK2 mutation) and control subjects were used to assess the different phenotypes between idiopathic and genetic PD. G2019S LRRK2 mutation displays increased mitophagy due to the activation of class III HDACs whereas idiopathic PD exhibits downregulation of clearance of defective mitochondria. This reduction of mitophagy is accompanied by more reactive oxygen species (ROS). In parallel, the acetylation protein levels of idiopathic and genetic individuals are different due to an upregulation in class I and II HDACs. Despite this upregulation, the total HDAC activity is decreased in idiopathic PD and the total HAT activity does not significantly vary. Mitophagy upregulation is beneficial for reducing the ROS-induced harm in genetic PD. The defective mitophagy in idiopathic PD is inherent to the decrease in class III HDACs. Thus, there is an imbalance between total HATs and HDACs activities in idiopathic PD, which increases cell death. The inhibition of HATs in idiopathic PD cells displays a cytoprotective effect.

Acetylome in Human Fibroblasts From Parkinson's Disease Patients.

Parkinson's disease (PD) is a multifactorial neurodegenerative disorder. The pathogenesis of this disease is associated with gene and environmental factors. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent genetic cause of familial and sporadic PD. Moreover, posttranslational modifications, including protein acetylation, are involved in the molecular mechanism of PD. Acetylation of lysine proteins is a dynamic process that is modulated in PD. In this descriptive study, we characterized the acetylated proteins and peptides in primary fibroblasts from idiopathic PD (IPD) and genetic PD harboring G2019S or R1441G LRRK2 mutations. Identified acetylated peptides are modulated between individuals' groups. Although acetylated nuclear proteins are the most represented in cells, they are hypoacetylated in IPD. Results display that the level of hyperacetylated and hypoacetylated peptides are, respectively, enhanced in genetic PD and in IPD cells.

ER-mitochondria signaling in Parkinson's disease.

Mitochondria form close physical contacts with a specialized domain of the endoplasmic reticulum (ER), known as the mitochondria-associated membrane (MAM). This association constitutes a key signaling hub to regulate several fundamental cellular processes. Alterations in ER-mitochondria signaling have pleiotropic effects on a variety of intracellular events resulting in mitochondrial damage, Ca2+ dyshomeostasis, ER stress and defects in lipid metabolism and autophagy. Intriguingly, many of these cellular processes are perturbed in neurodegenerative diseases. Furthermore, increasing evidence highlights that ER-mitochondria signaling contributes to these diseases, including Parkinson's disease (PD). PD is the second most common neurodegenerative disorder, for which effective mechanism-based treatments remain elusive. Several PD-related proteins localize at mitochondria or MAM and have been shown to participate in ER-mitochondria signaling regulation. Likewise, PD-related mutations have been shown to damage this signaling. Could ER-mitochondria associations be the link between pathogenic mechanisms involved in PD, providing a common mechanism? Would this provide a pharmacological target for treating this devastating disease? In this review, we aim to summarize the current knowledge of ER-mitochondria signaling and the recent evidence concerning damage to this signaling in PD.

PINK1 deficiency enhances autophagy and mitophagy induction.

Parkinson's disease (PD) is a neurodegenerative disorder with poorly understood etiology. Increasing evidence suggests that age-dependent compromise of the maintenance of mitochondrial function is a key risk factor. Several proteins encoded by PD-related genes are associated with mitochondria including PTEN-induced putative kinase 1 (PINK1), which was first identified as a gene that is upregulated by PTEN. Loss-of-function PINK1 mutations induce mitochondrial dysfunction and, ultimately, neuronal cell death. To mitigate the negative effects of altered cellular functions cells possess a degradation mechanism called autophagy for recycling damaged components; selective elimination of dysfunctional mitochondria by autophagy is termed mitophagy. Our study indicates that autophagy and mitophagy are upregulated in PINK1-deficient cells, and is the first report to demonstrate efficient fluxes by one-step analysis. We propose that autophagy is induced to maintain cellular homeostasis under conditions of non-regulated mitochondrial quality control.

mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines.

We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes.

IFDOTMETER: A New Software Application for Automated Immunofluorescence Analysis.

Most laboratories interested in autophagy use different imaging software for managing and analyzing heterogeneous parameters in immunofluorescence experiments (e.g., LC3-puncta quantification and determination of the number and size of lysosomes). One solution would be software that works on a user's laptop or workstation that can access all image settings and provide quick and easy-to-use analysis of data. Thus, we have designed and implemented an application called IFDOTMETER, which can run on all major operating systems because it has been programmed using JAVA (Sun Microsystems). Briefly, IFDOTMETER software has been created to quantify a variety of biological hallmarks, including mitochondrial morphology and nuclear condensation. The program interface is intuitive and user-friendly, making it useful for users not familiar with computer handling. By setting previously defined parameters, the software can automatically analyze a large number of images without the supervision of the researcher. Once analysis is complete, the results are stored in a spreadsheet. Using software for high-throughput cell image analysis offers researchers the possibility of performing comprehensive and precise analysis of a high number of images in an automated manner, making this routine task easier.

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.

G2019S LRRK2 mutant fibroblasts from Parkinson's disease patients show increased sensitivity to neurotoxin 1-methyl-4-phenylpyridinium dependent of autophagy.

Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. It is considered as a multifactorial disease dependent on environmental and genetic factors. Deregulation in cell degradation has been related with a significant increase in cell damage, becoming a target for studies on the PD etiology. In the present study, we have characterized the parkinsonian toxin 1-methyl-4-phenylpyridinium ion (MPP(+))-induced damage in fibroblasts from Parkinson's patients with the mutation G2019S in leucine-rich repeat kinase 2 protein (LRRK2) and control individuals without this mutation. The results reveal that MPP(+) induces mTOR-dependent autophagy in fibroblasts. Moreover, the effects of caspase-dependent cell death to MPP(+) were higher in cells with the G2019S LRRK2 mutation, which showed basal levels of autophagy due to the G2019S LRRK2 mutation (mTOR-independent). The inhibition of autophagy by 3-methyladenine (3-MA) treatment reduces these sensitivity differences between both cell types, however, the inhibition of autophagosome-lysosome fusion by bafilomycin A1 (Baf A1) increases these differences. This data confirm the importance of the combination of genetic and environmental factors in the PD etiology. Thereby, the sensitivity to the same damage may be different in function of a genetic predisposition, reason why individuals with certain mutations can develop some early-onset diseases, such as individuals with G2019S LRRK2 mutation and PD.

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.

Autophagy, mitochondria and 3-nitropropionic acid joined in the same model.

Huntington's disease (HD) is a neurodegenerative disorder caused by a mutation in the gene encoding the huntingtin protein. Although the precise mechanism by which neuronal degeneration occurs is still unclear, several elements are important to its development: (1) altered gene expression and protein synthesis, (2) mitochondrial damage and (3) improper regulation of the autophagy programme. In this issue of British Journal of Pharmacology, Galindo and co-workers provide the first evidence for a role of the mitochondrial permeability transition pore (mPTP) in mitochondrial fragmentation and autophagy activation. In a model of cell death induced by 3-nitropropionic acid (3-NP) in human neural cells, the authors describe clear functions for mPTP and Bax, but not the mitochondrial fusion/fission machinery, mitochondrial fragmentation and autophagy (mitophagy). This commentary summarises the significance of this relationship and suggests several points for future development.