The energetic brain - A review from students to students.

The past 20 years have resulted in unprecedented progress in understanding brain energy metabolism and its role in health and disease. In this review, which was initiated at the 14th International Society for Neurochemistry Advanced School, we address the basic concepts of brain energy metabolism and approach the question of why the brain has high energy expenditure. Our review illustrates that the vertebrate brain has a high need for energy because of the high number of neurons and the need to maintain a delicate interplay between energy metabolism, neurotransmission, and plasticity. Disturbances to the energetic balance, to mitochondria quality control or to glia-neuron metabolic interaction may lead to brain circuit malfunction or even severe disorders of the CNS. We cover neuronal energy consumption in neural transmission and basic ('housekeeping') cellular processes. Additionally, we describe the most common (glucose) and alternative sources of energy namely glutamate, lactate, ketone bodies, and medium chain fatty acids. We discuss the multifaceted role of non-neuronal cells in the transport of energy substrates from circulation (pericytes and astrocytes) and in the supply (astrocytes and microglia) and usage of different energy fuels. Finally, we address pathological consequences of disrupted energy homeostasis in the CNS.

UBR1 Promotes Sex-Dependent ACE2 Ubiquitination in Hypertension.

Background: Angiotensin (Ang)-II impairs the function of the antihypertensive enzyme ACE2 by promoting its internalization, ubiquitination and degradation thus contributing to hypertension. However, few ACE2 ubiquitination partners have been identified and their role in hypertension remains unknown. Methods: Proteomics and bioinformatic analysis were used to identify ACE2 ubiquitination partners in the brain, heart, and kidney from Ang-II-infused C57BL6/J mice from both sexes and validated the interaction between UBR1 and ACE2 in cells. Central and peripheral UBR1 knockdown was then performed in male mice to investigate its role in the maintenance of hypertension. Results: Proteomics analysis from hypothalamus identified UBR1 as a potential E3 ligase promoting ACE2 ubiquitination. Enhanced UBR1 expression, associated with ACE2 reduction, was confirmed in various tissues from hypertensive male mice and human samples. Treatment of endothelial and smooth muscle cells with testosterone, but not 17ß-estradiol, confirmed a sex-specific regulation of UBR1. In vivo silencing of UBR1 using chronic administration of small interference RNA resulted in the restoration of ACE2 levels in hypertensive males. A transient decrease in blood pressure following intracerebroventricular, but not systemic, infusion was also observed. Interestingly, UBR1 knockdown increased the brain activation of Nedd4-2, an E3 ligase promoting ACE2 ubiquitination and reduced expression of SGK1, the kinase inactivating Nedd4-2. Conclusions: These data demonstrate that UBR1 is a novel ubiquitin ligase targeting ACE2 in hypertension. UBR1 and Nedd4-2 E3 ligases appear to work synergistically to ubiquitinate ACE2. Targeting of these ubiquitin ligases may represent a novel strategy to restore ACE2 compensatory activity in hypertension.

Investigating serum extracellular vesicles in Cystic Fibrosis.

BACKGROUND: Extracellular vesicles (EVs) are emerging as biomarkers of disease with diagnostic potential in CF. With the advent of highly effective modulator therapy, sputum production is less common and there is a need to identify novel markers of CF disease progression, exacerbation and response to therapies in accessible fluids such as serum. METHODS: We used size exclusion chromatography (SEC) to isolate and characterise EVs from the blood of PWCF of different ages and compared to ultracentrifugation (UC). We used nanoparticle tracking analysis to measure the number of EVs present in serum obtained from children and adults with CF. Mass spectrometry based proteomics was used to characterise protein expression changes between the groups. RESULTS: EVs were successfully isolated in SEC fractions from 250 µl serum from PWCF in greater numbers (p <0.01) than density ultracentrifugation. There was not a significant difference in EV numbers between young children with CF and controls. However, there was significantly more EVs in adults compared to children (<6yrs) (p < 0.05). EVs from PWCF before and after Kaftrio treatment were also analysed. Significant protein expression changes were observed within all 3 group. The largest changes detected were between children and adults with CF (57 proteins had a 1.5 fold change in expression with 19 significant changes p < 0.05) and PWCF taking Kaftrio (24 significant changes in EV protein expression was observed 12 months post treatment). CONCLUSION: In this pilot study, we performed an initial characterisation of EVs in serum from PWCF demonstrating the potential of serum EVs for further diagnostic investigation.

Nedd4-2 up-regulation is associated with ACE2 ubiquitination in hypertension.

AIMS: Angiotensin-converting enzyme 2 (ACE2) is a critical component of the compensatory renin-angiotensin system that is down-regulated during the development of hypertension, possibly via ubiquitination. However, little is known about the mechanisms involved in ACE2 ubiquitination in neurogenic hypertension. This study aimed at identifying ACE2 ubiquitination partners, establishing causal relationships and clinical relevance, and testing a gene therapy strategy to mitigate ACE2 ubiquitination in neurogenic hypertension. METHODS AND RESULTS: Bioinformatics and proteomics were combined to identify E3 ubiquitin ligases associated with ACE2 ubiquitination in chronically hypertensive mice. In vitro gain/loss of function experiments assessed ACE2 expression and activity to validate the interaction between ACE2 and the identified E3 ligase. Mutation experiments were further used to generate a ubiquitination-resistant ACE2 mutant (ACE2-5R). Optogenetics, blood pressure telemetry, pharmacological blockade of GABAA receptors in mice expressing ACE2-5R in the bed nucleus of the stria terminalis (BNST), and capillary western analysis were used to assess the role of ACE2 ubiquitination in neurogenic hypertension. Ubiquitination was first validated as leading to ACE2 down-regulation, and Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) was identified as a E3 ligase up-regulated in hypertension and promoting ACE2 ubiquitination. Mutation of lysine residues in the C-terminal of ACE2 was associated with increased activity and resistance to angiotensin (Ang)-II-mediated degradation. Mice transfected with ACE2-5R in the BNST exhibited enhanced GABAergic input to the paraventricular nucleus (PVN) and a reduction in hypertension. ACE2-5R expression was associated with reduced Nedd4-2 levels in the BNST. CONCLUSION: Our data identify Nedd4-2 as the first E3 ubiquitin ligase involved in ACE2 ubiquitination in Ang-II-mediated hypertension. We demonstrate the pivotal role of ACE2 on GABAergic neurons in the maintenance of an inhibitory tone to the PVN and the regulation of pre-sympathetic activity. These findings provide a new working model where Nedd4-2 could contribute to ACE2 ubiquitination, leading to the development of neurogenic hypertension and highlighting potential novel therapeutic strategies.

ADAM17-Mediated Shedding of Inflammatory Cytokines in Hypertension.

The increase of Angiontesin-II (Ang-II), one of the key peptides of the renin-angiotensin system (RAS), and its binding to the Ang-II type 1 receptor (AT1R) during hypertension is a crucial mechanism leading to AD\AM17 activation. Among the reported membrane anchored proteins cleaved by ADAM17, immunological cytokines (TNF-α, IFN-γ, TGF-ß, IL-4, IL-10, IL-13, IL-6, FKN) are the major class of substrates, modulation of which triggers inflammation. The rise in ADAM17 levels has both central and peripheral implications in inflammation-mediated hypertension. This narrative review provides an overview of the role of ADAM17, with a special focus on its cellular regulation on neuronal and peripheral inflammation-mediated hypertension. Finally, it highlights the importance of ADAM17 with regards to the biology of inflammatory cytokines and their roles in hypertension.

Soluble epoxide hydrolase inhibitor, APAU, protects dopaminergic neurons against rotenone induced neurotoxicity: Implications for Parkinson's disease.

Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid, play a crucial role in cytoprotection by attenuating oxidative stress, inflammation and apoptosis. EETs are rapidly metabolised in vivo by the soluble epoxide hydrolase (sEH). Increasing the half life of EETs by inhibiting the sEH enzyme is a novel strategy for neuroprotection. In the present study, sEH inhibitors APAU was screened in silico and further evaluated for their antiparkinson activity against rotenone (ROT) induced neurodegeneration in N27 dopaminergic cell line and Drosophila melanogaster model of Parkinson disease (PD). In the in vitro study cell viability (MTT and LDH release assay), oxidative stress parameters (total intracellular ROS, hydroperoxides, protein oxidation, lipid peroxidation, superoxide dismutase, catalase, glutathione peroxidise, glutathione reductase, glutathione, total antioxidant status, mitochondrial complex-1activity and mitochondrial membrane potential), inflammatory markers (IL-6, COX-1 and COX-2), and apoptotic markers (JNK, phospho-JNK, c-jun, phospho-c-jun, pro and active caspase-3) were assessed to study the neuroprotective effects. In vivo activity of APAU was assessed in Drosophila melanogaster by measuring survival rate, negative geotaxis, oxidative stress parameters (total intracellular ROS, hydroperoxides, glutathione levels) were measured. Dopamine and its metabolites were estimated by LC-MS/MS analysis. In the in silico study the molecule, APAU showed good binding interaction at the active site of sEH (PDB: 1VJ5). In the in vitro study, APAU significantly attenuated ROT induced changes in oxidative, pro-inflammatory and apoptotic parameters. In the in vivo study, APAU significantly attenuates ROT induced changes in survival rate, negative geotaxis, oxidative stress, dopamine and its metabolites levels (p < 0.05). Our study, therefore, concludes that the molecule APAU, has significant neuroprotection benefits against rotenone induced Parkinsonism.

Evaluation of antiparkinson activity of PTUPB by measuring dopamine and its metabolites in Drosophila melanogaster: LC-MS/MS method development.

Soluble epoxide hydrolase (sEH) inhibition is reported to elevate endogenous epoxyeicosatrienoic acids (EET's), which are known to play an important role in neuroprotection by inhibiting oxidative stress and neuroinflammation. In the present study, PTUPB, a dual inhibitor of sEH and COX-2, has been tested for its antiparkinson activity against rotenone (ROT) induced neurodegeneration in Drosophila model of Parkinson's disease (PD). To determine the efficacy and brain bioavailability of PTUPB a simple, rapid and sensitive LC-MS/MS method was developed and validated for the estimation of PTUPB (Method-I), dopamine (DA) and its metabolites (Method-II) in fly head. Mass spectrometric acquisitions of analytes signals were performed in positive and negative electron spray ionization MRM mode by monitoring the daughter ions. The isocratic elution using formic acid (0.1% v/v) and acetonitrile (20:80v/v) (for method I), and acetic acid (0.1% v/v) and methanol (for method II) on Jones C18 was carried out to achieve the separation. The results of brain PTUPB, DA and its metabolites estimation shows a dose dependent increase in PTUPB concentration and a dose dependent prevention of ROT induced changes in DA and its metabolites levels (p<0.05), indicating a significant neuroprotection activity of PTUPB. In the present study, we have successfully developed and validated LC-MS/MS methods to identify and quantify PTUPB, DA and its metabolites using a UFLC-ESI-QqQ mass spectrometer for the screening of neuroprotective agents in Drosophila Melanogaster.

Possible role of Epoxyeicosatrienoic acid in prevention of oxidative stress mediated neuroinflammation in Parkinson disorders.

Parkinson's disease (PD) is a multifactorial neurodegenerative disease involving oxidative stress, neuroinflammation and apoptosis. Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites and they play a role in cytoprotection by modulating various cell signaling pathways. This cytoprotective role of EETs are well established in cerebral stroke, cardiac failure, and hypertension, and it is due to their ability to attenuate oxidative stress, endoplasmic reticulum stress, inflammation, caspase activation and apoptosis. The actions of EETs in brain closely parallel the effects which is observed in the peripheral tissues. Since many of these effects could potentially contribute to neuroprotection, EETs are, therefore, one of the potential therapeutic candidates in PD. Therefore, by increasing the half life of endogenous EETs in vivo via inhibition of sEH, its metabolizing enzyme can, therefore, constitutes an important therapeutic strategy in PD.