Class I PI3-kinase or Akt inhibition do not impair axonal polarization, but slow down axonal elongation.

PI3K proteins family have multiple and essential functions in most cellular events. This family is composed of class I, class II and class III PI3Ks, which upstream and downstream elements are not completely elucidated. Previous studies using the broad PI3K inhibitor, LY294002 allowed to propose that PI3 kinase>Akt pathway is a key element in the determination of axonal polarity in hippocampal neurons. Recently, new inhibitors with a higher selectivity for class I PI3K have been characterized. In the present study we have examined this widely accepted theory using a new class I PI3K inhibitor (GDC-0941), as well as Akt inhibitors, and PTEN phosphatase constructs to reduce PIP3 levels. Our present data show that both, class I PI3K inhibitor and Akt inhibitor did not alter axon specification in hippocampal neurons, but greatly reduced axon length. However, in the same experiments LY294002 effectively impeded axonal polarization, as previously reported. Our biochemical data show that both, class I PI3K and Akt inhibitors, effectively block downstream elements from Akt to S6K1 activity. Both inhibitors are stable in culture medium along the time period analysed, maintaining the inhibition better than LY294002. Besides, we found evidence that LY294002 directly inhibits mTORC1. However, further analysis using an mTORC1 inhibitor showed no change in neuron polarity. Same result was obtained using a general class III PI3K inhibitor. Interestingly, we found that either, wild-type PTEN, or a phosphatase-dead form of PTEN, disrupted axonal polarization, strongly suggesting that the role of PTEN in axonal polarity can be independent of PIP3.

Discovery of compounds that protect tyrosine hydroxylase activity through different mechanisms.

Pharmacological chaperones are small compounds that correct the folding of mutant proteins, and represent a promising therapeutic strategy for misfolding diseases. We have performed a screening of 10,000 compounds searching for pharmacological chaperones of tyrosine hydroxylase (TH), the tetrahydrobiopterin (BH4)-dependent enzyme that catalyzes the rate-limiting step in the synthesis of catecholamines. A large number of compounds bound to human TH, isoform 1 (hTH1), but only twelve significantly protected wild-type (hTH1-wt) and mutant TH-R233H (hTH1-p.R202H), associated to the rare neurological disorder TH deficiency (THD), from time-dependent loss of activity. Three of them (named compounds 2, 4 and 5) were subjected to detailed characterization of their functional and molecular effects. Whereas compounds 2 and 4 had a characteristic pharmacological chaperone (stabilizing) effect, compound 5 protected the activity in a higher extent than expected from the low conformational stabilization exerted on hTH1. Compounds 4 and 5 were weak competitive inhibitors with respect to the cofactor BH4 and, as seen by electron paramagnetic resonance, they induced small changes to the first coordination sphere of the catalytic iron. Molecular docking also indicated active-site location with coordination to the iron through a pyrimidine nitrogen atom. Interestingly, compound 5 increased TH activity in cells transiently transfected with either hTH1-wt or the THD associated mutants p.L205P, p.R202H and p.Q381K without affecting the steady-state TH protein levels. This work revealed different mechanisms for the action of pharmacological chaperones and identifies a subtype of compounds that preserve TH activity by weak binding to the catalytic iron. This article is part of a Special Issue entitled: Cofactor-dependent proteins: Evolution, chemical diversity and bio-applications.

Study of a fetal brain affected by a severe form of tyrosine hydroxylase deficiency, a rare cause of early parkinsonism.

Tyrosine hydroxylase (TH) deficiency is an inborn error of dopamine synthesis. Two clinical phenotypes have been described. The THD "B" phenotype produces a severe encephalopathy of early-onset with sub-optimal L-Dopa response, whereas the "A" phenotype has a better L-Dopa response and outcome. The objective of the study is to describe the expression of key synaptic proteins and neurodevelopmental markers in a fetal brain of THD "B" phenotype. The brain of a 16-week-old miscarried human fetus was dissected in different brain areas and frozen until the analysis. TH gene study revealed the p.R328W/p.T399M mutations, the same mutations that produced a B phenotype in her sister. After protein extraction, western blot analyses were performed to assess protein expression. The results were compared to an age-matched control. We observed a decreased expression in TH and in other dopaminergic proteins, such as VMAT 1 and 2 and dopamine receptors, especially D2DR. GABAergic and glutamatergic proteins such as GABA VT, NMDAR1 and calbindin were also altered. Developmental markers for synapses, axons and dendrites were decreased whereas markers of neuronal volume were preserved. Although this is an isolated case, this brain sample is unique and corresponds to the first reported study of a THD brain. It provides interesting information about the influence of dopamine as a regulator of other neurotransmitter systems, brain development and movement disorders with origin at the embryological state. This study could also contribute to a better understanding of the pathophysiology of THD at early fetal stages.

Bi-nasal sectors of ganglion cells complex and visual evoked potential amplitudes as biomarkers in pituitary macroadenoma management.

The study aimed to evaluate the retinal ganglion cell structure using optical coherence tomography and the visual pathway function employing visual evoked potentials in the diagnosis and monitoring of patients with pituitary macroadenoma. A descriptive, cross-sectional, and longitudinal study (3 and 12 months follow-up) was conducted on forty-two patients. Thirty-five age-matched healthy controls were used in the cross-sectional one. Full neuro-ophthalmological evaluation (structural and functional) was carried out including global and segmented retinal nerve fiber layer/ganglion cell complex analysis and amplitude and latency of P100 component in the electrophysiology. Statistical data analysis was conducted with R version 3.6.3 and Python version 3.8. Associations were evaluated using Spearman's correlations. Amplitude sensitivities were 0.999, and bi-nasal sectors of ganglion cell complex thickness specificities were 0.999. This structural parameter had the highest diagnostic value (area under curve = 0.923). Significant associations were found between bi-nasal sectors with amplitude at 12' (rho > 0.7, p < 0.01) and median deviation of the visual field (rho > 0.5, p < 0.01) at 3 months. Pre-surgical values of bi-nasal sectors and amplitude can predict post-surgically median deviation and amplitude (Oz, 12') at 3 months with r 2 > 0.5. Bi-nasal sectors of ganglion cell complex and visual evoked potentials P100 amplitude are efficient biomarkers of visual pathway damage for pituitary macroadenoma patients' management. Pre-surgical values of the bi-nasal sector and visual evoked potentials' amplitude could help to predict the restoration of parvocellular pathway traffic after decompression.

Solving patients with rare diseases through programmatic reanalysis of genome-phenome data.

Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP's Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics.

DDC expression is not regulated by NFAT5 (TonEBP) in dopaminergic neural cell lines.

The nuclear factor of activated T-cells 5 (NFAT5), also known as tonicity-responsive enhancer-binding protein (TonEBP), is a transcription factor that regulates osmoadaptive response in multiple tissues and is highly expressed in the developing central nervous system. A former study reported that NFAT5 activation through hypertonic stress increases the expression of the dopa decarboxylase enzyme (DDC), also known as aromatic-l-amino-acid decarboxylase (AADC), in human renal proximal tubule cells, leading to an increase of dopamine synthesis. In a previous study, we identified NFAT5 as a candidate gene for cocaine dependence, a complex psychiatric disorder in which dopaminergic neurotransmission plays an important role. Therefore, to test the hypothesis that NFAT5 may also affect dopamine levels in the nervous system through the regulation of DDC expression, we examined this regulation using two neural dopaminergic cell lines, SH-SY5Y and PC12. The effect of NFAT5 on the expression of the neuronal isoform of DDC was evaluated by qRT-PCR. Upon hypertonic stress, NFAT5 was activated and accumulated into the nuclei and, subsequently, the expression of NFAT5 and its known targets sodium/myo-inositol cotransporter 1 (SMIT) and sodium chloride/taurine cotransporter (TAUT) increased, as expected. However, the expression of DDC decreased. When silencing the expression of NFAT5 with a specific shRNA we observed that the downregulation of DDC is independent from NFAT5 in both cell lines and is due to hypertonic stress. In conclusion, NFAT5 does not regulate the expression of the neuronal isoform of DDC in neural dopaminergic cell lines and, consequently, it does not modulate dopamine synthesis through DDC.

The Influence of Gender Inequality in the Development of Job Insecurity: Differences Between Women and Men.

Job insecurity is an indicator of precarious work that refers to the fear of losing one's job. It is a relevant source of stress, with negative consequences on people's mental health. The main objective and contribution of this study is to identify how gender inequality and job insecurity are related, responding to the lack of consensus found in scientific literature in this field of study. To do so, a predictive study of job insecurity, broken down by gender, is developed, considering sociodemographic and labor variables as antecedents. The sample included 1,005 employees (420 men and 585 women) aged between 18 and 65, and a linear regression was conducted for each group. Results show that women perceive greater insecurity under precarious working conditions (temporary work, informal work, salary cuts, tenure), whereas in the case of men variables related to their professional careers (job category, education) and household incomes were relevant predictors. It is concluded that job insecurity affects both gender groups, but the conditions in which this perception grows are significantly impacted by gender inequality. These findings will allow for holistic and effective actions to decrease the effects of precarious work.

Job Insecurity and Mental Health: The Moderating Role of Coping Strategies From a Gender Perspective.

Job insecurity is a growing phenomenon, typical of an employment context characterised by high rates of temporary work and unemployment. Previous research has shown a direct relationship between job insecurity and mental health impairment. The present analysis goes into this relationship in depth, studying the moderating role of coping strategies and predicting that men and women implement different types of strategies. A sample of 1.008 workers is analysed, 588 women and 420 men. The Tobin CSI scale was used to analyse the coping strategies, in addition to JIS-8 to assess job insecurity, the MOS Perceived Social Support Survey and the GHQ-28 test to evaluate mental health. Then, a hierarchical linear regression was designed to study the moderating role of 8 coping strategies of job insecurity and 4 mental health subscales in men and women, separately. Results illustrate that coping strategies play a moderating role in the relationship between job insecurity and mental health. However, the aggravating role of disengagement coping strategies is more relevant than the buffering role of engagement strategies. On the other hand, women implement a greater number of coping strategies, with more positive results for mental health. Also, in the relationship between job insecurity and mental health the most important strategies are the ones related to social interaction inside and outside an organisation, and these are the main ones used by women. It therefore follows that strengthening rich social relationships inside and outside the working environment is a guarantee of well-being.

Assessment of Autophagy in Neurons and Brain Tissue.

Autophagy is a complex process that controls the transport of cytoplasmic components into lysosomes for degradation. This highly conserved proteolytic system involves dynamic and complex processes, using similar molecular elements and machinery from yeast to humans. Moreover, autophagic dysfunction may contribute to a broad spectrum of mammalian diseases. Indeed, in adult tissues, where the capacity for regeneration or cell division is low or absent (e.g., in the mammalian brain), the accumulation of proteins/peptides that would otherwise be recycled or destroyed may have pathological implications. Indeed, such changes are hallmarks of pathologies, like Alzheimer's, Prion or Parkinson's disease, known as proteinopathies. However, it is still unclear whether such dysfunction is a cause or an effect in these conditions. One advantage when analysing autophagy in the mammalian brain is that almost all the markers described in different cell lineages and systems appear to be present in the brain, and even in neurons. By contrast, the mixture of cell types present in the brain and the differentiation stage of such neurons, when compared with neurons in culture, make translating basic research to the clinic less straightforward. Thus, the purpose of this review is to describe and discuss the methods available to monitor autophagy in neurons and in the mammalian brain, a process that is not yet fully understood, focusing primarily on mammalian macroautophagy. We will describe some general features of neuronal autophagy that point to our focus on neuropathologies in which macroautophagy may be altered. Indeed, we centre this review around the hypothesis that enhanced autophagy may be able to provide therapeutic benefits in some brain pathologies, like Alzheimer's disease, considering this pathology as one of the most prevalent proteinopathies.

Specific roles of Akt iso forms in apoptosis and axon growth regulation in neurons.

Akt is a member of the AGC kinase family and consists of three isoforms. As one of the major regulators of the class I PI3 kinase pathway, it has a key role in the control of cell metabolism, growth, and survival. Although it has been extensively studied in the nervous system, we have only a faint knowledge of the specific role of each isoform in differentiated neurons. Here, we have used both cortical and hippocampal neuronal cultures to analyse their function. We characterized the expression and function of Akt isoforms, and some of their substrates along different stages of neuronal development using a specific shRNA approach to elucidate the involvement of each isoform in neuron viability, axon development, and cell signalling. Our results suggest that three Akt isoforms show substantial compensation in many processes. However, the disruption of Akt2 and Akt3 significantly reduced neuron viability and axon length. These changes correlated with a tendency to increase in active caspase 3 and a decrease in the phosphorylation of some elements of the mTORC1 pathway. Indeed, the decrease of Akt2 and more evident the inhibition of Akt3 reduced the expression and phosphorylation of S6. All these data indicate that Akt2 and Akt3 specifically regulate some aspects of apoptosis and cell growth in cultured neurons and may contribute to the understanding of mechanisms of neuron death and pathologies that show deregulated growth.

Análisis ambiental por activación neutrónica de sedimentos de la Bahía de La Habana / A study of environmental analysis of havana bay sediments using neutron activation analysis

RESUMEN Se realizó el análisis por activación neutrónica instrumental de sedimentos superficiales de la bahía de La Habana, Cuba. Se reportaron las concentraciones de 23 elementos (metales pesados y trazas), reportándose, por primera vez un grupo importante de elementos tierras raras (La, Ce, Nd, Sm, Eu, Tb, Yb y Lu). La normalización de los resultados a un metal de referencia demostró la presencia antropogénica de Sb, Ba, As, Cr y Zn producto de la descarga de residuales domésticos e industriales.

ABSTRACT Surface sediments from the Havana Bay (Cuba) were analyzed by instrumental neutron activation analysis (INAA). Concentrations of 23 heavy and trace elements in the sediments were reported, including for the first time, an important group of REE (La, Ce, Nd, Sm, Eu, Tb, Yb and Lu). The normalization of the results for a reference metal shows that an anthropogenic Sb, Ba, As, Cr and Zn inputs are due to discharges of sewage and industrial effluent.