Regulation of patterned dynamics of local exocytosis in growth cones by netrin-1

Axonal guidance and synaptic specification depends on specific signaling mechanisms that occur in growth cones. While several signaling pathways implicated in cone navigation have been identified, membrane dynamics in growth cones remains largely unknown. We took advantage of SynaptopHluorin and high-speed optical recordings to monitor the patterns of membrane dynamics in rat hippocampal growth cones. We show that exocytosis occurs both at the peripheral and central domains, including filopodia, and that SynaptopHluorin signals occur as spontaneous patterned peaks. Such transients average approximately two per minute and last ∼30 s. We also demonstrate that the chemoattractant Netrin-1 elicits increases in the frequency and slopes of these transients, with peaks averaging up to six per minute in the peripheral domain. Netrin-1-dependent regulation of exocytotic events requires the activation of the Erk1/2 and SFK pathways. Furthermore, we show that domains with high SynaptopHluorin signals correlate with high local calcium concentrations and that local, spontaneous calcium increases are associated with higher SynaptopHluorin signals. These findings demonstrate highly stereotyped, spontaneous transients of local exocytosis in growth cones and that these transients are positively regulated by chemoattractant molecules such as Netrin-1

Nystatin Regulates Axonal Extension and Regeneration by Modifying the Levels of Nitric Oxide

Nystatin is a pharmacological agent commonly used for the treatment of oral, mucosal and cutaneous fungal infections. Nystatin has also been extensively applied to study the cellular function of cholesterol-enriched structures because of its ability to bind and extract cholesterol from mammalian membranes. In neurons, cholesterol level is tightly regulated, being essential for synapse and dendrite formation, and for axonal guidance. However, the action of Nystatin on axon regeneration has been poorly evaluated. Here, we examine the effect of Nystatin on primary cultures of hippocampal neurons, showing how acute dose (minutes) of Nystatin increases the area of growth cones, and chronic treatment (days) enhances axon length, axon branching and axon regeneration post-axotomy. We describe two alternative signaling pathways responsible for the observed effects, and activated at different concentrations of Nystatin. At elevated concentrations, Nystatin promotes growth cone expansion through phosphorylation of Akt; whereas at low concentrations, Nystatin enhances axon length and regrowth by increasing nitric oxide levels. Together, our findings indicate new signaling pathways of Nystatin and propose this compound as a novel regulator of axon regeneration

Ubiquitination mediates Kv1.3 endocytosis as a mechanism for protein Kinase C-dependent modulation

The voltage-dependent potassium channel Kv1.3 plays essential physiological functions in the immune system. Kv1.3, regulating the membrane potential, facilitates downstream Ca2+ -dependent pathways and becomes concentrated in specific membrane microdomains that serve as signaling platforms. Increased and/or delocalized expression of the channel is observed at the onset of several autoimmune diseases. In this work, we show that adenosine (ADO), which is a potent endogenous modulator, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytic event that targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. Postsynaptic density protein 95 (PSD-95), a member of the MAGUK family, recruits Kv1.3 into lipid-raft microdomains and protects the channel against ubiquitination and endocytosis. Therefore, the Kv1.3/PSD-95 association fine-tunes the anti-inflammatory response in leukocytes. Because Kv1.3 is a promising multi-therapeutic target against human pathologies, our results have physiological relevance. In addition, this work elucidates the ADO-dependent PKC-mediated molecular mechanism that triggers immunomodulation by targeting Kv1.3 in leukocytes

Blockade of the SNARE Protein Syntaxin 1 Inhibits Glioblastoma Tumor Growth

Glioblastoma (GBM) is the most prevalent adult brain tumor, with virtually no cure, and with a median overall survival of 15 months from diagnosis despite of the treatment. SNARE proteins mediate membrane fusion events in cells and are essential for many cellular processes including exocytosis and neurotransmission, intracellular trafficking and cell migration. Here we show that the blockade of the SNARE protein Syntaxin 1 (Stx1) function impairs GBM cell proliferation. We show that Stx1 loss-of-function in GBM cells, through ShRNA lentiviral transduction, a Stx1 dominant negative and botulinum toxins, dramatically reduces the growth of GBM after grafting U373 cells into the brain of immune compromised mice. Interestingly, Stx1 role on GBM progression may not be restricted just to cell proliferation since the blockade of Stx1 also reduces in vitro GBM cell invasiveness suggesting a role in several processes relevant for tumor progression. Altogether, our findings indicate that the blockade of SNARE proteins may represent a novel therapeutic tool against GB

Blockade of the SNARE protein syntaxin 1 inhibits glioblastoma tumor growth

Glioblastoma (GBM) is the most prevalent adult brain tumor, with virtually no cure, and with a median overall survival of 15 months from diagnosis despite of the treatment. SNARE pro- teins mediate membrane fusion events in cells and are essential for many cellular process- es including exocytosis and neurotransmission, intracellular trafficking and cell migration. Here we show that the blockade of the SNARE protein Syntaxin 1 (Stx1) function impairs GBM cell proliferation. We show that Stx1 loss-of-function in GBM cells, through ShRNA lentiviral transduction, a Stx1 dominant negative and botulinum toxins, dramatically reduces the growth of GBM after grafting U373 cells into the brain of immune compromised mice. In- terestingly, Stx1 role on GBM progression may not be restricted just to cell proliferation since the blockade of Stx1 also reduces in vitro GBM cell invasiveness suggesting a role in several processes relevant for tumor progression. Altogether, our findings indicate that the blockade of SNARE proteins may represent a novel therapeutic tool against GBM

Voltage-dependent Na+ channel phenotype changes in myoblasts. Consequences for cardiac repair

Objective: Cellular cardiomyoplasty using skeletal myoblasts is a promising therapy for myocardial infarct repair. Once transplanted, myoblasts grow, differentiate and adapt their electrophysiological properties towards more cardiac-like phenotypes. Voltage-dependent Na + channels (Na v ) are the main proteins involved in the propagation of the cardiac action potential, and their phenotype affects cardiac performance. Therefore, we examined the expression of Na v during proliferation and differentiation in skeletal myocytes. Methods and results: We used the rat neonatal skeletal myocyte cell line L6E9. Proliferation of L6E9 cells induced Na v 1.4 and Na v 1.5, although neither protein has an apparent role in cell growth. During myogenesis, Na v1.5 was largely induced. Electrophysiological and pharmacological properties, as well as mRNA expression, indicate that cardiac-type Na v1.5 accounts for almost 90% of the Na + current in myotubes. Unlike in proliferation, this protein plays a pivotal role in myogenesis. The adoption of a cardiac-like phenotype is further supported by the increase in Nav 1.5 colocalization in caveolae. Finally, we demonstrate that the treatment of myoblasts with neuregulin further increased Na v 1.5 in skeletal myocytes. Conclusion: Our results indicate that skeletal myotubes adopt a cardiac-like phenotype in cell culture conditions and that the expression of Na v1.5 acts as an underlying molecular mechanism

Results of Nurse Case Management in Primary Heath Care: Bibliographic Review

Background: The new characteristics of today’s population, together with the presence of chronic diseases in the elderly, require a new approach to care, promoting coordination between different levels of care. In this sense, we find the figure of the nurse case manager (NCM) in primary health care mainly responsible for ensuring continuity of care in complex patients with chronic diseases. Objective: to describe the role of the NCM in care management, determining its effectiveness in addressing chronic disease (health outcomes and quality of life) and its efficiency in the health system. Methods: Bibliographic review of scientific evidence on case management applied to nursing. Between March and April 2020 a bibliographic search was carried out in the Dialnet, Scielo, Scopus and Pubmed databases. Inclusion criteria: articles written in the last 5 years, which analyze how this nursing rol influences the care and health of patients. Results: A total of 16 articles were selected. The NCM reduced the use of the emergency department, hospital admissions, readmissions, and the duration of these in the patients studied. Conclusion: The NCM is effective and efficient for both patients and health institutions, and a common practice model is needed that includes standardized protocols and evidence-based practices

FAIM-L regulation of XIAP degradation modulates Synaptic Long-Term Depression and Axon Degeneration

Caspases have recently emerged as key regulators of axonal pruning and degeneration and of long-term depression (LTD), a long-lasting form of synaptic plasticity. However, the mechanism underlying these functions remains unclear. In this context, XIAP has been shown to modulate these processes. The neuron-specific form of FAIM protein (FAIM-L) is a death receptor antagonist that stabilizes XIAP protein levels, thus preventing death receptor-induced neuronal apoptosis. Here we show that FAIM-L modulates synaptic transmission, prevents chemical-LTD induction in hippocampal neurons, and thwarts axon degeneration after nerve growth factor (NGF) withdrawal. Additionally, we demonstrate that the participation of FAIM-L in these two processes is dependent on its capacity to stabilize XIAP protein levels. Our data reveal FAIM-L as a regulator of axonal degeneration and synaptic plasticity

Development of a short questionnaire based on the Practice Environment Scale-Nursing Work Index in primary health care

Background: Professional nursing environments determine the quality of care and patient outcomes. Assessing the quality of environments is essential to improve and obtain better health outcomes. Simplifying and shortening the way to evaluate environments reliably is also important to help nurses better understand the strengths and weaknesses of their environments. In that sense, identifying essential elements of nursing environments would allow the construction of short assessment tools to improve such environments. Objective: To construct a short tool to assess primary health care (PHC) nursing environments based on the Practice Environment Scale-Nursing Work Index (PES-NWI) questionnaire. Methods: Observational, cross-sectional, analytical study (data collection February–April 2015). Tool: PES-NWI (31 items). Population: PHC nurses (three health districts in Valencia, Spain) with more than 3 months in the organization. The nurses were asked to select the 10 elements of the questionnaire (items) that they considered key to facilitate and improve professional care, establishing as a final selection criterion that they obtain a global election >40%. Variables: sociodemographic and 31 questionnaire items. Analysis: descriptive statistics, reliability, multidimensional scaling (ALSCAL), factor analysis, multiple linear regression. Finally, we have analyzed the concordance between both measurements (TOP10 score on the full scale score) using the Bland–Altman method. Results: Study sample = 269 (Response rate = 80.29%). A total of 10 elements were identified based on selection frequency of the questionnaire PES-NWI. A factorial analysis explained 62.1% of variance, internal structure of three dimensions: (1) Participation in leadership and management, (2) Nursing foundations for quality of care, (3) Adequacy of resources, with Accumulate Variance explained: Component (1): 24%; Component (2): 43.1%; Component (3): 62.1%. Reliability (Cronbach’s Alpha) was 0.816 for short questionnaire, and >0.8 for all measurements. Stress = 0.184 and RSQ = 0.793. Bland–Altman method: the scaling tends to be 1.92 points higher (equivalent to a maximum deviation of 1.54%) than the full-scale PES-NWI score (max score on PES-NWI = 124 points). Conclusions: It is possible to identify essential elements of environments to construct a short tool that simplifies the study of PHC environments. Conducting rapid studies of environments will provide managers with information about specific elements that require prioritization to enhance quality of care and safety

Caveolin interaction governs Kv1.3 lipid raft targeting

The spatial localization of ion channels at the cell surface is crucial for their functional role. Many channels localize in lipid raft microdomains, which are enriched in cholesterol and sphingolipids. Caveolae, specific lipid rafts which concentrate caveolins, harbor signaling molecules and their targets becoming signaling platforms crucial in cell physiology. However, the molecular mechanisms involved in such spatial localization are under debate. Kv1.3 localizes in lipid rafts and participates in the immunological response. We sought to elucidate the mechanisms of Kv1.3 surface targeting, which govern leukocyte physiology. Kv1 channels share a putative caveolin-binding domain located at the intracellular N-terminal of the channel. This motif, lying close to the S1 transmembrane segment, is situated near the T1 tetramerization domain and the determinants involved in the Kvβ subunit association. The highly hydrophobic domain (FQRQVWLLF) interacts with caveolin 1 targeting Kv1.3 to caveolar rafts. However, subtle variations of this cluster, putative ancillary associations and different structural conformations can impair the caveolin recognition, thereby altering channel's spatial localization. Our results identify a caveolin-binding domain in Kv1 channels and highlight the mechanisms that govern the regulation of channel surface localization during cellular processes